Reduced expression of renal Na + transporters in rats with PTH-induced hypercalcemia

【摘要】  The purpose of this study was to evaluate whether the natriuresis and polyuria seen in parathyroid hormone (PTH)-induced hypercalcemia are associated with dysregulation of renal Na transporters. Rats were infused with three different doses of human PTH [PTH ( 1 - 34 ); 7.5, 10, and 15 µg·kg -1 ·day -1 sc] or vehicle for 48 h using osmotic minipumps. The rats treated with PTH developed significant hypercalcemia (plasma total calcium levels: 2.71 ± 0.03, 2.77 ± 0.02, and 3.42 ± 0.06 mmol/l, respectively, P < 0.05 compared with corresponding controls). The rats with severe hypercalcemia induced by high-dose PTH developed a decreased glomerular filtration rate (GFR), increased urine output, reduced urinary osmolality, increased urinary Na excretion, and fractional excretion of Na. This was associated with downregulation (calculated as a fraction of control levels) of whole kidney expression of type 2 Na-P i cotransporter (NaPi-2; 16 ± 6%), type 3 Na/H exchanger (NHE3; 42 ± 7%), Na-K-ATPase (55 ± 2%), and bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1; 25 ± 4%). In contrast, an upregulation of the Ca 2+ -sensing receptor (CaR) was observed. Rats treated with moderate-dose PTH exhibited unchanged GFR but decreased urinary concentration. The whole kidney expression of NHE3 (52 ± 8%) and NaPi-2 (26 ± 5%) was persistently decreased, whereas BSC-1 and Na-K-ATPase protein levels were not altered. CaR expression was also increased. Moreover, rats treated with low-dose PTH showed very mild hypercalcemia but unchanged GFR, normal urinary concentration, and unchanged expression of Na transporters and CaR. In conclusion, the reduced expression of major renal Na transporters is likely to play a role in the increased urinary Na excretion and decreased urinary concentration in rats with PTH-induced hypercalcemia. Moreover, the increase in the CaR in the thick ascending limb (TAL) may indicate a potential role of the CaR in inhibiting Na transport in the TAL.

【关键词】  calciumsensing receptor hypercalcemia parathyroid hormone sodium transport urinary concentrating mechanism

THE KIDNEY IS ONE OF THE MAJOR target organs for parathyroid hormone (PTH). The principal physiological effect of PTH is on the regulation of renal tubular electrolyte and water transport ( 24 ). PTH hypersecretion is associated with adverse effects, such as polydipsia and polyuria, and this could be due to the hypercalcemia and/or hypophosphatemia caused by the hormone and to additional effects of elevated PTH level ( 40 ). Primary hyperparathyroidism is a common endocrine disorder causing hypercalcemia ( 8 ), and one major renal effect of hypercalcemia is the decrease in urinary concentrating capacity, which may explain some of the symptoms (such as polyuria) seen in patients and animals with hypercalcemia. This is also supported by our previous studies ( 44, 45 ) demonstrating that vitamin D-induced hypercalcemia in rats is associated with decreased urinary concentration and increased urinary sodium excretion. This is associated with reduced abundance of aqua-porin-2 (AQP2) in the collecting duct and of Na-K-2Cl cotransporter [rat type 1 bumetanide-sensitive cotransporter (BSC-1 or NKCC2)] in the thick ascending limb (TAL), whereas the abundances of the apical Na/H exchanger isoform NHE3 and the basolateral Na-K-ATPase are not reduced ( 44 ). It is therefore important now to clarify whether the reduced expression of selective renal sodium transporters represents a common mechanism mediating the urinary concentrating defect and increased urinary sodium excretion associated with different causes of hypercalcemia, such as elevated PTH-induced hypercalcemia.

PTH has multiple effects on the kidney glomeruli and tubules. Micropuncture studies of rat kidney have demonstrated that PTH reduced the plasma flow rate and glomerular ultrafiltration coefficient ( K f ) in superficial glomeruli ( 39 ). This may potentially reduce the glomerular filtration rate (GFR) in conditions with hyperparathyroidism-induced hypercalcemia. Moreover, previous studies have shown that PTH treatment is associated with increased natriuresis and diuresis ( 38, 49 ), which could be caused by changes in activity, trafficking, or expression of renal sodium transporters. In the proximal tubule, the apical Na/H exchanger (NHE3 isoform) is the main transporter by which hydrogen ions are secreted as well as sodium and bicarbonate are reabsorbed ( 4 ). The apical Na-P i cotransporter (NaPi-2 isoform) is involved in the reabsorption of 80% of the filtered phosphate ( 3, 28 ) and may also be involved in reabsorption of a minor fraction of filtrated sodium in the proximal tubule. There is evidence that PTH treatment is associated with decreased activity ( 6, 16, 28 ), rapid degradation ( 32 ), and redistribution/internalization of NHE3 and NaPi-2 ( 49 ). The Na-K-ATPase drives active transepithelial sodium reabsorption in the basolateral membrane ( 20 ), and in vitro and in vivo studies have demonstrated that PTH treatment decreases or inhibits Na-K-ATPase activity ( 37, 49 ). In the medullary and cortical thick ascending limbs (mTAL and cTAL), sodium reabsorption is accomplished by the apical Na-K-2Cl cotransporter (BSC-1 or NKCC2), NHE3, potassium channel (ROMK), and basolateral Na-K-ATPase ( 12, 14 ). Moreover, calcium reabsorption in this tubule segment is associated with concomitant sodium reabsorption. Recent studies have aimed at establishing the distribution of the G protein-coupled extracellular Ca 2+ -sensing receptor (CaR) along the nephron and collecting duct system ( 34 - 36, 48 ) and have demonstrated the potential role of the CaR as the mediator of the effects of extracellular Ca 2+ on several aspects of renal function ( 15 ). In particular, both CaR mRNA and protein are found in the TAL, indicating that CaR is important in the regulation of sodium and calcium transport in this segment of the kidney tubule. Consistent with this, it has been demonstrated that the basolateral CaR in the TAL inhibits the apical K + channel and thereby indirectly inhibits the Na-K-2Cl cotransporter ( 46, 47 ) and, in addition, chloride reabsorption by a different pathway ( 18 ).

Therefore, the purpose of the present study was to examine whether there are changes in the protein abundance of major renal sodium transporters as well as CaR protein to define the underlying mechanisms responsible for the polyuria and increased renal sodium excretion in kidneys of rats with hypercalcemia induced by PTH infusion.

MATERIALS AND METHODS

Experimental Protocols

Studies were performed in male Munich-Wistar rats, initially weighing 230-260 g. The animals had free access to standard rat chow (Altromin, Lage, Germany) and water throughout the experiment. Control rats and PTH-treated rats were chosen randomly and maintained in metabolic cages during the entire experiment. After 3 days of acclimatization, osmotic minipumps (Alzet 1003D, DURECT, Cupertino, CA) containing PTH were implanted into the subcutaneous tissue in the back area under halothane inhalation anesthesia (Halocarbon Laboratories, River Edge, NJ). The pumps were equilibrated in physiological saline solution for 4 h at 37°C before implantation. After surgery, all rats recovered from anesthesia within 5 min, and they were placed back in the metabolic cages.

Protocol 1: high-dose-PTH treatment. Rats were continuously infused with human PTH ( 1 - 34 ) (Bachem) dissolved in 2% cysteine-HCl solution with 150 mM NaCl, pH 1.5 ( 2 ), at a rate of 15 µg·kg -1 ·day -1 sc for 2 days ( n = 19). Controls were infused with the vehicle alone ( n = 19).

Protocol 2: moderate-dose-PTH treatment. This protocol was identical to protocol 1 except that moderate-dose PTH was administered. Rats were continuously infused with PTH ( 1 - 34 ) at a rate of 10 µg·kg -1 ·day -1 sc for 2 days ( n = 12). Controls were infused with the vehicle alone ( n = 12).

Protocol 3: low-dose-PTH treatment. This protocol was identical to protocol 1 except that low dose of PTH was administered. Rats were continuously infused with PTH ( 1 - 34 ) at a rate of 7.5 µg·kg -1 ·day -1 sc for 2 days ( n = 6). Controls were infused with the vehicle alone ( n = 6).

The rats were maintained in metabolic cages, and daily 24-h urine output and water intake were measured during the entire experimental period. Urinary volume, osmolality, sodium, potassium, calcium, phosphate, and creatinine concentrations were measured. Blood was collected from the abdominal aorta at the time of death for measurement of osmolality and sodium, potassium, calcium, phosphate, and creatinine concentrations.

After 48 h of PTH or vehicle infusion, all rats were killed under halothane inhalation anesthesia. In protocols 1 and 2, kidneys were rapidly removed, left kidneys were dissected into different zones and processed for membrane fractionation and semiquantitative immunobloting, while right kidneys were homogenized and processed for membrane fractionation for semiquantitative immunoblotting. Additional animals ( protocols 1 and 2 ) were used for immunocytochemistry. The kidneys were fixed by retrograde perfusion and processed, as described below. In protocol 2, kidneys were rapidly removed and whole kidneys were processed for semiquantitative immunoblotting.

Primary Antibodies

For semiquantitative immunoblotting and immunohistochemistry, previously characterized monoclonal and polyclonal antibodies were used as follows.

NHE3 (LL546AP). An affinity-purified polyclonal antibody to NHE3 was previously characterized ( 4, 22 ).

NaPi-2 (LL696AP). An affinity-purified polyclonal antibody to NaPi-2 has previously been characterized ( 3 ).

1 -Subunit of Na-K-ATPase. A monoclonal antibody against the 1 -subunit of Na-K-ATPase was a generous gift from Dr. Douglas Fambrough (Johns Hopkins University).

BSC-1 (LL320AP). An affinity-purified polyclonal antibody to the apical Na-K-2Cl cotransporter of the TAL has previously been characterized ( 7, 23, 29 ).

CaR polyclonal antibody. A CaR polyclonal antibody was purchased from Affinity Bioreagents (PA1-934, A. H. Diagnostics, Aarhus, Denmark).

Membrane Fractionation for Immunoblotting

Whole kidneys or dissected renal zones were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) using an ultra-turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany), and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membranes and intracellular vesicles. Gel samples (Laemmli sample buffer containing 2% SDS) were made of this pellet.

Electrophoresis and Immunoblotting

Samples of membrane fractions were run on 9% polyacrylamide minigels (Bio-Rad Mini Protean II) for BSC-1 and CaR or 12% polyacrylamide minigels for NHE3, NaPi-2, and Na-K-ATPase. An identical gel was run in parallel for each gel and subjected to Coomassie blue staining to ensure identical loading. The second gel was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na 2 HPO 4, 20 mM NaH 2 PO 4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with primary antibodies (see above). The labeling was visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies (P447 or P448, DAKO, Glostrup, Denmark) using the enhanced chemiluminescence system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).

Quantitation of Renal Abundance of Sodium Transporters

Enhanced chemiluminescence films with bands within the linear range were scanned using an AGFA scanner (ARCUS II) and Corel Photopaint Software to control the scanner. The labeling density was determined by blots where samples from PTH-treated rats were run on each gel with samples from control rats. The labeling density was corrected by densitometry of Coomassie blue-stained gels.

Preparation of Tissue for Immunocytochemistry

Kidneys from control rats and PTH-treated rats were fixed by retrograde perfusion via the aorta with 3% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). Kidney blocks containing all kidney zones were dehydrated and embedded in paraffin. The paraffin-embedded tissues were cut at 2-µm thickness on a rotary microtome (Leica, Heidelberg, Germany). The immunolabeling was performed as previously described ( 44 ). Double-immunofluorescence labeling was performed with polyclonal antibodies against rat BSC-1 and mouse monoclonal antibodies against the 1 -subunit of Na-K-ATPase. The labeling was visualized with Alexa 488- and Alexa 546-conjugated secondary antibodies (Alexa 488 anti-rabbit and Alexa 546 antimouse, respectively). Light microscopy was carried out by using a DMRE microscope and a TCS-SP2 laser confocal microscope (Leica).

Statistical Analyses

Values are presented in the text as means ± SE. Comparisons between groups were made by unpaired t -test. P values <0.05 were considered significant.

RESULTS

Rats Treated with High-Dose PTH (Protocol 1) Had Decreased GFR but Significant Polyuria and Natriuresis

In protocol 1, rats treated with high-dose PTH (15 µg·kg -1 ·day -1 sc for 2 days) developed significant hypercalcemia (plasma total calcium levels: 3.42 ± 0.06 vs. 2.59 ± 0.02 mmol/l in controls, P < 0.05, Table 1 ), consistent with previous reports ( 2 ). The PTH-treated rats exhibited polyuria and decreased urinary concentration. At day 2 of PTH treatment, urine output was significantly higher (79 ± 6 vs. 37 ± 4 µl·min -1 ·kg -1 in controls, P < 0.05, Table 1 ), accompanied by a significant decrease in urinary osmolality (851 ± 77 vs. 1,887 ± 198 mosmol/kgH 2 O in control rats, P < 0.05, Table 1 ). In parallel, the marked polyuria was associated with increased water intake: 112 ± 9 vs. 79 ± 6 µl·min -1 ·kg -1 in controls ( P < 0.05). Moreover, PTH-treated rats had significantly increased urinary sodium excretion rates (7.2 ± 0.3 vs. 5.5 ± 0.3 µmol·min -1 ·kg -1 in controls P < 0.05, Table 1 ). The fractional excretion of sodium to urine (FE Na ) was also significantly higher in PTH-treated rats (1.2 ± 0.1 vs. 0.5 ± 0.04% in controls, P < 0.05, Table 1 ), indicating that severe hypercalcemia induced by high-dose-PTH treatment is associated with a decrease in the tubular reabsorption of filtered sodium. In addition, PTH-induced hypercalcemia in this protocol was associated with a significant rise in urinary calcium and phosphate excretion ( Table 1 ).

Table 1. Functional data (protocol 1)

The plasma creatinine level in PTH-treated rats was increased (49 ± 5 vs. 27 ± 1 µmol/l in controls, P < 0.05, Table 1 ), and creatinine clearance was decreased ( Table 1 ). There was no indication of volume depletion in PTH-treated rats, as evidenced by high water intake and no differences in plasma osmolality ( Table 1 ). Thus this suggests that GFR was decreased in response to high-dose-PTH treatment, consistent with the previously observed effect of PTH to reduce the renal plasma flow rate and glomerular ultrafiltration coefficient in superficial glomeruli ( 39 ).

Decreased Renal Abundances of BSC-1, Na-K-ATPase, NHE3, and NaPi-2 in Rats Treated with High-Dose PTH (Protocol 1)

In protocol 1, we examined the renal abundance of the Na-K-2Cl cotransporter BSC-1 in PTH-treated rats and control rats using membrane fractions from whole kidney, cortex, and outer stripe of the outer medulla (OSOM; combined, cortex/OSOM) or the inner stripe of the outer medulla (ISOM). Semiquantitative immunoblotting demonstrated that whole kidney protein expression of BSC-1 in PTH-treated rats was significantly reduced to 25 ± 4% (100 ± 8% in controls, P < 0.05, Fig. 1, A and B, Table 2 ). Consistent with this finding, BSC-1 protein expression was significantly decreased in membrane fractions from the cortex/OSOM and ISOM, corresponding to 45 ± 11 (100 ± 10% in controls, P < 0.05, Table 2 ) and 34 ± 6% (100 ± 11% in controls, P < 0.05, Fig. 1, C and D, Table 2 ), respectively.

Fig. 1. Semiquantitative immunoblotting of membrane fractions of whole kidney and kidney inner stripe of the outer medulla (ISOM) of high-dose-PTH-treated rats (PTH) and control rats (Con; protocol 1 ). A and C : immunoblots were reacted with anti-bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) antibodies and recognized a strong, broad band of molecular mass 146-176 kDa, centered at 160 kDa. B and D : densitometric analyses revealed that BSC-1 protein expression in whole kidney as well as in ISOM was significantly decreased in PTH-treated rats compared with Con rats. * P < 0.05.

Table 2. Densitometric analysis of sodium transporter expression (protocol 1)

Next, we investigated the changes in the protein expression of the 1 -subunit of the Na-K-ATPase in membrane fractions from whole kidney or different kidney zones. Semiquantitative immunoblotting demonstrated that whole kidney protein expression of Na-K-ATPase in PTH-treated rats was significantly decreased to 55 ± 2% (100 ± 11% in controls, P < 0.05, Table 2 ). Furthermore, Na-K-ATPase protein expression was significantly decreased to 55 ± 13% (100 ± 7% in controls, P < 0.05, Table 2 ) in cortex/OSOM and 36 ± 6% (100 ± 11% in controls, P < 0.05, Fig. 2, A and B, Table 2 ) in ISOM of PTH-treated rats.

Fig. 2. Semiquantitative immunoblotting of membrane fractions of kidney ISOM of high-dose-PTH-treated rats and control rats ( protocol 1 ). A : immunoblots were reacted with anti-Na-K-ATPase ( 1 -subunit) antibodies and revealed a single 96-kDa band. B : densitometric analyses revealed that Na-K-ATPase expression in the ISOM was significantly reduced in PTH-treated rats compared with Con rats. * P < 0.05.

Double-labeling immunofluorescence microscopy for BSC-1 and Na-K-ATPase was performed. In the cTAL ( Fig. 3, A and C ) and mTAL ( Fig. 3, E and G ) of control rats, the intense immunofluorescence labeling of apical BSC-1 and basolateral Na-K-ATPase was seen, consistent with previous observations ( 7, 20 ). In contrast, TAL cells in PTH-treated rats exhibited clearly reduced labeling of BSC-1 and Na-K-ATPase ( Fig. 3, B, D, F, and H ), consistent with the decreased abundance determined by semiquantitative immunoblotting.

Fig. 3. Immunocytochemical analyses of BSC-1 and Na-K-ATPase in the thick ascending limb (TAL) of control rats ( A, C, E, and G ) and high-dose-PTH-treated rats ( B, D, F, and H; protocol 1 ). Whole kidney sections were incubated with polyclonal anti-BSC-1 and monoclonal anti-Na-K-ATPase ( 1 -subunit). Labeling was visualized with Alexa 488 anti-rabbit (red) and Alexa 546 anti-mouse (green), respectively. In normal controls, abundant apical BSC-1 (red) and basolateral Na-K-ATPase (green) labeling was seen on plasma membrane domains of the TAL cells in the cortex ( A and C ) and ISOM ( E and G ). In PTH-treated rats, labeling densities of BSC-1 (arrows) and Na-K-ATPase (arrowheads) in the TAL cells were markedly reduced in the cortex ( B and D ) and ISOM ( F and H ).

As shown in Fig. 4, densitometric analysis of all samples from PTH-treated and control rats revealed a dramatic decrease in whole kidney NHE3 protein expression to 42 ± 7% in PTH-treated rats compared with control rats (100 ± 10%, P < 0.05, Fig. 4, A and B, Table 2 ). In addition, the expression of NHE3 protein was significantly decreased in membrane fractions of cortex/OSOM (57 ± 8 vs. 100 ± 5%, P < 0.05, Table 2 ) and ISOM (53 ± 11 vs. 100 ± 10%, P < 0.05, Table 2 ) in PTH-treated rats. Immunocytochemical analysis confirmed the reduced expression of NHE3 in PTH-treated rats ( Fig. 5, A - D ). In control rats, anti-NHE3 antibodies strongly labeled the apical domains of the proximal tubule ( Fig. 5 A ) and of mTAL ( Fig. 5 C ) and cTAL cells (not shown). In contrast, the labeling of NHE3 in the proximal tubule ( Fig. 5 B ) and the mTAL cells ( Fig. 5 D ) was clearly reduced in PTH-treated rats, consistent with the results from immunoblotting. Therefore, the protein expression of BSC-1, NHE3, and Na-K-ATPase was reduced in the ISOM (i.e., in the TAL) in PTH-treated rats. Moreover, NHE3 and Na-K-ATPase were also reduced in the cortex (i.e., in the proximal tubule).

Fig. 4. Semiquantitative immunoblotting of membrane fractions of whole kidney of high-dose-PTH-treated rats and control rats ( protocol 1 ). A : immunoblots were reacted with anti-type 3 Na/H exchanger (NHE3) antibodies and revealed a single 87-kDa band. B : densitometric analyses revealed that NHE3 expression in whole kidney was significantly decreased in PTH-treated rats compared with Con rats. * P < 0.05.

Fig. 5. Immunocytochemical analyses of NHE3 in proximal tubule and TAL of control rats ( A and C ) and high-dose-PTH-treated rats ( B and D; protocol 1 ). In normal controls, abundant NHE3 labeling (arrows) was seen of apical plasma membrane domains of proximal tubule cells in cortex ( A ) and TAL in the ISOM ( C ), whereas the basolateral plasma membranes were unlabeled. In PTH-treated rat, the apical NHE3 labeling (arrows) of the proximal tubule cells and TAL cells was decreased in both the cortex ( B ) and ISOM ( D ). G, glomerulus. Magnification: x 250 ( A and B ) and x 650 ( C and D ).

Semiquantitative immunoblotting revealed a significant decrease in whole kidney NaPi-2 protein expression to 16 ± 6% in PTH-treated rats compared with control rats (100 ± 7%, P < 0.05, Fig. 6, A and B, Table 2 ). Consistent with this, immunocytochemistry also revealed that NaPi-2 labeling in the apical brush border of the proximal tubule cells was much decreased in PTH-treated rats ( Fig. 7, B and D ) compared with control rats ( Fig. 7, A and C ), suggesting that the reduced abundance of NaPi-2 may contribute to the increased urinary phosphate excretion in addition to the natriuresis.

Fig. 6. Semiquantitative immunoblotting of membrane fractions of whole kidney of high-dose-PTH-treated rats and control rats ( protocol 1 ). A : immunoblots were reacted with anti-type 2 Na-P i cotransporter (NaPi-2) antibodies and revealed an 85-kDa band. B : densitometric analyses revealed that NaPi-2 expression in whole kidney was significantly decreased in PTH-treated rats compared with Con rats. * P < 0.05.

Fig. 7. Immunocytochemical analyses of NaPi-2 in the cortex of control rats ( A and C ) and high-dose-PTH-treated rats ( B and D; protocol 1 ). In normal controls, abundant NaPi-2 labeling was seen of apical plasma membrane domains of proximal tubule cells (arrows) in the cortex ( A and C ). In PTH-treated rats, apical labeling of NaPi-2 in the proximal tubule cells (arrows) was markedly decreased or disappeared in the cortex ( B and D ). G, glomerulus. Magnification: x 250 ( A and B ) and x 650 ( C and D ).

Rats Treated with Moderate-Dose (Protocol 2) and Low-Dose (Protocol 3) PTH Had Hypercalcemia and Unchanged Creatinine Clearance

To examine the effect of lower doses of PTH, rats were treated with moderate-dose PTH (10 µg·kg -1 ·day -1 sc for 2 days) in protocol 2. Rats treated with moderate-dose PTH also developed a significant hypercalcemia (plasma total calcium levels: 2.77 ± 0.02 vs. 2.57 ± 0.02 mmol/l in controls, P < 0.05, Table 3 ). Plasma creatinine level and creatinine clearance, however, were not changed in rats treated with moderate-dose PTH ( Table 3 ), suggesting that GFR was maintained. In contrast to protocol 1, the urine output and urinary osmolality were not changed, whereas the urinary-to-plasma osmolality ratio was decreased ( Table 3 ), suggesting that urinary concentration was persistently decreased in PTH-treated rats with mild hypercalcemia. In addition, urinary sodium excretion rates and FE Na were not altered but urinary calcium excretion was increased ( Table 3 ). In protocol 3, low-dose-PTH treatment induced a mild but significant hypercalcemia (plasma total calcium levels: 2.71 ± 0.03 vs. 2.59 ± 0.02 mmol/l in controls, P < 0.05, Table 4 ). However, plasma creatinine level, creatinine clearance, urine output, urinary osmolality, and the urine-to-plasma osmolality ratio were not changed in rats treated with low-dose PTH ( Table 4 ).

Table 3. Functional data (protocol 2)

Table 4. Functional data (protocol 3)

Decreased Renal Abundance of NHE3 and NaPi-2, but Maintained BSC-1 and Na-K-ATPase Abundance, in Rats Treated with Moderate-Dose PTH (Protocol 2)

Consistent with the downregulation of NaPi-2 in rats treated with high-dose PTH, whole kidney protein expression of NaPi-2 was persistently decreased in response to moderate-dose-PTH treatment (26 ± 5 vs. 100 ± 18% in control rats, P < 0.05, Fig. 8, A and B, Table 5 ), indicating that the downregulation of NaPi-2 plays an important role in the increased urinary phosphate excretion in hyperparathyroidism. Moreover, the protein expression of whole kidney NHE3 in rats treated with moderate-dose PTH was significantly decreased (52 ± 8 vs. 100 ± 14% in control rats, P < 0.05, Fig. 8, C and D, Table 5 ), consistent with protocol 1. In contrast, the whole kidney protein expression of BSC-1 and Na-K-ATPase was unchanged ( Table 5 ).

Fig. 8. Semiquantitative immunoblotting of membrane fractions of whole kidney of moderate-dose-PTH-treated rats and control rats ( protocol 2 ). A : immunoblots were reacted with anti-NaPi-2 antibodies. B : densitometric analyses revealed that NaPi-2 expression in whole kidney was significantly decreased in PTH-treated rats compared with control rats. C : immunoblots were reacted with anti-NHE3 antibodies. D : densitometric analyses revealed that NHE3 expression in whole kidney was significantly decreased in PTH-treated rats compared with Con rats. * P < 0.05.

Table 5. Densitometric analysis of sodium transporter expression (protocols 2 and 3)

Unchanged Renal Abundance of Sodium Transporters in Rats Treated with Low-Dose PTH (Protocol 3)

Whole kidney protein expression of BSC-1, Na-K-ATPase, and NHE3 in rats treated with low-dose PTH was unchanged compared with controls ( Table 5 ). Although there was a tendency for reduced NaPi-2 expression and increased renal phosphate excretion, whole kidney protein expression of NaPi-2 was not significantly decreased (64 ± 11 vs. 100 ± 18% in controls, Table 5 ), consistent with unchanged urinary phosphate excretion (0.67 ± 0.1 vs. 0.48 ± 0.2 µmol·min -1 ·kg -1 in controls, P 0.05, Table 4 ).

Increased Renal Abundance of CaR in Rats Treated with High-Dose or Moderate-Dose PTH (Protocols 1 and 2), but Not in Rats Treated with Low-Dose PTH

The anti-CaR antibodies recognized a 120-kDa band in membrane fractions from the ISOM ( Fig. 9, A and C ) and the cortex/OSOM ( Fig. 9 E ). Densitometric analysis revealed a significant increase in CaR protein expression in ISOM to 281 ± 37% in high-dose PTH-treated rats ( protocol 1 ) compared with control rats (100 ± 12%, P < 0.05, Fig. 9 B and Table 6 ). Moreover, the expression of CaR was significantly increased in cortex/OSOM from high-dose PTH-treated rats (249 ± 59 vs. 100 ± 20%, P < 0.05, Table 6 ). Immunocytochemistry showed that CaR labeling was restricted to the TAL segment in the medullary ray and associated with the deep basolateral membrane infoldings in cTAL cells ( Fig. 10, A and C ), consistent with previous reports ( 34, 35, 48 ). In contrast to the previous observations ( 34, 35 ), there was no detectable immunolabeling of CaR along the other tubular segment of the kidney, as also examined by use of another CaR antibody (not shown). CaR immunolabeling in cTAL was much stronger in kidneys of high-dose PTH-treated rats ( protocol 1 ) ( Fig. 10, B and D ) than in kidneys from control rats ( Fig. 10, A and C ), indicating a potential role of CaR in the regulation of electrolyte transport in this tubule segment of kidney in response to PTH treatment. Semiquantative immunoblotting and densitometric analysis revealed significant increases in CaR protein expression in rats treated with moderate doses of PTH (protocol 2) (197 ± 23 vs. 100 ± 24% in ISOM and 203 ± 29 vs. 100 ± 17% in cortex/OSOM, P < 0.05, Table 6 ), compared with control rats ( Fig. 9 C-F, Table 6 ). Immunolabeling confirmed the increased expression of CaR in cTAL (not shown). In contrast, the expression of CaR was not changed in rats treated with low-doses of PTH compared with controls (97 ± 14 vs. 100 ± 11% in ISOM and 73 ± 12 vs. 100 ± 10% in cortex/OSOM, P 0.05, Table 6 ).

Fig. 9. Semiquantitative immunoblotting of Ca 2+ -sensing receptor (CaR) in membrane fractions of kidney ISOM and cortex/outer strip of outer medulla (OSOM) in PTH-treated rats and control rats [ protocols 1 ( A and B ) and 2 ( C - F )]. A, C, and E : immunoblots reacted with antibodies of CaR recognized an 120-kDa main band in membrane preparations from ISOM ( A and C ) and cortex/OSOM ( E ). B : densitometric analyses revealed a significant increase in CaR expression in high-dose-PTH-treated rats compared with control rats ( protocol 1 ). D : in moderate-dose-PTH-treated rats and control rats ( protocol 2 ), densitometric analyses revealed a significant increase in CaR expression in ISOM of PTH-treated rats compared with control rats. F : densitometric analyses revealed increased CaR expression in cortex/OSOM of PTH-treated rats compared with control rats ( protocol 2 ). Open bars, PTH-treated rats ( n =6); filled bars, corresponding controls ( n =6). * P < 0.05.

Table 6. Densitometric analysis of CaR expression

Fig. 10. Immunocytochemical analyses of CaR in TAL of control rats ( A and C ) and high-dose-PTH-treated rats ( B and D; protocol 1 ). In normal controls, CaR labeling was restricted to the basolateral part in cortical TAL segments in a pattern associated with the deep basolateral membrane infoldings (arrowheads, C ). In PTH-treated rats, CaR labeling was also seen at basolateral membrane of cTAL cells; importantly, labeling density was significantly increased (arrowheads, D ) compared with that seen in kidneys from control rats. P, proximal tubule. Magnification: x 250 ( A and B ) and x 650 ( C and D ).

DISCUSSION

We examined the effects of acute hypercalcemia induced by different doses of PTH on the protein expression of major renal sodium transporters and CaR in rat kidney. The severe hypercalcemia induced by high-dose-PTH treatment in rats was associated with a marked downregulation of NHE3, NaPi-2, Na-K-ATPase and BSC-1, and this is likely to play an important role in the associated urinary concentrating defect, natriuresis, and phosphaturia. Moreover, the observed upregulation of CaR in the TAL in response to severe hypercalcemia may suggest a potential role for CaR in mediating a decrease in NaCl reabsorption in the TAL. In contrast, mild hypercalcemia, which was induced by moderate-dose-PTH treatment, was associated with decreased urine concentration, but not polyuria, natriuresis, or any changes in GFR. The protein expression of NHE3 and NaPi-2 was also decreased, but BSC-1 and Na-K-ATPase protein levels were not altered. Vitamin D-induced hypercalcemia in rats has previously been shown to be associated with a significant decrease in the expression of BSC-1, but with no changes in the expression of NHE3 and Na-K-ATPase ( 44 ). Thus the observed reduction in BSC-1 expression in rats with hypercalcemia induced by treatment with relatively high doses of PTH but not in the rats treated with lower doses (and hence having milder hypercalcemia) indicates that the reduction in BSC-1 may be related to the degree of hypercalcemia. Consistent with this, plasma calcium concentration in the rats treated with vitamin D ( 44, 45 ) and with PTH at high doses was similar. In contrast, the reduction in NHE3 and Na-K-ATPase expression in PTH-induced hypercalcemia observed in response to high-dose-PTH treatment may be caused either by a direct effect of PTH itself or by other secondary factors, such as increased expression of CaR, or other regulating systems, but excluding a direct effect of the hypercalcemia per se, because such a reduction was not observed in vitamin D-induced hypercalcemia ( 44 ), a condition where plasma PTH levels are known to be decreased.

Expression of BSC-1, NHE3, and Na-K-ATPase Was Decreased in TAL of PTH-Treated Rats

The urinary concentrating process is largely dependent on generation of a hypertonic medullary interstitium by counter-current multiplication. This is dependent on the active reabsorption of NaCl by the mTAL ( 25, 26 ). The key sodium transporters responsible for the transport of NaCl in the mTAL are BSC-1 (or NKCC2) ( 7, 29 ), NHE3 ( 1 ), and basolateral Na-K-ATPase ( 20 ). The abundance of BSC-1 in the TAL is regulated, and this appears to play a significant role in the urinary concentration mechanism. An increase in the delivery of NaCl to the loop of Henle by chronic oral saline loading or vasopressin treatment (shown by use of DDAVP, a vasopressin V 2 -receptor selective agonist) is known to upregulate BSC-1 levels ( 7, 23 ), whereas hypokalemia-induced nephrogenic diabetes insipidus is associated with reduced BSC-1 expression ( 10 ). Because the V 2 receptor is coupled to activation of adenylyl cyclase, it is possible that the upregulation of BSC-1 by vasopressin is a result of elevated levels of cAMP. Consistent with this, a cAMP-regulatory element (CRE) was identified in the 5'-flanking region of the mouse NKCC2 gene ( 17 ). In contrast to vasopressin action, high extracellular calcium has been shown to decrease vasopressin-stimulated cAMP accumulation in isolated TAL segments from rat kidney ( 18 ) and mouse kidney ( 42 ).

Our data revealed that the protein abundances of BSC-1, NHE3, and Na-K-ATPase were markedly decreased in the ISOM (i.e., TAL) in high-dose-PTH-treated rats. Thus the downregulation of these major sodium transporters in the TAL could play a key role in the reduced reabsorption of sodium and chloride in the TAL segment in PTH-induced hypercalcemia, thereby reducing medullary interstitial tonicity and urinary concentration. Moreover, the decreased reabsorption of sodium chloride in the TAL could reduce the transepithelial voltage, thereby decrease calcium reabsorption secondarily ( 12 ). Because a large fraction of tubular calcium is reabsorbed in the TAL driven by NaCl reabsorption ( 12 ), this is consistent with the increase in urinary calcium excretion observed in the present study.

The decreased expression of BSC-1 in the mTAL in response to PTH-induced hypercalcemia is consistent with our previous data of vitamin D-induced hypercalcemia ( 44 ) showing downregulation of BSC-1 and other studies showing diminished NaCl reabsorption in the TAL and a decrease in medullary tonicity in hypercalcemia ( 30, 31, 42 ). However, in contrast to the findings in rats with vitamin D-induced hypercalcemia, PTH-induced hypercalcemia was associated with downregulation of all three major sodium transporters in the TAL (i.e., BSC-1, NHE3, and Na-K-ATPase). The difference is likely due to the effects of PTH itself because mRNAs coding for PTH/PTH-related protein receptors are present along the nephron including cTAL ( 35, 48 ). In contrast, rats with mild hypercalcemia that was induced by moderate-dose PTH ( protocol 2 ) were associated with downregulation of NHE3 in the TAL, but the expression of BSC-1 and Na-K-ATPase was not changed. Future studies are needed to understand the underlying mechanisms for this.

Decreased Expression of NaPi-2, NHE3, and Na-K-ATPase in the Proximal Tubule

The present study has also provided novel insights into the changes in the expression of major renal sodium transporters in the proximal tubule. It is well established that NaPi-2 is downregulated in response to acute PTH treatment (studies of 2 h of PTH treatment) ( 21 ), and here we demonstrated a significant downregulation of NaPi-2, NHE3, and Na,K-ATPase in the proximal tubule at 2 days after PTH treatment, which will be discussed below.

NaPi-2, located in the renal proximal tubule brush-border membrane, is the key player in renal phosphate reabsorption as well as in the overall homeostasis of phosphate ( 3, 21, 28 ). In protocols 1 and 2, PTH treatment was associated with downregulation of NaPi-2, suggesting a profound inhibitory effect of PTH on NaPi-2. The high-dose-PTH-infused rats in the present study showed a fourfold increase in urinary P i excretion compared with controls, and this was associated with a marked decrease in renal NaPi-2 expression, consistent with previous studies by Murer and associates ( 3, 21, 28 ). This suggests that increased urinary phosphate excretion is mediated by the decreased abundance of NaPi-2 in the proximal tubule. Recently, acute treatment with PTH (2 h) ( 21 ) was demonstrated to decrease the levels of NaPi-2 in the brush-border membrane in the proximal tubule and lead to accumulation of NaPi-2 in cytoplasmic vesicles, possibly due to endocytic withdrawal into a cytoplasmic pool. In contrast, the present study with 2 days of high-dose PTH treatment showed decreased labeling of NaPi-2 in both the apical membrane and subapical vesicles. This is probably because prolonged PTH treatment (2 days) ultimately causes NaPi-2 degradation after internalization into a cytoplasmic pool.

NHE3, which is expressed apically in the proximal tubule cells, mediates the major fraction of transcellular sodium and bicarbonate reabsorption, in conjunction with Na-K-ATPase and the electrogenic sodium-bicarbonate cotransporter, both present in the basolateral plasma membrane ( 14 ). We demonstrated here that both high-dose and moderate-dose PTH treatment were associated with a marked decrease in the protein expression of NHE3 as well as reduced labeling of the proximal tubule. A previous study has indicated that acute effects of PTH involve redistribution of NHE3 from the apical plasma membrane to endocytic vesicles ( 49 ). In contrast, chronic PTH treatment (for 8 days) was previously shown to be associated with decreased proximal tubular NHE3 protein and mRNA levels ( 13 ). The decrease in NHE3 expression is therefore likely to decrease sodium, bicarbonate, and fluid reabsorption in the proximal tubule. This was further supported by in vivo micropuncture analysis demonstrating an inhibitory effect of PTH on H + secretion at the apical membrane of the proximal tubule as well as on proximal tubule bicarbonate reabsorption ( 13, 16 ). However, elevated plasma PTH levels are not usually associated with metabolic acidosis, because H + secretion in the distal nephron is increased in this condition ( 41 ).

Besides the reduced expression of NHE3, the protein expression levels of the basolateral Na-K-ATPase protein was also decreased in both proximal tubule and TAL in response to high-dose-PTH treatment. This is consistent with previous studies, which revealed an inhibitory effect of PTH on Na-K-ATPase activity ( 11 ).

Expression of CaR Was Increased in the TAL of PTH-Treated Rats

Our data demonstrated that the abundance of the CaR was significantly increased in cortex/OSOM and ISOM in rats treated with either high or moderate doses of PTH, but not in rats with mild hypercalcemia induced by treatment with low doses of PTH. The CaR has been demonstrated to be localized in several renal tubule segments in rat kidney, especially at the basolateral membrane of TAL cells ( 34, 35, 48 ), where the receptor is likely to participate in the regulation of urinary concentration. Recently, it has been shown that CaR colocalizes with Ca 2+ -inhibitable adenylyl cyclase in cTAL cells of rat kidney and the CaR, which is activated by increasing concentrations of extracellular ionized calcium, inhibits AVP-dependent intracellular cAMP accumulation ( 19 ). This may suggest that the CaR-mediated reduction of cAMP levels in the cTAL cells probably contributes to the impaired cAMP-dependent reabsorption of NaCl in the TAL cells. Consistent with this, in the present study high-dose-PTH-induced hypercalcemia was associated with significant reduction of BSC-1 abundance (presumably due to an inhibition of intracellular cAMP levels) and increased urinary sodium excretion. This was accompanied by a significant increase in CaR protein expression in the TAL. Thus this finding supports the view that upregulation of the CaR may play an important role in mediating a decrease in NaCl reabsorption in the TAL in response to hypercalcemia.

Interestingly, in rats with PTH induced-hypercalcemia using moderate doses of PTH (without increased urinary sodium excretion), the expression of renal CaR was also significantly upregulated although it did not reach the same high levels as that seen in rats treated with high doses of PTH. In mild hypercalcemia, renal CaR expression levels were not changed. These findings raise the possibility that the degree of inhibition of NaCl reabsorption in the TAL depends on the magnitude of CaR activation. It has previously been shown by in vivo microperfusion of Henle's loop from thyroparathyroidectomized rats that calcium, being a more potent activator of the CaR than magnesium, induces reduction in NaCl reabsorption ( 33 ). A study in humans showed that under a PTH-clamp protocol and with strictly controlled NaCl balance, calcium infusion induces a modest increase in urinary NaCl excretion, with a saturation phenomenon at high plasma calcium concentrations ( 9 ). Moreover, the activating mutations of the CaR gene, seen in a patient with autosomal dominant hypocalcemia, were associated with a decrease in distal tubular fractional chloride reabsorption and a renal loss of NaCl with secondary hyperaldosteronism and hypokalemia ( 43 ). Taken together, it is therefore likely that CaR activation of sufficient magnitude is able to induce renal loss of NaCl due to reduction of NaCl transport in the TAL. In contrast, several other studies have suggested that activation of the CaR does not impair NaCl reabsorption or the transepithelial potential gradient ( 5, 27 ). Further studies are therefore warranted to define the role and the mechanism of CaR action in the regulation of sodium transport in the TAL.

Summary

We have demonstrated that hypercalcemia induced by treatment with a high dose of PTH was associated with downregulation of several major renal sodium transporters and upregulation of the CaR, in parallel with the development of polyuria, reduced urinary concentration, and increased urinary sodium, calcium, and phosphate excretion. In particular, rats treated with moderate-dose PTH exhibited unchanged GFR, but decreased urinary concentration. The protein expression of NHE3 and NaPi-2 was persistently decreased, but BSC-1 and Na-K-ATPase protein levels were not altered. Because vitamin D-induced hypercalcemia in rats was associated with a significant decrease in the expression of BSC-1 but with no changes in NHE3 and Na-K-ATPase expression ( 44 ), the reduced expression of NHE3 and Na-K-ATPase in the present study may be either a response to the PTH itself or a consequence of the increased expression of CaR.

GRANTS

The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Support for this study was provided by the Karen Elise Jensen Foundation, Human Frontier Science Program, Novo Nordic Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, the University of Aarhus, Dongguk University, the European Commission (QRLT 2000 00778 and QRLT 2000 00987), and the intramural budget of the National Heart, Lung, and Blood Institute.

ACKNOWLEDGMENTS

The authors thank Helle Høyer, Gitte Kall, Ida Maria Jalk, Lotte Vallentin Holbech, Inger Merete Paulsen, Merete Pedersen, Zhila Nikrozi, Mette Vistisen, and Dorte Wulff for expert technical assistance.

【参考文献】
  Amemiya M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, and Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney Int 48: 1206-1215, 1995.

Berdud I, Martin-Malo A, Almaden Y, Aljama P, Rodriguez M, and Felsenfeld AJ. The PTH-calcium relationship during a range of infused PTH doses in the parathyroidectomized rat. Calcif Tissue Int 62: 457-461, 1998.

Biber J, Custer M, Magagnin S, Hayes G, Werner A, Lotscher M, Kaissling B, and Murer H. Renal Na/Pi-cotransporters. Kidney Int 49: 981-985, 1996.

Biemesderfer D, Pizzonia J, Abu-Alfa A, Exner M, Reilly R, Igarashi P, and Aronson PS. NHE3: a Na + /H + exchanger isoform of renal brush border. Am J Physiol Renal Fluid Electrolyte Physiol 265: F736-F742, 1993.

Desfleurs E, Wittner M, Simeone S, Pajaud S, Moine G, Rajerison R, and Di Stefano A. Calcium-sensing receptor: regulation of electrolyte transport in the thick ascending limb of Henle's loop. Kidney Blood Press Res 21: 401-412, 1998.

Dolson GM, Hise MK, and Weinman EJ. Relationship among parathyroid hormone, cAMP, and calcium on proximal tubule sodium transport. Am J Physiol Renal Fluid Electrolyte Physiol 249: F409-F416, 1985.

Ecelbarger CA, Terris J, Hoyer JR, Nielsen S, Wade JB, and Knepper MA. Localization and regulation of the rat renal Na + -K + -2Cl - cotransporter, BSC-1. Am J Physiol Renal Fluid Electrolyte Physiol 271: F619-F628, 1996.

Slatopolsky E and Hruska KA. Disorder of phosphorus, calcium, and magnesium metabolism. In: Diseases of the Kidney and Urinary Tract, edited by Schrier RW. Philadelphia, PA: Lippincott Williams & Wilkins, 2001, p. 2607-2660.

El Hajj FG, Seifter J, Scott J, and Brown EM. Calcium-regulated renal calcium handling in healthy men: relationship to sodium handling. J Clin Endocrinol Metab 83: 2366-2372, 1998.

Elkjær ML, Kwon TH, Wang W, Nielsen J, Knepper MA, Frøkiær J, and Nielsen S. Altered expression of renal NHE3, TSC, BSC-1, and ENaC subunits in potassium-depleted rats. Am J Physiol Renal Physiol 283: F1376-F1388, 2002.

Feraille E and Doucet A. Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345-418, 2001.

Friedman PA. Codependence of renal calcium and sodium transport. Annu Rev Physiol 60: 179-197, 1998.

Girardi AC, Titan SM, Malnic G, and Reboucas NA. Chronic effect of parathyroid hormone on NHE3 expression in rat renal proximal tubules. Kidney Int 58: 1623-1631, 2000.

Greger R. Physiology of renal sodium transport. Am J Med Sci 319: 51-62, 2000.

Hebert SC. Extracellular calcium-sensing receptor: implications for calcium and magnesium handling in the kidney. Kidney Int 50: 2129-2139, 1996.

Hensley CB, Bradley ME, and Mircheff AK. Parathyroid hormone-induced translocation of Na-H antiporters in rat proximal tubules. Am J Physiol Cell Physiol 257: C637-C645, 1989.

Igarashi P, Whyte DA, Li K, and Nagami GT. Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-C1 cotransporter gene. J Biol Chem 271: 9666-9674, 1996.

Jesus Ferreira MC and Bailly C. Extracellular Ca 2+ decreases chloride reabsorption in rat CTAL by inhibiting cAMP pathway. Am J Physiol Renal Physiol 275: F198-F203, 1998.

Jesus Ferreira MC, Helies-Toussaint C, Imbert-Teboul M, Bailly C, Verbavatz JM, Bellanger AC, and Chabardes D. Co-expression of a Ca 2+ -inhibitable adenylyl cyclase and of a Ca 2+ -sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca 2+. J Biol Chem 273: 15192-15202, 1998.

Kashgarian M, Biemesderfer D, Caplan M, and Forbush B III. Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney Int 28: 899-913, 1985.

Kempson SA, Lotscher M, Kaissling B, Biber J, Murer H, and Levi M. Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 268: F784-F791, 1995.

Kim GH, Ecelbarger C, Knepper MA, and Packer RK. Regulation of thick ascending limb ion transporter abundance in response to altered acid/base intake. J Am Soc Nephrol 10: 935-942, 1999.

Kim GH, Ecelbarger CA, Mitchell C, Packer RK, Wade JB, and Knepper MA. Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop. Am J Physiol Renal Physiol 276: F96-F103, 1999.

Kimmel PL, Rivera A, and Khatri P. Effects of nonrenal hormones on the normal kidney. In: Principles and Practice of Endocrinology and Metabolism, edited by Becker KL. Philadelphia, PA: Lippincott Williams & Wilkins, 2001, p. 1885-1895.

Knepper MA, Kim GH, Fernandez-Llama P, and Ecelbarger CA. Regulation of thick ascending limb transport by vasopressin. J Am Soc Nephrol 10: 628-634, 1999.

Masilamani S, Knepper MA, and Burg MB. Urine concentration and dilution. In: The Kidney, edited by Brenner BM. Philadelphia, PA: Saunders, 2000, p. 595-635.

Motoyama HI and Friedman PA. Calcium-sensing receptor regulation of PTH-dependent calcium absorption by mouse cortical ascending limbs. Am J Physiol Renal Physiol 283: F399-F406, 2002.

Murer H, Hernando N, Forster I, and Biber J. Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 80: 1373-1409, 2000.

Nielsen S, Maunsbach AB, Ecelbarger CA, and Knepper MA. Ultrastructural localization of Na-K-2Cl cotransporter in thick ascending limb and macula densa of rat kidney. Am J Physiol Renal Physiol 275: F885-F893, 1998.

Peterson LN. Vitamin D-induced chronic hypercalcemia inhibits thick ascending limb NaCl reabsorption in vivo. Am J Physiol Renal Fluid Electrolyte Physiol 259: F122-F129, 1990.

Peterson LN, McKay AJ, and Borzecki JS. Endogenous prostaglandin E 2 mediates inhibition of rat thick ascending limb Cl reabsorption in chronic hypercalcemia. J Clin Invest 91: 2399-2407, 1993.

Pfister MF, Lederer E, Forgo J, Ziegler U, Lotscher M, Quabius ES, Biber J, and Murer H. Parathyroid hormone-dependent degradation of type II Na + /P i cotransporters. J Biol Chem 272: 20125-20130, 1997.

Quamme GA. Effect of hypercalcemia on renal tubular handling of calcium and magnesium. Can J Physiol Pharmacol 60: 1275-1280, 1982.

Riccardi D, Hall AE, Chattopadhyay N, Xu JZ, Brown EM, and Hebert SC. Localization of the extracellular Ca 2+ /polyvalent cation-sensing protein in rat kidney. Am J Physiol Renal Physiol 274: F611-F622, 1998.

Riccardi D, Lee WS, Lee K, Segre GV, Brown EM, and Hebert SC. Localization of the extracellular Ca 2+ -sensing receptor and PTH/PTHrP receptor in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F951-F956, 1996.

Sands JM, Naruse M, Baum M, Jo I, Hebert SC, Brown EM, and Harris HW. Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct. J Clin Invest 99: 1399-1405, 1997.

Satoh T, Cohen HT, and Katz AI. Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron. Am J Physiol Renal Fluid Electrolyte Physiol 265: F399-F405, 1993.

Schneider EG. Effect of parathyroid hormone secretion on sodium reabsorption by the proximal tubule. Am J Physiol 229: 1170-1173, 1975.

Schor N, Ichikawa I, and Brenner BM. Mechanisms of action of various hormones and vasoactive substances on glomerular ultrafiltration in the rat. Kidney Int 20: 442-451, 1981.

Spiegel AM. Pathophysiology of primary hyperparathyroidism. J Bone Miner Res 6, Suppl 2: S15-S17, 1991.

Suki WN, Lederer ED, and Rouse D. Renal transport of calcium, magnesium and phosphate. In: The Kidney, edited by Brenner BM. Philadelphia, PA: Saunders, 2000, p. 553-557.

Takaichi K, Uchida S, and Kurokawa K. High Ca 2+ inhibits AVP-dependent cAMP production in thick ascending limbs of Henle. Am J Physiol Renal Fluid Electrolyte Physiol 250: F770-F776, 1986.

Vargas-Poussou R, Huang C, Hulin P, Houillier P, Jeunemaitre X, Paillard M, Planelles G, Dechaux M, Miller RT, and Antignac C. Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome. J Am Soc Nephrol 13: 2259-2266, 2002.

Wang W, Kwon TH, Li C, Frøkiær J, Knepper MA, and Nielsen S. Reduced expression of Na-K-2Cl cotransporter in medullary TAL in vitamin D-induced hypercalcemia in rats. Am J Physiol Renal Physiol 282: F34-F44, 2002.

Wang W, Li C, Kwon TH, Knepper MA, Frøkiær J, and Nielsen S. AQP3, p-AQP2, and AQP2 expression is reduced in polyuric rats with hypercalcemia: prevention by cAMP-PDE inhibitors. Am J Physiol Renal Physiol 283: F1313-F1325, 2002.

Wang W, Lu M, Balazy M, and Hebert SC. Phospholipase A 2 is involved in mediating the effect of extracellular Ca 2+ on apical K + channels in rat TAL. Am J Physiol Renal Physiol 273: F421-F429, 1997.

Wang WH, Lu M, and Hebert SC. Cytochrome P -450 metabolites mediate extracellular Ca 2+ -induced inhibition of apical K + channels in the TAL. Am J Physiol Cell Physiol 271: C103-C111, 1996.

Yang T, Hassan S, Huang YG, Smart AM, Briggs JP, and Schnermann JB. Expression of PTHrP, PTH/PTHrP receptor, and Ca 2+ -sensing receptor mRNAs along the rat nephron. Am J Physiol Renal Physiol 272: F751-F758, 1997.

Zhang Y, Norian JM, Magyar CE, Holstein-Rathlou NH, Mircheff AK, and McDonough AA. In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition. Am J Physiol Renal Physiol 276: F711-F719, 1999.

作者单位：1 The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark; 2 Department of Biochemistry, School of Medicine, Kyungpook National University, Taegu 700-42 Korea; 3 Department of Medicine, Case Western Reserve University, Louis Stokes Veteran Affairs Medical Center, Clevelan