Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment

Abstract

The goal of this study was to identify alterations in mRNA and protein expression of various xenobiotic transport proteins in mouse kidney during cisplatin-induced acute renal failure. For this purpose, male C57BL/6J mice received a single dose of cisplatin (18 mg/kg, i.p.) or vehicle. Four days later, tissues were collected for assessment of plasma BUN, histopathological analysis of renal lesions, and mRNA and Western blot analysis of renal transporters including organic anion and cation transporters (Oat, Oct), organic anion transporting polypeptides (Oatp), multidrug resistance-associated proteins (Mrp), multidrug resistance proteins (Mdr), breast cancer resistance protein (Bcrp) and multidrug and toxin extrusion proteins (Mate). Cisplatin treatment caused necrosis of renal proximal tubules along with elevated plasma BUN and renal kidney injury molecule-1 mRNA expression. Cisplatin-induced renal injury increased mRNA and protein levels of the efflux transporters Mrp2, Mrp4, Mrp5, Mdr1a and Mdr1b. Uptake transporters Oatp2a1 and Oatp2b1 mRNA were also up-regulated following cisplatin. By contrast, expression of Oat1, Oat2, Oct2 and Oatp1a1 mRNA was reduced in cisplatin-treated mice. Expression of several uptake and efflux transporters was unchanged in cisplatin-treated mice. Apical staining of Mrp2 and Mrp4 proteins was enhanced in proximal tubules from cisplatin-treated mice. Collectively, these expression patterns suggest coordinated regulation of uptake and efflux pathways during cisplatin-induced renal injury. Reduced expression of basolateral and apical uptake transporters along with enhanced transcription of export transporters likely represents an adaptation to lower intracellular accumulation of chemicals, prevent their reabsorption and enhance urinary clearance.

Figure 1. Markers of cisplatin-induced nephrotoxicity

(A) Renal Kim-1 mRNA expression. Total RNA was isolated from kidneys of mice 4 days following injection with cisplatin (18 mg/kg, i.p.) or saline vehicle. RNA was analyzed by branched DNA assay for expression of Kim-1. The data are presented as mean relative light units (RLU) ± SE. (B) Plasma BUN Level. Plasma was isolated from mice treated with cisplatin or saline vehicle. The data are presented as mean plasma BUN (mg/dl) ± SE. (C and D) Kidneys from control (C) and cisplatin-treated (D) mice were fixed in formalin and stained with hematoxylin and eosin (x200). Asterisks (*) represent a statistical difference (p<0.05) from control.

Figure 2. Renal mRNA expression of Oat, Oct and Oatp transporters in cisplatin-treated mice

Total RNA was isolated from kidneys of mice 4 days following injection with cisplatin (18 mg/kg, i.p.) or saline vehicle. RNA was analyzed by branched DNA assay for expression of Oat1-3, Oct1-3, Oatp1a1, 14, 1a6, 2a1, 2b1 and 3a1. The data are presented as mean relative mRNA expression ± SE. Asterisks (*) represent a statistical difference (p<0.05) from control.

Figure 3. Renal mRNA expression of Mrp, Mdr, Bcrp and Mate transporters in cisplatin-treated mice

Total RNA was isolated from kidneys of mice 4 days following injection with cisplatin (18 mg/kg, i.p.) or saline vehicle. RNA was analyzed by branched DNA assay for expression of Mrp1-6, Mdr1a, 1b, 2, Bcrp, Mate-1 and Mate-2. The data are presented as mean relative mRNA expression ± SE. Asterisks (*) represent a statistical difference (p<0.05) from control.

Figure 4. Renal protein expression of Oct, Mrp, Bcrp and Pgp transporters in cisplatin-treated mice

Western immunoblots were performed using kidney crude membranes (60 µg protein/well) from mice at 4 days after treatment with cisplatin (18 mg/kg, i.p.) or saline vehicle. The data are presented as representative blots (A) and as mean relative Oct2, Mrp1, Mrp2, Mrp4, Mrp5, Mrp6, Pgp and Bcrp expression ± S.E. Blots were probed for β-actin staining to confirm equal protein loading. Asterisks (*) represent a statistical difference (p<0.05) from control.

Figure 5. Immunofluorescent staining of Mrp2 and Mrp4 transporters in kidneys from cisplatin-treated mice

Indirect immunofluorescence against apical Mrp2 protein (green) and actin (red) was conducted on kidney sections obtained at 4 days from control (A, B) and cisplatin-treated (C, D) mice. Indirect immunofluorescence against apical Mrp4 protein (green) and actin (red) was conducted on kidney sections obtained at 4 days from control (E, F) and cisplatin-treated (G, H) mice. Two magnifications are shown; x1000 (A, C, E, G) and x400 (B, D, F, H).