Deficiency of the calcium-sensing receptor in the kidney causes parathyroid hormone-independent hypocalciuria

Abstract

Rare loss-of-function mutations in the calcium-sensing receptor (Casr) gene lead to decreased urinary calcium excretion in the context of parathyroid hormone (PTH)-dependent hypercalcemia, but the role of Casr in the kidney is unknown. Using animals expressing Cre recombinase driven by the Six2 promoter, we generated mice that appeared grossly normal but had undetectable levels of Casr mRNA and protein in the kidney. Baseline serum calcium, phosphorus, magnesium, and PTH levels were similar to control mice. When challenged with dietary calcium supplementation, however, these mice had significantly lower urinary calcium excretion than controls (urinary calcium to creatinine, 0.31±0.03 versus 0.63±0.14; P=0.001). Western blot analysis on whole-kidney lysates suggested an approximately four-fold increase in activated Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). In addition, experimental animals exhibited significant downregulation of Claudin14, a negative regulator of paracellular cation permeability in the thick ascending limb, and small but significant upregulation of Claudin16, a positive regulator of paracellular cation permeability. Taken together, these data suggest that renal Casr regulates calcium reabsorption in the thick ascending limb, independent of any change in PTH, by increasing the lumen-positive driving force for paracellular Ca(2+) transport.

Figure 1.

Wild-type Casr gene, E3 targeting construct, and genotype analysis. (A) E2–E4 of the wild-type Casr gene. (B) A Sal1 restriction site in intron 2, the neomycin resistance gene (Neo; gray box) in intron 3 and LoxP sites (green arrowheads) were introduced into the targeting construct. Locations of genotyping primers are shown. (C) Excised allele. (D) Successful amplification with Postneo PCR (primers Casr-F/Postneo-R) indicates excision of E3 and Neo and presence of a Casr null (floxed) allele. Postneo PCR from Casr flox allele does not amplify due to large amplicon size. The absence of E3 amplification (primers Casr-E3-F/Casr-E3-R) indicates complete knockout of the Casr in targeted tissues. Neo PCR amplification (primers Neo-F/Neo-R) indicates presence of the Casr flox allele. (E) Amplification with primers Casr-F/Casr-E3-R followed by SalI digest indicates presence of the Casr flox allele.

Figure 2.

Casr expression in germline and conditional Casr-deficient models. (A) PCR amplification of whole-kidney cDNA generates E3-deleted amplicon of 200 bp compared with the nonexcised 500 bp amplicon. Whole-kidney cDNA from Casr^flox/- mice show both the intact and the E3-deleted amplicons. (B) QPCR from whole-kidney cDNA with Casr primers located in E2 and E3 shows no Casr expression in null mice compared with age- and sex- matched control animals (P<0.001). (C) Immunoblot analysis of whole-kidney homogenate with the ADD antibody (targeting a peptide sequence encoded by E4). No Casr protein was detected in Casr null mice and in Six2-Cre Casr floxed mice (on both +/+ and +/− backgrounds). CTL, control.

Figure 3.

Casr immunohistochemistry in control and Six2-Cre Casr floxed animals. (A) Immunohistochemistry with anti-Casr antibody shows Casr expression in renal tubular epithelia of control animals. (B) Six2-Cre Casr floxed animals show absence of Casr expression. (C) Magnified image of A suggests Casr expression in tubular structures consistent with TAL epithelia. (D and E) Immunohistochemistry of adjacent sections to C showing expression patterns of NKCC2 and Aquaporin 2, respectively, suggesting NKCC2 colocalization with Casr in TAL epithelia, but not the CD epithelia-specific Aquaporin 2. CTL, control.

Figure 4.

Selected serum chemistries in age- and sex-matched Six2-Cre Casr floxed animals. On regular chow 0.8% calcium diet (A–C) and with dietary calcium challenge (D–F), no significant differences were observed between Casr-deficient and control mice in serum Ca²⁺ (A and D), PTH (B and E), and serum phosphate levels (C and F). CTL, control.

Figure 5.

Urine calcium to creatinine ratios. (A) Urinary calcium to creatinine ratios were significantly lower in the Six2-Cre Casr floxed mice than in control animals (P=0.003). (B) Addition to drinking water of 1.5% calcium chloride increased the urine calcium to creatinine ratio in control animals much higher than in Six2-Cre Casr floxed mice (P=0.001). (C) Control mice and Six2-Cre Casr floxed mice were injected with furosemide (25 mg/kg body weight). The discrepancy in the urinary calcium to creatinine ratio between the two groups observed at baseline (P=0.03) was no longer evident 2 hours after intraperitoneal injection (P=0.73). (D) No significant differences were observed in serum Mg²⁺ levels between experimental and control mice. (E) Serum Mg²⁺ levels increased equally between both groups with dietary calcium challenge; Mg²⁺ levels in experimental mice remained slightly higher. (F) Mean urinary Mg²⁺ to creatinine ratios (0.035±0.008 versus 0.035±0.004; P=0.74) at baseline did not differ. (G) Serum 1,25 vitamin D levels were increased in experimental mice; however, the difference was not significant. (H) CYP27B1 (1-α hydroxylase) expression levels were approximately three-fold increased in Six2-Cre Casr floxed mice (P<0.001). CTL, control; i.p., intraperitoneal.

Figure 6.

NKKC2 expression. Quantitative PCR for NKCC2 from kidney tissue showed no difference in levels of mRNA (A) or protein (B). (C and D) Phospho-NKCC2 Western blot analysis revealed increased activation of NKCC2 in the same mouse kidney samples. Phospho-NKCC2 showed on average approximately four-fold increased expression in Six2-Cre Casr floxed mice (P<0.001). CTL, control.

Figure 7.

Expression of Claudins. (A) Expression data from whole-kidney RNA for Claudin14 showed approximately 80% downregulation in Six2-Cre floxed mice (P<0.001). (B) In contrast, Claudin16 expression showed a small but significant increase in experimental animals (P=0.002). (C) No difference was observed in Claudin19 expression (P=0.72). Expression levels of two microRNAs, miR-9 (D) and miR-374 (E), implicated in Claudin14 expression in in vitro studies, showed no significant difference in experimental versus control mice. CTL, control.