Tight junction (TJ) properties are determined by membrane protein complexes of neighboring cells that form both a barrier and a selective pathway for paracellular substrate transport. Our previous work supports the view that paracellular permeability changes in the thick ascending limb (TAL) may underlie the mechanism for familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), a rare autosomal recessive disease linked to mutations in claudin-16 (CLDN16) and claudin-19 (CLDN19). CLDN16 knockdown (KD) mice are lacking CLDN16 expression by transgenic RNA interference. We observed that the transport defect for Mg2+ and Ca2+ in this animal model is caused by a loss of paracellular cation selectivity. The permeability ratio for Na+ over Cl- in KD mice was lower by a factor of two without a change in paracellular conductance, compared to wild type (WT). This resulted in a collapse of the transepithelial voltage, which is the driving force for Mg2+ and Ca2+ absorption in TAL. Since CLDN16 KD mice revealed lower blood pressure and an increased aldosterone plasma concentration, we hypothesize that the reduction in paracellular selectivity could allow backflow of Na+ and Cl- into the lumen of the TAL, thus enhancing the distal NaCl load and challenging the organism with a latent NaCl loss.