前列腺素活化過氧化體增生活化接受體-α 在腎臟受缺血/再灌注及卡鉑傷害的分子及病生理機轉

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(1)臺北醫學大學醫學院臨床醫學研究所博士論文 Taipei Medical University College of Medicine Graduate Institute of Clinical Medicine Ph.D. Dissertation. 前列腺素活化過氧化體增生活化接受體-α 在腎臟受缺血/再灌注及卡鉑傷害的分子及病生理機轉 The Molecular and Pathophysiological Mechanisms of Prostacyclin Activating Peroxisome Proliferator-Activated Receptor-α in Ischemia/Reperfusion and Carboplatin Renal Injury.. 指導教授：陳振文 (Chen, Tzen-Wen) 共同指導教授：林. 恆 (Lin, Heng). 研究生：陳錫賢 (Chen, Hsi-Hsien) 撰學. 號：D102093015 中華民國九十九年六月 June, 2010.

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(3) 圖書館碩博士論文授權書.

(4) 致謝終於撐到這一天了，回想十一年前萌生攻讀研究所的念頭時，母親給我的鼓勵，終於做出重大的抉擇，從林口長庚醫院轉至北醫附醫任職。在陳培源主任的全力栽培及支持下，順利公費前往德州西南醫學中心、黃朝龍教授的實驗室學習基礎研究，奠定分子生物實驗理論與實務的基礎。回國後更在陳培源主任、陳盛煊主任及吳志雄院長的鼓勵下，考取博士班，前兩年的專題討論課程中，老師和同學的許多意見和指導，讓我更明顯的進步。但六年來要兼顧臨床工作、課業、及實驗，加上母親及陳培源主任先後病逝，身心俱疲，多次曾考慮休學。所幸朱元鏘醫師、賴怡君醫師、鄭仲益醫師及王梅美護理長協助分擔臨床工作；陳振文教授及林恒老師更提供充裕的資源及經費，讓我能無後顧之憂的進行實驗，並且時常和我討論實驗發展的方向，訓練我的邏輯思考及論文寫作能力；此外實驗室同事們不吝惜的幫忙，感謝侯欣翰及李孟紜在分生實驗上的指導與協助、金姵池及廖偉如在動物實驗方面鼎力相助；最重要的要感謝我的家人忍受我的忙碌及情緒，並在經濟上和情感上的支持。僅以個人的小成就，感謝所有曾經幫助過我的人。並以此告慰母親及陳培源主任，完成與您們的約定了。. 陳錫賢.

(5) 縮寫表(Abbreviations) AA, arachidonic acid; ARF, acute renal failure; AP-1, activator protein-1; CAY10441, a cAMP inhibitor; ChIP, chromatin immunoprecipitation; CM-H2DCFDA, chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate; COX, cyclooxygenase; DCF, dichlorodihydrofluorescein; DHA, docosahexaenoic acid; EMSA, Nuclear extracts and electrophoretic mobility shift assay; ERKs, extracellular signal-regulated kinases; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; HO, heme oxygenase; H/R, hypoxia-reoxygenation, IL, interleukin; I/R, ischemia/reperfusion; JNKs, c-jun N-terminal kinases; MAPKs, mitogen-activated protein kinases; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor-kappa B; NO, nitric oxide; PGs, prostaglandins PGH 2 , prostaglandin endoperoxide;.

(6) PGI 2 , prostacyclin; PGIS, prostacyclin synthase; PI3K, phosphatidylinositol 3-kinase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response element; RXRs, retinoid X receptors; ROS, reactive oxygen species; siRNA, small interfering RNA; SREBP-2, sterol responsive element-binding protein-2; STAT, signal transducer and activator of transcription; TNF-α, tumor necrosis factor-α; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VEGF, vascular endothelial growth factor;.

(7) CONTENTS 中文摘要. --------------------------------------------------------------- i. Abstract. --------------------------------------------------------------- iii. Introduction ----------------------------------------------------------- 1 Reserach objective (1) ------------------------------------------------ 5 Materials and Methods (1) --------------------------------------. 6. Results (1) --------------------------------------------------------- 14 Discussion (1) ----------------------------------------------------- 20 Reserach objective (2) ----------------------------------------------- 29 Materials and Methods (2) ------------------------------------- 30 Results (2) --------------------------------------------------------- 32 Discussion (2) ----------------------------------------------------- 38 Conclusion and Perspective ----------------------------------------- 49 References --------------------------------------------------------------- 50 Tables and Figures ----------------------------------------------------- 64 Appendix -------------------------------------------------------------. 105.

(8) 中文摘要我們假設環前列腺素(PGI 2 )可作為一個內生性的配體，來活化過氧化體增生活化接受體-α (PPARα)，並達到腎臟保護作用，我們利用缺血 /再灌注及卡鉑(Carboplatin)腎毒性兩種模式來測試我們的理論。首先在缺血/再灌注模式，以 PPAR-α 活化劑(DHA)預處理過的小鼠，顯著減少因缺血/再灌注所導致的腎臟功能不良、細胞凋亡反應和 NF-κB 的活化作用。且 PPAR-α 剔除鼠受到缺血/再灌注的傷害也更嚴重。我們更利用腺病毒使細胞過度表達環前列腺素，使得過氧化體增生活化接受體-α 往細胞核內移動，使得 caspase-3 的活化受到抑制；同時藉由與 NF-κB 結合來抑制 TNF-α 促進子(promoter)的活性。我們的結果，是第一個提出環前列腺素會使過氧化體增生活化接受體-α 往細胞核內移動，並減弱腎臟因缺血/再灌注傷害後，NF-κB 所導致的 TNF-α 活化作用。在卡鉑腎病模式，pravastatin可減少大鼠腎小管細胞株(NRK-52E) 受到卡鉑傷害後的反應性氧族(ROS)產生；增加血紅素氧酶-1 (HO-1)，環氧化酵素-2 (COX-2) 和 6-keto前列腺素(PG) F 1α 的產生；並促使得過氧化體增生活化接受體-α往細胞核內移動。我們並以染色質免疫沉澱反應證實在NRK-52E細胞中，過氧化體增生活化接受體-α會與血紅素氧酶-1 的促進子上的PPRE結合。小鼠注射卡鉑會導致急性腎衰竭及死 i.

(9) 亡，但此現象會因pravastatin的預處理而改善。野生型小鼠注射 pravastatin會使過氧化體增生活化接受體-α和血紅素氧酶-1 過分表現，卻不見於過氧化體增生活化接受體-α剔除鼠；而以COX-2 抑制劑NS-398 加入NRK-52E細胞中，pravastatin對過氧化體增生活化接受體-α 往細胞核內移動現象明顯減少、且血紅素氧酶-1 的活化作用亦明顯下降。我們的結果顯示pravastatin藉由增加血紅素氧酶-1，達到減少腎臟受到卡鉑的傷害；並暗示了環前列腺素/過氧化體增生活化接受體-α此一信息傳導路徑，牽涉在其中。我們結果證明前列腺素藉由活化過氧化體增生活化接受體-α來保護腎臟受缺血/再灌注及卡鉑的傷害，未來便可進一步發展以增加環前列腺素、過氧化體增生活化接受體-α、或此一信息傳導路徑之治療（例如pravastatin），將可治療腎臟因缺血/再灌注及卡鉑腎病之傷害。. 關鍵字: 環前列腺素、過氧化體增生活化接受體-α、血紅素氧酶-1、缺血/再灌注、卡鉑、Pravastatin. ii.

(10) Abstract To evaluate the prostacyclin (PGI 2 ) can function as an endogenous peroxisome proliferator-activated receptor-α (PPARα) ligand and mediate its renal protective effects, we examined the PGI 2 -enhanced protective effect of PPAR-α in ischemia/reperfusion (I/R) and carboplatin induced kidney injury. In the I/R model, pretreating mice with a PPAR-α activator, docosahexaenoic acid (DHA), significantly reduced I/R-induced renal dysfunction, apoptotic responses, and Nuclear Factor-kappa B (NF-κB) activation. By comparison, I/R-induced injury was exacerbated in PPAR-α knockout mice. Overexpression of PGI 2 using an adenovirus could also induce PPAR-α translocation from the cytosol into the nucleus to inhibit caspase-3 activation. This PGI 2 / PPAR-α pathway attenuated Tumor Necrosis Factor-α (TNF-α) promoter activity by binding to NF-κB. Taken together, our results demonstrate for the first time that prostacyclin induces the translocation of PPAR-α from the cytosol into the nucleus and attenuates NF-κB-induced TNF-α activation following renal I/R injury. In the carboplatin nephropathy model, pravastatin decreased production of reactive oxygen species (ROS), increased expression of heme oxygenase-1 (HO-1), cyclooxygenase (COX)-2, and 6-keto prostaglandin (PG) F 1α , and enhanced nuclear translocation of PPAR-α in carboplatin treated NRK-52E cells. Interaction of PPARα with peroxisome proliferator iii.

(11) response element (PPRE) on the HO-1 promoter was confirmed in NRK-52E cells by chromatin immunoprecipitation. Mice subjected to carboplatin injection developed acute renal failure and decreased survival, which were ameliorated by pretreatment with pravastatin. Pravastatin injection also induced overexpression of PPAR-α and HO-1 in wild-type mice, but the HO-1 expression was significantly attenuated in PPAR-α knock-out mice. When the COX-2 inhibitor NS-398 was added to pravastatin-treated cells, the nuclear translocation of PPAR-α and the increased in HO-1 level were also significantly attenuated. These results implied that PGI 2 / PPAR-α signaling pathway also involved in the pravastatin upregulates HO-1 and protects against carboplatin-induced renal dysfunction. Our result proved that PGI 2 induced nuclear translocation of PPAR-α to protect against renal I/R and Carboplatin injury. Treatments that can augment prostacyclin, PPAR-α, or the associated signaling pathways (such as pravastatin) may ameliorate conditions associated with renal I/R or carboplatin injury.. Key words: prostacyclin, peroxisome proliferator-activated receptor-α, heme oxygenase-1, ischemia/reperfusion, carboplatin, pravastatin.. iv.

(12) Introduction Renal ischemia and toxins induced renal tubular necrosis are the major causes of acute renal failure (ARF), which despite significant advances in critical care medicine, remains a major clinical problem, producing grave morbidity and mortality that has not decreased significantly over the last 50 years (Sheridan et al. 2001). To date, no specific therapy has been shown to alter the course or outcome of ARF (Lameire et al. 2001) and many reasons have been proposed for the inability of these interventions to improve the prognosis of ARF, including an incomplete understanding of the pathophysiology underlying the development of ARF.. Ischemia/reperfusion (I/R) Injury is an important cause of organ dysfunction often leading to ARF, causing high mortality among patients in intensive care who require dialysis (Deng et al. 2001). I/R is a stimulus for leukocyte–endothelial interaction (Granger et al. 1977).Which results in the accumulation of leukocytes (Heeman et al. 2000). Toxins account for 20 % of hospital acquired episodes of ARF, which include contrast dye, aminoglycosides, amphotericin B and cisplatin. Among them. carboplatin (cis-diammine-1,1-cyclobutanedicarboxylateplatinum II), a second generation platinum containing anticancer drug, is currently used in the treatment of lung and ovarian cancers, as well as head and neck cancers (Fujiwara et al. 2003; Pivot et al. 2001). Because carboplatin has fewer toxic effects than cisplatin, it can be used at higher doses to achieve optimal antitumor effects (Alberts et al. 1995). However, its byproduct, 1.

(13) platinum-ammine-DNA adduct, accumulates in the kidney and induces renal tubular damage (Kintzel et al. 2001); this limits its use.. Both I/R and toxins will result in the accumulation of ROS and activates the signaling mechanisms that culminate in tumor necrosis factor production (Donnahoo et al. 2000). Thus, correction of the oxidant/ antioxidant imbalance are important approach to reducing the risk of developing acute kidney failure.. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate a diversity of functions such as lipid, glucose metabolism, and adipogenesis. Recent evidence suggests that PPARs play an important role in the modulation of inflammatory responses (Daynes et al. 2002). PPARs regulate gene expression by binding, as heterodimers, with retinoid X receptors (RXRs) to specific PPAR response elements (PPRE) in the promoter regions of specific target genes. In the absence of a ligand, high-affinity complexes are formed between the PPAR–RXR heterodimer and nuclear receptor corepressor proteins, preventing transcriptional activation by sequestration of the nuclear receptor heterodimer from the promoter. Binding of a ligand to the heterodimer results in the release of the corepressor from the complex, which in turn results in the binding of the activated heterodimer to the response element in the promoter region ofthe relevant target genes, resulting in either the activation or suppression of a specific gene (Evans et al. 1988, Moras et al. 1998). 2.

(14) Three different isotypes of PPARs family have been identified: PPAR-α, PPAR-β/δ, and PPAR-γ. PPAR-α and PPAR-γ share potent anti-inflammatory properties (Delerive et al. 2001). They act as anti-inflammatory molecules by repressing the activity of transcription factors , such as NF-κB, signal transducer and activator of transcription (STAT), nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1) (Shenyang et al. 2005).. PPAR-α is highly expressed in liver, heart, renal proximal tubular cells, skeletal muscle, intestinal mucosa, and brown adipose, tissues that are considered metabolically very active (Beck et al. 1992). More recently, studies have revealed that PPAR-α ligands exert anti-inflammatory actions (Delerive et al. 2001). The mechanisms involve protein-protein interactions between PPAR-α and the p65 subunits of NF-κB, and an induction of the inhibitory protein IκBα that retains NF-κB in a cytoplasmic inactive complex. (Delerive et al. 1999 ; Delerive et al. 2000) Furthermore, the Wy-14643, a potent PPAR-a ligand, had been proved to reduce the gut I/R injury (Carmelo et al. 2006). Among the endogenous ligands for PPAR-α, prostaglandins (PGs) are extensively studied recently.. PGs are a diverse family of oxygenated fatty acids derived from arachidonic acid (AA). AA is converted to an intermediate, prostaglandin endoperoxide H 2 (PGH 2 ) by two isoforms of cyclooxygenase (COX), COX-1 and COX-2. The COX product PGH 2 is converted to one of several 3.

(15) biologically important prostanoids, including PGE 2 , PGD 2 , PGF 2α , thromboxane A 2 and prostacyclin (PGI 2 ) by specific synthases (Toshihisa et al. 2001). PGs have wide-ranging effects in regulating aspects of homeostasis and pathogenesis.. PGs function through cell surface G protein-coupled receptors linked to different cytoplasmic signaling pathways (Toshihisa et al. 2001) There have five major cell membrane spanning G-protein coupled receptors been defined pharmacologically. These correspond to each of the COX metabolites; DP for PGD 2 , EP for PGE 2 , FP for PGF 2α , IP for PGI 2 , and TP for thromboxane A 2 . (Timothy et al. 2004).. PGI 2 is synthesized by endothelial cells, macrophages, lung, and the kidneys. It is a potent vasodilator and an inhibitor of platelet aggregation that spontaneously hydrolyzes to form the stable but inactive metabolite 6-keto-PGF 1α (reviewed by James A.C. 2005). Nowadays, it is quite evident that not only does it play a key role in the vasculature but it also contributes to the maintenance of homeostatic functions of many organ systems and the pathogenesis of certain diseases. PGI 2 can potentially influence numerous pathomechanisms implicated in various aspects of renal disease, including renal hemodynamic changes, changes in GFR, oxidative stress, inflammatory processes, etc. (Rania et al. 2005). Despite the abundance of IP receptors in the kidney, the mild renal phenotype in the IP knockout compared with the prostacyclin synthase 4.

(16) (PGIS)-deficient mice (Yokoyama et al. 2002) suggests that PGI 2 interactions in the kidney are a great deal more elaborate and PGI 2 acting on IP-independent pathways, like activation of PPARs or other signaling systems, may play a more dominant role in renal PGI 2 biology. (Rania et al. 2005). Research Objective (1) : Recent studies have shown that PPAR-α contributes to the resolution of inflammation after renal I/R injury using extant ligands (Cha et al. 2007, Patel et al. 2009). PPAR-mediated modulation of gene transcription by PGI 2 may indicate a novel role for PPARs in the regulation of gene expression (Hertz et al. 1996). A stable analog of PGI 2 has been shown to activate PPARs (Hatae et al. 2001), but little is known about the relationship between PGI 2 and PPAR-α in the kidney. Thus we hope to characterize the effects of PGI 2 and PPAR-α in a renal I/R injury model and to investigate the influence of PGI 2 on PPAR-α induction and TNF-α-induced cell apoptosis following I/R injury.. 5.

(17) MATERIALS AND METHODS (1) Animal models Male C57/B6, H129 wild-type, and H129 PPARα–knockout (PPARα–/–) mice (aged 8–10 wk; 18–22 g) received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources (National Institutes of Health publication No. 86-23, revised 1985). The study protocol was approved by by the Institutional Animal Care and Utilization Committee, Academia Sinica.. Mice were anesthetized with intraperitoneal pentobarbital (50 mg/kg) and placed on a heating pad to maintain the core body temperature at 37°C. Both kidneys were exposed through the flank. The renal pedicles were clamped with vascular clamps (Roboz Surgical Instrument, Gaithersburg, MD) for 45 min and reperfused for 24 h (Lin et al. 2008). Animals received single intraperitoneal doses of vehicle (saline) or docosahexaenoic acid (DHA; 500 mg/kg body wt) 3 days before I/R surgery. The dose of DHA was modified from a previous study (Nguyen et al. 1999). Sham control animals underwent the same surgical procedures except for intraperitoneal saline injection.. Measurement of biochemical parameters Blood samples (500 μl) were collected via the tail vein. Samples were centrifuged (6,000 g, 3 min) to separate the serum from the cells. 6.

(18) Biochemical parameters were measured within 24 h of blood sampling.. Histopathologic preparations and immunocytochemistry Kidneys from all of the treated groups were fixed in 10% buffered formalin overnight at 4C and processed in paraffin wax. Sections (5-μm thickness) were stained with hematoxylin and eosin.. NRK-52E cells transfected with Flag-PPAR-α plasmid or infected with Adv-COX-1/PGIS were plated on 35-mm poly-L-lysine-coated glass-bottomed dishes (Matsunami Glass) and fixed for 20 min at room temperature with 4% paraformaldehyde and 0.4% Triton X-100 in PBS. The cells were incubated with an anti-FLAG antibody (rabbit polyclonal, Sigma) in PBST (PBS with 0.05% Tween 20) containing 2% horse serum. After three washes with PBS, the cells were incubated with Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) and Cy3-conjugated anti-mouse IgG (Amersham Biosciences) in PBST containing 2% horse serum for 1 h at room temperature. Fluorescent images were captured and analyzed with a μRadianceTM Laser Scanning Confocal Microscope System (Bio-Rad).. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) Mice were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Organs were dissected and postfixed overnight with 4% paraformaldehyde, embedded with paraffin, and sectioned in 5-µm 7.

(19) thickness. TUNEL assay followed the manufacturer’s protocol (Roche, Mannheim, Germany).. Replication-defective recombinant adenoviral vectors In replication-defective recombinant adenoviral (rAd) vectors, we constructed the following: a human phosphoglycerate kinase (PGK) promoter to drive COX-1 expression (Adv-PGK-COX-1 or Adv-COX-1); two separate PGK promoters (bicistronic) to drive COX-1 and PGIS, respectively (Adv-PGKCOX-1/PGIS or Adv-COX-1/PGIS); and PGK alone to serve as a control (Adv-PGK).. Replication-defective rAd vectors were generated by homologous recombination and amplified in 293 cells as described previously (Lin et al. 2002). rAd stocks were prepared by CsCl gradient centrifugation, aliquoted, and stored at -80°C. Viral titers were determined by a plaque-assay method. The 293 cells were infected with serially diluted viral preparations and then overlaid with low-melting-point agarose. The numbers of plaques that formed were counted within 2 wk.. Cell culture, infections, and transfections Renal tubule cells (NRK-52E) derived from rat kidney were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO 2 . NRK-52E cells were either infected or transfected on 8.

(20) the following day.. Infections were performed using a multiplicity of infection of adenovirus by adding the appropriate recombinant adenovirus, adv- COX-1/PGIS or Ade-HPGK, to the culture media for 2 days, followed by hypoxia for 24 h and reoxygenation for another 24 h.. Transfection was performed using lipofectamine with 1.5 μg of luciferase reporter plasmid and varying amounts of PPAR-α, PPAR-α short interference (si) RNA (5’-CCC TTA TCT GAA AA TTC TTA-3’; TRCN 0000025967, National RNAi Core Facility), and Flag PPAR-α expression vectors/6-cm2 culture dish. The amount of DNA was kept constant using an empty expression vector. Cells were harvested and assayed 16–24 h later.. Cytoplasmic and nuclear protein extraction For cytoplasmic and nuclear protein extraction, cell and kidney tissue proteins were extracted as nuclear and cytoplasmic fractions according to the manufacturer’s protocol (NE-PER Nuclear and Cytoplasmic Extraction Reagents, Pierce Chemical, Rockford, IL).. RNA extraction and RT-PCR analysis Total RNA was extracted from NRK-52E cells using the TRIzol method according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Total RNA extract was treated with 1 U of RQ1 RNase-free DNase (Promega) 9.

(21) per microgram of total RNA at 37°C for 1 h. Reverse transcription (RT) 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 (Invitrogen 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 Taq DNA Polymerase (Invitrogen). To control for the generation of PCR products due to residual contamination of genomic DNA, an aliquot of RNA without RT treatment was tested in parallel. PCR was performed for 30 cycles (94°C for 30 s, 57°C for 30 s, and 72°C for 30 s), and the products were visualized on 2% agarose gels by ethidium bromide staining. The PPAR-α primers were anti-sense 5’-CCA CCA TCG CGA CCA GAT-3’ and sense 5’-GAC GTG CTT CCT GCT TCA TAG A-3’. Each sample was run in triplicate and normalized to the level of GAPDH as a “housekeeping” gene.. Western blotting Western blotting was performed as previously described (Lin et al. 2002). Blots were incubated with antibodies against PPAR-α(1:500; Santa Cruz Biotechnology), caspase-3 (1:1,000; Cell Signaling, Beverly, MA), casapse-8 (1:5,000; Cell Signaling), NF-κB (p65, 1:500; Santa Cruz Biotechnology), I-κB (1:500; Santa Cruz Biotechnology), and COX-1 and PGIS (1:1,000; Cayman Chemical).. Nuclear extracts and electrophoretic mobility shift assays (EMSAs) 10.

(22) NF-κB DNA probes containing a consensus NF-κB enhancer element (5’-AGT TGA GGG GAC TTT CCC AGG C-3’) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For the PPRE primer, we designed a PPARα DNA-binding sequence (5’-GAG TTG TAA GGT CAT GGG AAA-3’) for use in electrophoretic mobility shift assays.. EMSA analysis of nuclear NF-κB was performed as described previously (Lin et al. 2008). After nuclear extracts were prepared, binding reactions were performed in 20-μl reaction mixtures with 3 μg of nuclear extracts in a buffer containing 12 mM HEPES (pH 7.9), 5 mM MgCl 2 , 60 mM KCl, 4 mM Tris-HCl (pH 7.9), 0.6 mM ethylenediaminetetraacetic acid (EDTA), 0.6 mM dithiothreitol (DTT), 0.5 mg/ml bovine serum albumin (BSA), 1 μg of poly[d(I-C)], 12% glycerol, and 20,000 dpm of radio-labeled double-stranded -120 bp ANF GATA probe or -21bp PPRE probe for 20 min at room temperature. Reactions were loaded on a 4% polyacrylamide gel and run at 200 V at room temperature in 0.25× Tris/Borate/EDTA buffer. The gel was dried and exposed to a PhosphorImager screen (Molecular Dynamics).. Immunoprecipitation Control and transfected cells were lysed at 4°C in lysis buffer [50 mM Tris, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, and protease inhibitors, Complete, Miniprotease inhibitor cocktail tablet (Roche)]. PPAR-α-associated proteins were collected with an 11.

(23) immunoprecipitation kit (Roche Diagnostics) that included anti-PPAR-α antibodies and protein Gagarose. The precipitates were subjected to Western blotting and hybridization with NF-κB (p65).. Eicosanoid measurement Following the overexpression of PGI 2 in NRK-52E cells using an adenovirus that contained the COX-1/PGIS plasmid, we measured eicosanoids in supernatants using immunoassay kits for PGE 2 , 6-keto-PGF1α, Thromboxin X 2 (R&D Systems, Minneapolis, MN), and PGD 2 (Cayman Chemical, Ann Arbor, MI). All procedures followed manufacturers’ protocols.. TNF-α measurement Culture medium was analyzed for mouse TNF-α by an ELISA kit according to the manufacturer’s protocol (RayBiotech, Norcross, GA).. TNF-α promoter assay A TNF-α reporter gene construct was created by cloning the human TNF-α promoter (-2,000 to +17) into the pGL2-basic promoterless plasmid vector from Promega (Madison,WI) and transfected into NRK-52E cells. Promoter luciferase activity was measured as previously described (Turner et al. 2007).. Statistical analysis 12.

(24) Continuous variables were compared by one-way analysis of variance (ANOVA). When there was a significant difference between groups, multiple comparisons of means were performed with the Bonferroni procedure and type-I error adjustment. All statistical assessments were two-sided and evaluated at the 0.05 level of significance. Statistical analyses were performed with SPSS 15.0 statistics software (SPSS Inc., Chicago, IL). Survival analysis was performed according to the Kaplan-Meier method, and between-group differences in survival were tested by log-rank test.. 13.

(25) RESULTS (1) Effects of PPAR-α on H/R-induced apoptosis. We first studied the effects of PPAR-α on hypoxia-reoxygenation (H/R) cells by constructing PPAR-α-Flag cDNA and then transfecting NRK-52E cells to induce this protein’s overexpression. Compared with controls or vector transformants, the level of PPAR-α was significantly elevated in cells with the PPAR-α construct (far right lane, Fig. 1A). During H/R treatment, cleaved caspase-3 and -8, which are important signals for TNF-α-induced apoptosis, were both significantly reduced by overexpression of PPAR-α (far right lane; Fig. 1B). Next, we employed gene knockdown using short interference RNA (siRNA) to evaluate any protective effects of PPAR-α on H/R-induced apoptosis. PPAR-α siRNA specifically and dose-dependently reduced the expression of PPAR-α in transfected NRK-52E cells (Fig. 1, C and D). After H/R, PPAR-α-transfected NRK-52E cells expressed less caspase-3 and -8 (lane 2 vs. lane 4, Fig. 1E). These effects were abolished by cotransfection with PPAR-α siRNA (lane 4 vs. lane 5, Fig. 1E).. Effects of PPAR-α on TNF-α expression. Previous studies have shown that following H/R, renal tubule cells are injured by TNF-α and that this causes an overt inflammatory reaction (Zager et al. 2005). Our results showed that H/R elevated TNF-α promoter activity and the concentration of TNF-α in NRK-52E cells and that these effects were only partially attenuated by infection with PPAR-α (Fig. 2, A and B). As these results were only partially due to the effects of PPAR-α, 14.

(26) there may be other I/R-induced TNF-α activation pathways involved (Minutoli et al. 2009). Coinfection with PPAR-α siRNA reversed the PPAR-α effect (Fig. 2B).. Effects of PPAR-α on the NF-κB-induced TNF-α pathway. TNF-α genes contain functional NF-κB binding sites that are essential for their induction following I/R injury (Nakao et al. 2002, Reiner et al. 1994). Thus we assessed NF-κB activation using EMSAs. H/R increased the NF-κB DNA binding activity in the nuclear extracts of renal tubular cells, but this was markedly suppressed in PPAR-α-transfected cells (Fig. 2C). We verified the identity of the gel shift band by competition analysis using unlabeled DNA fragments (lanes 25x and 50x, Fig. 2C). In H/R-treated PPAR-α-transfected NRK-52E cells, the level of nucleic NF-κB was reduced and cytosolic IκB, a protein that prevents translocation of NF-κB dimers into the nucleus, was elevated (top, far right lane, Fig. 2D). Taken together, these results suggest that PPAR-α plays a protective role in modulating H/R injury by inhibiting TNF-α expression via inactivation of NF-κB.. Effects of DHA on renal function and apoptosis. DHA, a long-chain fatty acid, is an activator of PPAR-α (Muia`et al. 2006). I/Rinjured mice showed increased serum levels of urea and creatinine compared with sham-operated mice (Fig. 3A). Treatment with DHA increased intracellular PPAR-α expression and lowered serum urea and creatinine levels compared with non-DHA-treated I/R mice (Fig. 3A),. 15.

(27) suggesting a marked reduction of renal I/R injury. Apoptosis has been implicated in the pathogenesis of renal I/R injury (Matthijsen et al. 2007, Terada et al. 2007). We used a TUNEL assay to evaluate any role for PPAR-α in renal tubular cell apoptosis following renal I/R. TUNEL-positive cells were virtually undetectable in the kidneys of sham-operated mice and DHA-treated mice without I/R (Fig. 3B). Following I/R, we observed large numbers of TUNEL-positive cells in untreated mice (primarily renal tubular cells) but significantly fewer TUNEL-positive cells in DHA-treated mice (Fig. 3B).. Caspases, a family of cysteine proteases involved in apoptosis, necrosis, and inflammation, are induced during renal I/R (Li et al. 2007). TNF-α has been implicated in TNF-α receptor-induced caspase-8 and -3 pathways in I/R-induced apoptosis (Qiao et al. 2005). Thus we examined the activations of caspase-8 and -3 in the kidneys of untreated and DHA-treated mice. Consistent with the results of our TUNEL assay, DHA-treated mice under I/R showed significantly less caspase-8 and -3 activation than untreated mice (Fig. 3C). These results suggest that PPAR-α is involved with renal tubular cell apoptosis during I/R injury.. Effects of IR on renal function in wild-type and PPARα-/- mice. We subjected wild-type (PPAR-α+/+) and mutant (PPAR-α-/-) mice to I/R and compared their renal functions and the levels of cleaved caspase-3 and -8. After I/R, PPAR-α-/- mice had increased levels of serum urea and creatinine, TUNEL positive cells, and expressions of caspase-3 and -8 (Fig.. 16.

(28) 4, A–C). We also assessed NF-κB activation by EMSA. I/R increased the NF-κB DNA binding activity in the renal nuclear extracts of WT mice, and this was further increased in the PPAR-α-/- mice (Fig.4D). These results suggest that PPAR-α has a protective role in renal I/R by inhibiting NF-κB binding.. Effects of PGI 2 on PPAR-α translocation. Prostaglandin derived products, such as PGI 2 , are ligands for PPARs (Bernardo et al. 2000). It is not known whether PGI 2 plays any physiological role in mice with I/R injury, although PGI 2 can serve as a ligand for the translocation of PPAR-α. We selectively overexpressed PGI 2 in NRK-52E cells using an adenovirus that contained COX-1/PGIS (Adv-COX-1/PGIS; Fig. 5, A and B). The levels of cytosolic PPAR-α were similar in HPGK- and COX-1/PGISinfected cells (Fig. 5C, top), but the nuclear level of PPAR-α was significantly elevated only in COX-1/PGIS-infected cells (lanes 5 and 7, Fig. 5C, bottom). In addition, neutralization of PGI 2 by a COX-1-specific inhibitor, SC560, attenuated the translocation of PPAR-α from the cytosol into the nucleus (Fig. 5D). Immunostaining also showed that Flag-PPAR-α was translocated from the cytosol into the nucleus after infection with Adv-COX-1/PGIS (Fig. 5E, bottom left). However, this phenomenon was significantly diminished after treatment with SC560 (Fig. 5E, bottom right). These results indicate that PGI 2 may play an important role in the translocation of PPAR-α into the nucleus.. 17.

(29) Effects of PGI2 on PPAR-α and NF-κB. The physical interaction between PPAR-α and the p65 subunit of NF-κB interferes with the activity of both transcription factors (Delerive P et al. 1999 and 2000). Thus we investigated whether PGI 2 and PPAR-α could inhibit H/Rinduced apoptosis via the NF-κB pathway. PPAR-α-transfected NRK-52E cells were immunoprecipitated with a PPAR-α antibody and then immunoblotted with an anti-p65 antibody. Our results (Fig. 6A) showed that after H/R, the levels of PPAR-α that coimmunoprecipitated with p65 were similar in control (lane 6) and HPGK cells (lane 7). However, either treatment with PPAR-α (lane 9) or Adv-COX-1/PGIS transfection (lane 8) significantly increased the level of the immunoprecipated protein. Simultaneous treatment with PPAR-α and transfection with Adv-COX/PGIS resulted in the highest level of immunoprecipitated protein (lane 10). PGI 2 is known to attenuate adriamycin-induced apoptosis (Vane et al. 1995). Thus we determined the levels of the cleaved form of caspase-3 in NRK52E cells following H/R (Fig. 6B). Following H/R, the level of caspase-3 was lower in cells receiving both treatment with PPAR-α and transfection with COX/PGIS (lane 5) than for cells receiving either of these treatments alone (lanes 3 and 4). Furthermore, gene knockdown with siRNA (lane 6) increased the level of caspase-3.. Effects of PGI 2 on TNF-α. A recent study showed that treatment with a PGI 2 analog, beraprost, reduced radio contrast- induced hypoxia injury via a PKA-dependent 18.

(30) CREB phosphorylation pathway (Yano et al. 2004). Thus we examined the signal transduction pathways for PGI 2 inhibition of TNF-α production following H/R. H/R-treated NRK-52E cells released large amounts of TNF-α into cell lysates (lane 2, Fig. 6C). However, Adv-COX-1/PGIS and PPAR-α transfections, both separately and synergistically, reduced the levels of TNF-α (lanes 3–5, Fig. 6C); these effects were reversed by PPAR-α siRNA (lane 6, Fig. 6C).. In addition, TNF-α promoter activity was attenuated in NRK-52E cells that were transfected either separately or synergistically with PPAR-α and/or Adv-COX-1/PGIS (lanes 6 and 7 vs. lanes 8–10, Fig. 7). These Adv-COX-1/PGIS or PPAR-α transfection effects were only partially reversed when cells were given CAY10441, a cAMP inhibitor (lanes 8–10 vs. lanes 13–15, Fig. 7) and an IP receptor antibody (lanes 8–10 vs. lanes 18–20, Fig. 7). Clearly, PPAR-α and cotransfection with Adv-COX-1/PGIS results in greater downregulation of TNF-α promoter activity than either of these alone, and these effects were only partially abolished by a cAMP inhibitor and an IP receptor antibody.. 19.

(31) DISCUSSION (1) Renal I/R injury is a clinically significant problem that can lead to ARF (Padanilam B.J. 2003). Recent studies of renal I/R injury have focused on the roles of neutrophils, inflammatory cytokines, the tissue factor thromboplastin, intercellular adhesion molecule-1, oxygen free radicals, vascular plugging, edema, and other mechanisms (Brown et al. 2003). TNF-α has been implicated in the pathogenesis of many inflammatory diseases of the kidney, including glomerulonephritis, septic acute renal failure, and renal I/R injury (Donnahoo et al. 1999). TNF-α binds to its membrane-bound receptor TNFR1, which activates the Fas-associated death domain (FADD), caspase-8, caspase-3, and ultimately causes cell death (Donnahoo et al. 1999).. All PPARs can regulate gene expression by forming heterodimers with the retinoid X receptor (RXR), which then binds to PPREs in the promoter regions of target genes (Cabrero et al. 2002). RXR also forms heterodimers with vitamin D, thyroid hormone, and other receptors (Ziouzenkova et al. 2008). PPARs are also known to bind free fatty acids and eicosanoids, including PGI 2 (Delerive et al. 2001).. In addition to regulating transcription, several studies have shown that PPARs interfere with inflammatory signaling pathways by interacting with either the activator protein (AP-1) complex (including c-fos, c-jun, and NF-κB) or with signal transduction and activation of transcription proteins (STATs) (Cabrero et al. 2002, Delerive et al. 1999, Delerive et al. 2000). A 20.

(32) study using the human colorectal carcinoma cell line SW620 showed that PPAR-α ligands inhibited tumor promoter PMA-mediated induction of genes associated with inflammation and tumor growth, including COX-2 and vascular endothelial growth factor (VEGF) (Grau et al. 2006). PPAR-α activators also reduced the transcriptional induction of COX-2 and VEGF by inhibiting AP-1-mediated transcriptional activation induced by PMA or by the overexpression of c-Jun (Grau et al. 2006).. PGI 2 is derived from -6 arachidonic acid and is rapidly broken down in the endothelium to 6-keto-PGF 1α , which has weaker vasodilator effects (Nasrallah et al. 2007). There is evidence that the interaction of PGI 2 with thromboxane is involved in cardiovascular homeostasis that effectively reduces vascular damage (Nasrallah et al. 200).. In this study, we demonstrated that increased PPAR-α expression can inactivate the NF-κB-dependent TNF-α gene and protect against renal I/R (H/R) injury both in vivo and in vitro. We also showed that PGI 2 will translocate PPAR-α from the cytosol to the nucleus where it binds with NF-κB to inhibit TNF-α activation. However, this effect could not be completely reversed by either a cAMP inhibitor or an IP receptor antibody. It means that there had an intracellular PGI 2 signaling pathway, which might translocate the PPAR-α to nucleus and exert its effects.. Taken together, our findings support the hypothesis that PGI 2 has potent antiapoptotic effects during renal I/R injury and that the PPAR-α and 21.

(33) IP/cAMP receptor pathways both mediate these effects.. Previous studies showed that renal I/R downregulated PPAR-α and that a PPAR-α agonist (clofibrate) was the most important factor for reducing renal I/R injury damage by PPAR-α (Patel et al. 2009, Sivarajah et al. 2002). Our results also indicate that PPAR-α inhibits H/R induced caspase-8-dependent apoptosis (Fig. 1).. The TNF-α induced inflammatory cascade for renal injury involves endothelial, epithelial, and infiltrating inflammatory cells (Sheridan et al. 2000). The infiltrating cells include macrophages and cytotoxic T cells, which increase the levels of E-selectin, TNF-α, and IFN-γ, resulting in renal dysfunction and apoptosis (Kurcer et al. 2007, Sandovici et al. 2007). TNF-α production is triggered by locally produced ROS, as well as by I/R injury, which activate transcription factor NF-κB and cause renal dysfunction (Kurcer et al. 2007).. Our results are in agreement with recent studies that showed that H/R-induced TNF-α activation in cultured renal epithelial cells is mediated by NF-κB and that inh ibition of PPAR-α by siRNA exacerbated H/R injury by enhancing NF-κB-mediated TNF-α induction (Fig. 2). In addition, an omega-3 fatty acid, DHA, which is a PPAR-α activator, can also inhibit leukocyte adhesion to cytokine-stimulated human umbilical vein endothelial cells (Sethi et al. 2002). Accordingly, we showed that renal. 22.

(34) dysfunction was markedly ameliorated in DHA treated mice in response to I/R injury (Fig. 3).. However, both TNF-α activation and cell apoptosis were only partially reduced when cells overexpressed PPAR-α or when mice were injected with DHA. The reason for these partial effects may be that there are multiple pathways involved in I/R-induced kidney injury via NF-κB-induced TNF-α activation (Frangogiannis et al. 2007). Thus overexpression of PPAR-α by a plasmid or DHA treatment can block only a part the of NF-κB-induced inflammatory pathway. In addition, the partial protective effects by PPAR-α in renal I/R injury may arise, as most tissues in humans and rodents express three PPAR subtypes: α, β/δ, and γ (Smith et al. 2000). Each member of this subfamily of nuclear transcription factors has antiatherogenic and anti-inflammatory functions (Di Paola et al. 2007). Experiments using PPAR-α, β/δ, γ-null mice as a model showed more severe cortical necrosis and renal dysfunction after I/R injury (Di Paola et al. 2007).. Although PPAR-α and PPAR-α gonists have shown therapeutic effects against I/R injury (Akahori et al. 2007, Inan et al. 2007), the effects of endogenous PPAR-α on conditions associated with I/R injury have not been investigated to date. A recent study of NRK-52E cells examined gentamicin-induced apoptosis, in which PGI 2 production was increased by infecting cells with an adenovirus carrying COX-1 and PGIS (Hsu et al. 2008). These authors concluded that elevated production of endogenous. 23.

(35) PGI 2 protected renal tubule cells from apoptosis by a PPAR-α-associated pathway (Hsu et al. 2008). Our study also provides strong evidence that renal I/R protection provided by PPAR-α is mediated by PGI 2 and that PGI 2 enhances PPAR-α translocation from the cytosol into the nucleus (Fig. 5).. PGI 2 is known to regulate growth, fibrosis, and apoptotic responses via the IP and PPAR pathways (Li et al. 2004). Hatae et al. (Hatae et al. 2001) demonstrated that activation of the PGI 2 /PPAR-α pathway was associated with apoptosis, whereas the PGI 2 /IP/cAMP pathway had antiapoptotic effects in human embryonic kidney (HEK- 293) cells. However, our study indicates that not only the PGI 2 /IP/cAMP pathway but also the PGI 2 /PPAR-α pathway has antiapoptotic effects in the kidney during I/R injury. This different results may explained by the effects of PGI 2 /PPAR-α pathway are cell dependent. Moreover, we known that PPAR-α is highly expressed in rodents and human kidneys, however, the roles and expression of PPAR-α in the immature kidney or in the embryonic kidney cells are still undefined. Thus, the renal tubular cells we tested should be more physiologically reasonable.. In the presence of endogenous COX-1/PGIS and PPAR-α in NRK-52E cells, transfection of PPAR-α or Adv COS-1/PGIS individually could partially reduce TNF-α activity. The presence of endogenous PPAR-α and the antiapoptotic PGI 2 /PPAR-α pathway also explains why a cAMP inhibitor or an IP receptor antibody could not completely reverse the 24.

(36) protective effects of COX-1/PGIS.. Furthermore, we demonstrated that the antiapoptotic PGI 2 /PPAR-α pathway is mediated by PGI 2 by enhancing PPAR-α translocation from the cytosol into the nucleus, which then inhibits NF-κB-induced TNF-α activation. However, it is not known whether there is a PGI 2 -PPAR-α complex that binds with a corepressor or another transcription factor that can protect against renal I/R. Also, it is not known whether another PPAR subfamily member might influence these results. There need more works to test the exist of direct protein-protein interaction between PGI 2 and PPAR-α.. In summary, our results indicate that PGI 2 -induced PPAR-α translocation plays an important role in protecting the kidney against I/R injury, in addition to its traditional IP/cAMP signaling pathway. Pharmacological modulation of renal PGI 2 and PPAR-α induction or its signal transduction may be a viable strategy for improving renal functional outcomes after I/R injury.. However, for PGI 2 is very labile and is rapidly metabolized into a nearly inactive product, 6-keto-PGF 1α . This rapid inactivation of PGI 2 has been a major limitation in the use of PGI 2 clinically. Several pharmacological analogs are now available, including cicaprost (CCP), which is a highly selective IP agonist, iloprost (ILP), which is less selective owing to its potency for the EP receptors, and beraprost, which is improved stability 25.

(37) (Wise et al. 1996, Olschewski et al. 2004, Clapp et al. 2002). However, them don’t have the intracellular effect as PGI 2 . Instead, use of PGI 2 activator or stimulator may resolve our problems. It has been report that prostacyclin can be induced by statins and mediate its cardioprotective effects(Yochai et al. 2005). The cardioprotective effects of atorvastatin are mediated by increased production of PGs achieved by upregulation of cPLA 2 , COX-2, PGI 2 synthase and 6-keto-PGF 1a (Birnbaum et al. 2005). This attract our interest to use the statin as a PGI 2 activator to evaluate the implication of PGI 2 /PPAR-α pathway to renal injury other than I/R.. Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. The resulting lower intracellular cholesterol concentration after statin treatment leads to a proteolytic activation of the transcription factor sterol responsive element-binding protein-2 (SREBP-2), which upregulates several genes controlling cholesterol homeostasis, including the LDL receptor (LDLr) (Brown et al. 1999).. A large number of clinical trials has shown beneficial effects of statins in the primary and secondary prevention of cardiovascular disease. Although it is generally assumed that these beneficial effects are directly related to the decrease of LDL-c, certain benefits of statin therapy may occur earlier and possibly to a larger extent than what might be expected from changes.

(38) in plasma LDL-c levels alone. These observations have led to the suggestion of effects beyond LDL-clowering, collectively termed “pleiotropic” effects. (Re´jane et al. 2007). The so-called pleiotropic effects of statins are though to be because of the inhibition of the parallel pathway of biosynthesis of isoprenoids, which constitute lipid attachments allowing membrane anchoring and activation of intracellular signaling molecules, such as the small GTP-binding proteins Rho, Ras, and Rac. These proteins then can activate various downstream signaling pathways including mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinases (ERKs) and p38-MAPKs, c-jun N-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3K) (Re´jane et al. 2007). Statins also exert antiinflammatory properties by inhibiting transcription factors pathways, such as nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) through the regulation of Heme oxygenase (HO)-1 ( Fiona et al. 2005).. HO is the key enzyme responsible for the degradation of heme to biliverdin, carbon monoxide and free iron. In mammals, biliverdin is further converted to bilirubin, an endogenous radical scavenger, through the action of biliverdin reductase. The release of free iron is rapidly sequestered into the iron storage protein ferritin. (reviewed by Sikorski et al. 2004).. 26.

(39) To date, there are three HO isoforms identified: HO-1, HO-2, and HO-3 (88-90). HO-1 is a 32 kDa protein which is induced by various stimuli such as cytokine, heavy metals, oxidants, and heme. (Danielle et al. 2005). There are many HO-1 inducers including ultraviolet A radiation; hydrogen peroxide; cytokines (interleukin [IL]-1, IL-6, IL-10, tumor necrosis factor, interferon-γ); endotoxin; growth factors (platelet-derived growth factor, transforming growth factor-β); oxidized lipids; hyperoxia; NO; NO donors; prostacyclin; PPAR-α and γ; angiotensin II; and glucose deprivation (reviewed by Ferrándiz and Devesa, 2008). Several of these stimuli play important roles in the pathophysiology of acute renal failure (Nath et al. 2001). There are many regulatory elements in the 5’ flanking region of human or mouse HO-1 gene, including cyclic AMP responsive element-binding site (CREB) (Danielle et al. 2005).. It has been shown that when animals are pretreated with HO-1 inducers, the damage and the acute inflammatory responses in various tissues as in oxidative stress- induced lung injury and endothoxin shock are markedly attenuated. Chronic renovascular hypertension and acute renal failure are exacerbated in HO-1 knockout mice. These findings provide evidance to support the cytoprotective role of HO-1. (Danielle et al. 2005, Lee et al. 2004). In the kidney, HO-1 has been shown to be upregulated and to ameliorate the severity of renal damage in a variety of kidney injury models, such as 27.

(40) kidney transplant rejection (Cuturi et al. 1999), and to inhibit the effects of nephrotoxins induced by cisplatin and cyclosporine (Agarwal et al. 1995; Li et al. 2004).. 28.

(41) Research Objective (2) Statins had been proved to protect tissues from various types of insult such as I/R injury in the kidney and heart (Gueler et al. 2007, Birnbaum et al. 2005, Cheng et al. 2008). Some studies have demonstrated that statins lead to activation of the promoter for the antioxidant defense protein HO-1, along with its transcription and accumulation (Lee et al. 2004), which may help to explain the pleiotropic antioxidant and anti-inflammatory actions of statins (Grosser et al. 2004).. Besides, HO-1 has been shown to be upregulated and to ameliorate the severity of renal damage in a variety of kidney injury models, such as kidney transplant rejection (Cuturi et al. 1999), and to inhibit the effects of nephrotoxins induced by cisplatin and cyclosporine (Agarwal et al. 1995). Li and colleagues also reported that pretreatment with Wy-14643, a ligand for PPARα, ameliorates cisplatin induced acute renal failure. (Li et al. 2004). However, whether statins can protect against carboplatin-induced renal injury via cholesterol-independent pathways is not clear. The effects of statins on PPARα are not completely defined, although another lipid-lowering agent, clofibrate, has been shown to be a ligand for PPARα (Forman et al. 1997). We hypothesized that the protective effects of statins against carboplatin-induced renal injury may be mediated by PGI 2 , PPARα and HO-1. To test this hypothesis, we investigated the effects of pravastatin on PPARα and HO-1 in a rat renal epithelial cell line and in mice models. 29.

(42) MATERIALS AND METHODS (2) Animal model of Carboplatin nephrotoxicity Mice were administered an intraperitoneal (i.p.) injection of a single dose of carboplatin (100 mg/kg) (Sigma-Aldrich), with (experimental group received) or without (control group) i.p. injection with pravastatin (5 mg/kg) (Sigma-Aldrich) 1 day before carboplatin treatment while the control group was treated with the same volume of normal saline (i.p.) instead of pravastatin.. Chromatin immunoprecipitation assay (ChIP) NRK-52E cells were transfected with FLAG-PPARα plasmid 1 day after treatment with pravastatin, fixed in 1% formaldehyde, and ChIP assay was performed according to the manufacture’s protocol (Millipore, Billerica, MA). Chromatin was immunoprecipitated with anti-FLAG antibody (Sigma-Aldrich) and anti-acetyl H4 antibody (Millipore). Purified DNA was detected by standard polymerase chain reaction.. HO-1 promoter and PPRE reporter assay The human HO-1-α promoter (positions –800 to +17) and three repeats of the PPRE-binding sequence were each cloned into pGL2-basic promoterless plasmid vectors (Promega, Madison, WI) and transfected into NRK-52E cells. Promoter-induced luciferase activity was measured as described previously (Chen et al., 2009).. Dichlorofluorescein assay for reactive oxygen species 30.

(43) The intracellular formation of ROS was detected with the fluorescent probe 5- (and 6-) chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes). NRK-52E cells (106 cells/ml) were cultured in the presence of 100 μM carboplatin for 24 h or pretreated for 30 min with 50 μM pravastatin and then loaded with 0.1 μg/ml CM-H2DCFDA at 37°C for 30 min in the dark. Cells were then washed twice with Hank balanced salt solution (HBSS) containing calcium and magnesium. Dichlorodihydrofluorescein (DCF) fluorescence was measured immediately with a charge-couple device (CCD) camera (DP72, Olympus, Melville, NY) coupled to a microscope system (BX51, Olympus) at 100× magnification.. Statistical analysis Continuous variables were compared by one-way analysis of variance (ANOVA). When there was a significant difference between groups, multiple comparisons of means were performed with the Bonferroni procedure and type-I error adjustment. All statistical assessments were two-sided and evaluated at the 0.05 level of significance. Statistical analyses were performed with SPSS 15.0 statistics software (SPSS Inc., Chicago, IL). Survival analysis was performed according to the Kaplan-Meier method, and between-group differences in survival were tested by log-rank test.. 31.

(44) RESULTS (2) Carboplatin stimulated apoptosis and caspase activation in vitro and in vivo. To determine if carboplatin-induced nephrotoxicity occurred via a caspase-dependent pathway in vitro, NRK-52E cells were treated with increasing carboplatin concentrations (50 ~ 800 μM), which resulted in a concentration-dependent increase in the level of caspase-3 (Fig. 8A); this increase continued for at least 48 h (Fig. 8B). We explored the effects of carboplatin in vivo by determining the expression of caspase-3 in kidney cells of mice injected with 100 mg/kg carboplatin. Immunoblotting results showed significantly increased staining for caspase-3 in the kidneys of carboplatin-treated mice (2 days after single treatment; Fig. 8C). Apoptosis in kidney cells of mice treated with carboplatin was also significantly increased to 16% compared to that of control mice (2%) (Fig. 8D).. Pravastatin attenuated carboplatin-induced ROS production, renal cell apoptosis, and caspase-3 expression. Exposure of NRK-52E cells to 100 μM carboplatin for 24 h induced production of ROS approximately 25-fold that in saline-treated cells, as shown by DCF staining (Fig. 9A). This increase in the level of ROS was significantly attenuated by pretreatment with 50 μM pravastatin for 30 min. Pravastatin also inhibited carboplatin-induced caspase-3 expression in NRK-52E cells (Fig. 9B).. In vivo experiments showed that injection of mice with pravastatin 32.

(45) reduced not only the rate of apoptosis in kidney cells, from 14% to 3% (Fig. 9C), but also the expression of cleaved, active caspase-3 compared to mice treated with carboplatin alone (Fig. 9D).. Pravastatin improved renal function and survival in mice treated with carboplatin. Carboplatin treatment resulted in abnormal renal function, as revealed by the levels of serum urea nitrogen and creatinine measured 5 days after injection. This effect was improved by pretreatment with pravastatin (Fig. 10B). In accordance with these markers of renal function, control mice and mice treated with pravastatin showed healthy, histologically comparable tubular systems (Fig. 10Ca, b). However, kidneys of mice treated with carboplatin showed substantial histopathologic changes such as tubular necrosis and dilation, protein casts, and loss of brush borders (Fig. 10Cc). Injection with pravastatin in addition to carboplatin produced a marked decrease in these features (Fig. 10Cd).. Survival of mice injected with carboplatin was markedly decreased compared to that of control mice. Treatment with pravastatin in addition to carboplatin significantly increased 7-day survival compared to mice treated with carboplatin alone, as determined by Kaplan-Meier analysis (Fig. 10A).. Pravastatin induction of HO-1 in NRK-52E cells involved cyclooxygenase-2 and a PPAR-α pathway. To test whether the renal protective effect of pravastatin was mediated 33.

(46) by HO-1, NRK-52E cells were treated with 20 μM pravastatin for 24 or 48 h, and the expression of HO-1 was determined by immunoblotting. As shown in Fig. 11A, the expression of HO-1 was increased after treatment with pravastatin for 24 h. However, HO-1 expression decreased when pravastatin treatment was continued for 48 h.. A similar temporal response was observed when we examined the effect of pravastatin on the expression of COX-2. To determine whether overexpression of COX-2 might increase the intracellular level of 15dPGI 2 , a ligand for PPARα, we examined the effect of 20 μM pravastatin on the level of 6-keto PGF 1α (the stable hydrolysis product of PGI 2 ) with an enzyme immunoassay detection system. As observed for the expression of COX-2, pravastatin also induced an increase in 6-keto PGF 1α at 24 h (Fig. 11B). Similarly, pravastatin increased the level of PPAR-α and HO-1 in NRK-52E cells (Fig. 11C).. We next examined whether inhibition of PPAR-α and COX-2 affected the expression of HO-1. When PPAR-α was inhibited by transfection of pravastatin-treated cells with plasmid containing PPAR-α siRNA, the expressions of PPAR-α and HO-1 were reduced (Fig. 11C). When the COX-2 inhibitor NS-398 (Cayman Michigan, USA) was added to pravastatin-treated cells, the increase in HO-1 level was again significantly decreased, and administration of NS-398 and PPAR-α siRNA in combination showed a synergistic reduction in the level of HO-1 (Fig. 11D). 34.

(47) Pravastatin enhanced PPAR-α nuclear translocation in NRK-52E cells. We next examined whether pravastatin enhanced nuclear translocation of PPAR-α. As shown in Fig. 12A, the level of cytosolic PPAR-α was not significantly affected by pravastatin in NRK-52E cells transfected with plasmid containing FLAG-PPAR-α. However, the level of nuclear PPAR-α was significantly increased in pravastatin-treated cells (Fig. 12A lane 3 vs lane 4). Immunostaining also showed that FLAG-PPAR-α translocated from the cytosol to the nucleus in NRK-52E cells treated with pravastatin (Fig. 12Bc). This effect was significantly decreased in cells treated with the COX-2 inhibitor NS-398 (Fig. 12Bd).. Pravastatin-activated HO-1 gene expression is involved in a PPAR-α-dependent pathway in NRK-52E cells. To clarify whether the increase in expression of HO-1 induced by pravastatin was dependent on translocation of PPAR-α, a PPRE primer for electrophoretic mobility shift assay was designed using a PPAR-binding sequence located in the HO-1 promoter (position –621 to –500) from rat DNA. PPAR-α DNA-binding activity was observed in nuclear extracts of pravastatin-treated NRK-52E cells, and this was markedly suppressed in cells pretreated with unlabeled primer (Fig. 13A). To further examine whether the PPAR-α protein was located in the nucleus and associated with PPRE, a ChIP assay was performed with control and pravastatin-treated cells. As shown in Fig. 13B, the association of PPAR-α with the PPRE region of the HO-1 promoter was increased in pravastatin-treated cells. 35.

(48) Finally, to determine the role of pravastatin-induced PPAR-α in activating PPRE and HO-1 promoter activity, NRK-52E cells were cotransfected with increasing amounts of a luciferase expression vector with the PPRE reporter or a 4.5-kb human HO-1 promoter-reporter construct, along with pcDNA3-RXR and pcDNA3-FLAG- PPAR-α or the PPAR-α agonist WY14643(Cayman Michigan, USA). As shown in Fig. 13C, PPAR-α or RXR-α alone slightly increased luciferase activity, but combined transfection with PPAR-α and RXR-α increased luciferase activity by approximately 50-fold in cells with the PPRE reporter and approximately 6-fold in cells with the HO-1 promoter construct. In addition, treatment with pravastatin augmented luciferase activity in both the PPRE and HO-1 promoters compared to cells untreated with pravastatin or transfected with PPAR-α or RXR-α alone. Treatment with WY14643 also increased luciferase activity by approximately 50-fold in cells with the PPRE reporter and approximately 8-fold in cells with the HO-1 promoter. Pravastatin cotreatment further increased luciferase activity in both the PPRE and HO-1 promoters compared to untreated cells.. Pravastatin induced HO-1 expression via a PPAR-α-dependent pathway in mice. We next subjected C57/B6, H129 wild type (PPAR-α+/+), and PPAR-α–/– mice to pravastatin injection. After injection of pravastatin for 2 days, expression of PPAR-α and HO-1 was increased in renal extracts of wild-type C57/B6 (Fig. 14A; mRNA and protein) and wild-type W129 36.

(49) mice (Fig. 14B; protein). However, the increase in HO-1 expression was markedly attenuated in PPAR-α–/– mice (Fig. 14B).. 37.

(50) DISCUSSION (2) It has been report that prostacyclin can be induced by statins and mediate its cardioprotective effects(Yochai et al. 2005). The cardioprotective effects of atorvastatin are mediated by increased production of PGs achieved by upregulation of cPLA 2 , COX-2, PGI 2 synthase and 6-keto-PGF 1a (Birnbaum et al. 2005). This attract our interest to use the statin as a PGI 2 activator to evaluate the implication of PGI 2 /PPAR-α pathway to renal injury other than I/R.. Statins are competitive inhibitors of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, which regulates the synthesis of cholesterol from mevalonic acid (Goldstein et al. 1990). Because mevalonate is the precursor not only of cholesterol but also of many nonsteroidal compounds; inhibition of HMG-CoA reductase by statins may result in pleiotropic effects such as anti-inflammatory and antiarteriosclerotic actions beyond lipid reduction (Faggiotto et al. 1999, Rosenson et al. 1998, Ni et al. 2001). The renoprotective effects of statins have been reported in ischemia-reperfusion injury (Joyce et al. 2001), subtotal renal ablation (Lee et al. 1993), diabetic nephropathy (Kim et al. 2000), and unilateral ureteral obstruction (Moriyama et al. 2001).. In a previous study, we showed that carboplatin-induced cardiotoxicity was characterized by marked increases in apoptosis (3- to 5-fold) and in the levels of caspase-3 and ROS in cardiomyocytes and that these effects could be ameliorated by treatment with pravastatin (Cheng et al. 2008). 38.

(51) Nevertheless, the benefits of statins in a mouse model of cisplatin-induced nephropathy have yet to be reported.. In the present study, we extended our work to include the effects of carboplatin in the kidney. Our findings of increased apoptosis and cleaved caspase-3 in cultured kidney cells and in vivo demonstrated that carboplatin had similar effects to those observed in the cardiac system. In addition, we showed that the levels of ROS also increased in kidney cells exposed to carboplatin, suggesting the involvement of ROS in carboplatin-induced apoptosis. Results of the present study indicate that pretreatment with pravastatin markedly decreased carboplatin-induced renal tissue damage and ameliorated renal dysfunction, consistent with the results of Li et al. (Li et al. 2004), who showed that pravastatin normalized serum creatinine levels in a rat model of chronic cyclosporine-induced nephropathy.. The cardioprotective effects of atorvastatin have been shown to involve the upregulation of COX-2 and increased production n of PGI 2 (Birnbaum et al. 2005). When we examined the effects of pravastatin on the expression of COX-2 and PGI 2 , we found that both were increased in response to pravastatin, suggesting that in kidney cells also, pravastatin stimulates overexpression of COX-2, resulting in increased PGI 2 .. PGI 2 is known to mediate the translation of PPAR-α (Chen et al. 2009). In the previous work, we showed that nuclear PPAR-α was increased in 39.

(52) NRK-52E renal epithelial cells in response to treatment with pravastatin and that this increase resulted from translocation of PPAR-α from the cytoplasm to the nucleus. The observation that translocation was blocked by the addition of the COX-2 inhibitor NS-398 supports the hypothesis that the translocation was mediated by PGI 2 .. Many lines of inquiry have pointed to a prominent cytoprotective role for HO-1 in modulating tissue responses to injury (Sikorski et al. 2004). In a rat model of renal ischemia-reperfusion injury, Gueler et al. (Gueler et al. 2007) showed that pretreatment with cervistatin decreased renal damage and dysfunction after ischemia-reperfusion. These authors found that HO-1 expression was increased after ischemia-reperfusion, that the increased expression was significantly greater in rats pretreated with cervistatin, and that the protective effect of statin was completely abolished by cotreatment with a competitive inhibitor of HO-1. Grosser et al. reported that treatment of endothelial cells with statins resulted in activation of the HO-1 promoter, along with accumulation of HO-1 transcript and protein (Grosser et al. 2004). In a study of vascular smooth muscles cells, Lee et al. reported that simvastatin increased the level of HO-1 and suggested that p38 and the phosphatidylinositol-3-kinase and protein kinase B (PI3K-Akt) pathway might be involved (Lee et al. 2004). Our present findings of significantly increased levels of HO-1 transcript and protein in the kidneys of pravastatin-pretreated mice after carboplatin-induced renal injury are consistent with these previous findings in rats. In addition, we demonstrated that this effect was mediated by PGI 2 and PPAR-α. This was 40.

(53) further corroborated by our in vitro data, in which we showed that pravastatin treatment increased expression of PGI 2 , PPAR-α, and HO-1 in cultured renal tubule cells. The work of Krönke et al. in human vascular cells showed that the HO-1 promoter contains a PPRE and that HO-1 is transcriptionally regulated by PPAR-α (Krönke et al. 2007). We showed PPRE-binding activity in nuclear extracts of pravastatin-treated NRK-52E cells, that PPAR-α was associated with the HO-1 promoter, and that the association was stimulated by pravastatin. Finally we showed that PPAR-α and RXR-α increased expression of a plasmid construct containing the HO-1 promoter and that this expression was stimulated by pravastatin.. Our in vitro results showing inhibition of pravastatin-induced HO-1 activation by NS-398 and PPAR-α siRNA further support the hypothesis that, in kidney cells, activation of HO-1 by pravastatin involves in a PPAR-α-dependent pathway. Our experiments in PPAR-α-knockout mice confirmed that in vivo, as in vitro, the increase in HO-1 expression stimulated by treatment with pravastatin was, to some extent, dependent on a PPAR-α pathway.. Taken together, our data support a relation between pravastatin, PGI 2 , PPAR-α, and HO-1. Pravastatin induces overexpression of COX-2, which upregulates PGI 2 . PGI 2 then promotes the translocation of PPAR-α to the nucleus, where it binds to the PPRE of HO-1 promoter and induces the expression of HO-1. 41.

(54) Because HO-1 plays a cytoprotective role in modulating the responses of many tissues to different types of injury and pathologic states, it is not surprising that there would be numerous complex tissue-specific inducers that lead to its increased expression. The cardioprotective effects of atorvastatin have been shown to be mediated by increased production of multiple prostaglandins, including cytosolic phospholipase A 2 (cPLA 2 ), PGI 2 , and PGE 2 , via upregulation of COX-2 (Birnbaum et al., 2005). Whether prostaglandins other than PGI 2 , such as PGE 2 and PGJ 2 , augment PPAR-α translocation and activate HO-1 expression in our carboplatin-induced renal injury model will require further study. Extension our study by useing PGIS knock-out animal model or PGIS inhibitor is carefully considered.. In addition, PPARs other than PPAR-α can be activated by statins. For example, Yano et al. reported that statins activate PPAR-γ in macrophages (Yano et al. 2007). We have not ruled out a role for other PPARs such as PPAR-β/δ or PPAR-γ in our model.. HO-1 degrades heme into CO and biliverdin, which have powerful anti-inflammatory, antiapoptotic, and antioxidant effects. This likely accounts for the beneficial effect of pravastatin in inhibiting carboplatin-induced nephrotoxicity. However, these heme degradation products are also potentially injurious. Indeed, simvastatin induction of HO-1 has been shown to be mediated by nuclear factor erythroid 2-related 42.

(55) factor 2 (Nrf2) in Neuro 2A cells, and its upregulation was significantly associated with increased apoptosis in that system (Hsieh et al. 2008).. To assay for possible iron toxicity induced by overexpression of HO-1, which can result in increased generation of ROS and inflammation, we overexpressed HO-1 by adenovirus infection in neuronal PC-12 cells. Results showed that overexpression of HO-1 induced ferric iron deposition, which was decreased by treatment with the iron chelator deferoxamine. We also assayed PC-12 cell cytotoxicity and showed that overexpression of HO-1 induced more cytotoxicity in PC-12 cells than in NRK-52 cells, indicating that the effect of HO-1 may be tissue dependent. This implies the limited use of HO-1 activator, or it may benefit to kidney but toxic to other tissues or organs. Thus, the multiple pathways for induction of HO-1 may work synergistically to optimize its expression under many different circumstances.. Besides, the pathway we have proposed is likely to be one of several, if not many, that regulates the expression of HO-1 and one of several that mediates the pleiotropic effects of statins. More evidences and data are required for conclusion.. Our present data showed that carboplatin at a single high dose (100 mg/kg) induced renal dysfunction in mice, as evidenced by elevated levels of blood urea nitrogen and creatinine. It is important to note that these parameters of nephrotoxicity were observed at least 5 days after 43.

(56) administration of carboplatin; whereas the survival of mice continued to decline until day 7. The decrease in survival produced by carboplatin in mice may be the result of both nephrotoxicity and cardiomyopathy (Cheng et al., 2008). In addition, impaired activity and suppressed expression of antioxidant enzymes in the kidneys of the mice may also be involved (Husain et al. 2004). Our present results showed that lower doses of carboplatin (50 mg/kg) did not alter the level of blood urea nitrogen or creatinine (data not shown) or 7-day survival in mice. Therefore, both dose and time of carboplatin exposure are probably important in causing nephrotoxicity.. An interesting observation from the present study is that pravastatin did not completely protect mice against carboplatin-induced nephrotoxicity (Figs. 2 and 3). Similar partial effects of other statins, including cervistatin and atorvastatin, on renal ischemia-reperfusion injury have been reported (Gueler et al. 2007; Gottman et al. 2007). Although the exact reason for the insufficient effect of pravastatin is not clear at present, one possible explanation involves an inhibitory activity of pravastatin on the mammalian target of rapamycin (mTOR) pathway (Roudier et al. 2006). Therefore, increases in the mTOR response as a consequence of pravastatin treatment may partially mask pravastatin’s beneficial effect on carboplatin-induced kidney injury. Additional studies of mTOR activity are needed to further explore whether these mechanisms are important for the antiapoptotic effect mediated by pravastatin in vitro and in vivo.. 44.

(57) In the present study, we administered pravastatin to mice at a dose of 1 mg/kg. This dose was lower than the daily therapeutic dose in humans (20–80 mg/kg/day) (McLean et al. 2008). However, when we increased the dose to 10 mg/kg in mice subjected to carboplatin treatment, the survival rate was significantly decreased compared to that in mice receiving doses of 5 mg/kg or 1 mg/kg (data not shown). This result is in conflict with that of a previous study that used pravastatin (100–150 mg/kg) to inhibit ischemia-reperfusion–induced nephrotoxicity (Sharyo et al. 2008). It is possible that the high dose of carboplatin used in our present study may have augmented the expression of inducible nitric oxide synthase (Ikeda et al. 2001) and contributed to more production of oxidative stress than that in the ischemia-reperfusion model. On the basis of our results, we conclude that low doses of pravastatin have the potential to provide effective protection against nephrotoxicity induced by carboplatin. However, higher doses must be administrated carefully. Further clinical evaluations in humans are needed to determine whether pravastatin would be an attractive drug for the treatment of acute renal injury.. Besides, the types and dosage of statins are major limiting factors in the use of these drugs, because of adverse effects such as myositis and rhabdomyolysis, especially when renal function was impaired. Unlike lipophilic HMG-CoA reductase inhibitors, pravastatin is hydrophilic, and its metabolism is independent of that of cytochrome P-450 3A4 in the liver (Williams et al. 2002). As a result, pravastatin may have fewer toxic effects than other statins. It has been reported to be efficacious and tolerated well 45.