The umbrella cell keratin network: organization as a tile-like mesh, formation of a girded layer in response to bladder filling, and dependence on the plectin cytolinker.

The keratin cytoskeleton and associated desmosomes contribute to the mechanical stability of epithelial tissues, but their organization in bladder umbrella cells and their responses to bladder filling are poorly understood. Using super-resolution confocal microscopy, along with 3D image reconstruction and platinum replica electron microscopy, we observed that the apical keratin network of umbrella cells was organized as a dense tile-like mesh comprised of tesserae bordered on their edges by cortical actin filaments, filled with woven keratin filaments, and crosslinked by plectin. A band of keratin was also observed at the cell periphery that was linked to the junction-associated actin ring by plectin. During bladder filling, the junction-localized desmosomal necklace expanded, and a subjacent girded layer was formed that linked the keratin network to desmosomes, including those at the umbrella cell-intermediate cell interface. Disruption of plectin led to focal keratin network dissolution, loss of the junction-associated band of keratin, perturbation of tight junction continuity, and loss of cell-cell cohesion. Our studies reveal a novel tile-like organization of the umbrella cell keratin cytoskeleton that is dependent on plectin, that reorganizes in response to bladder filling, and that likely serves to maintain umbrella cell continuity in the face of mechanical distension.

Expression and localization of the mechanosensitive/osmosensitive ion channel TMEM63B in the mouse urinary tract.

The epithelial cells that line the kidneys and lower urinary tract are exposed to mechanical forces including shear stress and wall tension; however, the mechanosensors that detect and respond to these stimuli remain obscure. Candidates include the OSCA/TMEM63 family of ion channels, which can function as mechanosensors and osmosensors. Using Tmem63bHA-fl/HA-fl reporter mice, we assessed the localization of HA-tagged-TMEM63B within the urinary tract by immunofluorescence coupled with confocal microscopy. In the kidneys, HA-TMEM63B was expressed by proximal tubule epithelial cells, by the intercalated cells of the collecting duct, and by the epithelial cells lining the thick ascending limb of the medulla. In the urinary tract, HA-TMEM63B was expressed by the urothelium lining the renal pelvis, ureters, bladder, and urethra. HA-TMEM63B was also expressed in closely allied organs including the epithelial cells lining the seminal vesicles, vas deferens, and lateral prostate glands of male mice and the vaginal epithelium of female mice. Our studies reveal that TMEM63B is expressed by subsets of kidney and lower urinary tract epithelial cells, which we hypothesize are sites of TMEM63B mechanosensation or osmosensation, or both.

PIEZO1 is a distal nephron mechanosensor and is required for flow-induced K+ secretion.

Ca2+-activated BK channels in renal intercalated cells (ICs) mediate luminal flow-induced K+ secretion (FIKS), but how ICs sense increased flow remains uncertain. We examined whether PIEZO1, a mechanosensitive Ca2+-permeable channel expressed in the basolateral membranes of ICs, is required for FIKS. In isolated cortical collecting ducts (CCDs), the mechanosensitive cation-selective channel inhibitor GsMTx4 dampened flow-induced increases in intracellular Ca2+ concentration ([Ca2+]i), whereas the PIEZO1 activator Yoda1 increased [Ca2+]i and BK channel activity. CCDs from mice fed a high-K+ (HK) diet exhibited a greater Yoda1-dependent increase in [Ca2+]i than CCDs from mice fed a control K+ diet. ICs in CCDs isolated from mice with a targeted gene deletion of Piezo1 in ICs (IC-Piezo1-KO) exhibited a blunted [Ca2+]i response to Yoda1 or increased flow, with an associated loss of FIKS in CCDs. Male IC-Piezo1-KO mice selectively exhibited an increased blood [K+] in response to an oral K+ bolus and blunted urinary K+ excretion following a volume challenge. Whole-cell expression of BKα subunit was reduced in ICs of IC-Piezo1-KO mice fed an HK diet. We conclude that PIEZO1 mediates flow-induced basolateral Ca2+ entry into ICs, is upregulated in the CCD in response to an HK diet, and is necessary for FIKS.