Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment

doi:10.1016/j.tox.2008.06.009

Toxicology

Volume 250, Issues 2–3, 4 September 2008, Pages 82-88

https://doi.org/10.1016/j.tox.2008.06.009 Get rights and content

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.

Introduction

Proximal tubule cells are sites of active secretion and reabsorption of xenobiotics and endogenous by-products into the urine and back into the body, respectively. Uptake of chemicals across basolateral membranes into the kidneys is mediated by secondary active transport systems including organic anion and cation transporters (Oats, Octs). Chemicals are subsequently effluxed across the brush border membrane into the urine by primary active transporters including multidrug resistance-associated proteins (Mrp) 2 and 4, multidrug resistance proteins (Mdr), breast cancer resistance protein (Bcrp) and multidrug and toxin extrusion proteins (Mate). P-glycoprotein (Pgp) is the protein product of Mdr genes. Reabsorption of chemicals from the filtrate can be accomplished by uptake carriers on the apical membrane including the organic anion transporting polypeptides (Oatps) as well as Oat2 (Klaassen and Lu, 2008). Retrograde transporters on the basolateral face of the plasma membrane, such as Mrp1, Mrp5 and Mrp6, reabsorb chemicals back into blood.

During periods of acute and chronic renal injury, glomerular filtration and renal excretion are reduced. This often necessitates reducing the dose and/or lengthening dosing intervals for pharmaceuticals that are cleared renally. While decreases in renal blood flow, metabolism and glomerular filtration are likely determinants of reduced renal filtration and consequently drug elimination, it is unknown whether the expression and/or function of drug transporters in the kidneys are altered during drug-induced renal damage.

Cisplatin is an effective antineoplastic drug for the treatment of solid tumors, although its clinical use is often limited because of adverse effects on renal function. Nephrotoxicity can be observed in as many as 38% of patients after a single dose of cisplatin (100 mg/m²) (Shord et al., 2006). This side effect often delays or precludes subsequent chemotherapy cycles, thereby reducing the overall antineoplastic efficacy of cisplatin. Rodents and humans develop renal injury at comparable doses (15–20 mg/kg in mice is equivalent to 45–60 mg/mm² in humans).

The initiation and progression of kidney injury by cisplatin is multifactorial, including biotransformation, adduct formation, oxidative stress, inflammation and changes in cell cycle. Humans and rodents exhibit similar histopathological changes and time profile for toxicity of cisplatin (Dobyan et al., 1980). Following cisplatin exposure, kidney sections demonstrate necrosis as well as apoptosis of renal proximal tubule cells in the S3 segment of the nephron, as well as occasional damage to distal tubules. The end result is compromised renal function. Because of the similar pathological changes among species, mice are routinely used to investigate mechanisms of cisplatin toxicity.

Previous reports demonstrate the affinity of cisplatin for cellular uptake via rat and human Oct2 (Ciarimboli et al., 2005, Yonezawa et al., 2005). Oct2 is localized to the basolateral surface of rat proximal tubule cells, predominantly in the S2 and S3 segments of the nephron (Karbach et al., 2000). Interestingly, administration of a single toxic dose of cisplatin to rats reduces Oct2 mRNA levels at 7 days, suggesting a defense response against subsequent exposure and renal uptake of cisplatin (Huang et al., 2001). Cisplatin treatment also increases expression of renal Mrp2 and Pgp in rats (Demeule et al., 1999, Huang et al., 2001). Mrp2 is overexpressed in a number of cisplatin-resistant cancer cell lines and tumors possibly implicating this transporter in the removal of cisplatin from cells (Cui et al., 1999, Kool et al., 1997). By contrast, Pgp does not transport cisplatin (Ishikawa and Ali-Osman, 1993). Therefore, induction of renal Pgp during cisplatin-induced nephrotoxicity suggests that the kidneys adapt to injury by up-regulating additional efflux transporters that aid in chemical elimination from the nephrotic kidney. Regulation of renal drug transporters in mice following cisplatin exposure has not been examined. Therefore, the purpose of this study was to investigate the effects of cisplatin on expression of uptake (Oat, Oct and Oatp) and export (Mrp, Mdr, Mate) xenobiotic transporters in mouse kidneys. Data generated from this study suggests that the kidney coordinately regulates the expression of xenobiotic transporters during drug-induced toxicity. Shifts in the expression of uptake and efflux transporters in the kidney may influence the pharmacokinetics and pharmacodynamics of co-administered pharmaceuticals.

Section snippets

Treatment regimen

Male C57BL/6J mice, aged 10–12 weeks old, were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice acclimated 1 week upon arrival. Animals were housed in a 12-h dark/light cycle, temperature- and humidity-controlled environment. The mice were fed laboratory rodent diet ad libitum. Cisplatin (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in saline (10 ml/kg). Groups of mice (4 control, 6 treated) were administered cisplatin (18 mg/kg, i.p.) or vehicle following overnight

Cisplatin-induced nephrotoxicity

Administration of cisplatin (18 mg/kg) to male C57BL/6J mice resulted in renal injury 4 days after treatment. Kim-1 is a novel renal biomarker of tubular damage that is measured in kidney or urine in addition to conventional serum chemistry markers. In the present study, cisplatin treatment increased renal Kim-1 mRNA levels by 42-fold over control values (Fig. 1A). This is in agreement with elevated plasma BUN levels (63 mg/dl) in cisplatin-treated mice compared to controls (26 mg/dl) (Fig. 1B).

Discussion

The present study investigated the effect of cisplatin toxicity (18 mg/kg) on the renal expression of numerous uptake and efflux xenobiotic transporters. Rodent models of acute renal failure by cisplatin are well-characterized. Injury is typically observed between 3 and 5 days following single dose exposure. Based on previous reports and our own pilot time-course studies, we selected 4 days following treatment as the time point to assess potential changes in expression of transport proteins. At

Conflict of interest

There are no conflicts of interest to report for the authors.

Acknowledgements

The authors would like to thank Steven Cohen and Angela Slitt for assistance with preliminary dose and time response studies. Lauren Aleksunes was a Howard Hughes Medical Institute Predoctoral Fellow. This work was supported by National Institutes of Health Grant DK069557.

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¹: Current address: University of Kansas Medical Center, Kansas City, KS, United States.

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Renal xenobiotic transporters are differentially expressed in mice following cisplatin treatment

Abstract

Introduction

Section snippets

Treatment regimen

Cisplatin-induced nephrotoxicity

Discussion

Conflict of interest

Acknowledgements

J. Biol. Chem.

Am. J. Pathol.

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

J. Urol.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Pharmacol.

Differential expression of mouse hepatic transporter genes in response to acetaminophen and carbon tetrachloride

Toxicol. Sci.

Tissue distribution and ontogeny of organic cation transporters in mice

Drug Metab. Dispos.

Prostaglandin transporter PGT is expressed in cell types that synthesize and release prostanoids

Am. J. Physiol. Renal Physiol.

Rat and mouse differences in gender-predominant expression of organic anion transporter (Oat1–3; Slc22a6-8) mRNA levels

Drug Metab. Dispos.

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