Isolation and purification of proximal kidney cells by centrifugal elutriation

Centrifugal elutriation has been used to prepare a proximal cell fraction (PCS) enriched in proximal tubule cells. Routinely an initial cell suspension (ICS) containing approximately 250 x 10(6) cells was isolated from four kidneys by EGTA/collagenase perfusion. Approximately 50% of the ICS were single, proximal tubule cells (13-19 mum diameter). The remaining small cells, cell debris, larger cells and undigested tubular fragments were removed by centrifugal elutriation. Approximately 60 x 10(6) PCS cells were obtained, of which 80% were proximal tubule cells. Histochemically, the proximal tubule enzymes gamma-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALKP) were located in the S(1) (ALKP), S(2) (GGT + ALKP) and S(3) (GGT) regions of the nephron. PCS cells exhibited GGT and ALKP staining and appear to be derived from the S(2) and S(3) regions. Enzyme specific activities (nmol/10(6) cells) for the PCS fraction were: GGT, 672 +/- 78 (n = 13); ALKP, 89 +/- 7 (n = 13); and hexokinase, 28 +/- 1 (n = 7). The last of these, a distal and loop of Henlé tubule marker enzyme, demonstrates that the PCS has some contamination from these cells. PCS cells were functionally competent as demonstrated by trypan blue exclusion (>90%) and succinate stimulation of cellular respiration, which was inhibited by cyanide. These studies demonstrate that centrifugal elutriation is a good isolation method for preparing proximal tubule cells for in vitro nephrotoxicity studies.