Metabolomic study of cisplatin-induced nephrotoxicity

We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H—nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-α (PPARα) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARα ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARα ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin-induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARα ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity. We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H—nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-α (PPARα) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARα ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARα ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin-induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARα ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity. Previous studies have documented the presence of glucose intolerance and insulin resistance, as metabolic abnormalities that coexist with the development of acute renal failure (ARF).1.Briggs J.D. Buchanan K.D. Luke R.G. et al.Role of insulin in glucose intolerance in uremia.Lancet. 1967; 1: 462-464Abstract PubMed Google Scholar, 2.Mondin E. Dolkas C.B. Reaven G.M. et al.The site of insulin resistance in acute uremia.Diabetes. 1978; 27: 571-576Crossref PubMed Scopus (41) Google Scholar, 3.Clark A.S. Mitch W.E. Muscle protein turnover and glucose uptake in acute renal failure.J Clin Invest. 1983; 72: 836-845Crossref PubMed Scopus (66) Google Scholar, 4.Feinstein E.I. Blumenkrantz M.J. Healy M. et al.Clinical and metabolic responses to parenteral nutrition in acute renal failure.Medicine. 1981; 60: 124-137Crossref PubMed Scopus (158) Google Scholar, 5.Kokot F. Kuska J. Infuence of extracorporeal dialysis on glucose utilization and insulin secretion in patients with acute renal failure.Eur J Clin Invest. 1973; 3: 105-111Crossref PubMed Scopus (10) Google Scholar The role of the kidney on the development of systemic metabolic alterations that accompany ARF has not been previously examined. In previous work, we and others have described the accumulation of free fatty acids and toxic long chain fatty acid metabolites in freshly isolated proximal tubules subjected to hypoxic injury, and in kidney tissue of animals subjected to ischemia/reperfusion injury. 6.Zager R.A. Scimpf B.A. Gmur D.J. et al.Phospholipase A2 can protect renal tubules from oxygen deprivation injury.Proc Natl Acad Sci. 1993; 1: 90:8297-90:8301Google Scholar, 7.Schonefeld M. Noble S. Bertorello A.M. et al.Hypoxia-induced amphiphiles inhibit renal NaK-ATPase.Kidney Int. 1996; 49: 1289-1296Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 8.Bonventre J.V. Weinberg J.M. Recent advances in the pathophysiology of ischemic acute renal failure.J Am Soc Nephrol. 2003; 14: 2199-2210Crossref PubMed Scopus (635) Google Scholar, 9.Feldkamp T. Kribben A. Roeser N.F. et al.Accumulation of nonesterified fatty acids causes the sustained energetic deficit in kidney proximal tubules after hypoxia/reoxygenation.Am J Phsyiol Renal Physiol. 2005; 290: F465-F477Crossref PubMed Scopus (46) Google Scholar We also documented the activation of intracellular calcium-independent phospholipase A2,10.Portilla D. Creer M.H. Plasmalogen phospholipids hydrolysis during hypoxic injury of rabbit proximal tubules.Kidney Int. 1995; 47: 1087-1094Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 11.Portilla D. Shah S.V. Lehman P.A. et al.Role of cytosolic calcium-independent plasmalogen selective phospholipase A2 in hypoxic injury to rabbit proximal tubules.J Clin Invest. 1994; 93: 1609-1615Crossref PubMed Scopus (77) Google Scholar and the inhibition of mitochondrial fatty acid oxidation as potential mechanism(s) responsible for the accumulation of free fatty acids in ischemic ARF.12.Portilla D. Role of fatty acid beta oxidation and calcium-independent phospholipase A2 in ischemic acute renal failure.Curr Opin Nephrol Hypertens. 1999; 8: 473-477Crossref PubMed Scopus (38) Google Scholar, 13.Portilla D. Dai G. Peters J.M. et al.Etomoxir-induced PPARalpha modulated enzymes protect during acute renal failure.Am J Physiol renal Physiol. 2000; 278: F667-F675PubMed Google Scholar Recent work by Zager et al14.Johnson A.C. Stahl A. Zager R.A. Triglyceride accumulation in injured renal tubular cells: alterations in both synthetic and catabolic pathways.Kidney Int. 2005; 67: 2196-2209Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 15.Zager R.A. Johnson A.C. Hanson S.Y. Renal tubule triglyceride accumulation following endotoxic, toxic, and ischemic injury.Kidney Int. 2005; 67: 11-121Abstract Full Text Full Text PDF Scopus (68) Google Scholar suggests that in addition to fatty acid accumulation, ARF is also accompanied by the accumulation of triglycerides (TG) and cholesterol as part of a ‘renal stress response’ to injury. We have found that the inhibition of mitochondrial and peroxisomal fatty acid oxidation during ARF results from decreased DNA-binding activity of the nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) to its target genes, and from reduced expression of its tissue-specific coactivator peroxisome proliferator activated receptor gamma-coactivator-1α.16.Portilla D. Dai G. McClure T. et al.Alterations of PPARalpha and its coactivator PGC-1 in cisplatin-induced acute renal failure.Kidney Int. 2002; 62: 1208-1218Abstract Full Text Full Text PDF PubMed Google Scholar, 17.Portilla D. Energy metabolism and cytotoxicity.Semin Nephrol. 2003; 23: 432-438Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar In addition, we recently demonstrated that in the ischemia/reperfusion injury and cisplatin models of ARF, the use of PPARα ligands such as fibrates ameliorates proximal tubule injury and renal function only in wild-type and not in PPARα-null mice, by directly inhibiting cellular mechanisms leading to apoptosis and necrosis of the proximal tubule.18.Li S. Wu P. Yarlagadda P. et al.PPARalpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.Am J Physiol Renal Physiol. 2004; 286: F572-F580Crossref PubMed Scopus (80) Google Scholar, 19.Li S. Bhatt R. Megyesi J. et al.PPAR-alpha ligand ameliorates acute renal failure by reducing cisplatin-induced increased expression of renal endonuclease G.Am J Physiol Renal Physiol. 2004; 287: F990-F998Crossref PubMed Scopus (87) Google Scholar, 20.Li S. Gokden N. Okusa M.D. et al.Anti-inflammatory effect of fibrate protects from cisplatin-induced ARF.Am J Physiol Renal Physiol. 2005; 289: F469-F480Crossref PubMed Scopus (118) Google Scholar, 21.Nagothu K.K. Bhatt R. Kaushal G.P. et al.Fibrate prevents cisplatin-induced proximal tubule cell death.Kidney Int. 2005; 68: 2680-2693Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar In addition to our work, experimental evidence from other laboratories also support the role of PPARα as an important modulator of metabolic abnormalities associated with the metabolic syndrome.22.Chinetti-Gbaguidi G. Fruchart J.C. Staels B. Role of PPAR family of nuclear receptors in the regulation of metabolic and cardiovascular homeostasis. New approaches to therapy.Curr Opin Pharmacol. 2005; 5: 177-183Crossref PubMed Scopus (81) Google Scholar, 23.Robitaille J. Brouillette C. Houde A. et al.Association between the PPARalpha-L162V polymorphism and components of the metabolic syndrome.J Hum Genet. 2004; 49: 482-489Crossref PubMed Scopus (98) Google Scholar PPARα is mainly expressed in the liver, kidney, muscle, and heart tissue,24.Braissant O. Foufelle O. Scotto C. et al.Differential expression of peroxisome proliferators activated receptors (PPARs): tissue distribution of PPARalpha, beta and gamma in the adult rat.Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1736) Google Scholar and it regulates many genes involved in the transport and oxidation of fatty acids. Ligands of PPARα include long chain fatty acids, eicosanoids, and hypolipidemic drugs such as fibrates. It has been well documented that fibrates lower plasma TG levels and promote elevation of high-density lipoprotein-cholesterol concentrations.25.Staels B. Dalloneville J. Auwerx J. et al.Mechanisms of action of fibrates on lipid and lipoprotein metabolism.Circulation. 1998; 98: 2088-2093Crossref PubMed Scopus (1409) Google Scholar Therefore, PPARα is a potential candidate that may influence the risk of developing the metabolic syndrome. Our previous work underscores the importance of PPARα on restoring fatty acid oxidation during ARF; however, our most recent studies suggest an additional role for this proximal tubule nuclear receptor in improving glucose utilization via regulation of pyruvate dehydrogenase complex (PDC) enzyme activity and pyruvate dehydrogenase kinase-4 in renal tissue.18.Li S. Wu P. Yarlagadda P. et al.PPARalpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.Am J Physiol Renal Physiol. 2004; 286: F572-F580Crossref PubMed Scopus (80) Google Scholar Using the model of cisplatin-induced ARF, we found that the inhibition of renal PDC enzyme activity preceded the inhibition of renal fatty acid oxidation.18.Li S. Wu P. Yarlagadda P. et al.PPARalpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.Am J Physiol Renal Physiol. 2004; 286: F572-F580Crossref PubMed Scopus (80) Google Scholar PDC is a mitochondrial enzyme responsible for the conversion of pyruvate to acetyl coenzyme A CoA under normal conditions. Inhibition of PDC during ARF is accompanied by increased expression and activity of pyruvate dehydrogenase kinase-4. As normal kidney tissue utilizes three carbon molecules such as acetyl CoA for the generation of ATP from glucose, this observation suggests that inhibition of glucose metabolism is affected early in the course of ARF. To further examine the metabolic alterations that occur in ARF, we have used high-resolution 1H-nuclear magnetic resonance (NMR) spectroscopy with pattern recognition to evaluate the metabolic profile of urine samples obtained from mice that developed ARF after a single dose of cisplatin. Our studies detected the early presence of metabolic abnormalities including glucosuria, aminoaciduria, as well as the presence of trichloroacetic acid cycle metabolites in urine within the first 24 h of cisplatin administration. The presence of this metabolic profile precedes the rise in serum creatinine and blood urea nitrogen (BUN) that occurs 3 days after cisplatin exposure. Serial biochemical determinations performed on body fluids of mice treated with cisplatin confirmed the early rise of serum glucose, fatty acids, and TG as well as increased levels of glucose and fatty acids in urine and the accumulation of glucose, fatty acids, and TG in kidney tissue. Pretreatment with WY, a fibrate class of PPARα ligand, prevented the rise of glucose, fatty acids, and TG in serum and urine, prevented the accumulation of glucose, fatty acids, and TG in kidney tissue and protected mice treated with cisplatin. Therefore, our study demonstrates that compared to mice treated with saline, cisplatin produced: (1) a unique NMR metabolic profile in urine of mice that developed ARF, (2) systemic metabolic alterations including an early rise of serum glucose, nonesterified fatty acid (NEFA), and TG, as well as accumulation of these metabolites in kidney tissue, and (3) confirms the protective effect of a fibrate class of PPARα ligands. These effects are likely the result of injury-induced alterations in metabolism that are reversed by PPARα ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of kidney injury. Kidney function was monitored by measuring BUN and serum creatinine for 3 days after intraperitoneal injection of saline (groups control and WY) or cisplatin (groups cisplatin and cisplatin+WY), Figure 1a and b present the changes on BUN and creatinine in mice treated with saline (control), cisplatin, and with WY in the presence of cisplatin. Mice treated with a regular diet and cisplatin developed ARF at day 3 (BUN went up from 28 to 135 mg/dl, and creatinine went up from 0.2 to 1.2 mg/dl). The group of mice that received the WY diet and cisplatin did not develop significant ARF when compared with mice treated with cisplatin alone (BUN went from 24 to 32 mg/dl, and creatinine was unchanged at 0.2 mg/dl after 3 days of cisplatin administration). By contrast, the protective effect of the ligand was lost in the PPARα-null mice (-/-). PPARα-null mice (-/-) pretreated with a WY diet before cisplatin administration developed ARF at day 3. BUN went from 30 mg/dl at day 1 to 186 mg/dl at day 3, and creatinine went from 0.3 mg/dl at day 1 to 1.5 mg/dl at day 3. Changes in kidney morphology are shown in Figure 2. There were no structural changes in the deep cortex of kidneys from control animals. As can be seen in Figure 2a, the S3 segment of proximal tubules contained regularly arranged brush-border microvilli and intact cell cytoplasm with abundant mitochondria. The thick ascending limbs and collecting ducts were well preserved. Figure 2b shows the major morphologic damage that occurs 3 days after cisplatin treatment. The S3 segments of the proximal tubules were extensively necrotic, the tubular basement membrane was often denuded and heavily damaged, some of them revealing necrotic cells with remnants of brush-border microvilli (arrowheads) and apoptotic cells, desquamated into the tubular lumen; at other sites, it was covered by very thin processes of epithelial cells, which display short irregular microvilli (arrows); thick ascending limb profiles are mostly intact (whereas in many CD profiles the epithelium is detached from the tubular basement membrane – not seen in this picture). The morphologic kidney damage caused by cisplatin was completely prevented in mice pretreated with fibrate. As shown in Figure 2c, the S3 segments of proximal tubules contained intact cytoplasmic and brush-border morphology occasionally, a few desquamated cells were visible in the lumen and apoptotic cells were rarely seen among intact epithelial cells (not seen in this picture). Our recent in vitro studies in proximal tubular cells in culture exposed to cisplatin showed an increased accumulation of intracellular fatty acids, which precedes the development of proximal tubule cell death.21.Nagothu K.K. Bhatt R. Kaushal G.P. et al.Fibrate prevents cisplatin-induced proximal tubule cell death.Kidney Int. 2005; 68: 2680-2693Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Therefore, in the present study, we examined whether intracellular lipids accumulate in vivo in kidney tissue by performing intracellular stain of neutral lipids using the oil red O dye. As shown in Figure 3c, there was a significant accumulation of neutral lipids, which was detected as an increased intracellular red stain after 3 days of cisplatin treatment when compared to control mice. This accumulation of neutral lipids was predominantly present in the region corresponding to the proximal tubules in the kidney cortex. Pretreatment of mice with PPARα ligand WY alone did not have a significant effect on neutral lipid staining (Figure 3b), but prevented the accumulation of neutral lipids induced by cisplatin as shown in Figure 3d. Figure 4 shows representative spectra from the urine of the same mouse obtained befor dosing, and for 3 days following injection with 20 mg/kg body weight (BW) cisplatin. Visual inspection of the spectra shows changes in various urine metabolites after the mouse had been given a dose of cisplatin, including increases in glucose, lactate, pyruvate, and 2-oxoglutarate 48 h postdosing. Principal component analysis (PCA) of the spectra integrals showed distinct clusters for control urine and for urine collected on days 1, 2, and 3 after cisplatin injection, indicating clear differences between the metabolic profiles, as shown in Figure 5. The loadings plot (data not shown) indicated that increases in chemical shifts resonance associated with 2-oxoglutarate, and creatinine were the major metabolites separating day 1 samples from control, whereas increases in the glucose and lactate chemical shift resonances drove the clustering of the day 2 and day 3 urine samples away from control samples. Metabolite analysis of the spectra showed that the concentration of 2-oxoglutarate increased over the first 48 h after dosing followed by a decrease from 48 to 72 h. Using PCA analysis, we were able to show differences in metabolic profiles not only between different days after cisplatin injection but also between the different study groups on the same day. Figure 6 shows the three-dimensional PCA plot obtained from analysis of day 2 urine samples from four groups of four mice each. These groups included control mice shown in blue, mice administered a single dose of cisplatin shown in red, and two groups, one maintained on a special diet of WY-14643 alone and the other one maintained in the fibrate diet for 7 days before cisplatin injection shown in yellow and green, respectively. The PCA plot shows that the four groups separate into distinct clusters, with the control group on the special diet clustered fairly close to the control group on a normal diet. The cisplatin group clustered below the three groups along the PC3 axis. Metabolite analysis shown in Table 1 indicated that cisplatin treatment for 2 days induced significant changes in the urine levels of glucose (200 fold increase), alanine (12-fold increase), lactate (fourfold increase), leucine (sevenfold increase), methionine (fourfold increase), 2-oxoglutarate (twofold increase), pyruvate (3.5-fold increase), and valine (eightfold increase). Pretreatment with the diet containing PPARα ligand WY provided protection from the metabolic changes induced by cisplatin by reducing the increased urine levels of glucose, lactate, methionine, oxoglutarate, and pyruvate. The data also indicate that the PPARα ligand provided changes in urine levels of amino acids proline and tyrosine when compared to control or saline-treated mice. In addition, metabolite analysis showed that 1-methylnicotinamide was elevated in both groups maintained on the diet containing PPARα ligand WY-16463. This metabolite has recently been shown to be a biomarker for peroxisome proliferation.26.Delaney J. Hodson M.P. Thakkar H. et al.Tryptophan–NAD pathway metabolites as putative biomarkers and predictors of peroxisome proliferation.Arch Toxicol. 2005; 79: 208-223Crossref PubMed Scopus (40) Google ScholarFigure 5Principal component (PC) scores plot showing the changes from control over a 72 h period following administration of cisplatin.View Large Image Figure ViewerDownload (PPT)Figure 63D Principal component (PC) scores plot indicating the effects of the PPARα ligand (WY) on cisplatin-induced ARF.View Large Image Figure ViewerDownload (PPT)Table 1Select urine metabolite concentrations measured by 1H NMR spectroscopy and normalized to the concentration of creatinine in the urineMetaboliteControl, n=7Cisplatin day 2, n=7WY, n=7WyWY±cisplatin day 2, n=7Alanine0.02±0.020.25±0.13*P-values.0.07±0.060.19±0.16*P-values.P=0.003P=0.037Glucose0.30±0.3060.98±34.82*P-values.0.66±0.6118.08±23.98P=0.004Lactate0.21±0.300.89±0.950.43±0.370.57±0.31Leucine0.03±0.020.22±0.10*P-values.0.04±0.010.28±0.36P=0.004Methionine0.03±0.010.13±0.07*P-values.0.02±0.010.05±0.07P=0.0091-Methylnicotinamide0.11±0.060.07±0.020.24±0.12*P-values.0.22±0.08*P-values.P=0.038P=0.0162-Oxoglutarate7.97±5.3715.79±2.78*P-values.15.60±3.76*P-values.6.77±3.21P=0.008P=0.010Proline0.26±0.180.41±0.220.46±0.420.50±0.41Pyruvate0.12±0.070.44±0.21*P-values.0.22±0.08*P-values.0.27±0.18P=0.008P=0.043Trimethylamine0.90±0.890.28±0.260.07±0.06*P-values.0.10±0.09P=0.048Tyrosine0.17±0.080.15±0.060.50±0.560.41±0.39Valine0.02±0.010.16±0.09*P-values.0.02±0.010.25±0.36P=0.006Values are reported as mg metabolite/mg creatinine.NMR, nuclear magnetic resonance.* P-values. Open table in a new tab Values are reported as mg metabolite/mg creatinine. NMR, nuclear magnetic resonance. As neutral lipid accumulation detected by oil red O stain represents the accumulation of NEFA, TG, diglycerides, and cholesterol esters, we next examined specifically the effects of cisplatin on NEFA levels measured in serum, urine, and kidney tissue. As shown in Figure 7a, cisplatin-treated mice exhibited a time-dependent increase in NEFA levels not only in serum and urine but also in kidney tissue. At day 3, there was a threefold increase in serum levels, and also at day 3 after cisplatin injection, there was a sevenfold increase in kidney tissue levels of NEFA. Our assay did not detect NEFA in the urine samples obtained from saline-treated mice. However, in cisplatin-treated mice, there was a detectable level of NEFA in the urine obtained at day 3 after cisplatin injection (0.15±0.01 mEq/l/mg protein). Our most recent studies have shown that the use of PPARα ligands prevents the development of cisplatin-induced proximal tubule cell death by preventing the inhibition of fatty acid oxidation.18.Li S. Wu P. Yarlagadda P. et al.PPARalpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.Am J Physiol Renal Physiol. 2004; 286: F572-F580Crossref PubMed Scopus (80) Google Scholar Therefore, we examined the effect of PPARα ligand on cisplatin induced increased NEFA levels in serum, urine, and kidney tissue. At day 3 after cisplatin injection, there was a threefold increase in the serum levels of NEFA. In contrast, the group of mice that received the diet containing PPARα ligand and cisplatin exhibited comparable levels of serum NEFA to the mice treated with saline alone, as shown in Figure 7b. We also measured NEFA levels in urine samples of mice treated with PPARα ligand and cisplatin, and again similarly to saline-treated mice, we were not able to detect measurable amounts of NEFA in the urine samples from these mice. We next examined the effect of PPARα ligand on cisplatin-mediated accumulation of NEFA in kidney tissue. In the group of mice that received the diet containing PPARα ligand and cisplatin, there was a 65% reduction in the levels of NEFA in kidney tissue, when compared to cisplatin-treated mice (Figure 7b). As TG represents the major component of neutral lipids in kidney tissue, we measured TG levels in serum, urine, and kidney tissue homogenates of mice treated with saline (control) and mice treated with cisplatin. As shown in Figure 8a, we were able to measure TG levels only in the serum and kidney tissue homogenates. Our assay was not able to detect measurable amounts of TG in urine samples obtained from control or cisplatin-treated mice. As shown in Figure 8a, cisplatin-treated mice exhibited a time-dependent increase in TG levels in serum, but also in kidney tissue. At day 3, there was a threefold increase in serum TG levels, and also at day 3 after cisplatin injection, there was a sixfold increase in TG levels in kidney tissue homogenates. We next examined the effect of PPARα ligand on cisplatin-mediated increased levels of TG in serum and kidney tissue. At day 3 after cisplatin injection, there was a threefold increase in the serum levels of TG. In contrast, the group of mice that received the diet containing PPARα ligand and cisplatin exhibited a 70% reduction in serum TG levels when compared to cisplatin-treated mice, as shown in Figure 8b. We also examined the effect of PPARα ligand on cisplatin-mediated accumulation of TG in kidney tissue. In the group of mice that received the diet containing PPARα ligand and cisplatin, there was a 55% reduction in TG levels in kidney tissue when compared to cisplatin-treated mice. These results are shown in Figure 8b. 1H-NMR analysis of urine samples revealed that glucose was present within the first 24–48 h postinjection of cisplatin. Therefore, to corroborate these findings, we next measured glucose levels in serum, urine, and kidney tissue by an enzymatic colorimetric assay as described in the Materials and Methods section. As shown in Figure 9a, cisplatininduced time-dependent increase in glucose levels in serum, urine, and kidney tissue. At day 3 after cisplatin injection, there was a 3.5-fold increase in serum glucose levels when compared to saline-treated mice. Cisplatin-treated mice also exhibited a remarkable 75-fold increase in urine glucose levels, and a 2.5-fold increase in kidney tissue levels of glucose, when compared to saline-treated mice. Our most recent studies have shown that the use of PPARα ligands prevent the development of proximal tubule cell death during ARF by preserving not only fatty acid oxidation but also by preventing the inhibition of PDC activity in the mitochondria.18.Li S. Wu P. Yarlagadda P. et al.PPARalpha ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.Am J Physiol Renal Physiol. 2004; 286: F572-F580Crossref PubMed Scopus (80) Google Scholar In addition to its stimulation of fatty acid oxidation and antiapoptotic effects, recent reports suggest that PPARα may also play a role in the regulation of metabolic abnormalities associated with the metabolic syndrome.22.Chinetti-Gbaguidi G. Fruchart J.C. Staels B. Role of PPAR family of nuclear receptors in the regulation of metabolic and cardiovascular homeostasis. New approaches to therapy.Curr Opin Pharmacol. 2005; 5: 177-183Crossref PubMed Scopus (81) Google Scholar Therefore, we examined the effect of PPARα ligand on cisplatin-induced increased levels of glucose in serum, urine, and kidney tissue. As shown in Figure 9b, serum glucose levels of wild-type mice receiving a regular diet were significantly increased by cisplatin treatment. At day 3, there was a 3.5-fold increase in serum glucose levels. By contrast, cisplatin-treated mice receiving a PPARα ligand in their diet, exhibited comparable levels of serum glucose to the mice treated with saline alone. Similarly to our NMR results, our biochemical analysis confirmed that cisplatin-treated mice exhibited remarkable increases in urine glucose levels (75-fold) when compared to saline-treated mice. In the group of mice that received the diet containing PPARα ligand and cisplatin, there was a 78% reduction in urine glucose levels when compared to cisplatin-treated mice. We next examined the effect of PPARα ligand on cisplatin-mediated accumulation of glucose in kidney tissue. In the group of mice that received the diet containing PPARα ligand and cisplatin, there was a 70% reduction in glucose levels in kidney tissue homogenates, when compared to cisplatin-treated mice (Figure 9b). In the next series of experiments, we measured the effects of cisplatin on serum insulin levels. Three days after one single injection of cisplati