Preparative free flow electrophoresis for the isolation of two populations of proximal cells from the rabbit kidney

Abstract

High voltage free flow electrophoresis is a carrier-free method used for analytical and preparative cell separation, based on charge surface properties of cells. Two cell populations from the proximal tubule of the rabbit kidney were isolated by free flow electrophoresis from a suspension of pure proximal cells. This single-cell suspension was obtained through an original method by the combination of a Ca-binder action and gentle mechanical treatment associated with several shifting steps, on a pure suspension of isolated proximal tubules. Before the electrophoretic separation, the proximal cell origin was confirmed by enzymatic marker measurements, and the metabolic capacity was assessed by the cell respiratory activity. The isolated cells were very poor in distal tubule marker enzymes and were enriched in proximal tubule marker enzymes. Respiratory measurement showed a high cell metabolic capacity. After the electrophoretic separation, the origin of the cell populations was assessed by measuring specific marker enzymes. The cells in the slow-moving electrophoresis fractions had a high gamma-glutamyl transpeptidase activity and a low glucose-6-phosphatase activity. The fast moving cells showed a high glucose-6-phosphatase content and a poor gamma-glutamyl transpeptidase activity. Cells isolated by free flow electrophoresis were shown to possess long microvilli. This new methodology, allowed for the first time, the separation of a fast-moving cell population originating from the convoluted portion of the proximal tubule and a slow-moving cell population originating from the straight part of the proximal tubule of the rabbit kidney.