[Primary culture and identification of rat glomerular podocytes].
OBJECTIVE
To establish an easy and feasible method for primary culture and identification of rat glomerular podocytes.


METHODS
Glomeruli from Sprague-Dawley (SD) rats weighing 60-100 gram were isolated by the method of different size combination of screen. Isolated glomeruli were appropriately digested with 2 g/L type IV collagenase and cultured in 25 cm2 plastic flask coated with rat tail collagen in K1-3T3 medium with ITS-X (containing insulin-transferrin-selenium). Subculture of primary cultured epithelial cells was performed at 9-10 days after implantation of collagenase digested glomeruli. Podocytes were identified by the morphology study with scanning electron microscope and inverted microscope, as well as the immunohistochemistry staining (SP methods) study for the expression of keratin, desmin and Wilms' tumor suppressor-1 (WT-1).


RESULTS
Epithelial cells outgrowth from isolated glomeruli appeared after 3 days primary culture and grew to confluence with cobblestone-appearance at 9-10 days. These cobblestone cells were subcultured at this point and gradually conversed into large, flat arborized cells with well-developed processes and microvilli. These arborized cells were negative expression with desmin staining and showed positive expression of cytokeratin and WT-1, which indicated that they were podocytes.


CONCLUSIONS
Implantating collagenase digested-glomeruli is an easy and feasible method for primary culture of rat glomerular podocytes. WT-1 may serve as a good marker to identify rat glomerular podocytes.
Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu, 610041, China.
Yu-Hong
YH
Tao
Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu, 610041, China.
Ya-Mei
YM
Wang
Department of Biochemistry and Molecular Biology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Wen-Zhen
WZ
Peng
D000818:Animals; D003470:Culture Media; D007678:Kidney Glomerulus / Q000166:cytology; D008297:Male; D050199:Podocytes / Q000166:cytology* / Q000648:ultrastructure; D061251:Primary Cell Culture / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D025721:WT1 Proteins / Q000378:metabolism
D003470:Culture Media; D025721:WT1 Proteins