The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2–null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2–null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.

Significance: Significant calcium absorption across renal and intestinal epithelia occurs via the paracellular pathway. However, the identity of the paracellular pore involved is unknown. Claudin-2 and claudin-12 contribute paracellular calcium permeability in cell models, but single knockout animals don’t have altered serum calcium or bone mineralization. To investigate this, Cldn2/12 double knockout mice were generated. They display decreased intestinal calcium absorption and renal calcium wasting, resulting in hypocalcemia and markedly reduced bone mineralization. Claudin-2 and claudin-12 don’t physically interact in vitro, and coexpression has an additive effect on calcium permeability. Our work identifies claudin-2 and claudin-12 as important constituents of the paracellular Ca ²⁺ pathway in intestine and kidney enabling calcium transport and highlights their important complementary roles in maintaining calcium homeostasis.

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (P_Ca). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with β-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and P_Ca was measured. P_Ca in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of P_Ca in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

Calcium and phosphate are critical for numerous physiological processes. Consequently, the plasma concentration of these ions are tightly regulated. Calcitriol, the active form of vitamin D, is a positive modulator of mineralization as well as calcium and phosphate metabolism. The molecular and physiological effects of calcitriol are well documented. Calcitriol increases blood calcium and phosphate levels by increasing absorption from the intestine, and resorption of bone. Calcitriol synthesis is a multistep process. A precursor is first made via skin exposure to UV, it is then 25-hydroxylated in the liver to form 25-hydroxyitamin D. The next hydroxylation step occurs in the renal proximal tubule via the 1-αhydroxylase enzyme (encoded by CYP27B1) thereby generating 1,25-dihydroxyvitamin D, that is, calcitriol. At the same site, the 25-hydroxyvitamin D 24-hydroxlase enzyme encoded by CYP24A1 can hydroxylate 25-hydroxyvitamin D or calcitriol to deactivate the hormone. Plasma calcitriol levels are primarily determined by the regulated expression of CYP27B1 and CYP24A1. This occurs in response to parathyroid hormone (increases CYP27B1), calcitriol itself (decreases CYP27B1 and increases CYP24A1), calcitonin (increases or decreases CYP24A1 and increases CYP27B1), FGF23 (decreases CYP27B1 and increases CYP24A1) and potentially plasma calcium and phosphate levels themselves (mixed effects). Herein, we review the regulation of CYP27B1 and CYP24A1 transcription in response to the action of classic phophocalciotropic hormones and explore the possibility of direct regulation by plasma calcium.

Plasma membrane Ca²⁺-ATPases (PMCAs) participate in epithelial Ca²⁺transport and intracellular Ca²⁺signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial transient receptor potential vanilloid 5 Ca²⁺channel. We therefore hypothesized that Pmca4 plays a significant role in transcellular Ca²⁺flux and investigated the localization and regulation of Pmca4 in Ca²⁺-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestines, whereas pan-specific Pmca antibodies detected Pmca1 in lateral membranes of enterocytes. In the kidney, Pmca4 showed broad localization to the distal nephron. In the mouse, expression was most abundant in segments coexpressing the epithelial ransient receptor potential vanilloid 5 Ca²⁺channel. Significant, albeit lower, expression was also evident in the region encompassing the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In the human kidney, a similar pattern of distribution was observed, with the highest PMCA4 expression in Na⁺-Cl⁻cotransporter-positive tubules. Electron microscopy demonstrated Pmca4 localization in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca²⁺balance, pointing to a housekeeping function of the pump in Ca²⁺-transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca²⁺-transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca²⁺transport.

Plasma calcium (Ca ²⁺ ) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca ²⁺ -sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca ²⁺ absorption by modulating 1,25-dihydroxyvitamin D ₃ production, bone resorption, and renal Ca ²⁺ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia, a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium; however, a direct role in regulating local intestinal Ca ²⁺ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca ²⁺ absorption. In Ussing chambers, increasing extracellular Ca ²⁺ or basolateral application of the calcimimetic cinacalcet decreased net Ca ²⁺ absorption across intestinal preparations acutely. Conversely, Ca ²⁺ absorption increased with decreasing extracellular Ca ²⁺ concentration. These responses were absent in mice expressing a nonfunctional TRPV6, TRPV6 ^D541A . Cinacalcet also attenuated Ca ²⁺ fluxes through TRPV6 in Xenopus oocytes when coexpressed with the CaSR. Moreover, the phospholipase C inhibitor U73122 prevented cinacalcet-mediated inhibition of Ca ²⁺ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca ²⁺ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.