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

B. A. Molitoris, C. Meyer, R. Dahl, A. Geerdes

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

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 μM, 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.

Original languageEnglish (US)
Pages (from-to)F907-F916
JournalAmerican Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume264
Issue number5 33-5
DOIs
StatePublished - 1993

Keywords

  • endocytosis
  • gentamicin
  • immunocytochemical techniques
  • immunogold labeling
  • phosphatidylinositol
  • phospholipids

ASJC Scopus subject areas

  • Physiology

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