Mitochondrial respiratory capacity in kidney podocytes is high, age-dependent, and sexually dimorphic.

Abstract

Whether and how podocytes depend on mitochondria across their long post-mitotic lifespan is unclear. With limited cell numbers and broad kidney distribution, isolation of podocyte mitochondria typically requires first isolating podocytes themselves. Disassociation of podocytes from their basement membrane, however, recapitulates an injured state and stresses mitochondria. Here, we devise a new strategy to examine mitochondria in podocytes. To address this, we crossed floxed hemagglutinin (HA)-mitochondria tagged (MITO-Tag) mice with those expressing Cre in either podocytes (NPHS2) or mixed tubules (CDH16), thus allowing for rapid, kidney cell-specific, isolation of mitochondria via immunoprecipitation. Mitochondrial respiration in fresh isolates from young (4-7 months) and aged (22-26 months) mice of both sexes demonstrated several previously unreported significant differences between podocyte and tubule mitochondria. First, although podocytes contain fewer mitochondria than tubule cells, mitochondria isolated from podocytes averaged twice the respiratory capacity of tubule mitochondria when normalized to mitochondrial content by citrate synthase levels. Second, age-related decline in respiration was detected only in podocyte mitochondria and only in aged male mice. Third, disassociating podocytes for cell culture initiates functional decline in mitochondria as those from cultured primary podocytes have half the respiratory capacity, but twice the hydrogen peroxide production, of podocyte mitochondria isolated directly from fresh kidneys. Finally, conformation of electron transport chain proteins differed between podocyte and tubule mitochondria, suggesting that cell-specific mitochondrial protein conformations dictate cell-specific mitochondrial function. Previous studies suggesting a limited role for mitochondrial regulation of podocytes relied on cell culture. This resulted in artifactual suppression of mitochondrial function and masks the roles of mitochondria in maintenance of podocyte health. Our approach shows that per organelle, podocytes maintain sexually dimorphic mitochondria with greater oxidative phosphorylation capacity than the mitochondria-dependent tubules.