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Am J Physiol Regul Integr Comp Physiol 280: R1524-R1533, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 5, R1524-R1533, May 2001

Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat

Craig Woda1, Susan E. Mulroney1, Nabil Halaihel2, Lijun Sun2, Paul V. Wilson2, Moshe Levi2, and Aviad Haramati1

1 Department of Physiology and Biophysics, Georgetown University School of Medicine, Washington, District of Columbia 20007 and 2 Department of Internal Medicine, University of Texas Southwestern Medical Center and Department of Veterans Affairs Medical Center, Dallas, Texas 75216


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To determine the tubular sites and mechanisms involved in enhanced renal phosphate (Pi) reabsorption seen in the juvenile animal, renal micropuncture experiments were performed in acutely thyroparathyroidectomized adult (>14 wk old) and juvenile (4 wk old) male Wistar rats fed either a normal Pi diet (NPD, 0.6% Pi) or low Pi diet (0.07% Pi) for 2 days, in the presence and absence of parathyroid hormone (PTH). Pi reabsorption was greater in proximal convoluted (PCT) and straight tubules (PST) of the juvenile compared with adult rats fed NPD, whether or not PTH was present. These findings were consistent with a greater Pi uptake in brush-border membrane (BBM) vesicles from both superficial (SC) and outer juxtamedullary (JMC) cortices of juvenile animals. Western blot analysis revealed a 2- and 1.8-fold higher amount of NaPi-2 protein in the SC and JMC, respectively, in juvenile rats. Immunofluorescence microscopy also indicated that NaPi-2 protein expression was present in the proximal tubule (PT) BBM to a greater extent in juvenile rats. Dietary Pi restriction in juvenile rats resulted in a significant increase in Pi reabsorption in the PCT and PST segments. NaPi-2 expression in the PT BBM was also increased, as was the expression of intracellular NaPi-2 protein. These studies indicate that Pi reabsorption in both the PCT and PST segments of the renal tubule contributes to the attenuated response to PTH in the normal juvenile animal. In addition, dietary Pi restriction in the juvenile rat upregulates BBM NaPi-2 expression, which is associated with a further increase in proximal tubular Pi reabsorption.

kidney; low dietary phosphate; parathyroid hormone; age; brush-border membrane; inorganic phosphate transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WE HAVE PREVIOUSLY REPORTED that rapidly growing juvenile animals have a significantly greater intrinsic renal capacity to reabsorb Pi compared with adult animals and display a blunted response to the phosphaturic effects of parathyroid hormone (PTH) (8, 17). Furthermore, despite the already high rate of Pi transport, juvenile animals can adapt to dietary Pi restriction by further enhancing the renal capacity to transport Pi (TmPi) (4, 26, 32). This enhanced TmPi facilitates the rapid restoration of Pi homeostasis and catch-up growth when Pi is replenished to the diet (26). However, the renal tubular sites contributing to the normally high rate of Pi transport in the juvenile rat, both during normal and low dietary Pi regimens, are not clear.

In adult animals, renal Pi uptake has been shown to depend on type II sodium-Pi transporters (NaPi-2) (11, 23-25), which are regulated by PTH (22), Pi diet (22, 24, 31), vitamin D (25, 36), and thyroid hormone (1), factors that are traditionally known to alter Pi reabsorption. Traebert et al. (36) reported that NaPi-2 transporters are present when the brush border develops and that expression was greater in 13- than 22-day-old rats. However, to what extent NaPi-2 transporters are an important mechanism contributing to the enhanced Pi reabsorption in the juvenile animal compared with the adult is unknown.

Previous studies have demonstrated that during states of dietary Pi restriction in the adult, the kidneys increase Pi retention to minimize urinary Pi losses (4, 28, 34, 35). In addition, the PTH-stimulated increase in urinary Pi excretion is blunted in Pi-deprived animals (3, 26, 31, 34). Both in vivo and in vitro studies in the adult animal have demonstrated that this adaptive increase in Pi uptake is due to enhanced Pi reabsorption within both the proximal convoluted tubules (PCT) (20, 21, 29, 37, 41) and segments beyond the PCT, most likely the proximal straight tubule (PST) (15, 29, 34, 41). Furthermore, dietary Pi deprivation in the adult animal is also associated with an increase in the maximal velocity (Vmax) of apical brush-border membrane (BBM) sodium-dependent Pi cotransport (NaPi) activity (4, 18, 20, 21, 24, 25). Levi et al. (25) have reported that this chronic adaptive increase in the Vmax of NaPi cotransport activity in response to low Pi diet (LPD) was mediated by enhanced levels of BBM type II (NaPi-2) transporter protein and mRNA.

Therefore, the aims of the present study were to determine the renal tubular sites of Pi transport and the role of NaPi-2 protein in the enhanced reabsorption of Pi in the juvenile rat in the presence and absence of PTH. We also examined the mechanisms associated with the age-related differences in the renal adaptation to alterations in dietary Pi intake.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental Animals

Experimental protocols were approved by the Georgetown University Animal Care and Use Committee. Male juvenile rats were obtained just after weaning at 21 days of age, acclimated in our animal care facility, and used at 28-30 days of age, while adult rats were obtained at 3-4 mo of age (Harlan Laboratories, Indianapolis, IN). All animals were fed a standard rodent chow (Purina Laboratories) and had free access to food and water.

Age-Related Differences in Tubular Pi Reabsorption

Micropuncture studies. To determine age-related differences in Pi transport, juvenile and adult rats were prepared for micropuncture studies. In addition, to evaluate the intrinsic and PTH-associated differences in Pi reabsorption, experiments were performed in the absence and presence of PTH, respectively. On the day of the acute experiment, juvenile and adult animals fed normal Pi diet (NPD; 0.6% Pi) were anesthetized with an intraperitoneal injection of Inactin (80 mg/kg) (Promonta, Hamburg, Germany) and placed on a heated table. Body temperature was maintained at 37 ± 0.5°C with a servo-controlled heat lamp (Yellow Springs Instruments, Yellow Springs, OH) and a rectal probe. Using a heat cautery, the animals were acutely thyroparathyroidectomized (TPTx) to remove the influence of endogenous PTH, and a tracheostomy was performed to allow the animals to breathe spontaneously. Polyethylene catheters were placed in the jugular vein for infusion of inulin, in the carotid artery for blood pressure measurements (Digimed blood pressure analyzer) and arterial blood sampling, and in the bladder for urine collections.

Briefly, the kidney was prepared by making a flank incision at the left subcostal margin and dissecting free the kidney from the surrounding fat tissue without disturbing the adrenal gland. Next, the mobile kidney was placed in a lucite cup and fixed with cotton to prevent any movement with each breath. The kidney was bathed in warmed saline to prevent drying, and the animals were infused with a 2.5% solution of inulin at 3% body wt/h to assess the glomerular filtration rate (GFR) and single nephron GFR (SNGFR). Animals were allowed to recover for 2 h to reach a steady state. Multiple tubular fluid samples were collected from the last accessible site of the proximal convoluted tubule (PCT) and the earliest accessible region of the distal convoluted tubule using micropipettes (5-8 µm diameter bore) containing light mineral oil dyed with Sudan black. Lissamine green (5%) was injected intravenously (0.1 ml) to facilitate the identification of the tubular segments. Delivery of Pi up to the PCT reflects reabsorption along the PCT, and because Pi permeability is very low in the loop of Henle, Pi delivery to the early distal tubule reflects reabsorption in the PST. After proximal and distal collections, several more tubular fluid samples were collected in the presence of PTH. PTH (rat 1---34, Bachem, King of Prussia, PA) was administered as a bolus (45 µg/100 g body wt), followed by a maintenance infusion (15 µg · 100 g body wt-1 · h-1) as previously reported (8, 17). Urine collections were made every 30 min throughout the experiment, and a blood sample was taken at midpoint.

BBM isolation. Adult and juvenile parathyroid-intact rats were anesthetized via an intraperitoneal injection of Pentothal (100 mg/kg ip), and the kidneys were removed for BBM isolation. The superficial cortex (SC) and outer juxtamedullary cortex (JMC) were dissected and homogenized in 15 ml of an isolation buffer consisting of (in mM) 300 mannitol, 5 EGTA, 1 phenylmethylsulfonyl fluoride, 16 HEPES, and 10 Tris, pH 7.5. The BBM from both regions were isolated from the homogenate by Mg2+ precipitation and differential centrifugation as described previously (23-25). The resulting BBM pellets were resuspended in a buffer of (in mM) 300 mannitol, 16 HEPES, and 10 mM Tris, pH 7.5, and aliquoted for Western blotting and Pi uptake studies.

BBM Pi transport in parathyroid-intact juvenile and adult rats. A group of parathyroid-intact adult and juvenile rats were used to determine BBM Pi uptake. Transport measurements were performed in freshly isolated BBM vesicles from both regions by radiotracer uptake followed by rapid Millipore filtration. To measure Na-gradient-dependent 32Pi uptake (NaPi cotransport), 10 µl of BBM preloaded in an intravesicular buffer (in mM) of 300 mannitol, 16 HEPES, and 10 Tris, pH 7.5, was vortexed at 25°C with 40 µl of an extravesicular uptake buffer of 150 mM NaCl, 100 µM K2H32PO4, 16 mM HEPES, and 10 mM Tris, pH 7.5. Uptake after 10 s (representing initial linear rate) was terminated by an ice-cold stop solution. All uptake measurements were performed in triplicate, and uptake was calculated on the basis of specific activity determined in each experiment and expressed as pmol 32Pi · 10 s-1 · mg BBM protein-1.

Age-Related Differences in NaPi-2 Expression

Immunofluorescence microscopy. Juvenile and adult rats fed NPD were anesthetized using thiopental (Pentothal, 100 mg/kg ip) and a catheter (PE-190 in adults and PE-60 in juveniles) was inserted into the abdominal aorta below the renal arteries. The kidneys were perfused with a fixative buffer in a retrograde fashion at 1.38 times hydrostatic pressure (25). The fixative buffer consisted of 3% paraformaldehyde, 2% Na-cacodylate, 3% sucrose, 10% pentastarch in 0.9% NaCl, 0.05% MgCl2, and 0.05% picric acid in distilled deionized H2O(ddH2O; pH 7.4). After 5 min the fixative was washed out by perfusing a 2% Na-cacodylate and 3% sucrose solution dissolved in ddH2O (pH 7.4, 300 mosmol/kgH2O). The kidneys were removed, sliced, and frozen in liquid propane cooled with liquid nitrogen. Sections (5 µm) were cut, containing both cortical and medullary areas, using a cryomicrotome (-20°C), mounted on chromalum-gelatin-coated glass slides, and incubated overnight at 4°C with an NaPi-2 primary antibody (11) diluted 1:500 in PBS containing 3% milk powder, 0.3% Triton X-100, and 0.01% sodium azide. The next day, the kidney sections were brought to room temperature, washed four times in PBS, and incubated in the dark for 1 h at 25°C with a secondary antibody (anti-rabbit IgG conjugated to fluorescein isothiocyanate; Dakopatts, Glostrup, Denmark) diluted 1:400 in PBS-milk powder-Triton. Next, the slides were rinsed four times in PBS and mounted under coverslips using DAKO-glycerol (Dakopatts) plus 2.5% 1,4-diazabicyclo[2.2.2]octane (Dabco, Sigma) as an anti-fading agent. The sections were examined for fluorescence using a laser scanning confocal microscope (Zeiss LSM 410, Germany).

SDS-PAGE and immunoblotting. Western blotting was performed on samples of cortical and juxtamedullary BBM that were prepared as previously stated. The samples were denatured for 2 min at 95°C in 2% SDS, 10% glycerol, 0.5 mM EDTA, and 95 mM Tris · HCl, pH 6.8 (final concentrations). BBM protein (10 µg) was loaded on 9% polyacrylamide gels and electrotransferred onto nitrocellulose paper. After blockage with 5% nonfat milk powder with 1% Triton X-100 in Tris-buffered saline (20 mM, pH 7.3), Western blots were incubated with antiserum against NaPi-2 (11) at a dilution of 1:4,000. Horseradish peroxidase-linked secondary antibody was used at a dilution of 1:500. Primary antibody binding was visualized using enhanced chemiluminescence (Pierce, Bradford, IL) and the signals were quantitated in a PhosphorImager with chemiluminescence detector and densitometry software (Bio-Rad, Richmond, CA). Western blots were reblotted with beta -actin for normalization of the NaPi-2 protein.

Northern blot analysis. NaPi-2 mRNA in kidneys was determined by Northern blotting as previously reported (24). Briefly, RNA size fractionated with 0.66% formaldehyde and 1% agarose gels (Bio-Rad). After electrophoresis, the gel was vacuum blotted onto GeneScreen Plus nylon membranes (DuPont-NEN) and RNA immobilized by ultraviolet irradiation. After prehybridization, blots were incubated with a [alpha -32P]dCTP cDNA probe to NaPi-2 mRNA. Densitometry was measured by PhosphorImager, and NaPi-2 signal was normalized by 18S RNA.

Effects of Dietary Pi Deprivation on Tubular Pi Transport and NaPi-2 Expression in Juvenile Rats

To determine the effects of dietary Pi restriction on Pi transport and NaPi-2 expression in the young, juvenile rats were fed an LPD (0.07% Pi). Acute micropuncture experiments and immunofluorescence microscopy were performed after 2 days of Pi deprivation. In addition, a separate group of adult and juvenile rats were fed a high Pi diet for 2 days, and comparisons of renal NaPi-2 expression by immunofluorescence were made between animals on low, normal, and high Pi diets.

Inulin and phosphate analysis. Inulin concentrations were measured in the plasma and urine using the anthrone method (13) and in the tubular fluid samples using the dimedone (5,5-dimethyl-1-3-cyclohexanedione) fluorometric method (12, 39). Phosphate concentrations in the plasma and urine were determined by the phosphomolybdate method of Chen et al. (7). Pi in the tubular fluid samples was measured in a flow-through microcolorimeter (38) using a modified method of Chen et al. (7). Briefly, 20-50 nl of tubular fluid were placed in a 2-in. piece of PE-60 tubing with 3 µl of a 10% ascorbic acid-10% H2SO4-10% ammonium molybdate solution. Both ends of the PE tubing were flame sealed and the sample was vigorously mixed for several minutes and then heated in a water bath for 2 h at 37°C to allow for the color reaction to take place. The sample was removed, injected into an injection port on a flow-through microcolorimeter, and run through the machine at a speed of 13.3 µl/min. The microcolorimeter was comprised of a glass cuvette with a light source on one side and a photodiode receptor and 640-nm wavelength filter on the other side. As the sample passed through the glass cuvette, a deflection in the amount of light hitting the photoreceptor occurred and registered as a change in voltage. The concentration of tubular fluid phosphate was assessed against a known standard curve.

GFR was equated with the clearance of inulin, and the fractional delivery of Pi [(TF/P)Pi/(TF/P)inulin] to the various tubular sites was assessed under the different experimental conditions, where TF/P is the tubule-to-fluid concentration ratio.

Statistical Analysis

Statistical comparisons between groups were made using unpaired Student's t-tests, whereas comparisons within groups were made using paired Student's t-tests. Results are reported as means ± SE with significance designated at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intrinsic Age-Related Differences in Tubular Pi Reabsorption

To assess the intrinsic capacity to reabsorb Pi in juvenile and adult rats, acute micropuncture experiments were performed after TPTx. Table 1 indicates that the absence of PTH did not affect basal mean arterial pressure (MAP) in either aged animal, and MAP was maintained throughout the study. Plasma Pi concentrations were significantly greater in the juvenile compared with adult rats and, as expected, the adults had a significantly higher GFR compared with juveniles. After TPTx, the fractional Pi excretion fell below 1% in both groups and was not significantly different between the groups.

                              
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  		Table 1.   Experimental parameters in the presence and absence of PTH in rats fed NPD and LPD

The SNGFR was significantly greater in the adult compared with juvenile animals. The tubular fluid-to-plasma inulin ratio values from both the late PCT and early distal tubule (Table 2) were not different between groups, indicating a similar fractional reabsorption of filtered water up to those puncture sites. The fractional delivery of Pi (FDPi) to both the late PCT and early distal tubule in the juvenile rats was significantly reduced compared with that observed in the adult rats (Fig. 1). This indicates that there is an intrinsic, PTH-independent adaptation to enhance Pi reabsorption in the juvenile animal in both the PCT and PST segments.

                              
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  		Table 2.   Micropuncture data obtained from the late proximal convoluted tubule and early distal tubule in the presence and absence of PTH in rats fed NPD and LPD



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  		Fig. 1.   Fractional delivery of Pi (FDPi) to the late proximal tubule (PT) and early distal tubule (DT) in the absence of parathyroid hormone (PTH). In rats fed normal Pi diet (NPD), thyroparathyroidectomy (TPTx) significantly reduced the delivery of Pi to both the late proximal convoluted tubules (PCT) and DT of juvenile (n = 9) compared with adult (n = 7) animals. *P < 0.05.

Intrinsic Age-Related Differences in NaPi-2 Expression

Immunofluorescence studies indicated that after acute removal of the parathyroid glands, NaPi-2 expression in proximal tubule BBM was greater in juvenile compared with adult rats (Fig. 2, A-TPTx and J-TPTx). The greater expression of BBM NaPi-2 protein in the tubules from the juvenile rat is consistent with the relatively greater amount of tubular Pi reabsorption observed by micropuncture.


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  		Fig. 2.   Immunofluorescence microscopy of the NaPi-2 transporter in adult (A) and juvenile (J) animals in the presence and absence of PTH. Whereas the brush-border membrane (BBM) NaPi-2 expression decreased in both adult and juvenile rats 1 h after the infusion of a pharmacological dose of PTH (TPTx + PTH), the level of BBM NaPi-2 expression was higher in the juvenile rats compared with adults both before and after PTH. (n = 5 in all groups)

Age-Related Differences in Tubular Pi Reabsorption: Effects of PTH

Adult and juvenile experimental parameters in the presence of PTH are shown in Table 1. MAP was significantly higher in the adult (P < 0.05) compared with the juvenile group animals and was maintained throughout the study. As expected from previous reports, plasma Pi levels were higher in the juvenile compared with adults rats and GFR was significantly greater in adult compared with the juvenile animals (P < 0.05). The urinary fractional excretion of Pi was significantly lower in the juvenile animals compared with adults (Table 1), confirming earlier reports of a blunted phosphaturic response to PTH in rapidly growing juvenile rats (8, 40).

Table 2 reports the results of micropuncture experiments of both the late proximal and early distal tubule. The SNGFR was significantly lower in the juvenile compared with the adult animals. The tubular fluid-to-plasma inulin ratio values from both the late proximal convoluted and early distal tubule were consistent between the groups, indicating a similar fractional reabsorption of filtered water up to those puncture sites. The FDPi to the late PCT was significantly reduced in the juvenile compared with the adult animal indicating enhanced Pi reabsorption along the PCT. Moreover, whereas the FDPi from the late PCT to the early distal tubule was not significantly different in adult rats, there was a further reduction in the delivery of Pi to the early distal tubule in the juvenile rats fed NPD. Figure 3 depicts the delivery of Pi to these segments.


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  		Fig. 3.   FDPi to the late PT and early DT in the presence of PTH. The delivery of Pi to the late PT and early DT was significantly reduced in the juvenile (n = 5) rat compared with adult (n = 6) animal when PTH was present. *P < 0.05 vs. adult; # P < 0.05 vs. delivery to PT.

Sodium-dependent Pi uptake into BBM vesicles (BBMV) prepared from both the SC and outer JMC increased 32 and 43%, respectively, in the juvenile compared with adult rats (P < 0.05) (Fig. 4). This finding is consistent with the results of the micropuncture study and helps localize the site to the BBM.


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  		Fig. 4.   BBM Pi uptake in the superficial cortex (SC) and juxtamedullary cortex (JMC) in parathyroid-intact adult and juvenile rats. There was a significant increase in BBM Pi uptake from both the SC and JMC of juvenile (n = 4) compared with adult (n = 4) rats. *P < 0.05.

Age-Related Differences NaPi-2 Expression: Effects of PTH

Immunofluorescence microscopy demonstrated that after a bolus injection of a pharmacological dose of PTH in TPTx rats, BBM NaPi-2 protein was internalized into the cell and away from the BBM to a greater extent in the adult rats compared with juveniles (Fig. 2, A-TPTx+PTH and J-TPTx+PTH). Western analysis in parathyroid-intact rats confirmed these findings (NaPi-2 signal normalized by beta -actin; Fig. 5, NPD). Further localization studies indicated that these age-related differences persisted with 2- and 1.8-fold differences in NaPi-2 protein from SC and JMC BBM, respectively, in the juvenile compared with adult rat in the presence of PTH (P < 0.05; Fig. 6, A and B).


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  		Fig. 5.   Effect of age on renal cortical NaPi-2 protein in parathyroid intact rats fed NPD and low Pi diet (LPD). NaPi-2 protein levels were elevated in the renal cortex of juvenile (n = 4) compared with adult (n = 3) rats, regardless of dietary Pi. Data are normalized by beta -actin.



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  		Fig. 6.   Western blot analysis of NaPi-2 protein levels in the SC and JMC in non-TPTx adult and juvenile rats. There was a significant increase in NaPi-2 protein from both the SC (A) and JMC (B) of juvenile rats (n = 4) compared with adults (n = 4). Representative blots are shown above the graphs.

Effects of Dietary Pi Deprivation on Tubular Pi Transport and NaPi-2 Expression in Juvenile Rats

The intrinsic capacity to reabsorb Pi was examined in juvenile rats after 2 days of dietary Pi deprivation. After acute TPTx, basal MAP (103 ± 4 mmHg) was similar to the TPTx juveniles fed NPD (Table 1) and was maintained throughout the study. Pi restriction significantly lowered plasma Pi levels (2.69 ± 0.14 mM) compared with TPTx juvenile controls (Table 1). GFR was not different between the juvenile groups, and fractional Pi excretion was virtually undetectable.

SNGFR and tubular fluid-to-plasma inulin ratio values in both the late proximal convoluted and early distal tubules are provided in Table 1. The values are not significantly different from those obtained from the TPTx juvenile rats fed NPD (Table 2). Dietary Pi restriction reduced Pi delivery to the late PCT compared with NPD-fed adult animals; however, delivery was not significantly different from juvenile rats fed NPD at either PCT or early distal tubule segments (Fig. 7).


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  		Fig. 7.   FDPi to the late PT and early DT in the absence of PTH. TPTx significantly reduced the delivery of Pi to both the late PCT and early DT of juvenile compared with adult animals. Delivery of Pi was not different between juvenile animals fed NPD (n = 9) or LPD (n = 15). *P < 0.05.

After PTH administration, basal MAP in Pi-deprived juvenile rats was similar to juvenile rats fed NPD and was maintained throughout the study (Table 1). Although plasma Pi levels were lower in the juvenile rats fed LPD compared with juvenile controls, they did not reach statistical significance. GFR was not significantly different between the juvenile groups. A complete inhibition of Pi excretion was observed in the Pi-deprived juvenile rats, despite the presence of PTH.

The micropuncture data from both the late proximal convoluted and early distal tubule of the Pi-restricted juvenile rat is depicted in Table 2. As expected, there was no difference in SNGFR and tubular fluid-to-plasma inulin ratio values from both the late proximal convoluted and early distal tubule in both juvenile groups. However, after 2 days of Pi deprivation, there was a further reduction in the delivery of Pi to both the PCT and early distal tubule compared with juvenile controls (Fig. 8). This indicates that dietary Pi deprivation enhances Pi reabsorption at both the PCT and PST nephron segments compared with juveniles fed NPD and contributes to the observed resistance to the phosphaturic effects of PTH in the juvenile Pi-restricted animal.


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  		Fig. 8.   FDPi to the late PT and early DT in the presence of PTH. PTH (n = 10) significantly reduced the delivery of Pi to both the late PT and early DT of juvenile (n = 5) compared with adult (n = 6) animals. Juvenile rats fed LPD displayed a further reduction in delivery of Pi to both the PT and DT, which was significantly lower than observed in juvenile rats fed NPD. *P < 0.05 vs. adult; #P < 0.05 vs. delivery to PT; @P < 0.05 vs. juvenile fed NPD.

Age-Related Changes in NaPi-2 Expression: Effect of Dietary Pi in Parathyroid-Intact Juvenile and Adult Rats

Immunofluorescence microscopy demonstrated that in intact animals fed an NPD, the juvenile animal has a greater expression of renal apical BBM and intracellular NaPi-2 protein compared with adult rats (Fig. 9, J-NPD and A-NPD). This was confirmed by Western analysis (Fig. 5, LPD). Under dietary Pi restriction, both the adult and juvenile rats have the capacity to increase BBM NaPi-2 protein levels (Fig. 9, A-LPD and J-LPD). This was also confirmed by Western analysis (data not shown). Interestingly, the juvenile rat shows not only a greater amount of BBM NaPi-2 protein expression by immunofluorescence compared with adults, but also the presence of intracellular vesicular NaPi-2 protein, which was not found in the adult animals. This is consistent with the micropuncture data above after 2 days of a LPD in the juvenile rat. Despite the increase in NaPi-2 protein, NaPi-2 mRNA (normalized by 18S RNA) was not significantly increased in response to LPD in either juvenile (155.7 ± 15.5 to 164.3 ± 11.7 densitometry units in LPD) or adult (107 ± 9.5 to 111.7 ± 15.4 densitometry units in LPD) kidney cortices. Of additional interest is the finding that NaPi-2 mRNA is significantly greater in juvenile compared with adult rat cortices under either diet (P < 0.05). Conversely, BBM NaPi-2 transporters in both the juvenile and adult rats were internalized and expression was subsequently reduced after 2 days of high Pi diet (HPD; Fig. 9, A-HPD and J-HPD). However, whereas the adult rat appears to have degraded most of the intracellular NaPi-2 transporters, the NaPi-2 transporters in the juvenile rats are still observed in the cytoplasm of the proximal tubular cells.


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  		Fig. 9.   Immunofluorescence microscopy of the NaPi-2 transporter in adult (A) and juvenile (J) animals as a function of dietary Pi intake. Under an NPD, the juvenile rats (n = 5) displayed a greater expression of BBM NaPi-2 transporters compared with adults (n = 4). Whereas the administration of a LPD for 2 days increased the expression of BBM NaPi-2 transporters in both the adult (n = 4) and juvenile (n = 5), the level of NaPi-2 expression remained greater in the juvenile animal. Conversely, whereas the administration of a high Pi diet (HPD) lowered the expression of BBM NaPi-2 in both adult (n = 4) and juvenile (n = 5) rats, the expression of NaPi-2 transporters remained greater in the juvenile animals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present micropuncture and BBMV uptake studies demonstrate that there is a PTH-independent elevation in phosphate reabsorption in both the proximal convoluted and proximal straight segments of cortical nephrons in juvenile compared with adult rats. Immunofluorescence studies and Western blot analysis indicate that the enhanced Pi reabsorption in the proximal tubular segments of the juvenile rat is due to an increase in the amount of BBM NaPi-2 cotransporters. These data confirm and expand our previous findings (22a), which reported that the Vmax for Pi transport into BBMVs prepared from proximal tubules of juvenile rats was twice that seen in the adult animal. In addition, during states of dietary phosphate restriction, the juvenile animal displays a further increase in the expression of proximal tubular BBM NaPi-2 transporters, enhancing Pi reabsorption in both PCT and PST segments and leading to a marked resistance to the phosphaturic effects of PTH. Of additional interest are the findings that basal NaPi-2 mRNA expression is significantly greater in kidneys from juveniles, compared with adults regardless of diet. Also, gene expression does not significantly increase in response to dietary Pi deprivation, despite the significant elevation in NaPi-2 protein. This indicates that the upregulation of NaPi-2 protein in kidneys from animals fed LPD for 2 days may be due to stabilization of the message or some other posttranscriptional modification, as opposed to direct increases in the message. This could be part of the initial early response to Pi deprivation, because longer periods of Pi restriction do elevate gene expression (24). Whereas feeding a high-Pi diet caused the decrease in BBM NaPi-2 transporters from the proximal tubule of both the adult and juvenile rat, the NaPi-2 appears to be degraded at a slower rate in the juvenile rat. Because these actions occurred in the absence of PTH, the implication is that factors other than PTH that are present in the developing animal contribute to the upregulation of Na-dependent Pi transport.

It has been well documented that there are age-related differences in the intrinsic capacity of the whole kidney to reabsorb phosphate (6, 16, 17, 26, 28). Haramati et al. (17) demonstrated that the maximum capacity to reabsorb Pi (TmPi) was relatively greatest in 3-wk-old rats and progressively declined throughout adulthood. The present micropuncture and BBMV studies demonstrate that the enhanced Pi retention seen in the juvenile animal is due in part to an increase in intrinsic Pi uptake within the proximal tubule. This is consistent with micropuncture studies in the rapidly growing guinea pig (3-14 days old), which indicated that the PCT had a greater fractional reabsorption of Pi compared with the adult (19). Interestingly, in the adult animal, reabsorption of Pi beyond the PCT is observed only during states of Pi conservation, such as thyroparathyroidectomy and dietary Pi restriction (15, 34, 41). Thus nephron sites past the PCT appear to be actively recovering filtered Pi and limiting urinary Pi losses in the juvenile animal under normal conditions. The present findings strongly suggest that an increase in BBM NaPi-2 expression is involved in the mechanism for the enhanced Pi uptake in the juvenile animals. Several factors may contribute to this increase in NaPi-2.

Growth hormone is one factor implicated in the enhanced Pi uptake in the juvenile animal. Acromegaly (30) and chronic administration of growth hormone (GH) to both humans and animals (5, 9, 10, 14) are associated with reduced urinary Pi excretion and elevated plasma Pi levels. In contrast, elimination of the pulsatile release of GH in the juvenile rat decreased whole kidney TmPi to levels observed in adult animals (16, 27). Our laboratory also has demonstrated that GH suppression results in a 30% reduction in the Vmax of BBM Pi transport (22a). Most recently, we have reported that GH suppression in juvenile rats reduces Pi reabsorption in PCT and abolishes Pi reabsorption in the PST segment (40a). The effect of GH was independent of PTH and was associated with a significant reduction in NaPi-2 expression in the superficial and juxtaglomerular proximal tubular preparations. This confirms that the enhanced Pi reabsorption in the juvenile animal is due to the effect of endogenous GH during this age period, which enhances proximal tubular NaPi-2 transporter expression.

Intracellular Pi concentrations may also stimulate NaPi-2 expression and enhanced Pi transport in the young. Barac-Nieto et al. (2) has reported that intracellular Pi concentrations are inversely proportional to Pi uptake and that intracellular Pi is lower in young compared with older animals. Thus the drive to reabsorb Pi may be fueled by the transcellular Pi gradient, which may directly regulate NaPi-2 expression.

The present study also has determined the nephron sites of regulation of Pi reabsorption by LPD and PTH. Despite an intrinsically high capacity to reabsorb Pi in the juvenile rat compared with adults, the kidneys of juvenile rats can adapt and reabsorb even more filtered Pi during dietary Pi restriction (26). Our findings of increased Pi reabsorption in the PCT of Pi-deprived juvenile rats extends the clearance data of Mulroney and Haramati (26) and appears to occur through the further upregulation of BBM NaPi-2 transporters. In addition, the present immunofluorescence data support findings by Levi et al. (24) who reported that proximal tubular BBM NaPi-2 protein expression is increased during Pi restriction in the adult rat. Therefore, the increase in Pi uptake by the proximal tubule of the juvenile rat during dietary Pi restriction seems to be explained by an increase in NaPi-2 transporters. It is also important to note that both the PCT and PST segments were resistant to the phosphaturic effects of PTH in the juvenile animal, and this was maintained during Pi deprivation. Thus an increase in Pi reabsorption in PCT and PST reabsorption contributes to the enhanced renal Pi reabsorption in juvenile animals. Immunofluorescence studies indicate that PTH reduces NaPi-2 expression in juvenile animals (Fig. 2); however, expression is still greater than observed in adult animals, consistent with the micropuncture findings.

In contrast to the upregulation of renal NaPi-2 expression in Pi-deprived animals, an HPD caused a reduction in proximal tubular BBM NaPi-2 protein expression. This is consistent with previous findings in our laboratory, indicating that HPD significantly decreases the transport maximum for Pi by the kidney in both the adult and the juvenile rat (26). Interestingly, whereas in the adult kidney there appears to be degradation of the intracellular NaPi-2 transporters, the transporters remain in intracellular vesicles in the juvenile rat tubules. The reason for this is unknown but may be due to the fact that the juvenile rat has a tremendous demand for Pi and may need to rapidly insert these transporters back into the BBM when needed.

In summary, the present findings indicate that the proximal convoluted and proximal straight segments of the cortical nephrons reabsorb a relatively greater amount of Pi in the rapidly growing juvenile compared with adult rats. The increased Pi uptake at these tubular sites results in the attenuated phosphaturic response to PTH observed in the juvenile animal. Immunofluorescence microscopy indicates that the increased Pi uptake in the proximal tubule of the juvenile rat is associated with an increase in the expression of apical BBM NaPi-2 transporters. Furthermore, the BBM NaPi-2 transporters are dramatically increased during dietary Pi restriction in the juvenile rat, which provides the animal with a complete resistance to the phosphaturic effect of PTH. These important adaptations in the juvenile kidney contribute to the avid renal Pi retention necessary for proper growth and development of the young animal.


    ACKNOWLEDGEMENTS

These studies were supported by the National Sciences Foundation Grant IBN-9516677 and an American Heart Association Established Investigator Award to S. E. Mulroney and Research Funds from Department of Veterans Affairs Merit Review to M. Levi.


    FOOTNOTES

Address for reprint requests and other correspondence: A. Haramati, Dept. of Physiology and Biophysics, Georgetown Univ. School of Medicine, 3900 Reservoir Rd., NW, Washington, DC 20007 (E-mail: haramata{at}georgetown.edu).

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.

Received 10 December 1999; accepted in final form 2 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 280(5):R1524-R1533
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