Organic Cation Transporter 2 Overexpression May Confer an Increased Risk of Gentamicin-Induced Nephrotoxicity

Abstract

Nephrotoxicity is a relevant limitation of gentamicin, and obese patients have an increased risk for gentamicin-induced kidney injury. This damage is thought to depend on the accumulation of the drug in the renal cortex. Obese rats showed substantially higher levels of gentamicin in the kidney than did lean animals. This study characterized the role of organic cation transporters (OCTs) in gentamicin transport and elucidated their possible contribution in the increased renal accumulation of gentamicin in obesity. The mRNA and protein expression levels of the organic cation transporters Oct2 (Slc22a2) and Oct3 (Slc22a3) were increased in kidney samples from obese mice fed a high-fat diet. Similarly, OCT2 (∼2-fold) and OCT3 (∼3-fold) showed increased protein expression in the kidneys of obese patients compared with those of nonobese individuals. Using HEK293 cells overexpressing the different OCTs, human OCT2 was found to transport [(3)H]gentamicin with unique sigmoidal kinetics typical of homotropic positive cooperativity (autoactivation). In mouse primary proximal tubular cells, [(3)H]gentamicin uptake was reduced by approximately 40% when the cells were coincubated with the OCT2 substrate metformin. The basolateral localization of OCT2 suggests that gentamicin can enter proximal tubular cells from the blood side, probably as part of a slow tubular secretion process that may influence intracellular drug concentrations and exposure time. Increased expression of OCT2 may explain the higher accumulation of gentamicin, thereby conferring an increased risk of renal toxicity in obese patients.

FIG 1

Effect of diet-induced obesity on organic cation transporter expression in kidneys. (A) Quantification of mRNA levels of genes associated with cationic drug transport in mouse kidney (n = 6 mice/group). Data are expressed as means ± SD. *, P < 0.05 (Mann-Whitney test). (B) Western blot analysis of Oct2 and Oct3 protein levels in kidney samples of chow- and HFD-fed mice. (C) Representative images of immunostaining for Oct3 on kidney paraffin sections from the two groups.

FIG 2

OCT2 and OCT3 expression in renal biopsy specimens from nonobese and obese human subjects with minimal change disease. Shown are representative images of immunostaining for OCT2 (A and B) and OCT3 (D and E) in kidney specimens obtained from nonobese (body mass index < 21 kg/m² [A and D]) and obese (body mass index > 25 kg/m² [B and E]) patients with overt proteinuria (>1.0 g/day). Also shown is quantitative analysis of the positive staining of OCT2 (C) and OCT3 (F) per high-power field. Data are expressed as means ± SD *, P < 0.05 (Student t test).

FIG 3

Inhibition of the ASP⁺ OCT-mediated influx by nonlabeled gentamicin. (A) Influx of 1 μM ASP⁺ was assessed in OCT1, OCT2, and OCT3 stably transfected HEK293 cells at pH 7.4 over 2 min in the absence (control) or presence of nonlabeled gentamicin at an extracellular concentration of 10 mM. Uptake in WT HEK293 cells was subtracted from that in transfected cells to define the OCT-specific transport. Data are expressed as a percentage of the control. (B) ASP⁺ influx at the indicated extracellular concentrations (1, 5, or 20 μM) as a function of increasing extracellular concentrations of nonlabeled gentamicin. The line is derived by plotting 1/v (velocity) against the concentration of gentamicin (inhibitor) (Dixon analysis). Data represent the means ± SD from three independent experiments.

FIG 4

Time course of the net uptake of [³H]gentamicin in OCT-transfected cells. Uptake of [³H]gentamicin at an extracellular concentration of 10 μM in OCT1-HEK293 (A), OCT2-HEK293 (B), and OCT3-HEK293 (C) cells was measured as the function of the time and compared with that in wild-type cells. Data represent the means ± SD from three independent experiments.

FIG 5

Kinetic analysis of [³H]gentamicin influx. (A) Initial uptake of [³H]gentamicin was assessed over 4 min at pH 7.4 in OCT2-transfected HEK293 cells. Data were corrected for uptake in wild-type cells. [³H]gentamicin influx was determined as a function of the substrate concentration. The data best fit to the sigmoidal allosteric plot derived from the equation Y = V_max × X^h/(K_prime + X^h), where V_max is the maximum enzyme velocity, K_prime is the affinity parameter, and h is the Hill slope. (B) Enlargement of the first part of the kinetic analysis. (C and D) Hanes-Woolf (C) and Eadie-Hofstee (D) plots of the data. Results are means ± SD from five independent experiments.

FIG 6

Inhibitory effect of metformin on OCT2-mediated transport. (A) Uptake of [³H]gentamicin at an extracellular concentration of 10 μM in OCT2-HEK293 cells was measured as a function of time in the presence or absence of nonlabeled metformin. The results were corrected for uptake in wild-type cells; results of a representative experiment are shown. (B) Uptake over 5 min of [³H]gentamicin at an extracellular concentration of 10 μM in the presence or absence of 10 mM nonlabeled metformin was measured in primary proximal tubular cells (PTCs) isolated from kidneys of C57/BJ mice. Data are expressed as the percentage of [³H]gentamicin transport in the absence of nonlabeled metformin and represent the means ± SD from four independent experiments. *, P < 0.05 (Student t test).