Pseudohypoaldosteronism type II (PHAII) is a hereditary disease characterized by hypertension, hypercalciuria and osteopenia. PHAII is caused by mutations in with-no-lysine kinase 1 (WNK1), WNK4, or the cullin RING ligase family members kelch-like 3 (KLHL3) or cullin 3 (CUL3). All mutations result in up-regulation of the WNK signalling pathway which activates thiazide-sensitive Na-Cl cotransporters (NCC) in renal distal tubules, leading to salt retention and hypertension in PHAII. The mechanism underlying hypercalciuria in PHAII is unknown. To better understand the mechanisms leading to osteopenia in PHAII, we used KLHL3R528H/+ knock-in mice carrying the same mutation as some PHAII patients. As expected, KLHL3R528H/+ mutants exhibited hyperkalemia, hypernatremia, renal calcium wasting and increased phosphorylation of NCC in the kidney. Furthermore, KLHL3R528H/+ mutants showed elevated serum parathyroid hormone (PTH), increased bone resorption as demonstrated by elevated urinary collagen crosslinks excretion and increased osteoclast numbers in femoral cancellous bone, and reduced distal femoral cancellous bone BMD and volume as evidenced by pQCT and μCT analysis. Analysis of the expression of proteins involved in renal calcium transport revealed elevated membrane abundance of the fully glycosylated epithelial calcium channel TRPV5, decreased TRPV6 abundance, and unchanged calbindin D28k expression in KLHL3R528H/+ mutants. In contrast to the upregulated TRPV5 protein expression, TRPV5 phosphorylation was reduced in KLHL3R528H/+ mutants, suggesting downregulated TRPV5 activity. In line with a crosstalk between NCC activity and PTH-mediated TRPV5 activation, we found by 2-photon microscopy that the PTH-mediated increase in Ca2+ uptake in mouse distal tubular mpkDCT4 cells was enhanced by the NCC blocker chlorothiazide or by knockout of NCC. Taken together, our study provides a mechanistic explanation for the hypercalciuria and bone loss found in PHAII patients: elevated NCC activity in KLHL3R528H/+ mice blocks PTH-mediated TRPV5 activation, leading to renal PTH resistance with subsequent renal Ca wasting and a counter-regulatory PTH-induced bone loss.
14 May 2016 - 17 May 2016