Molecular interactors that may define the localization of the Sodium Potassium-ATPase near mitochondria in the mouse kidney epithelial cells

The complex structural organization of renal epithelial cells enables them to perform the energetically demanding transport processes that are required to absorb the extremely large volumes of fluid and solutes that enter the renal tubule through glomerular filtration. We recently used enhanced Focused Ion Beam Scanning Electron Microscopy in combination with artificial intelligence-based segmentation tools to analyze and quantitate the distribution of organelles in 3D in mouse renal proximal tubule (PT) epithelial cells at up to 4 nm resolution. This analysis identified and quantitated inter-organelle interactions at membrane contact sites (MCS -defined as functional interactions established when organelles are <~40 nm apart). This analysis showed that mitochondria (MT) have a higher density in the basal and mid regions of PT cells in comparison with the apical region of the PT and that up to 99% of MT exhibited MCS with endoplasmic reticulum and 32% of MT exhibited MCS with the plasma membrane (PM). This extent of MT contact with the PM may be key in providing the large quantities of ATP demanded by the Na + ,K + -ATPase pump (Na,K-ATPase), the basolateral ion pump that drives the bulk of tubular fluid and solute absorption. We used Pan-expansion microscopy (pan-ExM) to determine at ultrastructural levels of resolution the distribution of the Na,K-ATPase in the basolateral PMs of renal tubule epithelial cells. pan-ExM permits uniform expansion of cellular structures up to 18 times in all 3 dimensions, and can be combined with immunofluorescence analysis to permit protein localization with far higher resolution than confocal microscopy. We found that the Na,K-ATPase is highly concentrated in regions of tubule cell basolateral PM that are in close contact with MT, colocalizing with MICOS60, a marker of MT cristae. We wondered whether the Na,K-ATPase could reside close enough to proteins of the outer MT membrane to make direct physical contact with them. To test this possibility, we employed proximity ligation assay (PLA) which detects proteins that reside within 40 nm of one another, a distance that is small enough to be permissive of direct physical and functional interactions. We used PLA to assess the proximity of the Na,K-ATPase to outer MT membrane proteins including the motor adaptor protein MIRO1, the transmembrane pore protein vDAC1 and TOMM20, which participates in protein import into the MT. We used primary antibodies against MIRO1, vDAC1, and TOMM20 and an antibody against the Na,K-ATPase with secondary antibodies conjugated to specific oligonucleotide arms to perform PLA on sections of mouse kidney. We find that the PM Na,K-ATPase resides within 40 nm of the MT proteins MIRO1, VDAC1 and TOMM20 and that these sites of close proximity exhibit differential distributions in cells of distinct tubule cell types, with a higher accumulation at the thick ascending limb in comparison with PT cells. A similar pattern of expression and distribution of MIRO1-Na,K-ATPase and TOMM20-Na,K-ATPase contacts was also found in epithelial cells of the pronephros of Xenopus tropicalis, an organism that will allow targeted and rapid modification of the expression of these MT proteins and assessment of their relevance in maintaining MT-PM contact sites as well as their role in organizing the distribution of the Na + ,K + -ATPase pump in kidney tubule cells. Funding: MJC:GR127175. OM'S:R43 DK137685-01. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.