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...