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Am J Physiol Renal Physiol 287: F990-F998, 2004. First published July 27, 2004; doi:10.1152/ajprenal.00206.2004
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PPAR-{alpha} ligand ameliorates acute renal failure by reducing cisplatin-induced increased expression of renal endonuclease G

Shenyang Li,1 Renu Bhatt,1 Judit Megyesi,1 Neriman Gokden,2 Sudhir V. Shah,1 and Didier Portilla1

1Division of Nephrology, Department of Internal Medicine, 2Department of Pathology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205

Submitted 3 June 2004 ; accepted in final form 23 July 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cisplatin injury to the kidney is characterized, in part, by inhibition of substrate oxidation, inflammation, and tubular cell death in the form of apoptosis and necrosis. Recently, we demonstrated that cisplatin-induced inhibition of substrate oxidation can be reversed by the administration of peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) ligands, resulting in amelioration of renal function. We therefore hypothesize that by improving fatty acid oxidation in vivo might protect renal function by reducing both apoptosis and necrosis in cisplatin-treated mice. Mice subjected to a single intraperitoneal injection of cisplatin developed acute renal failure (ARF) at days 3 and 4. At day 4 after cisplatin injection mRNA, protein levels and enzyme activity of proapoptotic renal endonuclease G (Endo G) were increased compared with saline-treated mice. In situ hybridization and immunohistochemical studies localized the increased expression of Endo G mRNA to the cytosolic compartment and Endo G protein to the nuclear compartment of proximal tubules in cisplatin-treated mice. Pretreatment of PPAR-{alpha} wild-type mice with PPAR-{alpha} ligand WY-14643 reduced significantly cisplatin-induced increased protein expression and enzyme activity of Endo G and prevented the nuclear translocation of mitochondrial Endo G. Morphological examination of tubular injury in the PPAR-{alpha} wild-type mice that received PPAR-{alpha} ligand and cisplatin did show significant amelioration of acute tubular necrosis, as well as a significant reduction in the number of apoptotic cells in the proximal tubule when compared with the cisplatin-treated group. In contrast, in PPAR-{alpha}-null mice treated with the ligand and cisplatin, Endo G protein expression was not reduced and this was accompanied by lack of protection of kidney function. We conclude that PPAR-{alpha} ligand protects against cisplatin-induced renal injury via a PPAR-{alpha}-dependent mechanism by reducing the expression and enzyme activity of proximal tubule Endo G, which results in amelioration of both proximal tubule cell apoptosis and necrosis.

peroxisome proliferator-activated receptor-{alpha}; fatty acid oxidation

NEPHROTOXICITY IS A COMMON side effect of cisplatin-based chemotherapeutic regimens (25). Our previous studies demonstrated decreased expression and reduced enzyme activity of several kidney peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) target genes in response to cisplatin nephrotoxicity (20, 21) and also that the use of PPAR-{alpha} ligands such as fibrates and WY-14,643 protected renal function by preventing proximal tubule cell death in the animal models of ischemia-reperfusion and cisplatin-induced acute renal failure (ARF) (14, 22). PPAR-{alpha} is a nuclear protein highly expressed in liver, heart, renal proximal tubular cells, skeletal muscle, intestinal mucosa, and brown adipose, tissues that are considered metabolically very active (3, 6, 10, 28). In liver tissue, the administration of PPAR-{alpha} ligands leads to the activation of PPAR-{alpha} and results in a pleiotropic response that includes increased peroxisome proliferation, upregulation of fatty acid oxidation, reduced inflammation, and suppression of apoptosis (4, 16, 26). The cellular mechanisms by which PPAR-{alpha} ligands ameliorate acute tubular necrosis in the model of cisplatin-induced ARF are not currently known.

Recent studies suggest that renal epithelial cell apoptosis contributes to the pathogenesis of cisplatin-induced renal injury (24, 27, 34). The apoptotic changes observed in cisplatin nephrotoxicity occur in proximal tubules as well as in distal tubular cells and relate to the activation of death receptors (34) or as a result of activation of a family of cysteine proteases called caspases (33). A mitochondrial step involving outer-membrane permeabilization is controlled by the pro- and antiapoptotic members of the Bcl-2 family and leads to the cytosolic release of mitochondrial intermembrane space proteins that can trigger either caspase activation or caspase-independent pathways (7, 17, 39). Mitochondrial proteins that cause caspase-dependent cell death include cytochrome c, which triggers caspase-9 activation by binding and activating the apoptosis protease-activating factor-1 (Apaf-1), and Smac/Diablo and HtrA2/Omi, which potentiate caspase activation by binding inhibitor of apoptosis proteins (IAPs) and blocking their caspase inhibitory activity (5, 8, 13, 32, 37, 38). Mitochondria also contain the caspase-independent death effectors apoptosis-inducing factor (AIF) (31) and endonuclease G (Endo G) (36). AIF induces chromatin condensation and large-scale DNA fragmentation when released into the cytosol. During apoptosis, Endo G, similar to AIF, translocates to the nucleus where it causes oligonucleosomal DNA fragmentation (11, 12, 35).

In the present study, we examined the mechanisms by which PPAR-{alpha} ligand ameliorate renal function in the model of cisplatin-induced ARF. We demonstrate in this study that cisplatin induces time-dependent increases of mRNA, protein levels, and activity of renal Endo G. In situ hybridization and immunohistochemical studies localized the increased expression of Endo G to mostly proximal tubules. Pretreatment of mice with PPAR-{alpha} ligand WY resulted in a significant reduction in the levels of expression of Endo G protein in the proximal tubule, and this effect was associated with reduction of both apoptosis and necrosis in cisplatin-treated mice. Renal function and Endo G expression were not improved in PPAR-{alpha}-null mice treated with the PPAR-{alpha} ligand and cisplatin. We conclude that increased expression of proximal tubule Endo G represents a cellular mechanism by which cisplatin leads to proximal tubule dysfunction and cell death. Further studies are needed to determine the mechanisms by which PPAR-{alpha} ligand(s) reduce the expression of renal Endo G.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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In vivo model of cisplatin-induced ARF. Male sv129 mice ~8–10 wk old, weighting 25 to 30 g, were assigned to treatment groups (8 animals per group). Animals received single intraperitoneal doses of vehicle (saline) or cisplatin (20 mg/kg body wt). After treatment, the animals were killed and the kidney tissue was frozen in liquid nitrogen for RNA or protein isolation. Mice were housed in a temperature- and light-controlled environment and provided food and water. Pelleted mouse chow was prepared containing either zero or 0.1% (wt/wt) WY-14,643. To investigate the effects of PPAR-{alpha} activation, mice were fed the WY-14,643-containing diet for 10 days. Blood urea nitrogen (BUN) and creatinine were measured by an enzymatic colorimetric assay as previously described (14). For histopathological evaluation, kidneys were collected in 10% neutral buffered formalin.

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’s 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-14,643. 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 the periodic acid-Schiff (PAS) method (18). The 12 morphological features described by Solez and co-workers (29) 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 (9).

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, 200 U of superscript II RNase H– reverse transcriptase (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); Endo G sense (5'-ACCGCATTTCTACTCGGATG-3') and antisense (5'-CCTCCTCGGTCAGAAATCTT-3') primers, 28s rRNA sense (5'-TGAGCTCTCGCTGGCCCT-3') and antisense (5'-ACATTGTTCCAACATGCCAGA-3') primers. The sense primer was end-labeled using [{gamma}-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 ImageQuantNT (Molecular Dynamic), and then subjected to autoradiography. Results are presented as the ratio of the signal for Endo G band to that of the 28s rRNA signal.

Western blots. Endo G protein levels were estimated by Western blot analysis of mouse kidney tissue extracts obtained from several experimental conditions, as described previously (12).

In situ hybridization for Endo G mRNA. Mouse kidney Endo G cDNA was generated by RT-PCR using freshly isolated mRNA and the following primers: sense (5'-ATGGACGACACCTTCTACCTGA-3') and antisense (5'-ATAGCCTTGAGGTTTCCAGCTC-3') resulting in a 413-bp Endo G gene fragment. This fragment was subcloned into pGEM-T Easy Vector (Promega), and the sequence and orientation of the insertion of Endo G cDNA were determined with an ABI Automated Prism 377 DNA Sequencer. The plasmid was linearized with NcoI and Endo G cRNA was made by in vitro transcription using Sp6 RNA polymerase and digoxigenin-labeled UTP according to the manufacturer's recommendations (Roche Diagnostics, Indianapolis, IN). The length of Endo G was shortened by alkaline hydrolysis, and the in situ hybridization on sections from untreated and treated animals was performed as previously described (19).

Immunohistochemistry. Immunohistochemical localization of Endo G was done in paraffin-embedded tissue sections using a microwave antigen retrieval technique (Antigen Unmasking solution: Vector Laboratories, Burlingame, CA). Endo G was detected using a rabbit polyclonal antibody (Chemicon International, Temecula, CA). Antigens were visualized using the ABC Elite Vectastain Kit (Vector Laboratories). The microscopic images within specific kidney areas were visualized and photographed at x20 magnification using a video camera (Cool Cam 2000 color).

Endonuclease activities assays. Isolation of mitochondria from mouse kidneys for mitochondrial endonuclease activity was done as previously described (15). All steps were performed at 4°C. The kidney tissues were homogenized in lysis buffer [30 mM Tris·HCl (pH 7.4), 20 mM EDTA, 150 mM NaCl, 10%(wt/vol) sucrose, 2 mM DTT, and 1 mM phenylmethylsulfonyl fluoride] and then centrifuged twice at 1,000 g for 5 min. The resulting supernatant was centrifuged again at 20,000 g for 20 min. The mitochondrial pellet was gently resuspended in lysis solution, followed by being briefly frozen in liquid nitrogen and then thawed on ice. An equal volume of lysis buffer was added to final concentration of 300 mM NaCl and 0.5% (wt/vol) Triton X-100. The resuspended mitochondria were incubated for 20 min on ice and then centrifuged at 30,000 g for 2 h at 4°C. The supernatant (fraction I) was precipitated with 30% ammonium sulfate (incubated for 1 h on ice before centrifugation for 30 min at 20,000 g at 4°C). The resulting supernatant was fractioned again using 60% ammonium sulfate (incubated for 1 h on ice before centrifugation for 30 min at 20,000 g at 4°C). The mitochondrial protein (fraction II) obtained after 60% ammonium sulfate precipitation was resuspended in 100 mM HEPES buffer (pH 7.5) containing 150 mM NaCl, 5 mM EDTA, 5 mM DTT, 10% (vol/vol) glycerol, and 1% (wt/vol) N-octylglucoside. The endonuclease activity in the fraction II of mitochondrial lysates was determined by agarose gel assays. We used a total of 5 µg of protein obtained from mitochondrial lysate fraction II and then added 0.5 µg of supercoiled plasmid (3.6 kb) into the reaction solution containing 30 mM Tris·HCl (pH 8.0), 2 mM DTT, 20 mM KCl, 1 mM Mg(OAc)2, and 0.2 µg/µl BSA into a final volume of 40 µl. The reactions were incubated for 30 min at 37°C and stopped by addition of 1% SDS. The DNA reaction products were separated by electrophoresis using 1% agarose gels.

Apoptosis detection and quantification. The terminal transferase-mediated dUTP nick end-labeling (TUNEL) technique (Apopkit-Trevigen) was used to detect apoptotic cells in situ according to the manufacturer's recommendations. All apoptotic nuclei within a section of the kidney were counted, and the results were expressed as the number determined per square millimeter of kidney tissue.

Statistics analysis. Results are presented as means ± SE. Statistical analysis was performed using unpaired Student t-tests. A P value of <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PPAR-{alpha} ligand WY protects kidney function during cisplatin-induced ARF. 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, present the changes in BUN and creatinine seen in mice treated with and without WY ligand in the absence (control) and presence of cisplatin. Comparison of the renal function between mice fed for 10 days with either a regular diet or 0.1% WY-containing diet did not show significant differences in BUN and creatinines vs. mice treated with a single injection of vehicle alone (saline IP). Mice treated with a regular diet and cisplatin developed ARF at day 4 (BUN went up from 27 to 264 mg/dl and creatinine went up from 0.2 to 2.1 mg/dl). The group of 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 38 mg/dl at day 4, and creatinine was unchanged at 0.3 mg/dl at day 4).



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  		Fig. 1. Effects of peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) ligand WY-14,643 (WY) on renal function in mice after single dose of cisplatin. Mice were fed with either a regular diet or a diet containing 0.1% WY for 10 days before cisplatin administration, as described in METHODS. Blood urea nitrogen (BUN) and creatinine levels were measured at day 4 after either saline (control and WY) or cisplatin (cisplatin and cisplatin + WY). Bars correspond to means ± SE of at least 6 independent experiments under each condition. A and B: changes in BUN and creatinine levels, respectively, after saline or cisplatin administration in PPAR-{alpha} wild-type mice. *P < 0.0005 when animals were compared with control by unpaired Student's t-test.

 
Histopathological alterations of mouse kidneys subjected to cisplatin-induced ARF in the absence and presence of PPAR-{alpha} ligand WY. Results of histopathological abnormalities in the kidneys are shown in Fig. 2. 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 were 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-14,643 consistently had a few scattered tubules with loss of their PAS-positive brush border (score = 0.5). Kidneys from mice treated with WY-14,643 also had occasional mitotic figures evident in the tubular epithelium (score = 0.5). All mice treated with cisplatin alone had moderate to marked tubular necrosis and loss of the epithelial cell brush border, and numerous casts. 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-14,643 together with cisplatin were readily distinguishable from the mice treated with cisplatin alone since they had only minimal loss of the brush border and no casts. Examples of a kidney from a control (untreated) mouse (Fig. 2A) and typical mice treated with WY-14,643 (Fig. 2C), cisplatin (Fig. 2B), and cisplatin+WY-14,643 (Fig. 2D) are shown in Fig. 2.



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  		Fig. 2. Histopathological alterations in PPAR-{alpha} +/+ wild-type mice kidneys treated with PPAR-{alpha} ligand and cisplatin. A histological section from a control (treated with saline) kidney (A) discloses a normal glomerulus and tubules, whereas a typical kidney from a mouse treated with WY (C) demonstrates loss of the periodic acid-Schiff (PAS)-positive brush border in some tubules. A typical kidney from a mouse treated with cisplatin (B) shows extensive necrosis of the tubular epithelium (*). Many of the tubules shown in B are necrotic and similar in appearance to that denoted with the star. A section from a typical cisplatin + WY-treated kidney (D) has only a rare loss of brush-border cells (arrow). All sections were stained with PAS reagent and photographed at the same magnification (x200).

 
Effects of cisplastin and PPAR-{alpha} on the expression of Endo G mRNA and protein levels. To examine the potential mechanism(s) by which PPAR-{alpha} ligand ameliorates cisplatin-induced ARF, we investigated the effects of cisplatin and PPAR-{alpha} ligand on the mRNA and protein expression of Endo G. Activation of this mitochondrial proapoptotic endonuclease can lead to translocation from the mitochondrial to the nuclear cellular compartment therefore contributing to oligonucleosomal DNA fragmentation and cell death (12). As shown in Fig. 3 by a representative autoradiogram (A) and by mRNA quantification (B), in mice treated with cisplatin we observed at least a threefold increased expression of mouse kidney Endo G mRNA levels. Treatment of mice with PPAR-{alpha} ligand significantly reduced cisplatin-induced expression of Endo G. We next examined in mouse kidney tissue by Western blot analysis the effects of cisplatin and PPAR-{alpha} ligand in protein levels of Endo G. Cisplatin led to a rapid increase in Endo G protein levels detected as early as 3 days after cisplatin injection (4-fold increase), and this effect was sustained at day 4 (6- to 7-fold) as shown in Fig. 4A. Pretreatment with PPAR-{alpha} ligand WY prevented cisplatin-induced increased expression of Endo G protein levels in PPAR-{alpha} wild-type mice (Fig. 4B). In contrast to the effects of WY on PPAR-{alpha} wild-type mice, pretreatment with WY did not affect cisplatin-induced increased expression of Endo G in PPAR-{alpha} null mice (Fig. 4C). These data, along with our previously reported studies showing that PPAR-{alpha} ligand did not ameliorate renal function in cisplatin-treated PPAR-{alpha}-null mice (23), suggest that the protective effects of PPAR-{alpha} ligand on Endo G protein expression are dependent on having an intact and functionally active PPAR-{alpha} gene.



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  		Fig. 3. Effect of cisplatin and PPAR-{alpha} ligand on the mRNA levels of renal endonuclease G (Endo G). A: representative autoradiographs of RT-PCR analysis performed using total RNA isolated from kidney tissue of mice who received a normal or WY-containing diet and then were treated with either saline (groups control and WY) or cisplatin (groups cisplatin and cisplatin + WY). B: quantitative densitometry of mRNA levels determined using labeled primers for Endo G and 28s rRNA, as described in METHODS. Bars represent means ± SE Endo G mRNA levels. Data were obtained from at least 4 independent experiments. *P < 0.005 compared with control by unpaired Student's t-test.

 


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  		Fig. 4. Effect of cisplatin and WY on Endo G protein levels in PPAR-{alpha} wild-type (+/+) and null (–/–) mice. A: time course of cisplatin-induced Endo G protein expression shows Western blot analysis of mouse kidney cell lysates from mice treated with saline (control) or cisplatin. Samples were collected after 1, 2, 3, and 4 days of single cisplatin injection. Western blot analysis was performed using polyclonal Endo G antibody as described in METHODS. B and C: Western blot analyses of Endo G in PPAR-{alpha} wild-type (B) and null (C) mice kidney tissues in control (saline treated), cisplatin, WY and WY + cisplatin experimental conditions. Bars correspond to Endo G protein levels ± SE of at least 4 independent experiments and the protein level in control was set to 100%. *P < 0.05 compared with control in unpaired Student's t-test.

 
Cisplatin increases Endo G expression in the mouse kidney. In situ hybridization for Endo G mRNA identified the cells of the proximal tubule as the principal sites of Endo G expression. Cisplatin caused an increase in the expression of Endo G on the proximal tubule, an effect that was apparent after 4 days of cisplatin treatment. PPAR-{alpha} ligand reduced the expression of Endo G mRNA expression in cisplatin-treated mice as shown in Fig. 5.



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  		Fig. 5. In situ hybridization for localization of Endo G in mouse kidney sections from animal treated with saline (A), WY (B), cisplatin (C), and WY + cisplatin (D). The hybridization of a Endo G RNA antisense probe can be only seen in cells of proximal tubules of the cisplatin-treated mouse kidneys (C) denoted by an arrow. The staining could not be detected in the saline-treated mouse kidneys (A; control), neither in WY (B) nor in WY + cisplatin (D)-treated mouse kidney.

 
Immunohistochemistry. Because our results demonstrate that the maximum expression of Endo G protein was observed at day 4 after cisplatin injection, we next examined the immunohistochemical localization of Endo G at day 4 of cisplatin treatment in the absence and presence of PPAR-{alpha} ligand WY. Minimal cytoplasmic Endo G staining was identified in sections from control untreated mice, as well as in WY- and WY plus cisplatin (4 days)-treated kidneys as shown in Fig. 6. In kidneys studied 4 days after cisplatin administration, positive nuclear staining for Endo G was seen in the outer cortex in proximal tubules which showed varying degree of injury (most likely S3 and S2 segments). There were also randomly appearing positively stained nuclei of individual cells in proximal tubules of the outer cortex, which showed only minimal vacuolization or no signs of injury (3 days after cisplatin, data not shown). There were only few Endo G-positive nuclei in the WY plus cisplatin kidneys. The glomeruli, corticomedullary junction, and medulla were negative for Endo G staining.



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  		Fig. 6. Immunolocalization of renal Endo G. Mouse kidney sections from animals treated with saline (A), WY (B), cisplatin (C), and WY + cisplatin (D) were processed and incubated with Endo G antibody as described in METHODS. Immunohistochemical staining of control (A), WY-treated (B), and WY + cisplatin-treated (D) mice was faint but present mostly in cytosolic fraction of proximal tubules. Cisplatin-treated mice reveal Endo G staining primarily in nuclear fractions of proximal tube (PT) cells as indicated by an arrow (C).

 
TUNEL. The presence of apoptotic cells was determined by the TUNEL technique. Using this in situ method, we observed very few stained nuclei in normal and WY-treated kidneys. In cisplatin-treated mice in the absence and presence of WY we observed the presence of apoptotic nuclei significantly increased compared with their controls. Appearance of positively stained nuclei could be seen primarily in the cortex; few apoptotic cells were detected in the medulla. The apoptotic cells in the cortex could be observed in both proximal (most likely S1 and S2 segments) and distal nephron segments. Pretreatment with WY almost completely eliminated the presence of necrosis caused by cisplatin administration (4 days) and also significantly (~60%) decreased the presence of apoptotic nuclei. The results of the quantitative evaluation of apoptosis are summarized in Fig. 7.



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  		Fig. 7. Quantitative evaluation of TUNEL-positive nuclei in cortex and medulla of mouse kidney. In situ detection of apoptosis in kidney tissue sections was performed as described in METHODS. Mouse kidney sections were obtained from mice treated with saline (A), cisplatin (B), WY (C), and WY + cisplatin (D). The TUNEL-positive nuclei (apoptotic cells) are noted by arrows. E: quantitative evaluation of TUNEL-positive nuclei (apoptotic cells) from A-D. Bars correspond to apoptotic cells per mm2. *P < 0.00001 compared with control. {dagger}P < 0.00001 compared with cisplatin in unpaired Student's t-test.

 
Endonuclease activity is increased in cisplatin-treated mouse kidney. In mammalian cells, Endo G represents the most abundant endonuclease present in the mitochondrial fraction. Therefore, the assays available to measure endonuclease activity are considered similar to evaluating Endo G activity. This enzyme is synthesized in the cytoplasm and then imported into mitochondria. The membrane disruption of isolated mitochondria is required to measure endonuclease activity. To test the nuclease activity of mitochondrial endonuclease, we performed an agarose gel assay. In this assay, endonuclease activity is estimated by its endonucleolytic nicking of supercoiled plasmid DNA, which is used as a substrate, and this reaction is followed by agarose electrophoresis. In this reaction, the nicked circular DNA is converted to supercoiled plasmid DNA, which then migrates more slowly during agarose gel electrophoresis due to the loss of superhelicity. Endonuclease activity in the mouse kidney crude extracts of mitochondria is sufficient to damage and nick supercoiled plasmid DNA as shown in Fig. 8 (lanes 2-5). Compared with the nicking reaction without mitochondrial lysate (lane 1), mouse kidney mitochondrial lysates from either saline-treated (control lane 2) or cisplatin-treated mice (lane 3) exhibit a 64% (0.64 ± 0.03) and 89% (0.89 ± 0.02) change in the supercoiled plasmid DNA which was converted into nicked circular DNA, respectively. In PPAR-{alpha} wild-type mice fed with the 0.1% WY-14643 diet, kidney mitochondrial lysates from either WY (lane 2) or cisplatin + WY-treated (lane 4) exhibit 67% (0.67 ± 0.03) and 65% (0.65 ± 0.03) of the supercoiled plasmid DNA converted into nicked circular DNA, respectively. In WY-treated, as well as WY-treated wild-type mice that received cisplatin, endonuclease activity had the same activity compared with control untreated mice. Endonuclease activity is significant higher in cisplatin-treated kidneys compared with that in cisplatin plus WY-treated kidneys (P < 0.005). Therefore, the results of this assay suggest that WY inhibits cisplatin-induced mitochondrial endonuclease activity in mouse kidney tissue. These effects of cisplatin and PPAR-{alpha} ligand WY on mitochondrial endonuclease activity, along with the effects of PPAR-{alpha} ligand on protein expression and immunolocalization of Endo G, suggest to us that PPAR-{alpha} ligand has a direct inhibitory effect on the activity of mitochondrial endonuclease during cisplatin-induced ARF.



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  		Fig. 8. Effect of cisplatin and PPAR-{alpha} ligand on the activity of endonuclease in renal mitochondria. Agarose gel assay of endonuclease activity was performed as described in METHODS. A: representative photograph of the agarose gel. The nicked circular (NC) and supercoiled (SC) plasmid DNA products are shown. Lane 1: incubation of plasmid DNA alone without mitochondrial lysate. Lanes 2-5: plasmid DNA in the presence of 5 µg of mitochondrial lysate obtained from mice treated without and with cisplatin in the absence and presence of WY ligand. B: quantitative densitometry of supercoiled plasmid DNA band. The densitometry of supercoiled plasmid DNA band reaction without mitochondrial lysate was set to a value of 1. The mitochondrial lysates were prepared from 3 to 5 mice from each experimental group. Bars represent means ± SE of supercoiled plasmid DNA. Data were obtained from at least 3 independent experiments from each group. *P < 0.005 compared with cisplatin + WY by unpaired Student's t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we extended our observations with regard to the mechanisms by which administration of PPAR-{alpha} ligand(s) ameliorate cisplatin-induced ARF (14,20–21). Here, we show that cisplatin significantly increased both mRNA and protein levels of mitochondrial Endo G, a protein which on activation is associated with cell death. In addition, our studies demonstrate for the first time in kidney tissue that the increased expression of Endo G occurs mostly in the proximal tubules. Administration of PPAR-{alpha} ligand significantly reduced cisplatin-induced expression of Endo G, and this response was associated with amelioration of proximal tubule cell apoptosis and necrosis, as well as protection of renal function measured as changes in BUN and creatinine. Furthermore, the finding that PPAR-{alpha} ligand did not reduce cisplatin-induced Endo G protein expression in kidneys of PPAR-{alpha}-null mice suggests also that an intact PPAR-{alpha} gene plays a direct role in the regulation of increased mitochondrial Endo G expression during toxic renal epithelial cell injury. This observation has not been previously described in any other tissue and will require additional studies to examine the nature of this interaction. In previous studies, we reported that PPAR-{alpha} ligands when administered to proximal tubule cells in culture reduced significantly cisplatin-induced activation of caspase-3 (23). More recent studies from our laboratory suggest also that the protective effects seen with PPAR-{alpha} ligands in cells in culture relate to the inhibition of cisplatin-induced cytochrome c release and also to an increased expression of anti-apoptotic Bcl2 protein in mitochondrial fractions (Nagothu KK and Didier P, unpublished observations). Therefore, based on those previous observations, we could speculate that the observed antiapoptotic effect of PPAR-{alpha} ligand reported in this study likely relates to the prevention of mitochondrial injury by cisplatin leading to inhibition of Endo G release.

Our results underscore the potential importance of reducing Endo G levels as a novel therapeutic tool to ameliorate ARF. In this regard, a recent study (40) evaluated the in vivo function of Endo G in cells and mice. These investigators found that Endo G homozygous mutant embryos die between embryonic days 2.5 and 3.5. Analysis of Endo G heterozygous-mutant mice suggests that Endo G function does not relate to mitochondrial DNA replication. The authors also evaluated the cellular responses of mouse embryonic fibroblasts isolated from Endo G+/– mice to TNF-{alpha} and staurosporine, and those results indicated that reduced Endo G expression rendered cells more resistant to cell death. Therefore, that study is the first to demonstrate that DNA fragmentation is reduced in Endo G+/– splenocytes and thymocytes compared with wild-type cells, and altogether their findings suggest that increased Endo G expression also facilitates DNA fragmentation and apoptotic cell death. Our study showing 1) that increased proximal tubule Endo G expression and activity correlated with increased apoptosis and necrosis and 2) that reduction of Endo G expression correlated with preservation of proximal tubular cell structure and improved renal function in PPAR-{alpha} wild-type but not on PPAR-{alpha}-null mice certainly supports the observations made in the previous study (40), where lower levels of Endo G expression correlated with protection against cell death.

Previous studies documented an increased activity in Endo G associated with translocation of mitochondrial Endo G to the nuclear compartment as the major mechanism of regulation of endo G during apoptosis (36). Our immunohistochemical studies demonstrate for the first time in kidney tissue that Endo G can be translocated from the cytosolic compartment to a nuclear compartment in proximal tubules of mice that received cisplatin. However, the observed increases in mRNA followed by increased protein levels also suggest that Endo G could be regulated at the transcriptional level, an effect not previously described as a mechanism of regulation of Endo G activity. Nevertheless, an example of this type of regulation has been demonstrated recently in kidney tissue for other proapoptotic molecules in the model of ischemia-reperfusion injury. In that model, Supavekin et al. (30) demonstrated that during ischemia- reperfusion injury the mRNA for proapoptotic molecules FADD, DAXX, P53, and BAD determined both by microarray analysis and by RT-PCR were increased as early as 3 h of reperfusion. Those findings were confirmed at the protein level by immunohistochemistry and positive TUNEL staining, which localized those proapoptotic molecules predominantly to distal tubule cells.

Recent studies have described the existence of various isoforms of endonucleases in kidney tissue including DNAse I, DNAse II, caspase-activated DNAse (CAD), enzymes which on activation could potentially play a role in the apoptotic form of cell death in renal epithelial cells during cisplatin injury (1). Basnakian et al. (2) demonstrated by DNA substrate gel analysis of extracts from normal rat kidney cortex the presence of a DNase-I like protein which could be implicated as one of the mechanisms involved in cell death induced by hypoxia/reoxygenation in vitro (2). Our studies presented here indicate the presence of a mitochondrial endonuclease, which, on activation by cisplatin, translocates from the mitochondrial to the nuclear compartment. Given the fact that the major mitochondrial endonuclease corresponds to Endo G and our data with Western blot analysis as well as inmmunohistochemical localization altogether suggest to us that Endo G represents one of the endonuclease isoforms that account for proximal tubule cell death during cisplatin-induced ARF.

Our new data lends support to the original observations made in our laboratory where PPAR-{alpha} activation ameliorated ARF in cisplatin-treated mice (14). Antiapoptotic effects of PPAR-{alpha} ligands have been described previously in liver tissue; however, our studies are the first ones to demonstrate the antiapoptotic effect of PPAR-{alpha} ligands on ARF. Likely mechanisms for these antiapoptotic effects include amelioration of cisplatin-induced increased lipotoxicity (21), accompanied by modulation of the metabolic responses of substrate utilization during ARF (20). Our studies suggest that cisplatin-induced release of Endo G occurs both in PPAR-{alpha} wild-type and null mice. The mechanisms that regulate the release of Endo G from mitochondria are not clear at all. A previous study (36) suggests that the formation of truncated Bid associated to a change in the lipid environment in the mitochondria could represent the likely mechanism for increased mitochondrial permeability and release of proapoptotic proteins such as Endo G and AIF. We demonstrated in previous studies (14, 21, 22) that pretreatment with a PPAR-{alpha} ligand of PPAR-{alpha} wild-type mice preserves fatty acid oxidation, which was inhibited by cisplatin or ischemia-reperfusion injury, an effect not seen in the PPAR-{alpha} null mice treated with the ligand. These observations suggest to us that preservation of both peroxisomal and mitochondrial fatty acid oxidation in the wild-type mice treated with the PPAR-{alpha} ligand in the presence of cisplatin is what associates with reduced expression of Endo G by the proximal tubule.

Our study, however, cannot conclusively establish cause and effect between PPAR-{alpha} activation and modulation of Endo G activity, although future in vitro studies using cotransfection of proximal tubules cells with cDNA including both PPAR-{alpha} and Endo G should allow us to examine the mechanisms of interaction of these two important proximal tubule molecules during acute injury.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grant PO1-DK58324–01A1 and a VA Merit Award to D. Portilla.


   ACKNOWLEDGMENTS
 
We acknowledge Dr. A. Basnakian for sharing preliminary data.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Portilla, Univ. of Arkansas for Medical Sciences, Dept. of Medicine, Slot 501, 4301 W. Markham St., Little Rock, AR 72205 (E-mail: portilladidier{at}uams.edu)

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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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