Mechanisms for Hypercalciuria in Pseudohypoaldosteronism Type II-Causing WNK4 Knock-In Mice

Abstract

The mechanisms underlying hypercalciuria in pseudohypoaldosteronism type II (PHAII) caused by WNK4 mutations remain unclear. In this study, we used Wnk4D561A/+ knock-in mice as a model of human PHAII for investigating the pathogenesis of hypercalciuria in PHAII. Serum and urine biochemistries were obtained from Wnk4+/+ and Wnk4D561A/+ littermates. Expression of the epithelial Ca2+ channels [transient receptor potential channel vanilloid subtype 5 (TRPV5) and TRPV6] and calbindin-D28k (CBP-D28k) in the distal nephron and two upstream Na+ transporters, Na+/H+ exchanger 3 and Na+-K+-2Cl− cotransporter 2 involved in paracellular Ca2+ reabsorption, were examined by real-time PCR, immunofluorescent staining, and immunoblotting. Compared with Wnk4+/+ littermate controls, Wnk4D561A/+ mice manifested hypercalciuria despite no significant differences in serum creatinine, ionized Ca2+, PTH, and 1,25 hydroxylvitamin D3 levels. There was no significant difference in TRPV5 expression, but a significant increase in TRPV6 and CBP-D28k was observed in Wnk4D561A/+ mice. Despite no significant change in Na+/H+ exchanger 3 expression, Na+-K+-2Cl− cotransporter 2 expression was significantly attenuated and urine Ca2+ excretion rate in response to furosemide was blunted in Wnk4D561A/+ mice. Decreased Ca2+ reabsorption in the upstream nephron, especially in the thick ascending loops of Henle, with a secondary adaptive increase in TRPV6 and CBP-D28k expression in the distal tubules might be involved in the hypercalciuria of PHAII.