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THIRST AND VOLUME, ELECTROLYTE HOMEOSTASIS
Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103
Submitted 10 March 2003 ; accepted in final form 14 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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fractional absorption; immunocytochemistry; phosphate uptake stimulator; sodium-glucose cotransporter; sodium-phosphate cotransporter
Proteins other than NaPi may also participate in the regulation of P balance. The Pi uptake stimulator (PiUS) or inositol hexakisphosphate kinase (34) has been shown to be involved in the absorption of dietary P. PiUS cRNA encodes a protein that markedly increased Na-dependent Pi-uptake when injected in Xenopus laevis oocytes (18, 26) but that is not a Pi transporter. PiUS mRNA has been isolated from rabbit (26) and rat (18) small intestine.
These findings have contributed to increases in our understanding of molecular mechanisms regulating intestinal and renal nutrient transport, especially in mammals. The rate of transport of many essential nutrients, including P in mammalian intestine and kidney, increases with decreasing dietary concentrations, and the adaptive mechanism is typically an increase in the number of transporters (11, 12). Dietary P restriction has been shown to upregulate Pi transport in rodents by translocation of renal NaPi-IIa protein to the apical surface of the epithelial cells independent of changes in NaPi-II transcription (reviewed in Ref. 24). Dietary P restriction also stimulates the recruitment of more distal regions of the rat renal tubules to participate in Pi reabsorption (32, 38).
In contrast, the adaptive patterns and mechanisms of transport of essential nutrients in other vertebrates are virtually unknown. Although we know that many genes are highly conserved among distantly related species, is regulation of those genes also highly conserved? This is an important question in regulatory biology. There have been advances in our knowledge regarding the evolutionary relationships of various NaPi isoforms in many animals, including fish (37). However, our knowledge of mechanisms regulating Pi metabolism and Pi transport in fish still remains rudimentary and lags far behind that of mammals. Moreover, dietary P is so poorly assimilated by fish that significant amounts eventually appear in effluents from fish farms and lead to severe pollution of nearby lakes and rivers, where P is the nutrient limiting primary productivity (5). These physiological, nutritional, and environmental roles of P have heightened the priority of P research in fish.
There has only been one study demonstrating dietary regulation of Pi transport in fish. In rainbow trout (Oncorhynchus mykiss), dietary P restriction increases intestinal Pi uptake (2). There have only been a few studies on the regulation of Pi absorption in vivo. Moreover, the molecular mechanisms regulating Pi transport have not been well studied in any fish species, except in an initial report indicating that trout renal and intestinal NaPi-IIb mRNA abundance increased under conditions of dietary P restriction and vitamin D excess (7). In rats, dietary P restriction increased intestinal PiUS mRNA abundance and Pi uptake rate by twofold (18). However, nothing is currently known about the regulation of PiUS in other species.
To increase our understanding of regulation of Pi transport, we studied the molecular mechanisms of Pi transport in rainbow trout by identifying the tissue distribution and localization of NaPi-IIb, PiUS, and the sodium-glucose cotransporter (SGLT1; negative control) and characterizing their regulation by dietary P in the intestine and kidney. Because oxygen concentrations in the water affect dietary P retention in trout (23), we also evaluated the effect of aqueous oxygen concentrations on NaPi and PiUS expression. Finally, we studied the in vivo regulation of P absorption by dietary P. We chose the rainbow trout as an animal model because it is one of the most important species in freshwater aquaculture and has been the subject of many studies on dietary P requirements and digestibility but unfortunately not on Pi transport. It has a relatively short gastrointestinal tract typical of carnivores and a glomerular nephron similar to mammals (40).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Tissue distribution of NaPi-II,
PiUS, and SGLT1 mRNA. A group of rainbow
trout (mean body wt 221 g) was fed a low-P diet at 2% body wt/day for 1 wk.
The purpose of the low-P diet was to stimulate NaPi-II and
PiUS mRNA expression in all tissues. To make a low-P diet, 50%
commercial low-P trout feed was mixed with 5% spray-dried egg white, 5% wheat
gluten, 25% wheat flour, 10% soy oil, and 5% vitamin and mineral premixture.
Preliminary digestibility measurements by Cr2O3
determined that the diet contained 0.35% available P, which is much less than
the known dietary P requirement of 0.6%. On day 7, five fish were
randomly sampled and analyzed for tissue distribution of NaPi-II,
PiUS, and SGLT1 mRNA. Two fish were examined by Northern blot and
the other three by RT-PCR for mRNA expression in the following tissues: heart
(whole), skin (no scales), muscle (skeletal, white), stomach, gills (filaments
only), scales (with mucus), blood (whole clotted blood), pyloric ceca (distal
from the intestinal-cecal junction), proximal intestine (distal to
pyloric ceca, light-colored region without folds, roughly 
of the
total length of the intestine), distal intestine (dark-colored intestine lined
with folds), kidney (posterior kidney), and liver (whole).
Immunolocalization of NaPi-II protein and regulation of NaPi-II, PiUS, and SGLT1 expression by dietary P and oxygen levels. Rainbow trout [initial mean body wt 188 ± 4.4 (SE) g] were randomly distributed in 12 tanks (1 m3) and pair fed either a low-P diet (0.46% available P; 0.60% total P) or high-P diet (0.74% available P; 0.90% total P) for 7 wk at two different oxygen levels (6 or 10 ppm). The moderately low-P diet and moderately high-P diet were chosen because these encompass the range of dietary P in commercial trout feeds, with the low-P diet expected to lead to moderate P deficiency and the high-P diet to P sufficiency. The modest differences in dietary P should not lead to marked changes in growth rate and therefore to nonspecific effects on NaPi-II expression. Moreover, severe hypophosphatemia is rarely encountered in cultured fish, whereas moderate hypophosphatemia is more prevalent.
Each treatment had triplicate tanks. Oxygen at 6 ppm was well above the lowest tolerance level for trout and similar to commercial trout ponds, whereas the high oxygen level was near saturation (31). Fish in all treatments were fed at 2% (1% at 8:00 AM and 1% at 2:00 PM) of their body weight daily. Diets were manufactured as dry pellets with the following composition (%): 18 soybean meal, 17.6 corn gluten, 12 soy oil, 10 blood meal, 10 fish meal, 6 fish protein hydrolysate, 2.4 pellet binder, 22.1 wheat flour, 0.4 L-lysine, 0.2 DL-methionine, 0.7 mineral mixture, and 0.6 vitamin mixture. The low-P diet and high-P diet had the same composition except that the high-P diet was supplemented with 1.35% NaH2PO4, replacing a portion of wheat flour in the low-P diet.
After 7 wk, 10 fish from each tank were randomly sampled for blood and carcass analysis. Tissues of the posterior kidney and the proximal intestine were sampled (2 fish/tank), pooled by tank, and kept in RNAlater solution (Ambion, Austin, TX) for mRNA analysis. Tissues collected for immunocytochemistry were rinsed in Kreb's Ringer bicarbonate buffer (KRB, in mM: 100 NaCl, 2 KCl, 2 CaCl2, 1 MgSO4, 1 NaHCO3, and 0.5 NaH2PO4, pH 7.4) and placed in buffered formalin (10% formalin, 75 mM sodium phosphate buffer, pH 7.4). After 24-48 h of fixation, tissues were rinsed in KRB solution and then transferred to 70% ethanol and stored at 4°C for 10-30 days until further processing. Blood samples were collected from caudal vessels of each fish into heparinized syringes 24 h after the last feeding, and the plasma was separated (1,500 g) immediately after collection.
In vivo fractional Pi absorption.
Rainbow trout [initial mean body wt 129 ± 2.7 (SE) g] were initially
fed either a low-P (0.18% total P) or high-P (0.92% total P) diet containing
0.5% Cr2O3 one time daily at 1% (dry basis) of their
body weight for 32 days. Except the dietary P level, compositions of these
diets were the same as those of diets used in the subsequent test period.
After the 32-day period, fish were switched to one of 10 test diets of varied
P concentrations for 12 days, and the feces were collected continuously for 24
h in an unstirred fecal collection tube attached to each tank (modified from
Ref. 14) at 3, 6, 9, and 12
days after the switch in diet for P and Cr analyses. The unstirred collection
tube minimized the leaching loss of P from feces after being voided by the
fish. Each tank contained 14 fish. All diets contained (%) 35 wheat gluten, 11
blood meal, 20 wheat flour, 25 fish oil, 0.9 K2SO4, 0.1
MgO, 0.5 Cr2O3, 3 vitamin and trace mineral mix, and
varied amounts of NaH2PO4 replacing 4.5
NaCl-
-cellulose mix. The analytical composition of the diets (dry
basis) was 42.8% crude protein, 0.054% calcium, and 5,260 kcal/kg diet
digestible energy. Total P levels of the test diets were (%dry diet): 0.18
(basal), 0.31, 0.44, 0.56, 0.69, 0.81, 0.92, 1.03, 1.14, and 1.25. The net
Pi absorption of the basal diet was 64.8% on average. Diets were
made low in calcium to minimize interaction (precipitation) of Pi
in the intestine. Dietary inclusion of wheat gluten prevented fecal
disintegration and leaching loss of P in the feces during collection. Compared
with the other two (tissue distribution and regulation) experiments, the
feeding rate was reduced to 1% daily because of the higher caloric content of
the diet.
The initial fish size differed among the three experiments conducted over a
2-yr period, because trout spawn one time a year, meaning that it is possible
to obtain fish of the same age only one time a year. However, trout grow
extremely fast between 3 and 12 mo of age, and we estimate the age difference
of fish used in this experiment (130-220 g) to be only 
3 mo. The rapid
growth of trout at this stage makes the fish sensitive to small changes in
dietary P concentrations. All fish were treated according to the guidelines of
the Institutional Animal Care and Use Committee of the University of Medicine
and Dentistry of New Jersey (animal care and use protocol no. 02038).
Analyses and Calculations
Northern blots and RT-PCR. Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA) from tissues previously stored in RNAlater solution, and mRNA abundance of tissues was quantified by Northern blot analysis as described by Shu et al. (35). In the tissue distribution study, Northern membranes were probed with a mixture of intestinal and renal cDNA (equimolar basis) for NaPi and SGLT1 since kidney and intestine sequences are different (64% homology for NaPi; 93% for SGLT1). For PiUS, intestinal cDNA was used for all tissues (PiUS sequences of other tissues were not studied). In addition to Northern analysis, semiquantitative RT-PCR was also performed to quantitate minor transcripts that were difficult to detect by Northern analysis. In the regulation study, the mRNA abundance (relative to 28S rRNA) of NaPi-II and PiUS in the intestine was compared with that of SGLT1. In the kidney, the abundance of SGLT1 mRNA was not quantified because its expression level was very low.
The cDNA probes for intestinal and renal NaPi-II,
PiUS, and SGLT1 were generated by RT-PCR using
oligo(dT)20 reverse-transcribed cDNA (Moloney murine leukemia virus
RT; Promega, Madison, WI) as the starting template and degenerate primers
designed from the consensus sequences of other animal species
(Table 1). Several primers were
designed and tested at various PCR conditions using a "touch-down"
procedure. ClustalW 1.8 and BoxShade programs were used for sequence
alignment. The PCR products were subcloned using plasmids (pDrive cloning
vector; Qiagen, Valencia, CA) and competent cells (DH5; Invitrogen).
The identity of the insert was verified by DNA sequencing (Dye terminator, ABI
377) and the standard BLAST program of NCBI. The cDNA probes were made by PCR
using purified, sequenced plasmids as the templates, followed by gel
purification.
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Immunocytochemistry. The distribution of NaPi-IIb in various tissues (proximal and distal intestine, pyloric ceca, kidney, gills, skin, scale, muscle, and stomach) was examined using a panel of three unique antibodies. TrIIb2 is a polyclonal rabbit antibody raised against a synthetic peptide (EDAPELLKVITEPVT) corresponding to the E62-T76 of trout kidney NaPi (accession no. AAG35800 [GenBank] ) linked to keyhole limpet hemocyanin (KLH), which was generated by Sigma Genosys. This peptide sequence comes from a highly conserved region of NaPi-IIb protein sequence and thus should recognize isoforms of NaPi-IIb other than those from which the original peptide sequence was derived. The corresponding intestinal peptide sequence has a homology of 64%, which when calculated including conservative amino acid substitutions increases to 93%. Polyclonal antibodies raised in rabbit against KLH-linked peptide sequences from zebrafish NaPi-IIb1 and NaPi-IIb2 isoforms were kindly supplied by Dr. Andreas Werner and have been characterized previously (13).
TrIIb2 serum was affinity purified by passing it through a column generated by covalently linking the NaPi peptide to beads (Aminolink; Pierce Endogen, Rockford, IL). After being washed thoroughly to remove weakly associated antibodies, those that specifically bound to the peptide were eluted in 1-ml fractions with glycine (100 mM, pH 2.5), neutralized (100 ml of 1 M Tris, pH 7.5/ml), and measured spectrophotometrically for protein concentration. Fractions with the highest concentration of protein were combined, dialyzed against PBS, and concentrated (Slide-A-Lyzer Concentrating Solution; Pierce Endogen). We then determined that the flow-through serum (that portion that did not bind to the peptide on the column beads) showed virtually no specific immunoreactivity in all tissues studied. In contrast, the eluted fraction (containing specifically bound antibodies) showed very clear staining patterns.
Zebrafish antisera were not affinity purified because of insufficient volume but were still used for the renal immunocytochemistry since they produced the most reproducibly clear staining pattern. Preliminary work indicated that nonspecific staining using unpurified zebrafish antisera in the intestine was restricted to mucous and goblet cells, and, since there are no mucous cells in the kidney, purification was considered unnecessary. Hence, zebrafish NaPi-IIb2 antisera were used in kidney sections, whereas affinity-purified trout antisera were used in all sections except the kidney. However, other than nonspecific staining in readily identifiable mucous and goblet cells, immunocytochemistry results between affinity-purified trout antisera and unpurified zebrafish antisera were qualitatively similar. Although goblet cell staining was virtually eliminated in sections incubated with affinity-purified antiserum, to rule out mucous as an epitope for the affinity-purified serum, we examined two tissues known to have mucous cells but that were unlikely to have NaPi (skin and stomach). Preimmune serum was also not affinity purified since immunocytochemistry using this serum showed no staining, even at high concentrations.
Paraffin sections (8 µm) were rehydrated and placed in a humidified
chamber at room temperature. Sections were incubated with blocking solution
(BS, 5% goat serum, 0.1% Tween 20 in PBS, pH 7.4) for 45 min and then
sequentially blocked with avidin and biotin each for 10 min (Vector
Laboratories, Burlingame, CA). Sections were incubated with primary antibody
(affinity-purified-TrIIb2 1:100, zebrafish antisera 1:1,000 in BS) for 2 h and
with secondary antibody (biotinylated goat anti-rabbit IgG diluted to 5
µg/ml in BS; Vector Laboratories) for 1 h. Sections were washed two times
with PBS and incubated for 10 min with 0.05% sodium azide and four drops of
Vector H2O2 stock in 5 ml PBS to block endogenous
peroxidase activity. Sections were then incubated with an
avidin-biotin-horseradish peroxidase complex (Vector Laboratories) containing
0.2 M -methyl mannoside and 0.4 M NaCl to block lectin binding and
ionic interaction, respectively. Slides were then washed and incubated with
substrate (3,3'-diaminobenzidine containing Ni2+; Vector
Laboratories), counterstained with Nuclear Fast Red, and mounted with Permount
(Fisher Scientific, Suwanee, GA).
Plasma and carcass P analysis. For plasma P analysis, plasma from five or six fish/tank were pooled, whereas, for carcass P analysis, four fish from each tank were individually analyzed following the Fiske and Subbarow method (Sigma Diagnostics, St. Louis, MO). The tank average was used for statistical comparison.
Calculation of in vivo fractional absorption. Absorption of
Pi (NaH2PO4) was determined in both total and
various fractions of dietary Pi levels in low-P and high-P fish at
days 3, 6, 9, and 12 according to the partial digestibility
method (19). Calculations were
as follows: fractional Pi absorption = (
I -
F)/I, where I is NaH2PO4Xg
(X = one incremental unit or fraction), and F is the fecal
excretion of the corresponding fraction. The F was determined based on
the standard digestibility protocol using Cr2O3 as an
indicator. The F of the basal diet represented the background, i.e.,
undigested dietary P + endogenous P. Any increase of F (F) above the
baseline (F of the basal diet) was assumed to be the nonabsorbed portion of
supplemented Pi (i.e., NaH2PO4).
Statistical Analysis
The experiment on dietary regulation was designed as a 2 x 2 factorial arrangement, and the data were analyzed by two-way ANOVA with dietary P and oxygen levels as independent variables. Each tank was considered as the experimental unit. When there was no significant difference (ANOVA P > 0.05), the data were reanalyzed by one-tailed t-test between low-P and high-P groups within each oxygen group. The in vivo fractional absorption data were analyzed for the effect of P status and the dietary P level using the following equation assuming that the fractional P absorption follows a standard sigmoid dose-response curve: Y = bottom plateau + [(top plateau - bottom plateau)/{1 + 10[(logEC50 - X)·HillSlope]}] where X = available dietary P, and Y = fractional P absorption. LogEC50 is the estimated X value when fractional absorption is halfway between the bottom and top plateaus, and the Hill-Slope describes the steepness of the curve. The two lines of low-P vs. high-P fish were compared by two-tailed paired t-test for each sampling day. Statistical calculations were performed with GraphPad Prism, version 3.03 (GraphPad Software, San Diego, CA). Treatment effects were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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NaPi-IIb mRNA. Subcloned trout intestinal and renal NaPi-IIb cDNAs had >99% sequence homology with known trout NaPi-IIb sequences (GenBank accession nos. AF297184 [GenBank] and AF297186 [GenBank] ). Tissue distribution of NaPi-IIb mRNA was determined by Northern analysis (Fig. 1) and by semiquantitative RT-PCR (data not shown). NaPi-IIb mRNA was found in proximal small intestine (most abundant), distal small intestine, pyloric ceca, and kidney but not in heart, skin, skeletal muscle, stomach, gills, scales, blood, or liver. Renal NaPi-IIb mRNA appears to be slightly larger in size than intestinal NaPi-II mRNA.
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NaPi-II protein. In the kidney of fish fed a low-P diet, NaPi-IIb expression was intense along the brush-border margin of cells lining the first segment of the proximal tubule (PI) and was faint or absent in the other regions of the nephron (Fig. 2, A-C). All three antisera produced similar staining patterns in the tubular kidney, suggesting overlapping affinities for the transporter, but the zebrafish NaPi-IIb2 antibody showed substantially better definition and intensity. Blood cells also stained with NaPi-IIb2 (Fig. 2C). Sections incubated with preimmune serum were completely devoid of NaPi-IIb2 signal (Fig. 2D). The distribution of NaPi-IIb2 within cells and along the nephron was independent of dietary P and of aqueous oxygen concentrations (data not shown). Moreover, the intensity of staining did not vary with dietary P or with aqueous oxygen concentrations.
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The distribution of NaPi-IIb within cells, among cells in the same villi, and between proximal and intestinal regions was independent of dietary P and of aqueous oxygen concentrations (data not shown). The intensity of staining also did not apparently vary with dietary P and with aqueous oxygen concentrations.
Because Pi homeostasis is tightly associated with that of calcium, and because fish absorb calcium via gill epithelium, we also examined gill tissue for the presence and distribution of NaPi-IIb. Sections showed strong staining along the apical margin of the ionocytes or mitochondria-rich cells in the gill epithelium (Fig. 3F). All sections incubated with preimmune serum were completely blank for all tissues (Fig. 3, B, D, and G).
Trout skin, scale, muscle, and stomach were also examined for NaPi-IIb protein expression by immunocytochemistry (only skin shown, Fig. 3H). Staining in these tissues was entirely absent when probed with affinity-purified TrIIb2 serum. Hence, with the exception of disconcordant results in the gill, NaPi protein expression determined by immunocytochemistry in various tissues paralleled exactly mRNA expression determined by Northern blots and RT-PCR.
PiUS mRNA. Subcloned trout intestinal PiUS cDNA (GenBank accession no. AY210434 [GenBank] ) showed 68% sequence homology with rabbit and rat intestinal PiUS and 68% with human inositol hexaphosphate kinase 2 (IP6K2). The base sequence did not significantly match any other sequences from any species. The translated protein sequence had 69-70% identity with rabbit and rat PiUS and human IP6K2. The PiUS mRNA had similar distribution to that of NaPi-IIb mRNA, but PiUS mRNA was also found in the heart, skin, muscle, stomach, gill, blood, and liver where NaPi-IIb mRNA was not detectable (Fig. 1).
SGLT1. The subcloned trout intestinal SGLT1 cDNA (GenBank accession no. AY210435 [GenBank] ) had 70-72% sequence homology with human, equine, and bovine SGLT1. The translated protein sequence had 74-76% identity with rat, human, mouse, bovine, and chicken SGLT1. The homology to other SGLT families was also high but significantly lower than for SGLT1. SGLT1 mRNA was abundant in pyloric ceca and the proximal small intestine, much less abundant in distal intestine and kidney, barely detectable in the stomach, and not detectable in the other tissues examined (Fig. 1).
Regulation of Renal and Intestinal NaPi-II, PiUS, and SGLT1 Expression by Dietary P and Ambient Oxygen Concentrations
Fish P status indicates that the low-P diet (0.46% available P) did not induce hypophosphatemia even after 7 wk of feeding, and the carcass P levels only slightly (P = 0.05) differed between low-P and high-P fish (Table 2). In addition, the growth rate did not differ significantly between the low-P and high-P fish. These results indicate that fish fed the low-P diet were only modestly, but not clinically, deficient in dietary P intake. Hematocrit values were not affected by dietary P but were markedly affected by ambient oxygen levels.
NaPi-IIb mRNA abundance was significantly higher in the low-P than high-P fish in both kidney (P = 0.04) and intestine (P = 0.03; Fig. 4). However, the effect of diet was observed only when ambient oxygen concentrations were high. In the low-oxygen group, where fish growth was decreased significantly, NaPi-II mRNA abundance in both intestine and kidney did not differ significantly between low-P and high-P fish. The mRNA abundance of renal PiUS was slightly but significantly higher (P = 0.04) in low-P than high-P fish in the high-oxygen group, whereas that of intestinal PiUS followed a similar pattern but was not significant (P = 0.08). There was no such difference in the low-oxygen group. Intestinal SGLT1 mRNA was similar among all treatments (P > 0.3). Northern analysis of renal SGLT1 was performed but not shown since its mRNA abundance was lower than the reasonable quantification limit.
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In Vivo Fractional Phosphate Absorption
Fractional Pi absorption, which represents the absorption efficiency of NaH2PO4 at various dietary P levels, was close to 100% when the dietary level (intake) was near the requirement (0.6%; Fig. 5 and Table 3). Fish previously fed high P before the switch to the test diets had significantly lower fractional Pi absorption at 3 days after the switch than fish previously fed low P (P = 0.003). However, the diet history of the fish had no effect on fractional Pi absorption 6, 9, or 12 days after the switch in diet. The absorption efficiency gradually decreased at higher (>0.85%) dietary available P levels in all sampling days in both low-P and high-P fish. However, the total absorption, which represents the overall absorption (often called digestibility), remained mostly above 90% even at high dietary P intakes (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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NaPi-II mRNA was found in the intestine and kidney as expected, but it was also detected in pyloric ceca, where the abundance was relatively low. Pyloric ceca are fingerlike diverticula that appear immediately posterior to the pyloric sphincter of the stomach along the first one-third to one-half of the proximal intestine. On average, trout have 56 pyloric ceca, which account for roughly 70% of the surface area of the entire gut (4). NaPi-IIb protein was also detected in the pyloric ceca. Given the presence of NaPi-IIb protein in the ceca and the tremendous surface area, this is likely to be an important site of Pi absorption in trout. Little is known, however, about the roles of this organ on dietary nutrient absorption. Our recent study showed that trout pyloric ceca had much higher luminal Pi concentration than other regions of the intestine and that Pi absorption in pyloric ceca was largely a passive process at postprandial luminal Pi concentrations (36).
The trout intestine can be divided into two morphologically and histologically distinct regions of roughly equal lengths. The proximal intestine is thin and lighter colored and has typical villi projecting in the lumen, whereas the distal intestine is thick and darker colored with mucosal folds. Localization of NaPi-IIb protein in the intestine was similar to that found in flounder and zebrafish (13, 20). Interestingly, expression appeared to diminish toward the distal end of the intestine. This is consistent with Pi uptake measurements in trout (2) that showed significantly lower Pi uptake from the distal intestine than from the proximal and with the present study that shows much higher abundance of NaPi-II mRNA in the proximal than distal region of the intestine (Fig. 1).
Rainbow trout have a glomerular nephron typical of freshwater fish, and the nephron consists of a Bowman's capsule, glomerulus, short neck segment, two morphologically distinct sections of proximal tubule, an intermediate segment, a distal tubule, and a collecting duct terminating in the mesonephric duct (1, 40). As shown in Fig. 2, PI consists of tall columnar cells with basal nuclei and a prominent apical brush border. PII has even taller columnar cells with central nuclei, a dense apical brush border, and a smaller lumen that often appears occluded by the brush border. The distal tubule consists of shorter columnar cells with basal nuclei, no brush border, a small lumen (smaller than PI), and a smaller overall diameter (1, 40).
In the flounder kidney, NaPi-IIb protein has been localized to the basolateral membrane of PII cells (9, 20) as well as the apical membrane of the collecting tubule (9). In the freshwater zebrafish, NaPi-IIb is found in the apical membrane of PII and the collecting tubule (13). In trout, another freshwater species, NaPi-IIb is also found at the apical pole but only in the proximal tubule PI. Hence, the trout NaPi-IIb is also likely to be involved in reabsorbing and recycling Pi secreted by the typically active glomerulus of freshwater fish, following the model presented by Graham et al. (13). However, the trout NaPi-IIb distribution along the nephron is different from that in zebrafish but similar to that in mammals fed normal dietary P where NaPi-IIa is located only in the early proximal region of the kidney. After the NaPi-II evolutionary tree (37), it is interesting to note that trout renal NaPi-IIb is much more closely related to the mammalian renal NaPi-IIa located in the PI region than to the zebrafish renal NaPi-IIb located in the PII region and may reflect an intermediate shift from distal (more primitive) to proximal (mammalian) Pi reabsorption (13). Although both live in freshwater habitats, differences in NaPi-IIb distribution along the nephron may be because of the fact that the zebrafish (order: Cypriniformes) and trout (order: Salmoniformes) are distantly related and diverged early in teleostean evolution.
The intensity of NaPi-IIb staining in trout kidney is less than that in the trout intestine. This finding is also supported by Northern blotting and RT-PCR results, suggesting that expression of NaPi-IIb, relative to other brush-border proteins, may be lower in the trout kidney.
PiUS mRNA was found in all tissues where NaPi-IIb mRNA was located. However, PiUS mRNA was also found in the gill, heart, blood, and other tissues where NaPi-IIb mRNA was absent. It is not clear why PiUS is present in those tissues where NaPi-IIb mRNA is not detectable. In those tissues, there could be other Pi transporters that coordinate their function with PiUS. Indeed, immunostaining of branchial ionocytes and blood cells with TrIIb2 strongly supports this possibility.
We examined gill tissue for NaPi because we had previously observed that P concentrations in inflow water (to fish tanks) were often higher than that in the outflow water when fish were fed low-P diets and because fish are known to absorb minerals, such as calcium, from water via gill epithelia (10). Functional studies measuring gill 32P uptake in vivo (29, 30, 41) did not show active uptake of 32P; however, these studies were conducted on fish with normal P status. It is possible that an inward-directed P transport exists in trout but is only active in fish with low-P status. Conversely, it is also possible that the transport of P is outward directed in fish with high-P status to assist the kidney in elimination of excess P. In the current study, NaPi-II staining was concentrated in the apical margin of ionocytes. Ionocytes are found in the gill epithelium and are involved with calcium uptake as well as ion and osmoregulation (for review, see Refs. 22 and 42). Gill ionocytes were identified based on their bulbous appearance and distribution along the gill filament of rainbow trout. There were also nonbulbous cells at the base of the gill filament that stained strongly. The latter are also ionocytes because salmonids have two types, one bulbous along the arterial blood supply of the gill filament and the other (not bulbous) along the venous blood supply at the base of the filaments (27, 40). The possibility of a Pi transport mechanism in these cells is consistent with its many functions and is also intriguing since Pi transport has not been well studied in the gill.
Regulation of Intestinal and Renal NaPi-II, PiUS, and SGLT1 Expression by Dietary P and Aqueous Oxygen Levels
Intestinal regulation. Moderate dietary P restriction for 6-8 wk causes a modest increase in abundance of NaPi-IIb mRNA and protein, as well as in rates of P uptake by juvenile goat jejunum (16). This observation is similar to our findings of diet-related changes in mRNA abundance (Fig. 4) and P uptake (2). In contrast, dietary P restriction had no effect on NaPi-IIb mRNA abundance in adult rat and mouse small intestine. In rats, PiUS mRNA abundance and Pi uptake increased even though NaPi-II and -III mRNA levels did not change (18). In mice, dietary P restriction or calcitriol [1,25-(OH)2 vitamin D] injection increased the abundance of NaPi-IIb protein and rates of Pi uptake in the brush-border membranes, independent of changes in mRNA abundance (15).
It was initially unclear to us why in rats and mice severe dietary P
restriction accompanied with hypophosphatemia did not increase intestinal
NaPi-IIb mRNA, whereas in trout, even when they were apparently not
clinically P deficient, NaPi-IIb mRNA expression increased. It
turns out that intestinal NaPi-II mRNA expression may be highly age
dependent. Calcitriol treatment increased intestinal brush-border membrane
vesicle Pi absorption by 2.5-fold in suckling rats and by 2.1-fold
in adult rats, whereas the same treatment increased NaPi-IIb mRNA
abundance by 2-fold in 14-day-old rats but had no effect on mRNA expression in
adults (39). The studies
reporting an absence of diet effects on intestinal NaPi-II mRNA
abundance were conducted using adults, whereas those reporting the presence of
diet effects were conducted using neonates or juveniles. For example, trout
used in this study (body wt 188 g) were juvenile and very small compared with
full-grown size (3 kg). The apparent discrepancy of intestinal mRNA
expression in dietary P restriction therefore might be dependent on the
developmental stage of the animal or on unidentified growth-related
mechanisms.
Unlike NaPi-II, the mRNA of trout PiUS did not increase significantly in response to reduced dietary P intake. However, in rat small intestine, Katai et al. (18) reported approximately twofold increases of both PiUS mRNA and maximal velocity (Vmax; Pi uptake) after 7 days of dietary P restriction (0.02% dietary P). The apparent disagreement in the PiUS response to dietary P restriction could be attributed to the degree of P deficiency. In our present study, dietary P was only moderately, but not severely, restricted; thus, the low-P fish had only slightly lower body P content than high-P fish. The plasma P level showed that fish fed a low-P diet were not hypophosphatemic. In most mammalian experiments, animals were fed nearly P-free rations and developed pronounced hypophosphatemia before studies on the expression of NaPi-II, PiUS, and other related genes. Thus direct comparison of our data with mammalian studies may be inappropriate.
Renal regulation. In rat kidney, changes of apical NaPi-IIa protein abundance and Vmax (within a few hours) precede the change of mRNA level (5-7 days), and the acute response is the result of the translocation of NaPi-IIa protein from the intracellular compartment to the apical membrane independent of NaPi-II transcription (21, 28, 32). In these studies, the renal NaPi-IIa mRNA indeed responded to dietary P levels but only in the chronic phase (5-7 days), and the chronic high-P rat still had one-half the level of NaPi-IIa mRNA compared with the chronic low-P rat. Coloso et al. (6) found that rainbow trout fed for 30 days with a 0.6% P diet had significantly higher renal NaPi-IIb mRNA abundance than those fed a 1% P diet. Our present results also showed upregulation of renal NaPi-IIb mRNA in response to dietary P restriction, which is in good agreement with mammalian "chronic" data and our previous data.
Dietary regulation of renal PiUS mRNA expression has not been studied in any species. Although trout renal PiUS mRNA was found to be upregulated in dietary P restriction, the difference was small compared with that of NaPi-IIb mRNA. Thus its functional role in renal P handling could be minor or indirect.
In rats and mice, severe dietary P restriction causes a drastic upregulation of NaPi-IIa protein expression in the early proximal tubule and also the appearance of NaPi-IIa protein in the middle and late proximal tubules where it was not previously visible (32, 38). This sort of change would be more difficult to see in the fish kidney because it lacks the three-dimensional organization that characterizes the mammalian kidney (3). However, we examined sections from areas throughout the kidney and identified the tubules that showed NaPi-II expression and those lacking expression, and we found no observable change with low dietary P.
Upregulation of NaPi-II and PiUS mRNA occurred only in fish reared in high-oxygen water and not in fish reared in waters with suboptimal oxygen concentrations. Because low oxygen resulted in a significant growth depression and because dietary P is required mainly for the growth of the skeletal system, the absence of upregulation in low-P fish could be attributed to the growth-related shift of dietary P requirement and the consequent relative sufficiency of dietary P intake for fish grown in low ambient oxygen concentrations. Nonetheless, a direct effect of hypoxia on NaPi expression has not been excluded.
In Vivo Fractional Phosphate Absorption
Dietary P restriction increases the efficiency of intestinal Pi absorption and renal Pi reabsorption (8, 24). Such an adaptive response has been shown in many mammals and in trout (2) by functional Pi uptake assay using either brush-border membrane vesicles or intact excised tissues. These in vitro assays are, however, determined under artificial conditions, and there still remains a question whether or not intact live fish are regulating Pi absorption as well. There have been few studies on the dietary regulation of Pi uptake in vivo.
In carp, intestinal Pi absorption in vivo increased linearly
from 0.35% up to 3.14% dietary P (dietary requirement is 0.6%), and the
absorption efficiency was constant and did not decrease over this range
(25). This demonstrated that
intestinal Pi absorption was not regulated in carp. In humans, the
Institute of Medicine (17)
expressed a similar view, as it wrote, "fractional phosphorus absorption
is virtually constant across a broad range of intakes." In the present
study, however, the in vivo absorption efficiency of
NaH2PO4 by trout is clearly regulated by diet, for two
reasons. First, in low-P fish, fractional absorption remained close to 100% up
to the known dietary requirement of 0.6%, but then decreased gradually to
70-90% at higher dietary P intakes, depending on the duration after the switch
in diet. In high-P fish, absorption efficiency decreased markedly from 90-100%
to 70-85% when dietary P increased from 0.6 to 1.2%. Second, at day 3
after the switch in diet, the maximum fractional absorption in vivo was 98.9%
for low-P fish but 93.7% for high-P fish. Hence, diet history modulates acute
regulation of intestinal Pi absorption.
At all dietary levels, fractional absorption in vivo continued to take
place with considerably high efficiency (70-100%). This observation may be
partly explained by the potentially significant passive Pi
transport in pyloric ceca
(36). Rodehutscord et al.
(33) reported a much lower
fractional absorption of Na2HPO4 (maximum 73%) in trout,
which peaked well below the dietary requirement and decreased thereafter. The
authors therefore suggested that the dietary P level, when testing dietary P
availability, must be 2.5 g/kg dry diet or lower. This is less than one-half
of the known minimum P requirement for fish. Conversely, our data showed the
highest fractional Pi absorption (100%) at around the
requirement level and the fractional absorption only gradually decreased at
higher dietary P levels, which kept overall Pi absorption nearly
constant across a broad range of dietary P intakes.
Perspectives
Currently, very little is known in fish regarding the molecular and physiological mechanisms of dietary P regulation. We have identified and localized NaPi-II, PiUS, and SGLT1 in selected tissues of rainbow trout and have shown that renal and intestinal NaPi-II and PiUS, but not SGLT1, are upregulated in response to moderate, chronic dietary P restriction. Adaptive upregulation of NaPi-II and PiUS expression paralleled that of in vivo fractional Pi absorption at low dietary Pi intakes. However, the severity of dietary P restriction, the time course of adaptive responses, the developmental stage of the fish, and the interactions among these factors on NaPi-II and PiUS expression are largely unknown. Future studies should focus on these mechanisms and on the involvement of other genes related to P transport.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, P-deficient fish (fish fed low-P diet until day
0); 
, P-excess fish (fish fed high-P diet until day 0).
Each point represents 1 tank containing 14 fish. See