Decreasing glycolysis increases sensitivity to mitochondrial inhibition in primary cultures of renal proximal tubule cells

We have previously shown that shaking the culture plates (SHAKE) of rabbit renal proximal tubule cells (RPTC) to maintain adequate aeration increased aerobic metabolism and decreased the induction of glycolysis compared to RPTC cultured under standard conditions (STILL). However, glycolysis in SHAKE RPTC remained elevated compared to glycolysis in proximal tubules in vivo. In the present study the contribution of culture medium sugar composition and concentration to glycolytic metabolism was assessed in RPTC. SHAKE and STILL RPTC cultured in 5 m M glucose contained lactate levels equivalent to the respective SHAKE and STILL RPTC cultured in standard culture medium which contains 17.5 m M glucose. Similarly, the activity of lactate dehydrogenase was unchanged by lowering the medium glucose concentration. Substituting 5 m M galactose for 5 m M glucose in the culture medium significantly reduced the lactate content of both SHAKE and STILL RPTC but had no effect on lactate dehydrogenase activity. Cell growth was equivalent under all culture conditions. Sensitivity to mitochondrial inhibition was determined for each culture condition by measuring cell death after exposure to the respiratory inhibitor antimycin A. The results showed a hierarchy of sensitivity to antimycin A (5 m M galactose SHAKE >5 m M glucose SHAKE >17.5 m M glucose SHAKE = 17.5 m M glucose STILL), which was generally inversely correlated with the level of glycolysis as measured by lactate content (17.5 m M glucose STILL >17.5 m M glucose SHAKE = 5 m M glucose SHAKE >5 m M galactose SHAKE).