Idiopathic hypercalciuria (IH) is the most common cause of

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1 Regulation of Renal Calcium Receptor Gene Expression by 1,25-Dihydroxyvitamin D 3 in Genetic Hypercalciuric Stone-Forming Rats Jim J. Yao,* Shaochun Bai,* Alexander J. Karnauskas,* David A. Bushinsky, and Murray J. Favus* *The University of Chicago Pritzker School of Medicine, Chicago, Illinois; and The University of Rochester School of Medicine, Rochester, New York Hypercalciuria in inbred genetic hypercalciuric stone-forming (GHS) rats is due, in part, to a decrease in renal tubule Ca reabsorption. Activation of the renal Ca receptor (CaR) may decrease renal tubule Ca reabsorption and cause hypercalciuria through suppression of Ca-sensitive potassium channel activity. Because the rat renal CaR gene is regulated by extracellular calcium and 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] and GHS rats have increased renal vitamin D receptor content, the current study was undertaken to determine the level of CaR gene expression in GHS rat kidney and whether CaR gene expression is regulated by 1,25(OH) 2 D 3. Male GHS and normal control (NC) rats were fed a Ca-sufficient diet (0.6% Ca). Western blotting revealed a four-fold increase in CaR protein in GHS rat renal tissue, and 1,25(OH) 2 D 3 administration increased renal CaR in both GHS and NC rats. Northern blot analysis of extracts of renal cortical tissue from GHS and NC rats revealed a major 7-kb transcript of CaR and a more modest 4-kb transcript, both of which were readily detectable. Both Northern blotting and real-time reverse transcription PCR revealed increased basal CaR mrna expression levels in GHS rat kidney. 1,25(OH) 2 D 3 administration increased renal CaR mrna levels 2.0- and 3.3-fold in GHS and NC rats, respectively. Despite the greater incremental increase by 1,25(OH) 2 D 3 in NC rats, CaR mrna levels remained higher in GHS rat kidney, and the elevation was more sustained. 1,25(OH) 2 D 3 increased CaR mrna through both elevated CaR gene expression and prolonged tissue half-life. These results demonstrate that GHS rats have high levels of CaR gene expression and CaR protein that may contribute to the hypercalciuria and calcium nephrolithiasis. J Am Soc Nephrol 16: , doi: /ASN Idiopathic hypercalciuria (IH) is the most common cause of Ca oxalate nephrolithiasis and contributes to kidney stone formation by increasing urine supersaturation with respect to Ca and oxalate (1 5). The sources of the excess urine Ca excretion are enhanced intestinal Ca absorption and increased bone resorption (1,3,6). Genetic hypercalciuric stone-forming (GHS) rats spontaneously form Ca-containing kidney stones (7 10) and share many other features with human IH. Both have normal serum Ca (6,11 14), intestinal Ca hyperabsorption (3,11,12,15 17), increased bone resorption (3,13,14,18), and defective renal tubule Ca reabsorption (13,19). Serum 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] levels are normal in 30 to 50% of IH patients (3,6,14,20 22) and all GHS rats (11,12). The normal serum Ca levels in both IH patients and GHS rats suggest that the hypercalciuria is accompanied by a normal filtered load of Ca and a defect in renal tubule Ca reabsorption (19,23). Indeed, in parathyroidectomized GHS rats with a filtered load of Ca maintained by Ca infusion, metabolic clearance studies reveal a marked inhibition of renal tubule Ca reabsorption when compared with similarly treated control rats (19). Received December 15, Accepted February 14, Published online ahead of print. Publication date available at Address correspondence to: Dr. Murray J. Favus, The University of Chicago, Pritzker School of Medicine, 5841 S. Maryland Avenue, MC 1027, Chicago, IL Phone: ; Fax: ; mfavus@medicine.bsd.uchicago.edu Elevated vitamin D receptor (VDR) levels in GHS rat intestinal mucosa, bone, and renal cortical tissue (12,13,24) strongly suggest that increased intestinal Ca absorption, enhanced bone resorption, and decreased renal Ca reabsorption may be mediated by the excess VDR through overexpression of vitamin D dependent genes that encode for proteins involved in epithelial and cellular Ca transport. The plasma membrane CaR is expressed in a variety of tissues, including parathyroid cells, kidney, gastrointestinal tract, placenta, pancreas, and brain (25). Sequencing of the human, rat, and mouse kidney CaR gene has revealed extensive homology with the parathyroid CaR (26,27). The CaR is a seven-membrane-spanning protein that belongs to the G-coupled protein family of plasma membrane receptors (27). Through its renal plasma membrane location, the CaR may regulate Ca transport through a CaR-responsive K-dependent voltage-gated chloride channel (28,29). In the thick ascending limb of the loop of Henle, CaR activation alters the luminal K channel, decreasing K migration from the cell into the lumen, decreasing the positive lumen potential, and thus decreasing paracellular Ca reabsorption. CaR gene expression is regulated by 1,25(OH) 2 D 3 and extracellular Ca (30), and GHS rats have increased VDR in renal tissue and increased urine Ca (12,31); therefore, this study was designed to test the hypothesis that (1) renal cell CaR gene expression is increased in GHS rats and (2) CaR is differentially regulated by 1,25(OH) 2 D 3 in GHS versus normal control (NC) rats. Copyright 2005 by the American Society of Nephrology ISSN: /

2 J Am Soc Nephrol 16: , 2005 Renal Ca Receptor in Hypercalciuric Rat 1301 Materials and Methods Animals and Diets Sprague-Dawley male rats (Harlan Teklad, Indianapolis, IN) were used as NC and were the same strain that was used to initiate the GHS colony. Specifically, the colony of GHS rats was created by mating spontaneously hypercalciuric male and female Sprague Dawley rats and selecting those with the highest urine Ca excretions for subsequent mating (7,8,11,12,32,33). Determination of urine Ca excretion was done at the time of weaning, when animals were placed in individual metabolic cages and fed a constant amount of a standard diet that contained 0.6% Ca, 0.65% phosphorus, 0.24% magnesium, 0.4% sodium, 0.43% chloride, and 2.2 IU vitamin D/g food (7,8,11,12,32,33). Deionized distilled water was given ad libitum. GHS rats were initially defined as those with urine Ca excretion of 2 SD above the mean of NC rats (usually 1.5 mg/24 h) on two successive 24-h urine collections after the animals had been equilibrated on the diet for 5 d. Male and female rats with the highest Ca excretion values were chosen for breeding to propagate the colony. The remaining male hypercalciuric rats with body weight of 190 to 240 g were used for these studies, and NC and GHS rats were fed a similar 0.6% Ca diet for at least 5 d before the study. Previously, we determined that NC and GHS rats grow at the same rate (Bushinsky DA, unpublished observations). Therefore, animals that were matched for weight were also matched for age. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Both the University of Chicago and the University of Rochester Animal Care Committees approved the protocol for the care and use of the animals. Experimental Protocols Four experimental protocols were conducted using rats that were fed a standard diet and weighed 190 to 240 g. Once randomized, the rats were given free access to deionized water and were killed in the fasting state. Experiment 1: Basal CaR Kidney Protein Levels and Gene Expression. NC and GHS rats received an injection of vehicle and then were killed. Kidney cortical tissue was removed for subsequent analysis including Western blotting and real-time reverse transcription PCR (RT-PCR) to quantify CaR protein and mrna expression levels, respectively. Experiment 2: 1,25(OH) 2 D 3 Regulation of CaR Gene Expression. NC and GHS rats received an intraperitoneal injection of either vehicle or 1,25(OH) 2 D 3 at 10, 50, or 200 ng/100 g body wt and were killed at various intervals thereafter. Experiment 3: In Vivo CaR mrna Half-Life. GHS rats were randomized into five experimental groups: Group 1 received vehicle injection but no actinomycin D and no 1,25(OH) 2 D 3 ; group 2 received 1,25(OH) 2 D 3 (30 ng/100 g body wt intraperitoneally) 8 h before being killed and no actinomycin D; group 3 received actinomycin D (400 g/100 g body wt intraperitoneally) 1 h before treatment with 1,25(OH) 2 D 3 (30 ng/100 g body wt intraperitoneally) and were killed 8 h later; group 4 received actinomycind8hbefore being killed and no 1,25(OH) 2 D 3 ; and group 5 received 1,25(OH) 2 D 3 (30 ng/100 g body wt intraperitoneally) 16 h before actinomycin D intraperitoneally and then were killed 8 h later. In preliminary experiments, actinomycin D (400 g/ 100 g body wt intraperitoneally) efficiently suppressed CaR mrna by 8 h. The majority of rats that were treated with actinomycin D survived for approximately 12 h but not to 24 h. Thus, rats were treated with actinomycin D (400 g/100 g body wt intraperitoneally) and killed at intervals over the ensuing 8 h. Kidney cortical total RNA was isolated at each time point, and CaR mrna levels were determined by Northern blot hybridization. CaR mrna on Northern blot was quantified, and the in vivo CaR mrna half-life then was calculated according to an established mathematical model (34). Experiment 4: Role of Protein Synthesis in CaR mrna Upregulation. GHS rats were given vehicle intraperitoneally, 1,25(OH) 2 D 3 (30 ng/100 g body wt intraperitoneally) 8 h before being killed, cycloheximide (4 mg/100 g body wt intraperitoneally) 1 h before 1,25(OH) 2 D 3 (30 ng/100 g body wt intraperitoneally) and killed 8 h later, or cycloheximide (4 mg/100 g body wt intraperitoneally) 8 h before being killed. Protein Extraction Kidneys were removed and cortical tissue was dissected from medulla by gross inspection. In preliminary studies, histologic inspection revealed minimal inclusion of medullary tissue in the cortical preparation. Cortical tissue then was homogenized in cold lysis 250 buffer (50 mm Tris/HCl [ph 7.4], 250 mm NaCl, 5 mm EDTA, 0.1% Nonidet P-40, 50 mm NaF, 1 mm PMSF, 1 g/ml leupeptin, and 1 g/ml antipain) and chilled on ice for 30 min. The tissue then was subjected to two cycles of rapid freezing and thawing, and then centrifuged for 30 min at 14,000 g at 4 C. The supernatant was removed and stored at 4 C. Protein concentration was determined using the Bio-Rad protein assay using a dye reagent (Bio-Rad, Hercules, CA). Western Blot Protein aliquots (30 g) were denatured in 6 SDS sample buffer (7 ml 4 Tris/Cl, SDS [ph 6.8], 3.0 ml of glycerol, 1 g of SDS, 0.93 g of dithiothreitol, and 1.2 mg of bromphenol blue in 10 ml of distilled deionized H 2 O) and then loaded onto SDS denaturing discontinuous gels. The proteins were transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) by electroblotting at 90 V for 1 h. The membranes then were blocked and washed with 5% nonfat dried milk in TBS that contained 0.1% Tween-20 (TBS-T). The primary polyclonal antibody (rabbit anti-car) was affinity purified (Chemicon International Inc., Temecula, CA) and then diluted with 1% nonfat dried milk in TBS-T at room temperature for 1 h. The membranes were washed with 5% nonfat milk in TBS-T and incubated with horseradish peroxidase conjugated anti-rabbit IgG for 1 h. Band intensities were developed using the ECL Chemiluminescence System (Amersham Life Science, Buckinghamshire, UK). The blots were quantified by scanning using One-Scan 1-D gel Analysis software (Scanalytics Inc., Fairfax, VA). RNA Isolation Animals were killed by exsanguination via the abdominal aorta while under light general anesthesia. Kidneys then were removed rapidly for RNA isolation. The kidney capsule was trimmed, medullary tissue was removed, and the remaining cortical tissue was rinsed with ice-cold PBS buffer. Twenty-five micrograms of tissue was minced, and total RNA was isolated from the solid tissue using an RNeasy Mini kit according to the manufacturer s protocol (Qiagen, Valencia, CA). Preparation of cdna Probe A 600-bp cdna probe detecting rat kidney CaR mrna used in this study was generated by RT-PCR amplification. A pair of 5 and 3 primers was designed on the basis of the mrna sequence of CaR published by Riccardi et al. (27). The sequence of the 5 primer was 5 -TTC CGT GGA TTC CGA TGG TTA CAA GC, and the 3 primer was 5 -TGG CCG TAG AGT TTT GGA TCA CTT CG. Approximately 5 g of total RNA was reverse-transcribed and amplified with the pair of primers using the GeneAmp System (Perkin Elmer Corp., Wellesley, MA). The 600-bp cdna was purified further and subcloned with the pgem-t Vector System (Promega, Madison, WI). The cdna sequence probe was verified using the Fmol DNA Sequencing Systems (Promega). Preliminary experiments demonstrated that this cdna probe detected a 7.0-kb and a 4.0-kb transcript of CaR mrna in total RNA from rat kidney cortex, similar to

3 1302 Journal of the American Society of Nephrology J Am Soc Nephrol 16: , 2005 Figure 1. Ca receptor (CaR) protein in normal control (NC) and genetic hypercalciuric stone-forming (GHS) kidney. Lanes are renal CaR protein from NC (lanes 1, 2, and 3; A) and GHS (lanes 4, 5, and 6; A) kidney under basal condition. CaR migrates at Actin is used to compare sample loading. Blots were scanned, and CaR for each sample was adjusted for loading (B). Values are mean SEM of three rats per group. *P versus NC. Figure 2. CaR mrna in NC and GHS kidney. (A) 7 kb is the dominant transcript of CaR mrna. Lanes are renal CaR mrna from NC and GHS kidney under basal conditions. (B) CaR mrna levels were determined using real-time PCR. Values are mean SEM of three rats per group. *P 0.05 versus NC. previous descriptions (27,35). Therefore, this cdna probe was used for all Northern hybridizations in this study. Reverse Transcription Reverse transcription with oligo-dt primers was performed with the Reverse Transcription System (Promega). One microgram of total RNA was used in all reverse transcription reactions. Reactions were performed in a final volume of 40 l that contained 0.5 g of Oligo(dT), 1 mm dntp, 5 mm MgCl 2, and 15 units of AMV reverse transcriptase. The reaction then was allowed to proceed at 42 C for 1 h. The sample was heated to 95 C for 5 min to terminate the reaction. Northern Blot Hybridization Tissue steady-state levels of CaR mrna were determined by Northern blot hybridization analysis as described previously (24,34,36,37). Approximately 15 g of total RNA was denatured in 50% formamide, 17.5% formaldehyde, and 1 MOPS buffer (20 nm 3-[N-morpholino]propane-sulfonic acid at ph 7.0, 5 mm sodium acetate, and 1 mm Na 2 -EDTA [ph 8.0]); electrophoresed in 1% agarose gel; and then transferred to GeneScreen Plus membranes (New England Nuclear, Boston, MA). The membranes were baked at 80 C for 4 h and then hybridized with a radiolabeled specific cdna probe at a concentration of cpm/ml. The cdna probe was labeled with [ 32 P]dCTP (specific activity 3000 Ci/mM; Amersham Corp., Piscataway, NJ) by random primer extension using the Multiprime DNA labeling system (Amersham Corp.). The specific activity of labeled probe ranged from 2to cpm/ g of cdna. After hybridization, the blots were washed and exposed overnight to XAR-5 film (Eastman Kodak, Rochester, NY) at 70 C. Conditions of prehybridization, hybridization, and washing procedures were the same as described previously (34,36,37). Real-Time PCR Real-time PCR reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and an ABI PRISM 770 apparatus (Perkin Elmer, Applied Biosystems). Thermocycling was done in a final volume of 25 l that contained 50 ng of cdna and 800

4 J Am Soc Nephrol 16: , 2005 Renal Ca Receptor in Hypercalciuric Rat 1303 nm of each of the forward and reverse primers. The sequences for the CaR forward and reverse primers were as follows: forward 5 -TTG TAG TAC CCA ACT TCC TTG AAC A-3 and reverse 5 -AAG CAC CTA CGG CAC CTG AA-3. The human housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used as a control, and the GAPDH primers sequences were as follows: forward 5 -GCC AGC CTC GTC TCA TAG ACA-3 and reverse 5 -AGA GAA GGC AGC CCT GGT AAC-3. PCR was performed using the following program: 95 C for 10 min; 40 cycles of 94 C for 5 s; 56 C for 30 s, 72 C for 20 s; one cycle of 95 C for 15 s; 62 C for 20 s; and 95 C for 15 s. Quantitative values were derived from the threshold cycle number at which the increase in the signal associated with exponential growth of PCR products was first detected. For each PCR product, the relative amount in each sample was calculated from standard curves generated from pooled RNA from control samples. For each sample, the GAPDH value was used to normalize the expression value. A relative gene expression was calculated by assigning the normal control a relative value of 1.0, with all other values relative to the normal control. Materials All reagents and chemicals, except where indicated, were purchased either from Sigma Chemical Co. (St. Louis, MO) or Life Technologies BRL (Carlsbad, CA), including reagents of molecular biology grade for gene expression studies. greater in GHS rat kidney. As the 7-kb CaR mrna is the predominant transcript, it was quantified in subsequent experiments. With the use of real time RT-PCR using isoform-specific primers, basal levels of renal cortical CaR mrna levels were increased 1.5-fold in GHS rats (P 0.05; Figure 2B). Although quantitative estimates of the fold increase in CaR mrna in GHS rat kidney differed between real-time RT-PCR (1.5-fold) and Northern blot (four-fold), both techniques are in agreement that the basal levels of CaR mrna were significantly elevated above NC. Effects of 1,25(OH) 2 D 3 The intraperitoneal administration of 1,25(OH) 2 D 3 (30 ng/100 g body wt) 24 h before killing increased renal cortical CaR mrna in kidneys from both NC and GHS rats (Figure 3). In a second experiment (Figure 4), 24 h after injection of 1,25(OH) 2 D 3 (30 ng/100 g body wt), CaR mrna increased 1.8-fold in GHS rat kidney and 3.3-fold in NC rat kidney. GHS CaR mrna remained higher (P 0.01) at this dose and at all doses studied. Lower (10 ng/100 g body wt) and higher (200 ng/100 g body wt) doses of 1,25(OH) 2 D 3 did not increase CaR mrna in either Quantitative Analysis After hybridization, radioactivity in the Northern blot bands of CaR mrna was quantified by scanning an autoradiogram of each gel (AMBIS; Scanalytics, Inc.). The intensity of the ribosomal RNA bands transferred onto GENEScreen membranes was visualized by ethidium bromide staining and analyzed densitometrically to ensure equal RNA sample loading. Values were normalized to the amount of RNA loaded and blotted. For optimizing group comparisons, samples of RNA extracted from GHS and NC rats were electrophoresed on the same gel and transferred onto the same membrane. Quantitative data were obtained from at least three separate experiments, each with four rats per experimental group. For permitting group comparisons and statistical analysis, densitometrically obtained intensity of the bands adjusted for RNA loading was expressed as relative RNA intensity. Statistical Analyses Data are presented as mean SEM. The significance of differences between the means of two groups was analyzed by t test, and statistically significant differences were taken at P When the means of more than two experimental groups were compared, significance of differences was determined by ANOVA. When ANOVA revealed significant group differences, post hoc testing for significance was performed using Tukey HSD procedure. Results Kidney CaR Protein Levels Semiquantitative analysis by Western blotting shows that CaR protein in kidney cortical tissue is four-fold higher in GHS rats (P 0.008; Figure 1). CaR mrna Both GHS and NC rats expressed low but readily detectable levels of the 7- and 4-kb transcripts of CaR mrna in kidney cortical tissue (Figure 2A). The 7-kb transcript was the dominant species of CaR mrna in both GHS and NC rats and was four-fold Figure 3. CaR mrna is induced by 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ]. Lanes are renal CaR mrna from wild-type control (N) and GHS (H) kidney at baseline ( ) and 24 h after administration of 1,25(OH) 2 D 3 ( 1,25D) 30 ng/100 g body weight intraperitoneally.

5 1304 Journal of the American Society of Nephrology J Am Soc Nephrol 16: , 2005 Figure 4. Dose response of CaR mrna to 1,25(OH) 2 D 3. CaR mrna from GHS (f) and NC ( ) kidney 24 h after a single intraperitoneal injection of 1,25(OH) 2 D 3 at doses of 10, 30, and 200 ng/100 g body wt. Values are mean SEM from three independent experiments. *P versus vehicle NC; **P versus vehicle GHS; P 0.01, 30 ng/100 g body wt. GHS or NC rats above vehicle-treated levels by 24 h after injection. The time course of response of CaR mrna levels to a single injection of 1,25(OH) 2 D 3 (30 ng/100 g body wt) is shown in Figure 5. Baseline CaR mrna relative intensities were set at 8 for GHS and NC groups. 1,25(OH) 2 D 3 significantly raised renal cortical CaR mrna in GHS rats as early as 4 h, reached peak levels by 24 h with a 4.5-fold increase, and returned toward baseline values at 48 h (Figure 5A). In NC rats, CaR mrna levels were increased above baseline by 6 h, reached peak levels by 24 h with a 3.3-fold increase above baseline, and returned to baseline by 48 h (Figure 5B). Thus, 1,25(OH) 2 D 3 stimulated CaR mrna in GHS and NC kidneys, and the incremental increases above baseline were not significantly different between the two experimental groups. The rise in CaR mrna seemed to be more sustained in GHS rats. For determining the role of gene transcription in the 1,25(OH) 2 D 3 mediated increase in CaR mrna in GHS rats, actinomycin D was administered to inhibit CaR gene expression. As shown in Figure 6, 8 h after 1,25(OH) 2 D 3 administration (30 ng/100 g body wt), the 7-kb CaR mrna was markedly upregulated (lane 2). Administration of actinomycin D1hbefore 1,25(OH) 2 D 3 blunted the anticipated 1,25(OH) 2 D 3 -induced rise in CaR mrna levels (lane 3). Eight hours after actinomycin D administration alone (lane 4), CaR mrna was reduced compared with control. However, 8 h of treatment with actinomycin D did not abolish the elevated CaR mrna levels induced by 24 h of 1,25(OH) 2 D 3 treatment (lane 5) These data indicate that 1,25(OH) 2 D 3 increased CaR mrna levels at least in part through new CaR gene transcription. The dependence of de novo protein synthesis on 1,25(OH) 2 D 3 - induced upregulation of renal CaR mrna was investigated in GHS rats. The 7-kb CaR mrna became undetectable by 8 h after the administration of cycloheximide (4 mg/100 g body wt intraperitoneally) alone (Figure 7, lane 4). The administration of cycloheximide 1 h before the administration of 1,25(OH) 2 D 3 (30 ng/100 g body wt) given 8 h before killing also suppressed the anticipated elevation of CaR mrna levels to below detection (Figure 7, lane 3). This experiment clearly shows that de novo synthesis of unidentified protein(s) participates in the expression of CaR mrna both at baseline and during 1,25(OH) 2 D 3 -induced upregulation. To estimate in vivo half-life of CaR mrna, gene transcription was inhibited by the administration of actinomycin D (see the Materials and Methods section for time of actinomycin D ad- Figure 5. Time course of CaR mrna response to 1,25(OH) 2 D 3. The administration of 1,25(OH) 2 D 3 30 ng/100 g body wt intraperitoneally increased CaR mrna levels in kidney from GHS (A) and NC (B) rats. Values are mean SEM for four rats per group. *P 0.05 versus baseline (0 time); **P 0.01 versus baseline.

6 J Am Soc Nephrol 16: , 2005 Renal Ca Receptor in Hypercalciuric Rat 1305 ministration), and CaR mrna levels were measured in the presence and absence of 1,25(OH) 2 D 3 at various time points. The in vivo half-life of CaR mrna was calculated on the basis of the decay of mrna levels using an established mathematical model (38). The in vivo half-life of renal CaR mrna under basal conditions was approximately 4 h in both GHS and NC rats (Figure 8). Twenty-four hours after treatment with 1,25(OH) 2 D 3, in vivo half-life of CaR mrna was significantly prolonged to longer than 8 h (Figure 9) in GHS rats. A more accurate estimate of half-life was unable to be determined because the experiment was stopped 8 h after actinomycin D administration, and the CaR mrna level had not fallen to 50% of baseline by that time. The experiment was not continued beyond 8 h because of the high mortality rate among the rats. Nevertheless, it is clear from inspection of the data that the administration of 1,25(OH) 2 D 3 markedly increased the stability of CaR mrna in GHS rats. Figure 7. Role of de novo protein synthesis in 1,25(OH) 2 D 3 - induced upregulation of CaR mrna. Lane 1, GHS control; lane 2, 1,25(OH) 2 D 3 30 ng/100 g body wt intraperitoneally and killed 8 h later; lane 3, cycloheximide 4 mg/100 g body wt 1 h before 1,25(OH) 2 D 3 and then killed 8 h later; lane 4, cycloheximide 8 h before killing. These values are representative of three independent experiments. Figure 6. Role of gene transcription in 1,25(OH) 2 D 3 upregulation of renal CaR mrna. Lane 1, GHS control; lane 2, 1,25(OH) 2 D 3 30 ng/100 g body wt intraperitoneally and killed 8 h later; lane 3, actinomycin D 4 g/100 g body wt (intraperitoneally) 1 h before 1,25(OH) 2 D 3 and killed 8 h later; lane 4, actinomycin D 4 g/100 g body wt (intraperitoneally) 8 h before killing; and lane 5, 1,25(OH) 2 D 3 24-h pretreatment and then treated with actinomycin D g/100 g body wt (intraperitoneally) 8 h before killing. Discussion This study clearly demonstrates that the cortical portion of the kidney of GHS rats contains elevated levels of CaR and CaR mrna. These observations support the potential role of CaR in the hypercalciuria of GHS rats. Breeding spontaneously hypercalciuric rats has established a colony of GHS male and female rats that have marked hypercalciuria and form upper urinary tract and parenchymal kidney stones composed of Ca salts (7,8,11,12,31 33,39,40). During adequate Ca intake, excess urine Ca is from both increased intestinal Ca transport and enhanced bone resorption (11,18). Dietary Ca restriction lowers urine Ca but not to the levels found in control rats (13). Significant stimulation of bone resorption during low and adequate Ca intakes is observed with 1,25(OH) 2 D 3, whereas parathyroid hormone (PTH)-stimulated bone resorption is not different between GHS and controls

7 1306 Journal of the American Society of Nephrology J Am Soc Nephrol 16: , 2005 Figure 8. In vivo half-life of renal CaR mrna. (A) Renal CaR mrna measured at baseline and up to 8 h after the in vivo administration of actinomycin D 4 g /100 g body wt intraperitoneally in wild-type control (NC, top) and GHS (GHS, bottom) kidney. (B) Each point is mean SEM of three independent experiments at each time point for NC (E) and GHS (F) rat kidney. No statistical comparisons were made between groups as they were analyzed on separate blots. Figure 9. Effect of 1,25(OH) 2 D 3 on in vivo half-life of renal CaR mrna in GHS rats. Each point is mean SEM of three independent experiments at each time point after the administration of 1,25(OH) 2 D 3 30 ng/100 g body wt (F) or vehicle control (E) at 0 time. *P 0.05 versus vehicle 4 h; **P 0.01 versus vehicle at6and8h. (18). Renal tubule Ca reabsorption is reduced in GHS rats during adequate and low Ca intakes and contributes to the hypercalciuria (13,19). The decreased tubule Ca reabsorption persists in the presence of fixed PTH blood levels and stable filtered load of Ca (19) and therefore is not due to a suppression of PTH. The mechanisms and nephron site(s) of the altered Ca reabsorption in GHS rats have not been identified. The elevated VDR levels in GHS rat intestine, bone, and kidney (12,18,24) have been demonstrated by standard saturation binding assay (12), semiquantitative Western blotting (12,13,24), and VDR ELISA (41). As serum 1,25(OH) 2 D 3 levels are normal and not elevated, the pathologic increases in VDR levels may be the major if not sole contributor to the intestinal Ca hyperabsorption and enhanced bone resorption. This study supports the potential role of VDR in mediating decreased renal tubule Ca reabsorption through elevated CaR protein and gene expression in GHS rat kidney. As the CaR gene contains vitamin D response elements in its promoter region (42), excess VDR and normal 1,25(OH) 2 D 3 may result in increased VDR-1,25(OH) 2 D 3 complexes and increased CaR gene expression in GHS rats. The CaR gene is one of numerous VDR-dependent genes that play an important role in modulating renal Ca transport in cortical and

8 J Am Soc Nephrol 16: , 2005 Renal Ca Receptor in Hypercalciuric Rat 1307 medullary nephron sites (35,42,43). Our study clearly demonstrates that the kidney of NC and GHS rats contains both 7- and 4-kb CaR gene transcripts and that GHS kidney tissue contains higher CaR mrna 7 kb. GHS kidney tissue contains four times more of the CaR protein compared with NC rats. In addition to increased CaR levels found in this study, GHS rat renal cortical tissue contains increased calbindin 9- and 28-kD mrna levels and increased calbindin 9- and 28-kD protein levels (41). The increased levels of proteins involved in renal Ca epithelial transport such as calbindin 28 kd and calbindin 9 kd, the elevated renal cortical VDR protein levels, and the elevated tissue levels of the CaR all suggest that the decrease in renal tubule Ca transport in GHS rats may be at least in part mediated by vitamin D regulated genes. Future studies are required in GHS rats to determine the regulation and function of CaR proteins and their location along the nephron. Previous studies have examined the effects of vitamin D status and 1,25(OH) 2 D 3 on CaR gene expression in normal rats. Basal renal CaR mrna is lower in vitamin D deficient rats (35,43), and 1,25(OH) 2 D 3 administration to vitamin D replete rats has been reported either to increase CaR mrna levels (35,42) or to have no effect (43). As described previously (27,42), our study observed a similar dominance of the 7-kb form of CaR mrna in renal cortical tissue from GHS and NC rats under basal conditions. Also, this study independently confirms that 1,25(OH) 2 D 3 significantly increases renal CaR mrna (27,35,42) and demonstrates that 1,25(OH) 2 D 3 -induced CaR gene upregulation is through both increase in CaR gene transcription (42) and prolongation of the CaR mrna half-life. Furthermore, de novo synthesis of as-yet-unidentified proteins is critical for the transcription and regulation of the CaR gene and its response to 1,25(OH) 2 D 3. We observed that 1,25(OH) 2 D 3 administration increased CaR mrna in both NC and GHS rats with similar incremental increases. As a result, because of the higher basal level, GHS rat renal CaR mrna remained higher after 1,25(OH) 2 D 3. One dose of 1,25(OH) 2 D 3 (30 ng/100 g body wt) increased renal CaR mrna in NC and GHS rats, whereas lower (10 ng/100 g body wt) and higher (200 ng/100 g body wt) doses failed to stimulate CaR mrna. However, the time course of response to 30 ng/100 g body wt included a rise in CaR mrna within 4 h that was followed by multiple measurements showing elevated levels over the 24 h after 1,25(OH) 2 D 3 injection. Therefore, at least the one dose of 1,25(OH) 2 D 3 is effective in raising CaR mrna, and this dose is equally effective in NC and GHS rat kidneys. In rat kidney, CaR gene expression has been identified in microdissected segments of proximal convoluted tubule, proximal straight tubule, thick ascending limb, distal convoluted tubule, and cortical collecting duct (44,45). As this study analyzed CaR mrna in cortical tissue in which these segments would be found, increases in CaR mrna levels in GHS and NC rats must occur in one or more of these segments. CaR mrna is also found in medullary thick ascending limb, outer medulla, and inner medullary collecting ducts (44). Functional studies in single in vitro perfused mouse cortical thick ascending limb demonstrate that activation of the CaR in this segment by Ca or the calcimimetic NPS R-467 directly inhibits PTH-dependent cellular Ca transport and indirectly inhibits passive Ca reabsorption (46). Although this mechanism may contribute to the decreased tubule Ca reabsorption, hypercalciuria, and inability to conserve Ca during dietary Ca restriction in GHS rats (13), the segmental localization of the increased CaR protein and CaR mrna and 1,25(OH) 2 D 3 -regulated CaR protein expression were not determined in this study. In summary, CaR and CaR mrna levels are increased in GHS rat kidney, and 1,25(OH) 2 D 3 increases CaR mrna through both gene transcription and stabilization of the mrna half-life. Pathologically elevated VDR levels in GHS rats may mediate the increased renal CaR mrna. Elevated CaR mrna increases CaR protein in the plasma membrane of Ca transporting tubule cells and thereby inhibits Ca transport in one or more nephron segments. These changes in Ca transport could contribute to the hypercalciuria and stone formation potential characteristic of GHS rats. Acknowledgment This work was supported by grant 1 PO1 DK56788 from the National Institutes of Health. 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