1Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205; 2Department of Biochemistry and Molecular Biology, Indiana University, Indianapolis, Indiana 46202; and 3Department of Pathology, Duke University, Durham, North Carolina 27710
Submitted 16 May 2003 ; accepted in final form 10 November 2003
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ABSTRACT |
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peroxisome proliferator-activated receptor-; pyruvate dehydrogenase complex; fatty acid oxidation
Previous studies in muscle tissue documented abnormalities in glucose homeostasis during ARF (4). The potential presence of similar metabolic abnormalities in kidney tissue during ARF has not been explored. PPAR has been recently shown to modulate glucose metabolism via regulation of pyruvate dehydrogenase complex (PDC) activity in skeletal muscle tissue (11). Regulation of the activity of PDC is an important component of the regulation of glucose homeostasis (8). PDC is a multienzyme complex that catalyzes the conversion of pyruvate to acetyl-CoA. This reaction is the first irreversible step in the oxidation of carbohydrate-derived carbon and regulates the entry of carbohydrate into the tricarboxylic acid cycle. PDC activity is regulated by reversible phosphorylation and dephosphorylation reactions catalyzed by an intrinsic kinase and phosphatase (8, 11, 15, 22). Phosphorylation of E1 catalytic subunits of PDC by pyruvate dehydrogenase kinase (PDK) causes inactivation of the enzyme, whereas pyruvate dehydrogenase phosphatase (PDP) removes phosphate and returns the enzyme to its active form (PDCa) (22). The relative activities of the PDPs and PDKs determine the proportion of the complex in its active form. There are four known isoforms of PDKs, which differ significantly in their tissue distribution, specific activity, and sensitivity to their effectors (26, 30). To date, PDK4 and PDK2 are the two most abundant PDK isoforms expressed in rodents and human kidney tissue (29, 31, 33).
In the present study, we examined the effects of PPAR ligand WY on cytoprotection in the toxic model of cisplatin-induced ARF. More specifically, we examined the effects of cisplatin on renal function and histological parameters of proximal tubule injury in the absence and presence of WY. To address the potential mechanisms of protection by PPAR
ligands, we also examined the effects of this ligand on medium chain acyl-CoA dehydrogenase (MCAD)-mediated FAO, PDC activity, and mRNA and protein levels of PDK isoforms in kidney tissue. Our studies corroborate previous observations that cisplatin-induced ARF is accompanied by significant attenuation of mRNA and enzyme activity of mitochondrial FAO enzyme MCAD (25). The use of PPAR
ligand WY ameliorated both proximal tubule cell injury and kidney function in the PPAR
wild-type mice but not in the PPAR
null mice. Increased mRNA and enzyme activity of renal FAO enzyme MCAD accompanied this protective effect. In addition, we observed that cisplatin induced a profound and sustained inhibition of PDC activity detected on the second day after cisplatin injection and before the development of acute tubular necrosis (ATN). PDC inhibition was accompanied by increased expression of protein and mRNA levels of renal mitochondrial PDK4 isoform. Pretreatment with PPAR
ligand WY reversed the inhibition of PDC activity by cisplatin and prevented the observed increased expression of kidney PDK4 protein levels in cisplatin-treated mice. Our results demonstrate for the first time in kidney tissue that cisplatin-induced ARF is accompanied by the presence of significant metabolic changes, which include inhibition of MCAD-mediated FAO, as well as the inhibition of glucose metabolism via inhibition of PDC activity. Pretreatment with PPAR
ligand WY reversed the inhibition of MCAD and PDC activities and correlated with protection of kidney function. Further studies are needed to delineate the nephron segments, and the mechanisms by which PPAR
activation modulate substrate oxidation, and protect kidney function during ARF.
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METHODS |
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All experimental procedures were approved by the Animal Care and Use Committee of the Central Arkansas Veterans Health Care System, Little Rock, AR, and were in accordance with the National Institutes of Health and American Physiological Society Guiding Principles in the Care and Use of Laboratory Animals.
Histopathological alterations. We evaluated histopathological alterations in the kidneys 4 days after the mice were treated with cisplatin with or without WY. Kidneys were bisected, fixed in 3.7% phosphate-buffered neutral formaldehyde, dehydrated with serial alcohols, and embedded in paraffin. We stained 3-µm-thick paraffin sections with hematoxylin and eosin and a periodic acid-Schiff (PAS) method (20). The 12 morphological features described by Solez and co-workers (28) were evaluated in a masked fashion: leukocyte accumulation in the vasa recta: tubular necrosis (presence of necrotic cells, apparently denuded areas of tubular basement membrane, or ruptured tubular basement membranes); tubular regeneration; mitotic figures in tubular cells; dilatation of Bowman's space with retraction of the glomerular tuft ("acute glomerular ischemia"); loss of PAS-positive tubular brush border; "tubularization" of the parietal epithelium of Bowman's capsule; tubular casts; interstitial inflammation; interstitial edema; tubular dilatation; and prominence of the juxtaglomerular apparatus. We graded the morphological changes on a scale from 0 to 2 where 0 = none; 1/2 = minimal; 1 = mild; 1 1/2 = moderate; and 2 = marked (14).
RNA isolation. Mice were killed following previously described experimental conditions, and the kidney tissue was rapidly snap-frozen in liquid nitrogen and stored at -75°C. Total RNA was extracted with TRIzol reagent (Invitrogene Life Technologies) according to the manufacturer's directions.
RT-PCR. Total RNA extract was treated with 1 U of RQ1 RNase-free DNase (Promega) per microgram of total RNA at 37°C for 1 h. Reverse transcription was performed at 42°C for 50 min in a total volume of 20 µl containing 5 µg RNA, 0.5 µg of oligo (dT)12-18, and 200 U of superscript II RNase H- RT (Invitrogene Life Technologies). Subsequently, RT was inactivated by incubation at 70°C for 15 min, followed by treatment with 1.2 U of RNase H at 37°C for 30 min. PCR was performed with 1/20 of the RT reaction in a total volume of 50 µl using the Taq DNA Polymerase (Invitrogene). To control for the generation of PCR products due to residual contamination of genomic DNA, an aliquot of RNA, not treated with RT, was also tested in parallel. Amplification was performed using the following primer pairs for 25 cycles (denaturation at 94°C for 30 s, annealing at 57°C for 25 s, and extension at 72°C for 30 s): PDK4 sense (5'-ACCGCATTTCTACTCGGATG-3') and antisense (5'-CCTCCTCGGTCAGAAATCTT-3') primers, GAPDH sense (5'-AACTTTGGCATTGTGGAAGG-3') and antisense (5'-agatccacgacggacacatt-3') primers, and MCAD sense (5'-GTACCCGTTCCCTCTCATCA-3') and anti-sense (5'-cgtgccaacaagaaatacca-3') primers. The sense primer was end-labeled using [-32P]ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs). Five microliters from the PCR reactions were resolved on a 4% acrylamide gel. The gels were dried, analyzed on a 445 SI PhosphorImager with ImageQuant (Molecular Dynamic), and then subjected to autoradiography. Results are presented as the ratio of the signal for PDK4 or MCAD band to that of the GAPDH signal.
Western blotting. PDK2 and PDK4 protein levels were estimated by Western blot analysis of mouse kidney tissue extracts obtained from several experimental conditions, as described previously (32).
PDC activity. Mice were killed after experimental conditions, and the kidney tissue was rapidly snap-frozen in liquid nitrogen and stored at -75°C. Frozen kidney tissue was extracted as described previously by Goodwin et al. (6) with modification. In brief, 50 mg tissue were homogenated in 0.5 ml of homogenization buffer with a Teflon homagenizer (7-10 strokes). The resulting homogenate was subsequently centrifuged at 10,000 g at 4°C for 10 min to remove tissue debris, and the supernatant was removed, placed on ice, and enzyme activity was assayed immediately. To determine the fraction of PDC in the active form (PDCa), tissue was extracted using a homogenization buffer containing 30 mM K-HEPES (pH 7.5), 3% Triton X-100, 5 mM EDTA, 10 mM EGTA, 10 mM DCA, 50 mM KF, 2% bovine serum, 0.01 mM TPCK, 10 µg/ml trypsin inhibitor, 1 µM, and 5 mM DTT. Under these conditions, both PDK and phosphatase were inhibited. To determine the total activity of PDC (PDCt), tissue was extracted under conditions where PDP was stimulated (and also with addition of exogenous PDP) and PDK was inhibited, using a homogenization buffer consisting of 30 mM K-HEPES (pH 7.5), 3% Triton X-100, 2% bovine serum, 0.01 mM TPCK, 10 µg/ml trypsin inhibitor, 1 µM leupeptin, 5 mM DTT, 20 mM DCA, 15 mM MgCl2, and 1 mM CaCl2. Enzyme activity assays of PDC in the above extracts were determined as described previously (20) with a SPECTRAmax 190 Micro plate Spectrophotometer (Molecular Devices, Sunnyvale, CA).
MCAD activity. MCAD activity assay was performed in kidney tissue extracts following protocol previously described by Lehman et al. (17) with minor modifications. Kidneys were homogenized in cold 100 mM HEPES with 0.1 mM EDTA (pH 7.6). The homogenates were then centrifuged briefly at 4°C and the MCAD activity was measured immediately on the supernatant at 37°C. Two microliters of supernatant were added to 200 µl of reaction solution of 100 mM HEPES buffer (pH 7.6), 0.1 mM EDTA, 200 µM ferricenium hexafluorophosphate, 0.5 mM sodium tetrathionate, and 50 µM octanoyl-CoA. The absorbance decrease at 300 nm in the ferricenium ion was determined by SPECTRAmax microplate spectrophotometer (Molecular Devices) over the initial 60-s period. The values were corrected by subtracting the background absorbance of a tissue blank, measured in the absence of octanoyl-CoA in the reaction solution. Results are presented as means ± SE of MCAD activity relative to that obtained for control mice, which was set arbitrary as 100% in each experiment and was calculated from at least three independent experiments.
Statistics analysis. Results are presented as means ± SE. Statistical analysis was performed using unpaired Student's t-tests. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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PPAR ligand WY protects kidney function during cisplatin-induced ARF in PPAR
wild-type mice but not in PPAR
null (-/-) mice. Kidney function was monitored for 4 days after intraperitoneal injection of saline or cisplatin, by measuring BUN and serum creatinine. Figure 1, A and B, presents the changes in BUN and creatinine seen in PPAR
wild-type mice treated with and without WY ligand in the absence (Control) and presence of cisplatin.
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Comparison of the renal function between PPAR wild-type mice fed for 2 wk with either regular diet or 0.1% WY-containing diet did not show differences in BUN and creatinines when treated with a single injection of vehicle alone (saline IP). Values for this control group C as shown in Fig. 1, A and B, were not significantly different on day 1 (BUN 24 mg/dl, creatinine 0.2 mg/dl) vs. day 4 (BUN 30 mg/dl, creatinine 0.2 mg/dl). Mice treated with a regular diet and cisplatin developed ARF on days 3 and 4 (BUN went up from 27 to 264 mg/dl and creatinine went up from 0.2 to 1.5 mg/dl on day 4 after single cisplatin injection). The group of PPAR
wild-type mice that received the WY diet and cisplatin did not develop significant ARF compared with mice treated with cisplatin alone (BUN went from 23 on day 1 to 40 mg/dl on day 4, and creatinine was unchanged at 0.3 mg/dl on day 4).
In contrast, PPAR null mice (-/-), pretreated with a WY diet before cisplatin administration, did not exhibit any protection in renal function on day 4 following cisplatin injection, BUN went from 30 mg/dl on day 1 to 250 mg/dl on day 4, and creatinine went from 0.3 on day 1 to 1.7 mg/dl on day 4. This is shown in Fig. 1, C and D. These observations suggest that the protective effect of PPAR
ligand WY on renal function seen only on PPAR
wild-type mice was dependent on having an intact PPAR
gene. PPAR
null mice treated with PPAR
ligand exhibit less FAO due to their lack of response to PPAR
ligands (16).
Histopathological alterations of mouse kidneys subjected to cisplatin-induced ARF in the absence and presence of PPAR ligand WY. Results of histopathological abnormalities in the kidneys are given in Table 1. Dilatation of Bowman's space with retraction of the glomerular tuft ("acute glomerular ischemia"), "tubularization" of the parietal epithelium of Bowman's capsule, interstitial inflammation, interstitial edema, tubular dilatation, and prominence of the juxtaglomerular apparatus was not seen in any of the kidneys (results not shown). The control mouse kidneys exhibited no pathological changes, whereas those from mice treated with WY consistently had a few necrotic epithelial cells (score = 0.5) and scattered tubules with loss of their PAS-positive brush border (score = 0.5). Three of four kidneys from mice treated with WY also had occasional mitotic figures evident in the tubular epithelium (score = 0.5). All four mice treated with cisplatin alone had moderate to marked tubular necrosis and loss of the epithelial cell brush border, numerous casts, and three of four had regenerative tubular changes manifest as flattened, hypereosinophilic epithelial cells. There was a minimal infiltrate of leukocytes in the vasa recta of one mouse and a mild infiltrate in one mouse (data not shown), but none had tubular epithelial cell mitoses. All four mice treated with WY together with cisplatin were readily distinguishable from the mice treated with cisplatin alone because they had only minimal epithelial cell necrosis, no or only minimal loss of the brush border, and no casts. The kidneys from mice receiving cisplatin and WY were distinguishable histologically from those treated with only WY by the absence of epithelial cell mitotic figures. An example of a kidney from a control (untreated) mouse and typical mice treated with WY, cisplatin, and cisplatin + WY is shown in Fig. 2. Also shown in this figure is the study of the effects of WY ligand on PPAR
null mice. A histological section from a PPAR
-/- mouse demonstrates the subtle loss of PAS-positive brush border in a few tubules, whereas a histological section of a PPAR
-/- mouse treated with WY and cisplatin shows necrosis of the tubular epithelium. Again, these histopathological alterations are consistent with the results shown in Fig. 1 on renal function. Altogether, our data suggest that PPAR
ligand WY protects PPAR
wild-type mice and not PPAR
null mice from cisplatin-induced ARF likely by a mechanism dependent on having an intact PPAR
gene.
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Effects of PPAR ligand WY on the expression of MCAD. To determine the mechanisms by which PPAR
ligand protects renal function and ameliorates histological parameters in cisplatin-induced ATN, we examined the effects of cisplatin and WY ligand on renal MCAD activities of both PPAR
wild-type and null mice. As shown in Fig. 3, A-C, by representative autoradiograms and PhosphorImaging analysis, cisplatin caused a progressive decline in the mRNA expression of MCAD, a rate-limiting enzyme in the metabolism of medium chain fatty acids by mitochondria in kidney tissue. At day 4 of renal failure, there was a 63% inhibition of MCAD expression compared with control mice (P < 0.005). In PPAR
null mice (see Fig. 3B), cisplatin at day 4 had a similar effect on MCAD mRNA levels. On day 4 following cisplatin injection, there was a 65% inhibition of MCAD activity (P < 0.005). Pretreatment with WY ligand reversed cisplatin-induced inhibition of renal MCAD activity in wild-type mice, but this effect was not observed in the PPAR
null mice (see Fig. 3C). These results are similar to our previously published observations in which the use of PPAR
ligands etomoxir and clofibrate resulted in upregulation of renal FAO during I/R. Therefore, again these observations further corroborate the cytoprotective role of PPAR
ligands on renal function during ARF and further underscore the importance of mitochondrial FAO on the preservation of structure and function of the proximal tubule during ARF.
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Effects of PPAR ligand WY on the enzyme activity of MCAD. Because PPAR
ligand WY prevented cisplatin-induced reduction of MCAD mRNA levels in the PPAR
wild-type mice but not in the PPAR
null mice, we next examined the effects of cisplatin and WY on MCAD enzyme activity. As shown in Fig. 4, cisplatin on day 4 caused a profound decline in the enzyme activity of renal MCAD in both PPAR
wild-type and null mice. Pretreatment with WY prevented cisplatin-induced reduction of renal MCAD activity in PPAR
wild-type mice. In contrast to the effects of WY on PPAR
wild-type mice, pretreatment with WY did not affect cisplatin-induced reduction of MCAD activity in PPAR
null mice. These data further support our previous results showing that the protective effect of PPAR
ligand WY on mRNA levels and activity of FAO enzyme MCAD were dependent on having an intact and functionally active PPAR
gene.
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Cisplatin-induced inhibition of PDC activity is prevented by PPAR ligand WY. Previous studies support the notion that ARF is accompanied by abnormal glucose oxidation in muscle tissue (4, 19). There is no experimental evidence that renal pathways involved in glucose homeostasis are affected during ARF. Therefore, we examined first the effect of cisplatin-induced ARF on PDC activity. Cisplatin led to a rapid inhibition of mitochondrial PDC activity detected within the first 48 h after cisplatin administration (P < 0.001; results not shown). This effect was sustained, and by day 4 as shown in Fig. 5, A and C, there was more than 70% inhibition of mitochondrial PDC activity by cisplatin (***P < 0.0005). Pretreatment of PPAR
wild-type mice with WY prevented cisplatin-induced inhibition of PDC activity as shown in Fig. 5C. In addition, WY treatment although alone decreased the fraction of both PDCa and PDCt in kidney tissue did not significantly affect PDC activity measured as a percentage of PDCa/PDCt as shown in Fig. 5C. These studies suggest that in addition to preventing cisplatin inhibition of FAO, WY also to some extent restores the capacity for the kidney to oxidize glucose. Therefore, our studies further support the role of PPAR
as a master regulator of substrate oxidation in kidney tissue.
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Effects of PPAR ligand and cisplatin on the expression of PDK4 mRNA levels. There are recent reports that PPAR
may play a role in the regulation of glucose metabolism (35). Previous studies showed that increased expression and activity of PDK account for the inhibition of PDC activity in pathophysiological states such as starvation, diabetes, and hyperthyroidism (10, 33). To determine the mechanisms by which PDC activity is inhibited by cisplatin, we examined the effects of cisplatin and WY ligand on PDK4 mRNA levels of both PPAR
wild-type and null mice. As shown in Fig. 6, A-C, by representative autoradiograms and PhosphorImaging analysis, cisplatin caused a progressive increase in the mRNA expression of PDK4 mRNA levels. As shown in Fig. 6, A and C, in PPAR
wild-type mice at day 4 of renal failure, there was a profound upregulation (8-fold) of PDK4 mRNA levels compared with control mice (**P < 0.005). In PPAR
null mice (see Fig. 6, B and C), cisplatin at day 4 had only a modest effect, causing a 2.8-fold stimulation of PDK4 mRNA levels. Pretreatment with WY ligand also led to a fivefold increase on PDK4 mRNA levels in wild-type mice, but this effect was again significantly lower (only 2-fold increase) in the PPAR
null mice (see Fig. 6C). We next examined the effects of WY on cisplatin-induced increased expression of PDK4. As shown in Fig. 6, A and C, cisplatin was unable to further increase PDK4 mRNA expression compared with cisplatin-treated mice. In contrast, in WY-fed PPAR
null mice cisplatin significantly increased PDK4 mRNA levels in kidney tissue of PPAR
null mice compared with control, cisplatin-, or WY-treated null mice.
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Effects of cisplatin and WY on the expression of renal PDK4 protein. Because changes in PDK4 mRNA levels induced by WY or cisplatin did not correlate with changes in PDC activity, in the next series of studies we investigated the effects of cisplatin and WY on the protein levels of PDK4 using Western blot analysis of mouse kidney tissue. As shown in Fig. 7 by a representative autoradiogram and by densitometric quantification, cisplatin induced a profound upregulation (8-fold) of PDK4 protein levels in wild-type mice. In WY-treated wild-type mice, as well as in WY-treated mice that received cisplatin, PDK4 protein was not expresssed. We conclude that increased protein levels of PDK4 induced by cisplatin in the wild-type mice represent one of the mechanisms by which PDC activity is inhibited and that the inhibition of the expression of PDK4 protein by PPAR ligand WY helps preserve PDC activity during ARF.
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DISCUSSION |
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Our histopathological analysis of mice treated with PPAR ligand WY also revealed the presence of proliferation of a few isolated proximal tubular cells, and minimal loss of brush border, effects that were not accompanied by significant changes in renal function. The significance of these findings is unknown but perhaps represents a toxic effect of this ligand on kidney tissue. The use of more potent PPAR
ligands that could be given at the time of cisplatin injection in future studies should allow us to avoid these side effects. In addition, our studies cannot completely rule out the possibility of a potential effect of the PPAR
ligand on preconditioning, an effect that can protect kidney tissue from further injury.
Because the observed protection in kidney function in PPAR wild-type mice that received a PPAR
ligand likely involves modification of various metabolic pathways, other than FAO in kidney tissue, we also examined the effect of PPAR
ligand on PDC actvity. Abnormalities in glucose metabolism have been previously described in skeletal muscle of rats subjected to ARF (4). Carbohydrate metabolism is deranged in ARF as a result of impaired insulin-mediated actions. Previous studies suggest that insulin resistance accounts for one of the hormonal abnormalities involved in the deranged muscle protein catabolism in ARF. Those studies demonstrated decreased insulin-mediated glucose uptake, glycogen synthesis, and glucose oxidation in the muscle of acutely uremic rats (19). Clark and Mitch (4) suggested that the abnormal net protein degradation in ARF may be linked to the altered carbohydrate metabolism and might be a consequence of defective glucose oxidation. In the present study, we examined the effects of cisplatin-induced ARF on PDC activity, the enzyme that catalyzes the conversion of pyruvate to acetyl-CoA in the mitochondria. Our studies demonstrate the inhibition of PDC activity as early as 48 h after cisplatin injection, a time point at which we could not detect significant changes in BUN or creatinine, or histopathological parameters of tubular injury. In addition, prior administration of PPAR
ligand WY reversed cisplatin-induced inhibition of PDC activity. The significance of this observation is great, because preservation of PDC activity perhaps represents an important metabolic adaptation of distal nephron segments to the lack of energy production during ARF.
The differential regulation of PDK and phosphatase is the key to the overall regulation of the activity of the PDC. Three specific serine residues in each -subunit of the E1 domain, namely, site 1 Ser264, site 2 Ser271, and site 3 Ser203, are subject to ATP-dependent phosphorylation and inactivation by PDKs (8). PDP dephosphorylates these three serine residues and reactivates PDC (3, 18). Mammalian PDK isoenzymes differ in their catalytic activity, responsiveness to modulators like NADH and acetyl-CoA, and tissue-specific expression. PDK1 is present mostly in the heart, whereas PDK2 is found in most tissues. PDK3 is predominantly expressed in testis, whereas heart, skeletal muscle, and kidney have the highest amount of PDK4 (2, 7, 31). In pathophysiological states such as starvation, diabetes, and hyperthyroidism (10, 29, 34), PDC activity is reduced and mRNA and protein levels of PDK4 are increased. Our studies showing a temporal relationship between cisplatin-induced inhibition of PDC activity and increased expression of renal PDK4 protein suggest a similar mechanism of regulation of PDC activity. In addition, our data also suggest that increased phosphorylation of PDC by free PDK4 could be a potential mechanism that accounts for the observed inhibition of PDC activity during ARF. However, our current data cannot conclusively establish a cause-effect relationship between these two metabolic abnormalities, because we also observed decreased PDC activity in cisplatin-treated PPAR
null mice and these mice did not express PDK4 protein when treated with cisplatin. Therefore, other potential pathways by which PDC activity could be downregulated during cisplatin could include decreased expression of PDPs, or changes in the levels of NAD, NADH. In fact, recent studies suggest that starvation and streptozotocin-induced diabetes cause decreases in PDP2 mRNA abundance, PDP2 protein amount, and PDP activity in rat heart and kidney. Refeeding and insulin treatment effectively reversed these effects of starvation and diabetes, respectively. Those findings indicate that opposite changes in expression of specific PDK and PDP isoenzymes contribute to hyperphosphorylation and therefore inactivation of the PDC in heart and kidney during starvation and diabetes (12). More studies will be needed to completely clarify all the mechanisms that contribute to renal PDC inhibition during ARF.
Our studies also suggest that by day 4 cisplatin inhibits gene expression of enzymes associated with FAO and pyruvate oxidation, two important metabolic substrates for the generation of acetyl-CoA, a three-carbon molecule that is used by normal kidney tissue via the TCA cycle for the generation of energy in the form of ATP. Metabolic abnormalities resulting from the inhibition of these two important sources of energy are likely to further contribute to the catabolic state of ARF and perhaps explain the metabolic need for increased protein degradation during ARF. Therefore, increased oxidation of amino acids by kidney tissue, as a maladaptive mechanism, is likely to provide the substrate needed for the generation of acetyl-CoA and energy production via the TCA cycle in the setting of decreased energy production. Of interest, a recent study (13) demonstrated that inhibition of protein degradation using a specific proteasome inhibitor results in cytoprotection during I/R injury to the kidney.
In the present study, we did not examine whether PPAR ligand had an effect on the apoptotic response to cisplatin. In preliminary studies not shown here, we only found positive TUNEL staining in the kidneys of PPAR
wild-type mice treated with cisplatin, which represented less than 5% of cellular injury seen in the corticomedullary junction, with necrosis representing the major form of cell death. These similar findings have been previously reported by other investigators (21). Of interest, our most recent studies done in proximal tubule cells in culture showed that PPAR
ligand inhibits caspase 3 activation by cisplatin, and this results in amelioration of proximal tubule cell death (25), suggesting that PPAR
-mediated inhibition of the caspase cascade might also represent a mechanism of cytoprotection.
In conclusion, our results clearly indicate the ability of PPAR ligand to ameliorate cisplatin-induced ARF. In addition, our studies further support the role of inhibition of MCAD-mediated FAO and inhibition of PDC activity in the pathogenesis of cisplatin-induced ARF. Further studies are needed to localize the nephron segment(s) where these abnormalities in PDC and FAO take place in the kidney, to further examine the cellular mechanisms of substrate inhibition, and to determine whether PPAR
ligands have similar effects on these cellular pathways, as our studies suggest it is the case in this in vivo study.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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