Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.

In epithelial cells, vinculin is enriched in cell adhesion structures but is in equilibrium with a large cytosolic pool. It is accepted that when cells adhere to the extracellular matrix, a part of the soluble cytosolic pool of vinculin is recruited to specialized sites on the plasma membrane called focal adhesions (FAs) by binding to plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We have previously shown that bradykinin (BK) induces both a reversible dissipation of vinculin from FAs, by the phospholipase C (PLC)-mediated hydrolysis of PtdIns(4,5)P2, and the concomitant internalization of vinculin. Here, by using an immunomagnetic method, we isolated vinculin-containing vesicles induced by BK stimulation. By analyzing the presence of proteins involved in vesicle traffic, we suggest that vinculin can be delivered in the site of FA reassembly by a vesicular endocytic recycling pathway. We also observed the formation of vesicle-like structures containing vinculin in the cytosol of cells treated with lipid membrane-affecting agents, which caused dissipation of FAs due to their deleterious effect on membrane microdomains where FAs are inserted. However, these vesicles did not contain markers of the recycling endosomal compartment. Vinculin localization in vesicles has not been reported before, and this finding challenges the prevailing model of vinculin distribution in the cytosol. We conclude that the endocytic recycling pathway of vinculin could represent a physiological mechanism to reuse the internalized vinculin to reassembly new FAs, which occurs after long time of BK stimulation, but not after treatment with membrane-affecting agents.

We have previously demonstrated that kidney embryonic structures are present in rats, and are still developing until postnatal Day 20. Consequently, at postnatal Day 10, the rat renal papilla contains newly formed collecting duct (CD) cells and others in a more mature stage. Performing primary cultures, combined with immunocytochemical and time‐lapse analysis, we investigate the cellular mechanisms that mediate the postnatal CD formation. CD cells acquired a greater degree of differentiation, as we observed that they gradually lose the ability to bind BSL‐I lectin, and acquire the capacity to bind Dolichos biflorus. Because CD cells retain the same behavior in culture than in vivo, and by using DBA and BSL‐I as markers of cellular lineage besides specific markers of epithelial/mesenchymal phenotype, the experimental results strongly suggest the existence of mesenchymal cell insertion into the epithelial CD sheet. We propose such a mechanism as an alternative strategy for CD growing and development.

It is known that bradykinin (BK) B2 receptor (B2R) is expressed in the collecting duct (CD) cells of the newborn rat kidney, but little is known about its role during early postnatal life. Therefore, we hypothesize that BK could participate in the mechanisms that mediate CD formation during the postnatal renal development. Performing primary cultures, combined with biochemical, immunocytochemical, and time-lapse analysis, we studied the role of BK in CD cell behavior isolated from renal papilla of neonatal rats. A reverse relationship was observed between B2R expression and the degree of CD epithelial cell sheet maturation. BK stimulation induced CD cell association upon B2R activation. The lack of B2R expression in cells showing mature adherens junctions suggested that BK is mostly involved in early adhesive events, thus favoring the initial formation of CD during development. Time-lapse analysis revealed that BK induced a high protrusive activity of CD cells, denoted by ruffle formation and lamellipodia extension. PI3K was involved in the BK-induced CD cell-cell association and the acquisition of the migratory phenotype since, when inhibited, membrane ruffles, and filopodia between cells diminished. Results indicate that the actions of BK mediated by PI3K activation were due to the downstream Akt and Rac pathways. This study, performed with CD cells that were not genetically manipulated, provides new experimental evidence supporting a novel role of BK in rat renal CD organization. As B2R blockade results in abnormal tubular differentiation, our results contribute to better understanding the etiology of human congenital renal malformation and diseases.

The renal collecting ducts (CD) are formed by a fully differentiated epithelium, and their tissue organization and function require the presence of mature cell adhesion structures. In certain circumstances, the cells can undergo de‐differentiation by a process called epithelial–mesenchymal transition (EMT), in which the cells lose their epithelial phenotype and acquire the characteristics of the mesenchymal cells, which includes loss of cell–cell adhesion. We have previously shown that in renal papillary CD cells, cell adhesion structures are located in sphingomyelin (SM)‐enriched plasma membrane microdomains and the inhibition of SM synthase 1 activity induced CD cells to undergo an EMT process. In the present study, we evaluated the influence of SM metabolism during the EMT of the cells that form the CD of the renal papilla during aging. To this end, primary cultures of renal papillary CD cells from young, middle‐, and aged‐rats were performed. By combining biochemical and immunofluorescence studies, we found experimental evidence that CD cells undergo an increase in spontaneous and reversible EMT during aging and that at least one of the reasons for this phenomenon is the decrease in SM content due to the combination of decreased SM synthase activity and an increase in SM degradation mediated by neutral sphingomyelinase. Age is a risk factor for many diseases, among which renal fibrosis is included. Our findings highlight the importance of sphingolipids and particularly SM as a modulator of the fate of CD cells and probably contribute to the development of treatments to avoid or reverse renal fibrosis during aging.