Establishment of Conditionally Immortalized Mouse Glomerular Parietal Epithelial Cells in Culture

Abstract

Parietal epithelial cells (PEC) are major constituents of crescents in crescentic glomerulonephritis. The purpose of these studies was to establish an immortalized PEC cell line with similar characteristics to PEC in vivo for use in future mechanistic studies. Glomeruli were isolated from H-2Kb tsA58 transgenic mice (ImmortoMouse) by standard differential sieving, and several candidate PEC cell lines were obtained by subcloning outgrowths of cells from capsulated glomeruli. One clone, designated mouse PEC (mPEC), was extensively characterized. mPEC exhibited a compact cell body with typical epithelial morphology when grown in permissive conditions, but the cell shape changed to polygonal after 14 d in growthrestrictive conditions. mPEC but not podocytes used as a negative control expressed claudin-1, claudin-2, and protein gene product 9.5, which are proteins specific to PEC in vivo, and did not express the podocyte-specific proteins synaptopodin and nephrin. The junctional proteins zonula occludens-1 and ␤-catenin stained positively in both mPEC and podocytes, but the staining pattern at cell-cell contacts was intermittent in mPEC and linear in podocytes. Finally, mPEC had thin bundled cortical F-actin filaments and no F-actin projections compared with podocytes, which exhibited thick bundled cortical F-actin filaments and interdigitating F-actin projections at cell-cell contacts. We conclude that immortalized mPEC in culture exhibit specific features of PEC in vivo and that these cells are distinct from podocytes, despite having the same mesenchymal origin. This mPEC line will assist in future mechanistic studies of PEC and enhance our understanding of glomerular injury.

SSeCKS sequesters cyclin D1 in glomerular parietal epithelial cells and influences proliferative injury in the glomerulus

“…23 Increasing cell-cell contact between PECs from 50 to 100% cell confluency induced SSeCKS expression in the cytoplasm, concomitant with the redistribution of cyclin D1 from nuclear to cytoplasmic compartments and marked downregulation of total cyclin D1 expression (Figure 2a and b), consistent with previous data. [13][14][15][16] This cytoplasmic cyclin D1 immunoprecipitated with SSeCKS (Figure 2b and Supplementary Figure 2), including after differentiation of fully confluent PECs over 14 days to mimic the state of mature PECs in normal glomeruli, 23 corroborating our findings of this binding interaction between SSeCKS and cyclin D1 at steady-state in vivo.…”

“…7,[19][20][21][22] Kidneys from male SSeCKS þ / þ and male SSeCKS À/À mice at 15 weeks of age were fixed in Karnovsky's solution for imaging glomerular ultrastructure by the University of Washington Pathology Research Service Laboratory on a Tecnai G2 Spirit Bio Twin Electron Microscope. Magnetic-bead isolation of capsulated and decapsulated glomeruli from wild-type mice following differential size sieving of digested kidneys was performed as described previously, 23 with isolated glomeruli immediately lysed in RIPA buffer (Teknova, Hollister, CA, USA) containing Complete Protease Inhibitor (Roche, Indianapolis, IN, USA), 50 mM sodium fluoride and 0.1 mM sodium orthovanadate at 41C to collect total protein. Passive NTN 24 was induced in cohorts of male SSeCKS þ / þ mice (n ¼ 5) and male SSeCKS À/À mice (n ¼ 5) at 15 weeks of age by intraperitoneal injection of nephrotoxic immunoglobulin (12.5 mg/20 g body weight), or an equivalent volume of vehicle (1 Â PBS, pH 7.4), on 2 consecutive days.…”

“…23,25 To study cell-cell contact inhibition in PECs, cells growing under conditions permissive for proliferation (331 with IFN-g) were analyzed at 50, 80, and 100% confluency, or following differentiation at 100% confluency for 14 days under nonpermissive conditions (371C without IFN-g). Total protein from cells was prepared by lysing cells in RIPA buffer containing Complete Protease inhibitor, 50 mM sodium fluoride, and 0.1 mM sodium orthovanadate at 41C.…”

Thrombomodulin domain 1 ameliorates diabetic nephropathy in mice via anti-NF-κB/NLRP3 inflammasome-mediated inflammation, enhancement of NRF2 antioxidant activity and inhibition of apoptosis

“…In vitro analyses of podocytes Conditionally immortalised mouse podocytes were obtained from S. J. Shankland (Department of Medicine, University of Washington, Seattle, WA, USA) and differentiation induced by culturing the cells for 13 to 15 days at 37°C in the absence of IFN-γ (Roche, Nutley, NJ, USA), as described previously [20].…”

Glomerular parietal epithelial cells in kidney physiology, pathology, and repair

“…This has been enabled by the generation of primary and immortalized PECs in culture [2,3], the establishment of reporter mice to cell fate map PECs [4], the identification of proteins and genes expressed by these cells in normal and diseased experimental and human conditions, and the application of molecular and cellular approaches that allow researchers to begin to probe how these cells may respond to certain interventions. The purpose of this review is to provide a recent literature update of our current understanding of how PEC biology underlies normal function in health (Table 1), and how in glomerular diseases PECs may serve a critical reparative role, or under different circumstances the response by PECs may lead to further glomerular damage.…”