Localization and Regulation of the Disease‐Causing H+‐ATPase B1 Subunit in the Early Distal Nephron of Human and Murine Kidney

Abstract

The H+‐ATPase B1 subunit is abundantly expressed in intercalated cells (IC) of the collecting system. Loss of function mutations in the gene results in distal renal tubular acidosis, due to insufficient acid secretion into urine. Curiously, mutations in the disease‐causing H+‐ATPase subunits has been reported to cause more severe forms of distal renal tubular acidosis, as compared to dominant mutations in AE1. Using 2 antibodies directed against the N‐terminal domain of the B1 subunit, we found abundant immunoreactivity in human kidney. As expected IC showed strongest signal, but significant signal was also observed in apical membrane aspects of the early distal nephron. Colocalization using known cellular markers confirmed expression of the B1 subunit in NCC positive distal convoluted tubules and NKCC2 positive cortical thick ascending limbs, but much less abundant in ENaC positive connecting tubule cells. Further immunohistochemical evaluation using an antibody directed against the other disease causing H+‐ATPase subunit A4, also showed localization to segments in the early distal nephron in human kidney. Expression was also detectable in macula densa for both subunits. In mouse and rat, B1 localization was investigated using antibodies directed against the N‐ or C‐terminal domains of the B1 subunit. Here subunit expression could be detected in apical membrane domains, beginning from the medullary thick ascending limb and stretching to the distal convoluted segment 2, in addition to the previously described IC. Using mice lacking the B1 subunit to validate the antibodies, complete loss of antigenicity was observed for two antibodies, while one showed minor staining in connecting tubules and IC, likely representing overlap with the B2 subunit epitope. To address whether the B1 subunit was subjected to regulation in the distal tubules, tissue from rats subjected to NH4Cl‐ and hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis were studied by immunohistochemistry. A mildly increased expression of the B1 subunit was noted in early distal nephron segments in kidneys of NH4Cl‐induced acidotic rats. In contrast, no change in expression could be observed in hypercapnia‐induced acidosis or NaHCO3‐ induced alkalosis. In conclusion, the B1 subunit is expressed in the early distal nephron in humans and rodents. The physiological consequences of H+‐ATPase expression in these segments remain to be delineated in detail.Support or Funding InformationThis work was funded by grants from the Novo Nordisk Foundation, the Carlsberg Foundation, the A.P. Møller Foundation, and the Lundbeck foundation.