Hepatocyte Growth Factor Ameliorates Renal Interstitial Inflammation in Rat Remnant Kidney by Modulating Tubular Expression of Macrophage Chemoattractant Protein-1 and RANTES
Rujun Gong*,
Abdalla Rifai,
Evelyn M. Tolbert*,
Purba Biswas*,
Jason N. Centracchio* and
Lance D. Dworkin*
*Division of Renal Diseases, Department of Medicine; and Department of Pathology, Rhode Island Hospital, Brown University School of Medicine, Providence, Rhode Island
Correspondence to Dr. Lance D. Dworkin, Division of Renal Diseases, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903. Phone: 401-444-6843; Fax: 401-444-6849; E-mail: LDworkin{at}lifespan.org
Hepatocyte growth factor (HGF) has been shown to reduce renalinjury in a variety of animal models of chronic renal disease.Suggested mechanisms to explain this action include preventionof tubular cell apoptosis, blocking epithelial-to-mesenchymaltransition, and promotion of extracellular matrix degradation.Inflammation is another common finding in kidneys that progressto end-stage renal failure; however, the effect of HGF on inflammationhas hardly been investigated. For examining this issue, beginning2 wk after subtotal nephrectomy, rats received a continuousinfusion of recombinant HGF, neutralization of endogenous HGFby daily injection of an anti-HGF antibody, or preimmune IgGfor an additional 2 wk. HGF infusion halted the progressionof proteinuria and decreased renal collagen accumulation. Renalinflammation in both glomeruli and tubulointerstitium was significantlyattenuated, associated with reductions in the tubular expressionof the chemokines macrophage chemoattractant protein-1 (MCP-1)and RANTES (regulated upon expression normal T cell expressedand secreted). In contrast, HGF neutralization worsened renalfibrosis, aggravated renal inflammation, and enhanced tubularexpression of MCP-1 and RANTES. In vitro, HGF suppressed basaland TNF-–induced expression of these chemokines at boththe mRNA and protein levels in a time- and dose-dependent mannerin proximal tubular epithelial cells. HGF also blunted TNF-–inducednuclear translocation and activation of NF-B, a pivotal transcriptionfactor that regulates chemokine expression. Immunohistochemistryshowed that activated NF-B was evident in tubules in remnantkidneys and increased remarkably with anti-HGF treatment. HGFinfusion markedly suppressed expression of activated NF-B inremnant kidneys. These findings suggest that the beneficialeffect of HGF in chronic renal disease is attributable, at leastin part, to a direct anti-inflammatory action, likely via NF-B,on tubular epithelial cells.
Influx of inflammatory cells into interstitium is an invariablepathologic finding in almost all forms of chronic renal diseaseregardless of the original cause (1,2). Infiltrating cells promoterenal scarring and loss of function by several mechanisms. Theysecrete extracellular matrix components (3,4) that directlycontribute to matrix accumulation. Leukocytes generate radicaloxygen species (5), lipid mediators, and proinflammatory cytokines(6) that promote tissue injury. Mononuclear cells, includinglymphocytes and macrophages, are important sources of profibroticmolecules such as TGF-1, basic fibroblast growth factor, andPDGF (6). These factors in turn activate resident fibroblasts(7,8) and promote epithelial-to-mesenchymal transition (9).Heterogeneous fibroblasts proliferate and generate excessiveextracellular matrix, resulting in renal scarring. Consistently,the extent of inflammation is well correlated with kidney functionand predicts long-term prognosis in a number of clinical settings(10). Suppression of renal inflammation by administration ofimmunosuppressive drugs is beneficial even in nonimmune modelsof renal disease (11) such as remnant kidney (12) and diabeticnephropathy (13).
Inflammatory infiltrates in chronic renal disease are composedof heterogeneous cell types (14). Although some cells are derivedfrom proliferation of resident macrophages (15), the majorityenter the kidney from the circulation. Chemokines activate leukocytesand mediate selective leukocyte trafficking at multiple stages,attracting specific populations to the site of injury (16–18).In the kidney, tubular epithelial cells (TEC) are consideredto be a prominent source of chemokines (19). In vitro, proteinoverload (20) as well as proinflammatory cytokines (19) suchas IL-1, TNF-, and interferon-, markedly induce TEC expressionof macrophage chemoattractant protein-1 (MCP-1) and RANTES (regulatedupon expression normal T cell expressed and secreted) (19),key chemokines that recruit mononuclear cells to the kidney(16,17).
HGF is a multifunctional, pleiotropic growth factor (21) thatameliorates chronic renal injury in a variety of models, includingremnant kidney (22–24), unilateral ureteral obstruction(25,26), and diabetic nephropathy (27). TEC are a major targetfor HGF in the kidney, and several mechanisms have been proposedto explain this beneficial action, including antiapoptosis (21,25,26),promoting TEC proliferation (21,25), prevention of epithelial-to-mesenchymaltransition (9,25,26), and increased activity of matrix degradationpathways (23,24). In many models, inflammatory infiltrationsubsides as well (28); however, the potential anti-inflammatoryeffects of HGF have been largely overlooked. In the presentstudy, we examined the suppressive effects of HGF on renal inflammationby modulating HGF activity in remnant kidney rats by infusingrecombinant HGF or by neutralizing endogenous HGF using an anti-HGFantibody. The mechanisms of this effect were also explored byexamining the actions of HGF on TEC expression of chemokinesin vivo and in vitro.
Animal Experimental Design
Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis,IN) that initially weighed 180 to 210 g were housed in an approvedanimal care facility and fed standard diet. Five-sixths renalablation was performed as described previously (23,24). Twoweeks after surgery, rats were randomly assigned to one of threegroups (n = 7): (1) HGF-treated group (HGF): rats received recombinanthuman HGF (Genentech, South San Francisco, CA) as a continuousintravenous infusion via an implanted osmotic minipump intothe jugular vein at a dose of 100 µg/kg per d for 14 d;(2) anti-HGF antibody–treated group (-HGF): rats receiveddaily injections of a high titer, rabbit polyclonal anti-HGFneutralizing antibody intravenously through the tail vein for14 d; (3) Preimmune IgG-treated group (Ctrl-IgG): rats receiveda continuous intravenous infusion of saline via minipump aswell as daily tail-vein injections of rabbit preimmune IgG.Sham-operated rats (Sham) with no renal ablation served as normalcontrols (n = 7). Two weeks after treatment, animals were killed.Kidneys were perfused with iced saline and excised. One portionof the kidney was fixed in 10% phosphate-buffered formalin formorphologic and immunohistochemistry analyses, and another partwas frozen immediately for cryostat sectioning. The remainderwas snap-frozen in liquid nitrogen and stored at –80°Cfor RNA and protein extraction.
Animal Studies
At the end of weeks 2 and 4, rats were weighed and placed inmetabolic cages, and urine was collected for 24 h. Urine volumewas measured, protein concentration was determined by precipitationwith 3% sulfosalicylic acid, and turbidity was assessed by detectingabsorbance at 595 nm with a spectrophotometer. Urinary proteinexcretion was calculated as milligrams of protein per 24 h.Blood was obtained from the tail vein. Creatinine levels inserum and urine were measured using a semiautomatic creatinineanalyzer (Beckman Instruments, Brea, CA). Creatinine clearancewas calculated using the standard formula and factored for bodyweight. On the day that the rats were killed, they were anesthetizedand placed on a heated table to maintain constant body temperature.A polyethylene catheter (PE-50) was inserted into the left femoralartery, and mean arterial pressure (MAP) was measured by a pressuretransducer connected to a computer.
Morphologic Studies
Formalin-fixed kidneys were embedded in paraffin and preparedin 3-µm-thick sections. For general histology, sectionswere processed for hematoxylin/eosin, periodic acid-Schiff,and Masson-Trichrome staining. The extent of glomerulosclerosiswas graded on periodic acid-Schiff–stained sections usingthe Banff criteria on a arbitrary scale from 0 to 3 and reportedas the mean of 20 glomeruli (29): 0, normal glomeruli; 1, mildexpansion of mesangial area up to the diameter of two mesangialcells; 2, moderate broadening of mesangial area by more thanthe diameter of two mesangial cells; and 3, severe mesangialsclerosis corresponding to at least 25% of the glomerular area.Severity of interstitial collagen deposition was scored on ascale from 0 to 3 and reported as the mean of 20 random high-power(x400) fields per trichrome-stained section (23): 0, absent;1, mild; 2, moderate; and 3, severe. Immunohistochemical stainingfor ED-1, MCP-1, and RANTES was performed as described previously(23). The antibodies for MCP-1 and RANTES were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA), and antibodies forED-1 were purchased from Serotec (Oxford, UK). Indirect immunofluorescentstaining of collagen I or phosphorylated NF-B p65 was carriedout on frozen kidney sections using rabbit polyclonal anti-rattype I collagen antibody (Chemicon, Temecula, CA) or mouse monoclonalanti-phosphorylated NF-B p65 antibody (Cell Signaling, Beverly,MA), respectively. The Alexa Fluor goat anti-rabbit or anti-mouseIgG (Molecular Probes, Eugene, OR) was used as secondary antibody.As a negative control, the primary antibody was replaced bynonimmune serum from the same species; no staining occurred.
Total Kidney Collagen Assay
Collagen is the only protein that contains hydroxyproline. Totalkidney collagen content was measured by indirect biochemicalanalysis of hydroxyproline in kidney hydrolysate as describedpreviously (23). Total collagen content was calculated on thebasis of the assumption that collagen contains 12.7% hydroxyprolineby weight.
Western Immunoblot Analyses
Rat kidneys were homogenized in RIPA buffer (1% Nonidet P-40,0.1% SDS, 100 µg/ml PMSF, 0.5% sodium deoxycholate, 1mM sodium orthovanadate, 2 µg/ml aprotin, 2 µg/mlleupeptin, 5 mM EDTA in PBS), and protein concentration wasassayed using BCA reagents (Sigma, St. Louis, MO). Tissue homogenateswith equal amounts of total protein were processed for immunoblotanalysis as described before (23).
Cell Culture
Human proximal TEC (HKC-8; courtesy of Dr. L. Racusen, JohnHopkins University, Baltimore, MD) and immortalized rat proximaltubular cells (IRPTC; provided by Dr. J. Ingelfinger, HarvardMedical School, Boston, MA) were maintained in DMEM/F12 supplementedwith 5% FBS. Cells were plated at 70% confluence in the mediumthat contained 5% FBS for 24 h and then underwent serum starvationfor another 24 h. Human recombinant HGF and recombinant humanor rat TNF- (hTNF-, rTNF-; R&D systems, Minneapolis, MN)were added to the culture with fresh serum-free medium at afinal concentration of 20 and 2 ng/ml, respectively, or otherwiseas indicated. For inhibition experiments, cells were treatedin the presence of n-tosyl-l-phenylalanine chloromethylketone(TPCK) or pyrrolidine dithiocarbamate (PDTC) at the indicateddose. At different time points, cell number was estimated byhemacytometer. Cell viability was assessed by Trypan blue exclusion.Cells and conditioned media were harvested for further investigation.
ELISA of Chemokines
The contents of MCP-1 and RANTES in kidney homogenates thatcontained equal amounts of total protein were determined usingspecific sandwich enzyme immunometric assay kits for rat MCP-1(Assay Design, Ann Arbor, MI) and rat RANTES (Biosource International,Camarillo, CA). The contents of MCP-1 and RANTES in conditionedmedia from HKC cells were determined by specific Quantikinesandwich ELISA kits for human MCP-1 and RANTES (R&D Systems).The results were normalized by total protein content in kidneyhomogenates or by cell numbers in culture.
Semiquantitative Reverse Transcription–PCR
Total RNA was extracted from 2 x 106 cells. Reverse transcription–PCR(RT-PCR) was performed as described previously (23) using primerslisted in Table 1. Glyceraldehyde-3-phosphate dehydrogenaseserved as an internal normalizing gene for RT-PCR. PCR productsresolved in 1.5 to 2% agarose gels were photographed under ultravioletlight.
Table 1. Nucleotide sequences of the primers used for RT-PCRa
Immunofluorescent Analyses of NF-B Translocation
HKC cells were plated on chamber slides for 24 h, then deprivedof serum for another 24 h. Quiescent cells were treated with20 ng/ml HGF, 2 ng/ml hTNF-, or the combination for 30, 60,and 120 min. Nontreated cells in serum-free medium served ascontrols. Cells were rinsed with PBS buffer and fixed in 4%paraformaldehyde for 20 min, followed by incubation with 1%Triton X-100. Cells were then sequentially incubated with apolyclonal antibody to NF-B p65 (Cell Signaling) and a FITC-labeleddonkey anti-rabbit antibody (Santa Cruz Biotechnology). Resultswere interpreted using a fluorescence microscope.
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay
Quiescent HKC cells were treated with HGF or hTNF- for the indicatedtimes. Nuclear and cytoplasmic extracts were prepared usingthe NE-PER extraction kit (Pierce, Rockford, IL). Protein concentrationwas measured using the BCA reagent (Sigma). For quantifyingthe nuclear translocation of NF-B, nuclear extracts were immunoblottedfor p65. Electrophoretic mobility shift assay was conductedusing LightShift EMSA kits (Pierce). Briefly, binding reactionsthat contained 5 µg of nuclear protein, 10 mM Tris, 50mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl2, 0.05% Nonidet P-40,and 15 pmol of biotinylated NF-B consensus double stranded oligonucleotideprobe (5'-agttgaggggactttcccaggc-3') were incubated in a finalvolume of 20 µl for 20 min at room temperature. Specificbinding was confirmed by adding 100-fold excess unlabeled probeas a specific competitor. After incubation, samples were separatedby 5% polyacrylamide nondenaturing gel electrophoresis, transferredto nylon membranes via electroblotting, and UV cross-linked.The DNA-protein complexes were visualized with a streptavidin-horseradishperoxidase followed by chemiluminescent detection. For supershiftassay, the binding reaction was carried out in the presenceof a rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology).
Statistical Analyses
One investigator in a blinded manner performed counting of ED-1–positivecells as well as histologic scoring. For immunoblot analysisand RT-PCR, bands were scanned and the integrated pixel densitywas determined using a densitometer and the NIH image analysisprogram. All data are expressed as mean ± SD. Statisticalanalysis of the data from multiple groups was performed by ANOVAfollowed by Student-Newman-Keuls tests. Data from two groupswere compared by t test. Linear regression analysis was appliedto examine possible relationships between two parameters. P< 0.05 was considered significant.
HGF Halts the Progression of Proteinuria and Attenuates Renal Fibrosis in Rat Remnant Kidneys
In rats with remnant kidneys, serum creatinine increased andproteinuria gradually worsened during the 2-wk treatment period.Continuous intravenous infusion of HGF significantly slowedprogression of proteinuria without altering MAP (Table 2). Renalfibrosis was also attenuated as demonstrated by a reductionin total kidney collagen content and type I collagen immunofluorescentstaining (Figure 1). Semiquantitative morphometric studies showedthat HGF also decreased fibrosis score in both glomeruli andtubulointerstitium. In contrast, neutralizing the endogenousHGF with the anti-HGF antibody significantly elevated the MAP,impaired renal function, and enhanced fibrosis.
Figure 1. Continuous intravenous infusion of hepatocyte growth factor (HGF) decreases interstitial type I collagen deposition in rat remnant kidney, whereas blocking the endogenous HGF increases its accumulation. Representative photomicrographs of type I collagen immunofluorescent staining in frozen kidney sections from sham-operated control rats (A and B) and nephrectomized rats that were treated with preimmune IgG (C), HGF (D), or anti-HGF antibody (E). As negative controls (A) for immunostaining, sections were processed with nonimmune serum from the same species as the primary antibody. All sections were counterstained with Evans’ blue. Magnification, x200.
HGF Ameliorates Renal Inflammation in Rat Remnant Kidney
Four weeks after renal ablation, an inflammatory infiltrateconsisting primarily of macrophages was prominent in both glomeruliand interstitium in the kidney of the preimmune IgG-treatedremnant control group (Figure 2B) compared with sham-operatedrats (Figure 2A). In addition to increasing tubular atrophyand interstitial and glomerular sclerosis, blockade of endogenousHGF significantly exacerbated the macrophage infiltration (Figure 2C).In contrast, HGF supplementation significantly attenuatedrenal inflammation, and this was associated with improvementin kidney morphology (Figure 2D). Counting the absolute numberof ED-1–positive cells revealed a marked increase in macrophageinfiltration after HGF blockade and a significant reductionin response to exogenous HGF in both glomeruli and interstitium(Figure 2E). In addition, the extent of interstitial macrophageinfiltration was highly correlated with total kidney collagencontent (Figure 2F). Because macrophages were not uniformlydistributed in remnant kidneys and for avoiding bias in selectingsections for counting, immunoblot analysis of ED-1 protein wasperformed on whole-kidney homogenates. These studies confirmedthat kidney ED-1 content was modulated by HGF (Figure 2, G and H).
Figure 2. HGF treatment ameliorates whereas neutralization of endogenous HGF worsens renal inflammatory infiltrates in rat remnant kidney. (A through D) Representative photomicroscopes of ED-1 immunohistochemistry staining in kidney sections from sham-operated normal control rats (Sham; A) and nephrectomized rats that were treated with preimmune IgG (Ctrl-IgG; B), anti-HGF antibody (-HGF; C), or HGF infusion (HGF; D). (E) Counting of ED-1–positive cells in glomeruli (grey bar) or tubulointerstitium (white bar) in kidney sections from each group; *P < 0.05, #P < 0.05 versus other groups; **P < 0.01, ##P < 0.01 versus Sham or -HGF. (F) Scatter plot of total collagen content versus ED-1 cell numbers in tubulointerstitium for each rat from Ctrl-IgG (white square), -HGF (white triangle), and HGF (white diamond) groups shows significant correlation between interstitial inflammation and renal fibrosis. (G) Representative pictures of Western immunoblot analysis of ED-1 molecules in kidney homogenates. (H) Arbitrary units of ED-1 abundance in immunoblot expressed as densitometric ratios of ED-1/actin as folds of normal control groups. *P < 0.05 versus other groups; #P < 0.05 versus Ctrl-IgG. In E, F, and H, n = 7 for each group. Magnification, x100 (x400 for inserts) in A through D.
HGF-Induced Remission of Renal Inflammation Is Associated with Suppression of Tubular Expression of MCP-1 and RANTES
Immunohistochemistry staining for MCP-1 showed very weak expressionin normal kidney (Figure 3A). In remnant kidney, renal expressionof MCP-1 increased focally primarily in tubular cells and, toa lesser extent, in glomeruli tufts (Figure 3B). Administrationof HGF antibody for 2 wk significantly enhanced tubular staining(Figure 3C) and increased MCP-1 content in kidney homogenatesdetermined by sandwich ELISA as compared with the preimmuneIgG-treated group or sham-operated rats (Figure 3E). Conversely,continuous HGF infusion markedly reduced MCP-1 staining anddecreased its absolute quantity in kidney homogenates (Figure 3, D and E).Consistent with its chemokine properties, MCP-1content was strongly correlated with the interstitial macrophageinfiltration (Figure 3F).
Figure 3. HGF supplement suppresses whereas HGF blockade enhances renal expression of macrophage chemoattractant protein-1 (MCP-1), mainly expressed by tubules, in rat remnant kidneys. (A through D) Representative pictures of MCP-1 immunohistochemistry staining (asterisks and arrowheads) in kidney sections from sham-operated rats (Sham; A) and nephrectomized rats that were treated with preimmune IgG (Ctrl-IgG; B), anti-HGF antibody (-HGF; C), or HGF (D). (E) MCP-1 content in kidney homogenates estimated by ELISA; *P < 0.05 versus other groups; #P < 0.05 versus Ctrl-IgG. (F) Scatter plot of ED-1 cell numbers in tubulointerstitium versus MCP-1 content for each rat from Ctrl-IgG, -HGF, and HGF groups shows significant correlation between MCP-1 expression and interstitial inflammatory infiltration. In E and F, n = 7 for each group. Magnification, x100 (x400 for inserts) in A through D.
Normal rat kidney showed a mild constitutive immunostainingfor RANTES in both tubules and in glomerular mesangium (Figure 4A).Renal ablation increased staining focally, most obviouslyin tubules (Figure 4B). Neutralization of endogenous HGF enhancedstaining and increased the number of positive tubules (Figure 4C).Conversely, HGF infusion suppressed RANTES to almost normallevels, with only sporadic staining noted in a few tubular cells(Figure 4D). Absolute content of RANTES (Figure 4E) determinedby ELISA on tissue homogenates was increased in remnant as comparedwith normal kidney. The level was further increased in antibody-treatedrats and markedly suppressed by HGF infusion. Linear regressionanalysis showed a strong correlation between RANTES expressionand the number of interstitial macrophages in individual rats(Figure 4F).
Figure 4. HGF infusion inhibits whereas HGF antibody elevates renal tubular expression of RANTES in rat remnant kidneys. (A through D) Representative micrographs of RANTES immunohistochemistry staining (asterisks and arrowheads) in kidney sections from sham-operated rats (Sham; A) and nephrectomized rats that were teated with preimmune IgG (Ctrl-IgG; B), anti-HGF antibody (-HGF; C), or HGF (D). (E) RANTES content in kidney homogenates determined by ELISA; *P < 0.05 versus other groups, #P < 0.05 versus Ctrl-IgG. (F) Scatter plot of renal ED-1 cell numbers in tubulointerstitium versus RANTES content for each rat from Ctrl-IgG, -HGF, and HGF group shows significant correlation between RANTES expression and interstitial inflammatory infiltration. In E and F, n = 7 for each group. Magnification, x100 (x400 for inserts) in A through D.
HGF Suppresses Constitutive and TNF- Induced Expression of MCP-1and RANTES in TEC Cells In Vitro
To examine further the effects of HGF on chemokine expression,we exposed HKC cells to HGF at different dosages and for differentintervals. HGF treatment for 24 h markedly suppressed constitutive(Figure 5A) as well as TNF-–induced (Figure 5B) MCP-1mRNA expression in a dose-dependent manner. The greatest inhibitoryeffect was observed at concentrations between 10 and 80 ng/ml.HGF-induced (20 ng/ml) suppression of MCP-1 mRNA levels in thepresence (Figure 5D) or absence (Figure 5C) of TNF- was timedependent, peaking at 12 to 24 h and declining at 48 h of exposure.MCP-1 content in the conditioned media from cultured cells asdetermined by ELISA revealed a similar pattern of responsesto HGF (Figure 6). At 24 h, maximal suppression of MCP-1 levelswas observed with HGF at concentrations between 10 and 80 ng/ml(Figure 6A). The greatest inhibition of TNF-–induced MCP-1production was observed at the concentration of 20 ng/ml (Figure 6B).HGF treatment significantly reduced MCP-1 production asearly as 24 h and produced a >25% decrement in MCP-1 contentat 48 h in HKC cells in the presence or absence of TNF- (Figure 6C).
Figure 5. HGF suppresses constitutive or TNF-–induced MCP-1 expression in HKC cells in a dose- and time-dependent manner. Representative pictures for semiquantitative reverse transcription–PCR (RT-PCR) studies in HKC cells that were treated by HGF with different dosage (ng/ml) for 24 h (A and C) or by HGF (20 ng/ml) for different time (B and D) in the presence (C and D) or absence (A and B) of hTNF- (2 ng/ml). Representative gels are shown at the top, and the results of densitometry are shown at the bottom. Values in the graph are expressed as densitometric ratios as folds over control from three separate experiments. In A and C, *P < 0.05 versus other treatment; in B and D, *P < 0.05 versus non-HGF treatment at the same time point.
Figure 6. Dose-dependent and temporal effect of HGF on the constitutive and the TNF-–induced expression of MCP-1 in HKC cells. HKC cells were treated in triplicate with HGF and/or hTNF- at different dosages and for different times. Conditioned media were harvested and subjected to ELISA analysis of MCP-1. (A) HKC cells were treated with HGF of indicated concentrations for 24 h; P < 0.001 by ANOVA; *P < 0.01 versus other groups with HGF, *P = 0.078 versus nontreated cells. (B) HKC cells were treated with HGF of indicated concentrations in the presence of TNF- (2 ng/ml) for 24 h; P < 0.001 by ANOVA, *P < 0.05 versus other groups with HGF. (C) HKC cells were treated with 20 ng/ml HGF in the presence or absence of 2 ng/ml TNF- for different indicated times; aP = 0.037, bP = 0.006, cP = 0.002 versus TNF-+HGF; dP = 0.012, eP < 0.001, fP = 0.002 versus HGF alone. For all values in the graph, n = 3.
HGF also suppressed expression of RANTES in HKC cells at bothmRNA and protein levels in a time- and dose-dependent manner.At 24 h, HGF at concentrations from 10 to 80 ng/ml HGF producedgreater suppression of RANTES mRNA levels than 5 ng/ml HGF (Figure 7A).In the presence of TNF-, HGF in concentrations from 10to 80 ng/ml suppressed the mRNA expression of RANTES to almostbasal levels (Figure 7B). Temporal studies showed that 20 ng/mlHGF inhibited RANTES expression beginning at 12 h with the maximaleffect seen at 48 h (Figure 7, C and D). RANTES protein levelswere modulated in a similar pattern. At 24 h, HGF inhibitedthe RANTES content in a dose-dependent pattern with the greatesteffect at 20 ng/ml (Figure 8A). In the presence of TNF-, HGFreduced RANTES content to the level produced by nontreated cells(Figure 8B). HGF also temporally inhibited the RANTES proteinlevels. TNF-–induced RANTES production was significantlydecreased after 24 h by HGF to a level comparable to that ofnontreated cells. Maximal suppression of RANTES production wasobserved at 48 h and was >50% for constitutive expressionand >60% for TNF-–induced expression (Figure 8C).
Figure 7. HGF inhibits constitutive or TNF-–induced RANTES expression in HKC cells in a dose- and time-dependent manner. Representative pictures for semiquantitative RT-PCR studies in HKC cells that were treated by HGF with different dosages (ng/ml; A and C) or by HGF at 20 ng/ml for different times (B and D) in the presence (C and D) or absence (A and B) of hTNF- (2 ng/ml). Representative gels are shown at the top, and the results of densitometry are shown at the bottom. Values in the graph are expressed as densitometric ratios as folds over control from three separate experiments. In A and C, *P < 0.05 versus other treatment; in B and D, *P < 0.05 versus non-HGF treatment at the same time point.
Figure 8. Dose-dependent and temporal effect of HGF on the constitutive and the TNF-–induced expression of RANTES in HKC cells. HKC cells were treated in triplicate with HGF and/or hTNF- at different dosages for different times. Conditioned media were then harvested and subjected to ELISA analysis of RANTES. (A) HKC cells were treated with HGF of indicated concentrations for 24 h; P < 0.001 by ANOVA, **P < 0.01 versus others, *P < 0.05 versus other groups with HGF. (B) HKC cells were treated with HGF of indicated concentrations in the presence of TNF- (2 ng/ml) for 24 h; P < 0.001 by ANOVA, *P < 0.05 versus other groups with HGF. (C) HKC cells were treated with 20 ng/ml HGF in the presence or absence of 2 ng/ml TNF- for different indicated times; aP = 0.017, bP = 0.008, cP = 0.002 versus TNF-+HGF, *P < 0.05 versus other groups with HGF; dP = 0.029, eP = 0.001, fP < 0.001 versus control. For all values in the graph, n = 3.
The effect of HGF on expression of MCP-1 and RANTES was alsoexamined in IRPTC. Although IRPTC were less responsive to TNF-stimulation than HKC, the results in IRPTC were remarkably similar.HGF downregulated the expression of MCP-1 and RANTES in IRPTC(Figure 9) in both the presence and the absence of TNF-.
Figure 9. HGF suppresses constitutive and TNF-–induced expression of MCP-1 and RANTES in rat tubular epithelial cells. Immortalized rat proximal tubular cells (IRPTC) were treated with HGF (20 ng/ml), rTNF- (10 ng/ml), or both for 24 h. RNA was then harvested for semiquantitative RT-PCR studies. Representative gels are shown at the top, and the results of densitometry are shown at the bottom. Values in the graph are expressed as densitometric ratios as folds over control from three separate experiments. *P < 0.05 versus nontreatment; #P < 0.05 versus TNF- treatment alone.
Influence of HGF on NF-B–Mediated Expression of MCP-1 and RANTES in TEC
To determine whether MCP-1 and RANTES induction in HKC by TNF-is regulated by NF-B activation, we examined the effects oftwo known inhibitors of NF-B, TPCK and PDTC. Both inhibitorsdecreased the expression of MCP-1 and RANTES (Figure 10A) atconcentrations that did not affect the HKC cell viability (Figure 10, B and C).
Figure 10. NF-B mediates the TNF-–induced expression of MCP-1 and RANTES in renal tubular epithelial cells. (A) Quiescent HKC cells were treated with hTNF- for 24 h in the presence or absence of n-tosyl-l-phenylalanine chloromethylketone (TPCK) or pyrrolidine dithiocarbamate (PDTC), specific NF-B inhibitors. RNA was harvested for semiquantitative RT-PCR studies. Representative gels are shown at the top, and the results of densitometry are shown at the bottom. Values in the graph are expressed as densitometric ratios as folds over control from three separate experiments. (B and C) No significant difference in the cellular viability was found between the cells that were treated with normal media and the cells that were treated with TPCK or PDTC at the concentration used in (A). In A, *P < 0.05 versus other treatments without TNF-; #P < 0.05 versus other treatments with TNF-; in B, *P < 0.05 versus other concentrations (n = 3).
Nuclear translocation is required for NF-B to mediate its proinflammatoryevents (30,31). Exposure of HKC to TNF- induced intense nuclearstaining for NF-B, suggesting that nuclear translocation waspromoted (Figure 11C). In contrast, NF-B was totally sequesteredin the cytoplasm, and translocation was not observed at anytime point in normal cells or cells that were treated with HGFalone (Figure 11, A and B). TNF-–induced translocationof NF-B was suppressed by HGF, as revealed by weak nuclear stainingin some cells (Figure 11D). HGF treatment for 30 or 120 minalso partially blocked TNF-–dependent nuclear translocationof NF-B (data not shown). These immunocytochemistry findingswere corroborated by immunoblot analysis of nuclear extractsfor NF-B p65 (Figure 11F). Quality of the nuclear extractionwas ensured by absence of cytoplasmic actin in nuclear extracts(Figure 11E) compared with an equal amount of total proteinof cytoplasmic extract. Densitometric analysis indicated thatTNF- induced nuclear translocation of NF-B, which was markedlydecreased by HGF at 60 min and 2 h (Figure 11F). Consistently,gel shift assay showed that TNF-–induced DNA binding byNF-B was abolished by HGF at 1 h (Figure 11G). These data suggestthat HGF suppresses TNF-–induced NF-B translocation andactivation, thereby inhibiting MCP-1 and RANTES expression inTEC.
Figure 11. HGF blunts the TNF-–induced NF-B activation in tubular epithelial cells. (A through D) HGF mitigates the TNF-–activated NF-B translocation in HKC cells. Quiescent cells were treated with HGF (20 ng/ml; B), hTNF- (2 ng/ml; C), or both (D) for 1 h, then were immunofluorescently stained for NF-B p65. Cells that were treated with serum-free media (A) served as normal control. (E and F) HKC cells were treated with HGF, TNF-, or both for different times before the nuclear and cytoplasmic extract was prepared. (E) The quality of the nuclear extract was ensured by the absence of the cytoplasmic actin on immunoblot compared with an equal amount of total protein of cytoplasmic extract. 1 through 4 represent four different experiments. (F) For quantifying the translocation of NF-B, nucleus extracts with equal amounts of total protein were immunoblotted with anti-p65 antibody. Representative blots are shown at the top, and the results of densitometry are shown at the bottom. Values in the graph are expressed as fold increase over cells at time 0 from three separate experiments. *P < 0.05 versus TNF- treatment alone at the same time point. (G) Gel shift assay reveals that HGF blunts the TNF-–activated DNA binding activity of NF-B. HKC cells were treated with HGF, TNF-, or both for 1 h before the nuclear extracts were prepared. Nucleus extracts with equal amounts of total protein were incubated with biotin-labeled oligonucleotides with NF-B consensus sequence, then subjected to gel shift assay in the presence or absence of anti-p65 antibody. ss, super shift band.
HGF Modulates the Activation of NF-B In Vivo
Phosphorylation of NF-B is a prerequisite of NF-B activation(32). We probed NF-B activation in vivo using a specific monoclonalantibody against phosphorylated NF-B p65 (p-p65). Normal kidneysfrom sham-operated rats had weak staining for activated NF-B,mainly located in nuclei of tubular epithelial cells (Figure 12A).This staining pattern was markedly increased after renalablation (Figure 12B) and was further significantly elevatedby HGF blockade with antibody (Figure 12C). In response to HGFsupplement, NF-B activation was suppressed to almost basal levels(Figure 12D). Immunoblots of kidney homogenates revealed attenuatedor enhanced expression of p-p65 after HGF infusion or blockade,respectively (Figure 12, E and F), corroborating the morphologyfindings. In addition, the ratio of activated p65 (p-p65) tototal p65 was markedly regulated by HGF, although the differencein total p65 expression did not reach a statistical significanceafter HGF infusion or blockade (Figure 12F).
Figure 12. HGF supplement suppresses whereas HGF blockade enhances the NF-B activation, mainly located in tubules, in rat remnant kidneys. (A through D) Representative micrographs of phosphorylated NF-B p65 immunofluorescent staining in frozen kidney sections from sham-operated rats (A) and nephrectomized rats that were treated with preimmune IgG (Ctrl-IgG; B), anti-HGF antibody (-HGF; C), or HGF (D). (E) Representative blots of Western immunoblot analysis of phosphorylated NF-B p65 (p-p65) and total p65 in kidney homogenates. (F) Arbitrary units of phosphorylated NF-B p65 and p65 abundance in immunoblot expressed as densitometric ratios over actin in Ctrl-IgG (blue bar), HGF (maroon bar), and -HGF (grey bar) groups as folds of sham-operated group (white bar). Ratios between phosphorylated NF-B p65 and p65 were also expressed as folds of sham-operated group. *P < 0.05 versus other remnant groups; #P < 0.05 versus other groups (n = 7 for each group). Magnification, x200 in A through D.
Renal inflammation is an important factor contributing to progressionof chronic renal disease (1,2,11). In the present study, HGFsuppressed renal inflammation and MCP-1 and RANTES expressionin TEC of the rat remnant kidney, attenuating renal fibrosis,and similarly inhibited chemokine expression in HKC and IRPTCin vitro. Conversely, neutralization of endogenous HGF by ananti-HGF antibody aggravated renal inflammation and enhancedtubular expression of chemokines, promoting tubular and glomerularinjury and fibrosis.
Several recent reviews (33–35) emphasized the antifibrogeniceffects of HGF as its major beneficial action in models of renalinjury. Our data provide three lines of experimental evidencefor another novel beneficial effect of HGF, suppressing renalinflammation. Thus, HGF treatment inhibited macrophage infiltrationin the remnant kidney model (Figure 2), suppressed expressionof proinflammatory chemokines MCP-1 and RANTES, and inhibitedthe activation of the NF-B transcription factor, the primarysignaling pathway of inflammation both in vivo and in vitro(30,31).
Although the precise mechanism whereby inflammatory cells causefibrosis is uncertain, most data suggest that macrophages promoterenal fibrosis (1,2,11,36,37). However, two notable studies(38,39) seem to contradict this hypothesis. One (38) reportedexacerbation in renal interstitial fibrosis associated withless macrophage infiltration at 14 d after unilateral ureteralobstruction (UUO) in lethally irradiated mice that receiveda transplant of angiotensin receptor type 1a–deficient(Agtr1–/–) bone marrow cells, as compared with Agtr1+/+bone marrow cell recipients. However, both groups failed toshow the florid histopathologic changes and renal dysfunctionthat are characteristic of the standard UUO model. In contrast,similar studies by Satoh et al. (40) and by Esteban et al. (41)demonstrated the essential role of macrophage and NF-B activationin the UUO model in wild-type (Agtr1+/+) mice. Consistently,Anders et al. (42) demonstrated that chemokine receptor-1 blockadereduced cellular infiltration and prevented renal fibrosis inthe UUO model.
In evaluating the role of urokinase receptor (uPAR) in regulatingthe phenotypic composition of interstitial cells (macrophage,myofibroblasts, and capillaries) in the UUO model, Zhang etal. (39) also noted decreased macrophage infiltration with increasedinterstitial myofibroblasts and endothelial cells associatedwith more renal fibrosis. Although Zhang et al. clearly demonstratedthat the uPAR deficiency decreased renal macrophage recruitment,they emphasized that the absence of this scavenger receptoris the main perpetrator of the fibrogenic response. Thus, theypostulated that uPAR-deficient macrophages delayed the clearanceof angiogenic/profibrotic molecules such as plasminogen activatorinhibitor-1. This may have resulted in aggressive myofibroblasticresponse and neovascularization with enhanced renal fibrosis.Thus, their findings underscore the phenotype of the macrophageas an important factor in interstitial injury that has beenrecently reviewed (36). Because of lack of specific reagentsand inability to similarly manipulate the genome of the experimentalrat model, we cannot ascertain the functional phenotype of themacrophage in the remnant kidney.
Chemokines are crucial in recruiting inflammatory cells fromthe intravascular compartment to the renal parenchyma (16,17).Our data suggest that the effect of HGF to downregulate MCP-1and RANTES expression in the tubulointerstitial compartmentwas largely mediated by TEC (Figures 3 and 4). Alternatively,infiltrating leukocytes may serve as a source of chemokines.For example, in a rat model of LPS-induced renal inflammation,Haberstroh et al. (43) demonstrated cross-talk between glomerularresident cells and monocytes/macrophages, whereby MCP-1 releasedby glomerular resident cells regulated monocyte/macrophage infiltrationthat then enhanced glomerular RANTES expression. Accordingly,we examined HGF modulation of chemokine production in rat alveolarmacrophages (CRL-2192, ATCC). HGF failed to modulate the constitutiveexpression of MCP-1 and RANTES in this cell line (data not shown),consistent with our view that effects on TEC predominate. However,further studies are necessary to resolve the question completely.
To model the proinflammatory milieu in damaged kidneys, we exposedTEC cells to the potent cytokine TNF-, which has been implicatedin the pathogenesis of renal inflammation and injury in theremnant kidney model (44). As predicted, TNF- induced expressionof MCP-1 and RANTES in both HKC cells and rat TEC cells, andthis was attenuated by HGF at both the mRNA and protein levels.However, there were noticeable differences in responses at themRNA and protein levels. Thus, marked inhibition of MCP-1 mRNAexpression (Figure 5) by HGF was not well correlated with thestatistically significant but marginal decreases in secretedMCP-1 protein (Figure 6). This discrepancy might be due to severalfactors, including (1) inherent differences in the turnoverrates of mRNA and protein, (2) the technical nature of the assaywhereby the cumulative levels of MCP-1 protein are comparedwith the mRNA transcription levels at a single-timed point,and (3) the lagged effect of HGF on NF-B inhibition (Figure 11F).This may have allowed for a window of significant MCP-1mRNA production in response to TNF-, resulting in the steepslope of MCP-1 protein levels during the first 12 h.
Cellular regulation of the expression of MCP-1 and RANTES iscomplex; however, the transcription factor NF-B is thought toplay a critical role in this process (30,31). More specific,TNF- has been shown to induce proinflammatory cytokines andchemokines via NF-B activation (45). We found that HGF mitigatesTNF-–provoked NF-B translocation and activation in TEC.In vivo, activated NF-B p65 was evident in tubules. HGF infusioninhibited whereas HGF blocking enhanced NF-B activation. Themechanism for HGF inhibition of NF-B activation is largely unknown.Although HGF clearly inhibited NF-B nuclear translocation at60 min, we observed lack of inhibition at the earlier time periodof 15 min. This may be due to the nature of the test systemwhereby both the activator (TNF-) and the inhibitor (HGF) wereadded simultaneously with the kinetics of the activator initiallysuperseding that of HGF action. Alternatively, the initial lagperiod may reflect HGF’s exerting an indirect inhibitoryeffect on NF-B translocation through end products of other signalingpathways activated by the HGF/c-Met axis. Detailed investigationis necessary to identify the mediators involved in the interactionof HGF and NF-B in tubular epithelial cells.
Another important finding in the present study is that HGF haltedthe progression of proteinuria and ameliorated glomerulosclerosisin remnant kidneys, demonstrating that HGF has beneficial effectson glomerular as well as interstitial injury (Table 2). Previously,we demonstrated that continuous infusion of HGF for 1 wk decreasedglomerular capillary pressure (24) in remnant rats, and thismight account for the antiproteinuric effect of HGF. Our findingsare also consistent with recent reports demonstrating beneficialeffects of HGF on glomerular injury in models of mesangial damage(46) and in diabetic glomerulopathy (27).
Alternatively, HGF may have reduced glomerular injury by attenuatingglomerular macrophage infiltration (Figure 2E). Although absolutemacrophage number was much lower in glomeruli than in interstitium,HGF administration substantially inhibited and HGF blockingmarkedly aggravated glomerular infiltration. The primary targetcell for HGF action in the glomerulus is still unclear. Mesangialcells seem to play a pivotal role in regulating glomerular infiltration;however, basal expression of c-met is extremely low (47) inthese cells, making them an unlikely target cell for HGF. Consistentwith this view, basal and TNF-–induced expressions ofchemokines were not regulated by HGF in cultured rat mesangialcells (CRL-2573, ATCC; data not shown). Nevertheless, data fromthis (47) and other laboratories (48) demonstrate that cytokinessuch as IL-6 and PDGF can induce expression of c-met in mesangialcells. It follows that increased production of these cytokinesin diseased glomeruli might render these cells susceptible tomodulation by HGF.
In conclusion, HGF reduces glomerular and tubulointerstitialinflammation and ameliorates proteinuria and renal fibrosisin the rat remnant kidney model. Attenuation of interstitialinflammation is associated with parallel reductions in tubularexpression of MCP-1 and RANTES as well as blunted NF-B activationin tubules. In vitro, HGF suppresses basal and stimulated expressionof these chemokines and inhibits NF-B activation in TEC. Ourfindings suggest that HGF exerts its beneficial effects on progressionof chronic renal disease at least in part by its anti-inflammatoryactions, likely via the NF-B pathway.
Acknowledgments
This work was supported by National Institutes of Health GrantR01-DK-52314 to L.D.D. R.G. was supported by a postdoctoralresearch fellowship from the Department of Medicine, Brown MedicalSchool.
We are grateful to Dr. Ingelfinger for providing the IRPTC cellline.
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Received for publication January 28, 2004.
Accepted for publication July 25, 2004.
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Abstract
Introduction
–induced expression of these chemokines at both the mRNA and protein levels in a time- and dose-dependent manner in proximal tubular epithelial cells. HGF also blunted TNF-
B, a pivotal transcription factor that regulates chemokine expression. Immunohistochemistry showed that activated NF-
1, basic fibroblast growth factor, and PDGF (6). These factors in turn activate resident fibroblasts (7,8) and promote epithelial-to-mesenchymal transition (9). Heterogeneous fibroblasts proliferate and generate excessive extracellular matrix, resulting in renal scarring. Consistently, the extent of inflammation is well correlated with kidney function and predicts long-term prognosis in a number of clinical settings (10). Suppression of renal inflammation by administration of immunosuppressive drugs is beneficial even in nonimmune models of renal disease (11) such as remnant kidney (12) and diabetic nephropathy (13). 
, markedly induce TEC expression of macrophage chemoattractant protein-1 (MCP-1) and RANTES (regulated upon expression normal T cell expressed and secreted) (19), key chemokines that recruit mononuclear cells to the kidney (16,17). 
180 to 210 g were housed in an approved animal care facility and fed standard diet. Five-sixths renal ablation was performed as described previously (23,24). Two weeks after surgery, rats were randomly assigned to one of three groups (n = 7): (1) HGF-treated group (HGF): rats received recombinant human HGF (Genentech, South San Francisco, CA) as a continuous intravenous infusion via an implanted osmotic minipump into the jugular vein at a dose of 100 µg/kg per d for 14 d; (2) anti-HGF antibody–treated group (
). Alternatively, infiltrating leukocytes may serve as a source of chemokines. For example, in a rat model of LPS-induced renal inflammation, Haberstroh et al. (43) demonstrated cross-talk between glomerular resident cells and monocytes/macrophages, whereby MCP-1 released by glomerular resident cells regulated monocyte/macrophage infiltration that then enhanced glomerular RANTES expression. Accordingly, we examined HGF modulation of chemokine production in rat alveolar macrophages (CRL-2192, ATCC). HGF failed to modulate the constitutive expression of MCP-1 and RANTES in this cell line (data not shown), consistent with our view that effects on TEC predominate. However, further studies are necessary to resolve the question completely. 
