Claudin-2 and claudin-12 form independent, complementary pores required to maintain calcium homeostasis.

Calcium (Ca2+) 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 Ca2+ pore remains to be delineated. Claudins (Cldn)-2 and -12 confer Ca2+ permeability, but deletion of either claudin does not result in a negative Ca2+ 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 Ca2+ absorption. Colonic Ca2+ permeability is also reduced in DKO mice and significantly lower than single-null animals, while small intestine Ca2+ permeability is unaltered. The DKO mice display significantly greater urinary Ca2+ 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 Ca2+ permeability in cell models with endogenous expression of the other claudin. We find claudin-2 and claudin-12 form partially redundant, independent Ca2+ permeable pores in renal and colonic epithelia that enable paracellular Ca2+ (re)absorption in these segments, with either one sufficient to maintain Ca2+ balance.

Kidney stones, hypercalciuria, and recent insights into proximal tubule calcium reabsorption.

PURPOSE OF REVIEW: Most kidney stones are composed of calcium, and the greatest risk factor for kidney stone formation is hypercalciuria. Patients who form kidney stones often have reduced calcium reabsorption from the proximal tubule, and increasing this reabsorption is a goal of some dietary and pharmacological treatment strategies to prevent kidney stone recurrence. However, until recently, little was known about the molecular mechanism that mediates calcium reabsorption from the proximal tubule. This review summarizes newly uncovered key insights and discusses how they may inform the treatment of kidney stone formers. RECENT FINDINGS: Studies examining claudin-2 and claudin-12 single and double knockout mice, combined with cell culture models, support complementary independent roles for these tight junction proteins in contributing paracellular calcium permeability to the proximal tubule. Moreover, a family with a coding variation in claudin-2 causing hypercalciuria and kidney stones have been reported, and reanalysis of Genome Wide Association Study (GWAS) data demonstrates an association between noncoding variations in CLDN2 and kidney stone formation. SUMMARY: The current work begins to delineate the molecular mechanisms whereby calcium is reabsorbed from the proximal tubule and suggests a role for altered claudin-2 mediated calcium reabsorption in the pathogenesis of hypercalciuria and kidney stone formation.

Gentamicin Inhibits Ca2+ Channel TRPV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway.

BACKGROUND: Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal Ca2+ wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. METHODS: We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient receptor potential vanilloid 5 (TRPV5), as determined by patch clamp in HEK293 cells. RESULTS: Gentamicin increased urinary Ca2+ excretion in wild-type mice after acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/- mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and chronic gentamicin administration downregulated distal nephron Ca2+ transporters. CONCLUSIONS: Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into Ca2+ wasting in patients treated with gentamicin.

Activation of the calcium sensing receptor increases claudin-14 expression via a PLC -p38-Sp1 pathway.

Activation of the basolateral calcium sensing receptor (CaSR) in the renal tubular thick ascending limb (TAL) increases claudin-14 expression, which reduces paracellular calcium (Ca2+ ) permeability, thus increasing urinary Ca2+ excretion. However, the upstream signaling pathway contributing to altered CLDN14 gene expression is unknown. To delineate this pathway, we identified and then cloned the CaSR responsive region including the promoter of mouse Cldn14 into a luciferase reporter vector. This 1500 bp sequence upstream of the 5' UTR of Cldn14 variant 1, conferred increased reporter activity in the presence of high extracellular Ca2+ (5 mM) relative to a lower (0.5 mM) concentration. Assessment of Cldn14 reporter activity in response to increased extracellular Ca2+ in the presence or absence of specific inhibitors confirmed signaling through PLC and p38, but not JNK. Overexpression of SP1 attenuated Cldn14 reporter activity in response to CasR signaling. SP1 is expressed in the TAL and phosphorylation was attenuated by CaSR signaling. Finally, activating mutations in the CaSR increased Cldn14 reporter activity while a dominant negative mutation in the CaSR inhibited it. Together, these studies suggest that basolateral activation of the CASR leads to increased Cldn14 expression via a PLC- stimulated p38 pathway that prevents Sp1 mediated repression.

Molecular mechanisms altering tubular calcium reabsorption.

The majority of calcium filtered by the glomerulus is reabsorbed along the nephron. Most is reabsorbed from the proximal tubule (> 60%) via a paracellular pathway composed of the tight junction proteins claudins-2 and -12, a process driven by sodium and consequently water reabsorption. The thick ascending limb reabsorbs the next greatest amount of calcium (20-25%), also by a paracellular pathway composed of claudins-16 and -19. This pathway is regulated by the CaSR, whose activity increases the expression of claudin-14, a protein that blocks paracellular calcium reabsorption. The fine tuning of urinary calcium excretion occurs in the distal convoluted and connecting tubule by a transcellular pathway composed of the apical calcium channel TRPV5, the calcium shuttling protein calbindin-D28K and the basolateral proteins PMCA1b and the sodium calcium exchanger, NCX. Not surprisingly, mutations in a subset of these genes cause monogenic disorders with hypercalciuria as a part of the phenotype. More commonly, "idiopathic" hypercalciuria is encountered clinically with genetic variations in CLDN14, the CASR and TRPV5 associating with kidney stones and increased urinary calcium excretion. An understanding of the molecular pathways conferring kidney tubular calcium reabsorption is employed in this review to help explain how dietary and medical interventions for this disorder lower urinary calcium excretion.

The intermembrane space protein Mix23 is a novel stress-induced mitochondrial import factor.

The biogenesis of mitochondria requires the import of hundreds of precursor proteins. These proteins are transported post-translationally with the help of chaperones, meaning that the overproduction of mitochondrial proteins or the limited availability of chaperones can lead to the accumulation of cytosolic precursor proteins. This imposes a severe challenge to cytosolic proteostasis and triggers a specific transcription program called the mitoprotein-induced stress response, which activates the proteasome system. This coincides with the repression of mitochondrial proteins, including many proteins of the intermembrane space. In contrast, herein we report that the so-far-uncharacterized intermembrane space protein Mix23 is considerably up-regulated when mitochondrial import is perturbed. Mix23 is evolutionarily conserved and a homolog of the human protein CCDC58. We found that, like the subunits of the proteasome, Mix23 is under control of the transcription factor Rpn4. It is imported into mitochondria by the mitochondrial disulfide relay. Mix23 is critical for the efficient import of proteins into the mitochondrial matrix, particularly if the function of the translocase of the inner membrane 23 is compromised such as in temperature-sensitive mutants of Tim17. Our observations identify Mix23 as a novel regulator or stabilizer of the mitochondrial protein import machinery that is specifically up-regulated upon mitoprotein-induced stress conditions.

Renal claudin-14 expression is not required for regulating Mg2+ balance in mice.

The kidneys play a crucial role in maintaining Ca2+ and Mg2+ homeostasis by regulating these minerals' reabsorption. In the thick ascending limb of Henle's loop (TAL), Ca2+ and Mg2+ are reabsorbed through the tight junctions by a shared paracellular pathway formed by claudin-16 and claudin-19. Hypercalcemia activates the Ca2+-sensing receptor (CaSR) in the TAL, causing upregulation of pore-blocking claudin-14 (CLDN14), which reduces Ca2+ and Mg2+ reabsorption from this segment. In addition, a high-Mg2+ diet is known to increase both urinary Mg2+ and Ca2+ excretion. Since Mg2+ may also activate CaSR, we aimed to investigate whether CaSR-dependent increases in CLDN14 expression also regulate urinary Mg2+ excretion in response to hypermagnesemia. Here, we show that a Mg2+-enriched diet increased urinary Mg2+ and Ca2+ excretion in mice; however, this occurred without detectable changes in renal CLDN14 expression. The administration of a high-Mg2+ diet to Cldn14-/- mice did not cause more pronounced hypermagnesemia or significantly alter urinary Mg2+ excretion. Finally, in vitro evaluation of CaSR-driven Cldn14 promoter activity in response to increasing Mg2+ concentrations revealed that Cldn14 expression only increases at supraphysiological extracellular Mg2+ levels. Together, these results suggest that CLDN14 is not involved in regulating extracellular Mg2+ balance following high dietary Mg2+ intake.NEW & NOTEWORTHY Using transgenic models and in vitro assays, this study examined the effect of Mg2+ on regulating urinary excretion of Ca2+ and Mg2+ via activation of the Ca2+-sensing receptor-claudin 14 (CLDN14) pathway. The study suggests that CLDN14 is unlikely to play a significant role in the compensatory response to hypermagnesemia.

Localization and regulation of claudin-14 in experimental models of hypercalcemia.

Variations in the claudin-14 (CLDN14) gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in the kidney is localized exclusively to the thick ascending limbs (TALs), mainly restricted to the cortical and upper medullary portion of the kidney. However, not all cells in the TALs expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16 but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14 showed a restricted localization pattern in the apical domain of select cells of the TAL.

Intestinal resection affects whole-body arginine synthesis in neonatal piglets.

BACKGROUND: Previous studies in piglets show a direct relationship between intestinal mass and arginine (Arg) synthesis. We aimed to study the effects of 75% intestinal resection on whole-body Arg synthesis. METHODS: Piglets were allocated to sham or jejunocolic (JC) surgery and to enteral nutrition (EN) at 20% [sham (n = 8), JC (n = 10)], or 40% [sham (n = 4), JC (n = 5)]. A gastric tube was placed for EN and a venous catheter for parenteral nutrition and blood sampling. On day 6, a primed bolus and constant infusion of Arg m + 2 label and proline m + 1 label was delivered. In addition, 40% EN piglets received a citrulline (Cit) m + 3 tracer. Blood sampling was undertaken and whole-body Arg synthesis was calculated. On day 7, intestinal length was measured, and samples were collected for gene expression (PCR quantification) and histopathology. RESULTS: On Day 7, sham piglets showed intestinal lengthening compared to JC (p = 0.02). Whole-body Arg synthesis was similar between groups (p = 0.50). Adjusting for absolute small intestinal length, JC piglets had greater Arg synthesis (p = 0.01). Expression of arginosuccinase was upregulated in the jejunum of JC compared to sham on 20% EN (p = 0.03). CONCLUSION: This demonstrates for the first-time adaptive changes in intestinal Arg synthesis following intestinal resection. IMPACT: The intestine makes a critical contribution to whole-body arginine synthesis, particularly in neonates, a human population at risk for short bowel syndrome. Therefore, we studied intestinal arginine synthesis in a neonatal piglet model of short bowel syndrome and demonstrated adaptive changes in the intestine that may preserve whole-body arginine synthesis, despite loss of intestinal mass. This research adds new information to our understanding of the effects a massive intestinal resection has on amino acid metabolism during neonatal development.

The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 gene, encoding the polycystic kidney disease protein polycystin-1 and the transient receptor potential channel polycystin-2 (also known as TRPP2), respectively. Polycystin-1 and polycystin-2 form a receptor-ion channel complex located in primary cilia. The function of this complex, especially the role of polycystin-1, is largely unknown due to the lack of a reliable functional assay. In this study, we dissect the role of polycystin-1 by directly recording currents mediated by a gain-of-function (GOF) polycystin-1/polycystin-2 channel. Our data show that this channel has distinct properties from that of the homomeric polycystin-2 channel. The polycystin-1 subunit directly contributes to the channel pore, and its eleven transmembrane domains are sufficient for its channel function. We also show that the cleavage of polycystin-1 at the N-terminal G protein-coupled receptor proteolysis site is not required for the activity of the GOF polycystin-1/polycystin-2 channel. These results demonstrate the ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex, enriching our understanding of this channel and its role in ADPKD.

Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells.

The tight junctional pore-forming protein claudin-2 (CLDN-2) mediates paracellular Na+ and water transport in leaky epithelia and alters cancer cell proliferation. Previously, we reported that tumor necrosis factor-α time-dependently alters CLDN-2 expression in tubular epithelial cells. Here, we found a similar expression pattern in a mouse kidney injury model (unilateral ureteral obstruction), consisting of an initial increase followed by a drop in CLDN-2 protein expression. CLDN-2 silencing in LLC-PK1 tubular cells induced activation and phosphorylation of guanine nucleotide exchange factor H1 (GEF-H1), leading to Ras homolog family member A (RHOA) activation. Silencing of other claudins had no such effects, and re-expression of an siRNA-resistant CLDN-2 prevented RHOA activation, indicating specific effects of CLDN-2 on RHOA. Moreover, kidneys from CLDN-2 knockout mice had elevated levels of active RHOA. Of note, CLDN-2 silencing reduced LLC-PK1 cell proliferation and elevated expression of cyclin-dependent kinase inhibitor P27 (P27KIP1) in a GEF-H1/RHOA-dependent manner. P27KIP1 silencing abrogated the effects of CLDN-2 depletion on proliferation. CLDN-2 loss also activated myocardin-related transcription factor (MRTF), a fibrogenic RHOA effector, and elevated expression of connective tissue growth factor and smooth muscle actin. Finally, CLDN-2 down-regulation contributed to RHOA activation and smooth muscle actin expression induced by prolonged tumor necrosis factor-α treatment, because they were mitigated by re-expression of CLDN-2. Our results indicate that CLDN-2 suppresses GEF-H1/RHOA. CLDN-2 down-regulation, for example, by inflammation, can reduce proliferation and promote MRTF activation through RHOA. These findings suggest that the initial CLDN-2 elevation might aid epithelial regeneration, and CLDN-2 loss could contribute to fibrotic reprogramming.

Claudin-12 Knockout Mice Demonstrate Reduced Proximal Tubule Calcium Permeability.

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 (PCa). 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 ß-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 PCa was measured. PCa 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 PCa 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.

Ces1d deficiency protects against high-sucrose diet-induced hepatic triacylglycerol accumulation.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Triacylglycerol accumulation in the liver is a hallmark of NAFLD. Metabolic studies have confirmed that increased hepatic de novo lipogenesis (DNL) in humans contributes to fat accumulation in the liver and to NAFLD progression. Mice deficient in carboxylesterase (Ces)1d expression are protected from high-fat diet-induced hepatic steatosis. To investigate whether loss of Ces1d can also mitigate steatosis induced by over-activated DNL, WT and Ces1d-deficient mice were fed a lipogenic high-sucrose diet (HSD). We found that Ces1d-deficient mice were protected from HSD-induced hepatic lipid accumulation. Mechanistically, Ces1d deficiency leads to activation of AMP-activated protein kinase and inhibitory phosphorylation of acetyl-CoA carboxylase. Together with our previous demonstration that Ces1d deficiency attenuated high-fat diet-induced steatosis, this study suggests that inhibition of CES1 (the human ortholog of Ces1d) might represent a novel pharmacological target for prevention and treatment of NAFLD.

NHE8 attenuates Ca2+ influx into NRK cells and the proximal tubule epithelium.

To garner insights into the renal regulation of Ca2+ homeostasis, we performed an mRNA microarray on kidneys from mice treated with the Ca2+-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 Ca2+ 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 Ca2+ balance, we measured changes in intracellular Ca2+ uptake by live cell Ca2+ imaging with the fluorophore Fura-2 AM. Inhibition of NHE8 with EIPA or by removing extracellular Na+-enhanced Ca2+ influx into NRK cells. Ca2+ influx was mediated by a voltage-dependent Ca2+ channel rather than directly via NHE8. NRK cells express Cav1.3 and display verapamil-sensitive Ca2+ influx and NHE8 inhibition-augmented Ca2+ influx via a voltage-dependent Ca2+ channel. Finally, proximal tubules perused ex vivo demonstrated increased Ca2+ 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 Ca2+ uptake into the proximal tubule epithelium.

H+-ATPase B1 subunit localizes to thick ascending limb and distal convoluted tubule of rodent and human kidney.

The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.

Ubiquitin COOH-terminal hydrolase L1 deletion is associated with urinary α-klotho deficiency and perturbed phosphate homeostasis.

Loss of ubiquitin COOH-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme required for neuronal function, led to hyperphosphatemia accompanied by phosphaturia in mice, while calcium homeostasis remained intact. We therefore investigated the mechanisms underlying the phosphate imbalance in Uchl1-/- mice. Interestingly, phosphaturia was not a result of lower renal brush border membrane sodium-phosphate cotransporter expression as sodium-phosphate cotransporter 2a and 2c expression levels was similar to wild-type levels. Plasma parathyroid hormone and fibroblast growth factor 23 levels were not different; however, fibroblast growth factor 23 mRNA levels were significantly increased in femur homogenates from Uchl1-/- mice. Full-length and soluble α-klotho levels were comparable in kidneys from wild-type and Uchl1-/- mice; however, soluble α-klotho was reduced in Uchl1-/- mice urine. Consistent with unchanged components of 1,25(OH)2D3 metabolism (i.e., CYP27B1 and CYP24A1), sodium-phosphate cotransporter 2b protein levels were not different in ileum brush borders from Uchl1-/- mice, suggesting that the intestine is not the source of hyperphosphatemia. Nonetheless, when Uchl1-/- mice were fed a low-phosphate diet, plasma phosphate, urinary phosphate, and fractional excretion of phosphate were significantly attenuated and comparable to levels of low-phosphate diet-fed wild-type mice. Our findings demonstrate that Uchl1-deleted mice exhibit perturbed phosphate homeostasis, likely consequent to decreased urinary soluble α-klotho, which can be rescued with a low-phosphate diet. Uchl1-/- mice may provide a useful mouse model to study mild perturbations in phosphate homeostasis.

Claudins and nephrolithiasis.

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.

Double Knockout of the Na+-Driven Cl-/HCO3- Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion.

We recently described a novel thiazide-sensitive electroneutral NaCl transport mechanism resulting from the parallel operation of the Cl-/HCO3- exchanger pendrin and the Na+-driven Cl-/2HCO3- exchanger (NDCBE) in ß-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na+ balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na+ balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na+ homeostasis and provide evidence that the Na+/Cl- cotransporter (NCC) compensated for the inactivation of NDCBE. To unmask the role of NDCBE, we generated Ndcbe/Ncc double-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K+ concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca2+-activated K+ channel BKCa. During NaCl depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCl depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K+ concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients.

A variant in a cis-regulatory element enhances claudin-14 expression and is associated with pediatric-onset hypercalciuria and kidney stones.

The greatest risk factor for kidney stones is hypercalciuria, the etiology of which is largely unknown. A recent genome-wide association study (GWAS) linked hypercalciuria and kidney stones to a claudin-14 (CLDN14) risk haplotype. However, the underlying molecular mechanism was not delineated. Recently, renal CLDN14 expression was found to increase in response to increased plasma calcium, thereby inducing calciuria. We hypothesized therefore that some children with hypercalciuria and kidney stones harbor a CLDN14 variant that inappropriately increases gene expression. To test this hypothesis, we sequenced the CLDN14 risk haplotype in a cohort of children with idiopathic hypercalciuria and kidney stones. An intronic SNP was more frequent in affected children. Dual luciferase and cell-based assays demonstrated increased reporter or CLDN14 expression when this polymorphism was introduced. In silico studies predicted the SNP introduced a novel insulinoma-associated 1 (INSM1) transcription factor binding site. Consistent with this, repeating the dual luciferase assay in the presence of INSM1 further increased reporter expression. Our data suggest that children with the INSM1 binding site within the CLDN14 risk haplotype have a higher likelihood of hypercalciuria and kidney stones. Enhanced CLDN14 expression may play a role in the pathophysiology of their hypercalciuria.