Isolation and culture of renal glomeruli from rats / 生理学报

ABSTRACT

The aim of the study was to establish a method for isolation and culture of rat renal glomeruli. The renal glomeruli of Sprague Dawley (SD) rats were isolated by a sieving method, and Bowman's capsule was removed by digesting the glomeruli in 0.5% type IV collagenase. The inverted phase contrast microscope was used to observe the structure of isolated renal glomeruli, and trypan blue staining was used to identify the activity of cells in glomeruli. Expressions of nephrin and α-smooth muscle actin (α-SMA) were observed by double-labeling immunofluorencence. Effects of Ang II on reactive oxygen species (ROS) generation were detected by dihydroethidium (DHE) staining. The levels of transforming growth factor-beta 1 (TGF-β1) and collagen IV mRNA were measured by real-time PCR. The results showed that the renal glomeruli with high purity and intact capillary structure were isolated by the modified protocol. The cells in the isolated glomeruli remained alive after 48 h of culture in DMEM. Confocal microscopy observations showed that nephrin and α-SMA were highly expressed in the isolated glomeruli. Treatment of the isolated renal glomeruli with Ang II enhanced ROS production. Furthermore, Ang II increased the mRNA levels of TGF-β1 and collagen IV. In conclusion, we have established a modified method that can isolate glomeruli from rat kidney, and the isolated glomeruli can be used for further observation in cultured condition. The protocol will provide a useful method for preclinical research on kidney diseases.