Effects of C3aR agonists on the phenotype of mouse primary renal tubular epithelial cell / 医学研究生学报

ABSTRACT

Objective The key points in the epithelial-mesenchymal transition ( EMT) procedure include the downregula-tion of epithelial protein (E cadherin) and the upregulation of cell activity and cell matrix generation .The aim of this study was to es-tablish a method for primary culture and identification of mouse renal tubular epithelial cells and to explore whether the activation of C3aR can induce epithelial-to-mesenchymal transition in mouse primary renal epithelial cells . Methods Murine renal tubular seg-ments were used for primary cell culture .Immunocytochemistry and immunofluorescence staining were used to identify the renal tubular epithelial cells.The experiment groups included control group , five different concentrations of C3aR agonist groups (0.1, 1, 100, 500, and 2000 ng/mL), and three different time-point groups.The mRNA levels of E-cadherin,α-smooth muscle actin (SMA) and colla-gen I in renal tubular epithelial cells were detected by Real-time PCR; the protein of E-cadherin, α-SMA were detected by Western blot.The cytoskeleton of epithelial cells was observed by phalloidin staining . Results Compared with the control group , the protein expression of E-cadherin deceased (0.950±0.901 vs 0.650±0.221) and the expression of α-SMA (1.380±0.062 vs 1.600±0.103) and collagen I increased in C3aR agonist group (500 ng/mL, after 48 hours) (P<0.05).In addition, the association between these changes and C3aR agonists was presented in a dose-and time-dependent man-ner, respectively.The cytoskeleton staining showed that treatment of renal tubular epithelial cells with C 3aR agonists induced the formation of actin stress fibers in a time-dependent manner . Conclusion The method for primary culture and identification of mouse renal tubular epithelial cells were successfully established .The activation of C3aR could induce epithelial-to-mesenchymal transition in mouse primary renal epithelial cells , which plays an essential role in the de-velopment of renal fibrosis .Moreover , this study indicated that C 3aR may become a new therapeutic target in kidney diseases .