Transgenic expression of proximal tubule peroxisome proliferator–activated receptor-α in mice confers protection during acute kidney injury
2009; Elsevier BV; Volume: 76; Issue: 10 Linguagem: Inglês
10.1038/ki.2009.330
ISSN1523-1755
AutoresShenyang Li, Kiran K. Nagothu, Varsha G. Desai, Taewon Lee, William S. Branham, Carrie L. Moland, Judit Megyesi, Mark D. Crew, Didier Portilla,
Tópico(s)Metabolism and Genetic Disorders
ResumoOur previous studies suggest that peroxisome proliferator–activated receptor-α (PPARα) plays a critical role in regulating fatty acid β-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARα in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARα expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia–reperfusion-induced acute kidney injury. Ischemia–reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARα activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARα ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury. Our previous studies suggest that peroxisome proliferator–activated receptor-α (PPARα) plays a critical role in regulating fatty acid β-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARα in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARα expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia–reperfusion-induced acute kidney injury. Ischemia–reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARα activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARα ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury. Peroxisome proliferator–activated receptors (PPARs) are transcription factors belonging to the ligand-activated nuclear hormone receptor superfamily.1.Issemann I. Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Crossref PubMed Scopus (2951) Google Scholar Through heterodimerization with retinoid X receptor (RXR) and binding to the PPAR response elements (PPREs) in the promoter of target genes, PPARs have been shown to have critical roles in regulating pleiotropic biological process, including lipid and glucose metabolism, adipogenesis, immune response, and cell growth and differentiation.2.Guan Y. Peroxisome proliferator-activated receptor family and its relationship to renal complications of the metabolic syndrome.J Am Soc Nephrol. 2004; 15: 2801-2815Crossref PubMed Scopus (145) Google Scholar,3.Shearer B.G. Hoekstra W.J. Recent advances in peroxisome proliferator-activated receptor science.Curr Med Chem. 2003; 10: 267-280Crossref PubMed Scopus (97) Google Scholar There are three major subtypes: PPARα, PPARβ/δ, and PPARγ. PPARα is predominantly expressed in metabolically very active tissues, such as liver, heart, renal proximal tubular cells (kidney), skeletal muscle, and brown fat.4.Kliewer S.A. Forman B.M. Blumberg B. et al.Differential expression and activation of a family of murine peroxisome proliferators-activated receptors.Proc Natl Acad Sci USA. 1994; 91: 7355-7359Crossref PubMed Scopus (1248) Google Scholar,5Smirnov A.N. Nuclear receptors: nomenclature, ligands, mechanisms of their effects on gene expression.Biochemistry. 2002; 67: 957-977PubMed Google Scholar Increased evidence suggests PPARα has an important role in the regulation of energy homeostasis.6.Lefebvre P. Chinetti G. Fruchart J.C. et al.Sorting out the roles of PPAR alpha in energy metabolism and vascular homeostasis.J Clin Invest. 2006; 116: 571-580Crossref PubMed Scopus (675) Google Scholar PPARα activates fatty acid catabolism by induction of target genes encoding fatty acid oxidation (FAO) enzymes in the mitochondria and peroxisomes, as well as proteins involved in cellular fatty acid import. In addition, PPARα not only stimulates gluconeogenesis and ketone body synthesis but also has a significant anti-inflammatory activity that seems to have a protective role and suppress apoptosis.7.Roberts R.A. Chevalier S. Hasmall S.C. et al.PPARα and the regulation of cell division and apoptosis.Toxicology. 2002; 182: 167-170Crossref Scopus (57) Google Scholar PPARα is a short-lived protein and is degraded by the ubiquitin–proteasome system. Its transcriptional activity is regulated by various factors at several levels. These include the regulation of its expression and stability, the nature and the level of endogenous or synthetic ligand, the co-activator and co-repressor proteins, and post-translational modifications of PPARα and associated activator and repressor. PPARα is highly expressed in the kidney, predominantly in the proximal tubule and in medullary thick ascending limb of Henle. Fatty acids constitute a major source of metabolic fuel for energy production in kidney cortex tissue, and activation of PPARα by various ligands can induce the expression of genes involved in controlling renal fatty acid β-oxidation. In previous studies, we have observed that the inhibition of peroxisomal and mitochondrial FAO enzymes in kidney tissue of mice undergoing ischemia/reperfusion (I/R)- and cisplatin (CP)-induced acute kidney injury (AKI) results from reduced transcriptional activity of PPARα.8.Portilla D. Dai G. Peters J.M. et al.Etomoxir- induced PPARalpha-modulated enzymes protect during acute renal failure.Am J Physiol Renal Physiol. 2000; 278: F667-F675PubMed Google Scholar, 9.Portilla D. Dai G. McClure T. et al.Alterations of PPARalpha and its coactivator PGC-1 in cisplatin-induced acute renal failure.Kidney Int. 2002; 62: 1208-1218Abstract Full Text Full Text PDF PubMed Google Scholar, 10.Li S. Wu P. Yarlagadda P. et al.PPARα ligand protects during cisplatin-induced acute renal failure by preventing inhibition of renal FAO and PDC activity.Am J Physiol Renal Physiol. 2004; 286: F572-F580Crossref PubMed Scopus (74) Google Scholar, 11.Portilla D. Energy metabolism and cytotoxicity.Semin Nephrol. 2003; 23: 432-438Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12.Li S. Basnakian A. Bhatt R. et al.PPAR-alpha ligand ameliorates acute renal failure by reducing cisplatin-induced increased expression of renal endonuclease G.Am J Physiol Renal Physiol. 2004; 287: F990-F998Crossref PubMed Scopus (80) Google Scholar, 13.Li S. Gokden N. Okusa M.D. et al.Anti-inflammatory effect of fibrate protects from cisplatin-induced ARF.Am J Physiol Renal Physiol. 2005; 289: F469-F480Crossref PubMed Scopus (107) Google Scholar, 14.Nagothu K.K. Bhatt R. Kaushal G.P. et al.Fibrate prevents cisplatin-induced proximal tubule cell death.Kidney Int. 2005; 68: 2680-2693Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Failure to oxidize long-chain fatty acids and long-chain acylcarnitines during AKI results in their accumulation and cellular toxicity, which further contributes to proximal tubule cell death.8.Portilla D. Dai G. Peters J.M. et al.Etomoxir- induced PPARalpha-modulated enzymes protect during acute renal failure.Am J Physiol Renal Physiol. 2000; 278: F667-F675PubMed Google Scholar,9.Portilla D. Dai G. McClure T. et al.Alterations of PPARalpha and its coactivator PGC-1 in cisplatin-induced acute renal failure.Kidney Int. 2002; 62: 1208-1218Abstract Full Text Full Text PDF PubMed Google Scholar We also documented that the administration of fibrate, a known PPARα ligand, before AKI (1) prevented the inhibition of FAO and the accumulation of non-esterified fatty acids and triglyceride in kidney tissue; and (2) fibrates ameliorated apoptotic and necrotic proximal tubule cell death, resulting in significant protection of renal function only in PPARα wild-type mice, and not in PPARα-null mice.11.Portilla D. Energy metabolism and cytotoxicity.Semin Nephrol. 2003; 23: 432-438Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12.Li S. Basnakian A. Bhatt R. et al.PPAR-alpha ligand ameliorates acute renal failure by reducing cisplatin-induced increased expression of renal endonuclease G.Am J Physiol Renal Physiol. 2004; 287: F990-F998Crossref PubMed Scopus (80) Google Scholar, 13.Li S. Gokden N. Okusa M.D. et al.Anti-inflammatory effect of fibrate protects from cisplatin-induced ARF.Am J Physiol Renal Physiol. 2005; 289: F469-F480Crossref PubMed Scopus (107) Google Scholar, 14.Nagothu K.K. Bhatt R. Kaushal G.P. et al.Fibrate prevents cisplatin-induced proximal tubule cell death.Kidney Int. 2005; 68: 2680-2693Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 15.Negishi K. Noiri E. Sugaya T. et al.A role of liver fatty acid-binding protein in cisplatin-induced acute renal failure.Kidney Int. 2007; 72: 348-358Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Altogether, these observations would suggest a critical role for PPARα in regulating fatty acid β-oxidation in kidney tissue that directly correlates with the preservation of kidney morphology and function during CP- and I/R-induced AKI. To further investigate the physiological role of PPARα in kidney, and to determine whether the upregulation of PPARα expression alone without the use of a synthetic ligand is sufficient to activate FAO in the kidney and to ameliorate renal function from CP- or I/R-induced AKI, we generated transgenic (Tg) mice that express mouse PPARα under the control of kidney androgen-regulated protein 2 (KAP2) promoter, which is androgen-responsive. This promoter has been used with success to generate mice that show proximal tubule and androgen-specific expression of human rennin.16.Lavoie J.L. Lake-Bruse K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in the renal proximal tubule.Am J Physiol Renal Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (94) Google Scholar More recently, KAP2 promoter was used to develop Tg mice that express proximal tubule-specific Cre recombinase activity.17.Li H. Zhou X. Davis D.R. et al.An androgen-inducible proximal tubule-specific Cre-recombinase transgenic model.Am J Physiol Renal Physiol. 2008; 294: F1481-F1486Crossref PubMed Scopus (18) Google Scholar In our KAP2-PPARα Tg mice reported here, renal PPARα expression was detectable in proximal tubules of female Tg mice and could be induced by testosterone treatment. In comparison with wild-type mice, without synthetic ligand, the upregulation of PPARα expression by testosterone treatment in the KAP2-PPARα female Tg mice prevented CP- and I/R-induced inhibition of FAO and protected kidney function and morphology from CP- or I/R-induced AKI. KAP2-PPARα Tg mice were generated to produce a tissue-specific and androgen-inducible expression of mouse PPARα in the proximal tubules of the kidney. Three founder Tg lines were obtained and all transmitted the KAP2-PPARα Tg to their progeny. Tg expression was evaluated in kidney, liver, heart, and brain tissues. Of the three founder Tg lines (#560, #561, and #562), all three lines were shown to express the Tg in the kidney by reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis. The highest level of KAP2-PPARα Tg expression was observed with line 562, therefore the studies presented in this study were performed in line 562. However, analogous results were obtained with line 561. As shown in Figure 1a, Tg FLAG-PPARα mRNA level was detected only in the kidney of KAP2-PPARα Tg mice. In addition, when KAP2-PPARα Tg mice were given testosterone, the expression of FLAG-PPARα was increased only in kidney but not in heart, liver, or brain tissue as shown in Figure 1b. As PPARα is normally expressed in heart, liver, and kidney tissue of wild-type mice, we then carried out real-time RT-PCR analysis using total RNA isolated from kidney, heart, and liver tissues of wild-type and Tg mice, and specific oligonucleotides to measure total PPARα mRNA levels (Tg +endogenous). As shown in Figure 1c, PPARα (Tg +endogenous) was increased about 2.4-fold only in kidney tissue of KAP2-PPARα Tg mice but not in the liver or heart tissue. To assess the inducibility of the KAP2 promoter, Tg female mice from line 562 were implanted with a testosterone pellet. After 14 days, the mice were killed, and tissues were collected for further analysis of the PPARα gene expression. When testosterone was given to either wild-type or KAP2- PPARα Tg mice, PPARα expression was increased only in kidney but not in liver or heart tissue. These results shown in Figure 1c further support the specificity of the expression of KAP2-PPARα Tg mice in kidney tissue, as well as the inducibility of PPARα gene in kidney tissue when testosterone was given. The increased mRNA expression of PPARα in kidney tissue of KAP2-PPARα Tg mice was also correlated with the increased protein levels of PPARα in kidney tissue. Our studies using anti-PPARα antibodies followed by immunoprecipitation and western blot analysis of kidney tissue obtained from KAP2-PPARα Tg mice showed that testosterone induced a 3.5-fold increase in PPARα protein levels (Figure 2).Figure 2Testosterone-induced PPARα protein expression in female KAP2-PPARα transgenic mice. (a) A representative autoradiogram of one single experiment of western blot analysis of immunoprecipitated peroxisome proliferator-activated receptor-α (PPARα) from kidney androgen-regulated protein 2 (KAP2)-PPARα transgenic (Tg) mouse kidney tissue. Immunoprecipitates (fractions F1, F2, F3) from mouse kidney tissues with (+ Testosterone) or without testosterone (− Testosterone) treatment. (b) Quantification of renal PPARα protein expression in the absence or presence of testosterone was carried out using kidney tissues of KAP2-PPARα Tg mice. This figure represents a summary of three separate experiments. Bars represent means±s.e. of three separate experiments.View Large Image Figure ViewerDownload (PPT) To determine the role of increased expression and activity of PPARα protein in the kidney proximal tubule, kidney function was monitored by measuring serum blood urea nitrogen (BUN) and creatinine for 2 days after single intraperitoneal injection of saline or CP. Figure 3a and b presents the changes in BUN and serum creatinine seen in KAP2-PPARα Tg or wild-type mice treated with saline or CP in the absence or presence of testosterone. Comparison of the renal function between wild-type mice pretreated with or without testosterone did not show differences in BUN and creatinine after saline injection (BUN: 7.85 and 8.39 mM, respectively; creatinine: 23.76 and 22 μM, respectively). Wild-type mice developed significant AKI in both groups at day 2 after CP injection (BUN: 100.35 and 94.59 mM, respectively; creatinine 233.2 and 205.04 μM, respectively). In contrast, KAP2-PPARα Tg mice, pretreated with testosterone before CP administration, showed protection in renal function on day 2 (BUN: 10.36 mM, creatinine: 33.44 μM). However, KAP2-PPARα Tg mice without testosterone pretreatment developed AKI at day 2 (BUN: 94.15 mM, creatinine: 193.6 μM) after CP injection. These observations suggest that the protective effect on renal function in KAP2-PPARα Tg mice was dependent on testosterone-mediated increased expression of proximal tubule PPARα. To investigate whether testosterone-induced PPARα expression had a similar effect in protecting kidney function during acute ischemic renal failure, we used an I/R model in both wild-type and KAP2-PPARα Tg mice. As shown in Figure 3c and d, the serum BUN and creatinine levels were much higher in wild-type mice (53.63 mM and 169.84 μM, respectively) that were subjected to I/R injury when compared with KAP2-PPARα Tg mice (13.99 mM and 49.28 μM, respectively), which further supports our previous observations of amelioration of renal function by increasing the expression of proximal tubule PPARα before I/R injury. We have assessed survival time using the model of 50 min of ischemia followed by reperfusion injury. In this model, serum BUN and creatinine were lower in our KAP2-PPARα Tg mice at 24 h of reperfusion when compared with wild-type mice as shown in Figure 3c and d. At 48 h of reperfusion, serum BUN and serum creatinine were already improved in wild-type mice when compared with sham-operated mice (results not shown). We did observe wild-type and KAP2-PPARα Tg mice for 5 days of reperfusion after 50 min of ischemia and did not see an increase in mortality in wild-type mice when compared with KAP2-PPARα Tg mice. In contrast, in the model of CP-mediated acute renal failure, we did see a significant improvement in survival rate in our KAP2-PPARα Tg mice when compared with wild-type mice. These data are shown in Figure 4. At 3 days after CP injection, the survival rate in wild-type mice was 60% when compared with 100% survival rate in KAP2-PPARα Tg mice. At day 4, survival rate for wild-type mice was only 28.6% when compared with 100% in KAP2-PPARα Tg mice. At day 5, survival rate for wild-type mice was 0% (all wild-type mice were dead at day 5 after CP injection), whereas the survival rate was reduced to 60% in KAP2-PPARα Tg mice that received the same dose of CP than the wild-type mice (P=0.00726). We subsequently observed KAP2-PPARα Tg mice for 7 days after CP injection, and the survival rate did not change and was still 60%. There was no mortality in the sham-operated or saline-treated control mice (data not shown). On the basis of these results, we believe that the course of CP-mediated acute renal failure in our hands was more severe than the injury seen in I/R injury, and we were able to establish significant differences in survival rate when comparing wild-type mice that received testosterone and then CP with KAP2-PPARα Tg mice that did also receive testosterone and then CP. On the basis of these results, we conclude that the observed increased survival rate in KAP2-PPARα Tg mice that received CP and developed acute renal failure further supports a true protection against acute renal failure. PPARα staining was primarily localized to the thick ascending limbs of loop of Henle and the early parts (S1 and S2 segments) of proximal tubules in untreated wild-type animals. Furthermore, occasional positive staining with random nuclear appearance could be seen in the S3 segment of proximal tubules. PPARα staining was significantly decreased throughout the kidney 2 days after CP administration in these wild-type mice (Figure 5a and b). In contrast, very strong diffuse staining pattern could be detected in the entire kidney cortex including the S3 segments of proximal tubules in untreated KAP2-PPARα Tg mice after testosterone induction (Figure 5c). This positive staining remained unchanged in testosterone-treated KAP2-PPARα Tg mice 2 days after CP treatment (Figure 5d). Morphologic kidney damage was also markedly reduced in testosterone-treated KAP2-PPARα Tg mice 2 days after CP administration. Evaluation of the morphological differences (Figure 5b) showed small necrotic foci; severe degeneration accompanied by the loss of brush border, tubular dilatation, and cast formation in wild-type mice. The analyzed parameters were significantly reduced in the KAP2-PPARα Tg group and only mild degeneration and brush border loss could be detected in some areas of the kidney (Figure 5d) in KAP2-PPARα Tg mice treated with CP and testosterone. Similar results were also observed in the I/R-induced AKI model. The sham mouse kidneys in some instances exhibited very mild pathological changes such as occasional loss of bush border and presence of few inflammatory cells (Figure 6a). All wild-type mice undergoing I/R injury had extensive tubular necrosis at the cortico-medullary junction (S3 segment of proximal tubules), loss off brush border, leukocyte infiltration, and numerous casts throughout the kidney (Figure 6b). In contrast, testosterone-induced PPARα Tg ischemic kidney showed significantly improved morphology. They had only mild loss of brush border and tubular dilatation, and only very few necrotic tubules were found occasionally in some kidney sections. There was only minimal leukocyte infiltrate and very few casts (Figure 6c).Figure 6Comparison of morphological damage in the cortico-medullary junction from control and ischemic kidneys 24 h after reperfusion. Representative photographs of periodic acid-Schiff (PAS)-stained sections of sham (a), testosterone-pretreated wild-type ischemia/reperfusion (b), and testosterone-induced peroxisome proliferator-activated receptor-α (PPARα) transgenic ischemia/reperfusion (c) mouse kidneys. The sham kidney shows normal kidney architecture (a), whereas extensive necrosis, loss of brush border, cast formation, and inflammatory cells can be seen in a typical wild-type ischemic kidney 24 h after reperfusion (b). A typical testosterone-induced ischemic PPARα transgenic kidney (c) appeared to be almost normal. Only some loss of brush border, tubular dilatation, and single, partially necrotic tubules could be seen occasionally. Asterisks: proximal tubules; arrowheads: thick ascending loop of Henle; arrow: necrotic foci, NT: necrotic tubule. Original magnification, × 125.View Large Image Figure ViewerDownload (PPT) The semi-quantitative analysis of morphological damages in CP-treated mice has shown statistically significant changes in proximal tubule cell necrosis (Figure 7a), and tubular degeneration when PPARα Tg mice were induced with testosterone treatment. In the I/R model of AKI, as shown in Figure 7b, there were statistically significant changes in proximal tubule cell necrosis, cast formation, and tubular degeneration when PPARα was induced with testosterone treatment when compared with wild-type mice subjected to I/R injury. To determine the mechanisms by which renal function was ameliorated in KAP2-PPARα Tg mice treated with CP, we examined the effects of CP and testosterone on the expression of PPARα target genes involved in renal FAO in both KAP2-PPARα Tg and wild-type mice. As shown in Figures 8 and 9, the mRNA expression levels of medium-chain acyl-coenzyme A dehydrogenase (MCAD), long-chain acyl coenzyme dehydrogenase (LCAD), very-long-chain acyl coenzyme dehydrogenase (VLCAD), and liver carnitine palmytoyltransferase-1 (L-CPT1) showed no significant changes (only 1.1- to 1.3-fold increase) in KAP2-PPARα Tg mice when compared with levels in wild-type mice. For both wild-type and KAP2-PPARα Tg mice, CP caused a significant decline (P<0.05) in the mRNA expression of MCAD (85 and 76%, respectively), LCAD (41 and 30%, respectively), VLCAD (68 and 61%, respectively), and L-CPT1 (79 and 62%, respectively). Pretreatment with testosterone prevented CP-induced inhibition of renal FAO expression in KAP2-PPARα Tg mice, but this effect was not observed in the wild-type mice. These results are similar to our previously published observations, in which the use of PPARα ligands etomoxir and fibrates resulted in the upregulation of renal FAO during I/R- and CP-induced AKI. Therefore, again these observations further support the cytoprotective role of PPARα on renal function during AKI and further underscore the importance of mitochondrial FAO on the preservation of structure and function of the proximal tubule during AKI. As testosterone prevented CP-induced reduction of MCAD mRNA levels in the KAP2-PPARα Tg mice but not in the wild-type mice, we next examined the effects of CP and testosterone on MCAD enzyme activity. As shown in Figure 8b, CP on day 2 caused a profound decline (P<0.05) in the enzyme activity of renal MCAD in both wild-type and KAP2-PPARα Tg mice (61 and 59%, respectively). Pretreatment with testosterone prevented CP-induced inhibition of renal MCAD activity in KAP2-PPARα Tg mice. In contrast to the effects of testosterone on KAP2-PPARα Tg mice, pretreatment with testosterone did not affect CP-induced reduction of MCAD activity in wild-type mice. Similar results were observed in I/R-induced AKI model, as shown in Figures 8c, d and 9d. Pretreatment with testosterone prevented I/R-induced inhibition of renal FAO expression and MCAD activity in KAP2-PPARα Tg mice, but not in the wild-type mice. These data indicate that the protective effect of testosterone on mRNA levels and activity of FAO enzyme MCAD were dependent on the activation of KAP2 promoter and the induction of PPARα expression in the proximal tubule of KAP2-PPARα Tg mice.Figure 9Effects of cisplatin (Cisp) and ischemia/reperfusion (I/R) injury on mRNA levels of fatty acid oxidation (FAO) genes. Pretreatment with testosterone prevented (a–c) Cisp- or (d) I/R-induced inhibition of the mRNA levels of renal FAO-related genes LCAD, VLCAD, and L-CPT1 in KAP2-PPARα transgenic (Tg) mice. Wild-type (WT) or KAP2-PPARα Tg female mice were administered Cisp in the absence or presence of 14-day testosterone pretreatment or underwent 50-min ischemia operation in the presence of 14-day testosterone pretreatment. Levels of renal long-chain acyl coenzyme dehydrogenase (LCAD), very-long-chain acyl coenzyme dehydrogenase (VLCAD), and liver carnitine palmytoyltransferase-1 (L-CPT1) were determined by quantitative real-time RT-PCR. Bars represent mean±s.e. mRNA levels for at least four mice in each group. *P<0.05 compared with control (WT+saline without testosterone) or sham, †P<0.05 compared with TG+Cisp without testosterone pretreatment or I/R-WT in unpaired Student's t-test.View Large Image Figure ViewerDownload (PPT) Microarray data analysis showed that CP treatment significantly reduced the expression of 454 (84%) of mitochondria-related genes with a false discovery rate of ≤0.05 in PPARα Tg mice. A noteworthy observation was downregulation of genes associated with oxidative phosphorylation (complexes I–V), mitochondrial DNA replication and repair, fatty acid metabolism, and tricarboxylic acid (TCA) cycle (Table S1, data not shown). Interestingly, however, pretreatment with testosterone of PPARα Tg mice was protective against CP-mediated inhibition of mitochondria-related gene expression to the extent that the expression levels of the majority of mitochondrial genes inhibited by CP was almost restored to baseline levels (Figure 10 and Table 1). Download .xls (.14 MB) Help with xls files Table S1Table 1Mitochondria-associated gene expression in line 562 KAP2-PPARα Tg miceGeneSalineCisplatinTestosterone+salineTestosterone+cisplatinNdufa71.000±0.0250.652±0.023*P<0.05 compared with control in unpaired Student's t-test.1.135±0.0450.926±0.029Mt-Nd4l1.000±0.0110.681±0.017*P<0.05 compared with control in unpaired Student's t-test.1.054±0.0390.920±0.054Cox7b1.000±0.0530.685±0.024*P<0.05 compared with control in unpaired Student's t-test.0.976±0.0220.911±0.022Atp5b1.000±0.0170.667±0.019*P<0.05 compared with control in unpaired Student's t-test.1.046±0.0280.957±0.040Atp5J1.000±0.0420.661±0.016*P<0.05 compared with control in unpaired Student's t-test.1.056±0.0450.926±0.043Polg1.000±0.0220.634±0.014*P<0.05 compared with control in unpaired Student's t-test.1.086±0.0150.906±0.037Aco21.000±0.0320.683±0.034*P<0.05 compared with control in unpaired Student's t-test.1.095±0.0841.011±0.044Mfn11.000±0.0360.607±0.005*P<0.05 compared with control in unpaired Student's t-test.1.115±0.0901.053±0.035Mfn21.000±0.0550.569±0.014*P<0.05 compared with control in unpaired Student's t-test.1.024±0.0290.894±0.045Values represent mean±s.e. mRNA levels determined by quantitative real-time RT-PCR for at least four mice in each group and normalized to that of control mice (=1.0) (saline without testosterone).* P<0.05 compared with control in unpaired Student's t-test. Open table in a new tab Values represent mean±s.e. mRNA levels determined by quantitative real-time RT-PCR for at least four mice in each group and normalized to that of control mice (=1.0) (saline without testosterone). To get a better insight into the effect of CP on expression levels of mitochondria-related genes in the presence and absence of testosterone
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