Influence of Oct1/Oct2-deficiency on cisplatin-induced changes in urinary N-acetyl-beta-D-glucosaminidase

Abstract

Purpose: This study aimed to test the influence of functional renal organic cation transporters (OCT2 in humans, Oct1 and Oct2 in mice) on biomarkers of cisplatin nephrotoxicity, such as urinary activity of N-acetyl-beta-D-glucosaminidase (NAG).

Experimental design: Temporal cisplatin-induced nephrotoxicity was assessed by histopathology and biomarkers. Cisplatin-mediated NAG changes and survival were determined in wild-type and Oct1/2(-/-) mice. Identification of OCT2 inhibitors was done in transfected 293Flp-In cells, and the NCI(60) cell line panel was used to assess contribution of OCT2 to cisplatin uptake in cancer cells.

Results: Classical biomarkers such as blood urea nitrogen and serum creatinine were not elevated until 72 hours after cisplatin administration and substantial kidney damage had occurred. Oct1/2(-/-) mice had 2.9-fold lower NAG by 4 hours (P < 0.0001) and 2.3-fold increased survival (P = 0.0097). Among 16 agents, cimetidine strongly inhibited uptake of tetraethylammonium bromide (P = 0.0006) and cisplatin (P < 0.0001), but did not have an influence on cisplatin uptake in SK-OV-3 cells, the cancer line with the highest OCT2 mRNA levels. In wild-type mice, cimetidine inhibited cisplatin-induced NAG changes (P = 0.016 versus cisplatin alone) to a degree similar to that seen in Oct1/2(-/-) mice receiving cisplatin (P = 0.91). Cumulative NAG activity of >0.4 absorbance units (AU) was associated with 21-fold increased odds for severe nephrotoxicity (P = 0.0017), which was linked with overall survival (hazard ratio, 8.1; 95% confidence interval, 2.1-31; P = 0.0078).

Conclusions: Cimetidine is able to inhibit OCT2-mediated uptake of cisplatin in the kidney, and subsequently ameliorate nephrotoxicity likely with minimal effect on uptake in tumor cells.

Figure 1

Measures of nephrotoxicity in adult male FVB mice after administration of cisplatin (17.5 mg/kg, i.p.; n=3 per time point). (A) Left panel, mean of blood urea nitrogen (BUN); Middle panel, mean of serum creatinine; Right panel, mean histopathology scores determined from the extent of acute renal tubular necrosis based on the percentage of observed damaged tubules: 0, absent; 1, <25% (rare); 2, 25–50% (mild); 3, 50–75% (moderate); 4, >75% (severe). Data are shown as mean (bars) and SE (error bars); (B) Representative kidney histopathology before and after administration of cisplatin. ***, P<0.001.

Figure 2

Influence of Oct1/Oct2-deficiency on cisplatin-induced changes in N-acetyl-β-D-glucosaminidase (NAG) and overall survival (n=9–19 per dose per genotype). (A) Cumulative urinary NAG activity in adult male FVB mice (closed symbols, solid line; n=12) and Oct1/2(−/−) mice (open symbols, dotted line; n=12) after administration of cisplatin (10 mg/kg, i.p.). Data are shown as mean (bars) and SE (error bars); (B) Overall survival curves in adult male FVB wildtype (dotted lines) and Oct1/2(−/−) mice (solid lines) after administration of cisplatin at a dose of 10 mg/kg (thin lines) or 20 mg/kg (thick lines). Symbols are shown for censored observations. ***, P<0.001.

Figure 3

Inhibition of OCT2-mediated transport of tetraethylammonium bromide (TEA) by various prescription drugs. Transport of [¹⁴C]TEA (2 µM) during a 30-min incubation was assessed in 293Flip-In cells transfected with an empty vector (VC) or with OCT2 in the presence and absence of the inhibitors at a substrate-to-inhibitor concentration ratio of 1:500 (1 mM of inhibitor) or 1:0.5 (1 µM of inhibitor). Data represent the extent of TEA uptake in OCT2-overexpressing cells corrected for nonspecific uptake in VC cells, and were expressed as a percentage of uptake in the absence of inhibitors, which was set at 100%. Data are shown as mean (bars) and SE (error bars) of at least three experiments performed in triplicate. Red, cancer drugs; Blue, supportive-care drugs; Green, known OCT2 inhibitors; Black, other. *, P<0.05 vs control; **, P<0.01 vs control; ***, P<0.001 vs control.

Figure 4

Effect of cimetidine (1 mM) on the accumulation of cisplatin (500 µM, for 30 minutes) in 293Flp-In cells transfected with empty vector (VC) or OCT2, or HEK293 cells transfected with empty vector (VC), wildtype OCT2, or the OCT2 p.270Ala>Ser variant (OCT2-Var). Open bars represent cisplatin alone, black bars represent cisplatin in the presence of cimetidine. Data are shown as mean (bars) and SE (error bars) of three experiments performed in triplicate. ***, P<0.001.

Figure 5

Cisplatin-induced (10 mg/kg, i.p.) changes in urinary NAG activity in Oct1(−/−) (n=6), Oct2(−/−) (n=6), Oct1/2(−/−) (n=12), or wildtype mice receiving no pretreatment (n=12), vehicle injection (saline, i.v.) (n=5), or cimetidine (30 mg/kg, i.v.) (n=6). Open bars represent pretreatment levels of urinary NAG activity, black bars are cumulative urinary NAG activity over 48 h. Data are shown as mean (bars) and SE (error bars).

Figure 6

Expression of the OCT2 gene, SLC22A2, in the NCI60 cancer cell lines and its influence on cisplatin transport. (A) Real-time PCR expression levels of SLC22A2 (normalized to GAPDH) in the NCI60 panel. Abbreviation: CNS, central nervous system; (B) Real-time PCR expression of the OCT1 gene, SLC22A1, and SLC22A2 in 293Flip-In cells transfected with an empty vector (VC), 293Flip-In cells transfected with OCT2, and SKOV-3 cells; (C) Influence of cimetidine (1 mM) on the uptake of cisplatin (500 µM) in SKOV-3 cells. Data are shown as mean (bars) and SE (error bars) of three experiments performed in triplicate.