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

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...

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na ؉ permeation. Removal of claudin-2 depressed the permeation of Na ؉ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na ؉ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl ؊ permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl ؊ and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na ؉ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na ؉ or as paracellular channels to Cl ؊ , depending upon the cellular background, to decrease the cation selectivity of the tight junction.Tight junctions are cell-cell interactions that provide the primary barrier to the diffusion of solutes through the paracellular pathway, creating an ion-selective boundary between the apical and basolateral extracellular compartments (see reviews in Refs. 1-3). The integral membrane proteins of the tight junction include occludin, a 65-kDa membrane protein bearing four transmembrane domains and two extracellular loops, and claudins, a family with at least 22 homologous proteins of 20 -28 kDa that share a common topology with occludin (4 -7).Claudins have been shown to confer ion selectivity to the paracellular pathway. In MDCK 2 cells, claudin-4, -5, -8, -11, and -14 selectively decrease the permeability of cation through tight junction, whereas the permeation of anion is largely unchanged (8 -12). MDCK cells express five endogenous claudins, claudin-1-4 and -7. LLC-PK1 cells express four endogenous claudins, claudin-1, -3, -4, and -7. In LLC-PK1 cells, claudin-2, -15, -16 selectively increase the permeability of cation through the tight junction with no significant effects on anions (13-14). When exogenous claudins are added to the tight junction, they constitute new charge-selective channels leading to a physiological phenotype that combines the contributions of both endogenous and exogenous claudins in the cell. A biochemical inventory of claudin-claudin interactions is not yet available, although the principle of specificity has been demonstrated in mouse L-fibroblasts (15). In addition, although efforts have been made to demonstrate the oligomerization p...

Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16. However, the molecular mechanisms underlining the renal handling of magnesium and its dysfunction causing FHHNC are unknown. Here we show that claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Using RNA interference, we have generated claudin-16-deficient mouse models. Claudin-16 knock-down (KD) mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Our data suggest that claudin-16 forms a non-selective paracellular cation channel, rather than a selective Mg 2؉ /Ca 2؉ channel as previously proposed. Our study highlights the pivotal importance of the tight junction in renal control of ion homeostasis and provides answer to the pathogenesis of FHHNC. We anticipate our study to be a starting point for more sophisticated in vivo analysis of tight junction proteins in renal functions. Furthermore, tight junction proteins could be major targets of drug development for electrolyte disorders.The human renal disorder, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC, 3 OMIM 248250), is characterized by progressive renal Mg 2ϩ and Ca 2ϩ wasting, leading to impaired renal function and chronic renal failure. FHHNC is genetically linked to mutations in the gene of claudin-16 (CLDN16, also known as paracellin-1; Ref. 1), which is expressed exclusively in the kidney (2). The claudins comprise a 22 gene family that encodes essential structural components of the tight junction, the principal regulator of paracellular permeability. In vitro studies have shown that ion selectivity of the paracellular conductance (see review: Ref.3) is a complex function of claudin subtype and cellular context (4, 5). Thus, in vivo models of FNNHC are essential to our understanding of its pathogenesis.To develop an in vivo model of FNNHC, we have employed transgenic RNA interference (RNAi), which is in theory more rapid and flexible than a conventional knock-out approach. While the use of transgenic RNAi has been limited thus far, it has been shown that an RNAi knockdown of Rasa1 recapitulates a null phenotype in mice (6). In addition, transgenic RNAi has been used to establish a role for Ryk in axon guidance (7) and a role for Nramp1 in controlling susceptibility to Type 1 diabetes (8). We used lentiviral transgenesis because it is more resistant than onco-retroviral transgenesis to epigenetic silencing during embryonic development (9 -11).In this study, we report the generation of CLDN16-deficient transgenic mouse lines using RNAi and have established physiological functions of CLDN16. We observed homeostatic changes of Mg 2ϩ , Ca 2ϩ , Na ϩ , and K ϩ resulting from RNAimediated knockdown. The lumen-positive transepithelial potential in the thick ascending limb (TAL) of ...

KCTD13 deletion leads to decreases in ubiquitination and increases in levels of ADSS KCTD13 deletion increases S-Ado levels, a metabolite observed in ADSL deficiency Treatment of KCTD13 deletion neurons with an ADSS inhibitor reduces S-Ado levels Human KCTD13 variants can alter ubiquitination of ADSS