Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is an inherited disorder caused by mutations in the genes encoding the TJ proteins claudin-16 (CLDN16) and CLDN19; however, the mechanisms underlying the roles of these claudins in mediating paracellular ion reabsorption in the kidney are not understood. Here we showed that in pig kidney epithelial cells, CLDN19 functioned as a Cl − blocker, whereas CLDN16 functioned as a Na + channel. Mutant forms of CLDN19 that are associated with FHHNC were unable to block Cl − permeation. Coexpression of CLDN16 and CLDN19 generated cation selectivity of the TJ in a synergistic manner, and CLDN16 and CLDN19 were observed to interact using several criteria. In addition, disruption of this interaction by introduction of FHHNC-causing mutant forms of either CLDN16 or CLDN19 abolished their synergistic effect. Our data show that CLDN16 interacts with CLDN19 and that their association confers a TJ with cation selectivity, suggesting a mechanism for the role of mutant forms of CLDN16 and CLDN19 in the development of FHHNC. IntroductionThe human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC; OMIM 248250) is characterized by progressive renal Mg 2+ and Ca 2+ wasting leading to impaired renal function and chronic renal failure. FHHNC has been genetically linked to mutations in the gene of claudin-16 (CLDN16, also known as paracellin-1) (1) and more recently to CLDN19 (2). The claudins comprise a 22-gene family that encodes essential structural proteins of the tight junction (TJ), which are the principal regulators of paracellular permeability. In vitro studies have shown that ion selectivity of the paracellular conductance (reviewed in ref.3) is a complex function of claudin subtype and cellular context (4, 5).In vitro analyses using cultured cell models show that CLDN16 plays a key role in maintaining the cation selectivity of the TJ and forms a nonselective paracellular cation channel (4). This hypothesis of a nonselective paracellular cation channel is supported by (a) a clinical study to correlate the cellular functions of CLDN16 mutations identified in FHHNC to the phenotypes of FHHNC patients, with a special focus on the progression of renal failure (6), and by (b) our mouse models using transgenic RNAi depletion of CLDN16 (7). Without CLDN16 expression in the kidney, TJs in the thick ascending limb (TAL) of the nephron lose cation selectivity, leading to the dissipation of the lumen-positive potential with a concomitant loss of the driving force for Mg 2+ reabsorption (7). While targeted deletion of CLDN19 in mice initially focused on its

The paracellular claudin channel of the thick ascending limb (TAL) of Henle is critical for Ca++ reabsorption in the kidney. Genome‐wide association studies (GWASs) have identified claudin‐14 associated with hypercalciuric nephrolithiasis. Here, we show that claudin‐14 promoter activity and transcript are exclusively localized in the TAL. Under normal dietary condition, claudin‐14 proteins are suppressed by two microRNA molecules (miR‐9 and miR‐374). Both microRNAs directly target the 3′‐UTR of claudin‐14 mRNA; induce its mRNA decay and translational repression in a synergistic manner. Through physical interaction, claudin‐14 blocks the paracellular cation channel made of claudin‐16 and ‐19, critical for Ca++ reabsorption in the TAL. The transcript and protein levels of claudin‐14 are upregulated by high Ca++ diet, while downregulated by low Ca++ diet. Claudin‐14 knockout animals develop hypermagnesaemia, hypomagnesiuria, and hypocalciuria under high Ca++ dietary condition. MiR‐9 and miR‐374 transcript levels are regulated by extracellular Ca++ in a reciprocal manner as claudin‐14. The Ca++ sensing receptor (CaSR) acts upstream of the microRNA‐claudin‐14 axis. Together, these data have established a key regulatory role for claudin‐14 in renal Ca++ homeostasis.

Tight junctions (TJs) play a key role in mediating paracellular ion reabsorption in the kidney. The paracellular pathway in the collecting duct of the kidney is a predominant route for transepithelial chloride reabsorption that determines the extracellular NaCl content and the blood pressure. However, the molecular mechanisms underlying the paracellular chloride reabsorption in the collecting duct are not understood. Here we showed that in mouse kidney collecting duct cells, claudin-4 functioned as a Cl -channel. A positively charged lysine residue at position 65 of claudin-4 was critical for its anion selectivity. Claudin-4 was observed to interact with claudin-8 using several criteria. In the collecting duct cells, the assembly of claudin-4 into TJ strands required its interaction with claudin-8. Depletion of claudin-8 resulted in the loss of paracellular chloride conductance, through a mechanism involving its recruitment of claudin-4 during TJ assembly. Together, our data show that claudin-4 interacts with claudin-8 and that their association is required for the anion-selective paracellular pathway in the collecting duct, suggesting a mechanism for coupling chloride reabsorption with sodium reabsorption in the collecting duct.C hloride is the predominant extracellular ionic constituent and thereby determines extracellular fluid volume (ECFV) and blood pressure (1-3). Although only responsible for the reabsorption of 2-3% filtered chloride, the aldosterone-sensitive distal nephron (ASDN) plays a vital regulatory role in renal handling of salt, ECFV control, and managing blood pressure (4). The ASDN comprises the distal convoluted tubule (DCT), the connecting tubule (CNT), and the collecting duct. The collecting duct is characterized by a heterogeneous epithelium-the principal cells and intercalated cells (5). Sodium reabsorption in the collecting duct is an active process driven by the basolateral Na + /K + -ATPase; acts through the apical epithelial sodium channel (ENaC) in the principal cell (6); and is responsible for generating the lumen-negative transepithelial potential. This electrogenic transport step creates a favorable electrical driving force for luminal reabsorption of chloride and secretion of potassium and proton. Chloride is transported by two major mechanisms (1). Chloride is actively reabsorbed by an electroneutral Cl − /HCO − exchanger (Slc26a4: pendrin) localized to the apical membrane of the β-type intercalated cell (7). Choride exits this cell via a basolateral Cl − channel (2) and diffuses passively down electrochemical gradients via the paracellular channel in the tight junction (TJ).The TJ is the most apical member of the junctional complex found in vertebrate epithelia responsible for the barrier to movement of ions and molecules between apical and basal compartments, the paracellular pathway (8). TJs are composed of three transmembrane proteins: occludin, claudins, and junctional adhesion molecule (JAM). The claudins (CLDNs) are a 28-member family of tetraspan proteins that range in m...

Claudins are tight junction integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and CLDN19 function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Previous studies showed that siRNA knockdown of CLDN16 in mice results in a mouse model for FHHNC. Here, we show that CLDN19-siRNA mice also developed the FHHNC symptoms of chronic renal wasting of magnesium and calcium together with defective renal salt handling. siRNA knockdown of CLDN19 caused a loss of CLDN16 from tight junctions in the thick ascending limb (TAL) without a decrease in CLDN16 expression level, whereas siRNA knockdown of CLDN16 produced a similar effect on CLDN19. In both mouse lines, CLDN10, CLDN18, occludin, and ZO-1, normal constituents of TAL tight junctions, remained correctly localized. CLDN16-and CLDN19-depleted tight junctions had normal barrier function but defective ion selectivity. These data, together with yeast two-hybrid binding studies, indicate that a heteromeric CLDN16 and CLDN19 interaction was required for assembling them into the tight junction structure and generating cation-selective paracellular channels.hypomagnesemia ͉ transgenic animal ͉ siRNA ͉ paracellular ionic channel ͉ renal calcium wasting T ight junctions (TJs) play a key role in mediating paracellular ion reabsorption in epithelia. TJs are composed of three transmembrane proteins, occludin, claudins, and junctional adhesion molecule (1). The claudins are a 24-member family of tetraspan proteins that range in molecular mass from 20 to 28 kDa (1, 2). Claudin and occludin are the major components of the branching and anastomosing network of tight junctional strands in the plasma membrane revealed by freeze-fracture microscopy (3, 4). It has been hypothesized that claudin oligomerization occurs before strand assembly on the basis of claudin-4 (CLDN4) expression studies in insect cells that do not form TJs (5) and exhibit Ϸ10-nm-sized multimers. After trafficking to the cell surface, it is believed that oligomerized claudins then assemble into the TJ strands where they interact with cognate claudins in the adjacent cell (1, 6). Assembly of claudins into TJ strands requires the TJ scaffold proteins ZO-1 or ZO-2, which interact with both claudin PDZ binding domains (7-10) and TJ peripheral proteins such as cingulin, .Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human hereditary disorder caused by mutations in the TJ proteins CLDN16 (14) and CLDN19 (15). The expression of CLDN16 is restricted to the thick ascending limb (TAL) of the nephron (16). CLDN16-deficient mice exhibit defects in paracellular cation selectivity and develop severe renal wasting of magnesium and calcium (17) similar to that seen in the human disease. In the kidney, CLDN19 is also exclusive to the TAL (15). In vitro, CLDN16 and CLDN19 interact and form a cation-selective TJ paracellular channel (18). On the basis of these observations, we hyp...

Tamm-Horsfall glycoprotein (THGP) or