Isolation of single mammalian proximal tubule cells: effects of hypotonic shocks on cell yield and function.

Abstract

Nord et al. [Am J Physiol 1986; 250:F539-F550] proposed a method to give a high yield of proximal tubule cells by exposing a suspension of rabbit cortical tubules to a hypotonic shock in calcium-free media. The present study describes the effects of both amplitude and duration of the hypotonic treatment on some transport-related characteristics of individual cells as compared to the starting tubule suspension. The averaged cell yield increased by an order of magnitude when the osmolality of the hypotonic solution was varied in four steps from 200 (C200 cells) to 70 mosm/kg H2O (C70 cells) while the proportion of trypan blue-positive cells progressively decreased from 33% for C200 cells to 9.5% for C70 cells. An increase in duration of the hypotonic shock from 0.5 to 6 min did not change the cell yield of C200 cells while it significantly increased that of C70 cells by 61%. Basal and ouabain-sensitive oxygen consumption (QO2) increased by 57 and 155%, respectively, from C70 to C200 cells but was approximately one order of magnitude smaller than the QO2 measured for tubule suspension. Intracellular ATP content averaged 5.5 +/- 0.8 nmol/mg for the starting tubule suspension, 4.6 +/- 0.8 nmol/mg for C70 cells but only 1.3 +/- 0.1 nmol/mg for C200 cells. The maximal velocity for phloridzin-sensitive alpha-methyl glucose transport averaged 13.7 +/- 1.7 nmol min-1 mg-1 for C70 cells and only 6.3 +/- 1.3 nmol min-1 mg-1 for C200 cells which is approximately one order of magnitude smaller than what can be expected from a tubule presenting a good access to luminal membrane. We conclude from these results that, in the process of isolating individual cells from a polarized epithelium, membrane transport rates have decreased by one order of magnitude and this reduction is intensified by a large hypotonic shock. In comparison with C200 cells, the cells obtained with a large hypotonic shock give a high yield, a larger proportion of trypan blue-negative cells and their lower overall transport rate allows the cells to maintain a better electrochemical gradient for Na and a higher intracellular ATP level.