AJP - Renal Physiology, Vol 264, Issue 5 907-F916, Copyright © 1993 by American Physiological Society

ARTICLES

Mechanism of ischemia-enhanced aminoglycoside binding and uptake by proximal tubule cells

B. A. Molitoris, C. Meyer, R. Dahl and A. Geerdes
Department of Medicine, University of Colorado Health Sciences Center, Denver.

Preceding ischemia or concurrent hypotension is known to enhance aminoglycoside nephrotoxicity; however, the underlying mechanisms responsible have not been determined. To investigate the effect of preceding mild ischemia on cellular gentamicin handling, brush-border membrane vesicle binding and in vivo cellular gentamicin uptake were quantified using [3H]gentamicin as a tracer. Fifteen minutes of ischemia resulted in a marked increase in apical membrane gentamicin binding (2.8 +/- 0.4 vs. 4.9 +/- 0.8 nmol/mg protein, P < 0.01). This increase was associated with an increased number of binding sites (3.7 +/- 0.3 vs. 9.1 +/- 2.3 nmol/mg protein, P < 0.01) and a reduced binding affinity (11.8 +/- 2.2 vs. 27.7 +/- 10.4 microM, P < 0.01). This increase in gentamicin binding was accompanied by alterations in apical membrane phospholipids including a doubling of phosphatidylinositol (PI) levels (13.8 +/- 0.4 vs. 27.5 +/- 3.1 nmol/mg protein, P < 0.01). Furthermore, treatment of apical membrane vesicles with PI-specific phospholipase C markedly reduced the difference in gentamicin binding between paired control and ischemic membrane fractions. Increased gentamicin binding was associated with increased in vivo uptake of gentamicin by S1/S2 and S3 cells. Outer cortical uptake of gentamicin increased from 2.18 +/- 0.39 to 2.68 +/- 0.27 nmol/mg protein (P < 0.01) after 15 min of ischemia and 4 h of reperfusion. Juxtamedullary uptake also increased from 1.39 +/- 0.31 to 1.75 +/- 0.12 nmol/mg protein (P < 0.01). Immunocytochemical techniques, utilizing immunogold labeling, showed gentamicin was taken up via the receptor-mediated endocytic pathway by S1/S2 and S3 cells. After ischemic injury gentamicin was localized in abnormal intracellular accumulations in S3 but not S1 or S2 cells. Taken together, these data indicate ischemia results in a marked increase in apical gentamicin binding due to increases in apical PI content. This is associated with increased internalization by S1/S2 and S3 cells and abnormal intracellular compartmentalization of gentamicin within S3 cells.

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