73 - Conditional Reprograming of urinary epithelial cells cultured from children with nephrotic syndrome

Conditional reprogramming (CR) media (DMEM media + condition media derived from irradiated murine fibroblasts) + 10 µM of the Rho-kinase inhibitor Y-27632 (RI), allows the unlimited proliferation of primary epithelial cells derived from the breast, colon, lung, nose, prostate, without exogenous genetic or viral manipulations. New methods are needed to culture primary podocytes from children with renal diseases (RD).  

Objective:

To determine how CR media + RI affect the transcriptome profile and proliferation of primary renal epithelial cells (REc) cultured from the urine of children with RD.  

Design/Methods: REc were isolated from the urine of a child with a primary nephrotic syndrome, and cultured in duplicates under different conditions: a) DMEM + 10% Fetal Bovine Serum (DMEM-FBS); b) DMEM-FBS + RI (10 µM); c) DMEM-FBS + RI + FGF-2 (10 ng/ml); d) DMEM-FBS + FGF-2; e) CR media alone;  f) CR media + RI; g) CR media + RI + FGF-2.  Cells were stained by immunohistochemistry using WT1, Pax-2, Vimentin, pan and 8/18 cytokeratin antibodies, and processed for bulk RNA-seq studies.   

Results: All urinary cells stained + for Vimentin and pan-cytokeratin, while ~ 85 % showed + staining for WT-1, Pax-2, and cytokeratin 8/18. Cells cultured in CR or DMEM-FBS alone, showed the slowest proliferation rate. Adding RI and /or FGF-2, increased cell proliferation. Cells exposed to DMEM-FBS + RI alone or with FGF-2, showed an up-regulation of the glomerular parietal epithelial cell mRNA transcripts ANXA3, CLDN1, AKAP12 and VCAM1, as well as the proximal tubular cells transcripts GPX3, SLC17A3 and SLC17A1, compared to those exposed to CR media with or without RI.  In contrast, cell exposed to CR + RI media  (with or without FGF-2) showed similar levels of podocyte mRNA transcripts (WT1, SYNPO, PLCE1, NPHS1, NPHS2) and proximal tubular epithelial cell transcripts (SLC17A3, SLC17A1, DCXR, SLC13A3, SERPINF2, SLC5A2,SLC5A12, SLCA22A7), as well as downregulation of the glomerular parietal epithelial transcripts described above, when compared to cells exposed to DMEM-FBS with and without RI and FGF-2.

Conclusion(s): A significant number of glomerular parietal epithelial cells are shed in the urine of children with primary nephrotic syndrome and proliferate in the presence of DMEM-FBS + RI and FGF-2. Our findings are consistent with the results of previous studies showing that glomerular epithelial cells underlying the Bowman capsule can generate podocytes and proximal tubular epithelial cells. We propose that CR media + RI can facilitate this transition.