Fibroblast growth factor-7 regulates stratification of the bladder urothelium.

PURPOSE: The cellular and molecular mechanisms that regulate the organization of bladder urothelium into basal, intermediate and superficial cell layers remain poorly understood. We tested the hypothesis that fibroblast growth factor (FGF)-7 is essential for generating a multilayered stratified bladder epithelium. MATERIALS AND METHODS: The morphological and molecular characteristics of bladder urothelium in age and sex matched FGF-7 +/+ wild-type and -/- null mice were evaluated. In addition, the effect of exogenous FGF-7 on the growth and differentiation of primary murine urothelial cells was assessed. RESULTS: Morphometric analyses demonstrate that FGF-7 null urothelium is markedly thinned compared with wild-type urothelium. Electron microscopy revealed that null urothelium lacks the intermediate cell layers and molecular marker analyses confirmed this observation. In vitro cell culture experiments indicated that FGF-7 regulates urothelial cell growth, differentiation and stratification. Primary urothelial cultures maintained without FGF-7 ceased to divide and expressed proteins characteristic of terminally differentiated umbrella cells. In contrast, cultures maintained with exogenous FGF-7 contained proliferating epithelial cells with protein expression patterns consistent with those of intermediate cells in addition to terminally differentiated, post-mitotic umbrella cells. Importantly, isolated urothelial cells maintained with exogenous FGF-7 formed a multilayered epithelium in vitro. CONCLUSIONS: Collectively these data indicate that FGF-7 is essential for normal bladder urothelial stratification, specifically the formation of the intermediate cell layers. Fibroblast growth factor-7 stimulates urothelial proliferation and delays the differentiation of these cells into post-mitotic umbrella cells.

Studies on the development and maintenance of epithelial cell surface polarity with monoclonal antibodies.

We examined epithelial cell surface polarity in subconfluent and confluent Madin-Darby canine kidney (MDCK) cells with monoclonal antibodies directed against plasma membrane glycoproteins of 35,000, 50,000, and 60,000 mol wt. The cell surface distribution of these glycoproteins was studied by immunofluorescence and immunoelectron microscopy. At the ultrastructural level, the electron-dense reaction product localizing all three glycoproteins was determined to be uniformly distributed over the apical and basal cell surfaces of subconfluent MDCK cells as well as on the lateral surfaces between contacted cells; however, after formation of a confluent monolayer, these glycoproteins could only be localized on the basal-lateral plasma membrane. The development of cell surface polarity was followed by assessing glycoprotein distribution with immunofluorescence microscopy at selected time intervals during growth of MDCK cells to form a confluent monolayer. These results were correlated with transepithelial electrical resistance measurements of tight junction permeability and it was determined by immunofluorescence that polarized distributions of cell surface glycoproteins were established just after electrical resistance could be detected, but before the development of maximal resistance. Our observations provide evidence that intact tight junctions are required for the establishment of the apical and basal-lateral plasma membrane domains and that development of epithelial cell surface polarity is a continuous process.

Analysis of epithelial cell surface polarity with monoclonal antibodies.

The hybridoma technique of Köhler and Milstein was utilized to isolate hybrid cell lines secreting monoclonal antibodies against cell surface proteins on the Madin-Darby canine kidney (MDCK) epithelial cell line. These antibodies were employed as high-affinity ligands to study the development and maintenance of epithelial cell polarity in MDCK cells and for the identification of nephron segment-specific proteins. Using standard procedures, we were able to immunoprecipitate glycoproteins with molecular weights of 25,000 ( 25K ), 35,000 ( 35K ), and 50,000 (50K). Immunofluorescence and immunoelectron microscopy of MDCK demonstrated that the 35K and 50K proteins could be localized on both the apical and basolateral membranes of subconfluent cells but primarily on the basolateral membranes of confluent cells. By determining the cell surface distribution of the 35K and 50K proteins on MDCK cells during growth into a confluent monolayer, and after the experimental disruption of tight junctions, evidence was obtained that the polarized distribution of these cell surface glycoproteins required the presence of tight junctions. We propose that confluent MDCK cells have a mechanism that is responsible for the establishment and maintenance of epithelial apical and basolateral membranes as distinct cell surface domains. These monoclonal antibodies were also used to localize the 25K and 35K glycoproteins in the kidney. The distribution of these proteins was mapped by immunofluorescence and immunoelectron microscopy and was determined to be on the basolateral membranes of epithelial cells in only certain tubular segments of the nephron. The possible functional implications of these distributions are discussed.

The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule.

Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues.