Neprilysin colocalizes with the V‐ATPase in kidney A‐type intercalated cells: possible role in urinary acidification

Intercalated cells (IC) in kidney collecting ducts are essential for acid‐base homeostasis. A proteomic analysis of these cells after purification by FACS revealed unexpectedly that they express high levels of neprilysin, a neutral protease, previously reported to be located in the brush border of proximal tubule cells. Therefore, we re‐examined the distribution of neprilysin in the kidney by immunohistochemistry and western blot analysis. Using anti‐neprilysin antibodies and two collecting duct markers: anti‐AQP2 antibody (principal cells) and anti‐A1 subunit v‐ATPase subunit (intercalated cells), we found that neprilysin was detectable not only in the proximal tubule, but also in ICs (but not principal cells) in all regions of the collecting duct. A similar distribution of neprilysin was seen in both mouse and rat kidneys. We then used an anti‐pendrin antibody to identify B‐type ICs in cortical collecting ducts. Only 35% of IC‐B population express neprilysin. Western blot analysis using an antibody against neprilysin showed that both cortical and medullary membrane preparations expressed neprilysin, and that female mice express more neprilysin than males in the cortex. However, this sex difference is not present in the medullary preparation, and we, therefore, attribute it mainly to a difference in proximal tubule expression. A similar result was observed in the kidneys of knockout mice that lack the IC‐specific B1 subunit of the V‐ATPase. In order to study the potential role of neprilysin in renal physiology, we treated male mice for 3 days with thiorphan, a selective neprilysin inhibitor. We measured a slight reduction of urine osmolality 1851 ± 160 to 1357 ± 240 (p=0.12) and an increase in urine volume 0.86 ± 0.21 to 1.48 ± 0.50, p= 0.23). In contrast, the change in urine pH was significant, increasing from 6.6 ± 0.1 to 6.9 ± 0.1, (n=3, p= 0.029) with the inhibitor, suggesting a role of neprilysin in acid excretion, possibly via IC function and V‐ATPase activity. In summary, we found that neprilysin is highly expressed in A‐ICs, where it colocalizes with the V‐ATPase. Its expression is higher in female mice, and it may have an as yet undetermined role in renal acid excretion. Support or Funding Information This work was supported by National Institutes of Health (NIH) grant DK096586 (D. Brown). The Zeiss LMS 800 confocal system in the Program in Membrane Biology (PMB) Microscopy Core was purchased using an NIH Shared Instrumentation Grant S10 RR031563‐01 (DB). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .