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Keywords:

  • peroxisome proliferator-activated receptor-γ;
  • pioglitazone;
  • cisplatin;
  • nephrotoxicity;
  • mice

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists not only improve metabolic abnormalities of diabetes and consequent diabetic nephropathy, but they also protect against non-diabetic kidney disease in experimental models. Here, we investigated the effect of PPAR-γ agonist pioglitazone against acute renal injury on a cisplatin model in mice. Nephrotoxicity was induced by a single intraperitoneal (i.p.) injection of cisplatin (10 mg kg–1). Pioglitazone was administered for six consecutive days in doses of 15 or 30 mg kg–1 day–1, per os (p.o.), starting 3 days before cisplatin injection. Cisplatin treatment to mice induced a marked renal failure, characterized by a significant increase in serum urea and creatinine levels and alterations in renal tissue architecture. Cisplatin exposure induced oxidative stress as indicated by decreased levels of non-enzymatic antioxidant defenses [glutathione (GSH) and ascorbic acid levels] and components of the enzymatic antioxidant defenses [superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx), glutathione reductase (GR) and and glutathione S-transferase(GST) activities)] in renal tissue. Administration of pioglitazone markedly protected against the increase in urea and creatinine levels and histological alterations in kidney induced by cisplatin treatment. Pioglitazone administration ameliorated GSH and ascorbic acid levels decreased by cisplatin exposure in mice. Pioglitazone protected against the inhibition of CAT, SOD, GPx, GR and GST activities induced by cisplatin in the kidneys of mice. These results indicated that pioglitazone has a protective effect against cisplatin-induced renal damage in mice. The protection is mediated by preventing the decline of antioxidant status. The results have implications in use of PPAR-γ agonists in human application for protecting against drugs-induced nephrotoxicity. Copyright © 2012 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Nephrotoxicity can be defined as renal dysfunction that arises as a direct result of exposure to external agents such as drugs and environmental chemicals. Many therapeutic agents have been shown to induce clinically significant nephrotoxicity (Arany and Safirstein, 2003). Cis-Diamminedichloroplatinum II (Cisplatin) is one of the most effective cancer therapeutic agents and it has become the drug of choice in the treatment of several solid tumors such as testicular and bladder tumors, head and neck, ovarian, breast and lung cancers and re-fractory non-Hodgkin's lymphomas (Giaccone, 2000). The therapeutic efficacy of this drug is often associated with severe toxic effects including nephrotoxicity (Arany and Safirstein, 2003). The nephrotoxicity induced by cisplatin is characterized by morphological destruction of intracellular organelles, cellular necrosis, loss of microvilli, alterations in the number and size of the lysosomes and mitochondrial vacuolization, followed by functional alterations including inhibition of protein synthesis, glutathione (GSH) depletion, lipid peroxidation and mitochondrial damage (Arany and Safirstein, 2003).

The importance of reactive oxygen species (ROS) in cisplatin-induced renal cell apoptosis has been documented in several previous studies (Hanigen and Devarajan, 2003; Richter et al., 1995; Ueda et al., 2006). It is well known that mitochondria continuously produce ROS such as superoxide. Also mitochondria continuously scavenge ROS via the action of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferrase (GST) (Richter et al., 1995). Cisplatin is known to accumulate in the mitochondria of renal epithelial cells (Gemba and Fukuishi, 1991). Several investigators have demonstrated that cisplatin induces ROS formation in renal epithelial cells primarily by decreasing the activity of antioxidant enzymes and depleting the intracellular concentration of GSH (Sadzuka et al., 1992).

Thiazolidinediones are a new class of antidiabetic drugs that improve insulin sensitivity and lipid metabolism, acting as agonists of peroxisome proliferator-activated receptor- γ (PPAR-γ) (Pereira et al., 2006). They change transcription of target genes that are involved in lipid metabolism, glucose homeostasis, cell proliferation and differentiation, as well as inflammatory responses (Xu et al., 2007). Pioglitazone is a synthetic ligand of PPAR-γ that has been used to treat patients with type 2 diabetes mellitus (Ito et al., 2004). In addition, pioglitazone may improve endothelial dysfunction in healthy humans with insulin resistance (Quinones et al., 2000), decrease anti-inflammatory cytokines (Haraguchi et al., 2008) and improve cellular antioxidant systems (Inoue et al., 2001). Furthermore, PPAR-γ agonists have been reported to reduce organ injury caused by ischemia/reperfusion (Shimazu et al., 2005) and attenuate drug-induced toxicity (Colletti et al., 1990).

The aim of the present study was to expand previous findings (Benigni et al., 2006; Toblli et al., 2009, 2011; Yoshimoto et al., 1997) by confirming with additional markers the protective effects of pioglitazone on renal injury induced by cisplatin in mice, and associating them with new mechanistic insights based on the modulation of oxidative stress, emphasizing enzymatic (SOD, CAT, GPx, GT and GST activities) and non-enzymatic parameters [reduced glutathione (GSH), ascorbic acid (AA), urea and creatinine levels] and histological benefits.

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Chemicals

Pioglitazone, cisplatin and all other reagents were purchased from Sigma (St. Louis, MO, USA).

Animals

Adult Swiss male mice, weighing 25–35 g, were obtained from a local breeding colony. The animals were kept in separate animal rooms, on a 12 h light/dark cycle, in an air conditioned room (22 ± 2 °C). A commercial diet (GUABI, Itaqui, RS, Brazil) and tap water were supplied ad libitum. The animals were used according to the guidelines of the Committee on Care and Use of Experimental Animal Resources, the Federal University of Santa Maria, Brazil.

In Vivo Experiments

Exposure

Mice were randomly assigned to six groups. The protocol of mouse treatment is given below:

  • Group (1) saline solution per os (p.o.) plus saline solution [10 ml kg–1, intraperitoneal (i.p.)] (n = 6);
  • Group (2) pioglitazone (15 mg kg–1, p.o.) plus saline solution (10 ml kg–1, i.p.) (n = 6);
  • Group (3) pioglitazone (30 mg kg–1, p.o.) plus saline solution (10 ml kg–1, i.p.) (n = 6);
  • Group (4) saline solution (p.o.) plus cisplatin (10 mg kg–1; i.p.) (n = 6);
  • Group (5) pioglitazone (15 mg kg–1, p.o.) plus cisplatin (10 mg kg–1; i.p.) (n = 6); and
  • Group (6) pioglitazone (30 mg kg–1, p.o.) plus cisplatin (10 mg kg–1; i.p.) (n = 7).

Groups 1 and 4 received daily p.o. normal saline solution and groups 2, 3, 5 and 6 received pioglitazone using an oral route (15 or 30 mg kg–1), respectively, for six consecutive days. Nephrotoxicity was induced in animals of groups 4, 5 and 6 using a single i.p. injection of cisplatin (10 mg kg–1). Groups 4, 5 and 6 received daily saline solution or pioglitazone for six consecutive days, starting 3 days before cisplatin injection (Fouad et al., 2008).

All mice were anesthetized for blood collection by heart puncture (hemolyzed plasma was discharged). After this procedure, mice were euthanized and the kidneys of the animals were removed, dissected and kept on ice until the time of assay. The samples of kidney were homogenized in 50 mM Tris–HCl, pH 7.5 (1/10, w/v), centrifuged at 2400 × g for 15 min. The resulting supernatant preserved the renal material for biochemical analysis. The supernatants (S1) were separated and used for biochemical assays.

Histopathological Analysis

Kidneys were fixed in 10% formalin. For light microscopy examination, tissues were embedded in paraffin, sectioned at 4 µm and stained with hematoxylin and eosin (Nath et al., 2000). Mice from all groups were examined by histopathology of the parameters: cellular vacuolization, loss of cellular architecture in the renal tubules and vascular congestion (n = 3 per group).

Renal Markers of Damage

Renal function was analyzed using a commercial Kit (Labtest; Diagnostica S.A., Minas Gerais, Brazil) by spectrophotometric assay of urea (Mackay and Mackay, 1927) and creatinine (Jaffe, 1886) levels in serum.

AA Determination

AA determination was performed as described by Jacques-Silva et al. (2001) with some modifications. Briefly, S1 was precipitated in 10% trichloroacetic acid solution. An aliquot of the sample (300 µl), at a final volume of 575 µl of the solution, was incubated at 38 °C for 3 h, then 500 µl H2SO4 65% (v/v) was added to the medium. The reaction product was determined using a color reagent containing 4.5 mg ml–1 dinitrophenyl hydrazine and CuSO4 (0.075 mg ml–1) at 520 nm. The content of AA is related to tissue amount (µmol ascorbic acid per g tissue).

Reduced GSH Levels

Levels of GSH were determined fluorometrically according to Hissin and Hilf (1976) using o-phthalaldehyde (OPA) as fluorophore. Briefly, the samples were homogenized in 0.1 M HClO4. Homogenates were centrifuged at 1957 g for 10 min and the S1 were separated for measurement of GSH. S1 (100 µl) was incubated with 100 µl of OPA (0.1% in methanol) and 1.8 ml of 0.1 M phosphate buffer (pH 8.0) for 15 min at room temperature in the dark. Fluorescence was measured with a fluorescence spectrophotometer at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. GSH levels were expressed as nmol g–1 of tissue.

CAT Activity

CAT activity was assayed spectrophotometrically according to the method of Aebi (1984), which involves monitoring the disappearance of H2O2 in the S1 presence at 240 nm. The enzymatic reaction was initiated by adding an aliquot of 40 µl of S1 and the substrate (H2O2) to a concentration of 0.3 mM in a medium containing 50 mM phosphate buffer, pH 7.0. The enzymatic activity was expressed in units (1 U decomposes 1 µmol of H2O2 per minute at pH 7 at 25 °C).

SOD Activity

SOD activity in S1 was assayed spectrophotometrically as described by Misra and Fridovich (1972). This method is based on the capacity of SOD in inhibiting autoxidation of epinephrine to adrenochrome. The color reaction was measured at 480 nm. One unit of enzyme was defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50% at 26 °C. The S1 was diluted 1: 1 0 (v/v) for determination of SOD activity in the test day. Aliquots of S1 were added in a Na2CO3 buffer 50 mM pH 10.3. The enzymatic reaction was started by adding epinephrine. One unit of enzyme was defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50% at 30 °C. The enzymatic activity was expressed as units (U) per mg protein.

GPx Activity

GPx activity in S1 of kidney was assayed spectrophotometrically according to the method of Wendel (1981), through the glutathione/NADPH/GR system, by the dismutation of H2O2 at 340 nm. In this assay, the enzyme activity is measured indirectly by means of NADPH decay. H2O2 is decomposed, generating oxidized glutathione (GSSG) from GSH. GSSG is regenerated back to GSH by the GR present in the assay media, at the expense of NADPH. The enzymatic activity was expressed in nmol NADPH min–1 mg–1 protein.

GR Activity

GR activity in S1 of renal tissue was determined as described by Calberg and Mannervik (1985). In this assay, GSSG is reduced by GR at the expense of NADPH consumption, which is followed at 340 nm. GR activity is proportional to NADPH decay. The enzymatic activity was expressed as nmol NADPH min–1 mg–1 protein.

GST Activity

GST activity, a phase II enzyme with antioxidant potential, was assayed through the conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB) at 340 nm as described by Habig et al. (1974). An aliquot of 100 µl of S1 of renal tissue was added in 0.1 M potassium phosphate buffer, pH 7.4, with CDNB, as substrate, and 50 mM of GSH. The enzymatic activity was expressed as nmol CDNB min–1 mg–1 protein.

Protein Quantification

The protein concentration was measured by the method of Bradford (1976), using bovine serum albumin as the standard.

Statistical Analysis

Data were analyzed by using a two-way analysis of variance (anova) (pioglitazone × cisplatin), followed by Duncan's Multiple Range Test when appropriate. All data of experiments were expressed as means ± standard error of the mean (SEM). Values of P < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Histopathological Analysis

Histological evaluation revealed a normal aspect of kidney structures in control and pioglitazone groups (Fig. 1A and 1C). Mice exposed to cisplatin presented intense loss of cellular architecture (Fig. 1B). Renal tissues from mice exposed to cisplatin revealed extensive injuries. Mice treated with cisplatin presented kidneys with severe proximal and distal tubular damage and marked vascular congestion between tubules. In addition, cisplatin-exposed mice presented cellular vacuolization (Fig. 1B). Evaluation of kidneys from mice exposed to cisplatin and pioglitazone at doses of 15 mg kg–1 (Fig. 1D) or 30 mg kg–1 (Fig. 1E) showed tubules with histological characteristics more preserved than those from the cisplatin group. Renal tubules displayed moderate damage. Pioglitazone, at all doses, markedly ameliorated the degree of kidney damage (Fig. 1D and E). It was observed that the higher dose (30 mg kg–1, p.o.) of pioglitazone caused appreciable protection in kidney histopathological analysis in this study (Fig. 1E).

image

Figure 1. Light microscopy of kidney tissues from mice with a detail on the left in: (A) the Control group shows normal glomerular cellularity and a normal tubular architecture. (B) The cisplatin group with intraluminal cast formation (*) in the distal tubules and edema interstitial. (C) The pioglitazone group shows mice with a normal aspect of the kidneys similar to the control group. (D) Pioglitazone (15 mg kg–1) + the cisplatin group. Tubules show an obvious decrease in intraluminal cast (arrow-head). (E) Pioglitazone (30 mg kg–1) + the cisplatin group. Note the renal tecidual destructuring and cytoplasmatic vacuolization (*) (arrow), proximal tubule; (arrow-head), Distal Tubule; g, glomerulus. Hematoxylin and eosin (H&E). 100× and 400×, respectively.

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Renal Markers of Damage

Serum urea and creatinine levels were unaltered after administration of pioglitazone (15 or 30 mg kg–1; p.o.) in mice (Table 1).

Two-way anova of urea levels revealed a significant pioglitazone × cisplatin interaction (F2,36 = 46.83; P < 0.001). Post-hoc comparisons showed that cisplatin exposure increased 240% urea levels (F1,36 = 77.15; P < 0.001) compared with the control group. Pioglitazone at the of dose of 30 mg kg–1 (F2,36 = 2.06; P < 0.05) attenuated the increase in urea levels caused by cisplatin exposure in mice (Table 1).

Two-way anova of creatinine levels yielded a significant pioglitazone × cisplatin interaction (F2,36 = 3.964; P < 0.027). Post-hoc comparisons revealed that cisplatin (F1,36 = 80.85; P < 0.001) increased (700 %) creatinine levels compared with the control group. Pioglitazone at a dose of 30 mg kg–1 (F2,36 = 3.49; P < 0.041) protected significantly against the increase in creatinine levels caused by cisplatin exposure in mice (Table 1).

Table 1. The effect of pioglitazone [mg kg–1, per os (p.o.)] administration on serum urea and creatinine levels of mice treated with cisplatin [10 mg kg–1; intraperitoneal (i.p.)]
GroupsUrea (mg dl-1)Creatinine (mgdl-1)
  1. Data are expressed as mean ± standard error (SE) of six or seven animals per group.

  2. a

    Denoted P < 0.05 as compared with the control group (anova and Duncan's Multiple Range Test).

  3. b

    Denoted P < 0.05 as compared with the cisplatin group (anova and Duncan's Multiple Range Test).

Control44.8 ± 3.00.22 ± 0.02
Pioglitazone 1546.0 ± 4.10.19 ± 0.03
Pioglitazone 3044.7 ± 3.50.24 ± 0.03
Cisplatin152.6 ± 12.6a1.5 ± 0.14a
Pioglitazone 15 + cisplatin139.7 ± 10.6a1.2 ± 0.12a
Pioglitazone 30 + cisplatin106.8 ± 8.9a, b0.82 ± 0.07a, b

AA Levels

AA levels were not altered in the kidneys of mice that received pioglitazone (15 or 30 mg kg–1, p.o.) (Table 2). Two-way anova of AA levels revealed a significant pioglitazone × cisplatin interaction (F2,36 = 3.14; P < 0.03). Post-hoc comparisons showed that cisplatin decreased (40%) significantly AA levels (F1,36 = 19.85; P < 0.001). Treatment with pioglitazone at a dose of 30 mg kg–1 (F2,36 = 2.44; P < 0.05) attenuated the decrease of AA levels induced by cisplatin in the kidneys of mice (Table 2).

Table 2. The effect pioglitazone [mg kg–1, per os (p.o.)] administration on ascorbic acid (AA) and glutathione (GSH) levels in the kidney after cisplatin treatment [10 mg kg–1; intraperitoneal (i.p.)] in mice
GroupsAA (µg AA per g tissue)GSH (nmol MDA equivalents per g tissue)
  1. Data are expressed as mean ± standard error (SE) of six or seven animals per group.

  2. a

    Denoted P < 0.05 as compared with the control group (anova and Duncan's Multiple Range Test).

  3. b

    Denoted P < 0.05 as compared with the cisplatin group (anova and Duncan's Multiple Range Test).

Control210.3 ± 15.60.96 ± 0.10
Pioglitazone 15194.7 ± 20.61.02 ± 0.12
Pioglitazone 30193.1 ± 20.50.96 ± 0.09
Cisplatin124.3 ± 10.4a0.49 ± 0.06a
Pioglitazone 15 + cisplatin144.3 ± 10.8a0.71 ± 0.05a, b
Pioglitazone 30 + cisplatin170.2 ± 15.2a, b0.82 ± 0.07b

GSH Levels

GSH levels were not altered in the kidneys of mice which received pioglitazone (15 or 30 mg kg–1, p.o.) (Table 2). Two-way anova of GSH levels revealed a significant pioglitazone × cisplatin interaction (F2,36 = 5.61; P < 0.001). Post-hoc comparisons showed that cisplatin significantly decreased (50%) GSH levels in the kidneys of mice (F1,36 = 22.44; P < 0.001). Treatment with pioglitazone at doses of 15 or 30 mg kg–1 (F2,36 = 2.49; P < 0.04) attenuated the decrease of GSH levels induced by cisplatin in the kidneys of mice (Table 2).

CAT Activity

CAT activity was unaltered after administration of pioglitazone (15 or 30 mg kg–1; p.o.) in mice (Table 3). Two-way anova of CAT activity revealed a significant pioglitazone × cisplatin interaction (F2,36 = 3.78; P < 0.046). Post-hoc comparisons showed that cisplatin significantly decreased (55 %) CAT activity (F1,36 = 42.94; P < 0.001) in renal tissue. Post-hoc comparisons demonstrated that pioglitazone at 30 mg kg–1 (F2,36 = 2.56; P = 0.09) attenuated CAT activity inhibition induced by cisplatin exposure in mice (Table 3).

Table 3. The effect of pioglitazone [mg kg–1, per os (p.o.)] administration on catalase (CAT) and superoxide dismutase (SOD) activities in the kidney after cisplatin treatment [10 mg kg–1; intraperitoneal (i.p.)] in mice
GroupsCAT (U mg–1 protein)SOD (U mg–1 protein)
  1. Data are expressed as mean ± standard error (SE) of six or seven animals per group.

  2. a

    Denoted P < 0.05 as compared with the control group (anova and Duncan's Multiple Range Test).

  3. b

    Denoted P < 0.05 as compared with the cisplatin group (anova and Duncan's Multiple Range Test).

Control4.7 ± 0.4928.6 ± 1.6
Pioglitazone 155.1 ± 0.4125.7 ± 1.8
Pioglitazone 304.9 ± 0.4725.6 ± 2.0
Cisplatin2.1 ± 0.18a17.6 ± 1.5a
Pioglitazone 15 + cisplatin2.8 ± 0.25a20.7 ± 1.2a
Pioglitazone 30 + cisplatin3.7 ± 0.32b22.9 ± 1.9a, b

SOD Activity

SOD activity was unaltered after administration of pioglitazone (15 or 30 mg kg–1; p.o.) in mice (Table 3). Two-way anova of SOD activity revealed a significant pioglitazone × cisplatin interaction (F2,36 = 5.61; P < 0.03). A significant main effect of cisplatin (F1,36 = 21.04; P < 0.001) in renal SOD activity was observed. Post-hoc comparisons demonstrated that mice exposed to cisplatin presented a reduction (40%) in SOD activity when compared with the control group. Post-hoc comparisons demonstrated that pioglitazone at 30 mg kg–1 (F2,36 = 3.59; P < 0.04) protected against SOD activity inhibition induced by cisplatin exposure in mice (Table 3).

GPx Activity

GPx activity was unaltered after administration of pioglitazone (15 or 30 mg kg–1; p.o.) in mice (Table 4). Two-way anova of GPx activity revealed a significant pioglitazone × cisplatin interaction (F2,36 = 3.12; P < 0.041). Post-hoc comparisons showed that cisplatin significantly decreased (30%) GPx activity (F1,36 = 12.45; P < 0.001). Post-hoc comparisons demonstrated that pioglitazone at 30 mg kg–1 (F2,36 = 0.985; P = 0.383) protected against GPx activity inhibition induced by cisplatin exposure in mice (Table 4).

Table 4. The effect pioglitazone [mg kg–1, per os (p.o.)] administration on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferrase (GST) activities in the kidney after cisplatin treatment [10 mg kg–1; intraperitoneal (i.p.)] in mice
GroupsGPx nmol NADPH min–1 mg–1 proteinGR nmol NADPH min–1 mg–1 protein)GST nmol CDNB conjugated min–1 mg–1 protein
  1. Data are expressed as mean ± standard error of the mean (SE) of 6 or 7 animals per group.

  2. a

    Denoted P < 0.05 as compared with the the control group (anova and Duncan's Multiple Range Test).

  3. b

    Denoted P < 0.05 as compared with the cisplatin group (anova and Duncan's Multiple Range Test).

Control195.3 ± 15.810.4 ± 1.192.4 ± 9.5
Pioglitazone 15196.5 ± 12.310.0 ± 0.996.5 ± 8.9
Pioglitazone 30186.7 ± 10.99.7 ± 0.997.6 ± 8.7
Cisplatin141.1 ± 10.8a5.6 ± 0.5a60.7 ± 6.0a
Pioglitazone 15 + cisplatin159.7 ± 11.5a8.3 ± 0.7b75.7 ± 6.2a
Pioglitazone 30 + cisplatin180.9 ± 12.9b9.0 ± 0.8b80.8 ± 7.1b

GR Activity

GR activity was unaltered after the administration of pioglitazone (15 or 30 mg kg–1; p.o.) in mice (Table 4). Two-way anova of GR activity showed a significant pioglitazone × cisplatin interaction (F2,36 = 3.11; P < 0.05). Post-hoc comparisons showed that cisplatin significantly decreased (45%) GR activity (F1,36 = 10.79; P < 0.002) in the kidneys of mice. Treatment with pioglitazone at doses of 15 or 30 mg kg–1 (F2,36 = 2.99; P < 0.03) protected against the decrease of GR activity induced by cisplatin in the kidneys of mice (Table 4).

GST Activity

GST activity was unaltered after the administration of pioglitazone (15 or 30 mg kg–1; p.o.) in mice (Table 4). Two-way anova of GST activity revealed a significant pioglitazone × cisplatin interaction (F2,36 = 5.65; P < 0.04). Post-hoc comparisons showed that cisplatin treatment significantly increased GST activity (F1,36 = 17.14; P < 0.002). Treatment with pioglitazone at 30 mg kg–1 (F2,36 = 1.81; P < 0.05) protected against the decrease of GST activity induced by cisplatin in the kidneys of mice (Table 4).

Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Results of this study indicate that pioglitazone rendered a partial protection against cisplatin-induced nephrotoxicity which was evident from the lowered urea and creatinine levels and pathology findings. Administration of cisplatin shows a significant increase in serum creatinine and urea levels compared with normal, which clearly indicated the renal acute renal failure. Pioglitazone (30 mg kg–1, p.o.) significantly protected the elevation of cisplatin-induced creatinine and urea levels. There are reports that pioglitazone diminishes renal injury in some models, such as in diabetes mellitus (Toblli et al., 2009), renal injury in aging (Yang et al., 2009) on tubular cell albumin uptake, proinflammatory and profibrotic markers of renal pathology (Zafiriou et al., 2004), immune-mediated glomerulonephritis (Benigni et al., 2006) and hypercholesterolemia (Omasu et al., 2007). Several mechanisms have been postulated to underlie pioglitazone-induced nephroprotection, such as antihypertensive (Yoshimoto et al., 1997), anti-oxidative and anti-inflammatory effects (Toblli et al., 2009, 2011). In the present study, pioglitazone treatment ameliorated both functional and histopathologic damage in a renal injury from nephrotoxic chemical cisplatin with antioxidant mechanisms. For the first time, we verified the involvement of GPx, GR and GST enzymes in the protective activity of pioglitazone in a renal injury. Moreover, this study was corroborated by other researchers (Chaudhry et al., 2007, Gumieniczek, 2003) with the involvement of CAT and SOD enzymes, GSH and AA levels in the activity of pioglitazone.

We observed that administration of cisplatin (10 mg kg–1, single dose) resulted in renal damage after 3 days. According to Kishore et al. (2000), the cisplatin-induced disturbances in water permeability regulation in the collecting ducts is associated with a decreased expression of renal aquaporins in the inner medulla that results in polyuria. Cisplatin nephrotoxicity is also characterized by discrete excretion of protein in urine (Camargo et al., 2001). The present study was carried out with higher dose of cisplatin (10 mg kg–1 body weight, i.p.) which corresponds to the dose of cisplatin currently being used in clinical practice. The corresponding dose in human beings (70 kg) is 35 mg–kg–1.

Animals treated with cisplatin presented an increase in histological alterations and the inflammatory cell. It is possible to observe an association with tubular cell necrosis. Treatment with pioglitazone in the cisplatin model of renal damage effectively inhibited these alterations, showing that pioglitazone may prevent the toxic effect of cisplatin in the kidneys in mice. Inflammatory cell infiltration was confirmed by histological analysis. There are several mechanisms by which leukocytes increase renal injury. Leukocytes are activated by inflammatory mediators, including cytokines, ROS, and eicosanoids, up-regulating adhesion molecules to engage counter-receptors on the activated endothelium (Bonventre and Zuk 2004).

GSH is one of the essential compounds for the regulation of a variety of cell functions, maintaining cell integrity, cell protection against a variety of toxins such as free radicals and their accompanied oxidative injury because of its reducing properties and participation in the cell metabolism. The thiol portion is very reactive with several compounds; mainly with alkylating agents such as cisplatin (Atessahin et al., 2005). The depletion in renal GSH has been observed in rodents in response to oxidative stress by cisplatin treatment (Atessahin et al., 2005). Reduced renal GSH can markedly increase the toxicity of cisplatin (Yilmaz et al., 2004). In the present study, the observed decline in the level of GSH and ascorbic acid in cisplatin-treated mice compared with the control group indicates that the depletion of GSH contributed to the nephrotoxicity (Karthikeyan et al., 2007). Experimental studies demonstrated that exogenous GSH could offer protection against cisplatin-induced renal injury (Hanigen and Devarajan, 2003). Treatment with pioglitazone restored protection owing to the preservation of GSH and ascorbic acid levels. Thus, pioglitazone protected against alterations of GSH and ascorbic acid levels induced by cisplatin in the kidneys of mice, suggesting that the protective effect may involve its antioxidant action.

A significant decline in antioxidant enzymes and an increase in free radicals in experimental models as well as in subjects are typical during the regimens of commonly used chemotherapy, and this is particularly related to cisplatin treatment regimens (Partibha et al., 2006). Signs of injury including the decrease of activities of renal SOD, CAT, GPx, GR and GST enzymes are proof of the oxidative stress caused by cisplatin treatment and have been previously reported in a number of previous studies (Ajith et al., 2007; Partibha et al., 2006; Wilhelm et al., 2012). These observations support the mechanism that nephrotoxicity induced by cisplatin in animals is partially related to the depletion of renal antioxidant system. The treatment of pioglitazone in the cisplatin model challenge could significantly prevent the depletion of these renal antioxidant systems. As SOD (Ajith et al., 2007), CAT (Brandão et al., 2009), GPx (Brandão et al., 2009), GR (Luchese et al., 2009) and GST (Casalino et al., 2004) are components of the antioxidant defense system of the cells, the increase caused by pioglitazone in these parameters could be one of the mechanisms by which pioglitazone acts as a protective agent in the kidneys of mice.

Kidneys are dynamic organs and represent the major control system maintaining the body homeostasis; they are affected by many chemicals and drugs (Ajith et al., 2007). Excretion of cisplatin is predominantly renal and it accumulates in the renal tubular cells approximately five times its extracellular concentration (Ali and Al-Moundhri, 2006). Consequently, the kidney is considered to be the primary target organ for cisplatin toxicity. Therefore, it is recognized as the main side effect and the dose-limiting factor associated with its use (Ajith et al., 2007). The mechanism for cisplatin nephrotoxicity remains uncertain. Cisplatin could cause decreased protein synthesis, membrane peroxidation, mitochondrial dysfunction and/or DNA injury and thereby cause tubular injury.

The thiazolidinedione pioglitazone is a high-affinity ligand for the PPAR-γ, and it is a member of a new class of antidiabetic drug that increases sensitivity towards insulin (Pereira et al., 2006). It was thought that PPAR-γ was only associated with metabolic diseases such as obesity, diabetes and atherosclerosis (Hwang et al., 2005). It has been reported that chronic treatment with pioglitazone prevented the marked increase in O2− production in cultured aortic smooth muscle cells that were chronically treated with high insulin with or without high glucose levels (El Midaoui et al., 2006). Other studies have shown that pioglitazone can relieve oxidative stress in diet-induced obese rats and alloxan-induced diabetic rabbits (Dobrian et al., 2004). Our group published that p-methoxyl-diphenyl diselenide, an antioxidant organoselenium compound, protected against cisplatin-induced renal toxicity in mice at doses of 50 and 100 mg kg–1 p.o. (Wilhelm et al., 2012). Luteolin, a flavone found in medicinal herbs and plants, and alpha-lipoic acid, ameliorated cisplatin-induced acute kidney injury in mice at doses of 50 and 100 mg kg–1 for 3 days, respectively (Kang et al., 2009, 2011). Thus, treatment with the PPAR-γ agonist pioglitazone demonstrated a nephroprotective effect in renal damage induced by cisplatin in mice at lower doses when compared with antioxidant therapies.

The study has several limitations that deserve acknowledgement. One limitation is that BUN and SCr as biomarkers of renal function were are not particularly sensitive. Although both biomarkers are largely utilized to demonstrate renal damage in mice (Atessahin et al., 2005; Brandão et al., 2009; Fouad et al., 2008; Wilhelm et al., 2012). Further studies are needed to determine the nephroprotective effect of pioglitazone in mice using more sensitive biomarkers such as albumin, α-GST, KIM-1, proteinuria, NAG and IL-18.

In conclusion, we have demonstrated the nephroprotective effect of PPAR-γ agonist, pioglitazone, on the cisplatin model in mice. Thus, PPAR-γ agonists may be a potent target for protecting the acute renal injury and the clinical application of PPAR-γ selective agonists can be considered in several clinical conditions such as kidney transplantation, glomerulopathies and chronic tubulointerstitial renal disease.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
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