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【摘要】 Growth hormone (GH) is an important factor in the developmental adaptation to enhance P i reabsorption; however, the nephron sites and mechanisms by which GH regulates renal P i uptake remain unclear and are the focus of the present study. Micropuncture experiments were performed after acute thyroparathyroidectomy in the presence and absence of parathyroid hormone (PTH) in adult (14- to 17-wk old), juvenile (4-wk old), and GH-suppressed juvenile male rats. While the phosphaturic effect of PTH was blunted in the juvenile rat compared with the adult, suppression of GH in the juvenile restored fractional P i excretion to adult levels. In the presence or absence of PTH, GH suppression in the juvenile rat caused a significant increase in the fractional P i delivery to the late proximal convoluted (PCT) and early distal tubule, so that delivery was not different from that in adults. These data were confirmed by P i uptake studies into brush-border membrane (BBM) vesicles. Immunofluorescence studies indicate increased BBM type IIa NaP i cotransporter (NaPi-2) expression in the juvenile compared with adult rat, and GH suppression reduced NaPi-2 expression to levels observed in the adult. GH replacement in the [ N -acetyl-Tyr 1 - D -Arg 2 ]-GRF-(1-29)-NH 2 -treated juveniles restored high NaPi-2 expression and P i uptake. Together, these novel results demonstrate that the presence of GH in the juvenile animal is crucial for the early developmental upregulation of BBM NaPi-2 and, most importantly, describe the enhanced P i reabsorption along the PCT and proximal straight nephron segments in the juvenile rat.
【关键词】 development sodiumphosphate cotransporters parathyroid hormone antagonist to growth hormonereleasing factor brushborder membrane vesicles
DURING STATES OF HIGH P i demand, such as growth, the kidneys play a central role in maintaining a positive phosphate (P i ) balance. This occurs through enhanced tubular P i reabsorption ( 20, 21, 29 ) and a blunted response to the phosphaturic effect of parathyroid hormone (PTH) ( 8, 21, 36 ). In the adult animal, renal P i uptake has been shown to depend mainly on type IIa NaP i cotransporters (NaPi-2) ( 12, 24 - 26 ), which are regulated by PTH ( 23 ), dietary P i content ( 23, 25, 31 ), vitamin D ( 26, 33 ), and thyroid hormone ( 1 ), factors that are traditionally known to alter P i reabsorption. However, whether regulation of type IIa NaP i transporters is an important mechanism in the juvenile animal is unknown. Recently, we reported that enhanced renal P i reabsorption in the proximal convoluted and proximal straight nephron segments was responsible for reclaiming the bulk of filtered P i in the juvenile rat and also accounted for the attenuated response to the phosphaturic effects of PTH ( 37 ). In addition, this enhanced proximal tubular P i uptake in the juvenile rat was associated with a significant increase in type IIa NaP i protein expression in the proximal tubular apical brush-border membrane (BBM) ( 37 ). However, the factor(s) responsible for the increases in type IIa NaP i protein expression and P i reabsorption in the juvenile rat remains unclear.
Growth hormone (GH) plays a central role during the growth process and has been shown to be a factor that increases the renal uptake of P i. When GH is elevated, or administered on a chronic basis to adult humans or adult animals, there is a reduction in urinary P i excretion and elevation in plasma P i levels ( 3, 9 - 11, 16 ). Conversely, removal of GH in the adult rat (through hypophysectomy) causes a significant decline in the maximum capacity to reabsorb P i (TmP i ) by the whole kidney and results in increased phosphaturia ( 4 ). However, this effect could not be attributed solely to GH because all pituitary hormones were removed. Furthermore, Hammerman et al. ( 16 ) reported that the effects of pharmacological doses of GH result in a selective stimulation of proximal tubular BBM NaP i transport systems.
Mulroney et al. ( 27, 28 ), using a peptidic antagonist to GH-releasing factor, [ N -acetyl-Tyr 1 - D -Arg 2 ]-GRF-(1-29)-NH 2 (GRF-AN), to suppress the pulsatile release of GH from the anterior pituitary, determined the physiological role of GH in P i homeostasis. Administration of GRF-AN to juvenile animals for 2 days led to doubling of the urinary excretion of P i and attenuation of somatic body growth ( 19 ). These effects were attributed to a decrease in the TmP i ( 19, 27 ) and highlighted an important interrelationship among GH, growth, and the renal reabsorption of P i. Interestingly, short-term GRF-AN treatment of adult rats had no effect on lowering the TmP i and increasing urinary P i excretion ( 19, 27 ). In addition, our laboratory has demonstrated that GRF-AN treatment for 48 h is associated with a 30% reduction in the V max of P i transport in proximal tubular BBM vesicles prepared from weanling rats, implicating GH in proximal tubule P i uptake. However, the nephron sites of GH action and the effect of GH on type IIa NaP i transporter expression in the proximal convoluted tubule (PCT) and proximal straight segments in the juvenile rat are unknown.
The present micropuncture and renal cortical BBM Na gradient-dependent P i (NaP i ) uptake studies were performed to determine the nephron sites of action of GH on renal tubular P i reabsorption seen in juvenile rats. In addition, using Western blot analysis and immunofluorescence microscopy, we assessed the role of GH in regulating the expression of proximal tubular BBM type IIa NaPi transporters in the juvenile rat.
MATERIALS AND METHODS
Animal models. To explore the role of GH on the nephron sites responsible for the increased renal phosphate reabsorption seen during growth, micropuncture studies were performed in adult (14- to 17-wk old) and juvenile (4-wk old) male Wistar rats. The rats were fed a normal-phosphate diet (0.7% P i ) and allowed access to diet and water ad libitum. In juvenile rats, suppression of the pulsatile release of GH was achieved through intravenous administration of the GH-releasing hormone antagonist GRF-AN (Bachem, Torrance, CA) at a dose of 100 µg/kg twice daily. Previous studies in this laboratory have demonstrated this dosage to be effective in completely blocking pulsatile GH release ( 19, 27, 28 ). Briefly, juvenile rats were anesthetized with Nembutal (0.1 ml/100 g ip), and a Silastic catheter (inner diameter, 0.020 in.; outer diameter, 0.037 in.; Dow Corning, Medfield, MA) filled with 500 U/ml heparinized saline was placed into the left jugular vein. Patency of the catheter was maintained with 200 µl of 500 U/ml heparinized saline daily. The animals were housed in separate cages and injected twice daily at 0900 and 1300 for 2 days with saline or GRF-AN. Body weight was measured throughout the experimental protocol, and the reduced growth rate was used as an indicator of the suppression of pulsatile GH release.
The acquisition of the data for the three experimental groups was performed in parallel. The data for the adult and juvenile rats have been previously published ( 37 ). This manuscript includes parts of the previously published data as controls to contrast the effects of GH suppression and replacement on the regulation of renal tubular reabsorption of P i in the juvenile rat.
In vivo micropuncture studies. On the day of the micropuncture experiments, the animals 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. The animals were acutely thyroparathyroidectomized (TPTX) using heat cautery to remove the influence of endogenous circulating PTH, and a tracheostomy was performed to allow the animals to breathe spontaneously. TPTX was considered successful when the basal urinary phosphate excretion was <1%. Polyethylene tubing was inserted into the left carotid artery (PE-50) for blood pressure measurements (Digimed BP analyzer) and arterial plasma sampling, the right jugular vein (PE-50) for infusions of inulin, and into the bladder (PE-90) for urine collections. The left kidney was prepared by making a flank incision at the left subcostal margin and dissecting the kidney free from the surrounding fat tissue without disturbing the adrenal glands. Next, the kidney was placed in a Lucite cup and fixed with cotton to prevent any movement with each breath. Warmed isotonic saline was dripped on the kidney (to prevent drying), and the animals were infused with a 2.5% inulin solution at 3% body wt (BW)/h and allowed to recover for 2 h to reach a steady state. Multiple tubular fluid samples were collected, in the absence of PTH, from the last accessible site of the proximal convoluted tubule and the earliest accessible region of the distal convoluted tubule. The micropipettes were made with a sharpened tip diameter of 5-8 µm and contained light mineral oil dyed with Sudan black. Lissamine green (5%) was injected intravenously (0.1 ml) to facilitate the identification of distal convoluted segments. After proximal and distal collections, 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 BW), followed by a maintenance infusion (15 µg·100 g BW -1 ·h -1 ) as previously reported ( 20, 28 ). While intravenous infusion of PTH caused a rapid fall in mean arterial pressure (MAP), tubular collections were made after MAP returned to control levels. Urine collections were made every 30 min throughout the experiment, and a blood sample was taken at the midpoint of each clearance.
BBM isolation. Intact and GRF-AN-treated juvenile rats were anesthetized via an injection of pentothal sodium (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 PMSF, 16 HEPES, and 10 Tris, pH 7.5. BBM from both regions were isolated from the homogenate by Mg 2+ precipitation and differential centrifugation as described previously ( 24, 26 ). The resulting BBM pellet was resuspended in a buffer of (in mM) 300 mannitol, 16 HEPES, and 10 Tris, pH 7.5, and aliquoted for NaP i transport measurement and Western blotting.
BBM phosphate transport activity measurement. Phosphate transport activity measurements were performed in freshly isolated SC-BBM and JMC-BBM vesicles by radiotracer uptake followed by rapid Millipore filtration. To measure Na gradient-dependent 32 P i uptake (NaP i cotransport), 10 µl of BBM preloaded in an intravesicular buffer comprising (in mM) 300 mannitol, 16 HEPES, and 10 Tris, pH 7.5, were vortexed at 25°C with 40 µl of an extravesicular transport buffer consisting of (in mM) 150 NaCl, 16 HEPES, and 10 Tris, as well as 100 µM K 2 HPO 4, pH 7.5. The final concentration of the buffer plus vesicles was (in mM) 120 NaCl, 80 P i, 12.8 HEPES, and 8 Tris, as well as 80 µM K 2 HPO 4, pH 7.5. All BBM vesicles were handled in the same manner. Uptake after 10 s (representing the 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 picomoles 32 P i per 10 seconds per milligram BBM protein.
SDS-PAGE and Western blot analysis. Samples of SC-BBM and JMC-BBM 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). Ten micrograms of BBM protein/lane were separated 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 type IIa NaP i protein ( 12 ) at a dilution of 1:4,000. 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).
Immunofluorescence microscopy. To assess the role of GH in the regulation of renal type IIa NaP i transporter protein, immunofluorescence microscopy was performed for adult, juvenile, and GRF-AN-treated juvenile male Wistar rats fed a normal-phosphate diet. In a separate group of chronically GH-suppressed juvenile rats, pulses of rat GH were exogenously administered at 1000, 1400, and 1800 daily to mimic three GH pulses. The animals were anesthetized using thiopental (pentothal sodium, 100 mg/kg BW 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 fixed in vivo by perfusion with a fixative buffer in a retrograde fashion into the renal arteries. The fixative buffer consisted of 0.1% glutaraldehyde, 3% paraformaldehyde, 2% Na cacodylate, 3% sucrose, 10% pentastarch in 0.9% NaCl, 0.05% MgCl 2, and 0.05% picric acid in ddH 2 O (pH = 7.4). After 5 min, the fixative was washed out by perfusing a 2% Na cacodylate and 3% sucrose solution dissolved in ddH 2 O (pH = 7.4, 300 mosmol/kgH 2 O). The kidneys were removed, sliced, and frozen in liquid propane cooled with liquid nitrogen. Five-micrometer sections were cut, containing both cortical and juxtamedullary areas, using a cryomicrotome (-20°C), mounted on chromalum-gelatin-coated glass slides, and incubated overnight at 4°C with a rabbit IgG NaP i primary antibody ( 12 ) diluted 1:500 in PBS containing 3% milk powder, 0.3% Triton X-100, and 0.01% sodium azide. The following day, the kidney sections were brought to room temperature, washed four times in PBS, and incubated in the dark for 1 h at 24°C with a secondary antibody (anti-rabbit IgG conjugated to FITC; Dakopatts, Glostrup, Denmark) diluted 1:400 in PBS-milk powder-Triton X-100 (see above). 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 a fading agent. The sections were examined using a Laser Scanning Confocal Microscope (Zeiss LSM 510, Jana, Germany) equipped for epifluorescence.
Analysis. Inulin concentrations were measured in plasma and urine using the anthrone method ( 14 ) and in the tubular fluid samples using the dimedone (5,5-dimethyl-1-3-cyclohexanedione) method ( 13, 35 ). Phosphate concentrations in the plasma and urine were determined by the phosphomolybdate method of Chen ( 6 ). P i in the tubular fluid samples was measured in a flow-through microcolorimeter ( 34 ) using a modified method of Chen ( 6 ). Briefly, 20-50 nl of tubular fluid are placed in a 5-cm piece of PE-60 tubing with 3 µl of a 10% ascorbic acid-10% H 2 SO 4 -10% ammonium molybdate solution. Both ends of the PE tubing are flame sealed, and the sample is vigorously mixed for several minutes and then finally heated in a water bath for 2 h at 37°C to allow for the color reaction to take place. The sample is removed, injected into an injection port on the flow-through microcolorimeter, and run through the machine at a speed of 13.3 µl/min. Inside the machine is a glass cuvette with a light source on one side and a photodiode receptor and 640-nm-wavelength filter on the other. As the sample passes through the glass cuvette, deflection of the amount of light hitting the photoreceptor occurs, and this is seen as a change in voltage. The concentration of tubular fluid phosphate is assessed against a standard curve.
The glomerular filtration rate (GFR) was equated with the clearance of inulin, and the fractional delivery of P i [(TF/P) P i ]/[(TF/P) inulin] at the two nephron sites was assessed under the different experimental conditions.
Statistical analysis. Statistical comparisons between groups were made using one-way of analysis of variance with Student-Newman-Keuls analysis. Results are reported as means ± SE with significance designated at P < 0. 05.
RESULTS
BBMV NaP i transport studies. NaP i transport activity was significantly higher in BBM prepared from SC and outer JMC of juvenile compared with adult rats. After 2-day GH suppression in the juvenile rat, there was a significant reduction in BBM NaP i uptake at both the SC and JMC to levels observed in the adults ( Fig. 1 ).
Fig. 1. Brush-border membrane vesicles (BBMV) NaP i uptake in superficial cortex (SC-BBM) and outer juxtamedullary cortex (JMC-BBM) in parathyroid gland intact adult, juvenile, and growth hormone (GH)-suppressed juvenile rats treated with [ N -acetyl-Tyr 1 - D -Arg 2 ]-GRF-(1-29)-NH 2 (Juvenile+GRF-AN). While the intact juvenile had a significantly greater amount of NaP i uptake into BBMV prepared from SC and JMC compared with adults, GH suppression in the juvenile rat significantly decreased NaP i uptake at both the SC-BBM and JMC-BBM to levels below, or similar to, that in adults.
In vivo experiments in the presence of exogenous PTH. Table 1 provides the experimental parameters in the presence of PTH. MAP was significantly higher in the adult rats compared with the intact- and GH-suppressed juvenile animals and was maintained throughout the study. Plasma P i concentrations were significantly higher in juvenile animals compared with adult animals, and GRF-AN treatment reduced plasma P i below levels observed in the adults ( Table 1 ). As expected, GFR was significantly greater in the adult rats compared with the juvenile rats. While the intact juvenile rats had a significantly lower fractional P i excretion compared with adult rats, fractional P i excretion was significantly elevated to levels observed in the adult rats when GH was suppressed in the juvenile animals ( Table 1 ).
Table 1. Parameters of renal function in GH-suppressed juvenile rats in the presence of PTH
Table 2 depicts the results of micropuncture experiments from both the late proximal and early distal tubule (eDT). While there were no differences in the single-nephron GFR (SNGFR) in the juvenile groups, it was significantly lower compared with that in adult animals. However, the tubular fluid-to-plasma inulin ratio values from both the late PCT and eDT were consistent between the groups, indicating a similar fractional reabsorption of filtered water up to those puncture sites. Suppression of GH led to a significant increase in the fractional delivery of P i to the late PCT compared with juvenile controls ( Table 2 ). This indicates a role for the normal pulsatile circulating GH in the enhanced reabsorption of PCT P i uptake in the juvenile rat. Moreover, while there was a significant decline in the delivery of P i to the eDT in the juvenile control rat, GH suppression in the juvenile rats blocked this effect. Interestingly, the delivery of P i to the eDT in the GRF-AN-treated juvenile rats was similar to that seen in the adult animals. This finding strongly supports a role for GH in the regulation of P i reabsorption along the proximal straight tubule, which contributes to the attenuation of the phosphaturic effect of PTH in the juvenile animal.
Table 2. Micropuncture data obtained from the late proximal convoluted tubule and early distal tubule in GH-suppressed juvenile rats in the presence of PTH
In vivo experiments in the absence of endogenous PTH. To assess the intrinsic P i transport in the GH-suppressed juvenile rat, micropuncture experiments were performed after acute TPTX. Table 3 provides values of various functional parameters in the absence of PTH. MAP was significantly higher in the adult rats compared with intact juveniles but was not significantly different vs. the GRF-AN-treated juvenile animals. Although the plasma P i concentration was significantly higher in the juvenile rats, there was no difference between the adult and GH-suppressed juvenile animals. As expected, after TPTX fractional P i excretion fell to 1% and was not significantly different between the groups.
Table 3. Parameters of renal function in GH-suppressed juvenile rats in the absence of PTH
Table 4 contains the results of micropuncture experiments from both the late PCT and eDT. Although adult rats had a significantly higher SNGFR compared with the juvenile groups, the tubular fluid-to-plasma inulin ratio values from both the PCT and eDT were consistent between the groups, indicating similar fractional water delivery. The significantly lower fractional delivery of P i to the late PCT of juvenile controls compared with adult animals was completely inhibited by GH suppression in the juvenile rats ( Table 4 ). This provides further evidence that pulsatile GH secretion modulates P i reabsorption in the PCT. In addition, while there was a significant decline in the delivery of P i to the eDT in the juvenile rats compared with adults, there was no change in the delivery of P i between the PCT and eDT of GH-deprived juvenile rats. Again, this finding supports the role of GH in increasing P i reabsorption in the proximal straight tubules of the juvenile rat.
Table 4. Micropuncture data obtained from the late proximal convoluted and early distal convoluted tubule in GH-suppressed juvenile rats in the absence of PTH
Immunofluorescence microscopy for NaP i expression in parathyroid-intact rats. Immunofluorescence microscopy was used to assess the regulation of type IIa NaP i transporter protein by GH in juvenile rats. Under normal physiological conditions, the expression of proximal tubular BBM NaP i protein was greater in the juvenile rats compared with the adult animals ( Fig. 2, A-NPD and J-NPD, respectively). Suppression of the pulsatile release of GH in the juvenile rats reduced proximal tubular BBM NaP i protein expression to levels seen in the adult ( Fig. 2, J-NPD+GRF-AN), a finding consistent with micropuncture studies in the GH-suppressed juvenile rats ( Table 2 ). To confirm the actions of GH on NaP i protein expression, exogenous administration of GH in the GRF-AN-treated juvenile rats increased the expression of BBM NaP i cotransporter protein in the proximal tubule to levels seen in the juvenile controls ( Fig. 2, J-NPD+GRF-AN+GH).
Fig. 2. Immunofluorescence microscopy of the type IIa NaP i cotransporter in parathyroid gland intact adult, juvenile, and GH-suppressed juvenile rats. Treatment of juvenile rats with GRF-AN (J-NPD+GRF-AN) reduced the expression of BBM NaPi protein to levels observed in the adults (A-NPD). Exogenous GH pulses to GH-suppressed juvenile rats (J-NPD+GRF-AN+GH) increased the expression of NaPi-2 transporters to levels seen in the juvenile controls. A-NPD, adult; J-NPD: juvenile; J-NPD+GRF-AN: juvenile+GRF-antagonist; J-NPD+GRF-AN+GH: juvenile+GRF-antagonist+growth hormone.
Western blot analysis of BBM NaP i protein abundance. BBM NaP i protein levels were significantly elevated in the juvenile rats at both the superficial (SC-BBM) and outer juxtamedullary (JMC-BBM) cortexes compared with adults. However, treatment of juvenile rats with GRF-AN for 2 days caused a dramatic, significant decrease in the amount of BBM NaP i transporter protein in both SC-BBM and JMC-BBM compared with juvenile controls ( Fig. 3, A and B ). The Western blotting data are in agreement with the immunofluorescence microscopic findings, and NaP i protein abundance correlates with BBM NaP i transport activity and micropuncture data with tubular reabsorption of P i.
Fig. 3. Western blot analysis of BBM type IIa NaPi transporters in parathyroid gland intact adult, juvenile, and GH-suppressed juvenile rats. BBM NaPi protein levels were significantly reduced in the GH-suppressed (GRF-AN) juvenile rats in SC-BBM ( A ) and JMC-BBM ( B ) to levels below that observed in the adult.
DISCUSSION
The present study demonstrates that pulsatile GH release is a key factor in the enhanced proximal tubular phosphate reabsorption observed in the juvenile rat. The micropuncture studies indicate for the first time that GH is responsible, independently of PTH, for the enhanced P i uptake in both the PCT and straight tubules of juvenile rats on a normal-P i diet. In addition, circulating GH contributes significantly to the blunted phosphaturic response to PTH in the juvenile rat. The mechanism for the enhanced NaP i cotransport activity appears to be through the action of GH on the expression of proximal tubular BBM type IIa NaP i transporter protein in the juvenile rat, because suppression of the pulsatile release of GH in the juvenile rat significantly decreased BBM NaP i protein expression. The present findings indicate that GH plays a central role in the renal adaptation to reabsorb P i seen during growth, by enhancing PCT and proximal straight tubule P i reabsorption, significantly extending previous findings by Mulroney et al. ( 27, 28 ).
Rapidly growing neonatal and juvenile animals have enhanced proximal tubular P i reabsorption, and it is postulated that the proximal straight tubule may contribute to the reclamation of filtered P i ( 22, 27, 28 ). The present study confirms that P i uptake in both the PCT and the proximal straight tubule is enhanced in the juvenile rat, and moreover, that the significant uptake in these segments is stimulated by circulating GH. This is in contrast to that observed in the adult animal, where these sites are only upregulated when P i is conserved, such as with TPTX, dietary P i deprivation, and respiratory alkalosis. Thus GH appears to selectively stimulate P i reabsorption in these segments in the young. While it is clear that this effect of GH on P i reabsorption is crucial for the proper growth and development of the young animal ( 19, 27, 28 ), the mechanism enhancing the sensitivity of the kidney to GH is unknown. GH receptor mRNA has been localized to the proximal straight tubule ( 7 ), but developmental differences in GH receptor expression have not been reported. Thus it is unclear whether an upregulation of GH receptors is responsible for the enhanced sensitivity of the juvenile kidney to the effects of GH.
The age-related differences in P i reabsorption within the proximal convoluted and proximal straight nephron segments are also consistent with previous micropuncture and BBM vesicle NaP i cotransport activity studies in our laboratory ( 37 ). In addition, the findings in the GH-suppressed juvenile rat confirm previous data showing that proximal tubular BBMV prepared from GRF-AN-treated juvenile rats had a significant reduction in V max of Na-dependent P i transport compared with juvenile controls. Na-proline uptake in these studies was unaffected by GRF-AN administration, highlighting the apparent specificity to NaP i cotransporters. Because both GH and IGF-I receptor mRNA have been localized on the apical surface of proximal tubular cells, this provides evidence for a direct and/or indirect action of the GH/IGF-I axis on P i uptake in the proximal segments ( 7, 15, 17, 32 ).
The fact that chronic suppression of pulsatile GH release in the juvenile rats significantly decreases P i reabsorption in the proximal tubule does not address whether GH acts directly or indirectly on the tubules. It is clear that both GH and IGF-I can enhance P i reabsorption, and it may be that GH acts both directly and indirectly through IGF-I to modulate renal P i reabsorption in the juvenile rat. In rabbit proximal convoluted tubular segments, Quigley et al. ( 24 ) found that IGF-I, but not GH, stimulated P i uptake. Furthermore, administration of IGF-I to the media of opposum kidney cells ( 2 ) or proximal tubular BBMV prepared from IGF-I-treated hypophysectomized rats ( 5 ) showed that this was associated with an increase in V max for Na-dependent P i transport. We have also reported that both GH and IGF-I were able to prevent the rapid decrease in TmP i observed in GRF-AN-treated juvenile rats. The notion that suppression of GH causes a rapid (within 3 h) reduction in P i transport suggests a direct action of GH on NaPi transporters, because an indirect route through reductions in renal IGF-I would probably take longer. Also, GH receptor mRNA has been localized to the proximal straight tubule ( 7 ), which may again point to a direct effect of GH on NaPi protein expression. The evidence from micropuncture, BBMV NaPi transport activity studies, and NaPi protein abundance clearly links circulating GH with the enhanced renal P i reabsorption in the growing animal and indicates that NaPi may be a target for GH. However, the intracellular mechanism by which GH/IGF-I stimulates NaPi in proximal tubular cells remains unknown.
In summary, phosphate is crucial to the proper development of the rapidly growing juvenile rat. The enhanced renal P i reabsorption seen during this state of high-P i conservation occurs predominately in the proximal convoluted and proximal straight tubule. Our findings indicate that pulsatile GH release plays a key role in the enhanced P i reabsorption at these segments through modulation of BBM type IIa NaPi transporter expression.
Perspectives. It is clear that GH is a key regulator of P i homeostasis in the juvenile animal by facilitating the avid reabsorption of P i. This is crucial for the rapidly developing animal, because limiting P i supply (via dietary deprivation) or transporter activity severely attenuates growth. Defining the sites of action of GH on the renal tubule is an important step in understanding the regulation of NaP i transporters and allows speculation as to the potential for GH to regulate NaP i in adults. Indeed, our findings that P i transporters and TmP i are dramatically reduced in the senescent animal ( 20, 24 ), coincidentally with decreases in circulating GH, suggest that one mechanism for the "anti-aging" effect of GH may be through increasing P i transporters in the kidney and perhaps other tissues. The current findings provide strong support for future work on the effects of GH on P i transport.
GRANTS
These studies were supported by National Science Foundation Grant IBN-95-11677, National Institute on Aging Grant NIAID-AG-18634-01, an American Heart Association Established Investigator Award (to S. E. Mulroney), and the Veterans Affairs Medical Research Service (to M. Levi).
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作者单位：1 Department of Physiology and Biophysics, Georgetown University School of Medicine, Washington, District of Columbia 20057; and 2 Division of Renal Diseases and Hypertension, Departments of Medicine, Physiology, and Biophysics, University of Colorado Health Sciences Center, Denver, Colorado 80262