Mechanism of ischemia-enhanced aminoglycoside binding and uptake by proximal tubule cells
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B A Molitoris1, C Meyer, R Dahl
1Department of Medicine, University of Colorado Health Sciences Center, Denver.
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 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.
Insights
Mild ischemia significantly increases gentamicin binding to kidney cells by altering phosphatidylinositol levels, leading to enhanced cellular uptake and abnormal accumulation in specific kidney cell types, contributing to nephrotoxicity.
Area of Science:
- Nephrology
- Cell Biology
- Pharmacology
Background:
- Aminoglycoside nephrotoxicity is exacerbated by preceding ischemia or hypotension.
- The precise mechanisms underlying this enhanced toxicity remain unclear.
- Understanding cellular handling of gentamicin during ischemic conditions is crucial.
Purpose of the Study:
- To investigate the impact of preceding mild ischemia on cellular gentamicin handling.
- To quantify changes in gentamicin binding to kidney brush-border membranes.
- To assess in vivo cellular gentamicin uptake following ischemic injury.
Main Methods:
- Utilized [3H]gentamicin as a tracer to quantify brush-border membrane vesicle binding.
- Measured in vivo cellular gentamicin uptake in kidney cortical and juxtamedullary regions.
- Employed immunocytochemical techniques with immunogold labeling for cellular localization.
Main Results:
- Fifteen minutes of ischemia markedly increased apical membrane gentamicin binding and phosphatidylinositol (PI) levels.
- Ischemia led to increased numbers of binding sites and reduced binding affinity for gentamicin.
- Increased gentamicin binding correlated with enhanced in vivo uptake in S1/S2 and S3 cells, with abnormal intracellular accumulation in S3 cells.
Conclusions:
- Ischemia significantly increases apical gentamicin binding, primarily due to elevated apical phosphatidylinositol content.
- This enhanced binding facilitates increased gentamicin internalization by proximal tubule cells (S1/S2 and S3).
- Ischemic injury results in abnormal intracellular compartmentalization of gentamicin within S3 cells, potentially contributing to nephrotoxicity.