Protective effect of a standardized Ginkgo extract (ginaton) on renal ischemia/reperfusion injury via suppressing the activation of JNK signal pathway.

Abstract

A new standardized Ginkgo extract (ginaton) destined of i.v. injection was investing in rats for its protective effect on renal ischemia/reperfusion injury. We report on the elucidation of the downstream mechanism of action of JNK on the renal ischemia/reperfusion injury, which can be explained as the decrease in JNK phosphorylation at 20 min and c-Jun phosphorylation (Ser63/73) at 3 h after renal ischemia. At the same time, ginaton attenuated the increased expression of FasL at 3 h and caspase3 immunoreactivity at 6 h after renal ischemia. Furthermore, ginaton significantly decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion, alleviating renal ischemia/reprefusion injury. These results cumulatively indicate that ginaton could suppress the JNK-c-Jun-FasL-caspase3 signaling cascade, protecting renal tubular epithelial epithelial cells against ischemia/reperfusion-induced apoptosis, which implies that antioxidants may be a potential and effective agent for prevention of the ischemic/reperfusion injury through the suppression extrinsic apoptotic signal pathway induced by JNK signal pathway. [C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Stand. Ginkgo biloba extract; Renal ischemia/reperfusion; JNK; C-Jun; Fas/FasL; Caspase 3; Apoptosis

Introduction

Renal ischemia/reperfusion injury is the major cause of morbidity and mortality in diseases such as acute renal failure, renal transplantation, trauma, and major surgery. Reperfusion f renal ischemia initiates the complex cellular events that results in renal injury and the eventual death of renal cells due to a combination of apoptosis and necrosis.

A primary factor in the initiation of the pathological response to reperfusion injury is the generation of high levels of ROS, which can covalently modify protein and lipid maeromolecules leading to cell damage, DNA mutaiton, and initiation of apoptotic and necrotic cascades (Greenlund et al., 1995). RPS-induced cell death has been reported in a wide variety of cells, including renal tubular epithelial cells (RTE) (Cuttle et al., 2001; Filipovic et al., 1999). ROS-induced cell injury has been attributed, in part, to the activation of intracellular signaling molecules, including mitogen-activated protein kinases (MAPKs), NF-kB, etc. (Lieberthal and Levine, 1996).

In mammals, signaling cascades culminating in apoptotic cell death are divided into two broad categories: "intrinsic" (i.e., mitochondria) and "extrinsic" (i.e., death receptor) pathways (Ashkenazi and Dixit, 1998; Green and Reed, 1998; Thornberry and Lazebnik, 1998). The extrinsic pathway directly activates the caspase cascade. For example, interaction of Fas with its ligand (FasL) triggers formation of a death-inducing signaling complex (DISC), which in turn recruits and activates caspase-8. Caspase-8 then activates other procaspases, culminating in cleavage of cellular substrates, and apoptosis.

Ginaton, extracted from ginkgo biloba leaves, has been used as a therapeutic agent in lung and brain ischemia (Reynolds and Hastings, 1995; Aoshima et al., 1997). The relationship between ginaton and signal molecules in renal ischemia has not been well elucidated, however.

Therefore, in our present study, ginaton, an antioxidant, was utilized to examine the relationship between the signal molecules and oxidative stress. We aimed to investigate whether ginaton can decrease renal tubular epithelial cell apoptosis-induced renal ischemia/reperfusion by inhibition of the downstream mechanism of INK-mediated apoptosis.

Materials and methods

Materials

Ginaton (Lot No. 050315, one ampoule with 5 ml (17.5 mg) extract of Ginkgo biloba leaves purified for injection, standardized to 4.2 mg Ginkgo flavonol glycosides (24%), 1.05 mg ginkgolides + bilobalide (6%), and the ginkgolic acids ([less than or equal to] 5 x [10.sup.-6] mg).) was purchased from the Dr. Willmar Schwabe, Karlsruhe, Germany. The following primary antibodies were used: rabbit polyclonal anti-p-JNK 1/2 ([Thr.sup.183], [Try.sup.185] rabbit polyclonal antibody) was from Promega Biotechnology. Rabbit polyclonal anti-JNK 1/2 antibody was from Sigma. Mouse monoclonal anti-p-c-Jun (sc-822), rebbit polyclonal anti-FasL (sc-6237), and rabbit polyclonal anti-Fas (se-1023) were purchased from Santa Cruz Biotechnology, rabbit polyclonal anti-cleaved caspase-3 (#9661) were obtained from Cell Signal Biotechnology and the secondary goat anti-rabbit IgG antibody used in our experiment were from Sigma (St Louis, MO, USA). Nitrocellulose filter was from Amersham. BCIP and NBT were form Promega. The ApopTag [R] Peroxidase In Situ Apoptosis Detection Kit (S7100) was purchased from Chemicon. All other chemicals were from Sigma unless indicated otherwise.

Animal surgical procedures and drug treatment

Male Sprague-Dawley rats (Certificate No. SYXK 2002-0038) weighing between 200 and 260g were divided into the following groups: (1) sham group; (2) I/R group; (3) saline-treated group; (4) I/R + ginaton 12.5 mg/kg; (5) I/R + ginaton 25 mg/kg; and (6) I/R + ginaton 50 mg/kg. The experimental procedures were approved by the local legislation for ethics of experiments on animals. Experiments were performed with some modifications of a previously described procedure (Wang et al., 2007; Guan et al., 2005). Following induction of anesthesia (chloral hydrate, 300 mg/kg intraperitoneally), bilateral clamping vascular clamp. During ischemia and reperfusion, rectal vascular clamp. During ischemia and reperfusion, rectal temperature was maintained at about 37 [degrees]C. The rats in ginaton-treated group and saline-treated group were pretreated with ginaton (12.5, 25, 50mg/kg) or equal volume saline every 24 h for 3 d by means of intravenous injection before surgery. The sham operation was performed using the same surgical exposure procedures except for occlusion of pedicles. Reperfusion was achieved by removing the clamp. The results of varying the duration of reperfusion were assessed by occluding the renal pedicles for 45 min and reperfusing the kidney for indicated periods *3, 6, and 24 h) following initiation of reperfusion. Rats were sacrificed upon completion of ischemia/reperfusion: blood samples were collected and analyzed for blood serum creatinine and blood urea nitrogen. Kidneys were immediately removed and rapidly frozen in liquid nitrogen or fixed in paraformaldehyde for immunohistochemical studies.

Sample preparation

Rats were harvested at specified time points of reperfusion after 45 min of ischemia, and renal tissues were rapidly frozen in liquid nitrogen. Cytosolic and nuclear fractions were extracted by modifications of previously described procedures (Wang et al., 2007; Guan et al., 2005). One hundred milligrams of renal tissue was homogenized 1:10 (w/v) in ice-cold homogenization buffer A containing 10mM HEPES, pH 7.9, 0.5 mM MgC[l.sub.2], 10 mM KC1, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM NaF, 5 mM dithiothreitol (DTT), 10 mM [beta]-glycerophosphate, 1 mM sodium orthovanadate, 1% NP40 and enzyme inhibitors [1 mM benzamidine, p-nitro phenyl phosphate (PNPP), phenylmethylsulfonyl fluoride (PMSF), and 5 [mu]g/m1 each of aprotinin, leupeptin, and pepstatin A] were centrifuged at 1000g for 10 min at 4[degrees] C. Supernatants were collected and denoted as the cytosolic fractions. Protein concentrations were determined by the method of Lowry et al. The nuclear pellets were extracted with homogenization buffer B containing 20 mM HEPES, pH7.9, 20% glycerol, 420 mM NaCL, 0.5mM MgC[l.sub.2], 1mM EDTA, 1mM EGTA, 1mM DTT, and enzyme inhibitors for 30 min at 4[degrees]C with constant agitation. After centrifugation at 12,000g for 15 min at 4[degrees]C, the supernatants were collected and denoted as the nuclear fractions. Protein concentrations were determined by the methods of Lowry et al. (1951). Samples were stored at -80[degrees]C and were thawed only once.

Immunoblotting 

Proteins were extracted from kidneys as previously described (Wang et al., 2006). Boiling at 100[degrees]C for 5 min in SDS-PAGE sample buffer eluted samples. Samples (100 [micro]g/lane) were separated on either a 10% or 12% SDS-polyacrylamide gel and electrotransferred onto a nitrocellulose membrane (Amersham, Buckinghamshire, UK). Membranes were blocked for 3h in Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% bovine serum albumin (BSA). Membranes were incubated with anti-p-JNK ([Thr.sup.183]/[Tyr.sup.185]1:3000), anti-JNK (1:10000), anti-p-c-Jun ([Ser.sup.63]/[Ser.sup. 73]1:100), anti-c-Jun (1:500), anti-FasL (1:1000), anti-Fas (1:200) overnight at 4 [degrees]C. Membranes were washed and incubated with 4[degrees]C. Membranes were washed and incubated with alkaline-phosphatase-conjugated secondary antibodies in TBST for 2h and developed using NBT/BCIP color substrate (Promega, Madison, USA). The density of the bands on the membrane was scanned and analyzed with an image analyzer (LabWorks Software, UVP Upland, CA, USA).

Assessment of renal function

Serum creatinine and BUN levels as markers of renal function were measured with Olympus automatic multi-analyzer.

Immunohistochemisty and TUNEL staining

Kidneys were perfusion-fixed with freshly prepared 4% paraformaldehyde in 0.1 M PBS (154m M NaCl, 50 mM sodium phosphate, and pH 7.3), removed rapidly, and further fixed with the same fixation solution at 4 [degrees]C overnight. Post-fixed tissue was embedded in paraffin; small sagittal sections were cut sectioned to 4 [micro]m thickness using a microtome (Leica RM2155, Nussloch, Germany) for immunohistochemistry and TUNEL assay.

Immunohistochemistry

Immunohistochemistry was determined by the avidin-biotin-peroxidase method. Briefly, sections were deparaffinized with xylene and rehydrated by ethanol at graded concentration and distilled water. High-temperature antigen retrieval was performed in 1 mM citrate buffer. Sections were incubated in [H.sub.2][O.sub.2] to block endogenous peroxidase activity. Sections were blocked with 5% (v/v) normal goat serum in PBS for 1 h at 37 [degrees]C, and incubated with rabbit polyclonal antibodies against cleaved caspase-3 protein (1:100) at 4 [degrees]C for 2 d. The sections were incubated with biotinylated goat-anti-rabbit/mouse secondary antibody overnight and subsequently with avidin-conjugated horseradish peroxidase for 1 h at 37 [degrees]C. Finally, sections were incubated with peroxidase substrate diaminobenzidine (DAB) until desired stain intensity develops. The sections were lightly counterstained with hematoxylin.

TUNEL staining

A number of recent studies have demonstrated a link between stress kinase activation and initiations of apoptotic cell death (Khan et al., 2001). To examine if there was evidence of apoptotic cell death in the rat kidney following stress kinase activation by ischemia/reperfusion, we examined apoptotic cell death in these tissues for TUNEL (TDT-mediated dUTP nick end labeling method). TUNEL staining was performed using an ApopTag[R] Peroxidase In Situ Apoptosis Detection Kit according to the manufacturer's protocol with minor modifications. The paraffin embedded tissue sections were deparaffinized and rehydrated and then treated with protease K at 20 [micro]g/ml for 15 min at room temperature. Sections were incubated with reaction buffer containing TdT enzyme and at 37[degrees]C for 1 h. After washing with stop/wash buffer, sections were treated with anti-digoxigenin conjugate for 30 min at room temperature and subsequently developed color in peroxidase substrate. The nuclei were lightly counterstained with hematoxylin. For each paraffin section, three fields were randomly selected and the frequency of TUNEL-positive cells was estimated at x 200 magnification.

Statistical evaluation

Values were expressed as means[+ or -]SD from at least four independent rats. Statistical analysis of the results was carried out by one way analysis of variance followed by Duncan's new multiple range method or Newman-Keuls test and p values < 0.05 were considered significant.

Results

Dose-dependent effect of ginaton treatment on renal function induced by reperfusion after renal ischemia

We examine the reduction of apoptosis by ginaton and the subsequent improvement of renal function after I/R injury; serum creatinine levels, an index of kidney function, increased to 170.10 mg/dl in the I/R group prior to enthanasia after 24 h of reperfusion, while in the ginaton-treated group, these levels declined to near-normal level by 24h of reperfusion (Fig. 1A). We also measured blood urea nitrogen as a second index of kidney function in these experimental groups. BUN levels in the I/R group increased to 22.04 mg/dl prior to euthanasia after 24 h of reperfusion. The BUN levels in the ginaton-treated group at a higher dose decreased to near-normal level by 24 h of reperfusion, compared to the I/R group (Fig 1B).

[FIGURE 1 OMITTED]

Dose-dependent effect of ginaton treatment on the activation of JNK1/2 induced by reperfusion after renal ischemia

We investigated the effects of ginaton on JNK activation by determining JNK phosphorylation with immunoblotting. As indicated in Fig. 2, JNK phosphorylation rapidly reached peak levels at 20 min as compared to the sham group. Administration of ginaton at a higher dose attenuated significantly the increase of p-JNK1/2 at 20 min after ischemia, as compared to the sham group. Administration of ginaton at a higher dose attenuated significantly the increase of p-JNK 1/2 at 20 min after ischemia, as compared to the I/R group. The same dose of saline has no influence on the increase of the activation of JNK.

Dose-dependent effect of ginaton treatment on activation and expression of c-Jun induced by reperfusion after renal ischemia

JNK activation phosphorylates nuclear substrates e.g. c-Jun, leading to cell death (Verheij et al., 1996). JNK activation induces neuronal cell death by c-Jun phosphorylation, promoting its transcription activity (Guan et al., 2005). The effects of ginaton on activation and expression of c-Jun subsequent to renal ischemia were examined with western blotting. C-Jun phosphorylation and expression were rapidly increased after ischemia and reached peak levels at 3 and 6h of reperfusion, respectively, compared to the sham group. Ginaton at a higher dose significantly attenuated the increase reperfusion compared to the I/R group, as demonstrated in Fig. 3. The same dose of vehicle did not affect the increase in the activation of c-Jun.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Dose-dependent effect of ginaton treatment on the increased expression of FasL induced by renal ischemia/reperfusion

In the present study, we examined the effect of ginaton on the expression of FasL and Fas. As presented in Fig. 4, results of western blotting revealed that the increased expression of FasL at 3h reperfusion was significantly suppressed by administration of ginaton at a higher dose, as compared to the I/R group. The same dose of vehicle did not increase FasL expression. The protein level of Fas was not affected by ginaton and vehicle.

[FIGURE 4 OMITTED]

Dose-dependent effect of ginaton treatment on the activation of caspase-3 induced by renal ischemia/reperfusion

Caspase-3 is one of the key executioners in extrinsic apoptotic pathways. It is a cytosolic protein as an inactive 32-kDa proenzyme and is activated by proteolytic cleavage into the 20 kDa (p20) and 11 kDa (p11) active subunits. In this study, we examined the effect of ginaton on activation of caspase-3. Immunohistochemical observation was carried out using anti-cleaved caspase-3 antibody. As shown in Fig. 5, weak cleaved caspase-3 immunoreactivity was detected in the cytosol in the sham group (a), cleaved caspase-3 immunoreactivity significantly increased 6h reperfusion after ischemia compared with the sham group (b). There was no inhibitory effect of vehicle on cleaved caspase-3 immunoreactivity 6h reperfusion after ischemia (c). Cleaved caspase-3 immunoreactivity was significantly inhibited at 6h reperfusion by administration of ginaton at a higher dose (e and f), as compared to the I/R group (b).

[FIGURE 5 OMITTED]

The protective role of ginaton against renal ischemia-induced apoptotic cell death

We investigated the ability of ginaton pretreatment to mediate protection against ischemia-induced apoptotic cell death. Adult Sprague--Dawley rats were subjected to 45 min ischemia followed by 24h reperfusion. Rats were pretreated with ginaton or vehicle for 3d before ischemia. After 24h reperfusion, rats were perfusion-fixed with paraformaldehyde and TUNEL staining was employed to determine apoptosis of renal tubular epithelial cells (Fig. 6). A significant number of TUNEL-positive cells were observed at 24h reperfusion after ischemia, predominantly located at the distal tubules of the outer medulla, a few at the proximal tubules of the cortex, some shed into the renal tubular cave. These cells demonstrated characteristic morphologies, e.g. shrunken and condensed nuclei. Few TUNEL-positive cells were observed in the sham group (a). A significant increase in the number of TUNEL-positive cells was displayed after 24h of reperfusion (b). Ginaton at a higher dose significantly attenuated TUNEL-positive cells (e and f) as compared to the renal ischemia-reperfusion group (b), and saline-treated group (c) did not demonstrate any protection.

[FIGURE 6 OMITTED]

Discussion

In the present study, our results demonstrated that ginaton blocked c-Jun/AP-1-mediated transcriptional regulation and the JNK activation, in turn, diminished the expression of FasL and the caspase3 activation, which attenuated renal ischemia-induced apoptosis, ultimately alleviated ischemia renal injury.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of renal diseases including ischemic and toxic acute renal failure (Basnakian et al., 2002; Nath and Norby, 2000). Mitogen-activated protein kinases (MAPKs) were presumably involved (Piantadosi and Zhang, 1996). Reactive oxidative species (ROS) such as [H.sub.2][O.sub.2] can induce the activation of the ASK1/JNK signaling cascade (Yoon et al., 2002; Gupta et al., 2003). Our results demonstrate that ischemia/reperfusion-induced activation of JNK and c-Jun. Our present study indicated that Ginaton significantly diminished the increase of p-JNK at 20 min and increased of p-c-Jun at 3h after renal ischemia.

Several synthetic free-radical scavengers have been evaluated in animal models of renal or cerebral ischemia/reperfusion. For example, N-acetylcysteine (NAC) could inhibit the activation of MAPK, and pyrrolidine dithiocarbamate has important neuroprotective effects against I/R-induced injury through down regulating the activation of NF-kB (Sekhon et al., 2003; Shen et al., 2003). Bilobalide, one pivotal component of EGb, could attenuate ROS-induced apoptosis, suggesting that bilobalide might be working as a free-radical scavenger. Bilobalide may block apoptosis in the early stage and then attenuate the elevation of c-Myc and activation of caspace-3 in cells (Zhou and Zhu, 2000). Our present study and others suggested that the action of antioxidants an suppression cell apoptosis was consistent with the activation of JNK signal pathway.

Extensive studies to elucidate the mechanisms by which apoptosis is induced and transduced have resulted in the generally accepted theory that intrinsic and extrinsic mechanisms (the receptor-dependent pathway) are involved (Fadeel and Orrenius, 2005; Kim et al., 2006). The extrinsic apoptotic pathway is initiated by the activation of death receptor, such as the TNF receptor and Fas, and caspase-8 (Kim et al., 2006). A JNK-dependent element in the Fas ligant promoter that binds c-Jun and ATFs has been identified; inhibition of c-Jun/AP-1 activation precents FasL-induced apoptosis (Faris et al., 1998; Kasibhatla et al., 1998). Our early study and other's strongly suggest through c-Jun/AP-1-mediated transcriptional regulation, activation of JNK could enhance the expression of FasL or Fas, which can ultimately contribute to Fasmediated apoptosis in renal or brain ischemia injury (Wang et al., 2007; Guan et al., 2005; Gao et al., 2005). Results from our current research indicated that pretreatment of gination could inhibit c-Ju/AP-1-mediated transcriptional regulation and the JNK activation, in turn, attenuated the expression of FasL, finally, blocking Fas-mediated apopotosis.

Recent data highlight the pivotal role of caspase-3 in the execution of ischemia-induced apoptosis (Zheng et al., 2003). Caspase-3 inhibitors can prevent delayed cell death after ischemia (Chang and Karin, 2001). Thus, caspase-3 is a key step in the execution process of apoptosis, and its inhibition can block apoptotic cell death. Our data implicate that extrinsic apoptotic cell death. Our data implicate that extrinsic apoptotic signal pathway results in activation of executioner caspase-3, and ginaton could decrease the caspase-3 activation, which were approved further by some who reported that ginkgo biloba extract can block apoptosis induced by brain ischemia via blocking Bax and capase-8, caspase-3 activity (Kaushal et al., 2004). These researches suggested that the pretreatment with ginkgo biloba extract could diminish the number of apoptotic renal epithelial tubular cells through blocking the activation of JNK-c-Jun-FasL-caspase-3 cascade, protecting kidney against renal ischemia.

In summary, our results clearly demonstrated that antioxidant ginaton could protect kidney against ischemic injury via decreasing the extrinsic signals of the JNK signal pathway, which provides a novel and effective strategy for prevention of the renal ischemic/reperfusion injury.

Acknowledgement

This work was supported by Xuzhou Science Research Foundation (No. XM07C068), and the Project of the Science Foundation of Affiliated Hospital of Xuzhou Medical College (No. 2006-37, 2007-21).

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Yan Wang (a), (1), Dong-Sheng Pei (c), (1), Huai-Xue Ji (a), Shu-Hua Xing (b), *

(a) Department of Pharmacy, Affiliated Hospital of Xuzhou Meical College, Xuzhou 221002, PR China

(b) Department of Fundamental Medicine of Xuzhou Medial College, Xuzhou 221002, PR China

(c) Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, PR China

* Corresponding author at: Department of Hospital Pharmacy, Affiliated Hospital of Xuzhou Medical College. 99 West Huai-hai Road, Xuzhou, Jiangsu 221002, PR China. Tel./fax: [phone]. E-mail address: [e-mail] (S.-H. Xing).

(1) These authors contributed equally to this work.

0944-7113/$-see front matter [C] 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2008.09.003