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Calcium (Ca 2+ ) homeostasis is maintained through coordination between intestinal absorption, renal reabsorption, and bone remodeling. Intestinal and renal (re)absorption occurs via transcellular and paracellular pathways. The latter contributes the bulk of (re)absorption under conditions of adequate intake. Epithelial paracellular permeability is conferred by tight-junction proteins called claudins. However, the molecular identity of the paracellular Ca 2+ pore remains to be delineated. Claudins ( Cldn )-2 and -12 confer Ca 2+ permeability, but deletion of either claudin does not result in a negative Ca 2+ balance or increased calciotropic hormone levels, suggesting the existence of additional transport pathways or parallel roles for the two claudins. To test this, we generated a Cldn2/12 double knockout mouse (DKO). These animals have reduced intestinal Ca 2+ absorption. Colonic Ca 2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca 2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca 2+ wasting than Cldn2 null animals. These perturbations lead to hypocalcemia and reduced bone mineral density, which was not observed in single-KO animals. Both claudins were localized to colonic epithelial crypts and renal proximal tubule cells, but they do not physically interact in vitro. Overexpression of either claudin increased Ca 2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca 2+ permeable pores in renal and colonic epithelia that enable paracellular Ca 2+ (re)absorption in these segments, with either one sufficient to maintain Ca 2+ balance., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

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.

To garner insights into the renal regulation of Ca 2+ homeostasis, we performed an mRNA microarray on kidneys from mice treated with the Ca 2+ -sensing receptor (CaSR) agonist cinacalcet. This revealed decreased gene expression of Na + /H + exchanger isoform 8 (NHE8) in response to CaSR activation. These results were confirmed by quantitative real-time PCR. Moreover, administration of vitamin D also decreased NHE8 mRNA expression. In contrast, renal NHE8 protein expression from the same samples was increased. To examine the role of NHE8 in transmembrane Ca 2+ fluxes, we used the normal rat kidney (NRK) cell line. Cell surface biotinylation and confocal immunofluorescence microscopy demonstrated NHE8 apical expression. Functional experiments found 5-( N -ethyl- N -isopropyl)amiloride (EIPA)-inhibitable NHE activity in NRK cells at concentrations minimally attenuating NHE1 activity in AP-1 cells. To determine how NHE8 might regulate Ca 2+ balance, we measured changes in intracellular Ca 2+ uptake by live cell Ca 2+ imaging with the fluorophore Fura-2 AM. Inhibition of NHE8 with EIPA or by removing extracellular Na + -enhanced Ca 2+ influx into NRK cells. Ca 2+ influx was mediated by a voltage-dependent Ca 2+ channel rather than directly via NHE8. NRK cells express Cav1.3 and display verapamil-sensitive Ca 2+ influx and NHE8 inhibition-augmented Ca 2+ influx via a voltage-dependent Ca 2+ channel. Finally, proximal tubules perused ex vivo demonstrated increased Ca 2+ influx in the presence of luminal EIPA at a concentration that would inhibit NHE8. The results of the present study are consistent with NHE8 regulating Ca 2+ uptake into the proximal tubule epithelium.

Purpose of Review: The greatest risk factor for kidney stone formation is increased urinary calcium excretion. Most filtered calcium is reabsorbed from the proximal tubule and the thick ascending limb (TAL) of Henle's loop via a paracellular pathway. Claudins are tight junction proteins that confer the permeability properties of an epithelium. We review the contribution of renal claudins to nephron calcium permeability and how perturbations in these pathways cause alterations in tubular calcium transport, hypercalciuria, nephrocalcinosis, or nephrolithiasis., Recent Findings: Claudin-16 and Claudin-19 form a complex with claudin-3 enabling divalent cation permeability in the TAL. Claudin-14 interacts with claudin-16 to attenuate calcium permeability through this pore. Intronic mutations in claudin-14 increase expression causing hypercalciuria and kidney stones. A different type of TAL tight junction pore is composed of claudin-10b, which does not preferentially permeate calcium. Deletion of claudin-10b results in increased expression of the claudin-16/claudin-19 complex expressed in the medullary TAL and nephrocalcinosis., Summary: Alterations to claudins expressed in the TAL tight junction greatly affects calcium homeostasis as highlighted by point mutations in claudin-16 or claudin-19 causing FHHNC or gain of function mutations in claudin-14 causing kidney stones.