Central control of renal sodium-phosphate (NaPi-2) transporters

【摘要】  Regulation of phosphate (P i ) reabsorption occurs through the up- and downregulation of the renal type-II sodium P i cotransporters (NaPi-2). Recently, renal NaPi2-type expression has been identified in areas of the brain. The present study determined whether brain NaPi-2 is regulated by dietary P i and whether the behavioral and renal adaptations to low-dietary P i are controlled centrally. NaPi-2-like expression in the third ventricle (3V) and amygdala of juvenile Wistar rats was regulated by dietary P i, as in the kidneys. When cerebrospinal fluid (CSF) P i concentration was elevated by 3V injections of Pi in rats fed low-P i diet (LPD), the behavioral and renal adaptations to LPD were abolished. Most importantly, NaPi-2 expression was markedly reduced not only in the brain, but also renal proximal tubules, despite the low plasma P i milieu. This was confirmed by the significant reduction in the transport maximum for P i (from 8.1 ± 0.2 in LPD + veh 3V to 1.7 ± 0.1 µmol P i /ml glomerular filtration rate in LPD + 3V P i, P < 0.001). These findings indicate that NaPi-2-like transporters in the brain are regulated by both dietary P i and CSF P i concentrations, and most significantly, that the central P i milieu can regulate renal NaPi-2 expression. We hypothesize that central 3V NaPi-2 transporters may act as P i sensors and help regulate both brain and whole body P i homeostasis.

【关键词】  juvenile rats behavior P i sensors

MAINTENANCE OF INORGANIC PHOSPHATE (P i ) homeostasis is critical to the proper growth and well-being of young and adult animals, because it is necessary for bone and tissue development and a myriad of metabolic functions requiring high-energy phosphates. The primary regulation of phosphate (P i ) homeostasis occurs at the kidney, through the insertion and removal of type II sodium-P i cotransporters from the membrane ( 6, 14, 21 ). These transporters (designated NaPi-2 in the rat) are regulated by factors that control P i homeostasis, such as parathyroid hormone (PTH) ( 7, 9, 14 ) dietary P i content ( 14, 16, 20 ), growth hormone ( 22 ), thyroid hormone ( 2 ), glucorticoids ( 8 ), metabolic acidosis ( 3 ), and age ( 18, 22 ). Interestingly, these renal-type transporters have been identified in rat brain ( 4, 5, 15 ), but their purpose and whether they respond to factors that traditionally regulate P i are unknown.

In response to dietary P i deprivation, renal NaPi-2 transporter expression increases ( 16, 20 ), significantly elevating the renal reabsorption of P i ( 10, 19 ). We recently reported that in addition to the renal adaptation, animals rapidly develop P i ingestive behavior and will seek out a source of P i when the dietary source is low ( 19 ). We hypothesize that this appetitive behavior is controlled centrally, through a reduction in cerebrospinal fluid (CSF) P i. This may regulate a central NaPi transporter expression and transduce signals to initiate the appetitive behavior and renal adaptations. Thus our objective was to determine whether central NaPi transporters are regulated by changes in dietary P i and whether the behavioral and renal adaptations to low-dietary P i are controlled centrally.

METHODS

Effect of dietary P i on NaPi-2 in the brain. To determine whether central NaPi-2 transporters respond to changes in dietary P i levels, juvenile (4 wk of age) male Wistar rats were fed diets of low (0.06% P i, n = 7)-, normal (0.7% P i, n = 4)-, or high (1.8% P i, n = 4)-P i content, as previously reported ( 10, 19 ). Food and water intake were monitored to ensure the animals were adequately fed, and after 2 days on the respective diets, the animals were anesthetized and perfused with a 4% paraformaldehyde solution followed by sodium nitrite as previously described ( 7, 9, 16, 18, 22 ). Brains and kidneys were flash-frozen until analysis. Brain tissues were sectioned at the level of the third ventricle (3V), and around the amygdala, and immunofluorescence microscopy was performed using a laser-scanning confocal microscope (Zeiss LSM 410) ( 7, 9, 16, 18, 22 ).

Effects of central P i injections on behavioral and renal adaptations to P i deprivation. For the integrated behavioral and renal studies, juvenile animals had stainless steel cannulas stereotaxically implanted into the 3V of the brain. This was confirmed by the presence of CSF in the cannula. Animals were allowed to recover for 4 days, and after their growth rates were consistent, they were fed normal- or low-P i diets, as previously stated. Food and water (distilled) intake were monitored daily to ensure the animals had not fasted. After 2 days, animals fed a low-P i diet (LPD) were randomly separated into two groups and received 2 µl of either vehicle (dH 2 O) or P i (20 nmol, dibasic potassium P i ) daily at 1100. Concomitantly, all animals were given free access to PiH 2 O (0.3 M dibasic potassium P i ) in addition to distilled water, as previously reported ( 19 ). PiH 2 O intake was monitored over the following 2 days. After 2 days of 3V injections, animals from each group were used for either acute renal clearance studies to determine the maximum capacity for P i reabsorption (TmP i ) or were perfused with fixative to assess NaPi-2 expression by immunofluorescence in brain and kidney tissues. Separate groups of animals were injected with P i or veh 3V and perfused with fixative at 15 min, 30 min, and 1 h post-3V injections to assess a potential time course for changes in NaPi-2 expression.

TmP i studies were performed as previously reported ( 10, 11, 13, 19 ). Briefly, animals were anesthetized with Inactin (Promonta) and underwent thyroparathyroidectomy (to remove the influence of endogenous PTH). Catheters were placed in the jugular vein for infusions of inulin and P i, the carotid artery for blood pressure monitoring and blood sampling, and the bladder for urine sampling. Blood and urine samples were obtained during a steady-state control clearance, and sequentially, as increasing concentrations of P i (3-9 µmol/min) were infused to elevate the filtered load of P i and facilitate determination of the transport maximum. Macro- and microphosphate analysis was performed as previously reported ( 10, 23 ). To collect CSF samples, injectors were placed in the guide cannula, and CSF was drawn into PE-10 tubing. The tubing was sealed at both ends by flame and stored at 4°C until microanalysis. Analysis was performed using a flow-through microspectrophotometer. CSF samples (100 nl) or phosphate standards (50 nl) were transferred to separate tubing containing 2 µl of reagent [10% ascorbic acid, 10% (8 M) H 2 SO 4, 10% (2.5%) ammonium molybdate in distilled water]. The samples were then sealed, mixed, and incubated in a 37°C water bath for 90 min. After incubation, the samples were injected into the spectrophotometer port, and the absorbance was read as a change in voltage. All samples were run in duplicate in two separate assays, and the phosphate concentration was determined against a known standard curve. Plasma phosphate concentration was measured by the phosphomolybdate method previously described ( 10, 20, 23 ). The TmP i was calculated as the average of the maximum reabsorbed P i normalized per milliliter of glomerular filtration rate (GFR) (RPi/GFR) from each animal. Statistical analysis between groups was performed using ANOVA, with Student-Neuman-Keuls post hoc tests. Differences between two groups were determined using unpaired Student's t -tests, whereas differences within groups were determined using paired Student's t -tests. Significance was designated as P < 0.05.

NaPi-2 immunofluorescence. NaPi-2 protein was detected by immunofluorescence techniques, as previously reported ( 7, 9, 16, 18, 22 ). Briefly, 5-µm cryosections of fixed frozen tissues were incubated overnight with a 1:500 dilution of rabbit anti-rat polypeptide antibody generated against the COOH-terminal peptide of rat NaPi-2 ( 2, 24 ). The staining we report is specific staining for NaPi-2 because there is complete protection in the presence of immune serum. This step is routinely used in our laboratory and was confirmed in a blind study by Dr. K. Adams. However, at this point, we refer to brain NaPi-2 staining as NaPi-2-like. The following day, sections were brought to room temperature, washed four times in PBS, and incubated in the dark for 1 h at 25°C with a 1:400 dilution of secondary antibody (anti-rabbit IgG/FITC, Dakopatts, Glostrup, Denmark). Slides were rinsed four times, mounted, and examined using a laser-scanning confocal microscope (Zeiss LSM 410).

RESULTS

Effect of dietary P i on NaPi-2 in the brain. Figure 1 A, middle, illustrates that there is discrete NaPi-2 staining in the ependymal cells surrounding the 3V of the brain in rats fed normal-P i diet (NPD). This staining was specific, as it was entirely blocked in the presence of NaPi-2 COOH-terminal peptide used to generate the antibody. Furthermore, as previously observed in the kidney, in rats fed LPD there was an upregulation of NaPi-2 expression in these cells ( Fig. 1, left ), whereas in rats fed high-P i diet (HPD), there was a downregulation of NaPi-2 expression ( right ). These findings reflected the differences in plasma P i levels (LPD 1.6 ± 0.04; NPD 3.3 ± 0.02; and HPD 3.8 ± 0.04 mM P i ). Interestingly, although there was some punctate staining around this area, only the cells directly lining the 3V expressed the vast majority of NaPi-2. NaPi-2 expression was also localized and regulated in the amygdala (data not shown). Both observations are in accordance with the original localization observation by Hisano et al. ( 4, 5 ). Furthermore, preincubation with immune serum abolished NaPi-2 staining in the 3V ependymal cells in animals fed NPD ( Fig. 1 B ).

Fig. 1. A : type II Na-P i cotransporter (NaPi-2)-like immunofluorescence (IF) around the 3rd ventricle (3V) of the brain. NaPi transporters (bright staining) were shown to be located in cells around the 3V of the brain and were upregulated in animals fed a low-P i diet ( n = 7) and downregulated in animals fed a high-P i diet ( n = 4), compared with normal ( n = 4) expression. Animals were fed diets for 2 days. NaPi-2 IF was performed as previously reported ( 7, 18 ). B : NaPi-2-like IF around the 3V of the brain in the presence and absence of preimmune serum. Preincubation with immune serum abolished the NaPi-2 staining around the 3V in animals fed a normal-P i diet (NPD).

Effects of central P i injections on behavioral and renal adaptations to P i deprivation. To further explore the role of the brain in P i homeostasis, the next objective was to determine whether increasing CSF P i content in the face of low systemic plasma P i levels would alter central NaPi-2 expression and the predicted P i appetitive behavior in rats fed LPD.

The P i levels in brain CSF were significantly greater in the LPD animals receiving 3V P i injections compared with those receiving 3V saline ( Table 1 ). This increase in CSF P i occurred without altering the low plasma P i. Figure 2 A indicates that the rats fed LPD + 3V vehicle had a significantly greater ingestion of the PiH 2 O, compared with animals fed NPD, illustrating the rapid P i appetitive behavior, consistent with our previous report ( 19 ). Most important, however, was the lack of P i appetite in the animals fed LPD + 3V P i. The physiological growth response in these groups of animals is illustrated in Fig. 2 B. Although both groups of animals exhibited the hallmark reduction in body weight gain when ingesting LPD without 3V injections, the animals fed LPD + 3V saline displayed an accelerated growth rate on days 3 and 4, commensurate with the ingestion of significant amounts of PiH 2 O ( Fig. 2 A ). In sharp contrast, the LPD + 3V P i rats did not exhibit the P i appetite ( Fig. 2 A ) and continued to lose weight over days 3 and 4 compared with days 1 and 2 ( Fig. 2 B ). Renal functional studies indicated that the renal TmP i was dramatically reduced in the LPD + 3V P i animals compared with that in LPD + 3V vehicle animals ( Fig. 3 ). This also could account for the loss of BW in these animals over days 3 and 4 ( Fig. 2 B ), because they had increased their urinary excretion of P i as a result of the low TmP i. There were no significant differences in GFRs or basal plasma P i levels; however, the basal fractional excretion of P i was significantly greater in animals with 3V P i injections (10 ± 5 vs. 0.1 ± 0.02% in rats with 3V vehicle injections, P < 0.1). Thus increasing CSF P i concentrations reversed the behavioral and functional adaptations to dietary P i restriction.

Table 1. Phosphate milieu in CSF and plasma

Fig. 2. A : phosphate (P i ) water intake in animals fed NPD, low-P i diet (LPD) + vehicle injected 3V, and LPD + P i injected 3V. The behavioral response to seek and ingest Pi was present in the animals fed LPD with vehicle injected 3V ( n = 5) compared with animals fed a NPD ( n = 5). Injection of P i into the 3V of animals fed LPD blocked the P i water ingestion ( n = 9). * P < 0.01 vs. NPD, by ANOVA and Student-Newman-Keuls post hoc tests. B : cumulative change in body weight in animals fed LPD with vehicle or P i injected into the 3V. Ingesting P i H 2 O resulted in an increased growth rate in the animals injected with vehicle 3V ( n = 5), whereas the animals receiving P i 3V continued to lose weight ( n = 9). This confirms the importance of the appetitive behavior in the P i -deprived animals. * P < 0.01 vs. LPD, by unpaired Student's t -tests.

Fig. 3. Renal transport maximum for P i (TmP i ) in animals fed NPD ( n = 5), LPD + veh injections in the 3V ( n = 5), or LPD + P i injections in the 3V ( n = 5). The intrinsic capacity to reabsorb P i in the absence of parathyroid hormone (PTH) is significantly elevated in animals fed LPD compared with NPD controls. However, in the P i -deprived rats with P i injected into the 3V, TmP i is significantly reduced compared even to normal levels. * P < 0.01 vs. NPD, by ANOVA and Student-Neuman-Keuls post hoc tests. # P < 0.01 vs. LPD, by ANOVA and Student-Neuman-Keuls post hoc tests.

NaPi-2 expression following central P i injections. Immunofluorescence microscopy was performed to determine NaPi-2 expression in the brain and kidneys from the animals fed LPD with and without 3V P i injections. Significant reductions in NaPi-2 immunoreactivity were observed after 1-2 days of central P i injections in both brain and kidney, commensurate with the behavioral and functional findings (data not shown). A time course was performed to assess the rapidity of the changes in NaP-2 activity following central P i injections. Figure 4 A is from tissues perfused 1 h post-3V injection, and it illustrates strong NaPi-2-like protein expression localized in the amygdaloidal area of the brain in the animals receiving 3V vehicle ( A, left ), whereas NaPi-2-like expression is almost undetectable in the same region in animals receiving 3V P i ( A, right ). Perhaps one of the most important findings, however, is that P i injections in the brains of animals fed LPD dramatically reduced NaPi-2 expression in the renal proximal tubular cells ( Fig. 4 B, right ) from the normally high expression observed during dietary P i deprivation ( Fig. 4 B, left ). Furthermore, removal of NaPi-2 transporters from membranes in 3V ependymal cells and kidney tubules was seen as soon as 15 min post-3V injection of P i ( Fig. 5 ). This is consistent with the dramatically reduced TmP i in these animals and continuous decline in growth during the 3V P i injections. Again, this occurred despite the continued low plasma P i levels, which should have stimulated renal NaPi-2 expression, and indicates that the brain may override the peripheral stimuli to regulate P i homeostasis.

Fig. 4. A : NaPi-2 IF in the amygdaloid region of the brain. Left : high NaPi-2 fluorescence in animals injected with vehicle into the 3V. Right : injection of P i 3V dramatically reduces NaPi-2 expression 1 h following injection. B : NaPi-2 IF in renal tubules. Left : high NaPi-2 expression in tubular cells of the renal cortex in the animals fed LPD with 3V vehicle injection. In sharp contrast, NaPi-2 expression is reduced to punctate staining in kidneys from animals with P i injected into the 3V 1 h postinjection of P i.

Fig. 5. A : NaPi-2 IF around the 3V of the brain 15 min post-3V injection of vehicle or Pi. Left : high NaPi-2-like fluorescence in animals injected with vehicle into the 3V. Right : injection of P i 3V dramatically reduces NaPi-2 expression only 15 min postinjection. B : NaPi-2 IF in renal tubules. Left : high NaPi-2 expression in tubular cells of the renal cortex in the animals fed LPD with 3V vehicle injection. In sharp contrast, NaPi-2 expression is reduced to punctate staining in kidneys from animals with P i injected into the 3V 15 min postinjection of P i.

DISCUSSION

Previous work in this laboratory focused on the importance of renal NaPi-2 transporters in normal growth, development, and aging. The present findings expand considerably on this work, by implicating central NaPi-2-like transporters in the regulation of renal NaPi-2 expression, and thus renal P i reabsorption.

The presence of the renal-type NaPi-2 transporters in discrete areas of rat brain and the fact that they can be regulated by dietary P i and CSF P i concentrations elevate them to a potential position to control P i homeostasis. We attempted to discern whether these are actually renal NaPi-2 transporters, and our initial findings suggest that they respond specifically to the NaPi-2 antibodies and can be blocked by incubation with preimmune serum. Although this, and the regulation by dietary and CSF P i, supports the idea that they are NaPi-2 transporters, they may represent some other isoform, and this issue will be studied further. Recent work by others describes a new NaPi/vesicular glutamate transporter that is localized to neuron-rich area of the brain. Brain-specific NaPi (DNPI) transporters have been localized in many diencephalic regions, including the amygdala and ependymal cells of the 3V ( 1, 4, 5 ). Functional studies have not been performed using the DNPI antibodies; however, our present work adds the important findings of regulation of brain NaPi-2-like transporters by diet and direct manipulation of CSF P i levels. This, in conjunction with the effects on the kidney NaPi-2 transporters, provides major novel mechanisms for the regulation of P i homeostasis.

One aspect of the potential central sensing and control over P i homeostasis is the induction of the P i appetitive behavior when animals are fed LPD. Our working hypothesis was that low CSF P i levels stimulate the behavioral response, and indeed, the present findings indicate that specific elevation of CSF P i (independent from plasma P i ) abolished the P i appetite ( Fig. 2 A ). This strongly suggests that areas in the brain may stimulate the appetitive behavior in response to the local P i environment. The observation that NaPi expression in the brain is also regulated by CSF P i adds to this concept. Other behaviors, such the sodium appetite ( 17 ), transduce their signals through the amygdaloidal area of the brain, where we also observed regulation of NaPi-2-like expression by CSF P i. Thus it is possible that the NaPi-2-like transporter is acting as a relay system in the transduction of the P i appetite through this area. Furthermore, because the cells around the 3V are in direct contact with CSF, it is likely that they may respond quickly to changes in CSF P i concentrations. The presence of a P i sensor that could control systemic P i transporters has been postulated but has not been demonstrated: we suggest that the circumventricular ependymal cells act as a P i sensor for the rest of the brain, as well as the periphery.

In support of this concept, when P i was added directly to the CSF in P i -deprived rats, NaPi-2-like expression was dramatically reduced in the ventricular ependymal cells and amygdaloid of the brain, as well as in the apical membranes of the renal proximal tubular cells ( Fig. 4 ). This occurred despite the continued low plasma P i environment ( Table 1 ). This strongly suggests that there is central control of renal P i homeostasis and thus may implicate central NaPi-2 transporters as P i sensors that could regulate brain, renal, and overall systemic P i homeostasis. Another possible mechanism could derive from changes in central CSF calcium, initiating the changes in central and peripheral P i transporters. Interestingly, the changes in renal NaPi-2 transporters observed appear to be independent of PTH, as the TmP i studies were performed in TPTX animals. This rules out a role of PTH in the central control of renal P i transport. Considering the present findings, we also hypothesize that significant regulation of renal NaPi-2 would also be observed following 3V P i injections in animals fed NPD.

Of tremendous impact is the notion that renal NaPi-2 transporters and hence renal function can be regulated by central P i concentrations. Our findings show a direct effect of central administration of P i on the reduction in renal NaPi-2, TmP i, and failure to grow, integrating these important mechanisms and linking them to a central pathway. This could have implications for normal growth and development, as well as in aging and disease. We hypothesize that central regulation would also be apparent in animals fed NPD and would contribute to the rapid day-to-day regulation of P i homeostasis, including the adaptations that enhance P i retention in the young animal ( 10 - 12 ). The role of the brain in renal P i regulation does not discount the role of local factors affecting the kidney but adds a potentially important aspect to the overall regulation of P i homeostasis.

Perspectives

The discovery of NaPi-2-like transporters in the brain and their ability to be regulated both by dietary P i and CSF P i content are an important step in understanding the metabolic needs of the brain for P i during growth, adulthood, and aging. Furthermore, the fact that the P i milieu of the brain regulates not only central but renal NaPi-2 expression indicates that there is central control of peripheral P i homeostasis through a NaPi transporter-related mechanism. These concepts provide a novel area for future studies centered on ways to target cellular aging and disease.

ACKNOWLEDGMENTS

The authors thank E. K. Bishop for excellent technical assistance.

GRANTS

This work was supported by National Institutes of Health Grant R03AG18634-01 and an Established Investigator Award 0040012N from the American Heart Association (AHA) to S. E. Mulroney and VA Merit Review, AHA, and Juvenile Diabetes Foundation grants to M. Levi.

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作者单位：1 Department of Physiology and Biophysics, Georgetown University School of Medicine, Washington, D.C. 20057; and 2 Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, Denver, Colorado 80262