Automated serial section electron microscopy and 3D reconstruction reveals role for nonmuscle myosin II in organelle structure and cargo transport regulation within the murine kidney thick ascending limb

We previously showed conditional knockout (cKO) of nonmuscle myosin II (NM2) isoforms Myh9&10 in adult mouse kidney epithelium results in severe progressive kidney disease of tubular origin. Reduction of the ion cotransporter NKCC2 and accumulation of uromodulin (UMOD) in the ER of epithelial cells of the thick ascending limb (TAL) were primary drivers of kidney disease, accompanied by ER expansion and upregulation of ER stress/UPR pathways. NKCC2 association with the apical plasma membrane (PM) has been shown by others to depend on the cycling of NKCC2-containing vesicles to and from the PM. UMOD has been shown to participate in the regulation of NKCC2 activity, and mutations in UMOD delaying its ER exit with upregulation of ER stress lead to kidney disease in human patients. To better understand the role of NM2 in the TAL, we employed immunostaining, light microscopy, and standard transmission electron microscopy (TEM). Unfortunately, common resident organelle proteins exhibit altered expression and/or localization by immunostaining in cKO kidney TAL cells, while individual TEM images provide limited information regarding three-dimensional (3D) properties, limiting comprehensive structural analysis. To explore subcellular structure in an unbiased manner, we performed 3D reconstruction and modeling using an innovative automated serial section scanning electron microscopy method (ATUM-SEM). Detailed 3D models of control TAL cells in situ show the ER to be an expansive, predominantly tubular network present throughout the cell. We also observe widespread presence of ER-associated vesicles and a distinct population of subapical vesicles, suggesting these cells tightly regulate ion reabsorption via UMOD and NKCC2 expression, transport, and localization. Furthermore, many TAL cell mitochondria are partially encapsulated within basolateral membrane infoldings which are positive for Na+ K+ ATPase by immunostaining and are continuous with apical tight junctions, while ER tubules are consistently observed in the narrow space between mitochondria and infoldings. This arrangement maximizes the surface area of Na+ K+ ATPase-rich PM in close contact with mitochondria and ER, representing an efficient method of meeting the relatively high energy demands of the TAL. While the PM appears intact in cKO TAL cells, the basolateral localization of mitochondria within membrane infoldings is perturbed and ER-mitochondria-PM contact appears reduced. Quantification of structural changes as well as 3D modeling of immunostained serial ATUM sections are ongoing as a means of further correlating TAL cell structure-function relationships with kidney physiology. Overall, our methods enable a detailed analysis of the distinct and complex ER-Mitochondria-PM architecture within TAL epithelia in situ, which we believe is necessary for the TAL to fulfill its physiological role in the kidney, and our results strongly implicate NM2 as being crucial for proper TAL cell function. 1R01DK131020-01A1 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.