Primary culture of rat glomerular podocytes and its specific expression of Nephrin

GU Jie; ZHANG Chun-jiang; JIA Lin; YANG Xiao-ping

doi:10.3969/j.issn.1001-9448.2017.06.003

Abstract：Objective To establish a repeatable and simple method for culturing rat glomerular podocytes in vitro and to study the significant expression of podocyte marker,Nephrin.Methods Sterile kidneys were extracted from male SD rats.The glumeruli were ground and separated by enzymatic digesting and sieving (100 mesh,150 mesh,and 200 mesh),and subsequently exposed in culture medium at a constant temperature incubator for 9 days.The podocytes growing from glumeruli were digested and passaged to different container according to the corresponding experimental demands.The distribution of Nephrin and WT-1 were detected by cell morphological observation and indirect immunofluorescence staining method.The purity of podocytes were determine by flow cytometry technology.The specific expression of Nephrin was demonstrated by immunostaining,Western blotting and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results Most of the glomeruli were attached to culture dishes and exhibited cellular outgrowths,which were identified as podocytes by their distinct staining for Nephrin and WT-1.The purity of podocytes was 94.7％ according to flow cytometry analysis.The Nephrin expression levels of primary cultured podocytes were higher than those of subcultured cells.Conclusion The isolation method with collagenase provides a number of glomeruli suitable for primary podocyte culture with stable expression of Nephrin.