Cationic substances like myeloma proteins, cationized albumin, and gentamicin are nephrotoxic at the level of the proximal tubule. Transport defects may occur rapidly, prior to the appearance of apparent biochemical or morphological damage. An early defect is bicarbonaturia, which is due to a reduction in Na/H antiporter activity in the luminal membrane of proximal tubular cells. The toxic cationic substances mentioned above enter the cells by endocytosis at the luminal membrane. We have recently shown that endosomes containing cationic material fail to fuse with lysosomes during early stages of uptake, and remain sequestered within the cytoplasm of proximal tubular cells. This raises the possibility that membrane transport proteins, including Na/H antiporters, may be taken up during the endocytic process and become trapped in a pool of endosomes that are not able to recycle normally. Preliminary evidence supports this interpretation. Calcium and magnesium have been reported to protect against gentamicin nephrotoxicity, and both inhibit binding of gentamicin to the luminal membrane. The purpose of this proposal is to examine the nature of early transport defects in the kidney due to cationic substances. The following hypothesis will be tested: endocytosis of cationic substances by renal proximal tubular cells causes the internalization of plasmalemmal carriers, which become trapped in a pool of cytoplasmic endosomes; calcium and magnesium protect against these charge-related events. Thus, the following specific aims are proposed: 1) to show that endocytosis of cationized albumin or gentamicin by renal proximal tubular cells inhibits fusion of endosomes to lysosomes, causing an accumulation of endosomal membrane in the cytoplasm during the early phase of toxicity; 2) to show that endocytosis of cationized albumin or gentamicin by renal proximal tubular cells results in the internalization and sequestration of Na/H antiporters in a pool of cytoplasmic endosomes, during the early phase of toxicity; 3) to show that calcium and magnesium reduce endocytosis of cationized albumin and gentamicin, diminish the endosomal-lysosomal fusion defect, and decrease the sequestration of Na/H antiporters observed in renal proximal tubular cells during the early phase of toxicity. The effects of cationized albumin and gentamicin on membrane shuttling and Na/H antiporter activity will be measured in microperfused proximal nephron segments and isolated renal membranes.
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