Hepatocyte Growth Factor Exerts Its Anti-Inflammatory Action by Disrupting Nuclear Factor-κB Signaling
2008; Elsevier BV; Volume: 173; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.070583
ISSN1525-2191
AutoresMyrto Giannopoulou, Chunsun Dai, Xiaoyue Tan, Xiao‐Yan Wen, George K. Michalopoulos, Youhua Liu,
Tópico(s)Phagocytosis and Immune Regulation
ResumoRenal inflammation, characterized by the influx of inflammatory cells, is believed to play a critical role in the initiation and progression of a wide range of chronic kidney diseases. Here, we show that hepatocyte growth factor (HGF) inhibited renal inflammation and proinflammatory chemokine expression by disrupting nuclear factor (NF)-κB signaling. In vivo, HGF gene delivery inhibited interstitial infiltration of inflammatory T cells and macrophages, and suppressed expression of both RANTES (regulated on activation, normal T cell expressed and secreted) and monocyte chemoattractant protein-1 in a mouse model of obstructive nephropathy. In vitro, HGF abolished RANTES induction in human kidney epithelial cells, which was dependent on NF-κB signaling. HGF did not significantly affect the phosphorylation or degradation of IκBα; it also did not influence the phosphorylation or nuclear translocation of p65 NF-κB. However, HGF prevented p65 NF-κB binding to its cognate cis-acting element in the RANTES promoter. HGF action was dependent on the activation of the phosphoinositide 3-kinase/Akt pathway, which led to the phosphorylation and inactivation of glycogen synthase kinase (GSK)-3β. Suppression of GSK-3β activity mimicked HGF and abolished RANTES expression, whereas ectopic expression of GSK-3β restored RANTES induction. HGF also induced renal GSK-3β phosphorylation and inactivation after obstructive injury in vivo. These observations suggest that HGF is a potent anti-inflammatory cytokine that inhibits renal inflammation by disrupting NF-κB signaling and may be a promising therapeutic agent for progressive renal diseases. Renal inflammation, characterized by the influx of inflammatory cells, is believed to play a critical role in the initiation and progression of a wide range of chronic kidney diseases. Here, we show that hepatocyte growth factor (HGF) inhibited renal inflammation and proinflammatory chemokine expression by disrupting nuclear factor (NF)-κB signaling. In vivo, HGF gene delivery inhibited interstitial infiltration of inflammatory T cells and macrophages, and suppressed expression of both RANTES (regulated on activation, normal T cell expressed and secreted) and monocyte chemoattractant protein-1 in a mouse model of obstructive nephropathy. In vitro, HGF abolished RANTES induction in human kidney epithelial cells, which was dependent on NF-κB signaling. HGF did not significantly affect the phosphorylation or degradation of IκBα; it also did not influence the phosphorylation or nuclear translocation of p65 NF-κB. However, HGF prevented p65 NF-κB binding to its cognate cis-acting element in the RANTES promoter. HGF action was dependent on the activation of the phosphoinositide 3-kinase/Akt pathway, which led to the phosphorylation and inactivation of glycogen synthase kinase (GSK)-3β. Suppression of GSK-3β activity mimicked HGF and abolished RANTES expression, whereas ectopic expression of GSK-3β restored RANTES induction. HGF also induced renal GSK-3β phosphorylation and inactivation after obstructive injury in vivo. These observations suggest that HGF is a potent anti-inflammatory cytokine that inhibits renal inflammation by disrupting NF-κB signaling and may be a promising therapeutic agent for progressive renal diseases. Renal inflammation, characterized by the influx of inflammatory cells, is one of the major pathological findings in a variety of chronic kidney diseases (CKD).1Segerer S Nelson PJ Schlondorff D Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies.J Am Soc Nephrol. 2000; 11: 152-176PubMed Google Scholar, 2Eddy AA Molecular basis of renal fibrosis.Pediatr Nephrol. 2000; 15: 290-301Crossref PubMed Scopus (552) Google Scholar, 3Remuzzi G Bertani T Pathophysiology of progressive nephropathies.N Engl J Med. 1998; 339: 1448-1456Crossref PubMed Scopus (1160) Google Scholar Although infiltration of the T cells and monocytes is a tissue-wounding response after injury that may be beneficial in combating various injurious insults, chronic inflammation is undoubtedly a detrimental process that is often linked to the fibrotic lesions and tissue scarring. Glomerular and interstitial inflammation, regardless of the initial causes, is believed to play a critical role in the genesis and progression of CKD.4Klahr S Morrissey JJ The role of vasoactive compounds, growth factors and cytokines in the progression of renal disease.Kidney Int Suppl. 2000; 75: S7-S14Crossref PubMed Scopus (182) Google Scholar Clinical studies also reveal that the decline in renal function in patients with CKD often correlates closely with the extent of inflammation.1Segerer S Nelson PJ Schlondorff D Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies.J Am Soc Nephrol. 2000; 11: 152-176PubMed Google Scholar Inflammatory cells contribute to tissue damage in many ways. They are capable of producing profibrotic cytokines such as transforming growth factor-β, which in turn induces the matrix-producing myofibroblast activation and tubular epithelial to mesenchymal transition,5Liu Y Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention.J Am Soc Nephrol. 2004; 15: 1-12Crossref PubMed Scopus (980) Google Scholar, 6Kalluri R Neilson EG Epithelial-mesenchymal transition and its implications for fibrosis.J Clin Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2151) Google Scholar, 7Yang J Liu Y Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis.Am J Pathol. 2001; 159: 1465-1475Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar thereby promoting fibrogenic process. In addition, inflammatory cells can elicit their activities by producing radical oxygen species and by releasing proinflammatory cytokines, and therefore modulate the response of renal residential cells to injurious stimuli. Finally, they are able to produce and secrete extracellular matrix components, matrix metalloproteinases, and their endogenous inhibitors, thereby contributing to a dysregulated extracellular matrix catabolism. Given the importance of inflammation in the pathogenesis of CKD, it is conceivable to speculate that inhibition of the inflammatory response may be beneficial attenuating fibrotic lesions after injuries. Indeed, the renal protective effect of interrupting the inflammatory process by different maneuvers has been well documented.8Vielhauer V Berning E Eis V Kretzler M Segerer S Strutz F Horuk R Grone HJ Schlondorff D Anders HJ CCR1 blockade reduces interstitial inflammation and fibrosis in mice with glomerulosclerosis and nephrotic syndrome.Kidney Int. 2004; 66: 2264-2278Crossref PubMed Scopus (125) Google Scholar, 9Anders HJ Belemezova E Eis V Segerer S Vielhauer V Perez de Lema G Kretzler M Cohen CD Frink M Horuk R Hudkins KL Alpers CE Mampaso F Schlondorff D Late onset of treatment with a chemokine receptor CCR1 antagonist prevents progression of lupus nephritis in MRL-Fas(lpr) mice.J Am Soc Nephrol. 2004; 15: 1504-1513Crossref PubMed Scopus (98) Google Scholar, 10Tamada S Nakatani T Asai T Tashiro K Komiya T Sumi T Okamura M Kim S Iwao H Kishimoto T Yamanaka S Miura K Inhibition of nuclear factor-kappaB activation by pyrrolidine dithiocarbamate prevents chronic FK506 nephropathy.Kidney Int. 2003; 63: 306-314Crossref PubMed Scopus (48) Google Scholar, 11Pérez De Lema G De Wit C Cohen CD Nieto E Molina A Banas B Luckow B Vicente AB Mampaso F Schlondorff D Angiotensin inhibition reduces glomerular damage and renal chemokine expression in MRL/lpr mice.J Pharmacol Exp Ther. 2003; 307: 275-281Crossref PubMed Scopus (43) Google Scholar, 12Anders HJ Vielhauer V Frink M Linde Y Cohen CD Blattner SM Kretzler M Strutz F Mack M Grone HJ Onuffer J Horuk R Nelson PJ Schlondorff D A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.J Clin Invest. 2002; 109: 251-259Crossref PubMed Scopus (211) Google Scholar, 13Romero F Rodriguez-Iturbe B Parra G Gonzalez L Herrera-Acosta J Tapia E Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats.Kidney Int. 1999; 55: 945-955Crossref PubMed Scopus (188) Google Scholar In several studies, administration of immunosuppressive agent mycophenolate mofetil prevents lymphocyte and macrophage infiltration and reduces progressive renal damage.13Romero F Rodriguez-Iturbe B Parra G Gonzalez L Herrera-Acosta J Tapia E Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats.Kidney Int. 1999; 55: 945-955Crossref PubMed Scopus (188) Google Scholar, 14Van den Branden C Ceyssens B Pauwels M Van Wichelen G Heirman I Jie N Verbeelen D Effect of mycophenolate mofetil on glomerulosclerosis and renal oxidative stress in rats.Nephron Exp Nephrol. 2003; 95: e93-e99Crossref PubMed Google Scholar Likewise, treatment with chemokine receptor CCR1 antagonist reduces interstitial macrophage and lymphocyte recruitment and ameliorates renal fibrosis in obstructive nephropathy.12Anders HJ Vielhauer V Frink M Linde Y Cohen CD Blattner SM Kretzler M Strutz F Mack M Grone HJ Onuffer J Horuk R Nelson PJ Schlondorff D A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation.J Clin Invest. 2002; 109: 251-259Crossref PubMed Scopus (211) Google Scholar, 15Anders HJ Vielhauer V Schlondorff D Chemokines and chemokine receptors are involved in the resolution or progression of renal disease.Kidney Int. 2003; 63: 401-415Crossref PubMed Scopus (225) Google Scholar Suppression of renal inflammation has been demonstrated to be advantageous even in nonimmune models of CKD such as angiotensin II-induced nephropathy and diabetic nephropathy.16Utimura R Fujihara CK Mattar AL Malheiros DM Noronha IL Zatz R Mycophenolate mofetil prevents the development of glomerular injury in experimental diabetes.Kidney Int. 2003; 63: 209-216Crossref PubMed Scopus (178) Google Scholar, 17Muller DN Shagdarsuren E Park JK Dechend R Mervaala E Hampich F Fiebeler A Ju X Finckenberg P Theuer J Viedt C Kreuzer J Heidecke H Haller H Zenke M Luft FC Immunosuppressive treatment protects against angiotensin II-induced renal damage.Am J Pathol. 2002; 161: 1679-1693Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar These observations establish that interfering with renal inflammation may represent a promising therapeutic strategy for progressive renal fibrotic disorders. The expression of the genes that are important to the inflammatory responses is primarily mediated by the transcription factor nuclear factor (NF)-κB activation, which leads to the production of cellular signaling proteins such as cytokines, growth factors, or chemokines. RANTES (regulated on activation, normal T cell expressed), also known as CC-chemokine ligand 5 (CCL5), is a potent chemoattractant that is a member of the CC chemokine family.1Segerer S Nelson PJ Schlondorff D Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies.J Am Soc Nephrol. 2000; 11: 152-176PubMed Google Scholar, 18Krensky AM Ahn YT Mechanisms of disease: regulation of RANTES (CCL5) in renal disease.Nat Clin Pract Nephrol. 2007; 3: 164-170Crossref PubMed Scopus (104) Google Scholar Numerous studies have described the expression and implication of RANTES in animal models of CKD.19Vielhauer V Anders HJ Mack M Cihak J Strutz F Stangassinger M Luckow B Grone HJ Schlondorff D Obstructive nephropathy in the mouse: progressive fibrosis correlates with tubulointerstitial chemokine expression and accumulation of CC chemokine receptor 2- and 5-positive leukocytes.J Am Soc Nephrol. 2001; 12: 1173-1187PubMed Google Scholar, 20Satriano JA Banas B Luckow B Nelson P Schlondorff DO Regulation of RANTES and ICAM-1 expression in murine mesangial cells.J Am Soc Nephrol. 1997; 8: 596-603PubMed Google Scholar It is believed that its production and accumulation on inflamed renal tubules and endothelial cells provides directional signals for the circulating leukocytes to undergo extravasation, leading to inflammatory cell infiltration.21Gröne HJ Weber C Weber KS Grone EF Rabelink T Klier CM Wells TN Proudfood AE Schlondorff D Nelson PJ Met-RANTES reduces vascular and tubular damage during acute renal transplant rejection: blocking monocyte arrest and recruitment.FASEB J. 1999; 13: 1371-1383Crossref PubMed Scopus (234) Google Scholar Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in the regulation of cell proliferation, survival, and differentiation in a variety of organs.22Liu Y Hepatocyte growth factor in kidney fibrosis: therapeutic potential and mechanisms of action.Am J Physiol. 2004; 287: F7-F16Crossref PubMed Scopus (217) Google Scholar, 23Matsumoto K Nakamura T Hepatocyte growth factor: renotropic role and potential therapeutics for renal diseases.Kidney Int. 2001; 59: 2023-2038PubMed Google Scholar A large body of evidence shows that HGF possesses a remarkable anti-fibrotic potential and ameliorates fibrotic lesions in a wide variety of CKD.24Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar, 25Mizuno S Matsumoto K Nakamura T Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy.Kidney Int. 2001; 59: 1304-1314Crossref PubMed Scopus (175) Google Scholar, 26Azuma H Takahara S Matsumoto K Ichimaru N Wang JD Moriyama T Waaga AM Kitamura M Otsuki Y Okuyama A Katsuoka Y Chandraker A Sayegh MH Nakamura T Hepatocyte growth factor prevents the development of chronic allograft nephropathy in rats.J Am Soc Nephrol. 2001; 12: 1280-1292PubMed Google Scholar This is corroborated by the observations that blocking endogenous HGF signaling with neutralizing antibody markedly exacerbates renal fibrosis and dysfunction.27Mizuno S Matsumoto K Kurosawa T Mizuno-Horikawa Y Nakamura T Reciprocal balance of hepatocyte growth factor and transforming growth factor-beta 1 in renal fibrosis in mice.Kidney Int. 2000; 57: 937-948PubMed Scopus (0) Google Scholar, 28Liu Y Rajur K Tolbert E Dworkin LD Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways.Kidney Int. 2000; 58: 2028-2043Crossref PubMed Google Scholar Interestingly, the antifibrotic action of exogenous HGF is often accompanied by an attenuation of renal inflammation in these models.29Gong R Rifai A Tolbert EM Biswas P Centracchio JN Dworkin LD 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. 2004; 15: 2868-2881Crossref PubMed Scopus (96) Google Scholar, 30Gao X Mae H Ayabe N Takai T Oshima K Hattori M Ueki T Fujimoto J Tanizawa T Hepatocyte growth factor gene therapy retards the progression of chronic obstructive nephropathy.Kidney Int. 2002; 62: 1238-1248Crossref PubMed Google Scholar However, the molecular mechanism underlying HGF inhibition of renal inflammation remains incompletely understood. We undertook the present study to test the hypothesis that HGF suppresses renal inflammation by inhibiting proinflammatory cytokine expression. Our findings in this study demonstrate that HGF is a potent anti-inflammatory cytokine that blocks proinflammatory RANTES expression in vivo and in vitro. Our results further indicate that the anti-inflammatory action of HGF is primarily mediated by disrupting NF-κB signaling. The primary antibodies were obtained from different sources: anti-RANTES (sc-1410), anti-tumor necrosis factor (TNF)-α (sc-8301), anti-IκBβ (sc-946), anti-interleukin (IL)-6 (sc-7920), and anti-actin (sc-1616) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-IκBα (Ser32/36), anti-IκBα, anti-phospho-p65 NF-κB (Ser536), anti-p65 NF-κB, anti-phospho-GSK-3β (Ser9), anti-GSK-3β (Cell Signaling Technology, Beverly, MA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX). Recombinant human HGF, TNF-α, and IL-1β were purchased from R&D Systems (Minneapolis, MN). Cell culture media, newborn calf serum, and supplements were obtained from Invitrogen (Carlsbad, CA). Chemical inhibitor wortmannin and cell-permeable inhibitor peptide NF-κB SN50 were purchased from Calbiochem (La Jolla, CA). All other chemicals were of analytic grade and were obtained from Sigma (St. Louis, MO) or Fisher (Pittsburgh, PA) unless otherwise indicated. Male CD-1 mice that weighed ∼18 to 22 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). Unilateral ureteral obstruction (UUO) was performed using an approved protocol by the Institutional Animal Care and Use Committee at the University of Pittsburgh, as described elsewhere.31Yang J Dai C Liu Y Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice.J Am Soc Nephrol. 2002; 13: 2464-2477Crossref PubMed Scopus (134) Google Scholar Briefly, under general anesthesia, complete ureteral obstruction was performed by double-ligating the left ureter using 4-0 silk after a midline abdominal incision. Sham-operated mice had their ureters exposed, manipulated but not ligated. Mice were randomly assigned into three groups: 1) sham normal control, 2) UUO control, and 3) UUO receiving HGF. Delivery of human HGF gene was achieved by intravenous injection of naked HGF plasmid (pCMV-HGF) at 1 mg/kg before (day − 1) and after (day 7) UUO, respectively, as described previously.32Dai C Yang J Bastacky S Xia J Li Y Liu Y Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice.J Am Soc Nephrol. 2004; 15: 2637-2647Crossref PubMed Scopus (107) Google Scholar, 33Yang J Chen S Huang L Michalopoulos GK Liu Y Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar Control UUO mice were injected with empty vector pcDNA3 plasmid at the same time points in an identical manner. At day 7 and day 14 after surgery, five mice from each group were sacrificed, respectively, and the kidneys were harvested for various analyses. Immunohistochemical staining of kidney sections was performed by an established protocol.34Tan X Li Y Liu Y Paricalcitol attenuates renal interstitial fibrosis in obstructive nephropathy.J Am Soc Nephrol. 2006; 17: 3382-3393Crossref PubMed Scopus (264) Google Scholar In brief, paraffin-embedded sections were stained with anti-CD3 (sc-20047, Santa Cruz Biotechnology), anti-F4/80 (14-4801-82; eBioscience Inc., San Diego, CA), and anti-RANTES (500-P118; PeproTech Inc., Rocky Hill, NJ) antibodies using the M.O.M. immunodetection kit, according to the protocol specified by the manufacturer (Vector Laboratories, Burlingame, CA). Indirect immunofluorescence staining was performed according to the procedures described previously.35Li Y Yang J Luo JH Dedhar S Liu Y Tubular epithelial cell dedifferentiation is driven by the helix-loop-helix transcriptional inhibitor Id1.J Am Soc Nephrol. 2007; 18: 449-460Crossref PubMed Scopus (74) Google Scholar Slides were viewed with an Eclipse E600 microscope equipped with a digital camera (Nikon, Melville, NY). Nonimmune normal rabbit IgG was used to replace the primary antibody as negative control, and no staining occurred. Nuclear staining for p65 NF-κB in HKC-8 cells after various treatments was counted and calculated. CD-3, F4/80, and RANTES staining were semiquantified by a computer-aided morphometric analysis (MetaMorph; Universal Imaging Co., Downingtown, PA). Briefly, a grid containing 117 (13 × 9) sampling points was superimposed onto images of cortical high-power field (×400). The number of grid points overlying positive area (except tubular lumen and glomeruli) was counted and expressed as a percentage of all sampling points. For each kidney, 10 randomly selected, nonoverlapping fields were analyzed in a blinded manner. For determination of RANTES and MCP-1 mRNA expression, a semiquantitative RT-PCR was used, as described previously.36Tan R Zhang J Tan X Zhang X Yang J Liu Y Downregulation of SnoN expression in obstructive nephropathy is mediated by an enhanced ubiquitin-dependent degradation.J Am Soc Nephrol. 2006; 17: 2781-2791Crossref PubMed Scopus (63) Google Scholar Total RNA was prepared from kidney homogenates of various groups of mice. After reverse transcription of the RNA, cDNA was used as a template in PCR reactions using gene-specific primer pairs. After quantifying band intensities by using densitometry, the relative steady-state level of mRNA was calculated after normalizing to β-actin. The sequences of the primer sets were as follows: RANTES, 5′-GTGCCCACGTCAAGGAGTAT-3′ (sense) and 5′-GGGAAGCGTATACAGGGTCA-3′ (antisense); MCP-1, 5′-CCCACTCACCTGCTGCTAC-3′ (sense) and 5′-TTCTTGGGGTCAGCACAGA-3′ (antisense). The sequences of the β-actin primer set were described previously.36Tan R Zhang J Tan X Zhang X Yang J Liu Y Downregulation of SnoN expression in obstructive nephropathy is mediated by an enhanced ubiquitin-dependent degradation.J Am Soc Nephrol. 2006; 17: 2781-2791Crossref PubMed Scopus (63) Google Scholar Human proximal tubular epithelial cell line (clone 8, HKC-8) was obtained from Dr. L. Racusen (The Johns Hopkins University, Baltimore, MD) and maintained in Dulbecco's modified Eagle's medium/F12 medium supplemented with 5% newborn calf serum. The HKC-8 cells were seeded onto six-well culture plates to ∼60 to 70% confluence in complete medium containing 5% newborn calf serum for 16 hours, and then changed to serum-free medium. After 24 hours of serum starvation, recombinant human IL-1β or TNF-α was added to the culture at a final concentration of 5 or 2 ng per ml, respectively. Thirty minutes before addition of TNF-α or IL-1β, recombinant human HGF was added at the concentration of 40 ng/ml unless otherwise indicated. The cells were typically incubated for 24 or 48 hours after addition of cytokines, before being subjected to Western blot or immunofluorescence staining, respectively. Cells were lysed with sodium dodecyl sulfate (SDS) sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mmol/L dithiothreitol, and 0.1% bromophenol blue, supplemented with protease and phosphatase inhibitor cocktails). Kidney homogenates were prepared essentially according to an established protocol, as described previously.31Yang J Dai C Liu Y Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice.J Am Soc Nephrol. 2002; 13: 2464-2477Crossref PubMed Scopus (134) Google Scholar Samples were heated at 100°C for 5 to 10 minutes before being loaded and separated on 10% SDS-polyacrylamide gels. The proteins were electrotransferred to Hybond-P polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ) in transfer buffer containing 48 mmol/L Tris-HCl, 39 mmol/L glycine, 0.037% SDS, and 20% methanol at 4°C for 1 hour. Nonspecific binding to the membrane was blocked for 1 hour at room temperature with 5% Carnation nonfat milk in TBST buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween 20). The membranes were then incubated for 16 hours at 4°C with various primary antibodies in blocking buffer containing 5% milk at the dilutions specified by the manufacturers. After extensive washing in TBST buffer, the membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 hour at room temperature in 5% nonfat milk dissolved in TBST. Membranes were washed with TBST buffer and the signals were visualized using the SuperSignal West Pico chemiluminescent substrate kit (Pierce Biotechnology, Rockford, IL). HKC-8 cells were seeded in complete medium in 12-well plates at a concentration of 1 × 105 cells per well. Twenty-four hours later, cells were changed to serum-free medium and kept in serum-free medium for an additional 24 hours, before addition of different cytokines. The supernatants of cell cultures were collected, and the levels of RANTES protein were determined by using enzyme-linked immunosorbent assay kit (Quantikine Immunoassay, R&D Systems) according to the manufacturer's protocol. ChIP assay was performed to analyze in vivo interactions of NF-κB and its cognate cis-acting element in RANTES promoter. This assay was performed essentially according to the protocols specified by the manufacturer (ChIP assay kit; Upstate, Charlottesville, VA). Briefly, HKC-8 cells, after various treatments as indicated, were cross-linked with 1% formaldehyde, and then resuspended in SDS lysis buffer containing protease inhibitors. The chromatin solution was sonicated, and the supernatant was diluted 10-fold. An aliquot of total diluted lysate was used for total genomic DNA as input DNA control. The anti-p65 NF-κB antibody was added and incubated at 4°C overnight, followed by incubation with protein A-agarose for 1 hour. The precipitates were washed and chromatin complexes were eluted. After reversal of the cross-linking at 65°C for 4 hours, the DNA was purified, and 1 μl of input control or ChIP samples were used as a template for PCR using the primer sets for human RANTES promoter regions (from −208 to −10) containing NF-κB response element.37Nelson PJ Kim HT Manning WC Goralski TJ Krensky AM Genomic organization and transcriptional regulation of the RANTES chemokine gene.J Immunol. 1993; 151: 2601-2612PubMed Google Scholar The sequences of primers used for ChIP assay were as follows: forward, 5′-TTGGTGCTTGGTCAAAGAGG-3′; and reverse, 5′-CCCTTTATAGGGCCAGTTGA-3′. All data examined were expressed as mean ± SEM. Statistical analysis of the data were performed using SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparison between groups was made using one-way analysis of variance, followed by Student-Newman-Keuls test. A P value of less than 0.05 was considered significant. HGF has been demonstrated to be able to ameliorate renal fibrotic lesions in obstructive nephropathy,30Gao X Mae H Ayabe N Takai T Oshima K Hattori M Ueki T Fujimoto J Tanizawa T Hepatocyte growth factor gene therapy retards the progression of chronic obstructive nephropathy.Kidney Int. 2002; 62: 1238-1248Crossref PubMed Google Scholar, 31Yang J Dai C Liu Y Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice.J Am Soc Nephrol. 2002; 13: 2464-2477Crossref PubMed Scopus (134) Google Scholar in which interstitial infiltration of inflammatory cells is a major pathological feature. To examine the potential effect of HGF on renal inflammation, we first investigated the infiltration of the CD-3-positive T cells in the obstructed kidney after UUO. As shown in Figure 1, ureteral obstruction induced substantial renal T-cell infiltration at 7 and 14 days after UUO, respectively, as illustrated by immunohistochemical staining for CD3 antigen. As reported previously,32Dai C Yang J Bastacky S Xia J Li Y Liu Y Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice.J Am Soc Nephrol. 2004; 15: 2637-2647Crossref PubMed Scopus (107) Google Scholar, 33Yang J Chen S Huang L Michalopoulos GK Liu Y Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar, 38Dai 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 delivery of the HGF gene via naked plasmid vector produced a substantial amount of exogenous HGF protein in the circulation, as well as in liver and kidney. The increase in renal human HGF protein was sustained to 7 days after injection, although it peaked at 16 to 24 hours.32Dai C Yang J Bastacky S Xia J Li Y Liu Y Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice.J Am Soc Nephrol. 2004; 15: 2637-2647Crossref PubMed Scopus (107) Google Scholar We found that HGF gene therapy effectively inhibited the infiltration of T cells in the obstructed kidney at different time points (Figure 1, C and E). Quantitative analysis also demonstrated a dramatic suppression of inflammatory T-cell infiltration by HGF at 7 and 14 days after UUO, respectively, in obstructive nephropathy (Figure 1F). We also examined the effect of HGF on monocyte/macrophage infiltration in the obstructed kidney. As shown in Figure 2, obstructive injury caused marked infiltration and accumulation of the F4/80-positive macrophages in renal interstitium. However, delivery of the HGF gene also dramatically inhibited macrophage infiltration at 7 and 14 days after obstructive injury, respectively (Figure 2, C, E, and F). Of note, as previously reported,24Yang J Liu Y Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis.J Am Soc Nephrol. 2002; 13: 96-107Crossref PubMed Google Scholar, 31Yang J Dai C Liu Y Hepatocyte growth factor gene therapy and angiotensin II blockade synergistically attenuate renal interstitial fibrosis in mice.J Am Soc Nephrol. 2002; 13: 2464-2477Crossref PubMed Scopus (134) Google Scholar delivery of the HGF gene significantly ameliorated t
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