Isolation of hog renal brush border membrane vesicles with application to the study of cadmium nephrotoxicity

Abstract

The epithelial cell of the kidney proximal convoluted tubule, like that of the small intestine, consists of two distinct plasma membrane surfaces referred to as the brush border, or luminal surface, and the basal-lateral, or contraluminal surface. Once isolated, membrane vesicles formed from the kidney cell can serve as models for the study of heavy metal mephrotoxin interaction with the two different plasma membrane surfaces. For ideal comparison between the two membranes, the isolation procedure should be identical for both. This has been accomplished by other investigators using differential centrifugation followed by free-flow electrophoresis or sucrose density centrifugation. Utilizing the self-forming gradient capabilities of Percoll, we have developed a less cumbersome procedure to effect simultaneous isolation of brush border and basal-lateral membrane vesicles. Brush border membrane vesicles isolated by this procedure exhibited 8 to 13-fold enrichment in marker enzyme activity but appeared to have impaired glucose uptake activity. Since the Percoll-prepared vesicles seemed unsuitable for uptake studies, brush border membrane vesicles isolated by a differential precipitation method were used to investigate the effect of cadmium on glucose uptake. Of the concentrations of cadmium tested, 0.1 mM CdCl slightly inhibited glucose uptake whereas 1 mM CdCl greatly reduced glucose uptake in these vesicles.