The Toxicologist - Society of Toxicology

740 EVALUATION OF MRP2 TRANSPORTER-MEDIATED
PROTECTION AGAINST CISPLATIN-INDUCED
NEPHROTOXICITY USING IN VITRO AND IN VIVO
APPROACHES.
X. Wen 1 , M. Goedken 2 , C. D. Klaassen 3 , J. E. Manautou 4 and L. M.
Aleksunes 1 . 1 Pharmacology and Toxicology, Rutgers University, Piscataway, NJ,
2 Pathology, Merck & Co., Inc., Lafayette, NJ, 3 Pharmacology, University of Kansas
Medical Center, Kansas City, KS and 4 Pharmaceutical Sciences, University of
Connecticut, Storrs, CT.
Use of the chemotherapeutic drug cisplatin is limited in part by nephrotoxicity.
Prior work has demonstrated that the organic cation transporter 2 (OCT2) is responsible
for uptake of cisplatin into renal tubule cells. We hypothesized that the
multidrug resistance-associated protein 2 (MRP2) effluxes cisplatin metabolites
into urine thereby limiting intracellular accumulation and subsequent nephrotoxicity.
In the present study, cisplatin disposition and toxicity were assessed in MRP2and
OCT2-transfected HEK293 cells (human embryonic kidney cells) and also in
male wild-type and Mrp2-null mice. Cisplatin induced cytotoxicity in HEK293
cells at 72 h with an IC50 value of about 4 μM. More potent toxicity was observed
in OCT2-expressing cells (IC50= 1 μM), whereas less toxicity (IC50=7 μM) was
found in MRP2-expressing cells. To study the in vivo role of MRP2 in the renal efflux
of cisplatin, serum and kidneys were collected from wild-type and Mrp2-null
mice between 10 min and 4 days after cisplatin treatment (15-23 mg/kg ip). At 3
and 4 days, plasma BUN levels were 2.4-fold higher in Mrp2-null mice than wildtype
mice. Histopathological analysis confirmed proximal tubule degeneration and
necrosis in cisplatin-treated mice, with more extensive injury observed in Mrp2null
mice. Increased susceptibility of Mrp2-null mice to cisplatin toxicity correlated
with higher renal platinum levels (27% and 47% at 24 and 48 h, respectively) compared
to wild-types. Notably, Oct2 protein expression was similar between wildtype
and Mrp2-null mice. Collectively, these data suggest that OCT2 and MRP2
are responsible for the renal secretion of cisplatin and deficiency in Mrp2 increases
platinum accumulation and susceptibility to cisplatin-induced kidney injury in
mice (Supported by DK-080774).
741 MODULATION OF CISPLATIN MEDIATED OXIDATIVE
STRESS BIOMARKERS BY RESVERATROL.
M. Valentovic, J. G. Ball and J. Brown. Pharmacology, Physiology, and Toxicology,
Marshall University School of Medicine, Huntington, WV.
Cisplatin is associated with a serious dose limiting nephrotoxicity as a major limiting
side effect. The development of interventions to retard or prevent cisplatin associated
renal toxicity would be of immediate human relevance. Resveratrol (RES)
is a naturally occurring phytochemical which displays both anticancer and antioxidant
properties. This study examined whether alterations in oxidative stress markers
were an early event of cisplatin toxicity that occurred prior to the loss of membrane
integrity. Male Fischer 344 rats (200-250 g) were anesthetized with isoflurane and
the kidneys were isolated, decapsulated and rinsed in 3 ml ice cold Krebs buffer.
Renal cortical slices were prepared and 50-100 mg of tissue was pre-incubated with
30 ul ethanol (VEH) or 30 ug/ml resveratrol (RES, final concentration) for 30 min
at 37oC under an oxygen atmosphere. The tissue was incubated for 120 min in 3
ml oxygenated Krebs containing 0, 75, or 150 ug/mL cisplatin in an oxygen atmosphere
under the same conditions. Loss of membrane integrity was evaluated as leakage
of lactate dehydrogenase (LDH). Cisplatin associated oxidative stress was evaluated
by measuring total, Cu/Zn and Mn Superoxide dismutase (SOD),
glutathione peroxidase and catalase enzyme activity in renal cortical slices. The protective
effect of RES was evaluated following a 30 min pre-exposure. LDH leakage
required a 120 min exposure to cisplatin. GSH peroxidase activity was diminished
by cisplatin when compared to vehicle control beginning at 60 min. Total SOD activity
was also diminished by cisplatin beginning at 60 min. A 30 min pre-incubation
with RES reduced oxidative stress by maintaining GSH peroxidase enzyme activity.
Total SOD enzyme activity was preserved by RES co-incubation with
cisplatin. These findings indicate that RES pre-incubation diminishes cisplatin
renal toxicity and early changes in oxidative stress prior to the onset of LDH leakage.
(Supported by NIH Grant INBRE 3P20RR016477-09S4).
742 URINARY BIOMARKERS FOR THE EARLY
PREDICTION OF CISPLATIN-INDUCED CHANGES IN
KIDNEY FUNCTION AND INJURY IN RATS.
Y. Chen, D. Brott, P. Bentley, D. Thurman, L. Kinter and R. Bialecki. Safety
Assessment, AstraZeneca Pharmaceuticals, Wilmington, DE.
Cisplatin (CDDP) is a common chemotherapy treatment for solid tumors; however,
nephrotoxicity remains a major adverse effect of its clinical use. Current diagnosis
of drug-induced acute kidney injury (DIKI) relies on detection of elevated
160 SOT 2011 ANNUAL MEETING
serum creatinine (SCr) and blood urea nitrogen (BUN) levels, delayed biomarkers
of acute insult. Our aim was to evaluate if CDDP-induced changes in multiple
DIKI biomarkers preceded decrements in renal hemodynamics (GFR, RPF) and
histopathology changes in the same animal using a novel integrated pharmacology
platform. The platform consists of an automated blood sampling and radio telemetry
(ABST) system with continuous infusion and quantitative urine collection capability
to measure renal hemodynamics and nephron site-specific DIKI biomarkers
(KIM-1, RPA-1, albumin, α-GST, GSTYb1, lipocalin, clusterin and
osteopontin) in surgically prepared conscious male Wistar rats (n=8). After a single
administration of CDDP (15 mg/kg, i.p.), renal hemodynamics, excretory function,
and DIKI biomarkers were measured on days 1, 2 and 3 via 24-hour urine
collection samples. Renal tissue was examined microscopically at study termination.
Cisplatin induced significant increases in SCr and BUN (5.9- and 7.2-fold vs.
baseline, respectively; P