Endothelium-Derived Relaxing Factor_ Nitric Oxide Effects on and

Document Sample
Endothelium-Derived Relaxing Factor_ Nitric Oxide Effects on and Powered By Docstoc
					Endothelium-Derived                                                         Relaxing Factor, Nitric Oxide:                                                                                        Effects
on and Production                                                         by Mesangial  Cells and the
Glomerulus1
Leopoldo                Raij and           Pamela            J. Shultz2


                                                                                                           long-acting               vasoconstrictor                       that      also affects                glomer-
L. Raij.     P.J.      Shultz,      Renal         Section,         VA      Medical            Center       ular mesangial                      cell tone and growth                            (2). At least             two
and University             of Minnesota,                  Minneapolis,             MN                      prostaglandins                       produced               by the endothelium                              have
                                                                                                           also been             described               to modulate                 vascular             tone.       Pros-
(J. Am. Soc. Nephrol.                       1993; 3: 1435-1441)
                                                                                                           taglandin             H2 is a vasoconstrictor,                               whereas              prostacy-
                                                                                                           din      iS    a vasodilator                     (3). Both            have         short         half-lives,
                                                                                                           and their              synthesis                is inhibited                by cyclooxygenase
                                                                                                           inhibitors.             In 1 980, Furchgott                          and Zawadski                    (4) iden-
ABSTRACT
                                                                                                           tified        another               vasodilating                  substance,                 which           was
The endothelium-derived                               relaxing          factor      nitric     oxide
                                                                                                           produced              by aortic              endothelium                   in response                  to ace-
(EDRF/NO)              is a labile, endogenous       vasodilator                               that is
                                                                                                           tylcholine,              even           in the presence                      of cyclooxygenase
important              in the control    of systemic     vascular                               tone.      inhibitors.             They called                 this labile            substance                endothe-
This review              focuses           on the effects      of EDRF/NO   on                             hum-derived                    relaxing             factor         (EDRF).              Although             It is
glomerular              mesangial            cells in vitro and on the role of                             likely that there                     are other,              as yet unidentified,                         types
EDRF/NO in mesangial           and glomerular      physiology                                              of EDRF,                extensive                 studies            have        established                 that
and pathophysiology          in vivo. It was concluded       that                                          nitric        oxide         (NO) is the major                          EDRF             (5). StudIes              in
EDRF/NQ can stimulate           increases    in cGMP,     inhibit                                          isolated          vessels            and cells in culture                        have        now shown
mesangial   cell contraction,         and inhibit growth     fac-                                          that       many           different               substances                 can         stimulate              the
tor-induced    proliferation      of mesangial    cells in cul-                                            endothelium                   to produce                 NO (4,6).            Furthermore,                    it is
                                                                                                           now apparent                    that NO can be produced                                   by cells other
lure.      Furthermore,                  incubation          with        endotoxin             or cy-
                                                                                                           than        the vascular                   endothelium                   (7- 1 0) and that                    this
tokines stimulates      mesangial   cells to produce       EDRF/
                                                                                                           substance,               in addition                to affecting              vascular             tone, can
NO, via an inducible      NO synthase    enzyme.     Therefore,
                                                                                                           inhibit         platelet            function              ( 1 1 ) and mediate                    cytostasis
it is likely that NO could play a role in the inflamma-                                                    and cytotoxicity                        (1 0, 1 2). Abnormalities                            in the syn-
tory response     within the glomerulus.      Finally, recent                                              thesis,         release,             or response                 of the smooth                    muscle          to
studies       providing            evidence             that EDRF/NO               is functional            EDRF/NO                 have          been          documented                   to accompany                      a
within       the       glomerulus             in vivo,        especially             during         en-     number            of experimental                           and       human              vascular             dis-
dotoxemia               and      inflammation                are    also         reviewed.                  eases       (1 3). including                  hypertension                   (1 4, 1 5), hypercho-
Key Words:             cGMP,      cell     contraction,          cell    proliferation,         endo-
                                                                                                            lesterolemia                (1 6), and atherosclerosis                                (17).
                                                                                                                The mesangial                       cell is a specialized                        pericyte           within
toxin,     cytokines.         nitrate,      nitrite
                                                                                                           the glomerulus,                          which            is located               adjacent              to the

I  n the last
   accumulated
                      10 yr. a large
                         demonstrating
                                              body
                                                that
                                                       of evidence
                                                       the vascular
                                                                              has
                                                                               en-
                                                                                                            capillary
                                                                                                            Mesangial
                                                                                                            substances
                                                                                                                                 endothelium
                                                                                                                                    cells
                                                                                                                                     via the same
                                                                                                                                                  respond
                                                                                                                                                                   and        the
                                                                                                                                                                       to a variety
                                                                                                                                                                    intracellular
                                                                                                                                                                                        afferent              arteriole.
                                                                                                                                                                                                     of vasoactive
                                                                                                                                                                                                 messenger               sys-
dothelium         is capable        of producing        substances           that                           tems        and        with          a similar               resultant              function             as do
modulate        the tone        of the vascular         smooth         muscle.                              vascular            smooth               cells.        For example,                     angiotensin                II
For example,          endothelin        is a peptide         first    purified                              (All) and endothelin                           stimulate              increases              in cytosolic
and characterized            from culture        media     conditioned          by                          calcium           in both              vascular              smooth           muscle             and       mes-
porcine     aortic     endothelial       cells (1). It is a potent            and                           angial        cells,       resulting              in constriction                   in blood vessels
                                                                                                            and reduction                   in the planar                 surface          area of mesangial
  ReceIved    August      24, 1992. Accepted      October  21, 1992.
                                                                                                            cells in culture                  (18). In contrast,                   the vasodilator,                    pros-
2Coffespondence           to Dr. P.J. Shultz, Renal SectIon (11 IJ),             VA MedIcal      Center,    taglandin             E2, increases                    cAMP          in both cell types                       and
I Veterans    Drive,    Minneapolis,       MN 55417.                                                        inhibits          or reduces                   the number                   of mesangial                    cells
1046-6673/0308-1435503.00/0                                                                                 contracting               in response                 to All (19). Thus,                   the question
Journal of the American     Society of Nephrology
Copyright    © 1993 by the American    Society of Nephrology                                                arose        as to whether                     mesangial               cells would                 also be a


Journal      of the American                Society       of Nephrology                                                                                                                                                 1435
EDRF/NO         and      Mesangial           Cells




target     cell for EDRF/NO             and whether            this substance                                     fore, it has been       thought        that  EDRF/NO            is produced
would       have      functions      within      the glomerulus           similar                                 by one cell type and acts locally                 upon     adjacent       cells.
to those         within        the systemic         vasculature.         Studies                                  However,       recent    studies       by Sweeney          et al. (25), as
regarding         the role of mesangial              cells    as a target        cell                             well    as work       in mesangial          cells     described         below,
for EDRF/NO              are reviewed        in this article.       In addition,                                  suggest    that EDRF/NO             may also be produced              by, and
evidence       that mesangial           cells can themselves             synthe-                                  act upon,        the same        cell in an “autocrine”               type       of
size NO and that                 NO may play a role in glomerular                                                 action,   as well.
physiology          and pathophysiology              in vivo is discussed.


                                                                                                                  MESANGIAL                     CELLS           AS       A      TARGET              FOR          EDRF
                                                                                                                      To examine                   the possibility                  that        glomerular                mes-
EDRF/NO               SYNTHESIS                 AND          ACTIONS                                              angial       cells         could        be a target                cell for the action                         of
    NO is synthesized                        from          the specific                substrate             L-   EDRF/NO,                rat mesangial                  cells in culture                    were         coin-
arginine          via NO synthase                     (NOS) (20). It is now known                                 cubated         with bovine                 aortic       endothelial                cells (26). Bra-
that there            are multiple               isoforms             of this enzyme.                   Sol-      dykinin          was used               to stimulate                EDRF            release         by the
uble      forms           of NOS           have          been         identified            in brain,             endothelial               cells,       and       cGMP            was         measured               in the
neutrophils,                macrophages,                      and         endothelium,                  and       mesangial            cells,         as a marker                of EDRF              action.         Brady-
particulate             forms         have        thus        far been            found         in mac-           kinin      caused             no changes               in mesangial                    cell cGMP               in
rophages           and aortic              endothelium                   (21). In addition                   to   the absence               of endothelial               cells, but when                    bradykinin
this classification                    based         on location                within         the cell,          was added                to the coincubations                            of endothelial                    and
functional             characteristics                   of NOS have                  been       used        to   mesangial               cells,        marked            increases               in cGMP               levels
distinguish              a constituitive                  and an inducible                       type of          were      detected               in the mesangial                        cells        (26).       The        in-
NOS. The constitutive                           type of NOS (cNOS)                           has been             crease       in mesangial                  cell cGMP               levels        in the endothe-
described           in vascular              endothelial               cells (4,20),             neutro-          lial-mesangial                 cell coincubation                     experiments                  was po-
phils      (7), and cerebellar                      cells (8). The cNOS is stimu-                                 tentiated          by superoxide                   dismutase               and was inhibited
lated to produce                  NO by agonists                    that elevate              intracel-           by hemoglobin,                      which          binds          NO, and              by L-NMMA,
lular      Ca2,          such        as acetylcholine,                       bradykinin,                and       which       inhibits            the production                  of NO, thus confirming
thrombin             (6,20).        The NO produced                           by cNOS             can be          that EDRF/NO                     produced            by the endothelial                        cells was
detected           within          seconds             and       is produced                 in nano-             responsible               for the rise in the level of mesangial                                            cell
molar        quantities.             cNOS          requires           NADPH,             fiavin,        and       cGMP.         NO dissolved                   in water             and added                 directly           to
calmodulin              as cofactors               (8). In contrast,                 the inducible                mesangial              cells        also      stimulated               intracellular                  cGMP
NOS enzyme                 (iNOS) is Ca2 and calmodulin                                    independ-              levels.      On the basis                    of these            observations,                   we con-
ent      and        has        been        demonstrated                      in macrophages                       cluded       that glomerular                      mesangial               cells were              a target
(9,22),       tumor          cells (10), and vascular                            smooth          muscle           cell for EDRF/NO                           produced                by endothelial                       cells.
cells      (23).       This        enzyme              requires             several          hours           of   Other       investigators                   confirmed               these          findings            using
exposure           to cytokines               or lipopolysaccharide                           (LPS), as           coincubation                 experiments               of bovine              glomerular              endo-
well as new                  RNA         and        protein           synthesis,              but ulti-           thelial      and mesangial                      cells (6). In addition,                         their        ex-
mately            produces                micromolar                   quantities                of       NO      periments               demonstrated                    that        glomerular                  capillary
(9,22,23).           Both types              of NOS are inhibited                           by L-argi-            endothelial              cells, similar              to vascular               endothelial               cells
nine analogs                 such       as L,N-monomethyl-L-arginine                                        (L-   from larger              vessels,          also had the capacity                            to produce
NMMA)            and        L-nitroarginine                    methylester                 (L-NAME),              EDRF/NO                in response                 to a number                    of agonists                in-
although           there         appears           to be some                difference             in the        cluding         bradykinin,                 ATP, thrombin,                      and platelet-ac-
potency          of these           inhibitors              for each           type of enzyme.                    tivating         factor.           No changes                in mesangial                    cell cGMP
NO is quickly                 inactivated               to the stable                end products                 were      detected             with       any of these                  agonists             in the ab-
nitrate        (NO3), nitrite               (NO2), and citrulline                          (9). These             sence      of endothelial                   cells.
end products                can be measured                       in vitro as markers                        of       In order          to demonstrate                     a biologic              effect        of NO on
NO production                   (9,24).                                                                           mesangial            cells, we measured                         the planar              surface          area
    EDRF/NO               stimulates             increases              in cGMP within                    the     of these        cells by digital                image         analysis            (26). Reduction
isolated         aorta         and in vascular                      smooth            muscle           cells      in mesangial                 cell planar             surface           area         has been             used
and       platelets            (11,20,23).                The        increase             occurs           via    as an in vitro corollary                         of mesangial                  cell contraction.
stimulation                of the         soluble,            or cytosolic,                guanylate              As previously                     reported           by other               investigators,                   All
cyclase.          Methylene                blue,         which           inhibits          guanylate              significantly               reduced           the planar              surface           area of mes-
cyclase,         prevents             the rise in cGMP                      and the action                   of   angial       cells in culture                   (18,19).            NO, at the same                       con-
NO in the target                   cell. NO diffuses                      readily        from        endo-        centrations               that are required                      to raise           cGMP,          signifi-
thelial        cells       to the adjacent                     vascular             smooth            cells.      cantly        inhibited              contractile             effect          of All and,               given
However,             it is rapidly             inactivated                 by superoxide                 an-      alone,        tended             to cause            an increase                    in the planar
ion, by oxygen,                 and by binding                   to hemoglobin.                   There-          surface          area        of the mesangial                        cells.        Thus,         we con-


1436                                                                                                                                                                          Volume          3’     Number          8’     1993
                                                                                                                                                                ..          ...   .   ..                Raij and          Shultz




                                                                                                                      120
cluded       that     EDRF/NO              inhibits        mesangial           cell con-
                                                                                                                                                                                                               #{149}SNAP
traction       induced        by All and therefore                  can be consid-
                                                                                                                      100                                                                                      #{149}SIN-i
ered     a mesangial            cell relaxing             agent,      similar       to its
                                                                                                                is
effect     in vessels.          Furthermore,                these      studies       raise
                                                                                                                E      80
the possibility           that      EDRF/NO              may counteract             other                       Cl)
effects      of All, such          as trafficking            of macromolecules                                         60
through         the mesangium                   (27).     Recent        work       by De
Nicola      et a!. (28) supports                  the concept           that     NO is a                               40

physiologic         antagonist            of All at the level of both the                                        C
                                                                                                                 a
glomerulus          and tubule.                                                                                  20
                                                                                                                 a
    An additional           effect       of EDRF/NO              may be its ability                             a’

to inhibit        mesangial          cell proliferation.             Garg and Has-                                            1%SERUM                 .6                5                          .4                     .3
 sid (29) first         demonstrated                that     S-nitroso-N-acetyl-                                                                            +   NO   Generating            Agent        (log   M)
penicillamine               (SNAP),   a substance                      that generates                  NO
slowly      over         several    hours,    and                   substances        that             re-       Figure 1. The antimitogenic             effects of the NO-generating
lease      NO within              the cell, such                as sodium                nitroprus-              agents SNAP and SIN-I on (3H)thymidine                  incorporation       in
side (SNP) and isosorbide                          dinitrate,           prevent            or reduce             human mesangial            cells are shown. Quiescent          cells were
serum-induced                   [3Hthymidine                  incorporation                    in mes-           incubated      with 1% serum alone or with SNAP or SIN-I at the                                      ,


angial       cells cultured                from       rats.      The antiproliferative                           concentrations       shown, for 28 h. The trichloroacetic             acid-
effects       of SNAP were inhibited                           by hemoglobin.                    SNAP,           precipitable      counts in wells incubated         with the NO-gener-
SNP,       and       isosorbide             dinitrate           also increased                     cGMP          ating agents       were calculated            as a percentage        of the
levels      in the rat mesangial                        cells at roughly                    the same             counts in wells receiving           serum alone. N = 3 experiments,
                                                                                                                 each condition       in triplicate;    ax P< 0.05 versus serum alone.
concentrations                   as needed               for the antiproliferative
effect.         In addition,                8-bromo-cGMP,                       an analog                 of
cGMP         that enters              the cell, also inhibited                         rat mesan-                as platelet-derived                    growth          factor         (32; unpublished
gial cell proliferation.                      From        these        studies,            they con-             observations).                These         concentrations                   of SNAP            and
cluded        that       EDRF/NO               inhibited          mesangial                cell mito-            SIN- 1 produced                  no significant                toxicity,         as assessed
genesis         and that this effect                     was mediated                   via cGMP.                by microscopic                  examination                or lactate          dehydrogen-
    We have              examined               the effect            of NO on human                             ase release.
mesangial               cell      proliferation.                 Previously,                  we       had           Interestingly,              in the human                    mesangial             cells,       we
shown          that this species                  of mesangial                 cells increases                   could      detect        no changes              in cGMP           with       SNAP,         SIN- 1,
DNA synthesis                 and cell number                    when         incubated              with        or SNP, and we found                      no inhibitory              effect      of 8-bromo-
serum         or platelet-derived                   growth          factor         (30) and that                 cGMP        on [3Hjthymidine                    incorporation                in these         cells.
calcium           channel           blockers           inhibit         this mitogenic                    ef-     Therefore,            unlike       what       had been reported                    in rat mes-
fect (3 1 ). Similar               to methods              described             in those           stud-        angial       cells,      It did not appear                  that      the antiprolifera-
ies, this study              used confluent,                  quiescent             human            mes-        tive effect           of NO required                 cGMP         as an intracellular
angial        cells (passages                 6 through             10), that            were        then        messenger              in the human                  cells.      Subsequent               studies
pulsed         with        [3H]thymidine                incorporation                  as a meas-                by Garg and Hassid                     (33) with BALB/c                   3T3 fibroblasts
ure of new DNA synthesis.                              When         NO itself was added                          also found            an antiproliferative                    effect      of SNAP,            with-
directly         to the cells,               no inhibition                of [3Hlthymidine                       out changes                 in cGMP.             Whatever              the intracellular
incorporation                 was         detected           (unpublished                     observa-           signal,       the antiproliferative                     effect       of NO could              mod-
tions).        We thought                 that      this      was        likely         due to the               ulate      or counteract                the proliferative                 effect       of PDGF
rapid      decomposition                   of NO when                exposed             to oxygen.              and other           growth         factors        that are released                 within        the
Therefore,             SNAP          and another                NO-generating                     agent,         glomerulus              during       injury        or inflammation.
 3-morpholino-sydnonionimine-HC1                                           (SIN-i;            provided               Other       actions          of NO have             been       described,           such        as
by D. Voegele,                   Cassella           AG, Frankfurt,                      Germany),                its interaction               with      superoxide             anion        to form         perox-
which         produce           NO continuously                     in solution                for sev-          ynitrite        (34). Generation                  of the latter            oxygen         radical
 eral     hours,          were        tested        for their            ability          to inhibit             has been             proposed           to cause           endothelial             cell injury.
 serum-induced                  mesangial             cell proliferation                     (32). The           However,            this and other               effects        of NO have yet to be
 percentage             of the serum-induced                          stimulation                 in [3HJ        explored         in mesangial               cells. Thus,           NO can inhibit                All-
 thymidine             that      was seen             with       SNAP           and SIN-i               are      induced             mesangial              cell      contraction               and         inhibit
 shown         in Figure           1. A dose-dependent                       inhibition              from        growth         factor-induced                   mesangial             cell proliferation.
  10-6 to iO               M was found,                  which         was significant                    at     Therefore,            it is possible           that NO could modulate                         renal
 the two highest                 concentrations                  of each           of these           sub-       function          acutely          by altering            the surface             area       of the
 stances.          SNAP        and SIN-i             also inhibited                  increases             in    mesangium.                In addition,            because          mesangial            cell pro-
 cell number              induced          by serum            over 6 days and inhib-                            liferation           may       lead to increased                   matrix          production
 ited [3Hthymidine                      induced           by other           mitogens,               such        and glomerulosclerosis,                           the antimitogenic                    effect        of


 Journal      of the American                Society        of Nephrology                                                                                                                                                      1437
EDRF/NO          and      Mesangial            Cells




NO may             prove        important               in attenuating                   the     develop-             ization         of the        glomerular                cNOS          mRNA            was       not      pos-
ment        of chronic              renal        failure         (27).                                                sible in that study,              although          it most likely          was in the
                                                                                                                      capillary       endothelial           cells.
EDRF/NO                PRODUCTION                          BY      MESANGIAL                      CELLS                   Thus,      whether         mesangial           cells possess          a cNOS iso-
                                                                                                                      form      remains         in question.            However,         it is well estab-
    In addition             to mesangial                  cells acting              as a target              cell
                                                                                                                      lished      at this point          that       they do have            the capability
for EDRF/NO,                    recent          studies            have        established                 that
                                                                                                                      to produce          NO via an inducible                   enzyme         that     is acti-
mesangial             cells themselves                       have the capacity                       to pro-
                                                                                                                      vated      by LPS and cytokines.                     Therefore,          NO could         be
duce        NO         (35-37).              Studies               by       Pfeilschifter                   and
                                                                                                                      produced          within       the glomerulus               directly        during       in-
Schwarzenbach                       (35) and                Marsden              and         Balierman
                                                                                                                      flammation            or sepsis       and mediate           alterations          in mes-
(36) demonstrated                        that          tumor          necrosis             factor           and
                                                                                                                      angial      function,        without         endothelial        injury      or infiltra-
interleukin-             1 caused              a dose-dependent                           increase              in
                                                                                                                      tion by other           cells.
cGMP        in rat mesangial                        cells.         This       increase            was po-
tentiated           by superoxide                    dismutase                and inhibited                    by
L-NMMA,             actinomycin                  D, and cycloheximide.                                At the
same       time,        studies           in vascular                 smooth            muscle            cells
                                                                                                                      ROLE          OF EDRF/NO   IN GLOMERULAR                                                 PHYSIOL-
in culture            and in isolated                      aortas          also demonstrated
                                                                                                                      OGY           AND  PATHOPHYSIOLOGY
stimulation              of cGMP             by LPS and interleukin-                                 1 sug-
                                                                                                        ,                 On the basis                     of the cell culture                          work         reviewed
gesting        the presence                  of an iNOS in these                              tissues          as     above,        it is clear              that       NO affects                several          mesangial
well (23,38).                                                                                                         cell functions                 and therefore                  could         potentially            have         a
     We undertook                  studies           in which             we simultaneously                           role in normal                    glomerular               physiology                and/or         patho-
measured             intracellular                 mesangial                cell cGMP               and ni-           physiology               in vivo.              To examine                  the        systemic            and
trite (NO2) and nitrate                      (NO3), two stable                      decomposition                     renal      hemodynamic                       effects        of NO in normal                    animals,
products           of NO, in the culture                             media         (37). We found                     several        investigators                   have given the animals                            NO syn-
that rat mesangial                      cells,        incubated              with either               Esch-          thesis       inhibitors               (43-46).           From          these        studies,         it has
erichia          coil        LPS        or gamma-interferon,                                  showed              a   been observed                    that systemic                  injection            of L-NMMA                or
marked          increase             in both             NO2+NO3               and cGMP.                 Both         L-NAME             causes           a marked              increase             in mean            arterial
the NO2+NO3                    and       cGMP             changes              induced             by LPS             pressure           and renal             vascular            resistance              (43,44).        When
required           several           hours          of incubation                    to occur,              and       L-NMMA               is infused                directly           into       the       renal       artery,
both were inhibited                        by L-NAME.                   Thus,         we concluded                    whole-kidney                   GFR is slightly                 decreased              or unchanged
that mesangial                  cells possess                 an iNOS that is activated                               and RBF is significantly                              reduced,            thus causing                a rise
to produce              NO in response                       to LPS or cytokines                            and       in filtration             fraction           (45). Micropuncture                        studies         after
that the NO generated                           could          then       act in an autocrine                         acute       administration                      of L-NMMA                  (46) demonstrated
manner          leading           to an increase                    in intracellular                 cGMP.            a marked              increase            in both           afferent            and efferent                ar-
Preliminary               studies           have         confirmed               this by demon-                       teriolar         resistance,                a decrease               in the ultrafiltration
strating          mRNA             for iNOS                in rat mesangial                        cells        in    coefficient             K,, and a marked                         increase             in glomerular
culture        after        incubation               with LPS (39).                                                   capillary            pressure.              Chronic            NO inhibition,                    induced
     Two brief             reports          have           suggested              that       mesangial                by 8 wk of L-NAME                             in the drinking                    water        of experi-
cells may also possess                           a cNOS.              The first            study         dem-         mental          rats,        also causes                systemic             hypertension,                  in-
onstrated            that       acetylcholine                    was found               to cause             an      creased          renal         vascular            resistance,              reduced           GFR, and
opening           of potassium                   channels                and a reduction                        in    reduced           K(4’7).          Thus,         it is clear that inhibition                         of NO
cytosolic         potassium,                which           was inhibited                 by L-NMMA                   production               in vivo has dramatic                           effects         on renal           and
and       methylene                blue        (40).         The        same          group          subse-           glomerular                 hemodynamics                       and         provides            good         evi-
quently          reported             positive            staining            with        antibody              to    dence       that endogenous                         NO is important                     in maintain-
cNOS in rat mesangial                            cells in culture                   (41). However,                    ing normal                  basal         tone        in the systemic                        and       renal
they were unable                      to demonstrate                      positive          staining            in    circulations.                It has also been shown                             that NO contrib-
whole       glomeruli,              thus raising                 the question               of whether                utes to the physiologic                            renal       vasodilation                and hyper-
the positive              findings            in the cultured                     cells were               arti-      filtration             that          accompanies                     amino             acid       loading
factual         or nonspecific.                      Furthermore,                     as previously                   (44,45).          Although               the        in vivo           glomerular                hemody-
discussed,             no changes                 in cGMP              could         be detected                in    namic        effects           of mesangial                cell shape              change         are still
the mesangial                  cells when                agonists            of the cNOS                 were         controversial,                  it has been                repeatedly                observed             that
added         directly           to mesangial                      cells       (6,26).           A recent             substances                that contract                 mesangial                cells in vitro              re-
study        has       examined                the localization                       of mRNA                 for     duce K1 in vivo, and likewise,                                   substances               that inhibit
cNOS in microdissected                             parts          of the kidney                (42). They             mesangial              cell contraction                     in vitro tend                 to cause           an
found        the largest               quantities                of this         mRNA             in inner            increase            in K1 in vivo,                 especially             if other          compensa-
 medullary            collecting            duct,        with lesser               amounts             in the         tory mechanisms                        are blocked                (48). The changes                      in K
glomeruli,             outer          medullary                  and        cortical            collecting            observed              with        NO inhibition                    in the micropuncture
ducts,       and inner               medullary                thin limb.             Further            local-        studies        described               above         are consistent                   with our find-


 1438                                                                                                                                                                             Volume              Number
                                                                                                                                                                                                  3#{149}                8’     1993
                                                                                                                                                                              -    .   --          Raij and   Shultz




ings in vitro demonstrating                                 that      NO can oppose                     vas-                                        LPS                                      Endothelial  cells
oconstrictor-induced                         mesangial               cell contraction.                                                          Cytokines               ACh                  or Macrophages
     A number                of recent              studies           have         demonstrated
impaired
animals
lesterolemia,
                     NO production
                  and humans
                            and atherosclerosis
                                                with
                                                        by systemic
                                                            hypertension,
                                                                         (14-17).
                                                                                     vessels
                                                                                            hypercho-
                                                                                             However,
                                                                                                       from

                                                                                                                               L-arginine
                                                                                                                                            -
                                                                                                                                                      III
                                                                                                                                                  inducible        Conntituitive
                                                                                                                                                                                            ) NO
                                                                                                                                                       NO Synthase
in most of these                   studies,           it was unclear                   whether             the
reduced          ability        to produce               or respond             to EDRF/NO                     is
                                                                                                                                 inhibition     of proliferation
a cause            for, or a result                      of, the          vascular             disease.
                                                                                                                                    opening
                                                                                                                                  #{149}         of K+ channels
Although             few studies                 have        examined               renal         vessels
specifically,              evidence             of a role for NO in renal                                 dis-
                                                                                                                                 inhibition     of contraction            (                 t cGMP
eases        is emerging.                 Studies            of ischemic                 acute        renal
failure         in rats         have        demonstrated                   a loss of normal
vasodilatory               response             to acetylcholine,                     suggesting                a   Figure 2. Diagram of the exogenous       and endogenous  path-
 loss of EDRF/NO                       production                or effect             (49). In rats                ways for the production     and action of EDRF/NO in glomeru-
with       experimental                   nephrotoxic                  nephritis             or active              lus and mesangial     cell. ACh, acetylcholine.
 Heymann               nephritis,             isolated           glomeruli               produce            in-
creased          amounts             of NO2, both basally                        and after            stim-         L-NAME          prevented           the rise in both                 the serum              and
 ulation        by LPS, compared                       with glomeruli                  from control                 urinary         NO2+NO3.           When          tissues        from        LPS-treated
animals            (50,51).          The authors                  of those            studies          have         rats were         incubated           ex vivo,        it was determined                    that
 suggested              that        infiltrating               macrophages                     are         the      the aorta,         whole       kidney,         and glomeruli,                but not the
 source         of the NO production                            in these           models.            How-          tubules,        were     sources         of NO production                  under        these
 ever,     it is also possible,                      on the basis                 of the in vitro                   experimental            conditions            (55). Isolated           giomeruli           pro-
 work       described             above,          that       mesangial               cells contrib-                 duced       the greatest            amount           of NO2+NO3                when         fac-
 ute to the glomerular                              production               of NO in these                         tored for tissue            weight.
 models.                                                                                                                From       these      studies,         it is apparent               that      LPS can
     Because            LPS is a potent                     stimulus            of NOS in vas-                      stimulate         endogenous              NO production                  in vivo,         both
 cular       smooth            muscle            and        mesangial               cells       and         be-     systemically          and within             the glomerulus,               and that NO
 cause         NO is an effective                          vasodilator,                 it has          been        exerts      a protective           effect       against        glomerular            throm-
 proposed            that       NO may              be a secondary                       mediator             in    bosis.      In addition,           we have           established             that     serum
 septic        shock          and       its associated                   renal          failure.          Kil-      and urinary           NO2 and NO3 can be used as markers                                        of
 bourn        and coworkers                    (52) have demonstrated                              in dogs          NO production                in vivo.         Recent         studies         in humans
 that     inhibitors             of NOS can correct                           hypotension                   in-     have      confirmed           that      NO2+NO3            can be measured                      in
 duced         by tumor             necrosis             factor,         which           is a known                 human         serum       and urine           as well (56). We believe                     that
 mediator             of endotoxemia.                        Others          have           confirmed               using      these      in vivo markers                   of NO production                     will
 that inhibition                of NO can attenuate                          or reverse              shock          greatly       enhance           our understanding                      of the        role of
 induced          by LPS (53). However,                           it has recently                 become            EDRF/NO             in experimental                  and      human            physiology
 clear that inhibition                      of NO synthesis                    in these            animal           and pathophysiology.
 models         is not always               beneficial              (54), perhaps               because
 local      production                of NO may                  play      a critical              role       in
                                                                                                                    SUMMARY
 maintaining                organ         perfusion              in some            situations.               In
 addition,          because            NO Inhibits               platelet         activation              and           In summary            (Figure        2), mesangial           cells are both            a
 adhesion,            Inhibition             of NO could               favor        thrombosis.                     target     cell for the action              of EDRF/NO            and a producer
     We have             recently           investigated                the role of endoge-                         of NO. Glomerular                   endothelial          cells     or Infiltrating
  nous      NO in endotoxin-induced                                   glomerular                 disease.           macrophages             could      be a source          of exogenous             EDRF/
 We found              that       rats given              a single          injection             of LPS,           NO within           the glomerulus.               In addition,            circulating
 which         caused          no histologic                 damage            by itself,            devel-         cytokines        or endotoxin            may act directly             on mesangial
 oped extensive                 glomerular                thrombosis               when         NO syn-             cells to stimulate             them      to produce         NO. EDRF/NO                can
 thesis        was Inhibited                 with        L-NAME            (55). This              throm-           stimulate        mesanglal           cell cGMP,         which        Inhibits       vaso-
  bosis      was        significantly                reduced             by giving             the rats             constulctor-induced                 mesangial          cell contraction               and
  excess        L-arginine.             Rats        treated           with       L-NAME              alone,         may have          other     undiscovered            actions        within       the cell
  without          LPS,        had very              few thrombosed                         glomeruli.              as well.       In addition,            EDRF/NO            can inhibit            growth
  Further         evidence            that the endogenous                            production                of   factor-induced               mesangial            cell     proliferation.             Evi-
  NO played            a role in the thrombosis                           was demonstrated                          dence      for a role for NO in basal                  renal      hemodynamics
  by the findings                 that serum                NO2+NO3              concentrations                     and      glomerular           function          in vivo        is accumulating.
  rose     4-fold         and        urinary            excretion            of NO2+N03                      in-    Furthermore,             recent        studies       suggest         that     NO may
  creased         3.5-fold         in these          rats after           LPS injection                  (55).      also be important                in glomerular            dysfunction            associ-


 Journal       of the       American           Society        of Nephrology                                                                                                                                       1439
EDRF/NO       and    Mesangial       Cells




ated with endotoxemia                    and inflammatory                  injury.       Ad-         rived vasoactive                substances            and the kidney.              The
ditional      studies        are needed            to determine            the role of               Kidney         1990;23:1-8.
NO in other           glomerular             diseases        and to explore              the   1 4. Luscher             TF, Raij L, Vanhoutte                         PM: Endothe-
                                                                                                     hum-dependent                    vascular          responses           in normo-
therapeutic          potential        of manipulating              the L-arginine-
                                                                                                     tensive         and hypertensive                   Dahl       rats.     Hyperten-
NO pathway            within       the glomerulus.              However,          it must            sion 1987:9:157-163.
be kept        in mind          that     altering         the natural            balance       1 5. Panza          JA,       Quyumi            AA, Brush             JE Jr. et at.:
between        circulating         or locally         produced        vasoconstric-                  Abnormal              endothelium-dependent                         vascular         re-
tor, procoagulant,                and       proproliferative             factors       and           laxation         in patients          with essential             hypertension.
                                                                                                     N Engl J Med 1990:323:22-27.
vasodilatory,           anticoagulant,               and     antimitogenic             fac-
                                                                                               1 6. Creager           MA, Cooke              JP, Mendelsohn                 ME, et at.:
tors      may      be both           beneficial          and     deleterious           and           Impaired            vasodilitation              of forearm             resistance
therefore        must      be undertaken                with caution.                                vessels        in hypercholesterolemic                        humans.          J Clin
                                                                                                     Invest       1990:86:228-234.
                                                                                               1 7. Frieman             PC, Mitchell               GC, Heistad              DD, Arm-
                                                                                                     strong         ML, Harrison                  DG: Atherosclerosis                    im-
REFERENCES
                                                                                                     pairs       endothelium-dependent                          vascular          relaxa-
                                                                                                     tion to acetylcholine                    and thrombin               in primates.
  1 . Yanagisawa        M, Kurihara           H, Kimura      S, et at. : A                           Circ Res 1986:58:783-789.
      novel potent     vasoconstrictor           peptide   produced      by                     1 8. Mene        P, Simonson               MS, Dunn            MJ: Physiology               of
      vascular    endothelial          cells.   Nature   (Lond)     1988:                             the       mesangial              cell.        Physiol          Rev       1989:69:
        332:411-415.                                                                                   1347-1424.
  2.   Kon V, Badr           KF: Biological          actions     and patho-                    1 9. Mene          P. Dunn           MJ: Eicosanoids                  and control            of
       physiologic       significance         of endothelin        in the kid-                        mesangial             cell contraction.                Circ      Res     1988:62:
       ney. Kidneylnt            1991:40:1-12.                                                        916-95.
  3.   Vane      JR, Angaard           EE, Botting         RM: Regulatory                      20. Moncada               S, Palmer          RMJ,       Higgs EA: Nitric oxide:
       function       of the vascular          endothelium.          N Engi J                         Physiology,            pathophysiology,                and pharmacology.
       Med 1990;323:27-36.                                                                            Pharmacol             Rev 1991:43:109-142.
  4.   Furchgott        RF, Zawadski            V: The obligatory            role              2 1 . Pollock          JS, Forstermann                  U, Mitchell           JA, et at.:
       of endothelial         cells    in the relaxation           of arterial                        Purification            and characterization                     of particulate
       smooth       muscle       by acetylcholine.           Nature      (Lond)                       endothelium-derived                       relaxing          factor       synthase
        1980:299:373-376.                                                                             from cultured              and native           bovine        aortic     endothe-
  5.   Ignarro        U,      Byrns       RE, Buga            GM, Wood              KS,               hal cells.           Proc       Natl       Acad       Sci USA            1991:88:
       Chaudhuri          G: Pharmacological                evidence         that en-                  10480-10484.
       dothelium-derived              relaxing       factor      is nitric      oxide:         22.    Xie Q-W, Cho HJ, Calaycay                                  J, et at. : Cloning
       Use of pyrogallol             and      superoxide           dismutase           to             and       characterization                  of inducible              nitric       oxide
       study     endothelium-dependent                      and nitric          oxide-                synthase             from        mouse           macrophages.                 Science
       elicited      vascular        smooth         muscle         relaxation.          J              1992:256:225-228.
       Pharmacol          Exp Ther         1 988:244:         1 8 1 -189.                      23.    Beasley           D, Schwartz             JH, Brenner               BM: Interleu-
  6.   Marsden         PA, Brock         TA, Ballerman               BJ: Glomer-                      kin       1 induces            prolonged             L-arginine-dependent
       ular endothelial            cells    respond         to calcium-mobi-                          cyclic guanosine                 monophosphate                   and nitrite          pro-
       lizing agonists         with release          of EDRF.          Am J Phys-                     duction          in rat vascular                smooth          muscle         cells.      J
       iol 1990;258:F1295-F1303.                                                                      Clin Invest            1991:87:602-608.
  7.   McCall      TB, Boughton-Smith             NK, Palmer                       RMJ,        24.    Shultz          PJ,      Tayeh         MA, Marletta                  MA,      Raij       L:
       Whittle      BJR,    Moncada       5: Synthesis         of                  nitric             Synthesis            and action             of nitric         oxide      in rat gb-
       oxide from L-arginine         by neutrophils:      release                     and             merular           mesangial             cells.       Am J Physiol                 1991;
       interaction      with superoxide       anion.   Biochem                        Bio-            261 :F600-F606.
       phys      Res     Commun           1989:261:293-298.                                    25.    Sweeney             C, Shultz          PJ, Raij L: Autocrine                       stim-
  8.   Bredt    DS, Hwang         PM, Glatt         CE, Lowenstein              C,                    ulation        of endothelial             cell (EC) cGMP by endothe-
       Reed    RR, Snyder         SH: Cloned           and expressed           ni-                    hum        derived         relaxing          factor,       nitric      oxide       (NO):
       tric oxide    synthase        structurally         resembles        cyto-                      Differential            effects       of EC injury             by two oxidants
       chrome      P-450      reductase.          Nature      (Lond)     1991:                        (OX) [Abstract].               J Am Soc Nephrol                     1991 :2:5 13.
       35 1:7 14-7 18.                                                                         26.    Shultz        P,.J, Schorer            AE, Raij L: Effects                   of endo-
  9.   Stuehr     DR, Marletta            MA: Induction            of nitrite/                        thelium-derived                 relaxing         factor        and nitric          oxide
       nitrate         synthesis         in        murine      macrophages                by          on rat mesangial                  cells.       Am J Physiol              1990:258:
       BCG infection,             lymphokines              or g-interferon.          J                Fi62-F167.
       Immunol          1987:139:518-525.                                                      27.    Shultz         PJ, Raij L: The gbomerular                             mesangium:
10.    Amber        IJ, Hibbs        JB Jr, Taintor              RR, Vavrin         Z:                Role in initiation               and progression                 of renal       injury.
       Cytokines         induce     an L-arginine-dependent                  effec-                   Am J Kidney               Dis 1991:1            7(suppl         1 ):8- 14.
       tor system         in nonmacrophage                 cells. J Leukocyte                  28.    Dc Nicola            L, Blantz         RC, Gabbai               FB: Nitric         oxide
       Biol 1988:44:58-65.                                                                            and        angiotensin              II. J Clin               Invest         1992:89:
11.    Radomski            M, Palmer          RMJ,        Moncada         S: Com-                      1248-       156.
       parative        pharmacology             of endothelium-derived                         29.    Garg       UC, Hassid             A: Inhibition               of rat mesangial
       relaxing       factor,     nitric     oxide      and prostacyclin            in                cell mitogenesis                by nitric        oxide-generating                 vaso-
       platelets.       Br J Pharmacol              1987:92:181-187.                                  dilators.        Am J Physiol                1989;257:F60-F66.
12.    Hibbs      JB, Taintor            RR, Vavrin            Z, Rachlin       EM:            30.    Shultz        PJ,     DiCorleto            P, Silver            B, Abboud             HE:
       Nitric     oxide:      A cytotoxic          activated        macrophage                        Mesangial      cells express    PDGF    mRNAs     and pro-
       effector       molecule.         Biochem          Biophys       Res Com-                       liferate   in response    to PDGF.   Am J Physiol     1988:
       mun    1988:157:87-94.                                                                         255:F674-F684.
13.    Shultz    PJ, Tolins                  JP,     Raij   L: Endothelial-de-                 31.    Shultz     PJ, Raij L: Inhibition     of human      mesan-


1440                                                                                                                                               Volume        3’    Number        8’    1993
                                                                                                                                                                       Raij and      Shulfz




      gial cell proliferation              by calcium       channel        block-                       lation      of renal        hemodynamic              responses.            Am J
      ers. Hypertension               1 990; 1 5(suppb       1 ):76-80.                                 Physiol        1 990:258:H655-H662.
32.   Shultz     P. Ruble       D, Raij L: S-nitroso-n              acetylpen-                   44.    King      AJ, Troy          JL, Anderson             S. Neuringer               JR,
      icillamine      (SNAP)        inhibits     mitogen-induced              mes-                      Gunning           M, Brenner           BM: Nitric        oxide:      A poten-
      angial     cell    proliferation.           Kidney       mt      1990:37:                         tial mediator           of amino        acid-induced          renal      hyper-
      203.                                                                                              emia       and      hyperfiltration.             J Am Soc              Nephrol
33.   Garg UC, Hassid              A: Nitric oxide-generating                vaso-                       1991:1:1271-1277.
      dilators     inhibit       mitogenesis          and proliferation             of           45.    Tolins       JP, Raij L: Effects              of amino       acid infusion
      BALB/c        3T3 fibroblasts             by a cyclic         GMP-inde-                           on renal           hemodynamics.                 Hypertension              1 99 1;
      pendent       mechanism.            Biochem        Biophys       Res Corn-                         17:1045-105             1.
      mun      1990:171:474-479.                                                                 46.    Zatz R, Dc Nucci                0: Effects        of acute      nitric      oxide
34.   Beckman          JS, Beckman              TW, Chen          J, Marshall                           inhibition          on rat gbomerular                 microcircubation.
      PA, Freeman               BA:      Apparent         hydroxyl        radical                       Am J Physiol              1991;26i:F360-F363.
      production           by peroxynitrite:              Implications            for            47.    Baylis       C, Mitruka           B, Deng        A: Chronic          blockade
      endotheliab         injury      from     nitric    oxide     and super-                           of nitric       oxide     synthesis         in the rat produces                sys-
      oxide.      Proc        Natl       Acad        Sd     USA        1990:87:                         temic       hypertension             and gbomerular              damage.            J
          1620-1624.                                                                                    Clin Invest           1992;90:278-281.
35.       Pfeilschifter        J, Schwarzenbach                        H: Interleukin            48.    Maddox           DA, Brenner             BM: Glomerular                ultrafil-
          1 and tumor         necrosis    factor          stimulate         cGMP for-                   tration.        In: Brenner            BM, Rector           FC, eds.           The
          rnation       in rat renal   mesangiab              cells.      FEBS      1990:               Kidney.        4th Ed. Philadelphia:                 WB Saunders                 Co:
          273:185-187.                                                                                   1991:205-244.
36.   Marsden            PA,      Ballerman              BJ:      Tumor          necrosis        49.   Conger       JD, Robinette               JB, Schner            RW: Smooth
      factor-to       activates          soluble         guanylate           cyclase        in         muscle      calcium          and endothelium-derived                     relax-
      bovine       glomerular            mesangial            cells via an L-argi-                     ing factor        in the abnormal             vascular         responses         of
      nine-dependent                 mechanism.               J Exp Med               1990:            acute      renal       failure.         J Clin         Invest        1988:82:
       172:1843-1852.                                                                                  532-537.
37.   Schultz          PJ, Tayeh             MA, Marietta                 MA, Raij          L:   50. Cattell       V. Cook           T, Moncada            5: Gbomeruli            syn-
      Synthesis           and action            of nitric         oxide      in rat gb-                thesize      nitrite      in experimental               nephrotoxic            ne-
      merular          mesangial            cells.       Am J Physiol                 1991:            phritis.     Kidney        Int 1 990;38:         1056-1060.
      F600-F606.                                                                                 5 1 . Cattell     V. Largen           P, de Heer E, Cook T: Gbomer-
38.   Beasley         D: Interleukin-              1 and endotoxin                activate             uli synthesize          nitrite      in active      Heymann            nephri-
      soluble        guanylate            cyclase          in vascular             smooth              tis: the source           is infiltrating         macrophages.              Kid-
      muscle.        Am J Physiol               1990:R38-R44.                                          ney Int 1991:40:847-851.
39.   Shultz        PJ, Archer            SL, Rosenberg                ME: Inducible             52. Kilbourn            RG, Gross           55, Jubran            A, et at. : NG
      nitric      oxide       (NO) synthase                mRNA          and     NO pro-               methyl-L-arginine                inhibits       tumor         necrosis       fac-
      duction         by rat mesangial                  cells      (MC). J Am Soc                      tor-inducedhypotension:                     Implications           for the in-
      Nephrol          1992,      in press.                                                            volvement          of nitric oxide.         Proc Natl Acad Sci USA
40.   Ganz       MB, Kasner                SE, Unwin              R: Acetylcholine                       1990:87:3629-3632.
      (ACh) induces              nitric     oxide       (NO)-dependent                open-      53.    Thiemermann                 C, Vane           J: Inhibition          of nitric
      ing of K channels                   in mesangial              cells (MC5) fAb-                    oxide synthesis             reduces        the hypotension             induced
      stract].      J Am Soc Nephrol                    1990:1:684.                                     by bacterial            lipopolysaccharides                 in the rat in
41.   Springall           D, Unwin             R, Ganz            M, Moncada                5,          vivo. Eur J Pharmacol                    1990:182:591-595.
      Polak       J: Rat mesangial                 cells (MC) in culture                 how     54.    Nava      E, Palmer           RMJ,      Moncada          S: Inhibition            of
      immunostaining                  for nitric         oxide      synthase          (NOS)             nitric    oxide synthesis             in septic      shock:        how much
      [Abstract].          J Am Soc Nephrob                    1991:2:512.                              is beneficial?           Lancet      1991:338:1555-1557.
42.   Terada         Y, Tomita            K, Nonoguchi                H, Marumo             F:   55.    Shultz         PJ,    Raij      L: Endogenously                 synthesized
      Pobymerase             chain      reaction         localization          of consti-               nitric     oxide     prevents        endotoxin-induced                  gbomer-
      tutive      nitric      oxide      synthase           and soluble            guanyl-              ular thrombosis.               J Clin Invest         1992,       in press.
      ate cyclase            messenger             RNAs        in microdissected                 56.    Hibbs       JB Jr. Westenfelder                  C, Taintor          R, et at.:
      rat nephron              segments.            J Clin Invest               1992:90:                Evidence          for cytokine-inducible               nitric      oxide     syn-
      659-665.                                                                                          thesis       from     L-arginine          in patients         receiving         in-
43.   Tolins       JP, Palmer            RMJ,       Moncadz            5, Raij L: Role                  terleukin-2           therapy.          J Clin        Invest         1992:89:
      of endothelium-derived                        relaxing         factor       in regu-              867-877.




Journal      of the    American       Society      of Nephrology                                                                                                                      1441

				
hkksew3563rd hkksew3563rd http://
About