Mechanism of ischemia-enhanced aminoglycoside binding and uptake by proximal tubule cells.
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.
Department of Medicine, University of Colorado Health Sciences Center, Denver.
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Molitoris
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Meyer
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Dahl
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Geerdes
D000617:Aminoglycosides / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005839:Gentamicins / Q000493:pharmacokinetics; D007150:Immunohistochemistry; D007511:Ischemia / Q000378:metabolism* / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000098:blood supply / Q000378:metabolism* / Q000473:pathology; D008297:Male; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015424:Reperfusion; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism / Q000494:pharmacology
D000617:Aminoglycosides; D005839:Gentamicins; D010716:Phosphatidylinositols; D010738:Type C Phospholipases