Loss of Ncoa7 leads to oxidative stress in kidney intercalated cells

Abstract

Oxidative stress (OS) results from excessive production of reactive oxygen species, relative to endogenous antioxidant defenses, leading to cellular damage. The metabolically active kidney is vulnerable to OS, which is a key step in the progression of many renal pathologies. Furthermore, many non-renal pathologies are characterized by OS. Therefore, although our work focuses on kidney epithelial cells, it has significant implications for understanding how OS is regulated at a molecular level in many organs and cell types.The V-ATPase, or proton pump, is required for the acidification of intracellular compartments. Recently, we identified a family of V-ATPase interacting proteins defined by their TLDc domain (Ncoa7, Oxr1, Tbc1d24, Tldc1, and Tldc2). TLDc proteins are protective against OS in the nervous system and mutations cause human neurological pathologies. However, little is known about the molecular mechanisms that provide these protective properties. The V-ATPase, like TLDc proteins, also protects against OS. Therefore, we hypothesize that TLDc proteins play a V-ATPase-dependent protective role against OS. This is supported by our previous findings that mutating an amino acid in Ncoa7 that is protective against OS abolishes the interaction with the V-ATPase, and Ncoa7 knockout (KO) decreases V-ATPase-dependent urinary acidification in mice.To test this, we focused on Ncoa7 because it showed the most robust protective effect against OS in previous studies and is highly expressed in the kidney where it is enriched in epithelial intercalated cells (ICs). RNA sequencing of isolated ICs from Ncoa7 KO mice revealed significant downregulation of antioxidant enzymes including glutathione peroxidase-3 (Gpx3) and carbonyl reductase-2 (Cbr2). Immunofluorescence (IF) showed Cbr2 enrichment in medullary ICs from control mouse kidneys and a decrease in Cbr2 fluorescent signal intensity in ICs from Ncoa7 KO mice. Furthermore, Cbr2 localizes to the mitochondria of ICs, a significant source of cellular OS.To investigate whether the loss of Ncoa7 promotes OS in ICs we examined changes in OS markers by IF. We monitored the expression of Advanced Glycation End products (AGEs) and malondialdehyde (MDA) in fixed kidney sections using specific antibodies. AGEs are generated by glycation events accompanying OS and their formation can be suppressed by Gpx3. MDA is a product of lipid oxidation due to OS and can be reduced by Cbr2. ICs in Ncoa7 KO mice exhibited a striking accumulation of both AGEs and MDA, often found in large V-ATPase-bound intracellular compartments. Furthermore, knockdown of Ncoa7 in cultured kidney cells increased expression of AGEs, supporting loss of Ncoa7 as the mechanism behind the increased accumulation of intracellular AGEs.Based on these data we conclude that loss of Ncoa7 promotes OS in renal intercalated cells potentially through a regulatory interaction with the V-ATPase, which leads to decreased expression of antioxidant enzymes. NIH/NIDDK T32DK007540-33 and NIH/NIDDK R01DK121848 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.