SPHINGOMYELIN ACCUMULATION IN PROXIMAL TUBULAR CELLS

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In human kidney proximal tubular (PT) cells incubated with gentamicin, we have found that there is increased cell surface binding and internalization of 125I-low density lipoproteins (LDL), but 125I-LDL degradation remains unchanged. This is accompanied by a 4 fold increase in the incorporation of [14C] acetate into sphingomyelin and the appearance of whorl-like "myeloid bodies" in the gentamicin-treated cells. Our overall objectives are: 1) to assess the contribution of exogenously derived lipids on plasma lipoproteins on the cellular sphingolipids in gentamicin-treated PT cells; 2) to assess the biochemical mechanisms responsible for the accumulation of sphingolipids in gentamicin-treated cells; 3) to isolate "myeloid bodies" from gentamicin-treated PT cells. First, the uptake of 3H-labeled sphingomyelin on low density lipoproteins (LDL) by control cells and gentamicintreated cells will be studied so as to investigate whether exogenously derived phospholipids on lipoproteins contribute directly to increased sphingomyelin levels in gentamicin-treated cells. The activities of ceramide choline phosphotransferases and palmitoyl-CoA: serine palmitoyl transferase will be measured next in order to assess whether gentamicin stimulated these enzymes, which in turn increased the endogenous synthesis of sphingolipids in RT cells. The levels of sphingomyelinase will be measured next in control and gentamicin-treated cells in order to assess whether inhibition of lysosomal degradation of sphingomyelin contributes to the increased cellular level of sphingomyelin. Finally, the myeloid bodies will be isolated from gentamicin-treated PT cells and characterized employing biochemical techniques and electron microscopy. Major hypothesis. Increased sphingomyelin in gentamicin-treated RT cells may be due to decreased sphingomyelin catabolism, increased uptake of exogenous sphingomyelin on lipoproteins or increased endogenous synthesis of sphingomyelin in gentamicin-treated PT cells. These studies will provide new insights in understanding sphingomyelin metabolism and lipoprotein metabolism in gentamycin-induced nephrotoxicity in human renal epithelial cells. Our studies on this culture system will provide a valuable tool in the extrapolation from animal data to man and can be used as an in vitro model for a host of future studies on drug-induced human nephrotoxicity.