MDR1 transporter protects against paraquat-induced toxicity in human and mouse proximal tubule cells

Abstract

Paraquat is a herbicide that is highly toxic to the lungs and kidneys following acute exposures. Prior studies have demonstrated that the organic cation transporter 2 and multidrug and toxin extrusion protein 1 contribute to the urinary secretion of paraquat in the kidneys. The purpose of this study was to determine whether the multidrug resistance protein 1 (MDR1/Mdr1, ABCB1, or P-glycoprotein) also participates in the removal of paraquat from the kidneys and protects against renal injury. Paraquat transport and toxicity were quantified in human renal proximal tubule epithelial cells (RPTEC) that endogenously express MDR1, HEK293 cells overexpressing MDR1, and Mdr1a/1b knockout mice. In RPTEC cells, reduction of MDR1 activity using the antagonist PSC833 or siRNA transfection increased the cellular accumulation of paraquat by 50%. Reduced efflux of paraquat corresponded with enhanced cytotoxicity in PSC833-treated cells. Likewise, stable overexpression of the human MDR1 gene in HEK293 cells reduced intracellular levels of paraquat by 50%. In vivo studies assessed the renal accumulation and subsequent nephrotoxicity of paraquat (10 or 30 mg/kg ip) in wild-type and Mdr1a/1b knockout mice. At 4 h after paraquat treatment, renal concentrations of paraquat in the kidneys of Mdr1a/1b knockout mice were 750% higher than wild-type mice. By 72 h, paraquat-treated Mdr1a/1b knockout mice had more extensive tubular degeneration and significantly greater mRNA expression of kidney injury-responsive genes, including kidney injury molecule-1, lipocalin-2, and NAD(P)H quinone oxidoreductase 1, compared with wild-type mice. In conclusion, MDR1/Mdr1 participates in the elimination of paraquat from the kidneys and protects against subsequent toxicity.

Keywords: ABCB1; MDR1; Mdr1a; P-glycoprotein; paraquat; proximal tubule.

FIG. 1.

Paraquat accumulation in MDR1-transfected HEK293 cells. HEK293 cells were stably transfected with empty vector (EV) or full-length human MDR1 plasmids. (A) Protein expression of MDR1 was detected by Western blot analysis. β-actin was used as a loading control. HEK-EV and MDR1 transfected cells were treated with rhodamine 123 (25 μM) or paraquat (100 μM) for 30 min (uptake period), washed, and then incubated in fresh culture medium for 60 min (efflux period). (B) Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) Intracellular paraquat accumulation was quantified by ELISA and normalized to protein lysate concentrations. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with HEK-EV cells.

FIG. 2.

Expression and function of MDR1 transporter in RPTEC cells. (A) Protein expression of MDR1 in RPTEC cells was detected by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period) and then treated with the culture media with or without PSC833 (2 μM) for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. Data are presented as mean ± SE (n = 4). Asterisk (*) represents statistically significant differences (p < 0.05) compared with RPTEC cells treated only with rhodamine 123 (no PSC833).

FIG. 3.

Paraquat accumulation in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with paraquat (100 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). (A) Intracellular paraquat accumulation was quantified by ELISA normalized to protein concentrations of the cellular lysates. (B) Paraquat concentrations in the culture media were quantified by ELISA and compared with levels detected in RPTEC cell lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated only with paraquat (no PSC833).

FIG. 4.

Paraquat accumulation in RPTEC cells following siRNA knockdown of MDR1. RPTEC cells were transfected with siRNA duplexes targeted against human MDR1. (A) Protein expression of MDR1 at 72 h was assessed by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). MDR1 siRNA transfected RPTEC cells were treated with rhodamine (25 μM) for 30 min (uptake period), washed, and then incubated in fresh culture media for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) RPTEC cells were treated as described in (B) using paraquat (100 μM) as a substrate. Intracellular paraquat accumulation was quantified by ELISA and normalized to protein concentrations of the cellular lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells without PSC833 or MDR1 siRNA.

FIG. 5.

Cytotoxicity of paraquat in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with different concentrations of paraquat (0–25 mM) for 3 h in the presence and absence of the MDR1 inhibitor PSC833 (2 μM) (uptake period) and then incubated in fresh culture media with or without PSC833 for 69 h (efflux period). (A) Cytotoxicity was assessed using the LDH assay and expressed as percent of LDH released into the cell culture media relative to total LDH activity. (B) RPTEC cells were pretreated with vehicle or PSC833 (2 μM) for 2 h and then treated with vehicle or paraquat (100 μM) for 24 h. Protein expression of Ho-1 was semi-quantified by Western blot. β-actin was used as a loading control. Data are presented as mean ± SE (n = 3). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated by paraquat (no PSC833).

FIG. 6.

Renal and serum concentrations of paraquat in WT and Mdr1a/1b KO mice. WT and Mdr1a/1b KO mice were treated with paraquat (10 mg/kg, ip). Kidney (A) and serum (B) were collected at different time points from 2 to 48 h and paraquat was quantified by LC/MS and ELISA, respectively. (C) Basal expression of Oct2 and Mate1 proteins in kidney homogenates from naive WT and Mdr1a/1 KO mice was detected by Western blot analysis. β-actin was used as a loading control. Data are presented as mean ± SE (n = 4–8). Asterisks (*) represent statistically significant differences (p < 0.05) compared with WT mice at the same time point.

FIG. 7.

mRNA expression of renal injury markers in WT and Mdr1a/1b KO mice treated with paraquat. WT and Mdr1a/1b KO mice were treated with saline vehicle or paraquat (10 and 30 mg/kg, ip). After 72 h, kidneys were collected and total RNA isolated. mRNA expression of Kim-1, Lcn-2, Nqo-1, Ho-1, and Tnfα was quantified by qPCR assay and normalized to the housekeeping gene, Rpl13a. Data are presented as mean ± SE (n = 4–8). Asterisks (*) represent statistically significant differences (p < 0.05) compared with paraquat-treated WT mice. Pounds (#) represent statistically significant differences (p < 0.05) compared with vehicle-treated mice.

FIG. 8.

Kidney histopathology of WT and Mdr1a/1b KO mice treated with paraquat. WT and Mdr1a/1b KO mice were treated with saline vehicle or paraquat (0 and 30 mg/kg, ip). After 72 h, kidneys were collected and fixed in zinc formalin prior to routine processing and paraffin embedding. Sections (5 μm) of kidneys were stained with hematoxylin and eosin and examined by light microscopy for the presence and severity of proximal tubule degeneration, apoptosis, and necrosis as well cast formation and neutrophil infiltration. 4× and 20× magnification.