Prevention of Neutrophil Extravasation by Hepatocyte Growth Factor Leads to Attenuations of Tubular Apoptosis and Renal Dysfunction in Mouse Ischemic Kidneys
2005; Elsevier BV; Volume: 166; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)62498-4
ISSN1525-2191
AutoresShinya Mizuno, Toshikazu Nakamura,
Tópico(s)Phagocytosis and Immune Regulation
ResumoIschemia and reperfusion (I/R) injuries occur in numerous organs under pathophysiological conditions. In this process, neutrophils play important roles in eliciting parenchymal injuries. Using a murine model of renal I/R, we show that hepatocyte growth factor (HGF) is a natural ligand that inhibits endothelial injuries and neutrophil extravasation. In mice after renal I/R, plasma HGF levels increased, along with c-Met/HGF receptor phosphorylation in the vascular endothelium. However, this c-Met activation was transient, associated with a decrease in endogenous HGF level, and followed by neutrophil infiltration and renal dysfunction. Suppression of endothelial c-Met phosphorylation by anti-HGF IgG led to rapid progressions of neutrophil extravasation, tubular apoptosis, and renal dysfunction. Inversely, enhancement of the c-Met activation by exogenous HGF blocked endothelial/tubular apoptotic injuries and acute renal failure. In this process, HGF prevented endothelial nuclear factor κB activation and inhibited induction of an adhesion molecule (ICAM-1), resulting in attenuated vascular edema and neutrophil infiltration. Thus, we conclude that 1) the HGF/c-Met system of endothelial cells confers an initial barrier to block neutrophil infiltration, and 2) transient and insufficient HGF production allows manifestation of postischemic renal failure. Our study provides a rationale for why HGF supplementation elicits therapeutic effects in ischemic kidneys. Ischemia and reperfusion (I/R) injuries occur in numerous organs under pathophysiological conditions. In this process, neutrophils play important roles in eliciting parenchymal injuries. Using a murine model of renal I/R, we show that hepatocyte growth factor (HGF) is a natural ligand that inhibits endothelial injuries and neutrophil extravasation. In mice after renal I/R, plasma HGF levels increased, along with c-Met/HGF receptor phosphorylation in the vascular endothelium. However, this c-Met activation was transient, associated with a decrease in endogenous HGF level, and followed by neutrophil infiltration and renal dysfunction. Suppression of endothelial c-Met phosphorylation by anti-HGF IgG led to rapid progressions of neutrophil extravasation, tubular apoptosis, and renal dysfunction. Inversely, enhancement of the c-Met activation by exogenous HGF blocked endothelial/tubular apoptotic injuries and acute renal failure. In this process, HGF prevented endothelial nuclear factor κB activation and inhibited induction of an adhesion molecule (ICAM-1), resulting in attenuated vascular edema and neutrophil infiltration. Thus, we conclude that 1) the HGF/c-Met system of endothelial cells confers an initial barrier to block neutrophil infiltration, and 2) transient and insufficient HGF production allows manifestation of postischemic renal failure. Our study provides a rationale for why HGF supplementation elicits therapeutic effects in ischemic kidneys. Neutrophil-endothelial interactions are the initial event involved in the onset of inflammatory diseases.1Beekhuizen H van-de Gevel JS Endothelial cell adhesion molecules in inflammation and postischemic reperfusion injury.Transplant Proc. 1998; 30: 4251-4256Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Especially in ischemia/reperfusion (I/R) injuries, circulating neutrophils selectively infiltrate ischemic tissues and contribute to tissue destruction.2Jordan JE Zhao ZQ Vinten-Johansen J The role of neutrophils in myocardial ischemia-reperfusion injury.Cardiovasc Res. 1999; 43: 860-878Crossref PubMed Scopus (563) Google Scholar, 3Martin TR Neutrophils and lung injury: getting it right.J Clin Invest. 2002; 110: 1603-1605Crossref PubMed Scopus (90) Google Scholar, 4Lauriat S Linas SL The role of neutrophils in acute renal failure.Semin Nephrol. 1998; 18: 498-504PubMed Google Scholar During the progression of I/R injuries, endothelial barrier is impaired within a few hours by hypoxia or neutrophil-released toxic products.1Beekhuizen H van-de Gevel JS Endothelial cell adhesion molecules in inflammation and postischemic reperfusion injury.Transplant Proc. 1998; 30: 4251-4256Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 4Lauriat S Linas SL The role of neutrophils in acute renal failure.Semin Nephrol. 1998; 18: 498-504PubMed Google Scholar, 5Scarabelli T Stephanou A Rayment N Pasini E Comini L Curello S Ferrari R Latchman D Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia and reperfusion injury.Circulation. 2001; 104: 253-256Crossref PubMed Scopus (294) Google Scholar Along with vascular damage, circulating neutrophils attach and transmigrate between injured endothelial cells, the result being peri-vascular edema (so called as extravasation). In neutrophil-infiltrated organs, tissue damages are accelerated in part via neutrophil-released radicals or proteinases such as elastases.1Beekhuizen H van-de Gevel JS Endothelial cell adhesion molecules in inflammation and postischemic reperfusion injury.Transplant Proc. 1998; 30: 4251-4256Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 4Lauriat S Linas SL The role of neutrophils in acute renal failure.Semin Nephrol. 1998; 18: 498-504PubMed Google Scholar Thus, current opinion defines neutrophils as a key mediator for inflammation under acute stress (including ischemic damage). In kidneys, ischemia is one of the most important causes of acute renal diseases.6Nissenson AR Acute renal failure: definition and pathogenesis.Kidney Int. 1998; 66: S7-S10Google Scholar Especially in renal transplantation, initial I/R injury affects long-term outcome of renal graft survival.7Hetzel GR Klein B Brause M Westhoff A Willers R Sandmann W Grabensee B Risk factors for delayed graft function after renal transplantation and their significance for long-term clinical outcome.Transpl Int. 2002; 15: 10-16Crossref PubMed Scopus (96) Google Scholar In I/R-undergone kidneys, tubular epithelial apoptosis participates in nephron destruction and renal dysfunction.8Schumer M Colombel MC Sawczuk IS Gobe G Connor J O'Toole KM Olsson CA Wise GJ Buttyan R Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia.Am J Pathol. 1992; 140: 831-838PubMed Google Scholar, 9Burns AT Davies DR McLaren AJ Cerundolo L Morris PJ Fuggle SV Apoptosis in ischemia/reperfusion injury of human renal allografts.Transplantation. 1998; 66: 872-876Crossref PubMed Scopus (136) Google Scholar In this process, infiltrated neutrophils (associated with endothelial injuries) play an important role in nephron destructions.4Lauriat S Linas SL The role of neutrophils in acute renal failure.Semin Nephrol. 1998; 18: 498-504PubMed Google Scholar, 10Klausner JM Paterson IS Goldman G Kobzik L Rodzen C Lawrence R Valeri CR Shepro D Hechtman HB Postischemic renal injury is mediated by neutrophils and leukotrienes.Am J Physiol. 1989; 256: F794-F802PubMed Google Scholar In other words, inhibition of neutrophil extravasation can be a target for abrogating tubular injuries and dysfunction.11Singbartl K Forlow SB Ley K Platelet, but not endothelial, P-selectin is critical for neutrophil-mediated acute postischemic renal failure.FASEB J. 2001; 15: 2337-2344Crossref PubMed Scopus (144) Google Scholar, 12Sato W Kadomatsu K Yuzawa Y Muramatsu H Hotta N Matsuo S Muramatsu T Midkine is involved in neutrophil infiltration into the tubulointerstitium in ischemic renal injury.J Immunol. 2001; 167: 3463-3469PubMed Google Scholar There may be an intrinsic defense system to suppress or retard initial neutrophil-endothelial interactions. If so, enhancement of this system may lead to avoidance of I/R-related renal injuries. However, it is still poorly understood how renal tissues are physiologically protected from postischemic challenge. Hepatocyte growth factor (HGF), originally identified and cloned as a mitogen for mature hepatocytes,13Nakamura T Nawa K Ichihara A Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats.Biochem Biophys Res Commun. 1984; 122: 1450-1459Crossref PubMed Scopus (1099) Google Scholar, 14Nakamura T Nishizawa T Hagiya M Seki T Shimonishi M Sugimura A Tashiro K Shimizu S Molecular cloning and expression of human hepatocyte growth factor.Nature. 1989; 342: 440-443Crossref PubMed Scopus (1987) Google Scholar has mitogenic, motogenic, and morphogenic activities in various cell types via c-Met/HGF receptor phosphorylation.15Matsumoto K Nakamura T Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases.Kidney Int. 2001; 59: 2023-2038PubMed Google Scholar, 16Balkovetz DF Lipschutz JH Hepatocyte growth factor and the kidney: it is not just for the liver.Int Rev Cytol. 1999; 186: 225-260Crossref PubMed Google Scholar, 17Funakoshi H Nakamura T Hepatocyte growth factor: from diagnosis to clinical applications.Clin Chim Acta. 2003; 327: 1-23Crossref PubMed Scopus (173) Google Scholar In renal tissues, HGF is protective and regenerative toward tubular epithelial cells (as well as glomerular cells).15Matsumoto K Nakamura T Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases.Kidney Int. 2001; 59: 2023-2038PubMed Google Scholar, 16Balkovetz DF Lipschutz JH Hepatocyte growth factor and the kidney: it is not just for the liver.Int Rev Cytol. 1999; 186: 225-260Crossref PubMed Google Scholar HGF prevents onsets of tubular injuries and acute renal failure in various animal models, while more importantly, nephron regeneration is accelerated by HGF even after the onset of acute renal failure.18Kawaida K Matsumoto K Shimazu H Nakamura T Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice.Proc Natl Acad Sci. 1994; 91: 4357-4361Crossref PubMed Scopus (393) Google Scholar, 19Miller SB Martin DR Kissane J Hammerman MR Hepatocyte growth factor accelerates recovery from acute ischemic renal injury in rats.Am J Physiol. 1994; 266: F129-F134PubMed Google Scholar, 20Dai C Yang J Liu Y Single injection of naked plasmid encoding hepatocyte growth factor prevents cell death and ameliorates acute renal failure in mice.J Am Soc Nephrol. 2002; 13: 411-422Crossref PubMed Google Scholar HGF is also cytoprotective for vascular endothelial cells.15Matsumoto K Nakamura T Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases.Kidney Int. 2001; 59: 2023-2038PubMed Google Scholar Nevertheless, whether and how HGF modulates neutrophil-endothelial interaction (before or during inflammation) remained to be determined. Surgical treatment of I/R induces neutrophil infiltration and tubular apoptosis in rodent kidneys,8Schumer M Colombel MC Sawczuk IS Gobe G Connor J O'Toole KM Olsson CA Wise GJ Buttyan R Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia.Am J Pathol. 1992; 140: 831-838PubMed Google Scholar, 11Singbartl K Forlow SB Ley K Platelet, but not endothelial, P-selectin is critical for neutrophil-mediated acute postischemic renal failure.FASEB J. 2001; 15: 2337-2344Crossref PubMed Scopus (144) Google Scholar, 12Sato W Kadomatsu K Yuzawa Y Muramatsu H Hotta N Matsuo S Muramatsu T Midkine is involved in neutrophil infiltration into the tubulointerstitium in ischemic renal injury.J Immunol. 2001; 167: 3463-3469PubMed Google Scholar which mimics ischemic renal diseases in humans. Using the well-documented model, we provide herein evidence that HGF is an intrinsic ligand to inhibit neutrophil infiltration and endothelial/tubular apoptosis, contributing to preventions of nephron destruction and dysfunction. Based on all available data, we discuss physiological and therapeutic functions of HGF to block vascular inflammatory response, a common event in ischemic organs. Eight-week-old female ICR mice (26–28 g; Slc, Hama-matsu, Japan) were anesthetized with ketamine chloride (80 mg/kg, subcutaneous (s.c.)) and xyladine sulfate (8 mg/kg, s.c.). Under general anesthesia, they underwent ischemia of the left renal artery with a vascular clamp (38°C for 30 minutes). After release of this ischemia, contra-lateral (ie, right) nephrectomy was done to elicit acute renal dysfunction.21Ishibashi N Weisbrot-Lefkowitz M Reuhl K Inouye M Mirochnitchenko O Modulation of chemokine expression during ischemia/reperfusion in transgenic mice overproducing human glutathione peroxidases.J Immunol. 1999; 163: 5666-5677PubMed Google Scholar For analysis of histopathological and biochemical changes during progression of renal I/R, 36 mice were killed at 3, 6, 12, and 18 hours after the surgical treatment of renal I/R. At the time of necropsy on six mice, renal tissues were fixed in 70% ethanol (or in part, 10% formalin) at 4°C overnight for immunohistochemistry, as described below. Remaining tissues were rapidly frozen in liquid nitrogen and stocked at −80°C for biochemical analysis. For neutralization of endogenous HGF, an anti-HGF antibody specific for rat HGF was used. This antibody shows cross-reactivity with mouse HGF (but not human HGF) and accelerates tissue injuries in kidneys.22Mizuno S Nakamura T Suppression of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy.Am J Physiol. 2004; 286: F134-F143Crossref PubMed Scopus (105) Google Scholar, 23Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar Thus, the renal I/R mice were randomly divided into two groups and injected with rabbit anti-rat HGF IgG (n = 6) or normal rabbit IgG (n = 6) (250 μg/mouse, intraperitoneal) immediately after the renal I/R treatment. To determine the role of endogenous HGF, mice were killed at 1 and 8 hours after injecting the anti-HGF IgG. To evaluate the therapeutic effects of exogenous HGF on the progression of renal injuries, 24 renal I/R mice were generated and injected with saline or recombinant human HGF (rh-HGF), which was purified from medium of chinese hamster ovary (CHO) cells transfected with a vector containing HGF cDNA of the 5-amino acid-deleted type, with >98% of purity on SDS-PAGE.18Kawaida K Matsumoto K Shimazu H Nakamura T Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice.Proc Natl Acad Sci. 1994; 91: 4357-4361Crossref PubMed Scopus (393) Google Scholar, 22Mizuno S Nakamura T Suppression of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy.Am J Physiol. 2004; 286: F134-F143Crossref PubMed Scopus (105) Google Scholar, 23Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar Immediately after the release of warm ischemia, mice were given saline or rh-HGF (500 μg/kg, every 6 hour, s.c.) and then killed at 3 and 18 hours after the renal I/R (n = 6 per group). Renal tissues were fixed in 70% ethanol and embedded in paraffin, as reported.22Mizuno S Nakamura T Suppression of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy.Am J Physiol. 2004; 286: F134-F143Crossref PubMed Scopus (105) Google Scholar, 23Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar Tissue sections were cut at a thickness of 4 μm, dewaxed, and stained with hematoxylin and eosin (H&E). To identify neutrophil infiltration, anti-mouse neutrophil Gr1 rat monoclonal IgG (BD Biosciences, San Jose, CA) was applied to the dewaxed sections as the primary reaction, followed by a second reaction with biotin-labeled anti-rat IgG goat IgG (Vector, Burlingame, CA). Finally, an avidin biotin coupling (ABC) reaction was done on sections, using a kit (Vectastain Elite; Vector). To detect apoptosis, an in situ end-labeling method was applied on renal sections, using a kit (ApoTag; Intergen, Purchase, NY).23Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar Furthermore, c-Met activation was evaluated on the kidneys, using rabbit IgG against phosphospecific c-Met (1:400) (pMet-1234/1235; Biosource, Camarillo, CA). To evaluate the endothelial inflammation, nuclear factor κB (NF-κB) and ICAM-1 were detected using anti-NF-κB (p65 subunit) rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-mouse ICAM-1 hamster IgG (BD Biosciences), respectively. These antigens were visualized using the ABC method, with 3–3′ aminobenzidine (ie, as brown signals). In the NF-κB-stained or TUNEL-stained sections, endothelial cells were identified using an anti-mouse-CD31 rat IgG (BD Biosciences) and an alkaliphosphatase-conjugated anti-rat IgG rabbit IgG (Vector). The CD31-positive signals were visualized as red signals, with a new Fuchsin kit (DAKO, Glostrup, Denmark). In the immunohistochemistry of phosphorylated c-Met and NF-κB, normal rabbit IgG (DAKO) was used as negative control. The degree of neutrophil infiltration was expressed as a Gr1-staining score, determined by counting numbers of polynuclear cells positive for Gr1, in >20 randomly chosen ×200 fields per mouse (n = 6). To evaluate the degree of tubular apoptosis, TUNEL score was determined by counting numbers of positive tubular cells in >20 ×200 fields. Immunoreactive signals for endothelial ICAM-1 were observed in >20 randomly chosen ×200 fields and scored on the scale of 0, no staining; 0.5, trace staining; 1, light staining; 2, moderate staining; and 3, intense staining.24Schwarz C Regele H Steininger R Hansmann C Mayer G Oberbauer R The contribution of adhesion molecule expression in donor kidney biopsies to early allograft dysfunction.Transplantation. 2001; 71: 1666-1670Crossref PubMed Scopus (56) Google Scholar To quantify edematous lesions, renal sections were examined under a light microscope, and the degree of renal expression was expressed as a mean value of the following histochemical scales evaluated in at least 20 randomly chosen ×200 fields: 0, absent; 1, mild; 2, moderate; and 3, severe. Furthermore, vascular apoptosis was checked in >20 chosen ×200 fields, and its degrees were expressed as TUNEL-positive cells per CD31-positive vascular lumen. These quantitative analyses were all made in a blinded fashion. To evaluate renal functions after renal I/R, blood urea nitrogen (BUN) and plasma creatinine levels were determined using a urease indophenol method and by Jeffe's method, respectively.22Mizuno S Nakamura T Suppression of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy.Am J Physiol. 2004; 286: F134-F143Crossref PubMed Scopus (105) Google Scholar, 23Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar The renal extract was obtained as reported22Mizuno S Nakamura T Suppression of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy.Am J Physiol. 2004; 286: F134-F143Crossref PubMed Scopus (105) Google Scholar, 23Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar, and c-Met expression and phosphorylation were detected as follows:25Wen J Matsumoto K Taniura N Tomioka D Nakamura T Hepatic gene expression of NK4, an HGF-antagonist/angiogenesis inhibitor, suppresses liver metastasis and invasive growth of colon cancer in mice.Cancer Gene Ther. 2004; 11: 419-430Crossref PubMed Scopus (67) Google Scholar total and phosphorylated c-Met were visualized on polyvinylidene difluoride (PVDF) membranes, with Western blotting. In the immunoblot analysis, β-actin was detected as an internal control. The c-Met expression and phosphorylation levels were quantified by a densitometric analysis, using computer software (NIH image). Endogenous HGF (ie, mouse HGF) levels in plasma and kidneys were determined, using an enzyme-linked immunosorbent assay (ELISA) for specifically detecting mouse (but not human) HGF.22Mizuno S Nakamura T Suppression of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy.Am J Physiol. 2004; 286: F134-F143Crossref PubMed Scopus (105) Google Scholar, 23Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-β1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar Furthermore, the human HGF level was monitored during rh-HGF administrations, using a human HGF-specific ELISA.25Wen J Matsumoto K Taniura N Tomioka D Nakamura T Hepatic gene expression of NK4, an HGF-antagonist/angiogenesis inhibitor, suppresses liver metastasis and invasive growth of colon cancer in mice.Cancer Gene Ther. 2004; 11: 419-430Crossref PubMed Scopus (67) Google Scholar To quantify the degree of infiltrated neutrophils, myeloperoxidase (MPO) activity before and after renal I/R was determined, as described previously.26Daemen MARC Veer C Denecker G Heemskerk VH Wolfs TGAM Clauss M Vandenabeele P Buurman WA Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation.J Clin Invest. 1999; 104: 541-549Crossref PubMed Scopus (494) Google Scholar To determine the effects of HGF on neutrophil-to-endothelial adhesion, an in vitro model was prepared, according to a reported method,27Taekema-Roelvink MEJ van Kooten C van der Kooij S Heemskerk E Daha MR Proteinase 3 enhances endothelial monocyte chemo attractant protein-1 production and induces increased adhesion of neutrophils to endothelial cells by up-regulating intercellular cell adhesion molecule-1.J Am Soc Nephrol. 2001; 12: 932-940PubMed Google Scholar with slight modifications. Umbilical vein endothelial cells (HUVECs) (Japan Health Sciences, Tokyo, Japan) were adjusted to a density of 1 × 104 cells/well and incubated at 37°C for 48 hours in 0.1% fetal bovine serum (FBS)-containing MCDB-131 (Gibco, Grand Island, NY). After washing twice with the medium, HUVECs were incubated in the presence of recombinant human tumor necrosis factor-α (TNF-α; 5 ng/ml) (R&D, Minneapolis, MN). Furthermore, HGF (3–30 ng/ml) or vehicle (0.1% bovine serum albumin (BSA)) was added into the HUVEC model 30-minute before adding TNF-α. The HUVECs were washed with medium 3 hour after adding TNF-α and used as an endothelial monolayer. Neutrophils were isolated from human peripheral blood with Ficoll-Conray gradients (P = 1.077 g/ml),27Taekema-Roelvink MEJ van Kooten C van der Kooij S Heemskerk E Daha MR Proteinase 3 enhances endothelial monocyte chemo attractant protein-1 production and induces increased adhesion of neutrophils to endothelial cells by up-regulating intercellular cell adhesion molecule-1.J Am Soc Nephrol. 2001; 12: 932-940PubMed Google Scholar with a >93% purity on Giemsa's staining. This neutrophil suspension was adjusted at 2 × 105 cells/well, placed onto the HUVEC monolayer, and kept for 30 minutes under static conditions to induce cell attachment. Each culture well was gently washed twice with medium to remove nonadherent cells, and then attached neutrophils were quantified using a MPO assay.27Taekema-Roelvink MEJ van Kooten C van der Kooij S Heemskerk E Daha MR Proteinase 3 enhances endothelial monocyte chemo attractant protein-1 production and induces increased adhesion of neutrophils to endothelial cells by up-regulating intercellular cell adhesion molecule-1.J Am Soc Nephrol. 2001; 12: 932-940PubMed Google Scholar For the immunoblot of ICAM-1, HUVEC lysates were obtained from whole cells (3 × 105 cells) and collected into lysis buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 10 mmol/L EDTA, and 0.5%-Triton X-100, pH 7.4). The lysates were subjected to SDS-PAGE, electrotransfer, and blotting procedures. Anti-human ICAM-1 mouse IgG (1:500; Sigma, St. Louis, MO) was applied on PVDF membranes as mentioned. After reaction with peroxidase-conjugated anti-mouse IgG (DAKO), immunoreactive signals were visualized on the PVDF membranes,22Mizuno S Nakamura T Suppression of chronic glomerular injuries and TGF-β1 production by HGF in attenuation of murine diabetic nephropathy.Am J Physiol. 2004; 286: F134-F143Crossref PubMed Scopus (105) Google Scholar using an ECL kit (Amersham Pharmacia, Little Chalfont, UK). Endothelial ICAM-1 and NF-κB were immunocytochemically detected after incubation for 3 hours in the presence of TNF-α or HGF plus TNF-α: these cells were fixed on a glass disk in cold 70% ethanol for 30 minutes, then subjected to the ABC technique, using a primary antibody against NF-κB or against ICAM-1, as mentioned. On the other hand, we obtained the nuclear extraction from remaining HUVEC cell suspension with a slow centrifugation and checked the nuclear NF-κB levels on ECL immunoblots. Finally, NF-κB activation was quantified using the TransAM NF-κB kit (Active Motif, Rixensart, Belgium), as reported previously.28Palomba L Persichini T Mazzone V Colasanti M Cantoni O Inhibition of nitric-oxide synthase-I (NOS-I)-dependent nitric oxide production by lipopoly-saccharide plus interferon-γ is mediated by arachidonic acid.J Biol Chem. 2004; 279: 29895-29901Crossref PubMed Scopus (36) Google Scholar Briefly, whole-cell extracts were prepared from 5 × 106 HUVECs, and protein concentrations of cell extracts were determined using a kit (DC protein assay; Bio-Rad, Hercules, CA). Ten micrograms per well of cell extracts was incubated in a 96-well plate on which have been immobilized double-strand oligonucleotides containing the sequence NF-κB DNA-binding site (5′-GGGACTTTCC-3′). The primary antibody used to detect NF-κB recognized an epitope on p65 subunit that is accessible only when NF-κB is activated and bound to its target DNA. After incubation with a peroxidase-conjugated secondary antibody and the developing solution, absorbance was read at 450 nm, according to a manufacturer's instruction. All data were expressed as mean ± SD. A Student's t-test or analysis of variance test for parametric values and Mann-Whitney U-test for non-parametric values were used to compare group means, with P < 0.05 accepted as significant. To determine changes in endogenous HGF levels during a process of postischemic renal injuries, we prepared a murine model of renal I/R, according to a previous report.21Ishibashi N Weisbrot-Lefkowitz M Reuhl K Inouye M Mirochnitchenko O Modulation of chemokine expression during ischemia/reperfusion in transgenic mice overproducing human glutathione peroxidases.J Immunol. 1999; 163: 5666-5677PubMed Google Scholar In our model, BUN levels rapidly increased within 24 hours (0 hour, 21.2 ± 2.28 mg/dl; 6 hours, 56.3 ± 7.13 mg/dl; 24 hours, 108.1 ± 6.23 mg/dl). In the renal tissues, infiltration of neutrophils (ie, Gr1-positive round cells) was noted around cortico-medullar areas especially from 3 hours after the I/R treatment (Figure 1A). Being in parallel with the enhanced neutrophil infiltration, tubular epithelial apoptosis (as evidenced by TUNEL staining) became severer, concomitantly with tubular dilatation and epithelial desquamation (Figure 1B). In the murine model, plasma and renal HGF levels were biphasic with peaks 1 and 24 hours after renal I/R treatment (Figure 1C). The later peak correlated well with G1/S progression in a cell cycle of the tubular cells (data not shown), indicating a renotropic role of HGF, as reported previously.29Igawa T Matsumoto K Kanda S Saito Y Nakamura T Hepatocyte growth factor may function as a renotropic factor for regeneration in rats with acute renal injury.Am J Physiol. 1993; 265: F61-F69PubMed Google Scholar In contrast, it is still unknown whether initial HGF elevation may alter fate of postischemic renal diseases. Thus, we hypothesized that the initial increases in HGF levels may be involved in protecting nephrons against I/R-induced challenges. To test our hypothesis, we injected an antibody specific to rodent HGF into mice immediately after renal I/R. In normal IgG-treated control mice, both the expression and phosphorylation of c-Met were evident at 1 hour after release from ischemia, whereas c-Met expression and activation were inhibited to below about 20% of basal levels by anti-HGF IgG treatment (Figure 2A). The loss of total c-Met expression levels with HGF neutralization may suggest that renal c-Met expression is in part dependent on ligand HGF, as reported previously.30Liu Y Tolbert EM Lin L Thursby MA Sun AM Nakamura T Dworkin LD Up-regulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure.Kidney Int. 1999; 55: 442-453Crossref PubMed Scopus (103) Google Scholar Under such HGF-neutralized conditions, there were significant increases in BUN and plasma creatinine levels at 8 hours postischemia in the HGF-neutralized mice (Figure 2B). Furthermore, anti-HGF IgG significantly enhanced tubular apoptosis at 8 hours after the renal I/R treatment (Figure 2C). The tubular TUNEL score of the anti-HGF IgG group increased to a twofold level of the placebo control group (P < 0.05). Consistently, t
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