Analysis and isolation of renal tubular cells by flow cytometry

Abstract

The cells of the renal cortex have rich heterogeneity of structure and function. Flow cytometry, the technique of rapid laser-based single cell analysis, can give information about cellular mixtures not obtainable by any other means. We examined a variety of fluorescent markers to identify populations of renal cells by flow cytometry. Cellular digests of rat cortex were fluorescently stained with either enzymatic activity probes, or polyclonal antibodies. Fluorescent staining for the proximal marker γ-glutamyl-transpeptidase (τ-GT) was an order of magnitude brighter than autofluorescence, and stained 71 ± 11% of the cells. Second, we colocalized enzymatic and antibody markers. There was tight colocalization of τ-GT enzyme activity, detected with fluorogenic substrates, with specific surface binding of τ-GT antibodies. Third, populations of fluorescently labelled cells can be rapidly isolated by flow cytometry sorting. Flow cylometry sorting isolated 107 cells positive for the proximal tubular marker τ-GT in a little under one hour. The sorted cells were viable with 99 ± 2% trypan blue exclusion (N = 8). Sodium-dependent phloridzin-inhibitable glucose uptake was present in sorted cells, with greater uptake/mg protein than in unsorted controls. The sorted cells grew in culture as a monolayer of tightly adherent cuboidal cells. Hence, flow cytometry allows us to quantitate the heterogeneity in mixed renal cellular digests. Flow cytometry allows us to rapidly isolate millions of cells according to fluorescently tagged markers. The isolated cells are viable, retain sodium-dependent transport properties, and grow in culture.