Adaptation of Epithelial Sodium-Dependent Phosphate Transport
in Jejunum and Kidney of Hens to Variations in Dietary Phosphorus Intake
K. Huber,*1 R. Hempel,† and M. Rodehutscord†
*Physiologisches Institut, Stiftung Tierarztliche Hochschule Hannover, Germany; and †Institut
¨ ¨ ¨
fur Ernahrungswissenschaften, Martin-Luther Universitat, Halle (Saale), Germany
ABSTRACT The objective of this study was to explore mRNA expression studies by northern analyses. Plasma
the homeostatic response of jejunal and renal epithelia Pi concentrations were additionally measured. The NaPi
regarding the inorganic phosphate (Pi) transport capacit- II transporter mRNA could speciﬁcally be detected in
ies to variations in dietary total phosphorus (tP) supply chicken jejunum and kidney. Functional parameters of
in hens. Adaptive processes were determined by quantita- Na+-dependent Pi transport indicated that these trans-
tive measures of intake and excretion, Pi transport studies porters were involved in chicken Pi transport across the
across brush border membranes, and semiquantitative apical membranes of jejunal and renal epithelia. Increased
detection of sodium-dependent phosphate transporters tP intake resulted in an increased overall tP excretion.
(NaPi II) based on mRNA expression in the jejunum and Correlating individual data from all animals by linear
kidney. Twelve hens (4/group) were adapted to 3 tP regression highlighted that the adaptive decrease of renal
feeding levels in a pair-fed manner (60 g/d): low P diet Pi transport capacity and NaPi IIa mRNA expression was
with 0.073% tP, medium P diet with 0.204% tP, and high associated with an increase in plasma Pi levels and re-
P diet with 0.343% tP. Excretion was measured during sulted in a higher tP excretion. Jejunal Pi transport capac-
the last 5 d of a 16-d feeding period. After slaughtering, ity and NaPi IIb mRNA expression did not react to varia-
jejunal mucosa and renal cortex were removed. Tissues tions in dietary tP supply. In conclusion, the homeostatic
were used for 32P uptake studies in brush-border mem- response was mainly based on the adaptive capacity of
brane vesicles by rapid ﬁltration technique and NaPi II the kidney in hens.
Key words: hen, inorganic phosphate transport, kidney, jejunum, NaPi type II cotransporter
2006 Poultry Science 85:1980–1986
INTRODUCTION with functional and structural studies on inorganic phos-
phate (Pi) transport mechanisms in respective organs
Balance studies with quantitative measurements of in- mainly relevant for P excretion promise a more detailed
take and excretion of total P (tP) are difﬁcult to interpret view of P homeostatic processes in chicken. In chicken
with regard to the effects of variable supply of dietary tP epithelia, the ﬁrst step of Pi absorption is a secondary
on P homeostasis because excreta contain tP from feces active, Na+-dependent process across the brush border
and urine in different proportions and different dietary membranes in the small intestine and the kidney (Renfro
ingredients differ in P availability. Rodehutscord et al. and Clark, 1984; Quamme, 1985). On molecular level, Na+-
(2002) studied the effects of supplementing an inorganic dependent Pi transporters of the solute carrier (SLC) 34
P source to a low-P basal diet in hens and compared tP family are detected in chicken kidney (NaPi IIa, SLC34A1)
ﬂow at the terminal ileum with tP excretion. Whereas the and in chicken small intestine (NaPi IIb, SLC34A2; Werner
prececal net absorption of supplemented tP in the small and Kinne, 2001). Effects of dietary tP restriction on P
intestines was very high (90%), supplemented P was al- homeostasis in chicken are not extensively studied with
most completely recovered in excreta in this study. Van a combination of quantitative, functional and structural
der Klis et al. (1997) found that the prececal net absorption approaches. Therefore, it was the aim of this study to
of supplemented tP in hens was 87%. These data suggest explore the overall P balance, the jejunal and renal Pi trans-
that the kidney plays the dominant role in hens in the port capacities and the expression levels of the regulatory
adaptation to variable P intake. However, the adaptive important Na+-dependent Pi transporters at different P
capacities of intestines and kidney cannot separately be feeding levels in hens.
examined in balance studies. Combining balance trials
MATERIALS AND METHODS
2006 Poultry Science Association Inc.
Diets, Animals, and Feeding
Received March 20, 2006.
Accepted May 4, 2006. Three diets were used. The ingredients of the diets were
Corresponding author: email@example.com chosen to allow for a very low P concentration in the basal
EPITHELIAL PHOSPHATE TRANSPORT IN CHICKENS 1981
Table 1. Composition of the basal diet Excreta were freeze-dried and ground through a 0.5-mm
Ingredient composition (%) Analyzed (g/kg) screen prior to analysis. Samples of feed and excreta were
hydrolyzed using H2SO4 and HNO3. Concentrations of tP
Rice ﬂour (51.6) Crude protein (206)
Dried sugar beet pulp (18.0) Crude fat (67) and Ca were determined with an Inductively Coupled
Dried egg albumen (15.0) Starch (373) Plasma Spectrometer (ICP-OES; JY 24, Jobin Yvon GmbH,
Soybean oil (5.00) Sugar (21) Grassbrunn, Germany) with details as given by Rodehut-
Sodium chloride (0.30) Phosphorus (0.73)
Premix1 (0.05) Calcium (36) scord and Dieckmann (2005).
Calcium carbonate2 (10.0)
Premix contained per kilogram: 29 g of Ca, 7.1 g of Mg, 1,200,000 Tissue Sampling
IU of vitamin A, 300,000 IU of vitamin D3, 4,200 mg of vitamin E, 650
mg of vitamin B2, 500 mg of vitamin B6, 200 mg of vitamin B1, 2,000 At the end of the experiment hens were slaughtered
mg of vitamin B12, 3,600 mg of nicotinic acid, 1,500 mg of D-calcium conventionally. Blood was collected during slaughtering,
pantothenate, 100 mg of folic acid, 15,000 g of biotin, 6,000 mg of Fe,
5,100 mg of Zn, 500 mg of Cu, 62 mg of I, 20 mg of Se, 12,000 mg of centrifuged at 600 × g at room temperature, and resulting
antioxidant. plasma was stored at −20°C until analyses. The gastrointes-
Calcium was provided either as limestone or as limestone plus re- tinal tract was removed from the body cavity completely.
spective amounts of calcium phosphate. Chymus samples were collected from the small intestines
if available. Jejunum of hens was removed, cut open,
washed in ice-cold saline, and shock-frozen in liquid N. A
diet (Table 1). Rice ﬂour, ground sugar beet pulp, and dried small piece of jejunum was separately dissected to isolate
egg albumen were the main ingredients with a presumably mucosa by scraping it off the underlying tissue for RNA
low P availability. Energy and all other nutrients were isolation. Kidneys were removed completely, were
contained at least at levels recommended by the Gesell- washed in ice-cold saline, and were also shock-frozen in
schaft fur Ernahrungsphysiologie (1999). In 2 other diets
¨ ¨ liquid N. Tissues were stored at −80°C until analyses.
the tP contents were increased by inclusion of monobasic
calcium phosphate [Ca(H2PO4)2] at levels of 5 and 10 g/ Determination of Plasma
kg at the expense of limestone. All ingredients with the and Chymus Pi and Ca Concentrations
exception of the variable ones were mixed in 1 batch to
ensure uniformity of the mix. This mix was divided in Plasma and chymus Pi was determined colorimetrically
3 portions, and monobasic calcium phosphate was then using the vanadate-molybdate method (Kruse-Jarres,
included at the respective level. The analyzed tP concentra- 1979) and Ca by the standard o-cresolphthaleine complex
tions in the diets were 0.73 g/kg (low P; LP), 2.04 g/kg method (Sarker and Chaunan, 1967).
(medium P; MP), and 3.43 g/kg (high P; HP).
Twelve 24-wk-old hens (Lohmann Brown) were kept Functional Characterization
individually in balance crates and allocated to 1 of the 3 of Epithelial Phosphate Transport
diets randomly (n = 4 hens per diet). For a period of 15
in Jejunum and Kidneys
d, the feed was placed into the crop in a 12-h interval
through a ﬂexible plastic tube after addition of tap water Brush border membrane vesicles (BBMV) were pre-
at a rate of 60 g of feed/d. This procedure was chosen pared from jejunal and renal epithelia by a modiﬁed Mg2+-
because we intended to avoid the high variation in feed EGTA precipitation method (Schro ¨der and Breves, 1996).
intake that was observed in previous trials and that had Enrichment of the brush border membrane in the ﬁnal
caused additional variation in tP intake. The feeding level vesicle preparations compared with the initial total ho-
was chosen on the basis of the voluntary intake of a tP- mogenates was determined by marker enzyme activities
deﬁcient diet by hens measured previously (Hempel et al., [alkaline phosphatase (AP) for brush border membrane,
2004). Hens responded to the low intake with a decrease in Na+-K+-ATPase for basolateral membranes]. Protein con-
laying performance, and no eggs were laid at the end of centration was assayed by applying the DC method from
the experiment. The mean BW of the hens at the end of BioRad (Hercules, CA).
the experiment was 1.46 kg. The experiment was approved The Na+-dependent Pi uptake into BBMV was quantiﬁed
by the animal welfare authorities [Approval No. 2-643 using the rapid ﬁltration technique as described by
MLU Hal, Landesverwaltungsamt Sachsen-Anhalt, Halle Schro¨der and Breves (1996) and Schro ¨der et al. (2000) for
(Saale), Germany] in accordance with the German Animal caprine intestinal and renal BBMV. In general, 20- L por-
Welfare Regulations. tions of vesicle suspensions containing 160 to 300 g of
protein were mixed with 80- L transport buffer containing
Excreta Collection and Analyses 1.0 Ci 32P and nonlabeled Pi to create an inwardly directed
Pi gradient. Extravesicular incubation buffers (100 mmol/
At the end of the experiment, excreta were quantitatively L of mannitol, 10 mmol/L of HEPES/Tris) contained 100
collected for 5 consecutive days from trays underneath mmol/L of NaCl to create an inwardly directed Na+ gradi-
the crates once daily. Excreta were bulk-stored at −18°C ent or 100 mmol/L of KCl to detect the diffusional compo-
for each hen separately. Thawed excreta were thoroughly nent of Pi uptake. The mixtures were incubated at room
mixed, weighed, and analyzed for dry matter content. temperature until the reactions were stopped by mixing
1982 HUBER ET AL.
them with ice-cold stop solution and immediately ﬁltered MMuLV (moloney murine leukemia virus) reverse
by vacuum suction. The ﬁlters were washed twice with transcription (MBI Fermentas, Vilnius, Lithuania),
5 mL of ice-cold stop solution to remove extravesicular poly(A)+RNA were transcribed into cDNA. The PCR was
radioactivity. The stop solution contained 10 mmol/L of performed at the following reaction conditions: 1× reaction
HEPES/Tris buffer, 150 mmol/L of KCl, and 1.0 mmol/ buffer (200 mmol/L of Tris-HCl, pH 8.4, 500 mmol/L of
L of KH2PO4 (pH 7.4 at 4°C). Filters with washed BBMV KCl), 0.4 mmol/L each dNTP, 1.5 mmol/L MgCl, 2.5 u
were then transferred into vials with 4 mL of scintillation Taq polymerase (Gibco BRL, Gaithersburg, MD), 20 pmol
liquid (Lumasafe plus, Lumac LSC B.V., Groningen, the of each primer, and 1.5 L template in a total volume of 25
Netherlands). Measurement of radioactivity was per- L. Using a Mastercycler gradient (Eppendorf, Wesseling-
formed in a liquid scintillation counter (TriCarb 2500 TR, Berzdorf, Germany), 34 cycles were run with denaturing
Packard Instruments Co., Meriden, CT). for 30 s (after an initial denaturing for 2 min at 94°C) at
94°C, annealing for 1 min at 60°C, and elongation for 2
Time-Dependent Pi Uptake min at 72°C. Finally, there was an elongation time of 15
Time-dependent accumulation of Pi in jejunal BSMV min at 72°C. Speciﬁc primers were derived from bovine
was determined from 10 s up to 4 h at 37°C in the presence kidney cell line NaPi IIb mRNA sequence (Acc. No.
of 100 mmol/L of Na+ or K+ to verify the integrity of X81699): sense 5′atggtggcctcctcactgctg3′ (nucleotides 609-
vesicles and the Na+-dependency of Pi transport. Time- 629), antisense 5′tggggtcatagcagacgtgaa3′ (nucleotides
dependent Pi uptakes of renal BBMV could not be per- 1406–1429). A band of 819 bp could be detected by agarose
formed because of low yield of BBMV. gel electrophoresis, which was identiﬁed by cloning and
sequencing (sequenced by Agowa, Berlin, Germany). This
Pi Concentration-Dependent Pi Uptake DNA fragment was used for detection of chicken-speciﬁc
NaPi IIb-mRNA in jejunum of the hens. For the detection
The Pi uptake was measured at 0.01, 0.05, 0.1, 0.3, 0.5, of chicken NaPiIIa in kidney, a goat-speciﬁc probe was
and 1.0 mmol/L of Pi in the extravesicular incubation used. This probe was created by reverse transcription-PCR
buffer in the presence of Na+. Additionally, at a Pi concen- using primers derived from the core region of murine
tration of 0.1 mmol/L, Pi uptake was measured in the NaPi IIa nucleotide sequence, which reﬂected the trans-
presence of 100 mmol/L K+ to estimate the extent of the
membrane regions of the corresponding NaPi IIa protein.
Na+-independent, diffusible part of Pi uptake at this Pi
Transmembrane regions of Pi transporters were strongly
concentration and to ensure by the difference between
conserved within the animal kingdom and also in chicken.
Na+-dependent and -independent Pi uptake rates the in-
High homology of NaPi IIa structure was conﬁrmed by
tegrity of the vesicle during uptake studies. This diffusible
detection of NaPi IIa protein using a murine NaPi IIa
Pi uptake value was not used to calculate the kinetic pa-
antibody (Dudas et al., 2002). Hybridization with this
probe resulted in a strong 2.4 kb band in northern analysis
The Na+/Pi uptake was calculated as total Pi uptake
that was the expected NaPi IIa mRNA size, indicating an
minus Na+-independent Pi uptake. The Na+-independent
adequate cross homology in chicken.
part of Pi uptake was calculated by using a modiﬁed equa-
tion according to Michaelis-Menten. The Michaelis-Men-
ten equation was extended by the term (+ A x) to introduce Northern Blot Analyses
A as the estimated diffusible part of Pi uptake. Kinetic
parameters Vmax (nmol Pi/mg protein/10 s) and Km ( mol Semiquantitative detection of speciﬁc NaPi IIa and IIb
Pi /L) were calculated ﬁtting the Pi uptake rates in the mRNA of jejunum and kidney was performed as described
presence of Na+ to y = Vmax x/(Km + x) + A x, where y is in detail by Huber et al. (2002). In brief, RNA was isolated
the Na+/Pi uptake in nmol Pi/mg of protein/10 s and x = by acid phenol/chloroform extraction, and jejunal
substrate (Pi) concentration in mmol/L. These values were poly(A)+RNA was enriched by afﬁnity chromatography
ﬁtted to all measured x and y values to create an optimal using an oligo-dT cellulose binding matrix. Renal RNA
curve with highest correlation coefﬁcient. The Vmax repre- (40 g/lane) or jejunal poly(A)+RNA (5 g/lane) were
sents the transporter capacity, and Km is the Pi concentra- fractionated in 1.0% formamide/agarose gels and trans-
tion at which the transporter is half-maximal saturated ferred by capillary blotting onto nitrocellulose membranes.
and that represents the afﬁnity of the transporter for Pi. Transferred RNA was ﬁxed on the nitrocellulose mem-
Fitting Pi uptake rates to the above-mentioned algorithm brane by heating at 80°C under vacuum. Fixed mRNA
was performed using Graphpad Prism software (Version were hybridized to radioactively labeled NaPi IIa-, -IIb
2.01, GraphPad Software Inc., San Diego, CA). and β-actin-speciﬁc probes. Labeling was performed with
50 Ci [α32P]dCTP per probe using Readyprime II random
Structural Characterization of Epithelial prime labeling kit according to the manufacturer’s proto-
Phosphate Transporters in Jejunum col (Amersham Biosciences Europe, Freiburg, Germany).
and Kidneys: NaPi IIb-Speciﬁc Reverse The membranes were analyzed after exposure to a phos-
phorus imager screen for 2 to 4 h with a phosphorus
Transcription-PCR in the Jejunum imager system (BioRad, Munich, Germany). The relative
The RNA from the jejunum of hens was isolated using amounts of speciﬁc mRNA were quantiﬁed by reference
a commercial kit (Qiagen, Hilden, Germany). Using 500 u to β-actin as an internal standard using the quantiﬁcation
EPITHELIAL PHOSPHATE TRANSPORT IN CHICKENS 1983
Table 2. The inorganic phosphate (Pi) and Ca concentrations in chicken plasma and intestinal chymus
Item LP MP HP
Pi (mmol/L) 0.55 ± 0.32 0.96 ± 0.35 0.98 ± 0.33
Ca (mmol/L 3.99 ± 0.52a 2.88 ± 0.44b 2.70 ± 0.17b
Pi (mmol/L) 1.65 3.43 ± 2.58 5.79 ± 3.77
Ca (mmol/L) 80.25 35.44 ± 33.42 38.93 ± 30.94
Signiﬁcance of difference is P < 0.01 within the row (1-way ANOVA with Tukey’s post test).
Values are given as mean ± SD; n = 4 animals/group for plasma data; n = 2 (LP)- 4 animals/group for
chymus data. LP = low P diet; MP = medium P diet; HP = high P diet.
software Quantity One (BioRad). Northern blots were per- account that determination of electrolytes in chymus is
formed at least in duplicate. difﬁcult because of strong variations in collectable
amounts of chymus. Therefore, electrolyte concentrations
Statistics could not reﬂect the physiological situation absolutely.
Hens from the LP group were in a negative P balance
Values are given as means ± SD. Statistical analyses (Table 3). With increased dietary tP intake, hens doubled
and linear regressions were performed with the software their tP excretion from the LP to HP group signiﬁcantly.
Graphpad Prism version 2.01. One-way ANOVA with Tu- The dependency of plasma Pi concentrations and tP excre-
key’s post test was used to compare means of LP, MP, tion on dietary tP intake is shown in Figure 1. From LP
and HP. Levels of signiﬁcance were set at P < 0.05, P < to MP plasma Pi concentrations and tP excretion increased
0.01, and P < 0.001. both, but from MP to HP the increase in tP concentration
was more pronounced, whereas plasma Pi concentrations
RESULTS were maintained at about 1 mmol/L. Overall, tP excretion
was strongly dependent not only on dietary tP intake but
Effect of Dietary High Ca and Low tP also on plasma Pi concentration (Figure 2).
Intake on Kidney Integrity
Parameters of Pi Uptake
Diets containing 3% Ca and less than 0.6% available P in Jejunal and Renal BBMV
(aP) could generate urolith formation in chickens caused
by alkalinization of the urine. Urolithiasis caused kidney Apical located alkaline phosphatase activity was en-
tissue damage, tubular degeneration, and interstitial ne- riched 6.9 ± 1.3-fold and 9.4 ± 1.0-fold in LP, 5.4 ± 0.8-fold
phritis, which could result in a high mortality of hens and 7.5 ± 1.2-fold in MP, and 6.2 ± 0.5-fold and 13.6 ± 2.4-
(Wideman et al. 1985, 1989). Although predisposition re- fold in the HP group in jejunal and renal BBMV, respec-
garding urolithiasis generation might have been relevant tively. The Na+K+ATPase activity located in the basolateral
with the experimental feeding design used, neither uro- membrane was enriched 1.3 ± 0.6-fold and 2.4 ± 1.4-fold
liths nor mineralization of renal medullary regions were in LP, 1.0 ± 0.2-fold and 1.7 ± 0.9-fold in MP, and 1.6 ±
found macroscopically. Also, morphology and size of kid- 1.1-fold and 1.3 ± 0.5-fold in HP group in jejunal and
neys was unchanged, indicating the lack of compensatory renal BBMV, respectively. These enrichment values were
hypertrophic growth of undamaged renal tissue. Histolog- comparable with those obtained in BBMV preparations of
ical examination of hematoxylin-eosin stained kidney tis- goats and pigs and were set to be adequate for studying
sue slices did not show signs of inﬂammation or tissue Pi transport processes across the apical membranes. Con-
lesion. taminations by basolateral membranes were comparably
low in all feeding groups. Integrity of BBMV was con-
Effects of Dietary tP Intake on Pi ﬁrmed by measuring time-dependent Pi uptakes into the
and Ca Plasma or Chymus vesicles of 1 hen of each group (Figure 3). Intravesicular
Concentrations and tP Excretion Pi accumulations were observed in the presence of 100
mmol/L Na+ in the incubation buffer, whereas incubation
Plasma Pi concentrations in hens increased with in- with 100 mmol/L K+ resulted in low linear Pi uptakes into
creased dietary tP intake from LP to MP, reaching a plateau jejunal BBMV. These results conﬁrmed the presence of a
at about 1 mmol/L in MP and HP groups. Plasma Ca Na+-dependent Pi transport system in the apical mem-
concentrations were signiﬁcantly enhanced in LP com- branes of jejunal enterocytes whereas K+-dependent Pi up-
pared with MP and HP groups, indicating a P depletion take reﬂected only the diffusible part of Pi uptake. This
status only in the LP group (Table 2). Concomitantly, Pi diffusible part was low and uninﬂuenced by dietary P
concentrations in small intestinal chymus increased with supply. Time- and Na+-dependent uptake of Pi into the
increasing dietary tP intake, whereas Ca concentrations vesicles showed an overshoot phenomenon in each BBMV
seemed to be constant (Table 2). But it has to be taken into preparation. Elongation of incubation time up to 4 h did
1984 HUBER ET AL.
Table 3. Daily dietary total P (tP) intake, tP excretion, inorganic phosphate (Pi) transport capacities (Vmax) in
jejunum and kidney and expression level of jejunal and renal NaPi type II transporters1
Item LP MP HP
tP intake (mg/d) 39 111 187
tP excretion (mg/d) 44 ± 7.2ac 56 ± 5.3a 88 ± 5.6bd
Vmax jejunum (pmol/mg of protein/10 s) 64.0 ± 29.0 46.0 ± 28.0 43.0 ± 20.0
NaPi IIb mRNA expression level3 (NaPi IIb/β-actin) 2.66 ± 0.48a 3.13 ± 1.29a 4.97 ± 1.42b
Vmax kidney (nmol/mg of protein/10 s) 1.87 ± 0.56 1.01 ± 1.16 0.49 ± 0.85
NaPi IIa mRNA expression level3 (NaPi IIa/β-actin) 1.46 ± 1.02 0.65 ± 0.14 0.38 ± 0.09
Signiﬁcance of difference is P < 0.05 and c,dSigniﬁcance of difference P < 0.01 within the row (1-way ANOVA
with Tukey’s post test).
Means ± SD are given; n = 4 animals/group. LP = low P diet; MP = medium P diet; HP = high P diet.
The P uptake values are given without standard deviation because hens were fed by a crop tube.
Expression level values are given as result of 1 characteristic blot of a total of 2 to 4. Values are dimensionless
because the ratio of NaPi II to β-actin was given.
not result in equilibrium because of the low incubation were in the expected size range for these transporters,
temperature at about 18°C. indicating the speciﬁcity of the detection. Whereas the
The Pi concentration-dependent Pi uptake in jejunal and NaPi IIb mRNA level signiﬁcantly increased from the LP
renal BBMV was performed to calculate Vmax and Km val- to HP group, NaPi IIa mRNA level decreased successively
ues of Pi transport as dependent on dietary tP supply. from LP to HP (Table 3). However, this decrease in NaPi
Mean Vmax values representing the mean Pi transport ca- IIa mRNA expression was not signiﬁcant.
pacity were highest in the LP group and tended to decrease
to HP group in the jejunum (64, 46, and 43 pmol/mg of DISCUSSION
protein/10 s) but more pronounced in the kidney (1.87,
The Pi homeostasis in chickens inﬂuenced by dietary
1.01, and 0.49 nmol/mg of protein/10 s; Table 3). However,
tP supply is affected by the adaptive capacities of small
in both organs these decreases were not signiﬁcant. Mean
intestines and kidney, like in mammalian species. The aim
Km values representing mean transporter afﬁnity were not
of this study was to characterize the basic functional and
affected by the dietary tP supply. In jejunum and kidney,
structural principles of Pi transport in the jejunum and
the mean Km values were 45 and 104 mol/L in LP, 43 kidney. Inﬂuenced by variations of dietary tP supply, the
and 75 mol/L in MP, and 38 and 92 mol/L in HP adaptation of epithelial Pi transport processes was com-
group, respectively. bined with balance data and plasma Pi concentrations to
assess the physiological role of each of these components
NaPi Type II mRNA Expression Levels of Pi homeostasis.
in Jejunum and Kidney
Epithelial Pi Transport in Hens
Hybridization of mRNA with a chicken-speciﬁc NaPi
IIb probe created on chicken jejunal cDNA resulted in a Apical-located NaPi IIb and NaPi IIa transporters repre-
strong signal at about 4 kb. Detection of renal NaPi IIa sent the rate-limiting steps for transepithelial Pi transport
mRNA with a goat-speciﬁc probe revealed a strong band
at about 2.4 kb in northern analysis. Both mRNA signals
Figure 2. Relation between plasma inorganic phosphate (Pi) and total
P (tP) excretion. One hen of the medium group P was omitted because
Figure 1. Dependency of plasma inorganic phosphate (Pi) concentra- of its low tP excretion despite high plasma Pi concentration. Linear
tions (ﬁlled circles) and total P (tP) excretion (open circles) on dietary regression resulted in r2 = 0.68, P < 0.01 signiﬁcance of deviation of the
tP intake. LP = low P diet; MP = medium P diet; HP = high P diet. slope from zero.
EPITHELIAL PHOSPHATE TRANSPORT IN CHICKENS 1985
tubuli mediated by NaPi IIa is only one factor for inﬂuenc-
ing renal Pi excretion. From elegant studies on renal Pi
excretion in birds inﬂuenced by parathyroid hormone
(PTH) and variations in dietary aP supply, a tubular Pi
secretion was detected that is not expressed in mammalian
species. This mechanistically unknown Pi secretion was
stimulated by PTH irrespective of dietary aP supply
(Wideman, 1987; Stanton et al., 1989). At which extent this
Pi secretion participates in the adaptation to variations in
dietary aP is still unclear, but PTH levels should be low
at the relatively high dietary Ca intake in hens of this
study. Therefore, adaptation to variations in dietary aP
should be modulated by tubular Pi reabsorption mediated
by NaPi IIa. To summarize, the assumption that NaPi II-
Figure 3. Time-dependent inorganic phosphate (Pi) uptake in jejunal mediated Pi transport mechanisms exist in kidney and
brush border membrane vesicles of 3 hens in the presence of 100 mmol/ jejunum of hens was proven by the presence of NaPi IIa
L Na+ (closed symbols) or K+ (open symbol) in the incubation buffer. and IIb mRNA and the concordance of functional charac-
Each point represents the mean of 3 measurements of 1 individual hen;
= low P diet (LP) hen, ▲ = medium P diet (MP) hen, ▼ = high P teristics of Na+-dependent Pi transport with those known
diet (HP) hen, = mean values of K+ dependent Pi uptake of LP, MP, from NaPi IIa- or IIb-mediated Pi transport processes in
and HP hens (standard deviation was <0.016 nmol/mg of protein/10 s). mammalian species.
Adaptation of Epithelial Pi Transport
in jejunal and renal epithelia. Physiological regulation of
Pi transport capacities of renal and jejunal epithelia is medi-
Inﬂuenced by Dietary P Restriction
ated by changes in the abundance of these transporters Regulation of P homeostasis in chicken should be analo-
(Murer et al., 2004). In hens, NaPi IIb and NaPi IIa mRNA gously based on adaptive changes in NaPi II expression
could be detected in jejunum and kidney speciﬁcally. Be- and Na+/Pi transport capacity, like in mammals (Werner
cause antibodies against the respective proteins are not et al., 1994; Murer et al., 2000; Huber et al., 2002; Radanovic
available, BBMV uptake studies were performed to charac- et al., 2005). However, functional (Vmax) and structural
terize jejunal and renal Na+-dependent Pi transport func- (NaPi II mRNA expression) parameters were not changed
tionally. The Km value for Pi is characteristically low in signiﬁcantly by dietary P restriction when the experimen-
Na+-dependent Pi transport mediated by NaPi IIb at about tal groups were compared. Neither chymus nor plasma
50 mol/L, indicating a high Pi afﬁnity (Murer et al., 2004). Pi concentrations inﬂuenced intestinal Pi absorption. It is
In chicken jejunum, Km(Pi) values of all groups were equally noticeable that plasma Pi levels lowest in LP group were
low at about 40 mol/L, indicating that Na+-dependent combined with hypercalcemia, indicating strong P restric-
Pi transport is mediated by NaPi IIb in this species also. tive feeding. Plasma levels of MP and HP group were only
In kidney, apparent Pi afﬁnity was 100 mol/L for Na+- slightly higher than in LP but did not reach the level of
dependent Pi transport mediated by NaPi IIa (Murer et adequate tP-fed chickens (1.2 to 1.8 mmol/L; Boorman
al., 2004). In chicken kidney a comparable Pi afﬁnity was and Gunaratne, 2001). This could be due to the restricted
detected indicating that renal Na+-dependent Pi transport amount of feed that was to be given in this study to stan-
was also mediated by NaPi IIa. The existence of NaPi dardize tP intake. This restriction in feed also caused a
IIa in chicken should be associated with the existence of decrease in egg production of the hens, which also interacts
mammalian-type nephrons in chicken kidneys because, with the homeostasis of Ca and P. Therefore, plasma Pi
hypothetically, the evolution of NaPi IIa was paralleled levels did not reach higher values.
by development of this type of nephrons (Werner and However, increased plasma Pi levels signiﬁcantly
Kinne, 2001). But Pi reabsorption capacity in the proximal caused increased tP excretion, which could be the result
Table 4. Correlations between renal and jejunal inorganic phosphate (Pi) transport capacities (Vmax), sodium-
dependent phosphate transporters (NaPi IIa and IIb) mRNA expression levels, plasma Pi concentrations, and
total P (tP) excretions
Item r2 Slope P-value1
Vmax kidney/plasma Pi 0.60 −2.0 <0.01
Vmax jejunum/plasma Pi 0.05 −0.01 NS
NaPi IIa mRNA expression level/plasma Pi 0.34 −1.8 <0.05
NaPi IIb mRNA expression level/plasma Pi 0.005 0.33 NS
Vmax kidney/tP excretion 0.36 −14 <0.05
Vmax jejunum/tP excretion 0.10 −296 NS
The P value gives the signiﬁcance level of slope different from zero. Linear regressions were performed
using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). Correlation coefﬁcients (r2) at about
0.3 were set to be signiﬁcant.
1986 HUBER ET AL.
of a decreased intestinal or renal Pi absorption or both. Hempel, R., E. Strobel, and M. Rodehutscord. 2004. Inevitable
Correlations of individual data of all animals between phosphorus losses of laying hens. Proc. Soc. Nutr. Physiol.
renal and jejunal Pi transport capacities, NaPi IIa and IIb Huber, K., C. Walter, B. Schro ¨der, and G. Breves. 2002. Phosphate
mRNA expression, plasma Pi levels, and tP excretion by transport in the duodenum and jejunum of goats and its
linear regression highlighted signiﬁcant relations between adaptation by dietary phosphate and calcium. Am. J. Physiol.
renal functional and structural parameters of Pi transport Regul. Integr. Comp. Physiol. 283:R296–R302.
and plasma Pi or tP excretion (Table 4). The Vmax of renal Kruse-Jarres, J. D. 1979. Klinische Chemie, vol II, Spezielle klin-
isch-chemische Analytik. Fischer Verlag, Stuttgart, Germany.
Na+-dependent Pi transport as well as NaPi IIa mRNA Murer, H., I. Forster, and J. Biber. 2004. The sodium phosphate
expression was signiﬁcantly reduced by increased plasma cotransporter family SLC34. Pﬂugers Arch.—Eur. J. Physiol.
Pi levels, which gave rise to decreased renal Pi absorption 447:763–767.
capacity. As a consequence, renal Pi excretion should in- Murer, H., H. Hernando, I. Forster, and J. Biber. 2000. Proximal
crease, which was supported by the signiﬁcant negative tubular phosphate reabsorption: Molecular mechanisms.
Physiol. Rev. 80:1373–1409.
correlation between renal Vmax and tP excretion (Table Quamme, G. A. 1985. Phosphate transport in intestinal brush-
4). In the jejunum Pi transport capacity, NaPi IIb mRNA border membrane vesicles: Effect of pH and dietary phos-
expression, plasma Pi levels, and tP excretion were gener- phate. Am. J. Physiol. Gastrointest. Liver Physiol.
ally not correlated with each other. Therefore, Pi absorp- 249:G168–G176.
tion and NaPi IIb expression was obviously not modulated Radanovic, T., C. A. Wagner, H. Murer, and J. Biber. 2005. Regula-
tion of intestinal phosphate transport I. Segmental expression
by dietary tP supply in the jejunum of hens. In the study and adaptation to low Pi diet of the type IIb Na+-Pi cotrans-
by Rodehutscord et al. (2002) an increased intake of tP porter in mouse small intestine. Am. J. Physiol. Gastrointest.
resulted in an increased tP excretion of hens, but differ- Liver Physiol. 288:G496–G500.
ences in the amount of tP measured in the terminal ileum Renfro, J. L., and N. B. Clark. 1984. Parathyroid hormone effect on
were hardly found. The authors had suggested that the chicken renal brush-border membrane phosphate transport.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 247:R302–R307.
kidneys rather than the intestines play a major role in Rodehutscord, M., and A. Dieckmann. 2005. Comparative studies
adaptation to variable tP intake, which is conﬁrmed by with 3 wk-old chickens, turkeys, ducks, and quails on the
the results from the present study. response in phosphorus utilization to a supplementation of
In the duodenum of cockerels, Na+-dependent Pi trans- monobasic calcium phosphate. Poult. Sci. 84:1252–1260.
port was increased by dietary P restriction (Quamme, Rodehutscord, M., F. Sanver, and R. Timmler. 2002. Comparative
study on the effect of variable phosphorus intake at two differ-
1985). This difference to the ﬁndings from the present ent calcium levels on P excretion and P ﬂow at the terminal
study could be due to the fact that that study was per- ileum of laying hens. Arch. Anim. Nutr. 56:189–198.
formed in younger, rapidly growing cockerels, in which Sarker, B. C., and U. P. S. Chaunan. 1967. A new method for
disturbances of P homeostasis by dietary P restriction determining microquantities in biological materials. Anal. Bio-
could be more relevant due to the higher P requirement. chem. 20:155.
Schro¨der, B., and G. Breves. 1996. Mechanisms of phosphate
Additionally, another segment of the small intestines was uptake into brush border membrane vesicles from goat jeju-
examined, which could result in location-dependent ef- num. J. Comp. Physiol. [B] 166:230–240.
fects on Pi absorption. Schro¨der, B., C. Walter, G. Breves, and K. Huber. 2000. Compara-
It is still unclear why jejunal NaPi IIb mRNA expression tive studies on Na-dependent Pi transport in ovine, caprine
in the hens increased with increased dietary tP intake. and porcine renal cortex. J. Comp. Physiol. [B] 170:387–393.
Stanton, T. S., R. P. Glahn, and R. F. Wideman, Jr. 1989. The
In conclusion, the adaptive capacity of the kidney re- effects of dietary phosphorus and parathyroid hormone (PTH)
garding the Pi transport had the most important role for infusion rates on the avian phosphaturic response to PTH. J.
regulation of P homeostasis in these hens. Plasma Pi level Exp. Biol. 144:521–533.
seemed to have a relevant modulatory inﬂuence on renal Van der Klis, J. D., H. A. J. Versteegh, P. C. M. Simons, and A.
Pi reabsorption capacity in chickens as it was observed in K. Kies. 1997. The efﬁcacy of phytase in corn-soybean meal-
based diets for laying hens. Poult. Sci. 76:1535–1542.
mammals (Widiyono et al., 1998). Werner, A., S. A. Kempson, J. Biber, and H. Murer. 1994. Increase
of Na/Pi-cotransport encoding mRNA in response to low Pi
REFERENCES diet in rat kidney cortex. J. Biol. Chem. 269:6637–6639.
Werner, A., and R. K. H. Kinne. 2001. Evolution of the Na-Pi
Boorman, K. N., and S. P. Gunaratne. 2001. Dietary phosphorus cotransport systems. Am. J. Physiol. Regul. Integr. Comp.
supply, egg shell deposition and plasma inorganic phospho- Physiol. 280:R301–R312.
rus in laying hens. Br. Poult. Sci. 42:81–91. Wideman, R. F., Jr. 1987. Renal regulation of avian calcium and
Dudas, P. L., A. R. Villalobos, G. Gocek-Sutterlin, G. Laverty, phosphorus metabolism. J. Nutr. 117:808–815.
and J. L. Renfro. 2002. Regulation of transepithelial phosphate Wideman, R. F., Jr., J. A. Closser, W. B. Roush, and B. S. Cowen.
transport by PTH in chicken proximal tubule epithelium. Am. 1985. Urolithiasis in pullets and laying hens: Role of dietary
J. Physiol. Regul. Integr. Comp. Physiol. 282:139–146. calcium and phosphorus. Poult. Sci. 64:2300–2307.
Gesellschaft fur Ernahrungsphysiologie. 1999. Energie- und Nah-
¨ ¨ ¨ Wideman, R. F., Jr., W. B. Roush, J. L. Satnick, R. P. Glahn, and
rstoffbedarf landwirtschaftlicher Nutztiere. 7. Empfehlungen N. O. Oldroyd. 1989. Methionine hydroxyl analog (free acid)
zur Energie- und Nahrstoffversorgung der Legehennen und
¨ reduces avian kidney damage and urolithiasis induced by
Masthuhner (Broiler). DLG-Verlag, Frankfurt a. M., Germany.
¨ excess dietary calcium. J. Nutr. 119:818–828.