Nephrol. Dial. Transplant.-1999-NDTAbstracts-A17-31

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Nephrol. Dial. Transplant.-1999-NDTAbstracts-A17-31 Powered By Docstoc
					                                                           Immunology, immunopathology and pathology
                                                                       (basic and clinical)                                                                    Abstracts

 E: Immunolgy, immunopathology and                                                                  HEPATITIS        C      VIRUS        (HCV)
                                                                                                    IMMUNECOMPLEX (IC) GLOMERULAR DISEASE IN PA-
                                                                                                                                                ASSOCIATED

 pathology (basic and clinical)                                                                     TIENTS (PTS) COINFECTED WITH HIV
                                                                                                    G. Barbiano di Belgiojoso, A. Genderini, N. Landriani, M.T.
                                                                                                    Barone, D. Scorza, S. Bertoli, M. Trezzi
CORRELATION BETWEEN TNM STATUS AND IMMUNOMORPHOLOGY                                                 Renal Unit, L. Sacco Hospital, Milan, Italy
OF RENAL CELL CARCINOMA
D Brasanac1, J Markovic-Lipkovski1, GA Müller2, CA Müller3                                          The presence of HCV infection in pts with HIV coinfection and
1
 Institute of Pathology, University Medical School, Belgrade, Yugoslavia; 2De-                      glomerular involvement has been indicated as responsible of
partment of Nephrology and Rheumatology, Georg August University, Göttingen,                        nephropathy. The association of HCV infection and IC GN in
Germany; 3Section of Transplantation, Immunology and Immunohematology,                              HIV-infected pts has been reviewed. 9 cases have been selected,
Eberhard-Klaus University, Tübingen, Germany                                                        all submitted to renal biopsy. All patients were white, 8 intrave-
                                                                                                    nous drug abusers (IDA), one homosexual. Age was between
TNM status is one of the most reliable parameters for tumor prognosis. In this                      33 and 37 years (mean 34), 4 were males. 7 pts presented with
work we investigated possible associations between TNM status and various                           nephrotic syndrome, 2 with proteinuria and microscopic
immunological characteristics of renal cell carcinoma (RCC).                                        hematuria. Low C4 was present in 3 cases, cryoglobulins (cryo)
Cryostat sections of 37 RCC (25 clear cell type, 10 granular and 2 chromophobe)                     in 1/7 tested cases. At light microscopy 5 pts showed
were analyzed with indirect immunoperoxidase technique using monoclonal                             membranoproliferative GN (among which 3 lupus-like), 2 dif-
antibodies to: HLA class I (HLA-ABC) and class II (HLA-DR, -DP, -DQ) anti-                          fuse endocapillary proliferative GN, one IgAGN, one
gens, ICAM-1 (CD54), CD3, CD14, CD4 and CD8 molecules.                                              immunotactoid GN. At immunofluorescence (performed in all
Comparing to T1/T2 cases, T3/T4 tumors showed higher level of HLA class II                          cases) 5 with membranoproliferative GN had intense parietal
antigens (especially HLA-DQ), more frequently reduced HLA class I level (50%                        deposits of IgG, IgM and C3. Endocapillary forms had promi-
of T3/T4 versus 14% of T1/T2), widespread ICAM-1 expression, prevalence of                          nent parietal C3 deposits, IgA-GN mesangial deposits of IgA,
T lymphocytes over monocytes among tumor infiltrating mononuclear cells                             IgG and C3. Tactoid GN had IgG deposits in multiple sites,
and CD4/CD8 ratio about one. Similar differences were observed between M1                           mainly subepithelial. Electron microscopy (performed in all cases)
and M0 RCC, with even more pronounced ICAM-1 presence and absence of                                confirmed the presence of large dense deposits in multiple sites,
CD8+ T lymphocytes domination in all M1 tumors (comparing to 15% of cases                           without cryo-type structurated deposits. The clinical and histo-
with such domination among M0 tumors). Possible explanations for aforemen-                          logical aspects of these 9 pts were different from mixed cryo
tioned findings could be anergy of T lymphocytes induced by tumor cells due to                      GN, HCV related: no history of purpura or Raynaud phenom-
a lack of necessary co-stimulatory molecules (e.g. B7), blocking activity of sheded                 ena, absence of monocytes and structurated deposits, rarity of
ICAM-1 molecules, or reduced antitumor action of CD8+ (presumably cyto-                             C4 reduction and serum cryo.
toxic) T lymphocytes on cells with reduced level of HLA class I antigens.                           In conclusion, the clear-cut difference between IC GN in HCV-HIV
Immunohistochemical analysis of RCC suggests that immune response in T3/                            pts and cryo-GN indicates for the former a mechanism other
T4 and M1 tumors is altered both quantitatively and functionally comparing to                       than cryo formation, possibly related to HIV viral antigens. The
T1/T2 and M0 cases.                                                                                 strict correlation of HCV with drug abuse suggests that HCV
                                                                                                    may represent a marker of such risk group rather than having a
                                                                                                    pathogenetic role.



EFFECT OF PENTOXIFYLLINE ON AUTOIMMUNE GLOMERULONEPHRI-                                             GLUTATHIONE S-TRANSFERASE ISOENZYME PROFILE IN
TIS IN BROWN NORWAY RATS.                                                                           NORMAL HUMAN KIDNEY AND RENAL CELL CARCI-
A Wystrychowski1, F Kokot1, H I Trzeciak2 .                                                         NOMA
1 Dept. of Nephrology, Endocrinology and Metabolic Diseases, 2 Dept. of                             T Simic1, J Mimic-Oka1, Z Reljic1, D Dragicevic2, P Dragicevic2
                                                                                                    1
Pharmacology, Silesian University School of Medicine, Katowice, Poland                               Institute of Biochemistry and 2Institute of Urology, Faculty of
                                                                                                    Medicine, University of Belgrade, Yugoslavia.
Pentoxifylline (PTX), a non-selective phosphodiesterase inhibitor, shows
immunosuppressive properties. It reduces the production of TNFα, IL 12 and                          The exposure of kidney cells to the toxic metabolites of various
IFNγ as well as ICAM-1 and IL 2 receptor expression and inhibits proliferation                      origins, which may have carcinogenic potential, is supposed to
of mesangial cells and formation of collagen.                                                       play role in the initiation of renal cell carcinoma. Glutathione S–
Thus it may be expected that PTX could exert a beneficial effect on glomerulone-                    transferases (GSTs), a family of detoxification enzymes, play a
phritis. To test this hypothesis we compared the severity of autoimmune glomeru-                    critical role in protecting the kidney by catalyzing conjugation of
lonephritis (AGN) induced in male Brown Norway (BN) (230-340 g) rats treated                        different carcinogens with glutathione.
with PTX (100 mg/kg b.w., s.c., every 8 hours for 2 weeks) with a control group,                    GST isoenzyme profile was investigated in normal human kid-
which received the same volume of saline. AGN was evoked following a stand-                         ney, different renal tumors and corresponding kidney tissue
ard protocol (HgCl , 1 ml/kg b.w., s.c., every 2nd day for a total of 2 weeks).                     adjacent to human renal tumors. Enzyme purification was per-
                        2
After 2 weeks of HgCl and PTX treatment (as proteinuria reached a plateau                           formed by using affinity chromatography and isoelectric
                          2
level) several renal function parameters [plasma urea concentration (P , mmol/                      chromatofocusing.
                                                                        urea
l), proteinuria (U , mg/24 h/100 g b.w.), albuminuria (U , mg/24 h/100 g                            Purification of normal kidneys’ GSTs by affinity chromatogra-
b.w.), albumin clearance (C , µl/min/100 g b.w.), urine protein-to-creatinine
                     prot                                       alb
                                                                                                    phy revealed the presence of two GST fractions: flow–throught
ratio (U /1 µl C ) and kidney weight (g/100 g b.w.)] were assessed. Two rats
                               alb
                                                                                                    GST (26-28%), with lower affinity for GSH linked to epoxy–
         prot        cr
of the control group died in metabolic cages and 5 rats of the PTX group had                        activated agarose affinity resin and GST fraction tightly bound
cystitis with retention of urine. These animals were excluded from the statisti-                    to affinity matrix. Further purification of bound GSTs resulted
cal analysis.                                                                                       in a rich profile of different GST isoenzymes with balanced ex-
group      N     U          U         C         kidney P         U µmol/    P        U              pression of both anionic and cationic forms. The results ob-
                     prot       alb       alb         wt urea      Ca         Ca      prot
                                                g/100g           24h/100g   mmol/l    /1µl C
                                                                                               Cr   tained suggest efficient cellular anti-carcinogenic potential in
Control   13     68         25        2,17 0,85          25,4    6,2        2,12     171            normal kidney. However, kidney tissue adjacent to human renal
          (15)   ±31        ±13       ±1,46 ±0,17        ±9,4    ±3,7       ±0,10          ±124
                                                                                                    tumors had substantially less flow-through GST fraction (1-
PTX       10     21^* 7 *             0,38^ 0 , 7 3 #    13,8    15,6^      2,37#       52          4%), whereas renal tumors did not express flow-through GST at
          (15)   ±19 ±8               ±0,55 ±0,11        ±13,6   ±10,2      ±0,06    ±45            all. Isoelectric focussing indicated significantly smaller number
                                                                                                    of GST isoenzymes in non-tumor kidney regions when com-
MEAN±SD # p<0,05, * p<0,01, ^ p<0,002 (U Mann-Whitney test) (N)=initial numbers
                                                                                                    pared to normal kidney, with anionic forms being dominant.
                                                                                                    Isoenzyme profile of renal carcinoma tissue differed significantly
As seen on the table PTX alleviates the severity of the disease. These findings
                                                                                                    from both non-tumor adjacent renal tissue and normal kidney.
suggest that PTX could be of benefit in the treatment of glomerulonephritis.
                                                                                                    The GST pattern in renal cell carcinoma showed the predomi-
                                                                                                    nance of anionic GST forms.
                                                                                                    Based on the results obtained, it can be concluded that patients
                                                                                                    with renal cell carcinoma have defective anti-carcinogenic po-
                                                                                                    tential due to the qualitative changes in GST expression.




Nephrology Dialysis Transplantation                                           Vol. 14 n.9                          1999                                                   A17
                                     Immunology, immunopathology and pathology
Abstracts                                        (basic and clinical)


      LIPOPOLYSACCHARIDE-INDUCED CHEMOKINE GENE                             CAPTOPRIL VERSUS HYDRALAZIN AND MORPHOLOGIC CHANGES IN
      EXPRES-SION IN MESANGIAL CELLS                                        SHRWITHADRIAMYCINNEPHROPATHY
      1
       H Rim, 2JW Park                                                      D.B. Jovanovic1, Lj. Djukanovic1, J. Dimitrijevic1, A. Starevic1, M. Jerkic3, Dj.
      1
       Dept. of Internal Medicine, Kosin Univ. Gospel Hospital,             Jovovic3, J. Varagic3, Z. Dragojlovic2
      Pusan, Korea, 2Dept. of Immunology, Keimyung Univ.,                   Clinic of Nephrology1 and Medical Biochemistry2, Clinic Center of Serbia, and
      Taegu, Korea                                                          Institute for Medical Research3,Belgrade, Yugoslavia

      This study was designed to investigate the molecular mecha-           Antihypertensive therapy has been shown as one of the ways of slowing down
      nism of chemokine induction by lipopolysaccharide (LPS) in the        the progression of chronic renal failure (CRF). The most important factor for
      courses of infections. Chemokine gene expression was evalu-           slowing down CRF progression is to regulate blood pressure independently of
      ated by the RT-PCR assay using RNAs isolated either from              kind of antihypertensive drugs, as shown in our recent study (Clin Nephrol
      kidneys of LPS-injected mice or from the mesangial cells stimu-       1998;50(6):390-392). The aim of this study was to compare effects of captopril
      lated with LPS. Chemokine biological activity was also demon-         (C) and hydralazin (H) on morphologic changes in spontaneously hypertensive
      strated by the chemotaxis assay. LPS was shown to induce di-          rats (SHR) with adriamycin (ADR) nephropathy.
      rectly IFN-γ inducible protein 10 (IP-10) and monokine induced        Adult female SHR were divided into four groups: 1. Control group: 12 SHR; 2.
      by interferon gamma (MIG) through mesangial cells. As the LPS         ADR group: 27 SHR treated with ADR (2mg/kg i.v. twice in 20 days); 3. ADR-
      induction of IL-12 gene expression was shown previously in the        C group: 30 SHR treated with ADR and thereafter with C (60mg/kg/day); 4.
      mesangial cells, chemokine cascade could be induced also              ADR-H group: 17 SHR treated with ADR and thereafter with H (6mg/kg/day).
      through IL-12 pathway. To evaluate this pathway, normal and           Rats were sacrificed at week 18 after second ADR injection and hystologic analy-
      gamma interferon knock-out (GKO) mice were injected with IL-          sis was semiquantitatively performed by calculation of glomerular index, vas-
      2 and/or IL-12. MIG and IP-10 gene expression was detected in         cular index and index of interstitial fibrosis and tubular atrophy.
                     ,
      normal kidney however, absent in the GKO mouse kidney indi-           Both antihypertensive agents normalized systemic blood pressure, but failed to
      cating that chemokine induction was mediated through                  prevent proteinuria. H was significantly effective than C in slowing down
      mesangial IL-12 and IFN- γ. It was also demonstrated that IFN-        glomerular changes in ADR SHR (ADR vs ADR-C vs ADR-H vs Control = 5.00
      γ alone induce these chemokines in the mesangial cell culture         vs 3.66 vs 2.23 vs 0.14) (p<0.001). The more severe changes in arterial blood
      system. Furthermore, sodium salicylate, wortmanin and pip-            vessels in week 18 occurred in control group and the difference was significant
      erazine were added to the mesangial cell culture system to evalu-     as compared to all other groups (p<0.001). In ADR-H group changes in arterial
      ate LPS signalling pathway. All of these agents blocked LPS           blood vessels were significantly lower than in ADR-C group and similar to
      mediated chemokine induction suggesting the activation of nu-         ADR group (2.20 vs 2.90 vs 2.14 vs 3.40). C failed to prevent tubular atrophy in
      clear factor-κ B pathway.                                             ADR SHR, but H reduced tubular changes significantly (2.44 vs 2.20 vs 1.64 vs
      It is concluded from this study that mesangial cells are the          1.00). H was significantly effective than C in slowing down interstitial changes
      target of LPS in the renal failure resulting from the systemic        in ADR SHR (3.22 vs 2.40 vs 1.45 vs 0.14).
      infections. LPS induces chemokines directly and/or IL-12 in the       So, we can conclude that hydralazine reduced morphological changes in ADR
      mesangial cells. Mesangial IL-12 may activate T cells which           SHR more significantly than captopril.
      induce also chemokine cascade and inflammation ultimately.




      CLINICAL SIGNIFICANCE OF THE RENAL EXPRESSION                         URINARY EXCRETION OF VASCULAR ENDOTHELIAL
      OF ICAM-1 IN IgA NEPHROPATHY (IgAN)                                   CELL GROWTH FACTOR (VEGF) IN MEMBRANOUS
      P Arrizabalaga, C Ascaso, M Solé, X Cuevas1, J Soler2, J Pascual3,    GLOMERULONEPHRITIS (MGN)
      A Darnell
                                                                            E. Honkanen, A-M. Teppo, C. Grönhagen-Riska
      S. of Nephrology, Pathology, Biostatistical Unit, Hospital Clinic,
      Barcelona; Hospital of Tarrasa1, Manresa2, Clinica Gerona3. Spain.    Helsinki University Central Hospital, Department of Medicine,
                                                                            Division of Nephrology, Helsinki, Finland
      Introduction: abnormal ICAM-1 expression on proximal tubule
      epithelium is associated with infiltration of T-cells and             VEGF is a dimeric glycoprotein which increases vascular perme-
      macrophages in IgAN (Arch Pathol Lab Med 1998; 122: 817-822).         ability, stimulates angiogenesis, and protease activity. It is nor-
      Objective: to analyze the relation between the tubular and inter-     mally expressed by podocytes and glomerular endothelial cells
      stitial expression of ICAM- I and the renal disfunction in the
                                                                            but its role in the pathophysiology of proteinuria and glomeru-
      IgAN.
      Material and methods: in 32 patients with IgAN, 39 ± 16 (x ±          lar diseases is unclear.
      SD) years old, protenuria (Pr) 2 ± 1.8 g/24h and creatinine (Cr)      By using a novel sandwich enzyme immunoassay we measured
      in serum 1.76 ± 1.12 mg/dI, we have assessed the tubular and          urinary VEGF excretion in 30 patients with idiopathic MGN, 9
      interstitial expression of intercellular adhesion molecule-1          with minimal change (MC) glomerulopathy, 8 with necrotizing
      (ICAM-1)       with     monoclonal       antibody      CD54     by    glomerulonephritis associated with systemic vasculitis (VAS),
      avidin-biotin-peroxidase and we analyzed their relation with          12 with diabetic nephropathy (DNP), and 33 normal controls.
      the Pr and Cr at the moment of renal biopsy and after 2.4 ± 2
                                                                            The mean (±SE) urinary VEGF was significantly lower in MGN
      years. An increase ≥ 50 % over the initial levels was considered as
      progressive disease.                                                  (16±3 [95%CI 10 to 23] ng/mmol crea) than in the normal con-
      Results: The ICAM-1+ stain on proximal tubule epithelium was          trols (68±10 [CI 49 to 88] ng/mmol crea; P< 0.0001 ANOVA).
      seen in 13 patients, the median value being 0. 11 ± 0. 18 mm2 /       In MC and DNP the excretion was unchanged (55±14 [CI 24 to
      mm2 of tubule. The number of ICAM-1+ interstitial leukocytes          86] and 101±25 [CI 45 to 156] ng/mmol crea, respectively;
      was 234 ± 307 / mm 2 of interstitium. The Pr was 2.7 ± 1.5 g/         P=NS) whereas VAS patients had elevated VEGF excretion
      24 h in the patients with ICAM-1+ tubular expression versus 1.5       (184±68 [CI 24 to 344] ng/mmol crea; P<0.01) compared to the
      ± 1.8 g/24 h (U=44, p=0.005) in the patients without. Correla-
                                                                            normal controls. In 17 MGN patients followed up for 12 months
      tion was found between the ICAM-1+ tubular expression and
      the Pr (r=0.4059, p=0.02). ICAM-1 + interstitial leukocytes were      decreasing proteinuria paralelled increasing urinary VEGF while
      379.3 ± 371 / mm2 in the patients with HBP and 108 ± 164.3 /          persistent nephrotic syndrome was associated with stable or
      mm2 in the patients without (U=52, p=0.03). Correlation was           decreasing VEGF excretion (D proteinuria vs. D VEGF: r=-0.51,
      found between the ICAM-1+ interstitial expression and the Cr          P=0.003).
      (r=0,6343, p<0,001), ICAM-1+ interstitial leukocytes were 516 ±       In conclusion, urinary VEGF excretion is decreased in MGN but
      3 60 / mm2 in the patients with increase ≥ 50 % in Cr and 66          not in the other glomerular diseases studied and decreasing
      ±87,8 / mm2 in the patients without (U= 16, p=0. 004).
                                                                            clinical activity (proteinuria) is associated with increasing VEGF
      Conclusions: the tubular and interstitial ICAM-1+ expression
      would reflect the seventy of the renal disturbance in IgAN.           excretion. Changes in VEGF excretion may indicate reversible
      ICAM-1 interstitial more than ICAM-1 tubular can play a role          podocyte injury in MGN.
      as marker of progression in this disease.



A18                                               Nephrology Dialysis Transplantation                        Vol. 14 n.9                             1999
                                   Immunology, immunopathology and pathology
                                               (basic and clinical)                                                            Abstracts

HEMORRHAGIC FEVER WITH RENAL SYNDROME                                 PROTECTIVE MECHANISM OF HEME OXYGENASE
S. Susa, Lj. Susa
Serbian Academy of Sciences and Arts, Belgrade, Yugoslavia
                                                                      (HO)-1 IN HEMIN-INDUCED CELL INJURY
                                                                      T Toma, A Yachie, K Ohta, Y Kasahara, S Koizumi
Hemorrhagic fever with a renal syndrome (HFRS) is spread over         Dept. of Pediatr. and Dept. of Lab. Sci., Faculty of Med.,
many countries of the world. In Yugoslavia hemorrhagic fever          Kanazawa University, Kanazawa, Japan
has been found and described some time ago. We detected the
first epidemic in our country in 1961 in a military unit. In our
country hemorrhagic fever with renal syndrome which corre-            HO-1 is an inducible form of HO and it is known to protect
sponds to Corean type of hemorrhagic fever is most frequently         renal damage through its anti-oxidant effect. However, pre-
met. Our to date investigations of the HFRS in 350 pts
pathogenesis point out that immunological mechanisms are
                                                                      cise protective role of HO-1 in stress-induced cell damage is
moss important in development of this disease. We have noted          largely unknown. We established a lymphoblastoid cell line
the increase of IgG, IgM, IgD and IgA in many of our patients.        (LCL) from a patient with congenital HO-1 deficiency and
The level of IgA has increased more quickly than the level of IgG.    the mechanism of cell protection by HO-1 was studied.
We have found the highest circulating immune complexes in
98,2% of our patients with severe forms of the HFRS, at the           LCLs were established from the patient and controls by
acute stage, i.e. in the first 30 days since the beginning of the     immortalization with EB virus. Hemin-induced cell injury
disease. After the 8th week all patients had a negative titre. We     was evaluated by a flow cytometry using FITC-conjugated
also found immunological disturbances at early stages of dis-
ease, especially in the febrile phase. These findings made us
                                                                      Annexin V binding. Apoferritin or bilirubin was added to
aware of the fact that the immunity complexes are being laid          the culture at various concentrations. Retrovirus-based HO-
down in the kidneys in the form of deposits which causa kidney        1 vector or vehicle control was transfected to HO-1 defi-
impairment of varying intensity followed by extensive proteinu-       cient LCL. HO-1 expressions by these cells were detected
ria of the glomerular type, which indirectly points at various
forms of glomerular lesions. The clinical picture of HFRS is of       by immunoblotting and flow cytometry.
varying severity and depends on many factors, primarily on the        HO-1 deficient LCL was extremely sensitive to hemin-in-
degree of kidney impairment. When renal biopsies were done            duced cell injury, whereas LCLs from controls were resist-
histopathological changes were found in all such patients, as-
suming various forms of mesangio-proliferative, membrana-
                                                                      ant. Apoferritin or bilirubin did not inhibit the cell injury.
proliferative, focal segmental, and membranous glomerulone-           HO-1 gene transfection resulted in constitutive expression
phritis with evident interstitial changes in the sense of secondary   of HO-1 and nearly complete reversal of the cell death.
interstitial nephritis. Prevention and treatment should be carried    These results suggest the existence of unknown protective
out on the basis of unique criteria. There is today no specific
treatment of the HFRS. From the point of view of the latest           mechanism in HO-1-mediated protection of the cell injury,
achievements and knowledge about fast diagnosing and use of           in addition to the effects of ferritin or bilirubin. Whether
modern technology in the treatment of the HFRS mortality over         HO-1 induces a novel cytoprotective molecule, or it plays a
5% cannot be tolerated.
                                                                      direct role in hemin-stimulated cultures, is yet to be evalu-
                                                                      ated.


FLOW CYTOMETRIC DETECTION OF ANTI-ENDOTHELIAL                         HEME OXYGENASE (HO)-1 EXPRESSION BY RENAL TUBU-
CELL ANTIBODIES (AECA) IN RENAL VASCULITIC DIS-                       LAR EPITHELIUM IN RENAL DISEASES
EASE                                                                  K Fujimoto, H Kaneda, A Seno, T Toma, K Ohta, Y Kasahara, A
P R Evans, S Terry, T Carr, M Rogerson, M Waldron, P Bass             Yachie, S Koizumi
Depts. of Pathology, Immunology, and Division of Medicine             Dept. of Pediatr. and Dept. of Lab. Sci., Faculty of Med.,
and Paediatric Nephrology, Southampton University Hospital,           Kanazawa University, Kanazawa, Japan
UK
                                                                      Tubular epithelial cells are constantly exposed to various
Endothelial cells are the foremost targets in renal vasculitis. The   oxidative stresses and are vulnerable to the injury whenever
possible pathogenic role of AECA in adult and paediatric renal        these insults are overwhelming. HO-1 protects these cells by
vasculitis is controversial. Techniques to measure AECA often         neutralizing the noxious agents, including heme proteins. We
have lacked standardization. We have used flow cytometry (FC)         compared HO-1 expression in renal tissue in various renal dis-
to detect low levels of antibody directed to the cell surface of      eases and analyzed the pattern of HO-1 expression in relation to
endothelial cell lines to give a more relevant assessment of in       clinical symptoms.
vitro antibody mediated damage. Eighteen patients with biopsy         Renal biopsy specimens were obtained, dewaxed and stained
proven renal vasculitis were studied. Of the adults (N=14) there      for HO-1 using rabbit anti-human HO-1 antibody. Distribution
were 6 cases of Wegeners Granulomatosis (WG), 5 Systemic              of HO-1 staining was analyzed and the intensity was graded
Lupus Erythematosis (SLE) and 3 Henoch Schonlein Purpuras             into four different scales.
(HSP); in the paediatric group there were 2 HSP’s, 1 WG and 1         Pathological diagnosis and clinical symptoms, including pro-
SLE. 130 serum samples were examined using flow cytometry             teinuria and hematuria were compared with the result of immu-
and a panel of endothelial, epithelial, hybrid cell lines and renal   nohistochemistry.
proximal tubular cells. Carefully screened selected negative con-     HO-1 expression was minimum in normal kidney. Regardless of
trol sera were used. AECA were found to be present in signifi-        the type of renal pathology, HO-1 staining within the proximal
cant amounts in the adult vasculitic group compared to normal         tubules tended to be more intense with greater degree of
adults, particularly in adult WG (p=0.005). AECA in adult SLE         hematuria. Similar, but less remarkable tendency was observed
was of borderline significance (p=0.05). The binding intensity of     for the distal tubules. HO-1 expression was virtually absent in
AECA reflected the clinical course in WG. No antibodies were          HO-1 deficiency, although gross hemolysis with persistent
detected in the paediatric group. These findings suggest AECA         hematuria was associated with severe injury and atrophy of
has a significant pathogenic role in a range of renal vasculitides.   tubular epithelium.
                                                                      These results indicate that HO-1 expression by tubular epithe-
                                                                      lium may serve as a significant protective barrier in the case of
                                                                      glomerular injury and subsequent hematuria.



Nephrology Dialysis Transplantation                  Vol. 14 n.9                     1999                                                 A19
                                     Immunology, immunopathology and pathology
Abstracts                                        (basic and clinical)


      GLOMERULAR AND INTERSTITIAL T CELL SUBESTS IN                         DETECTION AND PREVENTION OF HEPATITIS C IN A
      PRIMARY CRESCENTIC GLOMERULONEPHRITIS AND                             NEPHROLOGY UNIT. ALONG TIME FOLLOW UP        .
      CLASS IV LUPUS NEPHRITIS                                              Almroth G, Ekermo B, Svensson G, Månsson A, Widell A,
      L. Grcevska, M. Polenakovik
                                                                            Sweden.
      Department of Nephrology, Clinical Center, Skopje, R.Macedonia

      A remarkable mononuclear cell infiltrate may be associated with       Sera from 21 patients, who had ben in hemodialysis (HD)
      primary and secondary glomerulonephritides. Class IV lupus            during 1988-91, known to be positive (or close to the cut off
      nephritis (diffuse proliferation)(LN) may have similar histopatho-    level, one) in ELISA, were tested in 1991 for antibodies to
      logical features as primary crescentic glomerulonephritis(CGN).       hepatitis C virus (anti-HCV) with recombinant immunoblot
      In the present study monoclonal antibodies to T-cell subsets          assay 2 (RIBA 2) and for hepatitis C virus ribonucleic acid
      were used to analyze interstitial cellular infiltrates and            (HCV-RNA) with polymerase chain reaction (PCR). RIBA
      intraglomerular cells in primary CGN and class IV LN. 20 renal
                                                                            was positive in 9 cases and indeterminate in 6, while HCV-
      biopsy specimens of primary CGN and 15 of class IV LN were
      analyzed, 10 renal tissues from accidental authopsies were
                                                                            RNA was present in 12/15 tested sera.
      used as control group. Ten microscopic fields at magnification        In Oct 1991; 9/58 patients (16 %) from our HD unit were
      500 were analyzed for each patient, and the number of positive        considered as positive for PCR or RIBA. By the end of 1996
      cells was expressed as M+-SEM. The number of glomerular               there were 4 out of 37 (11%) HD patients in the unit who
      CD3 cells (3,7+-0,3) was increased in class IV LN, compared to        were considered as possible HCV-carriers as determined
      CGN (2,3+-0,4, p<0,01) and controls (0,04+-0,00, p<0,001).            by ELISA, RIBA or PCR. The condition in the whole neph-
      Intraglomerular CD4/CD8 ratio was elevated in LN(1,8) com-            rology unit (HD, peritoneal dialysis, transplanted patients)
      pared to CGN(1,2 p<0,05) and controls (0,93 p<0,01). The situ-
                                                                            was also equal between Oct.-91 and the end of 1996 (10 vs 9
      ation was different with the interstitial infiltration, CD3 cells
      were increased in CGN (324+-126), compared to LN (282+-69,
                                                                            % possible carriers).
      p<0,05) and controls (3,8+-0,5, p<0,01). But, CD4/CD8 ratio           Preventive measures may limit the number of carriers in a
      was higher in LN (2,1) compared to CGN (1,9 p<0,05) and               nephrology unit.
      controls (1,3 p<0,01). We can conclude that CD3 positive              Indeterminate RIBA-results should be regarded with cau-
      intraglomerular and interstitial cells were significantly increased   tion due to the relative immunodeficiency of uremic pa-
      in both CGN and class IV LN, intraglomerular values were higher       tients.
      in LN and interstitial in CGN. Interstitial and intraglomerular
      CD4/CD8 ratio was also higher.




      EXPRESSION AND DISTRIBUTION OF ADHESION MOL-                          INFLUENCE OF HAEMODIALYSIS ON MARKERS OF
      ECULES IN RENAL CELL CARCINOMA                                        NEUTROPHILS APOPTOSIS IN VITRO
      G. Basta-Jovanovic, Z. Jovanovic, S. Kovacevic, L. Kovacevic          E. Majewska*, Z. Sulowska**, Z. Baj*, J. Rysz***, M. Luciak***
                                                                            *Department of Pathophysiology and Clinical Immunology,
      Institute of Pathology, Medical Faculty Belgrade, Yugoslavia
                                                                            MMA, **Microbiology and Virology Center, Polish Academy of
                                                                            Sciences,***2and Department of Internal Medicine, MMA, Lodz,
      The aim of our study was to analyse the level of expression and       Poland
      the distribution of different adhesion moleculs in different types
      of renal cell carcinoma (RCC), to compaire the expression of          Interaction of cell surface with haemodialysis membranes, dia-
      adhesion molecules with the distribution of different compo-          lysate-derived bacterial contamination, complement activation,
      nents of extracellular matrix and to compaire adhesion mol-           IL-1 and TNFα synthesis can influence the neutrophil apoptosis.
                                                                            We tested 12 patients with end-stage renal disease on a long-
      ecules expression with the degree of tumor malignancy. Tumor
                                                                            term haemodialysis (HD) treatment. The blood samples were
      tissue from 14 renal cell carcinomas that were classified accord-     drawn before, at 20th min and 4th hr of HD. Using the flow
      ing to nuclear gradus G1-G3 and to cell type: 9 clear cell, 2         cytometry, we examined the ability of neutrophils to undergo
      chromophilic (1 eosinophilic and 1 basophilic), 1 chromophobe         apoptosis by estimating percentage of apoptotic neutrophils
      and 2 spindle cell. In addition, 5 normal human kidneys were          (based on Annexin V and propidium iodide binding), apoptosis–
      analysed as controls. On cryostat sections the monoclonal anti-       related (Fas,p53) and survival–related (bcl-2) proteins expres-
      bodies which defined different adhesion molecules (cadherins E,       sion in the whole blood neutrophils cultured for 18 hrs. All data
                                                                            are presented as mean fluorescence intensity ± standard error.
      N, P; integrins alpha 1-6, alphaV; and ICAM-1) were applied by
                                                                            Evaluation of statistical significance was performed by
      indirect immunoperoxidase method. The results of our investi-         Wilcoxon’s signed rank test and Spearman rank correlation analy-
      gations showed that in renal cell carcinoma there is either ex-       sis. P values ≤ 0.05 were considered significant. The percentage
      pression of N or E cadherins, or their combined expression which      of apoptotic neutrophils in cultured whole blood sampled be-
      does not depend on the cell type of renal cell carcinoma or their     fore HD (61.0±15%) is higher compared with 20th min
      nuclear gradus. Cadherin P was not detected in any investi-           (42.1±8,3%) and 4th hr of HD (54.8±14,9%). The changes in
      gated type of renal cell carcinoma. Expression of alpha1, 2 and       percentage of apoptotic neutrophils positively correlate with
                                                                            Fas molecule expression. HD does not affect the expression of
      4 integrins was variable in different cases. Alpha 3, alpha 5 and
                                                                            p53 and bcl-2 cytoplasmic expression in neutrophils, but we
      alpha V expression was found in almost all our investigated           have revealed the positive and negative correlation between the
      cases. There was no correlation between alpha1-alpha 5 integrin       percentage of apoptotic neutrophils and p53 and bcl-2 proteins
      expression and the cell type or nuclear gradus of renal cell carci-   expression at each time point, respectively. Presented results
      noma, while alpha V integrin expression was found to be in            suggest a transient selected sequestration of potentially apoptotic
      correlation with nuclear gradus of renal cell carcinoma. Inten-       neutrophils.
      sive expression of ICAM 1 was found in all cell types of renal cell
      carcinoma except in chromophobe type where it was absent.




A20                                                Nephrology Dialysis Transplantation                 Vol. 14 n.9                      1999
                                            Immunology, immunopathology and pathology
                                                        (basic and clinical)                                                                      Abstracts

INTERSTITIAL MYOFIBROBLASTIC ACTIVITY IN MEMBRANOUS NEPH-                               NO-INDUCED APOPTOSIS AS A MECHANISM OF
ROP ATHY(MN)ASAPROGNOSTICFACTORANDITSRELATIONSHIPWITH                                   MESANGIOLYSIS IN ANTI-THY 1.1 NEPHRITIS.
THE EXPRESSION OF PDGF AND TGF-β.                                                       Z. Hruby, M. Rosinski, B. Tyran, W. Kopel
S Mezzano1, ME Burgos1, A Droguett1, C Aros1, L Ardiles1, I Caorsi1 , J Egido 2.
1
 Div. Nephrology, Univ. Austral, Valdivia, Chile; 2 Fundación Jiménez Díaz,
                                                                                        Dept. Of Nephrology, Wroclaw University of Medicine, Wroclaw,
Madrid, España.                                                                         Poland

The myofibroblast (Mf) is recognized as an important participant in progres-            Apoptotic cell death has been regarded as a mechanism of
sive renal fibrosis. In order to know whether the presence of Mfs predicts pro-         homeostatic control of mesangial hypercellularity in the prolif-
gression of the MN and its relationship with the expression of the profibrogenic        erative phase of anti-Thy 1.1 nephritis in the rat. Nonetheless,
cytokines PDGF-BB and TGF-β, we have studied renal biopsies of 25 patients              since administration of an iNOS inhibitor in the earliest,
with idiopathic MN. 13 patients developed progressive disease while 12 showed
a non-progressive variant. Mfs were identified by immunohistochemistry (IMH)
                                                                                        mesangiolytic phase of nephropathy notably reduced mesangial
using monoclonal anti α-SMA antibodies. Detection of PDGF-BB and TGF-β                  cell damage, we assessed the extent of NO-linked glomerular
(protein and/or mRNA) was done by IMH and by in situ hybridization (ISH).               apoptosis on days 0-4 after i.v. injection of nephritogenic anti-
IMH coupled to ISH was performed to detect simultaneously α-SMA positive                Thy 1.1 antibody in Wistar rats.
cells, and cells expressing TGF-B and PDGF-BB. Monocyte/macrophages were                On day 2 after induction, the apoptotic index in glomeruli (as-
identified using monoclonal anti CD68 antibodies.                                       sessed by TUNEL method) reached peak values (0.41+-0.03),
α-SMA interstitial positive cells were mainly detected in patients with a pro-          accompanied by enhanced generation of nitric oxide in renal
gressive disease (13 out 13 progressive versus 4 out 12 with non-progressive;
p=0.0004); the staining distribution was mainly in peritubular and
                                                                                        tissue (Griess method on slices of renal cortex: 1.83+-0.49 µmol)
periglomerular cortical interstitium, with a stronger expression in patients            and high expression of glomerular iNOS mRNA assessed by the
with a progressive MN. The presence of Mfs was significantly associated with            in situ RT-PCR. On days 0-3 mesangial cell proliferation was
the tubular interstitial cell infiltration (p=0.02), interstitial fibrosis (p=0.005),   undetectable, determined by immunostaining for glomerular
and with the severity of proteinuria (p=0.03). In 4 progressive and 3 non-pro-          PCNA. Similarly, we have not revealed any presence of infiltrat-
gressive cases, we observed α-SMA positive cells at the glomerular level that           ing macrophages (staining with labeled ED-2 antibody) in ne-
represent a phenotypic modulation of activated mesangial cells. Double IMH              phritic glomeruli at this phase of the disease. Administration of
coupled with ISH was performed in some progressive cases, observing α-SMA
immunostaining mainly in the peritubular intestitium, and the TGF-B and PDGF-
                                                                                        l-NAME, an iNOS inhibitor from day 0 of nephropathy resulted
BB mRNA expression in the surrounding tubular epithelial cells. The IMH for             in marked reduction of apoptotic index (day 2, 0.19+-0.05) and
CD68 was negative or weak in the majority of cases.                                     no demonstrable production of nitrite in kidney tissue
On the whole, in patients with MN, the expression of interstitial α-SMA stain-          In conclusion, apoptosis of mesangial cells linked with the au-
ing indicates myofibroblastic activation and is associated to inflammatory cell         tologous activity of iNOS, may at least in part, be responsible
infiltrates and progression of renal disease. The increase in PDGF and TGF-β            for mesangiolysis at the earliest stage of anti-Thy 1.1 nephritis.
staining suggest that they could be involved in the myofibroblast activity. By
contrast, the glomerular expression of α-SMA, did not correlate with the degree
of glomerulosclerosis. ( Supported by Fondecyt 1970628 ).



                                                                                        SURFACE MARKER EXPRESSION, IN VITRO LYMPHOCYTE
CHARACTERIZATION              OF       RENAL         AND
                                                                                        SURVIVAL AND SOLUBLE ACTIVATION MARKERS DUR-
CARDIOVASCULAR           APOPTOSIS        FOLLOWING                                     ING HAEMODIALYSIS SESSION
ENDOTOXEMIC SHOCK IN RATS.                                                              N. Ursea, L. Petrescu
ME Grossman1, AH Campos1 MF Franco 2 and N Schor1.                                      “Dr Carol Davila” Teaching Hospital of Nephrology, Bucharest,
Nephrology1 and Pathology2 Divisions, UNIFESP, SP, Brazil.                              Romania

Apoptosis may be involved in organ dysfunction observed                                 Objective. In order to evaluate the influence of HD session on T
                                                                                        cell and monocyte surface markers expression, lymphocyte acti-
during endotoxic shock. However, its involvement in renal failure
                                                                                        vation, and in vitro peripheral blood lymphocyte survival, we
consequent to sepsis was not clarified. We addressed this question                      investigated 20 HD pts [age: 45-55 yrs (51±5.7); HD duration
employing a model of murine endotoxic shock. Male Wistar rats                           4±1.5yrs.; M=10, F=10; primary renal disease: GN (n=14), IN
were treated with E. coli lipopolysaccharide (LPS, 10 mg/kg of                          (n=3) and nephroangiosclerosis (n=3)]; free of intercurrent in-
body weight; i.p.) and the animals were sacrificed at different                         flammatory diseases and without immunosupresive drugs dur-
time intervals. Heart, aorta and kidney apoptosis was evaluated                         ing last three months. HD (3x4.5hrs/week) was performed with
by means of DNA agarose gel electrophoresis (AGE), Hoechst                              polysulphone membranes and bicarbonate dialysate. Ten healthy,
                                                                                        age and gender-matched controls were used.
33342 DNA staining, hematoxilin/eosin (HE), and DNA nick
                                                                                        Methods. Blood samples were drown at the beginning, at 15, 30,
end labeling (TUNEL). Mortality rate among animals treated                              60 min and at the end of the HD session. Flow-cytometry was
for 24 h was higher than 40%. Neither DNA AGE nor HE staining                           performed with CD3/CD4, CD3/CD8, CD11b/CD18 two-color
were able to detect apoptosis in survivors after 6, 24 or 48 h in                       monoclonal antibodies (MAbs). Peripheral blood lymphocytes
any of the studied organs. In addition, no inflammatory reaction                        (PBL) survival in vitro was assessed by Trypan blue exclusion
was disclosed by HE. On the other hand, preliminary data from                           method at 24, 48 and 72 hours of culture. Electrophoresis of
Hoechst 33342 fluorescent staining showed signs suggestive of                           DNA extracted from in vitro cultured PBLs was performed to
                                                                                        show the apoptosis pattern of internucleosomal cleavage.
apoptosis in aortic endothelium, myocytes and renal cells after
                                                                                        Results. The percentage of CD3/CD4 lymphocytes increased
24 h, and to a lesser extent, after 6 h. TUNEL confirmed and                            during HD session (r=0.902, p<0.05). CD3/CD8 remained con-
extended this findings, showing that renal apoptosis was                                stant on lymphocytes as well as CD11b/CD18 on monocytes. In
concentrated mainly in the tubular region, sparing relatively the                       vitro survival of PBL decreased faster during the culture time in
glomeruli, and being present even after 48 h. We conclude that,                         patients than in controls (p<0.05 at 48h and 72h) and during the
besides its participation in the cardiovascular compromise                              HD session (r=0.995, p<0.001 at 72h), but was similar to con-
observed in endotoxic shock, apoptosis may also be involved in                          trols at baseline in the 24 h culture. Cell death was mainly
                                                                                        apoptotic, as suggested by morphological criteria and DNA
the genesis of sepsis-related renal dysfunction. Support:
                                                                                        ladder in electrophoresis.
FAPESP                                                                                  Conclusions. HD session had little or no influence on T cell and
                                                                                        monocytes phenotypes, respectively. CD4 positive T cell popu-
                                                                                        lation transiently increased during the HD session starting with
                                                                                        the first 15 minutes. PBLs of HD patients seem to be more prone
                                                                                        to die in vitro, mostly by apoptosis, but only during the HD
                                                                                        session.



Nephrology Dialysis Transplantation                                Vol. 14 n.9                         1999                                                  A21
                                              Immunology, immunopathology and pathology
Abstracts                                                 (basic and clinical)


      COMBINED BLOCKADE OF ANGIOTENSIN II - ENDOTHELIN 1 PATHWAY                               COMPARATIVE EFFECTS OF FOUR IMMUNOSUPPRESOR
      PRESERVED RENAL FUNCTION AND REDUCED INTERSTITIAL FIBRO-                                 DRUGS ON RENAL FUNCTION AND INTERSTITIAL FIBRO-
      SIS IN AN EXPERIMENTAL MODEL OF CHRONIC PROLIFERATIVE                                    SIS IN THE LATE PHASE OF EXPERIMENTAL CHRONIC
      GLOMERULONEPHRITIS.                                                                      PROLIFERATIVE NEPHRITIS.
      Segarra A, Arbós MA, Buscà B,Argelaguer X, Alvarez E, Griera E, Quiles MT,               Segarra A, Arbós MA, Buscà B, Argelaguer X, Alvarez E, Quiles
      Schwartz S and Piera LL.                                                                 MT, Schwartz S and Piera LL.
      Research Unit. Nephrology Department. Hospital Vall d’ Hebrón. Barcelona.                Research Unit. Nephrology Department. Hospital Vall d’ Hebrón.
                                                                                               Barcelona
      Aim: To analyze the efectiveness of angiotensin II –endothelin 1 pathway block-
      ade in preventing the progression of interstitial fibrosis in an experimental            Aim: To compare the effect of four immnunossupresor drugs on
      model of chronic proliferative glomerulonephritis.                                       interstitial infiltrates, interstial fibrosis and renal function in an
      Methods: Eighty male Wistar rats weighing 225 to 250 g,were used for the experi-         experimental model of chronic proliferative glomerulonephritis.
      ments. Eight weeks after having induced a chronic anti Thy1 proliferative                Methods: Eight weeks after having induced a chronic anti Thy1
      glomerulonephritis, animals were randomly assigned to one of four groups.                proliferative glomerulonephritis, 50 Wistar male rats weighing
      One control group (group 0) which received no therapy and three intervention             225 to 250 g were randomly assigned to one of five groups of 10
      groups (groups 1,2,3) receiving therapy with the specific endothelin 1 receptor          animals. Group 1 received no therapy and groups 2 to 5 received
      antagonist FR 139137 plus enalaprilate, enalaprilate and FR 139137 respec-               therapy with prednisone (0,25 mg/day), azathioprine (0,20 mg/
      tively from week 10 to week 20. At week 20, we compared the severity of renal            day), tacrolimus (0,02 mg/day) and micophenolate (0,01 mg/
      failure, interstitial infiltrate and interstitial fibrosis among the different groups    day) respectively from week 8 to week 16. At week 16, animals
      using immunohistochemical techniques, RT PCR and Northern Blot for colla-                were sacrified and we compared the severity of renal failure,
      gen III, TGF beta, angiotensinogen, ET1 receptor and pre-proendotelin.                   interstitial infiltrate and interstitial fibrosis among the different
      Results: Eight weeks after the first dose of antithy1, blood pressure increased          groups using immunohistochemical techniques and RT PCR for
      significantly, all animals developed proteinuria and progresive renal failure.           collagen III and TGF beta.
      At week 8, serum creatinine, blood pressure and proteinuria were similar in all          Results: At week eight all experimentals groups showed renal
      study groups. At the end of follow-up, the three intervention groups showed              failure, proteinuria and high blood pressure with no significant
      lower levels of proteinuria, serum creatinine and low blood pressure than con-           differences among them. During follow-up, groups 1 to 4 showed
      trol group. There were no differences between groups 2 and 3. When compared              progressive renal failure and presistent proteinuria whereas in
      with groups 2 and 3, blood pressure , urinary protein excretion and serum cre-           group 5, renal function remained stable. At week 16, serum
      atinine were all significantly lower in animals treated with both drugs (group           creatinines were 2.16 (0.3), 2.3 (0.6), 2.11 (0.4) and 1.98 (0.1)
      1). This later group showed also significantly lower intensity of interstitial           and 1.68 (0.47) respectively (p<0.001) and urinary protein ex-
      fibrosis, and lowerTGF beta, ET1 receptor and col III mRNA expression.                   cretion was 0.52 (0.17), 0.82 (0,21), 0.72 (0.22), 0.45 (0.11) and
      Conclusions: Long – term combined blockade of angiotensin II and endotelin A             0.21 (0.1) respectively (p<0.001). When compared with the other
      receptors reduced proteinuria,prevented the progression of interstitial fibrosis         groups, group 5 showed significantly lower interstitial fibrosis
      and preserved renal function in a model of chronic proliferative glomerulone-            and lower m RNA TGF beta/B actin and col III/B actin ratios.
      phritis. These preventive effects could be attributed to direct actions on intersti-     Conclusions: In the chronic phase of renal interstitial inflamma-
      tium but were also related to more effective reduction of blood pressure.                tion and fibrosis micophenolate mofetil was the only drug able
                                                                                               to slow down the development of progressive renal failure. This
                                                                                               effect was related to a significant reduction of renal expression
                                                                                               of TGF beta and collagen III.




      BLOCKADE OF LFA-ICAM1 INTERACTIONS AND MCP1 DID NOT PRE-                                 DIFFERENTIAL        EXPRESSION      AND       REGULATION        OF
                                                                                               ENDOTHELIN 1 mRNA AND MDR mRNA IN RENAL TUBULAR
      VENT THE DEVELOPEMENT OF INTERSTITIAL FIBROSIS IN EXPERI-                                CELL LINES.
      MENTALCHRONIC PROLIFERATIVE GLOMERULONEPHRITIS.                                          F Arrebola, F O’Valle, A Olmo, M Aguilar, B Espigares, M Guillen, D
      Segarra A, Arbós MA, Buscà B, Argelaguer X, Alvarez E, Quiles MT, Schwartz               Aguilar, RG Del Moral
      S and Piera LL.                                                                          Dept. de Anatomía Patológica, Hospital Universitario San Cecilio,
      Research Unit. Nephrology Department. Hospital Vall d’ Hebrón. Barcelona                 18012 Granada, Spain

                                                                                               P-glycoprotein (P-gp) has an important role in protection against
      Aim: To analyze whether the specific blockade of LFA-ICAM interactions and               xenobiotics. Endothelin 1 (Et1) is a powerful vasoconstrictor and its
      MCP1, reduced interstitial fibrosis and prevented chronic renal failure in an            overexpression in renal cells can be one of the mediators of drug-
      experimental model of chronic proliferative glomerulonephritis.                          induced tubular lesions. Transcriptional regulation of the multi-drug
      Methods. Eight weeks after having induced a chronic anti Thy1 proliferative              resistance (MDR1) and Et1 genes share some activation mechanisms
      glomerulonephritis, 60 Wistar male rats weighing 250 g were randomly as-                 through protein kinase C and the AP1 promoter. In this study we show
      signed to one of six groups. Group 1 received no therapy and served as a control.        the different behaviours of MDR and ET1 expression in renal cells in
                                                                                               vitro, their modulation by immunosuppressive (IS) and
      Groups 2 to 6 were the intervention groups each of them receiving therapy with           chemotherapeutic (CT) drugs, and their relationship with the cell cycle.
      blocking monoclonal antibodies against both ICAM1 and MCP1 administered                  In vitro assays were done with renal tubule-derived MDCK and LLC-
      ip in increasing doses from week 10 to week 14. Specific antibody binding was            PK1 cell lines maintained in MEM culture medium with 5% IFBS.
      determined by flow cytometry. At week 14, all animals were sacrified and the             Pharmacological induction was achieved by incubation for 15 days
      severity of renal failure, interstitial infiltrate and interstitial fibrosis among the   with sublethal doses (IC ) of the different nephrotoxic drugs (IS and
                                                                                                                         30
      different groups were compared using immunohistochemical techniques and                  CT). Levels of expression of the MDR1 and endothelin 1 genes were
                                                                                               determined by RT-PCR. Flow cytometry was used to measure the
      RT PCR for collagen III and TGF beta.                                                    following parameters: P-gp expression (JSB-1 clone), percentage of
      Results: At week eight all experimentals groups showed renal failure, proteinu-          positive cells (%POS) and mean specific fluorescence intensity (MSFI).
      ria and high blood pressure with no significant differences among them. During           Cell cycle and growth fraction were also determined.
      follow-up, all animals developed progressive renal failure and increasing pro-           Under basal conditions MDR1 mRNA expression was 0.1 ± 0.01 in
      teinuria. At week 14, all animals showed advanced renal failure with serum               MDCK and 0.11 ± 0.007 in LLC-PK1; the %POS and the MSFI of P-gp
      creatinine levels ranging from 1.9 mg/dL to 4 mg/dL and urinary protein ex-              were also similar in the renal tubule cell lines (%POS MDCK 83.65 ±
                                                                                               22.71, LLC-PK1 81.28 ± 20.5; MSFI MDCK 24.8 ± 3.1, LLC-PK1 23.9 ±
      cretion higher than 500 mg/day. Serum creatinine, proteinuria, interstitial in-          5.9). Under these conditions, ET1 mRNA expression was greater than
      filtrate, interstitial fibrosis and m RNA TGF beta/B actin and col III/B actin           MDR1 mRNA levels (MDCK 0.69 ± 0.01, LLC-PK1 0.51 ± 0.03). Incuba-
      ratios were all similar in treated and untreated groups independently of the dose        tion with drugs (IS or CT) increased MSFI of P-gp and MDR1 mRNA
      of monoclonal antibody administered.                                                     expression in both lines (p < 0.001, ANOVA). The drugs that induced
      Conclusion. High doses of blocking antibodies against LFA-ICAM1 and MCP1                 the greatest MDR1 mRNA overexpression induced the greatest reduc-
      failed to reduce the amount of interstitial mononuclear infiltrate and did not           tion in ET1 mRNA expression in renal tubule cell lines. RT-PCR stud-
                                                                                               ies showed that ET1 and MDR1 mRNA expression were inversely
      prevent the development of progressive interstitial fibrosis when tested in a            related (Pearson coefficient r= -0.488, p<0.05). P-gp and MDR1 mRNA
      chronic model of proliferative glomerulonephritis.                                       levels were directly related to the percentage of cells in S and G M
                                                                                                                                                                     2
                                                                                               phases (Pearson coefficient r= 0.465 and 0.576, respectively), while ET1
                                                                                               mRNA levels showed no statistical correlation.
                                                                                               In conclusion, our findings suggest that one of the keys to nephrotox-
                                                                                               icity control is the imbalance in MDR1 and ET1 mRNA expression.




A22                                                            Nephrology Dialysis Transplantation                           Vol. 14 n.9                        1999
                                               Immunology, immunopathology and pathology
                                                           (basic and clinical)                                                                                       Abstracts

RELATIONSHIP OF HYALINE ARTERIOPATHY WITH mRNA OF INTRARENAL P-                               DOES HLA-DR14 ALLELE CONFER SUSCEPTIBILITY TO
GLYCOPROTEIN AND ENDOTHELINS IN CHRONIC CYCLOSPORIN-INDUCED NE-                               CRYOGLOBULINEMIC GLOMERULONEPHRITIS?
PHROTOXICITY IN RATS
                                                                                              S. Pasquali, W. Mantovani*, A. Zucchelli, S. Casanova**, P. Zucchelli
A Olmo, C Ramírez, M Aguilar, F Arrebola, ME Reguero, F Revelles, F O’Valle, RG Del Moral.
Dept. de Anatomía Patológica, Hospital Universitario San Cecilio, 18012 Granada, Spain.       Divisone di Nefrologia e Dialisi Malpighi, Laboratorio Analisi*, Istituto di
                                                                                              Anatomia Patologica**, Azienda Ospedaliera S.Orsola-Malpighi, Bologna,
P-glycoprotein (P-gp) acts physiologically as an efflux pump to expel hydrophobic sub-        Italia.
stances from cells. Our group and others have shown that cyclosporin A (CsA), among
other actions in the kidney, induces P-gp overexpression (Am J Pathol,1995). Endothelin       Although a clear association between hepatitis C virus (HCV) and essential
1 (Et1) is widely expressed in the kidney in a variety of physiological or pathological
                                                                                              mixed cryoglobulinemia (EMC) has been established, it is not yet clear why
situations, the latter of which can progress to sclerosis. Exposure to cyclosporin A (CsA)
of mesangial, endothelial and renal tubule cells induces Et1 overexpression. The patho-       only some HCV+ cryoglobulinemic patients develop renal damage.
physiological function of Et3 in the kidney is currently being debated; Et3 may be involved   In an attempt to differentiate these 2 subtypes of cryoglobulinemic patients,
in the regulation of water reabsorption through the action of type B tubular receptors.       with and without renal disease, we have studied 23 HCV+ type II
110 male Sprague-Dawley rats fed with a maintenance diet were divided into three              cryoglobulinemic patients (8 males, 15 females, mean age 63.2 years), 16 with
groups: two control groups, one inoculated with 0.9% of sodium chloride (SC) and the          and 7 without cryoglobulinemic glomerulonephritis (CGN) followed-up for
other with the solvent used for CsA injection, and an experimental group treated with
                                                                                              more than 4 years.
25 mg CsA per kilogram of body weight per day during 28 or 56 days. We evaluated the
expression levels of P-gp mRNA using the RT-PCR technique. Prepro Et1 and Et3 mRNA            The genetic characteristics of the HCV and of the host were analyzed in the 2
was determined by northern blot (NB).                                                         groups of patients.
Chronic treatment with CsA induced an increase in the expression of P-gp mRNA in a            HCV genotype was identified in 20 patients using type-specific oligonuclectide
dose and time dependent manner, more evident by 58 days (0.725 vs 0.251, p<0.01,              probes after hybridization. All the patients were typed for HLA (Classes I and
Newman-Keuls test.) (Table). A remarkable finding was that the upregulation of P-gp           II) by the standard lymphocytotoxicity technique. HLA results were compared
mRNA was inversely related to the incidence of hyaline arteriopathy (Spearman´s test,
                                                                                              with normal controls.
r=-0.3819, p<0.01). Prepro Et3 mRNA levels were greatly increased from posttreatment
day 28, whereas prepro Et1 mRNA levels were increased from posttreatment day 56               The genotype distribution in patients with CGN was no different from the one
(Table). On day 28 renal lesions clearly correlated with levels of Et3 mRNA. However, on      found in patients without CGN while the presence of HCV subtype 1b correlated
day 56 the key finding was the strong correlation of prepro Et1 mRNA levels in CsA            with the presence of liver damage.
nephrotoxicity with the most important analytical, histological and immunohistochemi-         Class I HLA-88 was present in 57.1% (4/7) of the patients without renal damage
cal alterations.                                                                              compared to 10.1% (38/377) of normal controls (p = 0.003, pc = n.s.). Only 1 out
Table              28 Days (x±SD)         56 Days (x±SD)           Significance
                                                                                              of 16 (6.2%) patients with renal damage exhibited this antigen.
Group:             CsA         SC         CsA          SC          Two Way
                                                                   ANOVA                      As regards the expression of class II HLA alleles, a signifiicant association was
P-gp (RT PCR) 0.47±0.33 0.24±0.07 0.72±0.36 0.25±0.22 P<0.01                                  found with HLA-DR14, detected in 5 out of the 16 (31.2%) nephropathic patients
Et1-NB             0.20±0.10 0.23±0.05 0.34±0.09 0.15±0.05 P<0.05                             compared to 6.4% (24/377) of the normal controls (p = 0.0038; pc < 0.05). None
Et3-NB             0.48±0.30 0.07±0.11 0.36±0.10 0.10±0.04 P<0.01                             of the patients without renal damage exhibited this antigen.
                                                                                              The results suggest that: 1) the genetic heterogeneity of HCV does not influence
The increased expression of P-gp mRNA in CsA-treated compared with untreated rats
                                                                                              renal involvement while it appears to influence liver damage; 2) the develop-
and the inverse relation of P-gp mRNA levels with hyaline arteriopathy are in agreement
with previous analytical, histological and immunohistochemical findings, suggesting an        ment of CGN requires a genetic predisposition of the host associated with the
important role for P-gp in the prevention of pharmacological nephrotoxicity by CsA            HLA-DRl4 marker.
through its action as a detoxicant in renal cells. Our results support the hypothesis that
clinical and morphological phenomena related to CsA nephrotoxicity are related to the
hypersecretion of endothelins in the progression to CsA-induced interstitial fibrosis. The
changes are first evident in Et3 expression and angiotensin II accumulation, and are later
reflected in Et1 expression.




PREVALENCE AND DIAGNOSTIC SIGNIFICANCE OF ANTIBODIES TO THE                                   MYCOPHENOLATE MOFETIL (MMF) REDUCES APOPTOSIS IN THE RE-
HIGH MOBILITY GROUP NON-HISTONE CHROMOSOMAL PROTEINS                                          NAL ABLATION MODEL IN THE RAT.
HMG1 AND HMG2.                                                                                J Rincón, Y Quiroz, F Romero, G Parra and B Rodriguez-Iturbe
Radice A, *Bianchi M E, °Covini G, *Bonaldi T, °Bredi E, Sinico R A and D’Amico               Dept. of Immunobiology, Inbiomed and Renal Service, Hospital Universitario,
G.                                                                                            Universidad del Zulia, Maracaibo, Venezuela.
Dept of Nephrology S. Carlo Hospital, *Genetics & Biol of Microorganisms                      .
University, °Gen Med Humanitas Inst - Milan, Italy.                                           The role of apoptosis in the development of glomerulosclerosis is controversial.
                                                                                              Deletion of glomerular cells could be a physiologic response to proliferation or,
Recently, two novel antigens, HMG1 and HMG2 proteins, have been described                     alternatively, a pathogenetic mechanism, resulting in scarring. Since MMF
as possible targets for ANCA in non vasculitic diseases. However, their preva-                ameliorates the sclerosis and prevents renal failure in the 5/6 nephrectomy (Nx)
lence in different disease groups needs to be ascertained yet. Aim of our work                model (Kidney Int March, 1999) we examined the effects of MMF in the prolif-
was to study the prevalence of antibodies to HMG1 and HMG2 in selected cat-                   erative activity (mAb anti-PCNA) and apoptosis (TUNEL method) in this ex-
egories of ANCA-associated diseases.                                                          perimental model. We studied 40 male Sprague-Dawley rats after 5/6Nx that
Anti-HMG1 or HMG2 antibody was assayed by ELISA using recombinant                             received MMF 30 mg.Kg-1.day-1 by gastric gavage (n=20) or vehicle (n=20). Five
HMG1 or HMG2 from E. Coli as antigen in solid phase.                                          additional rats were sham operated. Rats were sacrificed 3, 7, 28 and 56 days
The anti-HMG1 level (mean OD±SD) was significantly increased in lupus erythe-                 after renal ablation (5 rats from each group at each time interval). Serum creati-
matosus systemic (SLE) (0.653±0.430, p=0.02), ulcerative colitis (UC)                         nine and proteinuria were determined weekly.
(0.668±0.359, p=0.04), autoimmune colangitiis (AC) (0.798±0.414, p=0.03), pri-                Results. MMF treatment prevented the progressive rise in serum creatinine and
mary biliary cirrhosis (PBC) (0589±0.334, p=0.0001) and type II autoimmune                    proteinuria (p<0.001). Eight weeks after surgery, segmental sclerosis was present
hepatitis (AH-II) (0.594±0.130, p=0.001) but not in primary sclerosing colangitiis            in 48.4 ± 8.35% of the glomeruli of vehicle treated rats and in 25 ± 10.5% in the
(PSC) (0.692±0.384), type I autoimmune hepatitis (AH-I) (0.718±0.597), ANCA-                  MMF-treated rats (p<0.001). Apoptosis and proliferation are shown in Tables
associated systemic vasculitis MPO/PR3 positive (AASV) (0.525±0.255 and                       1 and 2.
0.331±0.239) in comparison to normal controls (NC) (0.374±0.150).
Anti-HMG1 were frequently detected in sera from patients with UC (6/18, 33%,                  Table 1. Proliferation (PCNA positive cells/glomerular cross section)
p=0.04), AC (4/7, 57%, p=0.007) and PBC (14/45, 31%, p= 0.04) but were not                                        3 days      7 days            28 days       56 days
statistically significant in SLE (5/17, 29%), PSC (3/7, 43%), AH-1 (3/7, 43%),                5/6Nx               0.47 ± 0.11 1.16 ± 1.32       1.51 ± 0.58 2.17 ± 1.85
AH-II (1/8, 12.5%), AASV (6/27, 22%); in normal controls only 1 out of 26 sera                5/6Nx+MMF           0.40 ± 0.06 0.65 ± 0.48       0.89 ± 0.37 0.60 ± 1.77
(3.8%) were positive.
Anti-HMG2 antibody was detected with the same disease distribution but with                   Table 2. Apoptosis (TUNEL positive cells/glomerular cross section)
a lower prevalence. Anti-HMG1 and anti-HMG2 antibodies were detected more                                       3 days      7 days             28 days        56 days
frequently in sera with positivity for P-ANCA by immunofluorescence on etha-                  5/6 Nx            0.24 ± 0.20 0.79 ± 0.38        0.43 ± 0.30 0.66 ±
nol-fixed granulocytes.                                                                       5/6 Nx+MMF        0.17 ± 0.10 0.56 ± 0.12        0.39 ± 0.43 0.07 ± 0.08
In conclusion, our results confirm that HMG1 and HMG2 are new ANCA anti-
gens and specific autoantibodies can be found in a variety of ANCA- associated                (All values are mean ± SD). MMF treatment reduces proliferation by about 15%
diseases, ranging from rheumatic to inflammatory bowel and hepatic diseases.                  at 3 days to 72% at 56 days and reduces apoptosis by 29% to 5% at the same time
This spread distribution decreases their diagnostic significance in comparison                intervals. Decreased proliferation and apoptosis is likely involved in the reduc-
with other ANCA antigens.                                                                     tion in glomerulosclerosis resulting from MMF-treatment.



Nephrology Dialysis Transplantation                                     Vol. 14 n.9                             1999                                                               A23
                                     Immunology, immunopathology and pathology
Abstracts                                        (basic and clinical)


      CIRCULATING SOLUBLE ADHESION MOLECULES IN                              TGF-α1 AND APOPTOSIS IN GLOMERULONEPHRITIS
      ANCA-ASSOCIATED VASCULITIS.                                            D Goumenos, A Tsamandas*, S Tsakas, F Sotsiou+, D Bonikos*,
      J. Ara, A. Saurina, E. Mirapeix, P. Arrizabalaga, R. Rodriguez,        J Vlachojannis.
      R. Abellana*, C. Ascaso*, A. Darnell.
      Nephrology Service, Biostatistical and Epidemiological Unit*
                                                                             Departments of Nephrology and Pathology* University of Patras,
      Hospital Clínic. Barcelona. Catalonia. Spain.                          and Department of Pathology+ Evangelismos Hospital, Greece.

      The aim of this study was evaluate whether changes in concen-          TGF-α1 is considered the most fibrogenic growth factor.
      trations of soluble (s) E-selectin, sP-selectin, sL-selectin, sICAM-   Apoptosis, programmed cell death, has been implicated in
      1 and sVCAM-1 reflect disease activity in patients with ANCA-          pathogenesis of wound healing and scarring. Bcl-2 protein pro-
      associated vasculitis. A sandwich ELISA was used to measure            longs life of rapidly proliferating cells while bax protein pro-
      these soluble adhesion molecules in sera from 20 patients with
      ANCA associated vasculitis during active and remission phases
                                                                             motes apoptosis. The aim of the study was to evaluate the
      (ten patients with Wegener´s granulomatosis and ten patients           potential relation among TGF-α1, bcl-2, and bax expression and
      with microscopic polyangiitis.                                         apoptosis in various types of glomerulonephritis (GN).
      At the time of diagnosis, sE-selectin, sICAM-1 and sVCAM-1             Renal biopsies were obtained from 40 patients (M/F=25/25,
      levels (mean+/-sd) (88+/-42,27 ng/ml, 437,25+/-184,46 ng/              age 50.5±17.6 yr, serum creatinine 1.4±0.2mg%). Streptavidin-
      ml, 1720,36+/-1174,05 ng/mL, respectively) were significantly          biotine technique was employed on paraffin sections using spe-
      higher in patients with ANCA associated vasculitis than in             cific antibodies against TGF-α1, bcl-2, and bax protein. Presence
      healthy controls (p<0,0001, p=0,002, p=0,001 respectively). sP-
      selectin values did not differ from those obtained in normal
                                                                             of apoptotic cells was assessed using TUNEL method.
      donors. In contrast, sL-selectin levels (940,8+/-349,09 ng/mL)         Quantitation of immunostained cells was performed using
      were significantly lower in patients than those recorded in healthy    morphometric analysis.
      controls (p<0,0001). A significant decrease in concentration of        TGF-α 1 was localized in the cytoplasm of tubular epithelial
      sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 was observed            cells and renal interstitium. Apoptotic nuclei were also detected
      between active and remission phases (p<0,0001, p=0,002,                in tubular cells and interstitium. Bcl-2 and bax were present in
      p=0,001, p=0,001 respectively). No significant differences was         tubular epithelial cells. Glomerular bax expression was more
      observed in sL-selectin levels between active and remission
      phases. sL-selectin concentration (802,34+/-306,7 ng/mL) dur-
                                                                             evident than bcl-2. TGF- α1 expression was positively correlated
      ing the remission phase remained lower than those observed in          with TUNEL(+)cells, and bax expression and reversibly with
      healthy controls (p<0,0001). A positive correlation was observed       bcl-2 expression (p<0.05). TUNEL(+)cells were correlated posi-
      between VCAM-1 and creatinine levels during the active phase           tively with bax and reversibly with bcl-2 expression (p<0.05).
      (0,51,p=0,002). No correlation was observed between other ad-          TGF α1 and tubular bax expression were correlated with serum
      hesion molecules and this variable.                                    creatinine values (p<0.05)
      The increase in sE-selectin, ICAM-1 and VCAM-1 levels on ac-           In conclusion, TGF-α1 and bax expression in human GN seem to
      tive phase and the return to normal values during the remission
      phase suggest that the concentration of these adhesion mol-
                                                                             be related to enhanced apoptotic process and progressive renal
      ecules reflect disease activity in patients with ANCA associated       disease. Bcl-2 expression may provide a survival advantage to
      vasculitis.                                                            tubular epithelium.



                                                                             RENAL AND SYSTEMIC INVOLVEMENT IN MIXED
                                                                             CRYOGLOBULINEMIASASSOCIATEDWITHHEP               ATITISCVIRUSCHRONIC
                                                                             INFECTION
      LYMPHOCYTE SUBSETS IN CHLDHOOD IgA NEPHROPA-                           D. Rosca, E. Mota, M. Mota, M. Georgescu
      THY (IgAN)                                                             Clinical Hospital no. 1 Craiova, Nephrology and Dialysis Deprtment, Faculty
      M Ekim1, A Günlemez1, A Ikinciogullari2, N Kara3, E Babacan2, N        of Medicine, Craiova, Romania
      Tümer1.
      University of Ankara School of Medicine, Pediatric Nephrology1         In the renal damages associated with hepatitis C virus (HCV) infection are in-
      and Immunology-Allergy2, Social Insurance Hospital Pediatric           volved immune mechanisms, mainly being considered the mixed
      Nephrology3, Ankara, Turkey.                                           cryoglobulinemias (MC). MC are type II or III, according with Brouet classifi-
                                                                             cation, in use yet. In the same time, MC are involved in the occurrence of another
      The objective of the study is to determine whether or not periph-      local and systemic manifestations.
      eral blood (PB) total T, B lymphocytes and their subsets play a        Our goal was to identify the renal involvement and systemic disorders in the
      role in the pathogenesis of IgAN in childhood. 13 patients (age        chronic infected patients with HCV, with detectable cryoglobulinemia. In this
      range; 9-20 year) and 18 age matched healthy controls (age             prospective and retrospective study were included 297 patients with HCV chronic
      range; 7-18 year) were taken into the study. All patients have         infection documented by the presence of anti-HCV antibodies (using ELISA II);
      normal renal function, blood pressure and no previous therapy          these patients was tested for cryoglobulis and measured cryocit. All was renal
      have been applied. Among all, one have persistant microscopic          and immunological investigated and some of they benefit by renal or/and skin
      hematuria, one have mild proteinuria (<0.5 gr/m2/day), 6 have          biopsy.
      both persistent microscopic hematuria and proteinuria (<0.5gr/         Cryoglobulins was detected in 115 (38,7%) of the patients chronic infected with
      m2/day), while 5 have normal urine. Neither macroscopic                HCV, type II in 31 (26,9%) and type III in 84 (73,1%) patients. Chronic liver
      hematuria nor infectious episode have been detected when study         disease was documented in 118 patients (39,7%), being present in these situa-
      was performed. PB total T lymphocyte (CD3+), B lymphocyte              tions in the on set of the infection diagnostic. The most frequent features of the
      (CD20+),T lymphocyte subsets (CD4+, CD8+) and CD40+ B                  renal involvement was proteinuria and microscopic hematuria with nephritic
      cells were analysed using dual color direct immunofluorescence         syndrome in 17 patients, nephrotic syndrome in 3 cases, isolated proteinuria
      method by flowcytometry (Coulter-EPICS XL-MCL). CD3+,                  and/or hematuria in 12 cases, respectively 27,8% with renal involvement. These
      CD4+, CD8+, CD20+ and CD40+ measurements were as fallows               disorders was detected in only 8 cases in the moment of cryoglobulinemia detec-
      (mean±SD).                                                             tion, in the rest of the cases during the time. The evolution to chronic renal failure
                  CD3+% CD4+ % CD8+ % CD20+% CD40+%                          was noted in 3 cases, in one of these to the uremia, needing dialysis. Extrarenal
      Patients    68.9±5     38.7±9     27.6±7     13±4       19.8±3         involvement were palpable purpura in 68 patients (59,1%), arthralgias in 61
      Controls 67.8±6        38.2±5     25.8±7     13±3       20.9±3         (53%), peripheral neuropathy in 29 (25,2%) and general features (weakness,
                                                                             increased body temperature, weight loss) in 103 (89,5%). Immune disorders
      No difference have been detected in PB total T, B lymphocytes          were: detectable rheumatoid factor in 43 patients (37,4%), decrease of the C4 and
      and their subset levels in children with IgAN compared to con-         C3 serum levels and the increase of liver transaminases in 2/3 of cases, sugges-
      trols (p>0.05). Therefore our preliminary results failed to sup-       tive for active liver disease.
      port the major role of celluler immune responce in the pathogenesis    In conclusion, the renal manifestations in the MC HCV associated are mainly of
      of IgAN; however, forworded for further investigations, espe-          glomerular type, rising later in the course of disease, but hepatic involvement is
      cially during clinical exacerbation of the diseases.                   present early.



A24                                                Nephrology Dialysis Transplantation                          Vol. 14 n.9                                1999
                                          Immunology, immunopathology and pathology
                                                      (basic and clinical)                                                                                             Abstracts

RENAL FIBROSIS PRECEDES INFLAMMATION IN A RAT                                      IgA NEPHROPATHY: A CLINICOPATHOLOGICAL CORRELATIVE AND FOLLOW-
MODEL OF OBESITY, A FEATURE AGAINST THE PARA-                                      UP STUDY OF 33 CASES
                                                                                   S Güçer1, K Tinaztepe1, M Güllülü2, K Dilek2, G Gönüllü2, M Yavuz2, B Can3, C Güven3,
DIGM OF INFLAMMATION IN NEPHROPATHY.
                                                                                   M Yurtkuran2,
J Chevalier, B Poirier, S Lavaud, C Mandet, MF Bélair, J Bariéty,                  Ped. Nephropathology Unit, Hacettepe Univ. Faculty of Medicine Ankara1, Dept. of
I Myara.                                                                           Nephrology, Uludag Univ. Faculty of Medicine Bursa2, Dept. of Histology-Embryology,
Inserm U430 and C. Bernard Ass., Broussais Hosp., Paris,                           Ankara Univ. Faculty of Medicine, Ankara3, Turkey
France.
                                                                                   Since primary IgA nephropathy(IgAN) presents with a highly variable clinical course and
                                                                                   histopathologic features in individual patients(pts) there have been still ongoing debates
Macrophages have been proposed as key factor in the process of
                                                                                   on the predictive histologic indicators. In this study, clinicopathological correlations were
renal injury. We examined the role of inflammation in the onset                    investigated in 33 pts with IgAN (ages 18-47 yrs, M:16,F:17) at the time of biopsy diagnosis
and progression of interstitial fibrosis in Zucker obese (fa/fa) ZO                and after a mean follow-up period of 33 months (range:2-120 mos). Treatment consisted
rats, which rapidly develop kidney lesions in absence of hyper-                    of prednisolone (Pred) alone, if unresponsive, Pred+Azathioprine, Pred+ Cyclophospha-
tension and hyperglycemia. Lean (Fa/fa) ZL rats act as an useful                   mide or Pred+CycAadministered for 2-50 mos (mean 20 mos).Two pts received no therapy.
internal control. Type I and III collagens were quantified using                   Renal biopsies were evaluated by light(LM) and immunofluorescence microscopy (IF) in
                                                                                   all and additionally by electron microscopy (EM) in three. Five pts had second renal
polarized light and computer-assisted image analyzer. The ki-
                                                                                   biopsies with two yrs. intervals. Hematuria (mainly gross) was present in 92% of the pts
netics of expression of mRNA encoding matrix components,                           and hypertension in 70%. LM showed histologic sublasses; minimal glomerulopathy (Class
adhesion molecules, chemokines and growth factors was fol-                         I) (no case), focal glomerulosclerosis type in 19 pts (Class II), focal proliferative GN (Class
lowed by reverse transcriptase polymerase chain reaction on                        III) in 3, diffuse proliferative GN (Class IV) in 5 and advanced sclerosing GN in 6. Histo-
pieces of kidney cortex. The presence of synthesized proteins as                   logic scores (HS) [Glomerular(1-6), interstitial (1-13), vascular (0-9) and total (2-32)] were
well as macrophages/ED +-cells was followed by immunohis-                          determined as previously described and their clinical correlations were analyzed. IF re-
                           1                                                       vealed heavy mesangial IgA deposition in all with C3(75%), IgM(40%), IgG (14%) in a
tochemistry. Fibrosis developed in two phases. The first one
                                                                                   lesser degree and no C1q deposition. EM showed paramesangial deposits with alterations
occured as early as 3 months and resulted from a neosynthesis                      in GBM. Conclusions of clinicopathological correlations were as follows: 1)There was no
of Type III collagen and fibronectin and a reduction of extracel-                  correlation between hypertension or a high serum IgA level on admission and prognosis
lular matrix (ECM) catabolism as shown by an increased ex-                         (p > 0.05) 2)Clinical improvement was obtained in 16 pts (10 in Class II, two each in other
pression of the metalloproteinase inhibitor TIMP-1. ECM and                                                                            ,
                                                                                   classes) while clinical worsening in 7 (4 in Class V 2 in Class IV and 1 in Class I), unchanged
TIMP-1 overexpression was probably under the control of TGF-                       clinical course in (5 in Class II and one in Class I). End-stage renal failure developed in 5
                                                                                   pts (3 in Class V and 2 in Class IV) and three were lost to follow-up. 3)Of five pts rebiopsied,
β1 which is synthesized at 3rd-month-onwards in ZO rats. These
                                                                                   two showed a correlative clinical and histologic improvement but the remaining three
events occured independently of any macrophage infiltration.                       had persistent pathologic findings despite clinical improvement. 4)Cases with high
The second phase started at 6 month-onwards: interstitial fibro-                   glomerular score (≥5) and total HS (≥18) showed a poor clinical course(p<0.05). 5)There
sis worsened with a large accumulation of Type I collagen. By                      was no prognostic effect of additional IgG and/or IgM depositions in glomeruli (p > 0.05).
contrast, this process appeared associated with ED1+-cell infil-                   6)These preliminary results might indicate the value of rebiopsy studies in determining
tration. Thus, in absence of hypertension and hyperglycemia,                       the prognostic indicators in larger series and longer follow-up periods.
inflammation cannot explain the onset of interstitial fibrosis
which develops in young obese Zucker rats. Once the lesions and
the renal impairment were launched, macrophages probably
aggravated the process.




MYOFIBROBLASTIC TRANSDIFFERENTIATION OF TUBULAR CELLS: A                           THE USE OF DIALYSATE FILTER U 8000 S CANNOT PRE-
POSSIBLE ROLE IN THE PROGRESSIVE RENAL SCLEROSIS                                   VENT INDUCTION OF IL-6 PRODUCTION DURING DIALY-
A Amore, P Cirina, G Conti, and R Coppo                                            SIS WITH CUPROPHAN/HEMOPHAN-MEMBRANES.
Nephrology and Dialysis Department, Regina Margherita Children’s Hospital,         H-J Guth, S Gruska, H Preez, G Kraatz (intr. by B Osten)
Torino, Italy                                                                      Department of Internal Medicine A, Ernst-Moritz-Arndt-Uni-
                                                                                   versity, Loefflerstr. 23a, 17489 Greifswald, Germany
The relentless fibrosclerosis in evolutive nephropathies is characterized by a
progressive expansion of newly formed matrix, in which fibroblastic-like cells     Activation of monocytes and other immuno-competent cells
are included. Renal tubular cells (TC) and fibroblasts share a common mesen-       during hemodialysis is due to contact with membrane surface,
chymal origin; the differentiation during the embryogenesis is regulated by the    blood line tubing systems and endotoxins. The use of cartridges
suppression or transcription of different genes: among those TGFβ3 and type I      (sodium bicarbonate column) and dialysate filter can reduce
collagen are tought to play a role.                                                endotoxins in dialysate. The kind of membrane can also play an
Aim of this study was to investigate the possibility to instigate in vitro the     important role in biocompatibility.
progressive fibroblast-like transdifferentiation of TC. Cells were conditioned     Method: 24 patients, undergoing standardized hemodialysis
with stimuli that in previous studies of our group were able to modulate several   including sodium bicarbonate cartridge system with
functional activities of TC, including Cyclosporin (CyA) (from 250 to 1000 ng/     Cuprophan/Hemophan (N=12)- and Polyamide (N=12)-mem-
ml), native human serum albumin (HSA) and fatty-acid-free HSA (faf-HSA)            branes, were immediately investigated before and after treat-
(from 2 to 20 mg/ml). As positive controls TC were incubated with 5-10 ng/ml       ment using a whole blood stimulation assay for IL-6 (“Dynamix”
TGFβ3 and 0.1-1 ng/ml type I collagen. We evaluated the following parameters:      Il 6 -DIA, Biosource Diagnostics, Germany). Before the next
the phenotype by light microscopy, some specific mesenchymal (vimentin, α          dialysis treatment all AK 200 hemodialysis machines were con-
actin -αSMA) and epithelial markers (cadherin) by immunoperoxidase and             verted to dialysate filter U 8000 S (all devices from Gambro
PCR. Moreover, the synthesis of TGFβ (ELISA and western blot) and its specific     Group, Lund, Sweden) and the same patients were investigated
mRNA (PCR) were detected. The results are reported in the table:                   again. In both groups (Cuprophan/Hemophan and Polyamide)
                          Vimentin       E-cadherin     α-SMA         TGFβ3        baseline concentrations and production capacity for Il 6, ob-
                                                                      (pg/ml)      tained during dialysis with and without U 800 S filter, were
TC basal                 10% (trace)     100% (+++)     —             31±4.2
                                                                                   compared (student’s t-test).
TC + HSA 10 mg/ml        25% (++)        30% (+)        30% (+)       41±7.8
TC+ faf-HSA 10 mg/ml     8% (trace)      97% (+++)      —             34±3         Results: In the Polyamide-group the IL-6-concentrations and
TC + CyA 500 ng/ml       50% (++)        40% (+)        45%           132±34       the production capacity for IL-6 were not markedly changed (p
TC+ TGFβ3 10 ng/ml       55% (+++)       60% (trace)    50% (+++)     __           > 0.05) before and after dialysis with or without dialysate filter.
                                                                                   In the Cuprophan/Hemophan-group IL-6-concentrations were
The synthesis of TGFβ3 was confirmed in western blot. In PCR we observed a         increased (p = 0.049) and the IL-6-production capacity was
modulation in the expression of the specific mRNA of the different markers         significantly elevated after dialysis (p = 0.003). The use of U
evaluated. A 25-45% of TC treated with HSA and CyA dysplayed a progressive         8000 S cannot prevent the induction of IL-6-production capac-
fibroblast phenotype transformation. The transition of TC to a fibroblastic        ity in peripheral blood mononuclear cells after dialysis (IL-6-
phenotype, detected by light microscopy, correlated with: a) decreased expres-     baseline p = 0.027; IL-6 stim. p = 0.033).
sion of the epithelial markers; b) increased expression of mesenchymal mark-       Conclusions: The use of dialysate filter U 8000 S cannot reduce
ers; c) synthesis and reorganization of actin fibers.                              immune response in Cuprophan/Hemophan dialysis, meas-
We conclude that different stimuli may elicit in TC a myofibroblastic transfor-    ured by IL-6-production capacity before and after dialysis.
mation: this mechanism could be relevant in the progressive sclerosis of neph-
ropathies.



Nephrology Dialysis Transplantation                             Vol. 14 n.9                              1999                                                                         A25
                                              Immunology, immunopathology and pathology
Abstracts                                                 (basic and clinical)


      EXPRESSION OF METALLOTHIONEIN IN VARIOUS                                                 PHARMACOLOGIC MODULATION OF THE EXPRESSION OF THE CELL
      GRADES OF RENAL CELL CARCINOMA. AN IMMUNO-                                               CYCLE REGULATORY PROTEINS IN AN EXPERIMENTAL MODEL OF
      HISTOCHEMICAL STUDY.                                                                     MESANGIAL NEPHRITIS
      H. Paraskevakou, S.E. Theocharis, L. Soubassi, A. Athanasiades,                          E. Menegatti§, M. Chiara§, G. De Rosa#, D. Bellis#, L.M. Sena§ and D. Roccatello*
      P.S. Davaris.                                                                            CMID* e Servizio di Anatomia Patologica#, ASL4, Dipartimento di Medicina
      Dept of Pathology, University of Athens, Medical School, Ath-                            ed Oncologia Sperimentale dell’Università di Torino§, Italy.
      ens Greece.
                                                                                               The response of mesangial cells to a phlogistic challenge includes cell prolifera-
      Metallothionein (MTs) are cytosolic proteins rich in cysteine ap-                        tion and mesangial matrix expansion. Cell proliferation is a highly regulated
      pearing to have a physiological role in the absorption, transport                        process which includes enhancing factor such as cyclins (C) and cyclin-depend-
      and metabolism of trace elements, mainly of zinc and copper.                             ent kinases (Cdk) and inhibitory proteins, such as p27. Cs and Cdks promote cell
      Recent reports have linked over-expression of cellular MT with                           entering into G1-phase and progression through cell cycle.
      the progression of malignant tumors.                                                     Aim of this study was to evaluate the effects, on the cell cycle regulatory system,
      The aim of this study was to investigate the possible signifi-                           of the purine analogue roscovitine (R), administered in the florid proliferation
      cance of MT expression in Renal Cell Carcinoma (RCC) in asso-                            phase of an experimental model of mesangial proliferative nephritis induced by
      ciation with the grading of tumors. Forty-five patients (33 M, 12                        the monoclonal antibody (MoAb) anti Thy-1 antigen.
      F) who underwent nephrectomy due to RCC consisted the group                              Twenty Wistar male rats (200-220 g body weight) were i.v. given 0.25 µl/kg BW
      of this study. Their age ranged from 36 to 82 years old. Sections                        of MoAb anti Thy-1 (OX-7, Cederlane). Three days after nephritis induction, 5
      of paraffin embedded tissues were stained immunohistochemi-                              rats were given roscovitine (2.12 mg/kg BW, Alexis) suspended in dimethylsul-
      cally by the streptavidin-biotin peroxidase technique, using a                           phoxide (DMSO) and 5 received the veicole alone. Samples of cortical and med-
      mouse (IgG ) monoclonal antibody (Zymed, San Francisco, Calif,                           ullary tissues were separately processed by RT-PCR for the gene expression of
                  1k
      USA) that recognized a common epitope for both MT isoforms                               cyclins B, D1, D2, D3 and the inhibitory protein p27.
      (I, II).                                                                                 The MoAb anti Thy-1 induced, one day after administration, focal aspects of
      From 45 tumors examined, 22 (49%) were characterized posi-                               mesangiolysis and tuft collapse and, after 7 days, a remarkable mesangial
      tive and 23 (51%) negative to MT expression. Specifically MT                             hypercellularity in 50 per cent of glomeruli, with aspects of neutrophil exuda-
      expression was detected only in 4 from 24 (17%) cases classified                         tion.
      as grade I. The frequency became higher in 11 from13 (84%)                               The analysis of the amplification products of the reverse transcribed RNA, sepa-
      cases of grade II whilst, all of the 8 (100%) cases of more ana-                         rately extracted from cortical and medullary tissues, showed, at the cortical
      plastic neoplasms (grades III, IV) showed positivity to MT. The                          level, a pronounced increase in gene expression of cyclin B (involved in the mi-
      intensity of staining was graded as mild to intense. MT distribu-                        totic process), D1, D2 and, even more, D3 (regulating cell entering into cell cy-
      tion was observed almost exclusively in the cytoplasm of                                 cle), and the inhibitory protein p27 one day after the MoAb administration.
      neoplastic cells and in the tubular epithelium in normal tissue.                         Cyclin hyperexpression persisted, weakened, at the 7th day.
      In conclusion, the difference in MT expression between grades II,                        Roscovitine, but not its vehicle, DMSO, markedly reduced cyclin B and D expres-
      III, IV and I, may indicate that MT overexpression in more                               sion and induced, at the cortical level, a remarkable increase in the expression
      anaplastic RCC is associated with poor prognosis of the dis-                             of the cell proliferation inhibitory protein p27.
      ease.                                                                                    The present study suggests the possibility to pharmacologically handle the regu-
                                                                                               latory system of cell proliferation, in response to a phlogistic challenge in an
                                                                                               experimental model of mesangial glomerulonephritis.


      QUANTITATIVE AND GENOTYPIC ANALYSIS OF THE HEPATITIS C VIRUS
      (HCV) DETECTED IN LYMPHOCYTES AND MONOCYTES FROM PATIENTS                                TUBERCULIN AND ANERGY SKIN TESTING OF PATIENTS
      WITH CRYOGLOBULINAEMIC GLOMERULONEPHRITIS
                                                                                               RECEIVING LONG-TERM HEMODIALYSIS
      E. Menegatti#, V. Ghisetti§, D. Rossi*, A. Barbui§, G. Marchiaro§, M. Chiara#,
      L.M. Sena# and D. Roccatello*.
                                                                                               H Taskapan, FS Oymak, A Dogukan, C Utas,
      CMID, Ospedale L. Einaudi*, Laboratorio di Microbiologia, Ospedale                       Nephrology and Respiratory Disease Dept. of Erciyes Univer-
      Molinette§, Dipartimento di Medicina ed Oncologia Sperimentale, Università               sity Medical Faculty, Kayseri, Turkey
      di Torino#, Italia.
                                                                                               The incidence of tuberculosis (TB) in patients on regular
      The hepatitis C virus (HCV) RNA is frequently found in sera and is concentrated          hemodialysis (HD) is higher than that of general population.
      in crioglobulins from patients with mixed crioglobulinaemia. It is widely ac-
                                                                                               The tuberculous skin test (TST) is an imperfect test for detecting
      cepted that the HCV infection is pathogenetically related to the crioglobulinaemic
      syndrome. HCV has a special tropism towards hepatocytes, but also mononu-
                                                                                               latent TB. End stage renal failure is known to be a risk factor for
      clear cells of the peripheral blood and bone marrow. Mononuclear cell infection          skin test anergy, but the rate of anergy in HD patients is unclear.
      could be etiologically relevant to the development of some crioglobulinaemic             In this study, the frequency of TB reactions and anergy in 65 HD
      manifestation.                                                                           patients (Male 35, female 30) were examined. The mean age was
      In the present study, limphocytes and monocytes from 8 patients with glomeru-            38.24±14.90 with range 14 to 70. All patients were tuberculin
      lonephritis associated with mixed crioglobulinaemia (type II in 6 and type III in        tested using the Mantoux tecnique with 0.1 mL (5 tuberculin
      other 2 cases) were separately isolated from the peripheral blood by means of
                                                                                               units) of purified protein derivative intradermally injected into
      centrifugation on Ficoll gradients and cell incubation on Petri’s plates (37°C for
      60 min.)
                                                                                               the volar surface of the forearm that did not have the arteriov-
      Total RNA was extracted according to Trizol ® procedure (Life Technologies,              enous shunt. Antigen for candida was injected by similar tech-
      Italy). RNA total amount and purity were evaluated by spectrophotometric                 nique, separated by 30 mm of skin. Results were interpreted 48
      analysis. Viral load was determined in cell extracts by RT-PCR (Amplicor                 hour later. Anergy was accepted as less than 2mm of induration
      Monitor Roche) and results were expressed as genomes/µg total RNA. HCV                   to candida antigen. Tuberculin positivity was defined as an
      genotype was identified in the two cell types and in sera by RT-Nested PCR               induration of ≥ 10 mm. The duration of HD ranged 4 to 126
      (Innolipa Immunogenetics, Belgium)
                                                                                               months with mean 43.12±28.47 months. No reaction to candida
      Each patient was viremic. Moreover, viral genome was revealed in 6 out of 7
      monocyte extracts and 4 of 7 lymphocyte extracts. Viral load in monocytes was
                                                                                               antigens was found in 24 (36.9%) patients. However, one of
      found to be higher than in lymphocytes [ 2.70 (0 - 10.00) vs 0.24 (0 - 1.26) genomes/    these patients reacted to TST. 14 of 24 patients considered to
      µg total RNA , p< 0.05)] The same genotype detected in serum was revealed in             have a positive candida antigen test were tuberculin positive. Of
      lymphocytes and monocytes from 6 out of 8 patients. In 2 monocyte extracts,              the 65 participants, 15 (23.1%) were TST positive. There was no
      beside the genotype determined in serum (1b), another genotypes was detected             significant relationship between age, the duration of HD and
      (3a and 2a/2c, respectively).                                                            anergy. Even with a high rate of anergy, TST test appears to be
      These results suggest that HCV has a definite tropism to the peripheral blood
                                                                                               useful test in HD patients. Anergy testing may be helpful to
      monocytes, which are more frequently infected (with higher viral load) than
      lymphocytes. The occasional presence in mononuclear cells of genotypes other
                                                                                               determine the predictive value of a negative TST.
      than those detected in sera suggests either a co-infection by different viral subtypes
      (which could use monocyte as a “reservoir”) or a high potential of viral muta-
      tion.



A26                                                            Nephrology Dialysis Transplantation                               Vol. 14 n.9                              1999
                                                Immunology, immunopathology and pathology
                                                            (basic and clinical)                                                                                           Abstracts

RENAL PROTEINASE-ACTIV       ATED RECEPTOR-2 (PAR-2) EXPRESSION CORRELATES                      TWO LATENT FORMS OF TGF-b1 ARE EXPRESSED IN THE NORMAL
WITH TUBULOINTERSTITIAL DAMAGE IN IgA NEPHROPATHY (IgAN).                                       HUMAN KIDNEY AND ARE DIFFERENTIALLY UP-REGULATED IN
G. Grandaliano, R. Monno, E. Ranieri, G. Cerullo, C. D’Altri, L. Gesualdo, F.P. Schena.
                                                                                                GLOMERULONEPHRITIS (GN)
Div. of Nephrology, Dept of Emergency and Organ Transplantation, University of Bari,
Bari, Italy.                                                                                    Z. I. Niemir, E. Pawliczak, P. Olejniczak, G. Dworacki, M. Kurpisz, R. Waldherr,
                                                                                                & S. Czekalski
Several studies have suggested the involvement of mast cells in the pathogenesis of tissue      University of Medical Sciences and Polish Academy of Sciences, Poznan, Po-
fibrosis in different organs, although the mechanisms underlying their potential fibrogenic     land, and University of Heidelberg, Germany.
role is still largely unclear. Tryptase is one of the main components of mast cell secretory
granules and can be released upon cell activation. PAR-2 is a G protein-coupled receptor
                                                                                                Results concerning the expression of latent TGF-1 forms in glomerular diseases
that is cleaved and activated by trypsin-like proteolytic enzymes, including tryptase.
The aim of the present study was to evaluate mast cell infiltration and PAR-2 gene expres-      are rather scarce.
sion in IgAN renal biopsies and to investigate the potential role of tryptase-PAR-2 inter-      We performed an immunocytochemistry study on renal biopsy specimens with
action in the pathogenesis of glomerular and interstitial fibrosis. To this purpose the pres-   features of IgA-GN (n=37), membrano-proliferative GN (MPGN; n=4), idiopathic
ence of tryptase-positive cells were investigated by histochemistry in 17 biopsies from         membranous GN (IMGN; n=14), focal-segmental glomerulosclerosis (FSGS;
IgAN patients and 10 apparently normal portions of kidneys removed for renal carci-             n=12), and minimal change disease (MCD; n=11), looking for the expression of
noma. PAR-2 gene expression was evaluated in the same tissue samples by RT-PCR,
                                                                                                the large latent TGF-b1 complex (LLTC), small latent TGF-b1 complex (TGF-b1
quantified by computerized densitometry and normalized to GAPDH expression. PAR-2
gene expression was also evaluated, by RT-PCR, in cultured human mesangial and proxi-           LAP), an active form of TGF-b1, and TGF-b receptor type II (TGF-bRII).
mal tubular cells under basal conditions and upon stimulation with IL-1, a pro-inflamma-        Normal human kidney served as control (n=4). The generation of the active form
tory cytokine mainly produced by monocytes. Finally, gene and protein expression of TGF-        of TGF-b1 in the kidney samples was also analysed by immunoblotting of de-
β, a powerful fibrogenic factor, was evaluated in trypsin-stimulated human mesangial            tergent-buffer extracts from these specimens, separated by SDS-PAGE under
and proximal tubular cells by northern blot and ELISA, respectively.                            non-reducing conditions.
Tryptase-positive cells were not detected either in the glomeruli or in the interstitium of
                                                                                                Our results show that in the normal kidney LLTC is expressed in mesangial
normal kidneys, whereas in IgAN biopsies they were present mainly at the interstitial level
and scantly within the glomerular tuft. In normal kidneys, PAR-2 gene expression was            areas, whereas TGF-b1 LAP localises to podocytes. The expression of TGF-bRII
barely detectable by RT-PCR, whereas in the biopsy samples obtained from IgAN patients          is predominantly noted in association with LLTC. Up-regulation of LLTC is
the mRNA levels for this protease-activated receptor were strikingly increased (PAR-2/          observed in MCD, early stages of IMGN and FSGS, as well as in IgA-GN with
GAPDH ratio: Control 0.37+0.06; IgAN 1.6+0.2, p<0.01). Interestingly, PAR-2 gene expres-        mild mesangial cell proliferation. The expression of LLTC decreases, however,
sion was directly correlated with the extent of interstitial fibrosis (r=0.778, p<0.001) and    in MPGN, IgA-GN with marked proliferative response of mesangial cells, and
mononuclear infiltrate (r=0.65, p=0.02). PAR-2 mRNA was expressed by cultured human
                                                                                                in advanced stages of FSGS. The podocyte expression of TGF-b1 LAP increases
mesangial and proximal tubular cells and its expression was induced in a time-dependent
manner by IL-1 in tubular, but not in mesangial cells. Finally, the activation of PAR-2 by      particularly in IgA-GN with intermittent macroerythrocyturia and low-range
trypsin, in both mesangial and tubular cells, induced a significantly upregulation of TGF-      proteinuria, while diminishing in nephrotic patients. The intensity of
β gene expression with a peak at six hours, followed by an increase of TGF- β protein           immunostaining for the active form of TGF-b1, confirmed by Western-blot, seems
synthesis and release.                                                                          to correlate with the expression of LLTC.
Our data suggest that tryptase released from mast cells may play a significant role in the      Our results seem to indicate a link between up-regulation of LLTC and the pres-
pathogenesis of extacellular matrix expansion in IgAN through the interaction with its
                                                                                                ervation of the glomerular architecture. A negative relationship between the
specific cell-surface receptor and the subsequent increase of TGF- β expression
                                                                                                podocyte expression of TGF-b1 LAP and the glomerular filter permeability
                                                                                                may also be suggested.




FAS AND BCL-2 EXPRESSION ON LYMPHOCYTE SUBPOPULATIONS IN                                        CYTOKINE INDUCED DIFFERENTIAL EXPRESSION OF ADHESION MOL-
IgA NEPHROPATHY                                                                                 ECULES AND HLA-DR IN CULTURED HUMAN GLOMERULAR ENDOT-
J Gascó, J Iglesias, R Bernabeu, N Matamoros and X Bestard.                                     HELIAL CELLS (HGEC).
Dept. of Nephrology and Immunology. Hospital Son Dureta. Palma de Mallorca.                     S Park, WS Yang, SK Lee, JH Kim, HH Jung, H Ahn, JD Lee.
Spain.                                                                                          Dept. of Internal Medicine, Urology, Biochemistry, University of Ulsan, Seoul,
                                                                                                Korea
Abnormal T and B lymphocyte function and aberrations in lymphocyte mark-
ers have been recognized in IgA Nephropathy. We study the peripheral blood                      Glomerular endothelial cells should participate in the process of glomerular
lymphocytes (PBL) spontaneous apoptosis and Fas and Bcl-2 expression in                         disease by expressions of HLA antigens and adhesion molecules. However, few
patients diagnosed by renal biopsy of Idiopathic Mesangial IgA Nephropathy.                     has been known about the regulation of the expression of these molecules in the
PBL of 18 p diagnosed of Idiopathic IgA Nephropathy were studied. As normal                     glomerular endothelial cells. In this study, we investigated the effect of cytokines
controls, 14 healthy subjects. MoAb used: CD8-PerCP, CD4-PE, CD19-PE and                        (IL-1β, TNF-α, IFN-λ) on the expression of ICAM-1, VCAM-1, and HLA-DR in
Simultest control from Beckton Dickinson (BD), CD95-FITC (UB-2) by Coulter                      cultured HGEC. HGECs were isolated from kidneys resected for renal cell car-
Immunotech. Two or three colour analysis on a FACScan flow cytometer. For the                   cinoma, and confirmed by homogenous positive staining of factor VIII and
intracellular Bcl-2 expression, staining of surface antigens was performed first.               homogenous uptake of fluorescent-labeled acetylated LDL (DiI-Ac-LDL). Cel-
Whole blood was permeabilized using FACS permeabilizing solution from                           lular expressions of ICAM-1, VCAM-1, and HLA-DR were quantified by ELISA
BD.Bcl-2 expression was measured using direct staining with Bcl-2 MoAb (FITC-                   on fixed adherent cells. The results are as followings;
labelled; Dako). PBM were obtained by Ficoll density gradient centrifugation                                                   ICAM-1 (n=9)1                         VCAM-1 (n=6)1
and cultured for 72 hours in complete medium. Apoptoic cells were evaluated                                    6h            24h           48h         6h        24h         48h
                                                                                                                           2
                                                                                                control        3.01±0.19 3.23±0.23 3.39±0.25 1.37±0.22           1.49±0.19 1.60±0.29
by flow cytometry using 7-aminoactinomycin D (7-AAD, Sigma) as DNA                                                        *           **                      *            *
                                                                                                IL-1β(5ng/ml)  3.74±0.03 3.73±0.04 3.75±0.04 2.23±0.21 2.04±0.31 1.72±0.29
marker,after staining with surface antigens with diferent monoclonal antibod-                   TNF-α(10ng/ml) 3.45±0.03* 3.77±0.03* 3.76±0.03 2.49±0.19* 2.56±0.23* 2.30±0.36*
ies for specific populations. Data were analyzed using the t-Student test (Berger               IFN-σ(20ng/ml) 3.44±0.39* 3.77±0.02* 3.73±0.03 1.34±0.18         1.42±0.14 1.44±0.17
vs Control patients). 1)CD95 expression on cell subsets. Percentage: CD4+ T                                   HLA-DR (n=3-4)1
cells, 29+/-10% vs 31+/-8%; CD8+ T cells, 21+/-13% vs 19+/-10%; CD19+,                                         24h            48h             72h         1: each n is the mean of 4-
12+/-4% vs 7+/-3% (p=0.004). Mean Fluorescence Intensity (MFI): CD4+ T cells,                   control        0.38±0.05      0.49±0.09       0.45±0.04   8 well experiment
                                                                                                IL-1β(5ng/ml) 0.54±0.04       0.65±0.14       0.45±0.06   2: the value of optical den
27+/-6% vs 27+/-3%; CD8+ T cells, 21+/-6% vs 19+/-3%; CD19+, 28+/-8 vs
                                                                                                TNF-α(1ng/ml) 0.44±0.05       0.71±0.04       0.47±0.04   sity mean ± standard error
29+/-11. 2)Bcl-2 expression on cell subsets (Berger vs Controls patients). Per-                 IFN-γ(10ng/ml) 0.76±0.16 *
                                                                                                                              2.11±0.34  *
                                                                                                                                              2.40±0.23*  *: P<0.05, compared to
centage: 100% of the cells studied in the diferent subsets were Bcl-2+.MFI:CD14+                IFN-γ(20ng/ml) 0.85±0.15 *
                                                                                                                              2.13±0.24  *
                                                                                                                                              2.67±0.12 *
                                                                                                                                                          control by paired t test
Tcells, 113+/-29 vs 108+/-14; CD8+ T cells, 95+/-23% vs 109+/-16 ; CD19+,                                                          **: P=0.053
79+/- 18 vs 75 +/-16. 3)Percentage of spontaneous apoptosis on cell subsets:
CD4+ T cells, 23+/-7 vs 10+/-6 (p<0.0001); CD8+ T cells, 30+/-13 vs 11+/-6                      The results showed that 1. ICAM-1 was increased by IL-1β, TNF-α and IFN-γ. 2.
(p<0.0001); CD19+, 41+/-13 vs 35+/-13.                                                          VCAM-1 was increased by IL-1β and TNF-α, not by IFN-γ. 3. IFN-γ only increased
Spontaneous apoptosis in peripheric CD4+ and CD8+ T cells from IgA neph-                        expression of HLA-DR in HGEC. 4. Basal expression of ICAM-1 was higher
ropathy patients were increased significantly, comparing with controls.                         than VCAM-1 and HLA-DR in cultured HGEC. 5. The time course of expression
Normal levels of spontaneous apoptosis were observed in B lymphocytes                           was different according to adhesion molecule. In conclusion, HGECs expressed
(CD19+). Conversely, the expression of CD95 were raised in CD19+ cells. We                      adhesion molecules and HLA-DR, which were regulated differentially by
have not found diferences in the Bcl-2 expression between IgA Nephropathy                       inflammatory and immune-regulatory cytokines.
patients and normal controls.




Nephrology Dialysis Transplantation                                       Vol. 14 n.9                              1999                                                                 A27
                                            Immunology, immunopathology and pathology
Abstracts                                               (basic and clinical)


      MORPHOLOGICAL INVESTIGATION OF DNA-FRAGMENTATION/                                     SECRETION OF PDGF, TNF-α, IL-6 AND IL-8 BY UROEPITHELIAL CELLS
      APOPTOSIS IN KIDNEY BIOPSIES OF PATIENTS WITH CHRONIC                                 STIMULATED BY E. COLI AND CITROBACTER
      GLOMERULONEPHRITIS (GN)                                                               R Fünfstück1, S Franke1, M Hellberg1, B Knöfel2, E Straube2, J. Hacker3, G Stein1
      Ott, U.1, A. Koscielny1, D. Kinder1, R. Fünfstück1, A. Aschoff2, G. Jirikowski2, G.   1
                                                                                             Dpt. of Internal Medicine and 2Institute of Medical Microbiology, University of
      Stein1                                                                                Jena; 3Institute of Molecular Infectional Biology, University of Würzburg; Ger-
      1
       Dpt. of Internal Medicine IV and 2Dpt. of Anatomy II, Friedrich-Schiller-Uni-        many
      versity of Jena, Germany
                                                                                            Urinary tract infections induce a local response of mucosal cells and systemic
      In many glomerular diseases a hypercellular, proliferative state changes to a         inflammatory reactions including the activation of polymorpho-nuclear cells
      hypocellular, sclerotic phase. The mechanism responsible for terminating              and release of various immunoregulatory cytokines, which are important for
      glomerular cell proliferation is not clearly understood. It is speculated that        the initiation and maintenance of renal inflammation.
      regulation and clearance of cell excess as well as cell proliferation is balanced     Cytokine release (PDGF, TNF-α, IL-6, IL-8) of T 24 bladder cells as stimulated
      by apoptotic mechanisms.                                                              by the noninvasive the E. coli HB 101 (K: 12), the uropathogenic E. coli 0: 18 and
      In renal biopsies of 24 patients with proliferative GN (IgA nephropathy,              the highly invasive Citrobacter CB 3009 were determined. Analyses were per-
      mesangioproliferative and membranoproliferative GN), 14 patients with                 formed after incubating 20.000 cells/ml with 20.000-000, bacteria/ml in the
      nonproliferative GN (focal segmental glomerulosclerosis, minimal change GN,           supernatants for 5, 15, 30, 60 and 120 min.
      membranous GN) and 7 patients with rapid progressive glomerulonephritis               Cytokine production was induced by all microorganisms investigated. The
      (RPGN), in situ end labeling (ISEL) of fragmented DNA were performed for              processes were time-dependent, with a significant increase in cytokine activity
      characterizing cells with different degrees of DNA damage probably leading to         after 60 min. The release of PDGF and IL-8 was found to be higher due to the E.
      apoptosis. For ISEL staining of semithin sections bromodesoxyuridine (BrdU)           coli HB 101 than by the other bacteria. Citrobacter CB 3009 induced the highest
      and terminal transferase were used. Incorporated BrdU was visualized by an            IL-6 level. A stimulatory effect of TNF-a was detectable 240 min after starting
      anti-BrdU antibody and by peroxidase-antiperoxidase. The rate of apoptotic            the experiment.
      cells could be evaluated by the Quantimet-System (Fa. LEICA).                         Cytokine levels after 120 min of interaction in FCS (pg/ml)
      DNA-fragmentation was mainly found in the nuclei of distal tubular epithelial                                     PDGF         IL-6          IL-8         TNF-α
      cells, but also in the nuclei of mesangial cells in the glomerulus.                   time (min)                  120          120           120          240
      DNA-fragmentation in nuclei per 15 000 µm2:                                           -----------------------------------------------------------------------------
                                     glomerulus               tubulus                       CB 3009                     176          436           4386         16
      RPGN                           29,0+/-4,9               23,6+/-5,6                    E. coli 0: 18               118          84            7794         44
      proliferative GN               27,6+/-3,7               17,0+/-6,8                    E. coli HB 101              262          101           8592         33
      non-proliferative GN           19,0+/-4,2               22,0+/-3,4
                                                                                            In all analyses, a significant correlation was found between the timedependent
      The difference between proliferative and non-proliferative GN was significant         release of IL-6 and IL-8 (p<0.05). In case of Citrobacter infection, the PDGF level
      (p < 0,05). The degree of DNA-damage did not correlate with the concentration         significantly correlated with IL-6 and IL-8 values (p<0.001).
      of serum creatinine or proteinuria and no correlation was found for urinary           Conclusion: The data demonstrate that urinary tract epithelial cells are able to
      excretion of albumin, a1-microglobulin and IgG. As one sign of progressive            produce different cytokines in response to an incubation with E. coli and
      damage to the cells, DNA-fragmentation could be detected in proliferative GN.         Citrobacter strains. The virulence of the pathogens does not seem to play a role.
      In mesangial cells apoptosis is important for the clearance of proliferating cells,   This epithelial cell cytokine response can modify immunoregulatory processes
      but in the distal tubule it could indicate a cell turnover depending on the degree    caused by an infection responsible for parenchymal injury.
      of inflammation.



                                                                                            CYTOKINE PROFILES DEFINES DIFERENT RESPONSES TO
                                                                                            INTERFERON-α TREATMENT IN PATIENTS WITH CHRONIC HEPATITIS C
      DETECTION OF SELECTED ENZYMATIC PATTERNS IN
                                                                                            VIRUS INFECTION (HCV) AND END STAGE RENAL DISEASE (ESRD)
      KIDNEY BIOPSY MATERIAL FROM PATIENTS WITH                                             G.L. Kissinger, J. Stachowski*, M. Pollok, H. Steffen, K. Jihlke, B. Kneissl, U. Töx,
      GLOMERULONEPHRITIS                                                                    T. Goeser, C.A. Baldamus;*
      M. Gliesing2, U. Ott1, R. Fünfstück1, K.-J. Halbhuber2, J. Schubert3,                 University of Posen, Poland; Medical Clinic IV, University of Cologne, Köln,
      G. Stein                                                                              Germany
      Depts. of Internal Medicine IV 1, Anatomy II 2 and Urology3,
      Friedrich-Schiller-University of Jena, Germany                                        Cytotoxic T lymphocytes, natural killer cells (NK) and immunoregulatory
                                                                                            cytokines may be important in the clearance of hepatitis C virus (HCV) infected
                                                                                            cells. T-helper type 1 (Th1) cytokines (Interleukin-2, Interferon-γ) are required
      Glomerulonephritis (GN) is not only characterized by changes of                       for antiviral immune response, including cytotoxic T cells and NK activation.
      the glomeruli but also by disturbances of the tubulointerstitium.                     In contrary T-helper type 2 (Th2) cytokines (Interleukin-4, Interleukin-10) in-
      Enzymatic patterns of tubular cells in kidney biopsy material                         hibit the effective antiviral defense. However there are only a few studies that
      from 18 pats. with GN were evaluated and compared with                                specifically analyzed the levels of immunregulatory cytokines under a specific
      normal renal tissue. The activities of monoamine oxidase                              therapy in chronic HCV infection and there are no data available of patients
      (MAOX), cytochrom c oxidase (COX), succinate dehydrogenase                            with HCV and ESRD. For these reasons we have studied the effects of Interferon-a
                                                                                            (INF-α) monotherapy on Th1 and Th2 cytokine levels in ESRD patients with
      (SDH) and glucose-6-phosphatase (G6Pase) were analyzed his-
                                                                                            HCV compared to patients with HCV without ESRD (n=20) and to normal con-
      tochemically by lightmicroscopical metal precipitation, in case                       trols (n=20).
      of SDH by tetrazolium technique. After quantifying enzymatic                          10 consecutive anti-HCV and HCV-RNA positiv patients on maintenance
      activities by Quantimet 500+ an index for the area of enzymatic                       hemodialysis (mean age 45.4±12.1 years) were treated with INF-α2b (5MU 3
      reaction to total tubule area in correlation to the mean grey value                   times/week, INTRON, Essex). Studies were undertaken before, 12 weeks during
      was determined (Tab.).                                                                and after INF-α therapy.
      Enzymatic activities:                                                                 Cytokines were evaluated in culture supernatants of PBMC cultured for 48 hours
                                                                                            in Costar plates. The levels of cytokines were measured by means of a solidphase
                       MAOX           COX          SDH       G6Pase
                                                                                            enzyme-linkedimmunosorbent-assay (ELISA), specific for human IL-2, IFN-γ
      GN               0.2-8.9        0.5-9.8      0.5-6.3   0.1-8.5                        and IL-4, IL-10 (GenzymeCorporation, Cambridge, USA). Response was de-
      controls         11.4           15.6         8.1       19.3                           fined after 12 weeks of therapy (HCVRNA negativ). 4 patients were treatment
                                                                                            responder. Before therapy all patients had a TH2 pattern compared to normal
      No difference between proliferative and non-proliferative GN                          controls. There was no difference between the patients with or without ESRD.
      was detectable, but the interrelationship between enzyme activi-                      INF-α therapy decreased the TH2 cytokines and increased TH1 cytokines. But
      ties and the degree of tubular damage was significant. SDH was                        Nonresponder (NR) had significantly lower TH1 and higher Th2 levels (p<0.01).
                                                                                            Our results suggest that persistently high TH2 patterns indicate NR to therapy.
      reduced more focally in contrast to the general reduction of
                                                                                            Responder showed a stronger switch to TH1 cytokine profiles compared to NR.
      G6Pase, MAOX and COX. The reduced enzymatic activities                                There was no difference between patients with ESRD and without ESRD. As in
      reflect tubular damage and correlate with proteinuria, espe-                          patients without ESRD the stronger modulation towards Th1 pattern may be
      cially with a1-microglobulin and albumin excretion.                                   one mechanism of HCV clearance.



A28                                                          Nephrology Dialysis Transplantation                               Vol. 14 n.9                               1999
                                      Immunology, immunopathology and pathology
                                                  (basic and clinical)                                                                 Abstracts

DISTRIBUTION OF TYPE IV COLLAGEN α1 TO α6 CHAINS                            THE INFLUENCE OF HEMODIALYSIS ON PERIPHERAL
IN PRIMARY IgA NEPHROPATHY.                                                 BLOOD LYMPHOCYTE APOPTOSIS
S Palle1, B Laurent1-2, Y Sado3, C Deprele1-2, JM Koller1-2, F Berthoux1-   1
                                                                             J. Rysz, 2E. Majewska, 1J. Mudyna, 1Z. Zbrog, 2Z. Baj, 1M. Luciak,
2                                                                           1
                                                                              P. Bartnicki.
1                                                                           1
  Res. Group on Glomerulonephritis and Renal Transplantation                 2nd Dept. of Internal Medicine, WAM and 2Dpt. of Pathophysi-
(UPRES-EA n°773), 2Dpt. of NDT Univ. of Saint-Etienne, France.              ology, WAM, Lodz, Poland.
3
  Div. of immunology, Shigei Med. Res. Inst., Okayama, Japan
                                                                            The percentage of apoptotic lymphocyte cell was evaluated in
In several nephropathies, variations in the thickness of glomeru-           10 chronic uremic patients before and after hemodialysis ses-
lar basement membrane (GBM) were generally associated with                  sion and following 24-hr culture. The control group consisted of
alterations of extracellular components. Abnormal thinning of               10 healthy persons. Apoptosis was investigated by means of
GBM (<275nm in M ; <265nm in F) was found in 40% of patients                Annexin V-FITC and TUNEL flow cytometry methods. The re-
with IgA nephropathy (IgAN) leading to hypothesize a defect in              sults are presented as apoptotic cell percentage of the whole cell
the type IV collagen network.                                               population (X+SEM):
Using immunolabelling technics, we investigated the distribu-                             Healthy                         Patients
tion of the α1 to α6 type IV collagen chains in thin- (n=4) and                                              before HD            after HD
normal-GBM (n=4) of IgAN patients as compared with normal                   Annexin       2.38±0.69          4.23±2.37            5.43± 8.06
kidneys (n=4).                                                              TUNEL         10.78 ± 5.90      8.58 ± 0.66           11.85 ± 9.17
No significant changes were observed in α1 to α6 chains between
IgAN and normal kidneys. We failed to find a significant in-                                           24-hr culture
crease of α3 chain despite an increased labelling in IgA patients.                       Healthy                            Patients
On the contrary, a significant increase of α2 chain was evidenced                                          before HD        after HD
in the GBM of IgAN with normal-GBM; no modifications were                   Annexin      8.98±15.3         5.25±2.12 a      4.10±2.54 a
found in the mesangium between the three groups but a signifi-              TUNEL        14.10±24.50       4.65±0.92 a      11.03±14.60 a
                                                                            a
cant increase was also noted in the Bowman’s capsule in both                  p<0.05 in relation to healthy persons
normal- and thin-GBM groups. For α4 and α5 chains, a signifi-
cant weaker labelling was found in two patients independently               It seems that hemodialysis does not immediately numbers of
of thin- or the normal-GBM, while others are comparable to                  apoptotic cells. However, following 24-hr culture lymphocytes
normal.                                                                     taken from patients before and after hemodialysis were lower in
In conclusion, our study failed to evidence defect or significant           comparison with those from healthy persons.
alteration in the distribution of collagen IV αchains in IgAN and
therefore Thin-GBM could not be explained by abnormal colla-
gen IV network.



ROLE OF INTERLEUKIN-18/ IFN-γ INDUCING FACTOR (IL-                          PROGNOSIS MARKERS OF TUBULOINTERSTITIAL INJURY
18/IGIF) IN IgA NEPHRITIS                                                   IN MESANGIOCAPILLARY GLOMERULONEPHRITIS TYPE
Napodano P, *Libetta C, *Centore F, °Meloni F, Marzorati A,                 I
Ferrario F, *Dal Canton A, D’Amico G.                                       L. Petrica1, M. Raica2, A. Schiller1, S. Velciov1, Gh. Gluhovschi1,
                                                    ,
Renal Immunopathology Center, Division of Nephrology S. Carlo               V Trandafirescu1, Gh. Bozdog1, C. Gluhovschi1, F. Bob1
                                                                              .
                                                                            1
Borromeo Hospital, Milan and *Departement of Internal Medi-                   - Dpt. of Nephrology, 2 - Dpt. of Histology, County Hospital,
cine and Nephrology and °Istitute of Respiratory diseases,                  University of Medicine and Pharmacy, Timisoara, Romania
S.Matteo Hospital, Pavia, Italy.
                                                                            A group of 32 patients (p) with mesangiocapillary type I
IL-18/IGIF is a recently characterized cytokine, structurally re-           glomerulonephritis (MCGN) [M-19p (59.37%); F-13p (40.63%);
lated to IL-1β and produced by monocytes/macrophages, that                  mean age - 42.31 ± 11.28 y] was assessed concerning the rela-
induces IFN-y synthesis by T cells and synergizes with IL-12 in             tionship between the severity of the tubulointerstitial (TI) le-
T helper type1 (Th1) cell development, while it inhibits the pro-           sions and blood pressure (BP), proteinuria and serum creatinine
duction of some anti-inflammatory cytokines, such as IL-10.                 (SC).
Moreover, some recent studies have suggested a potential role of            All p underwent kidney biopsies which were processed in light
IL-18/IGIF in immunoregulation by mediating immune cell in-                 microscopy (LM - hematoxylin-eosin, Masson’s trichrome, PAS),
filtration into the inflammed tissues and it is already well known          immunofluorescence, immunohistochemical (IH) procedures
the important role of leukocyte infiltration in the renal damage            with monoclonal antibodies [performed with the EPOS
of human proliferative nephritis, such as IgA nephritis.                    system-DAKO: antismooth muscle cell actin (a-SMA),
In order to investigate the potential role of IL-18/IGIF in IgA             anti-desmin (D), anti-cytokeratin (CK), anti-proliferating cell
nephritis we measured, by ELISA, the serum levels of IL-18/                 nuclear antigen (PCNA)].
IGIF (IL-18 S) in 6 healthy subjects and, at biopsy time, in 16             Results were analysed by statistical methods (Student’s un-
IgA patients: 9 pts with necrotizing/crescentic IgA nephritis               paired ttest, Pearson’s test, Spearman’s rank order test); signifi-
(NC-IgAN) (% of crescents 25.2±10.6; score of interstitial dam-             cance was considered as P<0.05.
age: 3.2±0.9, range 0-4) and 7 pts with non necrotizing/crescentic          The evaluation of the TI lesions in LM revealed in 23 p (71.88%)
IgA nephritis (NNC-IgAN)(score of intersitial damage: 1.7±0.7,              severe TI injury, which correlated significantly with the intersti-
range 0-4).                                                                 tial IH data: - the extent of the a-SMA positive cells
IL-18 S in IgA pts were significantly higher than those observed            (myofibroblasts) infiltrates (P<0.001), PCNA (P<0.05), D (proxi-
in control subjects (26.2±20.2 vs 4.1±2.5 pg/ml, p<0.01). Moreo-            mal tubular necroses) (P<0.01), CK (distal tubular necroses)
ver, NC-IgAN pts exhibited significantly elevated IL-18 S when              (P<0.001). Proteinuria correlated with a-SMA (P<0.01), PCNA
compared with NNC-IgAN pts (28.1±15.4 vs 10.1±4.3 pg/ml,                    (P<0.05), D (P<0.01), CK (P<0.01); SC with a-SMA (P<0.001),
p < 0.02).                                                                  PCNA (P<0.01), D (P<0.01), CK (P<0.05). BP did not correlate
In conclusion, our results suggest that IL-18/IGIF might play a             with these parameters.
potential role in the renal damage of IgA nephritis, especially in          TI lesions in MCGN type I imply important myofibroblasts infil-
those forms characterized by extracapillary proliferation and               trates and severe proximal and distal tubular involvement. These
marked leukocyte intraglomerular and interstitial infiltration.             changes are consistent with the level of proteinuria and the rate
                                                                            of decline of the renal function.




Nephrology Dialysis Transplantation                       Vol. 14 n.9                      1999                                                   A29
                                                 Immunology, immunopathology and pathology
Abstracts                                                    (basic and clinical)


      IMMUNOLOGICAL CHANGES IN ANCA POSITIVE NE-                                                       THE BOOSTER PHENOMENON IN TUBERCULIN SKIN TESTING (TST) OF
                                                                                                       HEMODIALYSIS (HD) PATIENTSAND THE RELATIONSHIP WITH SOLU-
      PHROPATHY                                                                                        BLE INTERLEUKIN 2 RECEPTOR (sIL-2R) LEVELS
      R. Vladimirova, V. Draganov, B. Kenarova, K. Plochev, R.                                         A. Akçay, Y. Erdem, B. Altun, S. Ulusoy, C. Usalan, Ü. Yasavul, Ç. Turgan, S.
      Penkov                                                                                           Çaglar and S. Kirazli
      Military Medical Academy, Sofia, Bulgaria                                                        Hacettepe University School of Medicine, Ankara-Turkey

                                                                                                       The booster effect is defined as an initially negative TST in a patient which is
      The aim of the study is to assess the parameters of humoral                                      then boosted to a positive test by the testing procedure itself. The significance of
      and cellular immunity and levels of some cytokines in se-                                        the booster effect in sequential testing of this patients has not been determined.
                                                                                                       It is suggested that elevated serum sIL-2R levels may decrease the bioavailability
      rum and urine of 11 patients with clinicolaboratory                                              of IL-2 for T cell response to antigenic stimuli in HD. We examined the frequency
      constelation of systemic vasculitis with nephropathy. Pa-                                        of the booster effect and anergy, and the relationship with serum sIL-2R levels
      tients: Included in the study were 3 patients with poliarteritis                                 in HD patients.
      nodosa, 3 with SchonleinHenoch nephropathy, 3 with                                               Fifty three HD patients, aged 20-72yr, M:F 35:18 were studied. Ten (18.8%) pa-
                                                                                                       tients had a history of active tuberculosis. Skin tests were placed by the Mantoux
      pulmo-renal syndrome of Goodpasture, 2 with other forms                                          method, using 0.1 mL of tuberculin (5 TU) and candidin control antigen (10.000
      of systemic vasculitis, and 10 healthy persons as controls.                                      BE/ml). Patients with <10 mm induration to the initial TST had a repeat tuber-
      Methods: The parameters of cellular immunity were deter-                                         culin test 7 days later. Nineteen (35.8%) of 53 patients demonstrated a signifi-
                                                                                                       cant tuberculin reaction (>10 mm) on the initial TST, and 10 (18.8%) additional
      mined through the flowcytometry method. The titers of                                            patients showed a significant reaction (booster effect) to a second TST (p<0.001).
      antineurophil cytoplasmic antibodies (ANCA) were meas-                                           All the patients with a history of tuberculosis had a strongly positive tuberculin
      ured through indirect immunofluorescense and the levels                                          reaction (>15 mm) on the initial TST. Ten (18.8%) of 53 patients did not response
      of IL-2, IL6, and INF-gamma were measured through the                                            to either candidin or two-step TST, and this patients were considered completely
                                                                                                       anergic. Serum sIL-2R levels were measured in 10 healthy subjects (age 22-62,
      ELISA method. Results: Total T-cell population and T-helpers                                     5M, 5F), 10 anergic (age 24-56, 4M, 6F) and 10 non-anergic (age 28-61, 6M, 4F)
      in ANCA positive patients were significantly increased in                                        HD patients. The mean values of serum sIL-2R were significantly increased in
      contras to T-suppresors and B-cells, which were decreased.                                       the patients as compared to healthy subjects (12.65±5.64 vs. 4.13±0.76 pg/mL,
                                                                                                       p<0.001). The levels of serum sIL-2R were significantly higher in the anergic
      The levels of IL-2 and INF-gamma were significantly in-                                          group than in the non-anergic group (17.98±5.69 vs. 7.72±1.24 pg/mL, p<0.005).
      creased, especially in the urine of the patients as compared                                     Our findings suggest that the booster effect is not uncommon phenomenon in
      to the controls, but there was no difference in IL-6 levels.                                     end-stage renal disease and the elevation of serum sIL-2R levels may play an
      Conclusion: Our finding in this survey supports the possi-                                       important role in the pathophysiological mechanisms of cutaneous anergy in
                                                                                                       uremic patients.
      bility of Th1 way of immune caskade playing important or
      maintanance of glomerular barrier dysfunction.



      THE COMPARISON OF ANTIBODY RESPONSE TO INFLUENZA VACCINATION IN                                  INTERFERON-Α (IFN) THERAPY IN HEMODIALYSIS PATIENTS (HD) WITH
      CAPD, HD AND RENAL Tx PATIENTS                                                                   ASSOCIATED HEPATITIS C VIRUS INFECTION (HCV): INFLUENCE ON THE TH1
      C. Cavdar, M. Sayan, A. Sifil, C. Artuk, N. Yýlmaz, S. Kavukcu, H. Bahar, T. Camsarý             AND TH2 BALANCE USING INTRACELLULAR CYTOKINES ANALYSIS
      Dokuz Eylül University Hospital & Ankara Refik Saydam Hýfzýssýhha Instýtute - Türkiye            Stachowski J., Kissinger G.L., Steffen M., Pollok M., Jilke K., Kneissel B., Eilers R., Baldamus
                                                                                                       C.A.
      The immune system in CAPD, hemodialysis (HD) and renal transplanted (Tx) patients                Medical Clinic IV, University of Cologne, Germany & Clinic of Pediatrics Poznan, Poland
      have been supressed. Generally these patients give weakly antibody (Ab) response to
                                                                                                       Th1 activity correlates with the expression of IFN-γ and IL-2 whereas Th2 with IL-4 and
      vaccination in comparison to healthy adults.
      The aim of this study is to detect Ab response to influenza vaccine as an indicator of the       IL-10 production. Th1 cells promote delayed type hypersensitivity reaction including cell
                                                                                                       mediated cytotoxicity, whereas Th2 exert suppressive effects and induce immune toler-
      level of immunity in CAPD, HD and Tx patients and then to investigate the difference
                                                                                                       ance. The clearance of HCV infected cells depends on an adequate cytotoxic activity (T
      of these groups. In this study 48 patients including 17 Tx, 16 CAPD and 15 HD were
                                                                                                       and NK cells) and Th1/Th2 balance. Influence of INF therapy on Th1/Th2 balance in
      vaccinated. 10 healthy adults were incorporated to this study as control group. During
      October - November 1997, a purified, split-virus, commercial trivalent influenza vaccine         HD patients with HCV infection is not well known. The study was undertaken in HD
      (VAXIGRIP - Pasteur Merieux Connaught) was given to both patients and control group              patients (n=15) with associated HCV infection before and during IFN-therapy (5MU 3
                                                                                                       x /week for 3 month and 3MU 3x/week for 9 month) therapy in patients non-responding
      in the dosage of 0.5 mL one times to deltoid muscle intramuscularly. Vaccine contained 15
      µg of each hemagglutinin of A/Johannesburg/82/96(H1N1),A/Nachang/933/                            to IFN. Non-response (NR; n=7) was defined after the first 3 month of ineffective IFN-
                                                                                                       therapy (HCV-RNA+). FACS technique was used in analysis of intracellular cytokines in
      95(H3N2)and B/Harbin/07/94 strains. Serum samples were collected before and 1 month
                                                                                                       peripheral blood lymphocytes (PBL). Cytokine expression in activated CD4+ lymphocytes
      after vaccination for Ab determinations. Determination of Ab response was performed
      with hemagglutination-inhibition test (HAI). Wilcoxon paired t test and Mann - Whitney           was estimated by using 3-colour fluorescence. Activated PBL were stained with anti-CD4
      U test were used for statistical analysis. p < 0.05 was accepted as statistically significant.   (Cy-Chrome), fixed, permeabilized (saponine) and then labelled with anti-IL-2 and anti-
      All of the results related with study groups are shown in Table 1.                               IFN-γ (FITC) or anti-IL-10 and anti-IL-4 (PE). Results are presented as % of cytokine
                                                                                                       CD4+cells. Data of NR to IFN and subsequent responders (R) or NR to the long-lasting (9
      Table 1: The Results of the Study Groups
      Ab Response              Tx               CAPD              HD             Control               month) IFN-therapy were compared to healthy donors (controls). *p<0.05; +p<0.01;
                                                                                                       #
                                                                                                         p<0.001 (Table)
      B (Before)               32,35±4,97       27,50±4,33        34,00±5,84     46,00±7,92
      B (After)                96,47±34,82*     62,67±8,92*       74,67±12,72* 88,00±16,65*                                Th1                                 Th2
      ∆B                       2,88±0,52        2,53±0,29         1,93±0,20      2,00±0,26                                  IL-2                IFN-γ              IL-4             IL-10
      H1N1 (Before)            30,59±9,01       25,00±5,70        19,67±4,59     28,00±6,63
      H1N1 (After)             75,29±17,62*     60,00±12,19*      40,67±5,89* 102,00±28,67*            IFN-α-therapy        NR
      ∆H1N1                    3,06±0,51        3,67±1,07         2,23±0,30      3,80±0,76
      H3N2 (Before)            38,82±4,61       36,25±7,0         32,00±6,91     48,00±13,73           Before               ↓20+       ↓21+     ↓22+     ↓24+      33      ↑34*     ↑40*    30
      H3N2 (After)             67,06±12,33*+    58,67±12,07*+     75,33±15,18*+ 204,00±61,81*
      ∆H3N2                    1,68±0,25+       2,30±0,51         2,32±0,52      5,00±1,46             5 MU                 ↑43#       ↑43#     ↑47#     ↑49#      ↓12*    ↓10*     ↓15*    ↓12*
      * Ab levels were increased statistically significant after influenza vaccine
      + Ab response against to H N was significantly lower in Tx, CAPD and HD                          3 MU                 ↑42*       ↑42#     ↑45*     ↑45*      24      18       31      25
      ∆B, ∆H1N1, ∆ H N : The magnitude of Ab rising
                                    3 2

                        3 2                                                                            Direct after IFN-α NR           R        NR       R         NR      R        NR      R
      In conclusion, Ab levels after vaccination in Tx, CAPD and HD patients increased statis-
      tically significant and there were no differences between them ; this resulted in increasing                          ↑48#       ↑↑62 #   ↑52#     ↑↑↑68 ↓↓12# ↓↓↓8# ↓14*             ↓↓↓9#
      the proportion of patients having protective Ab titration however these proportions were
      found lower than those of control group. Although the ratio of patients having protective        3 month later         ↓21+    ↑30#     ↓21+    ↑33+     31      24      ↑41* 28
      Ab titration with vaccination increased in Tx, CAPD and HD patients, the magnitude of            The patients with double NR to IFN after 3 month and subsequent 9 month therapy
      Ab rising against to H3N2 and the levels at first month were detected as lower than that         revealed very low activity of Th1 cells (Th1<<<Th2) before the therapy: in the course of
      of control group. Tx, CAPD and HD patients should be vaccinated every year because of            IFN therapy the Th1/Th2 balance was moved into Th1 activity. In NR the reversed Th1/
      medical complications caused by influenza infection. Trial of high dose vaccination protocols    Th2 (Th1>Th2) activity in the course of long-term IFN-therapy was significantly lower
      may be useful to increase Ab response.                                                           comparing to R to IFN therapy (Th1>>>Th2). Conclusion: Long-lasting IFN therapy is
                                                                                                       useful in order to eliminate HCV in HD patients but the high Th1 activity is only observed
                                                                                                       during the therapy.




A30                                                                  Nephrology Dialysis Transplantation                                        Vol. 14 n.9                                     1999
                                            Immunology, immunopathology and pathology
                                                        (basic and clinical)                                                                       Abstracts

IMPACT OF HEPATITIS B AND/OR C ON THE OCCURRENCE OF NEPH-                               1,25-(OH) VIT.D INHIBITS iNOS mRNA EXPRESSION AND
                                                                                                  2     3
ROPATHY IN PATIENTS WITH HEPATO-SPLENIC SCHISTOSOMIASIS                                 NO PRODUCTION FROM LPS-STIMULATED RAW 264.7
H El-Ghoneimy*, ZA Shaker #, MG Saadi *, I Shaltout*, A El-Shamaa*, A El-               JM Chang, SJ Hwang, JC Tsai, JH Tsai, YH Lai
Bassiouni#, KA Maher#, MM Kamel#.                                                       Division of Nephrology, Kaohsiung Medical College, Kaohsiung,
Departments of int. medicine and nephrology*, and clinical pathology #, faculty         Taiwan.
of medicine, Cairo University, Egypt.
                                                                                        The activity of inducible nitric oxide synthase (iNOS) of
Nephropathy has been proved to occur with infestation by Schistosoma man-               macrophages is greatly enhanced during infection. 1-α-hydroxy-
soni, particularly following porto-systemic shunting. The effect of associated          lase activity and 1,25-(OH) Vit.D (Vit.D) synthesis are also in-
                                                                                                                       2      3
infection with viral hepatitis B and/or C on the tendency of occurrence of this         creased. The increase of Vit.D may in some occasion induce
nephropathy has not been studied.                                                       hypercalcemia and lead to clinical illness. The significance of the
Forty eight patients with Schistosoma mansoni hepatosplenomegaly (HSS) were             increased amount of Vit.D is, however, not clear. The aim of this
studied, including 13 with -ve HCV antibodies and HBsAg (gp I), 11 with +ve             study is to clarify the effect of Vit.D on the iNOS mRNA expres-
HBsAg (gp II), 12 with +ve HCV (gp III), and 12 with +ve HCV and HBsAg (gp              sion and the nitric oxide (NO) production.
IV). In addition 24 were included as controls 12 with -ve virology and 12 with          Mouse macrophage-like cell line, RAW 264.7, was used in this
+veHCV     .                                                                            study. They were cultured in DMEM/F12 supplemented with
The assessment included clinical data, laboratory investigations for the urine,         5% FCS. 24 hours before study, the medium was changed to
stools, liver and kidney functions, and abdominal ultrasonography. Upper en-            DMEM/F12 and charcoal-treated bovine serum in order to re-
doscopy and occasionally a liver biopsy were done for the patients. The immu-           move the influence of Vit.D in the serum. After quiescent, vari-
nological assessment included testing for circulating Schistosomal antigens             ous doses of endotoxin — E coli lipopolysaccharide (LPS,
(CSA), circulating immune complexes (CICs), complement C3, C4 and C3d.                  O127:B8) — and Vit.D were added to RAW 264.7 for 24 hours.
Microalbuminuria was tested as an indicator of nephropathy.                             NO gas production into the medium was measured by NO
CSA and CICs were significantly higher in all groups than reference values of           detector, ISO-NO MARK II (World Precision Instruments), and
controls. CICs were highest significantly in group II 68.714 ± 36.142 mg/ml p<          cells were collected to assess the expression of iNOS mRNA,
0.01 and group IV 79.458 ± 32.806 mg/ml p< 0.001. C3 and C4 were signifi-               using GAPDH as internal control.
cantly lower in the patients groups than in controls, and C3d was +ve in most           After total extraction of RNA, RT-PCR was used to assess iNOS
patients being more in groups II and IV, 72.72% and 75% respectively.                   mRNA expression. Vit.D, ≥10-10 M, showed an inhibitory effect
Microalbuminuria was present in a greater percentage (25%) and higher value             on iNOS mRNA expression and the inhibition was dose-de-
(72.8 ± 25.67 mg/ml in patients of group IV than the other groups.                      pendent. The inhibition even became evident from ≥10-11 M in the
The risk of developing nephropathy in HSS patients is increased by concomitant          lower dose of LPS stimulation. The change of NO gas produc-
HCV or HBV infections, particularly when both are present together in these             tion generally followed that of iNOS mRNA. NO production
patients.                                                                               was gradually decreased by the increase of Vit.D and the de-
                                                                                        crease became significant when Vit.D concentration was ≥10-11
                                                                                        M. Under very low dose of LPS stimulation, as low as 10-12 M
                                                                                        Vit.D was able to decrease NO production.
                                                                                        Our study revealed the inhibitory role of Vit.D on the iNOS
                                                                                        expression and NO production from LPS-stimulated
                                                                                        macrophages. Vit.D may be one of the rescue systems to salvage
                                                                                        the cell from the oxidation injury due to the abrupt production
                                                                                        of NO during infection.
EVIDENCE THAT AN ENHANCED TH2, TRANSFORMING GROWTH FAC-
TOR BETA-1 (TGF-β1) AND TUMOR NECROSIS FACTOR α (TNF-α) IMMUNE
RESPONSE IS PRESENT IN PATIENTS WITH CHRONIC HEPATITIS C VIRUS
(HCV) INFECTION.
Garinis G1,2, Spanakis N1,2, Ioannidis T1, Theodorou V1, Manolis E 1,4, Valis
D 1,2, Karameris A3
1. MEDICANALYSIS Institute of Molecular Biology Applications, Athens,
Greece. 2. HYGEIA Therapeutic Center, Unit of Nephrology, Athens, Greece. 3.
401 General Army Hospital, Dept. of Pathology, Athens, Greece. 4. University of
Athens, School of Nursing, Dept. of Anatomy, Histology and Embryology, Ath-
ens, Greece

Objective. In this study, we report serum circulating levels of IL-1β, 2, 4, 6, TNF-α
and TGF-β1 in two different HCV related groups: HCV (+) haemodialysis (HD)
patients and HCV (+) non-HD patients. Both groups were subcategorized in a)
patients with active chronic hepatitis and b) HCV carriers without liver dam-
age. Results were compared with HCV viraemia profiles and liver biochemis-
try parameters. Methods. IL-2, 4, 6, 1β, TNF-α and TGF-β1 serum levels were
determined in 92 individuals: 52 HCV (+) individuals and 40 controls: [20 HCV
(-) HD patients and 20 healthy volunteers (HCV (-), non-HD)]. Patients were
divided into the following four groups: 1.HCV (+) HD active chronic hepatitis
patients (n=10), 2. HCV (+) HD carriers with no liver damage (n=12), 3. HCV (+)
non-HD active chronic hepatitis patients (n=11), 4. HCV (+) non-HD carriers
with no liver damage (n=12). Results. IL-2, 4, 6, TNF-α and TGF-β1 but not IL-1β
serum levels (P>0.05) were significantly higher (<0,03) in HD active chronic
HCV patients versus HD HCV carriers with no liver damage: [100.1±26.30,
113.4±36.8, 135±33.64, 81.4±76.12, 193.4±60.3, 48.8±19.2 vs. 69.45±22.49,
76.90±19, 82.81±10.39, 145.18±26.8, 104.18±48.71, 35±12.107 pg/mL] and in
non HD active chronic HCV patients vs. non HD HCV carriers with no liver
damage [87.92±36, 98.08±35.65, 11.25±33.4, 203±69.4, 142±28.49, 11.46±3.44
vs. 33.61±15.93, 40.83±11.73, 45.41±19, 92,66±26, 51.25±17.58, 13.30±5.08 pg/
mL]. Elevated serum levels of both IL-4 and 6 were greater than IL-2 cytokine
in all patient groups versus controls. A substantial number of HD and non-HD
patients (n=5) with chronic hepatitis C were shown to have normal or near
normal alanine aminotransferase/ aspartate aminotransferase/
γ-glutamyltransferase (ALT/AST/γ-GT) values. No correlation was observed
between cytokines serum concentrations and HCV viraemia (>0.05). Conclu-
sion. Our results suggest that irrespective of HCV viraemia and liver enzyme
levels an enhanced Th2, TGF-β1 and TNF-α immune response is present in chronic
HCV infection.



Nephrology Dialysis Transplantation                                Vol. 14 n.9                         1999                                                   A31

				
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