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HGF–MSP chimera protects kidneys from ischemia–reperfusion injury
Biochemical and Biophysical Research Communications 363 (2007) 451–456 www.elsevier.com/locate/ybbrc
HGF–MSP chimera protects kidneys from ischemia–reperfusion injury Feng Xue a,e, Yoshitaka Isaka a,b,*, Terumi Takahara d, Ryoichi Imamura c, Chigure Suzuki b, Naotsugu Ichimaru c, Paolo Michieli f, Shiro Takahara a a
Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan b Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Japan c Department of Urology, Osaka University Graduate School of Medicine, Suita, Japan d Third Internal Medicine, Faculty of Medicine, University of Toyama, Toyama, Japan e Organ Transplantation Center, Renji Hospital Affiliated to Shanghai Jiao-tong University, Medical School, Shanghai, China f Division of Molecular Oncology, University of Torino Medical School, Candiolo, Italy Received 24 May 2007 Available online 14 September 2007
Abstract Renal ischemia–reperfusion (I/R) injury is inevitable in transplantation and is related to long-term graft function. MF-1, a bifunctional hepatocyte growth factor (HGF)–macrophage-stimulating protein (MSP) (HGF–MSP) chimera was recently reported to prevent apoptosis. We therefore hypothesized that treatment with MF-1 would protect kidneys from I/R injury by inhibiting tubular epithelial apoptosis. MF-1 directly guarded cultured proximal tubular epithelial cells from hypoxia-induced necrosis and apoptosis in vitro. In addition, the therapeutic effects of MF-1 were evaluated using a rat I/R injury model in vivo. Saline-treated kidneys had increased creatinine and BUN, and exhibited tubular epithelial apoptosis with activated caspase 3 expression. In contrast, MF-1 treatment up-regulated Akt phosphorylation, and inhibited caspase 3 activation and tubular apoptosis, thereby ameliorating renal dysfunction. Of particular interest is that macrophage infiltration was suppressed in the MF-1-treated kidney. In conclusion, we identified a novel therapeutic approach using MF-1 to protect kidneys from I/R injury. 2007 Elsevier Inc. All rights reserved. Keywords: Ischemia–reperfusion; Hypoxia; Apoptosis; Kidney; Macrophage
Renal ischemia–reperfusion (I/R) injury, which is unavoidable in renal transplantation and is frequently associated with shock or surgery, is a major cause of acute renal failure [1]. Despite decades of laboratory and clinical investigations and the advent of renal replacement therapy, the overall mortality rate due to acute tubular necrosis has changed little. Moreover, long-term graft function in renal transplantation is associated with the initial intensity of I/ R injury [2]. Therefore, due to the clinical importance of renal I/R injury, specific therapy for such injury should be considered.
* Corresponding author. Address: Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. Fax: +81 6 6879 3749. E-mail address: [email protected] (Y. Isaka).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.229
Although I/R injury is a major cause of acute renal failure, reperfusion itself causes additional cellular injury that results in the eventual death of renal cells in a sequential combination of both apoptosis and necrosis [3]. Apoptotic cell death has been documented in animal models and human biopsies after renal I/R injury [4], and inhibition of apoptotic signaling has been shown to ameliorate the associated injury and inflammation [5]. Renal tubulointerstitial damage, including interstitial fibrosis, tubular atrophy, and macrophage infiltration, is the main cause of end-stage renal failure, regardless of the primary etiology of renal disease. Recruitment of macrophages, which accompany interstitial matrix accumulation, is correlated with tubular atrophy [6]. Chemokines activate leukocytes and mediate selective trafficking at multiple stages, attracting specific populations to the site of injury [7,8]. In the kidney, tubular epithelial cells are
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considered to be a prominent source of chemokines [9]. Therefore, protecting tubular epithelial cells against I/R injury makes it possible to reduce interstitial fibrosis. Hepatocyte growth factor (HGF) and macrophagestimulating protein (MSP) have an intrinsic dual nature; while they are protective cytokines that prevent apoptosis and support regeneration, they also promote invasion. For example, HGF administration prevents cyclosporineinduced acute renal failure [10] and ameliorates renal ischemic injury [11]. On the other hand, HGF antagonists were found to inhibit tumor invasiveness [12]. Similarly, HGF-like factor, MSP, is a pleiotropic factor capable of stimulating resident peritoneal macrophages [13], but prevents apoptosis of epithelial cells [14]. HGF and MSP each have a large molecular mass and require proteolytic processing to be active. Studies in mice have indicated that injected precursors are not activated by endogenous proteases [15]. In addition, endogenous proHGF and pro-MSP are ubiquitously present in plasma and in the extracellular matrix of tissues at high concentrations, and thus injection of recombinant precursors simply increases the concentration of inactive factors. Therefore, to obtain a biological response, pre-activated factors must be activated. The activation of scatter factors in vitro by proteases involves the re-purification of factors after proteolysis, and activated HGF and MSP are less stable than their precursors, at least in vitro. It was recently shown that a recombinant chimera between selected domains of HGF and MSP (MF-1) (i) has a lower molecular mass than either of the parent molecules; (ii) does not require proteolytic activation; (iii) binds independently to both the Met and Ron receptors, while eliciting biological signals only through MetRon heterodimerization; (iv) promotes proliferation and protection against apoptosis at nanomolar concentrations in cells co-expressing the two receptors; (v) is devoid of pro-invasive activity; and (vi) prevents drug-induced acute renal damage in mice [16]. Here, we examined the therapeutic effects of MF-1 on I/R injury. Materials and methods Purification of MF-1. The MF-1 gene was transduced in MDA-MB 425 cells by the lenti viral vector. Secreted MF-1 protein was purified by dualstep affinity chromatography using a heparin-Sepharose and Ni2+-chelate column (Amersham Pharmacia, Uppsala, Sweden) [16]. Cell culture. Human renal proximal tubular epithelial cells (human kidney-2, HK-2; ATCC, Manassas, VA) were cultured in DMEM with (5%) FCS under a 5% CO2 and 95% air atmosphere at 37 C. To examine the renoprotective effects of MF-1, we added MF-1 to the culture medium (final concentrations of 0, 50 or 100 ng/ml). Hypoxia was then induced by incubating the plate under low-oxygen conditions. Oxygen concentration was maintained at 1% using the compact gas oxygen controller APM-36 (ASTEC, Fukuoka, Japan) with a residual gas mixture composed of 94% N2 and 5% CO2. Lactate dehydrogenase (LDH) assay and apoptosis assay were conducted after incubation for 15 h under low-oxygen condition. In order to evaluate cell viability, LDH activity was measured in cell culture medium and cell lysates by quantitative biochemical assay according to the manufacturer’s instructions (Roche Diagnostics). Cell
viability was assessed as the percentage of total LDH activity released into the medium. We also confirmed hypoxia-induced apoptosis by apoptosis assay. Briefly, a cell-death enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics, Indianapolis, IN) was used to assess hypoxia-induced apoptosis in cultured HK-2 cells with/without MF-1 in accordance with the kit manufacturer’s protocol. HK-2 cells were dispensed in 96-well plates at a density 5 · 103 cells/well. With/without MF-1 co-incubation, HK-2 cells were then exposed to hypoxia (1% O2) for 15 h. The relative decrease in apoptosis was determined by comparing results in MF-1treated cells with those in untreated controls. Experimental design. Male Sprague–Dawley rats weighing 150 g were purchased from Japan SLC Inc. (Hamamatsu, Japan) and were maintained under standard conditions until experiments were completed. All studies were performed in accordance with the principles of the Guidelines on Animal Experimentation of Osaka University. Rats were randomly allocated into 2 groups: (1) the saline-treatment group (control group; n = 12) and (2) the MF-1-treatment group (MF-1 group; n = 12). Rats were then subjected to I/R injury. All rats were anesthetized with an intraperitoneal injection of sodium thiopentone (30 mg/kg), and were allowed to stabilize for 30 minutes (min) before they were subjected to 45 min of bilateral renal occlusion using artery clips to clamp the renal pedicles. Occlusion was visually confirmed by a change in kidney color to a paler shade. Reperfusion was initiated with the removal of artery clips and was visually confirmed by noting the subsequent blush. Prior to the removal of the artery clips, rats received saline or MF-1 (20 lg in 500 ll of saline) via the tail vein. Rats were sacrificed 72 h after reperfusion. Blood samples (1 ml) were collected from the tail veins of anesthetized rats for measurement of serum creatinine and blood urea nitrogen (BUN) levels at 72 h after reperfusion. Antibodies. In order to detect the pathways that protect kidneys, we used the following antibodies for Western blotting: polyclonal phosphoAkt (Ser473) antibody (1:1000, Cell Signaling Technology, Beverly, MA); polyclonal Akt antibody (1:1000, Cell Signaling); and Caspase 3 (1:1000 dilution; BD Biosciences San Jose, CA). Macrophage infiltration. In order to examine the effects of MF-1 on macrophage infiltration, we used the monoclonal antibody ED1 (macrophage marker; Serotec, Oxford, UK). Tissue samples were fixed in 4% (wt./vol.) of buffered paraformaldehyde (PFA) for 16 h, and were then embedded in paraffin. Sections (4 lm) were mounted on silane (2% 3-aminopropyltriethoxysilane)-coated slides (Muto Pure Chemicals, Tokyo, Japan) and were deparaffinized with xylene. Immunohistochemical staining was performed using the Envision system (Dako), according to the manufacturer’s instructions. Endogenous peroxidase activities were blocked with 3% H2O2 for 10 min. The monoclonal antibody ED1 was diluted in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T), and was then incubated for 24 h at 4 C. This was followed by incubation with secondary antibodies. All incubations were performed in a humidified chamber. Chromogenic color was developed with 3,3 0 diaminobenzidine tetrahydrochloride (DAB; Dako). Negative controls omitted the first antibodies and were prepared for each reaction. All histological slides were examined by light microscopy using a Nikon Eclipse 80 i (Nikon, Tokyo, Japan); pictures were taken with the Nikon ACT-1 ver. 2.63. ED-1-positive cell cells were counted in 10 random areas per rat, and the number of ED-1-positive cells per mm2 was then calculated. Terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining. TUNEL staining was performed using the in situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan), according to the manufacturer’s instructions. Briefly, sections were deparaffinized and subjected to antigen retrieval in preheated 10 mmol/l sodium citrate (pH 7) using a steamer for 40 min. Sections were then incubated with 3% H2O2 for 10 min, followed by incubation with TdT enzyme solution for 90 min at 37 C. The reaction was terminated by incubation in a stop/wash buffer for 30 min at 37 C. The number of TUNEL-positive cell nuclei and the total numbers of cell nuclei stained with hematoxylin were counted in 10 random areas, and the percentage of TUNEL-positive nuclei against total cell nuclei was then calculated.
F. Xue et al. / Biochemical and Biophysical Research Communications 363 (2007) 451–456 Western blot analysis. Kidney tissue was homogenized in a radioimmunoprecipitation (RIPA) Lysis Buffer with phenylmethylsulfonylfluoride (PMSF) solution, sodium orthovanadate solution, and protease inhibitor (Santa Cruz). Homogenates were centrifuged (12,000g for 10 min at 4 C), and total protein in the supernatant was measured by Lowry protein assay (Bio-Rad, Hercules, CA). Total protein lysate (15 lg) containing 1:1 denaturing sample buffer was boiled for 3 min and resolved on 5.0–10.0% SDS–polyacrylamide gels and was electrophoretically transferred onto an immobilon PVDF membrane (Millipore, Bedford, MA). The membrane was blocked with 5% (wt./vol.) nonfat milk or 1% BSA in 10 mM Trisbuffered saline with 0.1% Tween 20 (TBS-T), followed by overnight incubation at 4 C with diluted primary antibodies in TBS-T or blocking buffer. After washing with TBS-T 5 times, the membrane was incubated with secondary antibody (1:1000) (Cell Signaling) in TBS-T for 45 min at room temperature and was developed using ECL reagents (Amersham Bioscience Corp., Piscataway, NJ) to detect specific protein bands. Band density was analyzed by NIH Image software. Statistical analysis. Data are expressed as means ± SD. Statistical analysis was performed by unpaired t-test or ANOVA for multiple comparisons, followed by Scheffe’s F-test.
Results Effects of MF-1 on hypoxia-induced injury in cultured proximal tubular epithelial cells In cultures of HK-2 cells, 15-h exposure to hypoxia decreased viability, as assessed by released LDH activity (Fig. 1A). Incubation with MF-1 (50 and 100 ng/ml) significantly reduced the release of LDH in a dose dependent manner, thus suggesting that MF-1 directly protects tubular epithelial cells from hypoxia-induced necrosis. In addition, cell-death ELISA revealed that HK-2 cells exhibited an increase in DNA fragmentation under hypoxic conditions. Treatment with MF-1 (50 and 100 ng/ml) significantly inhibited DNA fragmentation, which suggests that MF-1 suppressed tubular epithelial apoptosis under hypoxia (Fig. 1B).
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Effects of MF-1 on renal function Saline-treated rats demonstrated a significant increase in serum creatinine (0.95 ± 0.46 mg/dl) and BUN (61.8 ± 32.9 mg/dl) at 72 h after I/R injury. In contrast, MF-1 treatment significantly suppressed the increases in creatinine (0.41 ± 0.13 mg/dl; p < 0.05, vs. control group) and BUN (25.4 ± 5.9 mg/dl; p < 0.05, vs. control group). Effects of MF-1 on tubular apoptosis in I/R-injured kidney In order to elucidate the protective mechanisms of MF-1, we performed TUNEL immunostaining to quantify the number of apoptotic cells. In the control group, TUNEL positive, apoptotic cells increased among tubular epithelial cells at 72 h after I/R injury (percent of TUNEL-positive cells, 7.2 ± 2.5%) (Fig. 2A and C). In contrast, apoptotic cells were significantly decreased in the MF-1 group (3.4 ± 1.1%; p < 0.05, vs. saline group) (Fig. 2B and C). Western blot analysis demonstrated that active (cleaved) caspase 3 was increased in saline-treated I/R injury model rats at 72 h, while active caspase 3 was decreased in MF-1treated rats (Fig. 3A). These data indicate that I/R of kidney induces apoptosis in tubular epithelial cells, and this is markedly prevented by MF-1. To elucidate the intracellular MF-1 signaling implicated in tubular protection, we examined the expression and activation of Akt (Fig. 3B). Western blot analysis demonstrated that treatment with MF-1 increased phosphorylation of Akt at 72 h after I/R injury, while phosphorylated Akt was not observed in the saline group.
Fig. 1. Effects of MF-1 on hypoxia-induced cell death. (A) HK-2 cells, when exposed to hypoxia for 15 h, showed decreased cell viability, as assessed by LDH assay, while incubation with MF-1 (50 and 100 ng/ml) significantly reduced the release of LDH (*p < 0.01, vs. control). (B) HK-2 cells exhibited increased DNA fragmentation under hypoxic conditions. Treatment with MF-1 (50 and 100 ng/ml) significantly inhibited DNA fragmentation (*p < 0.01, **p < 0.001 vs. control).
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Fig. 2. Effects of MF-1 on apoptosis at 72 h. Representative TUNEL immunostaining results for saline group (A), and MF-1 group (B) are shown. Dark brown dots correspond to representative TUNEL-positive nuclei. (C) MF-1 treatment significantly decreased TUNEL positive, apoptotic cells (*p < 0.05 vs. control group) (magnification, 200·).
Fig. 3. Western blot analysis of I/R-injured kidney. (A) Western blot analysis confirmed that the cleaved (active) form of caspase 3 (18 kD) was suppressed in MF-1-treated rats when compared to saline treatment. (B) Western blot analysis demonstrated that treatment with MF-1 increased Akt phosphorylation at 72 h after I/R injury, whiel 500 IU EPO or CEPO had no effect on Akt activation.
Effects on interstitial macrophage infiltration in I/R-injured kidney In order to detect interstitial macrophage infiltration, which is associated with interstitial damage and fibrosis, expression of ED-1 was examined immunohistochemically. Interstitial expression of ED-1 increased at 72 h after I/R injury in saline-treated rats (Fig. 4A and C), while MF-1 treatment significantly suppressed interstitial ED-1 expression and macrophage infiltration (Fig. 4B and C). Discussion In this paper, we demonstrated that MF-1 has therapeutic effects in a rat model of I/R injury. This is based on the fact that MF-1 directly protects tubular epithelial
cells from hypoxia-induced necrosis and apoptosis. We then investigated whether treatment with MF-1 is able to suppress tubular apoptosis and macrophage infiltration in a rat I/R injury model. Untreated kidneys exhibited increased creatinine and BUN with tubular apoptosis and interstitial macrophage infiltration. On the other hand, MF-1 treatment ameliorated renal dysfunction, and inhibited tubular apoptosis and macrophage infiltration. We also demonstrated that protection of the proximal tubular epithelial cells by MF-1 was attenuated by Akt phosphorylation. Once activated, Akt activates multiple targets with anti-apoptotic effects, including phosophorylation of Bad, Bax, caspase 9, and GSK-3b [17]. Thus, MF-1 appears to protect proximal tubular epithelial cells against I/R injury by phosphorylation of Akt, resulting in the inhibition of tubular apoptotic cell death, which leads to the attenuation of macrophage recruitment. Chimeric MF-1, derived from the a-chains of HGF and MSP, concomitantly activates the HGF receptor (Met) and the MSP receptor (Ron). Although the trophic effects of MF-1 would be restricted to tissue expressing both Met and Ron, various tissues, including tubular epithelial cells, express both Met and Ron [15,18]. HGF and MSP have an intrinsic dual nature; they are trophic cytokines that prevent apoptosis, and are scatter factors that promote invasion. For example, HGF/Met signaling activates proteases and mediates cellular inva-
Fig. 4. Effects on macrophage infiltration at 72 h. Interstitial macrophage infiltration was assessed by immunohistochemical staining of ED-1 in the control group (A), and MF-1 group (B). (C) Quantification (per mm2) of ED-1 positive macrophages in kidney sections. Data shown are means ± SD for 10 independently performed experiments (*p < 0.001 vs. control group).
F. Xue et al. / Biochemical and Biophysical Research Communications 363 (2007) 451–456
sion and metastasis [19]. Therefore, separation of the favorable effects of HGF and MSP from their adverse effects is a promising technique. MF-1 offers both biochemical and biological advantages over HGF and MSP. From a biochemical point of view, HGF and MSP have a large molecular mass and require proteolytic processing to be active [16]. In contrast, MF-1 has a lower molecular mass than either HGF or MSP and does not require proteolytic activation [16]. From a biological point of view, the ability of MF-1 to promote cell survival without inducing invasion is important to its potential therapeutic application [16]. Based on our results, MF-1 appears to have antiapoptotic function against I/R injury. We demonstrated that MF-1 treatment markedly suppresses I/R injuryinduced tubular epithelial apoptosis when compared to saline treatment. Furthermore, MF-1 inhibited hypoxiainduced apoptosis. One possible signal transduction pathway by which tubular apoptosis could be suppressed involves the activation of Akt. In fact, on Western blot analysis, we found that MF-1 treatment induced Akt phosphorylation at 72 h after I/R injury. It has been reported that suppression of c-Met phosphorylation by anti-HGF IgG leads to rapid progression of tubular apoptosis, while enhancement of c-Met activation by exogenous HGF blocks tubular apoptotic injury [20]. In this process, HGF prevents nuclear factor kappa B (NF-jB) activation, which has been implicated in events downstream of Akt. In addition, MSP-induced prevention of apoptosis is reportedly mediated by the Akt pathway [14]. Taken together with our observations, these data suggest that MF-1 protects tubular cells from I/Rinduced apoptosis via Akt phosphorylation. Although we demonstrated that MF-1 treatment reduced macrophage infiltration in the interstitium of I/R-injured kidneys, MSP is reported to stimulate resident peritoneal macrophages [13]. It was hypothesized that MSP released by tubular cells in the course of tubulointerstitial inflammatory disorders stimulates resident macrophages and recruits circulating monocytes, thus amplifying the inflammatory response [18]. In contrast, the HGF–MSP chimera MF-1 suppressed macrophage infiltration. One explanation for this observation is that HGF itself inhibits macrophage recruitment by suppressing the expression of proinflammatory chemokine MCP-1 (macrophage chemoattractant protein-1) and RANTES (regulated upon expression normal T cell expressed and secreted), and inhibits activation of the NF-jB transcription factor [9]. Another explanation is that MF-1 binds to Met and Ron with lower affinity than does HGF or MSP, and is able to mediate a biological signal only through Met–Ron heterodimers [16], by which MF-1 might behave as an antagonist of scatter factors. In conclusion, MF-1 treatment up-regulated the phosphorylation of Akt, and resulted in the inhibition of caspase 3 activation and tubular apoptosis, thereby ameliorating
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renal dysfunction in a rat I/R-induced renal injury model. In addition, macrophage infiltration was suppressed in MF-1-treated kidneys. These observations suggest that MF-1 is a novel therapeutic approach for protecting kidneys from I/R injury. References [1] F. Gueler, W. Gwinner, A. Schwarz, H. Haller, Long-term effects of acute ischemia and reperfusion injury, Kidney Int. 66 (2004) 523–527. [2] D.A. Shiskes, P.D. Halloran, Delayed graft function in renal transplantation: etiology, management and long-term significance, J. Urol. 155 (1996) 1831–1840. [3] S.C. Weight, P.R. Bell, M.L. Nicholson, Renal ischaemia–reperfusion injury, Br. J. Surg. 83 (1996) 162–170. [4] M.A. Daemen, C. Van’t Veer, G. Denecker, V.H. Heemskerk, T.G. Wolfs, M. Clauss, P. Vandenabeele, W.A. Buurman, Inhibition of apoptosis induced by ischemia–reperfusion prevents inflammation, J. Clin. Invest. 104 (1999) 541–549. [5] W. Lieberthal, S.A. Menza, J.S. Levine, Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells, Am. J. Physiol. 274 (1998) F315–F327. [6] S. Mizuno, K. Matsumoto, T. Nakamura, Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy, Kidney Int. 59 (2001) 1304–1314. [7] H.J. Anders, V. Vielhauer, D. Schlondorff, Chemokines and chemokine receptors are involved in the resolution or progression of renal disease, Kidney Int. 63 (2003) 401–415. [8] S. Segerer, P.J. Nelson, D. Schlondorff, Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies, J. Am. Soc. Nephrol. 11 (2000) 152–176. [9] R. Gong, A. Rifai, E.M. Tolbert, P. Biswas, J.N. Centracchio, L.D. Dworkin, Hepatocyte growth factor ameliorates renal interstitial inflammation in rat remnant kidney by modulating tubular expression of macrophage chemoattractant protein-1 and RANTES, J. Am. Soc. Nephrol. 15 (2004) 2868–2881. [10] H. Amaike, K. Matsumoto, T. Oka, T. Nakamura, Preventive effect of hepatocyte growth factor on acute side effects of cyclosporin A in mice, Cytokine 8 (1996) 387–394. [11] S.B. Miller, D.R. Martin, J. Kissane, M.R. Hammerman, Hepatocyte growth factor accelerates recovery from acute ischemic renal injury in rats, Am. J. Physiol. 266 (1994) F129–F134. [12] K. Date, K. Matsumoto, K. Kuba, H. Shimura, M. Tanaka, T. Nakamura, Inhibition of tumor growth and invasion by a fourkringle antagonist (HGF/NK4) for hepatocyte growth factor, Oncogene 17 (1998) 3045–3054. [13] A. Skeel, T. Yoshimura, S.D. Showalter, S. Tanaka, E. Appella, E.J. Leonard, Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity, J. Exp. Med. 173 (1991) 1227–1234. [14] A. Danilkovitch, S. Donley, A. Skeel, E.J. Leonard, Two independent signaling pathways mediate the antiapoptotic action of macrophagestimulating protein on epithelial cells, Mol. Cell. Biol. 20 (2000) 2218–2227. [15] K. Matsumoto, T. Nakamura, Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases, Kidney Int. 59 (2001) 2023–2038. [16] P. Michieli, S. Cavassa, C. Basilico, A. De Luca, M. Mazzone, C. Asti, R. Chiusaroli, M. Guglielmi, P. Bossu, F. Colotta, G. Caselli, P.M. Comoglio, An HGF–MSP chimera disassociates the trophic properties of scatter factors from their pro-invasive activity, Nat. Biotechnol. 20 (2002) 488–495. [17] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science 296 (2002) 1655–1657.
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[18] T. Rampino, C. Collesi, M. Gregorini, M. Maggio, G. Soccio, P. Guallini, A. Dal Canton, Macrophage-stimulating protein is produced by tubular cells and activates mesangial cells, J. Am. Soc. Nephrol. 13 (2002) 649–657. [19] M. Jeffers, S. Rong, G.F. Vande Woude, Enhanced tumorigenicity and invasion-metastasis by hepatocyte growth factor/scatter factor-
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HGF–MSP chimera protects kidneys from ischemia–reperfusion injury Feng Xue a,e, Yoshitaka Isaka a,b,*, Terumi Takahara d, Ryoichi Imamura c, Chigure Suzuki b, Naotsugu Ichimaru c, Paolo Michieli f, Shiro Takahara a a
Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan b Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Japan c Department of Urology, Osaka University Graduate School of Medicine, Suita, Japan d Third Internal Medicine, Faculty of Medicine, University of Toyama, Toyama, Japan e Organ Transplantation Center, Renji Hospital Affiliated to Shanghai Jiao-tong University, Medical School, Shanghai, China f Division of Molecular Oncology, University of Torino Medical School, Candiolo, Italy Received 24 May 2007 Available online 14 September 2007
Abstract Renal ischemia–reperfusion (I/R) injury is inevitable in transplantation and is related to long-term graft function. MF-1, a bifunctional hepatocyte growth factor (HGF)–macrophage-stimulating protein (MSP) (HGF–MSP) chimera was recently reported to prevent apoptosis. We therefore hypothesized that treatment with MF-1 would protect kidneys from I/R injury by inhibiting tubular epithelial apoptosis. MF-1 directly guarded cultured proximal tubular epithelial cells from hypoxia-induced necrosis and apoptosis in vitro. In addition, the therapeutic effects of MF-1 were evaluated using a rat I/R injury model in vivo. Saline-treated kidneys had increased creatinine and BUN, and exhibited tubular epithelial apoptosis with activated caspase 3 expression. In contrast, MF-1 treatment up-regulated Akt phosphorylation, and inhibited caspase 3 activation and tubular apoptosis, thereby ameliorating renal dysfunction. Of particular interest is that macrophage infiltration was suppressed in the MF-1-treated kidney. In conclusion, we identified a novel therapeutic approach using MF-1 to protect kidneys from I/R injury. 2007 Elsevier Inc. All rights reserved. Keywords: Ischemia–reperfusion; Hypoxia; Apoptosis; Kidney; Macrophage
Renal ischemia–reperfusion (I/R) injury, which is unavoidable in renal transplantation and is frequently associated with shock or surgery, is a major cause of acute renal failure [1]. Despite decades of laboratory and clinical investigations and the advent of renal replacement therapy, the overall mortality rate due to acute tubular necrosis has changed little. Moreover, long-term graft function in renal transplantation is associated with the initial intensity of I/ R injury [2]. Therefore, due to the clinical importance of renal I/R injury, specific therapy for such injury should be considered.
* Corresponding author. Address: Department of Advanced Technology for Transplantation, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. Fax: +81 6 6879 3749. E-mail address: [email protected] (Y. Isaka).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.05.229
Although I/R injury is a major cause of acute renal failure, reperfusion itself causes additional cellular injury that results in the eventual death of renal cells in a sequential combination of both apoptosis and necrosis [3]. Apoptotic cell death has been documented in animal models and human biopsies after renal I/R injury [4], and inhibition of apoptotic signaling has been shown to ameliorate the associated injury and inflammation [5]. Renal tubulointerstitial damage, including interstitial fibrosis, tubular atrophy, and macrophage infiltration, is the main cause of end-stage renal failure, regardless of the primary etiology of renal disease. Recruitment of macrophages, which accompany interstitial matrix accumulation, is correlated with tubular atrophy [6]. Chemokines activate leukocytes and mediate selective trafficking at multiple stages, attracting specific populations to the site of injury [7,8]. In the kidney, tubular epithelial cells are
452
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considered to be a prominent source of chemokines [9]. Therefore, protecting tubular epithelial cells against I/R injury makes it possible to reduce interstitial fibrosis. Hepatocyte growth factor (HGF) and macrophagestimulating protein (MSP) have an intrinsic dual nature; while they are protective cytokines that prevent apoptosis and support regeneration, they also promote invasion. For example, HGF administration prevents cyclosporineinduced acute renal failure [10] and ameliorates renal ischemic injury [11]. On the other hand, HGF antagonists were found to inhibit tumor invasiveness [12]. Similarly, HGF-like factor, MSP, is a pleiotropic factor capable of stimulating resident peritoneal macrophages [13], but prevents apoptosis of epithelial cells [14]. HGF and MSP each have a large molecular mass and require proteolytic processing to be active. Studies in mice have indicated that injected precursors are not activated by endogenous proteases [15]. In addition, endogenous proHGF and pro-MSP are ubiquitously present in plasma and in the extracellular matrix of tissues at high concentrations, and thus injection of recombinant precursors simply increases the concentration of inactive factors. Therefore, to obtain a biological response, pre-activated factors must be activated. The activation of scatter factors in vitro by proteases involves the re-purification of factors after proteolysis, and activated HGF and MSP are less stable than their precursors, at least in vitro. It was recently shown that a recombinant chimera between selected domains of HGF and MSP (MF-1) (i) has a lower molecular mass than either of the parent molecules; (ii) does not require proteolytic activation; (iii) binds independently to both the Met and Ron receptors, while eliciting biological signals only through MetRon heterodimerization; (iv) promotes proliferation and protection against apoptosis at nanomolar concentrations in cells co-expressing the two receptors; (v) is devoid of pro-invasive activity; and (vi) prevents drug-induced acute renal damage in mice [16]. Here, we examined the therapeutic effects of MF-1 on I/R injury. Materials and methods Purification of MF-1. The MF-1 gene was transduced in MDA-MB 425 cells by the lenti viral vector. Secreted MF-1 protein was purified by dualstep affinity chromatography using a heparin-Sepharose and Ni2+-chelate column (Amersham Pharmacia, Uppsala, Sweden) [16]. Cell culture. Human renal proximal tubular epithelial cells (human kidney-2, HK-2; ATCC, Manassas, VA) were cultured in DMEM with (5%) FCS under a 5% CO2 and 95% air atmosphere at 37 C. To examine the renoprotective effects of MF-1, we added MF-1 to the culture medium (final concentrations of 0, 50 or 100 ng/ml). Hypoxia was then induced by incubating the plate under low-oxygen conditions. Oxygen concentration was maintained at 1% using the compact gas oxygen controller APM-36 (ASTEC, Fukuoka, Japan) with a residual gas mixture composed of 94% N2 and 5% CO2. Lactate dehydrogenase (LDH) assay and apoptosis assay were conducted after incubation for 15 h under low-oxygen condition. In order to evaluate cell viability, LDH activity was measured in cell culture medium and cell lysates by quantitative biochemical assay according to the manufacturer’s instructions (Roche Diagnostics). Cell
viability was assessed as the percentage of total LDH activity released into the medium. We also confirmed hypoxia-induced apoptosis by apoptosis assay. Briefly, a cell-death enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics, Indianapolis, IN) was used to assess hypoxia-induced apoptosis in cultured HK-2 cells with/without MF-1 in accordance with the kit manufacturer’s protocol. HK-2 cells were dispensed in 96-well plates at a density 5 · 103 cells/well. With/without MF-1 co-incubation, HK-2 cells were then exposed to hypoxia (1% O2) for 15 h. The relative decrease in apoptosis was determined by comparing results in MF-1treated cells with those in untreated controls. Experimental design. Male Sprague–Dawley rats weighing 150 g were purchased from Japan SLC Inc. (Hamamatsu, Japan) and were maintained under standard conditions until experiments were completed. All studies were performed in accordance with the principles of the Guidelines on Animal Experimentation of Osaka University. Rats were randomly allocated into 2 groups: (1) the saline-treatment group (control group; n = 12) and (2) the MF-1-treatment group (MF-1 group; n = 12). Rats were then subjected to I/R injury. All rats were anesthetized with an intraperitoneal injection of sodium thiopentone (30 mg/kg), and were allowed to stabilize for 30 minutes (min) before they were subjected to 45 min of bilateral renal occlusion using artery clips to clamp the renal pedicles. Occlusion was visually confirmed by a change in kidney color to a paler shade. Reperfusion was initiated with the removal of artery clips and was visually confirmed by noting the subsequent blush. Prior to the removal of the artery clips, rats received saline or MF-1 (20 lg in 500 ll of saline) via the tail vein. Rats were sacrificed 72 h after reperfusion. Blood samples (1 ml) were collected from the tail veins of anesthetized rats for measurement of serum creatinine and blood urea nitrogen (BUN) levels at 72 h after reperfusion. Antibodies. In order to detect the pathways that protect kidneys, we used the following antibodies for Western blotting: polyclonal phosphoAkt (Ser473) antibody (1:1000, Cell Signaling Technology, Beverly, MA); polyclonal Akt antibody (1:1000, Cell Signaling); and Caspase 3 (1:1000 dilution; BD Biosciences San Jose, CA). Macrophage infiltration. In order to examine the effects of MF-1 on macrophage infiltration, we used the monoclonal antibody ED1 (macrophage marker; Serotec, Oxford, UK). Tissue samples were fixed in 4% (wt./vol.) of buffered paraformaldehyde (PFA) for 16 h, and were then embedded in paraffin. Sections (4 lm) were mounted on silane (2% 3-aminopropyltriethoxysilane)-coated slides (Muto Pure Chemicals, Tokyo, Japan) and were deparaffinized with xylene. Immunohistochemical staining was performed using the Envision system (Dako), according to the manufacturer’s instructions. Endogenous peroxidase activities were blocked with 3% H2O2 for 10 min. The monoclonal antibody ED1 was diluted in 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T), and was then incubated for 24 h at 4 C. This was followed by incubation with secondary antibodies. All incubations were performed in a humidified chamber. Chromogenic color was developed with 3,3 0 diaminobenzidine tetrahydrochloride (DAB; Dako). Negative controls omitted the first antibodies and were prepared for each reaction. All histological slides were examined by light microscopy using a Nikon Eclipse 80 i (Nikon, Tokyo, Japan); pictures were taken with the Nikon ACT-1 ver. 2.63. ED-1-positive cell cells were counted in 10 random areas per rat, and the number of ED-1-positive cells per mm2 was then calculated. Terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining. TUNEL staining was performed using the in situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan), according to the manufacturer’s instructions. Briefly, sections were deparaffinized and subjected to antigen retrieval in preheated 10 mmol/l sodium citrate (pH 7) using a steamer for 40 min. Sections were then incubated with 3% H2O2 for 10 min, followed by incubation with TdT enzyme solution for 90 min at 37 C. The reaction was terminated by incubation in a stop/wash buffer for 30 min at 37 C. The number of TUNEL-positive cell nuclei and the total numbers of cell nuclei stained with hematoxylin were counted in 10 random areas, and the percentage of TUNEL-positive nuclei against total cell nuclei was then calculated.
F. Xue et al. / Biochemical and Biophysical Research Communications 363 (2007) 451–456 Western blot analysis. Kidney tissue was homogenized in a radioimmunoprecipitation (RIPA) Lysis Buffer with phenylmethylsulfonylfluoride (PMSF) solution, sodium orthovanadate solution, and protease inhibitor (Santa Cruz). Homogenates were centrifuged (12,000g for 10 min at 4 C), and total protein in the supernatant was measured by Lowry protein assay (Bio-Rad, Hercules, CA). Total protein lysate (15 lg) containing 1:1 denaturing sample buffer was boiled for 3 min and resolved on 5.0–10.0% SDS–polyacrylamide gels and was electrophoretically transferred onto an immobilon PVDF membrane (Millipore, Bedford, MA). The membrane was blocked with 5% (wt./vol.) nonfat milk or 1% BSA in 10 mM Trisbuffered saline with 0.1% Tween 20 (TBS-T), followed by overnight incubation at 4 C with diluted primary antibodies in TBS-T or blocking buffer. After washing with TBS-T 5 times, the membrane was incubated with secondary antibody (1:1000) (Cell Signaling) in TBS-T for 45 min at room temperature and was developed using ECL reagents (Amersham Bioscience Corp., Piscataway, NJ) to detect specific protein bands. Band density was analyzed by NIH Image software. Statistical analysis. Data are expressed as means ± SD. Statistical analysis was performed by unpaired t-test or ANOVA for multiple comparisons, followed by Scheffe’s F-test.
Results Effects of MF-1 on hypoxia-induced injury in cultured proximal tubular epithelial cells In cultures of HK-2 cells, 15-h exposure to hypoxia decreased viability, as assessed by released LDH activity (Fig. 1A). Incubation with MF-1 (50 and 100 ng/ml) significantly reduced the release of LDH in a dose dependent manner, thus suggesting that MF-1 directly protects tubular epithelial cells from hypoxia-induced necrosis. In addition, cell-death ELISA revealed that HK-2 cells exhibited an increase in DNA fragmentation under hypoxic conditions. Treatment with MF-1 (50 and 100 ng/ml) significantly inhibited DNA fragmentation, which suggests that MF-1 suppressed tubular epithelial apoptosis under hypoxia (Fig. 1B).
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Effects of MF-1 on renal function Saline-treated rats demonstrated a significant increase in serum creatinine (0.95 ± 0.46 mg/dl) and BUN (61.8 ± 32.9 mg/dl) at 72 h after I/R injury. In contrast, MF-1 treatment significantly suppressed the increases in creatinine (0.41 ± 0.13 mg/dl; p < 0.05, vs. control group) and BUN (25.4 ± 5.9 mg/dl; p < 0.05, vs. control group). Effects of MF-1 on tubular apoptosis in I/R-injured kidney In order to elucidate the protective mechanisms of MF-1, we performed TUNEL immunostaining to quantify the number of apoptotic cells. In the control group, TUNEL positive, apoptotic cells increased among tubular epithelial cells at 72 h after I/R injury (percent of TUNEL-positive cells, 7.2 ± 2.5%) (Fig. 2A and C). In contrast, apoptotic cells were significantly decreased in the MF-1 group (3.4 ± 1.1%; p < 0.05, vs. saline group) (Fig. 2B and C). Western blot analysis demonstrated that active (cleaved) caspase 3 was increased in saline-treated I/R injury model rats at 72 h, while active caspase 3 was decreased in MF-1treated rats (Fig. 3A). These data indicate that I/R of kidney induces apoptosis in tubular epithelial cells, and this is markedly prevented by MF-1. To elucidate the intracellular MF-1 signaling implicated in tubular protection, we examined the expression and activation of Akt (Fig. 3B). Western blot analysis demonstrated that treatment with MF-1 increased phosphorylation of Akt at 72 h after I/R injury, while phosphorylated Akt was not observed in the saline group.
Fig. 1. Effects of MF-1 on hypoxia-induced cell death. (A) HK-2 cells, when exposed to hypoxia for 15 h, showed decreased cell viability, as assessed by LDH assay, while incubation with MF-1 (50 and 100 ng/ml) significantly reduced the release of LDH (*p < 0.01, vs. control). (B) HK-2 cells exhibited increased DNA fragmentation under hypoxic conditions. Treatment with MF-1 (50 and 100 ng/ml) significantly inhibited DNA fragmentation (*p < 0.01, **p < 0.001 vs. control).
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Fig. 2. Effects of MF-1 on apoptosis at 72 h. Representative TUNEL immunostaining results for saline group (A), and MF-1 group (B) are shown. Dark brown dots correspond to representative TUNEL-positive nuclei. (C) MF-1 treatment significantly decreased TUNEL positive, apoptotic cells (*p < 0.05 vs. control group) (magnification, 200·).
Fig. 3. Western blot analysis of I/R-injured kidney. (A) Western blot analysis confirmed that the cleaved (active) form of caspase 3 (18 kD) was suppressed in MF-1-treated rats when compared to saline treatment. (B) Western blot analysis demonstrated that treatment with MF-1 increased Akt phosphorylation at 72 h after I/R injury, whiel 500 IU EPO or CEPO had no effect on Akt activation.
Effects on interstitial macrophage infiltration in I/R-injured kidney In order to detect interstitial macrophage infiltration, which is associated with interstitial damage and fibrosis, expression of ED-1 was examined immunohistochemically. Interstitial expression of ED-1 increased at 72 h after I/R injury in saline-treated rats (Fig. 4A and C), while MF-1 treatment significantly suppressed interstitial ED-1 expression and macrophage infiltration (Fig. 4B and C). Discussion In this paper, we demonstrated that MF-1 has therapeutic effects in a rat model of I/R injury. This is based on the fact that MF-1 directly protects tubular epithelial
cells from hypoxia-induced necrosis and apoptosis. We then investigated whether treatment with MF-1 is able to suppress tubular apoptosis and macrophage infiltration in a rat I/R injury model. Untreated kidneys exhibited increased creatinine and BUN with tubular apoptosis and interstitial macrophage infiltration. On the other hand, MF-1 treatment ameliorated renal dysfunction, and inhibited tubular apoptosis and macrophage infiltration. We also demonstrated that protection of the proximal tubular epithelial cells by MF-1 was attenuated by Akt phosphorylation. Once activated, Akt activates multiple targets with anti-apoptotic effects, including phosophorylation of Bad, Bax, caspase 9, and GSK-3b [17]. Thus, MF-1 appears to protect proximal tubular epithelial cells against I/R injury by phosphorylation of Akt, resulting in the inhibition of tubular apoptotic cell death, which leads to the attenuation of macrophage recruitment. Chimeric MF-1, derived from the a-chains of HGF and MSP, concomitantly activates the HGF receptor (Met) and the MSP receptor (Ron). Although the trophic effects of MF-1 would be restricted to tissue expressing both Met and Ron, various tissues, including tubular epithelial cells, express both Met and Ron [15,18]. HGF and MSP have an intrinsic dual nature; they are trophic cytokines that prevent apoptosis, and are scatter factors that promote invasion. For example, HGF/Met signaling activates proteases and mediates cellular inva-
Fig. 4. Effects on macrophage infiltration at 72 h. Interstitial macrophage infiltration was assessed by immunohistochemical staining of ED-1 in the control group (A), and MF-1 group (B). (C) Quantification (per mm2) of ED-1 positive macrophages in kidney sections. Data shown are means ± SD for 10 independently performed experiments (*p < 0.001 vs. control group).
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sion and metastasis [19]. Therefore, separation of the favorable effects of HGF and MSP from their adverse effects is a promising technique. MF-1 offers both biochemical and biological advantages over HGF and MSP. From a biochemical point of view, HGF and MSP have a large molecular mass and require proteolytic processing to be active [16]. In contrast, MF-1 has a lower molecular mass than either HGF or MSP and does not require proteolytic activation [16]. From a biological point of view, the ability of MF-1 to promote cell survival without inducing invasion is important to its potential therapeutic application [16]. Based on our results, MF-1 appears to have antiapoptotic function against I/R injury. We demonstrated that MF-1 treatment markedly suppresses I/R injuryinduced tubular epithelial apoptosis when compared to saline treatment. Furthermore, MF-1 inhibited hypoxiainduced apoptosis. One possible signal transduction pathway by which tubular apoptosis could be suppressed involves the activation of Akt. In fact, on Western blot analysis, we found that MF-1 treatment induced Akt phosphorylation at 72 h after I/R injury. It has been reported that suppression of c-Met phosphorylation by anti-HGF IgG leads to rapid progression of tubular apoptosis, while enhancement of c-Met activation by exogenous HGF blocks tubular apoptotic injury [20]. In this process, HGF prevents nuclear factor kappa B (NF-jB) activation, which has been implicated in events downstream of Akt. In addition, MSP-induced prevention of apoptosis is reportedly mediated by the Akt pathway [14]. Taken together with our observations, these data suggest that MF-1 protects tubular cells from I/Rinduced apoptosis via Akt phosphorylation. Although we demonstrated that MF-1 treatment reduced macrophage infiltration in the interstitium of I/R-injured kidneys, MSP is reported to stimulate resident peritoneal macrophages [13]. It was hypothesized that MSP released by tubular cells in the course of tubulointerstitial inflammatory disorders stimulates resident macrophages and recruits circulating monocytes, thus amplifying the inflammatory response [18]. In contrast, the HGF–MSP chimera MF-1 suppressed macrophage infiltration. One explanation for this observation is that HGF itself inhibits macrophage recruitment by suppressing the expression of proinflammatory chemokine MCP-1 (macrophage chemoattractant protein-1) and RANTES (regulated upon expression normal T cell expressed and secreted), and inhibits activation of the NF-jB transcription factor [9]. Another explanation is that MF-1 binds to Met and Ron with lower affinity than does HGF or MSP, and is able to mediate a biological signal only through Met–Ron heterodimers [16], by which MF-1 might behave as an antagonist of scatter factors. In conclusion, MF-1 treatment up-regulated the phosphorylation of Akt, and resulted in the inhibition of caspase 3 activation and tubular apoptosis, thereby ameliorating
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