Proceedings of The Physiological Society

University College London (2011) Proc Physiol Soc 24, C11 and PC11

Oral Communications

Disruption of ClC-5 is not associated with defective proton secretion by the collecting duct

S. Bourgeois1, H. Belge2, N. Mohebbi1, C. A. Wagner1, O. Devuyst1,2

1. Institut of Physiology, University of Z


Mutations in CLCN5 gene cause Dent’s disease, an X-linked disorder characterized by proximal tubule dysfunction including low-molecular-weight proteinuria, hyperphosphaturia, and hypercalciuria. CLCN5 codes for ClC-5, is a Cl-/H+ antiporter expressed in the proximal tubule, the thick ascending limb, and the α-type intercalated cells (αIC) of the collecting duct (CD). At the subcellular level, ClC-5 has been localized to apical endosomes together with the vacuolar H+-ATPase. Patients harbouring mutations of ClC-5 may show defective urinary acidification and modifications in the polarity and/or expression of vacuolar H+-ATPase in the proximal tubule and in αIC. Here we investigated the role of ClC-5 in the function of αIC using Clcn5 knock-out KO) mice which are a faithful model of Dent’s disease. We challenged these mice with a chronic NH4Cl load to establish their physiological response and studied H+-ATPase activity and trafficking in isolated CDs. Metabolic studies under baseline condition confirmed the presence of hypercalciuria and higher titratable acid excretion which are features of Dent’s disease, without any difference in blood parameters. Clcn5 KO mice also exhibited more acidic urine than control (WT) littermates. Mice were anesthetized by a mixture (s.c. 0.1ml/g body weight) of ketamine (10%) and xylazine (5%) and the right carotid artery catheterized. Fresh arterial blood was collected from conscious, mildly constricted mice 3 h after anesthesia. Venous blood samples were obtained from the inferior vena cava immediately after killing. For acid-loading experiments, a solution of 300 mM NH4Cl (in 2% sucrose) was given to mice in drinking water for 2 or 6 days. Urine was collected daily (16h) under mineral oil before acid-loading experiments (day 0) and after 2 or 6 days of ingestion of NH4Cl. The body weight, fluid intake and urine flow rate were monitored. After a chronic NH4Cl load, both Clcn5 WT and KO mice were able to adequately increase their ammoniuria. As expected, WT mice increased the excretion of titratable acid, whereas KO mice did not increase titratable acid above their baseline level. Clcn5 KO mice showed a lower blood pH and bicarbonate compared to WT mice. They also exhibited hyperchloremia and hyperkalemia. We then assessed apical NH3 uptake and proton secretion by the cortical and outer medullary CDs isolated from chronic NH4Cl loaded mice (300 mM NH4Cl 2% sucrose solution as beverage, 6 days before the experiment). Mice were anesthetized with xylazin/ketamin i.p (as above). The CDs were microperfused in vitro (kidneys were cooled in situ for 1 min and then removed) and apical NH3 uptake and H+ secretion were measured after cell acidification via an apical NH4Cl prepulse. Both apical NH3 entry and H+ excretion were drastically increased in CDs from Clcn5 KO mice. Furthermore, immunostaining revealed that the α4 subunit of the vacuolar H+-ATPase was more present at the luminal plasma membrane in outer medulla αICs from Clcn5 KO mice compared to WT controls. For immunostaining, mice were anesthetized with ketamine/xylazin (as above) and perfused through the left ventricle with PBS followed by fixative. These data reveal that the lack of ClC-5 in mouse is not reflected by defective acid secretion in the CD. On the contrary, mice lacking ClC-5 show a major increase in acid excretion, mediated by increased trafficking of vacuolar H+ATPase to the apical plasma membrane of αIC, probably to compensate for the generalized proximal tubule defect.

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