Counteractive effects of HGF on PDGF-induced mesangial cellproliferation in a ra - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:38 |只看该作者 |倒序浏览 |打印

作者：KazuhikoBessho, ShinyaMizuno, KunioMatsumoto,  ToshikazuNakamura作者单位：Division of Molecular Regenerative Medicine, Course ofAdvanced Medicine, Osaka University Graduate School of Medicine,Osaka 565-087 Japan 7 |; Z+ `& R: m8 t0 g8 |* C: }  ]- E

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      【摘要】
   Activationand proliferation of glomerular mesangial cells play an important rolein the development of mesangioproliferative glomerulonephritis. Weinvestigated the role of hepatocyte growth factor (HGF) in regulatingactivated mesangial cell proliferation. In glomeruli of normal rats,mesangial cells barely expressed the c-Met/HGF receptor. However, whenmesangioproliferative glomerulonephritis was induced in rats by theadministration of an anti-Thy 1.1 antibody, glomerular HGF expressiontransiently decreased along with mesangiolysis, and activation ofmesangial cells was associated with upregulation of the c-Met receptor.Activated mesangial cells in culture also expressed the c-Met/HGFreceptor. Although addition of HGF to cultured mesangial cells did notincrease DNA synthesis, HGF did diminish PDGF-induced DNA synthesis.PDGF induced activation of ERK, which continued for at least 48 h.When PDGF and HGF were simultaneously added, HGF inhibited theprolonged activation of ERK, which suggests that early inactivation ofPDGF-induced ERK may be involved in the inhibitory effect of HGF onmesangial cell proliferation. Furthermore, administration of HGF torats with anti-Thy 1.1 nephritis resulted in a selective suppression of activated mesangial cell proliferation, and this suppressive effect wasassociated with attenuation of phosphorylated glomerular ERK. Theseresults indicate that HGF counteracts PDGF-induced mesangial cellproliferation and functions as a negative regulator of activated mesangial cell proliferation. : K1 \, ^0 r5 w8 \" z; x
      【关键词】 hepatocyte growth factor myofibroblastlike cell cmet ERK
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GLOMERULAR MESANGIAL CELLS mechanically support glomerular capillary loopsand control the rate of plasma ultrafiltration under physiologicalconditions. On the other hand, mesangial cell proliferation is akey feature of a variety of human glomerular diseases, includingIgA nephropathy, lupus nephritis, variants of idiopathic focalsclerosis, and amyloid or diabetic nephropathy ( 12 ). Inthese diseases, mesangial cell proliferation is associated with aphenotypic change into myofibroblast-like cells (we here refer these to"activated" mesangial cells) ( 17 ). In experimental models of nephritis, mesangial cell proliferation frequently precedes and is linked to an increase in the extracellular matrix in the glomerulus ( 9 ). Although some proliferation and phenotypic changes in mesangial cells are likely to benefit the host in the physiologically reparative response to tissue injury, excessive proliferation of activated mesangial cells leads to the development ofglomerulosclerosis. Therefore, a better understanding of regulatory mechanisms involved in mesangial cell proliferation would provide useful insights into therapeutic strategies to treat humans with progressive glomerular diseases.

Hepatocyte growth factor (HGF), originally identified and cloned as amitogen for mature hepatocytes ( 31, 32 ), exhibits multiplebiological activities in a wide variety of cells ( 22, 27, 38 ) through the c-Met receptor tyrosine kinase ( 6 ). Several lines of evidence indicate an important role for HGF in themorphogenesis of organs during development ( 3, 35 ).Physiologically, HGF has organotrophic functions in the regenerationand protection of various organs from injuries ( 22, 27 ).Biological activities of HGF are likely to support tissue organizationduring development and regeneration. In the kidney, biological andrenotropic roles of HGF have been well noted. HGF has mitogenic,morphogenic (induction of branching tubulogenesis), and anti-cell deathactions on renal tubular epithelial cells ( 3, 28 ). In theglomerulus, HGF induces a mitotic response in glomerular endothelialcells ( 37 ). Cyclosporine A-induced apoptosis inglomerular epithelial cells was prevented by HGF ( 1 ). Inresponse to acute renal injury, the expression of HGF increases and HGFplays a role in renal regeneration and protection from injuries( 3, 28 ). Furthermore, the renotropic role of HGF afterrenal injury led to therapeutic approaches for treatment of renaldiseases, including acute and chronic renal diseases in experimentalmodels ( 7, 20, 29 ). Past approaches shed light onbiological and renotropic roles of HGF in renal epithelial andendothelial cells; however, the potential functions of HGF inregulating mesangial cell proliferation and behavior have beendiscussed in only a few studies ( 19, 21, 37 ).

In the present study, we investigated the role of HGF and the c-Metreceptor in the proliferation of activated mesangial cells, using themost widely studied experimental model for mesangioproliferative glomerulonephritis, anti-Thy 1.1 glomerulonephritis ( 11 ).Our findings indicate that HGF is a negative regulator of activated mesangial cell proliferation.
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MATERIALS AND METHODS, t( e* s, ^8 M# |+ b: u& e; w5 V
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Animals and materials. Male Wistar rats (7-8 wk old; 190-210 g) were purchased fromSLC Japan (Hamamatsu, Japan). All animal experiments were done inaccordance with National Institute of Health guidelines, as dictated bythe animal care facility at Osaka University Graduate School ofMedicine. Recombinant human HGF was purified from cultured media ofChinese hamster ovary cells transfected with cDNA, which encodes avariant type of human HGF with five deleted amino acid residues in thefirst kringle domain ( 20, 29, 30 ). The purity of HGFexceeded 98%, as determined by SDS-PAGE plus protein staining. Recombinant human PDGF-BB was purchased from R&D Systems (Minneapolis, MN). B cell hybridoma (OX-7), which produces anti-rat Thy 1.1 mousemonoclonal IgG 1, was a gift from the Cell Resource Center for Biomedical Research, Tohoku University. Hybridoma cells were administered intraperitoneally into male BALB/c mice (SLC Japan), andascites was obtained from these mice./ r1 d, X! `3 d- x' d, O& ~: G
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Experimental mesangioproliferative glomerulonephritis. To induce anti-Thy 1.1 glomerulonephritis in rats, the ascites obtainedfrom the mice was given intravenously to rats at 25 µl/rat. Rats weredivided into two groups so that the average concentration of urinaryprotein collected over 34-46 h after anti-Thy 1.1 antibodytreatment was equal in each group. HGF (600 µg/kg body wt in 0.5 mlsaline) or an equal volume of saline alone was given subcutaneouslytwice a day from 48 h after anti-Thy 1.1 treatment. Afteradministration of HGF, plasma human HGF levels increased to 12.3, 13.4, and 7.2 ng/ml at 1, 2, and 6 h, whereas human HGF levels in renaltissue were 11.2, 12.6, and 5.8 ng/g tissue at 1, 2, and 6 h,respectively (data not shown). 5-Bromo-2'-deoxyuridine (BrdU; NacalaiTesque, Kyoto, Japan) was given intraperitoneally (100 mg/kg body wt)2 h before the rats were killed.
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Real-time quantitative RT-PCR. For analysis of changes in glomerular gene expression in anti-Thy 1.1 glomerulonephritis, glomeruli were isolated from kidneys of nephriticrats. In brief, renal cortexes of each rat were subjected todifferential sieving ( 25 ) at 4°C, then 200-300glomeruli/rat were collected under microscopy and stored at 80°Cuntil use. Total RNA was extracted from purified glomeruli using anRNeasy Mini kit and RNase-Free DNase Set (Qiagen), according to themanufacturer's instructions. Ten microliters of total RNA solutionwere subjected to reverse transcription into first-strand cDNA with arandom hexaprimer using Superscript II RT (Invitrogen, Carlsbad, CA). Taq Man quantitative PCR was done using the ABI PRISM 7700 Sequence Detector System (PerkinElmer Biosynthesis, Foster City, CA),as described elsewhere ( 30 ). Sequences for primers and Taq Man fluorogenic probes were as follows. For rat HGF, theforward primer was 5'-AAG AGT GGC ATC AAG TGC CAG-3'; the reverseprimer was 5'-CTG GAT TGC TTG TGA AAC ACC-3'; and the probe was5'(FAM)-TGA TCC CCC ATG AAC ACA GCT TTT TG -(TAMRA) 3'. For rat PDGF-Bchain, the forward primer was 5'-TCC AGA TCT CGC GGA ACC T-3'; thereverse primer was 5'-CTG CAC ATT GCG GTT ATT GC-3'; and the probe was 5'(FAM)-CGA TCG CAC CAA TGC CAA CTT CC-(TAMRA) 3'. For GAPDH, theforward primer was 5'-CCA TCA CTG CCA CTC AGA AGA C-3'; the reverseprimer was 5'-TCA TAC TTG GCA GGT TTC TCC A-3'; and the probe was5'(FAM)-CGT GTT CCT ACC CCC AAT GTA TCC GT-(TAMRA) 3'. Experimentalsamples were matched to a standard curve generated by amplifyingserially diluted products, using the same PCR protocol. To correct forvariability in RNA recovery and for efficiency of reversetranscription, GAPDH cDNA was amplified and quantitated in each cDNA preparation.
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To analyze effects of HGF on expression of PDGF-B chain mRNA incultured mesangial cells, the cells were seeded in six-well cultureplates (3 × 10 4 cells/well), cultured for 24 h,serum-starved for 48 h, and treated with 30 ng/ml HGF for 6 and12 h. Total RNA from the cells was prepared, using Iso-Gen (NipponGene, Toyama, Japan), and 3 µg of total RNA were reverse transcribedinto first-strand cDNA with a random hexaprimer using Superscript IIRT. Taq Man quantitative PCR for rat PDGF-B chain was doneusing the same primers and probes as above.
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Histological analysis. Tissues were fixed in neutral buffered formalin (pH 7.4), embedded inparaffin, and then sections were stained with hematoxylin and eosin.Forty-eight glomerular cross sections from each rat were examined, andthe mean number of nuclei per glomerular cross section was measured.For immunohistochemical staining, tissues were fixed in 70% ethanolfor detection of HGF. The primary antibodies used were polyclonalanti-rat HGF ( 30 ). Tissue sections were subjected to theavidin-biotin coupling technique, using a commercial kit (VecstastainElite ABC, Vector Laboratories, Burlingame, CA), according to themanufacturer's instructions. To identify activated mesangial cells,peroxidase-conjugated monoclonal anti-human -smooth muscle actin( -SMA; DAKO, Glostrup, Denmark) was used. Glomerular staining for -SMA was evaluated by the scoring method principally described byFloege et al. ( 13 ). In each sample, 48 glomerular crosssections were examined, and two individual observers made evaluationsof all the slides.* p! b7 q: [! J7 l
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For double immunohistochemical staining of c-Met and Thy 1.1 or c-Metand -SMA, tissues were fixed in 70% ethanol and then incubated withpolyclonal anti-mouse c-met (SP260, Santa Cruz Biotechnology, SantaCruz, CA) plus anti-Thy 1.1 ascites (see above) or monoclonalanti-human -SMA. After being washed with PBS, tissue sections weresequentially incubated with Alexa Fluor 546 anti-mouse IgG conjugateand Alexa Fluor 488 anti-rabbit IgG conjugate (Molecular Probes,Eugene, OR). Tissue sections were examined under a laser-scanning microscope.4 ?: Q8 k7 {. v9 w! z
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For immunohistochemical staining for BrdU, tissues were fixed in 70%ethanol and incubated with murine anti-BrdU monoclonal IgG (Takara Bio,Kyoto, Japan). The sections were then incubated withperoxidase-conjugated anti-murine IgG, and BrdU-positive cells wereidentified based on the enzymatic reaction using Vecstastain Elite ABC.In the doublestaining for BrdU and -SMA, tissue sections stained forBrdU were successively incubated with a commercially available blockingreagent (MOM Immunodetection kit, Vector Laboratories) and monoclonalanti-human -SMA. The sections were next incubated with alkalinephosphatase-conjugated anti-murine IgG, and -SMA-positive cells weredetected using a commercial kit (Vecstastain ALP ABC, VectorLaboratories). In each sample, 48 glomerular cross sections wereexamined, and the mean numbers of double-positive cell per glomerularcross section were determined. All antibodies and control IgG were usedat 1 µg/ml, and no significant signal was obtained on substitution ofthe primary antibody with equivalent concentrations of normal rabbit ormouse IgG and with the c-Met primary antibody preabsorbed with anexcess of c-Met blocking peptide (SP260P, Santa Cruz Biotechnology).

Cell culture and measurement of cell growth. Mesangial cells were prepared from 8-wk-old male Wistar rats by adifferential sieving method and characterized as described ( 25 ). Cells were cultured in DMEM supplemented with 20黃, 0.5 µg/ml streptomycin, 100 U/ml penicillin G, and 0.25 µg/ml amphotericin B. For all experiments, we used cells of passages 4-10. For measurement of DNA synthesis, cells were seeded in96-well culture plates (5 × 10 3 cells/well) andcultured for 24 h. Under these conditions, a cell confluence of~95% was reached. Cells were serum-starved for 48 h in mediumsupplemented with 0.5% FCS, and then the medium was replaced withfresh medium and cells were further cultured for 48 h in theabsence or presence of PDGF and/or HGF. The cells were pulse-labeledwith [ 3 H]thymidine (1.0 µCi/ml) for 5 h, and theamount of [ 3 H]thymidine incorporated into nuclei wasmeasured, as described elsewhere ( 32 ). Six wells were usedfor each condition.

Western blotting. To detect c-Met receptor expression in the cultured cells, the cellswere seeded in 90-mm culture dish (2 × 10 5 cells/dish), cultured for 4 days, serum-starved for 24 h, then lysed in sample buffer for SDS-PAGE. The lysate was subjected toSDS-PAGE on a 6% polyacrylamide gel, and proteins were electroblotted on polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Afterblocking, the membrane was sequentially incubated with the anti-mousec-Met antibody and horseradish peroxidase-conjugated anti-rabbit IgG.Signals were visualized using an enhanced chemiluminescence reagent(Amersham Pharmacia Biotech).

For analysis of ERK phosphorylation in cultured mesangial cells, cellswere seeded in six-well culture plates (3 × 10 4 cells/well) and cultured for 24 h. Cells were serum-starved for 48 h then treated with 20 ng/ml PDGF-BB and/or 30 ng/ml HGF for the indicated periods. Cells were washed once with ice-cold PBS, snap-frozen, thawed, and lysed with lysis buffer composed of (in mM) 20 mM Tris · HCl (pH 7.5), 150 NaCl, 10 EDTA, 2 Na 3 VO 4, 10 NaF, and 1 PMSF as well as 1.0%Triton X-100, 0.1% SDS, and 10 µg/ml each of leupeptin, antipain,and pepstatin. The cell lysate was centrifuged at 12,000 g for 30 min. Protein concentration in the cell lysate was measured,using a DC protein assay kit (Bio-Rad). The extract (20 µg protein)was resolved by SDS-PAGE on a 12% polyacrylamide gel under reducingconditions and transferred onto polyvinylidene difluoride membrane.After blocking, the membrane was sequentially incubated withanti-phospho-p44/p42 MAPK (ERK1/2) monoclonal antibody (E10; NewEngland BioLabs, Beverly, MA), biotinylated anti-mouse IgG (VectorLaboratories), and horseradish peroxidase-conjugated streptavidin(Amersham Pharmacia Biotech). To detect total ERK protein, the samemembrane was stripped and reprobed with anti-ERK1 antibody (K-23; SantaCruz Biotechnology) as the primary antibody. The band densities ofimmunoblots were analyzed using National Institutes of Health Imagesoftware (Wayne Rasband Analytics, NIH, http://rsb.info.nih.gov/nih-image/ ).: g  S1 j  Y: k
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For analysis of glomerular ERK phosphorylation, rats were perfused withice-cold PBS containing (in mM) 10 EDTA, 2 Na 3 VO 4, 50 NaF, and 2 PMSF as well as 10 µg/ml each of leupeptin, antipain, and pepstatin. Glomeruli wereisolated using the differential sieving method and were immediatelylysed with RIPA buffer composed of (in mM) 50 Tris · HCl (pH 7.5), 150 NaCl, 1 EDTA, 2 Na 3 VO 4, 50 NaF, and 2 PMSF as well as 0.1%SDS, 1% Nonidet P-40, 0.1% sodium deoxycholate, and 10 µg/ml eachof leupeptin, antipain, and pepstatin. The lysate was centrifuged at12,000 g for 30 min, and the supernatant containing 100 µgof protein was subjected to SDS-PAGE, followed by immunoblot analysis,as described above.

ELISA for glomerular PDGF-BB. For measurement of glomerular PDGF levels, glomeruli were isolated fromthe kidney 4 days after anti-Thy 1.1 treatment. Glomeruli wereextracted in buffer composed of 20 mM Tris · HCl(pH 7.5), 2 M NaCl, 0.1% Tween 80, 1 mM EDTA, 1 mM PMSF, and 10 µg/ml each of leupeptin, antipain, and pepstatin. Glomerular PDGFlevels were measured using a commercial kit (Quantikine human PDGF-BB, R&D systems), according to the manufacturer's instruction.

Statistical analysis. For statistical analysis, we used Student's unpaired t -testor ANOVA to determine the statistical significance. A P value significant.9 c& f8 j( I2 G0 r  J8 N) l
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RESULTS
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Change in glomerular HGF expression in anti-Thy 1.1 glomerulonephritis. After administration of the anti-Thy 1.1 antibody, glomeruli showedchanges typical of mesangioproliferative glomerulonephritis, asreported ( 11 ). A loss of mesangial cells associated with disruption of the mesangial matrix (mesangiolysis) was seen on days 1-2, and mesangial cell proliferation began from day 2. Expression of -SMA, a marker of activatedmesangial cells, was undetectable in normal glomeruli but wasdetectable from 2 days after anti-Thy 1.1 treatment (data not shown).The overshooting mesangial cell growth was observed on days4 and 8. To determine the potential involvement of HGFin pathophysiological changes in anti-Thy 1.1 nephritis, we firstanalyzed changes in glomerular expression of HGF during progression ofglomerulonephritis (Fig. 1 ). Glomerular HGF mRNA levels decreased on day 2 (~29% compared withthe value at day 0 ), the time when glomerular cell loss wasmost prominent (Fig. 1 A ). The HGF mRNA level then increasedtoward day 8 and returned to ~80% of the pretreatmentlevel. To determine the localization of HGF, immunohistochemicalexamination was made on the glomeruli of anti-Thy 1.1 glomerulonephritis. In normal glomeruli, HGF was identified mainly inthe capillary luminal areas composed of endothelial cells, whereas weaksignals were sparse in mesangial areas (Fig. 1 B, arrowheadand inset, day 0 ). On day 2 afteranti-Thy 1.1 treatment, glomerular cells markedly decreased, and HGFexpression was noted also on remaining mesangial-like cells, which weremutipolar and located between clusters of capillary loops (Fig. 1 B, arrow, day 2 ). HGF expression becamepredominant in mesangial areas rather than capillary areas, from 4 daysafter anti-Thy 1.1 treatment. On day 8, HGF expressionbecame extensive, accompanied by glomerular hypercellularity, and thiswas consistent with the recovery of HGF mRNA levels. At this time,HGF-positive signals were mainly noted in mesangial areas (Fig. 1 B, arrow and inset, day 8 ). These results suggest that the temporal decrease in HGF mRNA levels in thismodel may be due to glomerular injuries of intrinsic cells (possiblyincluding endothelial and mesangial cells) caused by mesangiolyticevents. On the other hand, plasma HGF levels did not changesignificantly during the progression of glomerulonephritis.* _& a8 [. n9 m" L
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Fig. 1. Change in hepatocyte growth factor (HGF) mRNA expression( A ) and localization ( B ) in glomeruli duringdevelopment of anti-Thy 1.1 glomerulonephritis. A : rats werekilled before ( day 0 ) and on days 2, 4, and 8 after anti-Thy 1.1 antibody injection.Total RNA was obtained from purified glomeruli of each rat at each timepoint, and HGF mRNA levels were determined by real-time quantitativeRT-PCR. Values (% of day 0 ) are means ± SD( n = 6). B : immunohistochemical findings ofHGF in glomeruli. Before onset of injury, endothelial-like cells,forming capillary loops (arrowhead and inset ), were stainedfor HGF, and HGF-positive signals were detectable sparsely also in themesangial area. On the other hand, on days 2, 4,and 8, HGF was identified in multipolar mesangial-likecells, located between clusters of capillary loops (arrows and insets ). Magnification: ×200 and ×800( insets ).; m- r) M% Q4 ~9 g. S
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Induction of the c-Met receptor in activated mesangial cells. Next, we analyzed expression of the c-Met/HGF receptor in glomeruli(Fig. 2 A ). In the normalglomerulus, c-Met receptor expression was evident in cells, presumablyepithelial and endothelial cells based on their localization andmorphology (Fig. 2 A, center top ). Whenc-Met staining was merged with Thy 1.1 staining, a marker of mesangialcells, c-Met-positive cells and Thy 1.1-positive cells were mostlynoncolocalized but not exclusively, thereby indicating that mesangialcells in normal glomeruli are mostly c-Met negative (Fig. 2 A, top ). On the other hand, whenglomerular c-Met expression was analyzed in rats with anti-Thy1.1 nephritis, based on double immunohistochemistry of c-Met and -SMA (Fig. 2 A, bottom center and left ), -SMA-positive cells were mostly c-Met positive,thus indicating that the -SMA-positive activated mesangial cellsexpressed the c-Met receptor (Fig. 2 A, bottom right ). These results indicate that the c-Met receptor isinducible in mesangial cells during activation towardmyofibroblast-like cells, and hence HGF may play a role in regulatingactivated mesangial cell behavior. On the other hand, when we measuredthe change in c-Met mRNA expression in isolated glomeruli usingquantitative PCR, glomerular c-Met mRNA expression did not changesignificantly.) [7 K/ e6 b5 K8 x' Z

Fig. 2. Colocalization of c-Met receptor expression in activatedmesangial cells in glomeruli of rats with anti-Thy 1.1 nephritis andexpression of the c-Met receptor in rat mesangial cells in culture. A : expression of Thy 1.1, a marker of mesangial cells( top left ), and -smooth muscle actin (SMA), a marker ofactivated mesangial cells ( bottom left ), and the c-Metreceptor ( middle, top and bottom ) wereanalyzed in normal rat glomerulus ( top ) and glomerulus on day 4 after anti-Thy 1.1 treatment ( bottom ). Top and bottom right : merged images of Thy 1.1 and c-Met at day 0 and -SMA and c-Met at day4, respectively (merged areas are in yellow). Magnification:×200. B : expression of c-Met receptor in cultured mesangialcells, as detected by Western immunoblotting.

To obtain further evidence for induction of the c-Met receptors inmesangial cells, mesangial cells isolated from glomeruli of the normalrat kidney were used. During cultivation, mesangial cells autonomouslyunderwent a phenotypic change into -SMA-positive activated mesangialcells (data not shown), as reported elsewhere ( 8 ). Whenexpression of the c-Met receptor was analyzed by Western blotting,activated mesangial cells in culture clearly expressed the c-Metreceptor (Fig. 2 B ), which means that activated mesangialcells may be targets of HGF.  k: U$ j+ S$ C' Y* C" F1 a
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Reduction of PDGF-induced DNA synthesis by HGF in culturedmesangial cells. Because induction of the c-Met receptor in activated mesangial cellsand regulation of HGF expression suggested the potential involvement ofHGF in the pathophysiology of mesangioproliferative glomerulonephritis, we next asked whether HGF would regulate the proliferation of activated mesangial cells in culture. Because PDGF isthe most potent known mitogen for mesangial cells ( 12 ) andPDGF induced in glomeruli with anti-Thy 1.1 nephritis is involved inthe proliferation of mesangial cells ( 10, 16 ), mesangial cells were cultured in the absence or presence of HGF, PDGF, or theircombination and subjected to measurement of DNA synthesis (Fig. 3 A ). Consistent with reporteddata ( 14 ), addition of PDGF stimulated DNA synthesis ofmesangial cells and the stimulatory effect of PDGF was maximal atconcentrations over 20 ng/ml (Fig. 3 A and data not shown).HGF alone up to 100 ng/ml had no significant effect on DNA synthesis ofmesangial cells. However, the simultaneous addition of HGF and PDGFdose dependently inhibited PDGF-induced DNA synthesis, and 10-30ng/ml HGF inhibited DNA synthesis to the basal level seen without PDGF.
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Fig. 3. Effects of PDGF and HGF on DNA synthesis and change inPDGF-B mRNA expression by HGF treatment in cultured rat mesangialcells. A : cultured rat mesangial cells were growth arrestedand stimulated with PDGF (20 ng/ml), HGF (3-100 ng/ml), or acombination of PDGF (20 ng/ml) and HGF (1-30 ng/ml). Cells werecultured for 48 h, and DNA synthesis was measured based on[ 3 H]thymidine incorporation into nuclei. Values aremeans ± SD of 6 measurements. * P P B : total mRNA was prepared fromcultured mesangial cells treated with PBS (filled bars) or 30 ng/ml HGF(open bars), and PDGF-B chain mRNA levels were determined byquantitative RT-PCR. Values are means ± SD; n = 6 wells/condition.2 C8 k4 I& q  l3 e! Z! D

Because a previous study showed that mesangial cells expressed PDGF inthis model ( 15 ), regulation of PDGF expression by HGFmight be involved in a mechanism by which HGF suppressed PDGF-induced cell proliferation. We thus analyzed the effect of HGF on PDGF expression in cultured mesangial cells, using real-time quantitative RT-PCR (Fig. 3 B ). At both 6 and 12 h after HGFtreatment, there was no significant change in PDGF B-chain mRNAexpression between untreated cells and cells treated with HGF. We nextanalyzed changes in the activation of ERK in mesangial cells (Fig. 4 ), because ERK plays a central role inproliferation of cells downstream of both PDGF receptor and the c-Metreceptor tyrosine kinases, and inhibition of ERK in cultured mesangialcells potently diminishes PDGF-induced proliferation ( 5 ).In growth-arrested mesangial cells, ERK was marginally phosphorylatedor mostly unphosphorylated, whereas it was strongly phosphorylated 10 min after stimulus with either PDGF or HGF. It is noteworthy, however,that activation/phosphorylation of ERK continued for at least 48 hafter the PDGF stimulus, whereas activation/phosphorylation of ERK byHGF was transient. ERK was strongly phosphorylated by the HGF stimulusat 10 min, which rapidly decreased after 10 min, and only marginalphosphorylation remained 2 h after the HGF stimulus. When PDGF andHGF were simultaneously added, HGF significantly inhibited thePDGF-dependent prolonged phosphorylation of ERK, although levels ofphosphorylated ERK were higher than seen in the case of HGF alone.Levels of phosphorylated ERK in cells treated with PDGF plus HGF weresignificantly lower than those in PDGF-treated cells later than 2 h after treatment, and weakly phosphorylated ERK was seen 24-48 hafter treatment with PDGF plus HGF.
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Fig. 4. Time course of phosphorylation of ERK, induced by PDGF,HGF, and PDGF plus HGF in cultured mesangial cells. A :changes in phosphorylated ERK ( top ) and total ERK protein( bottom ) were detected by Western immunoblotting, usinganti-phosphorylated ERK antibody and anti-ERK antibody. Proteins ineach cell lysate were separated by SDS-PAGE and subjected to Westernblotting. The same membrane used in anti-phosphorylated ERK wasreprobed with an anti-ERK antibody. B : densitometricanalysis of ERK phosphorylation as detected by Western blotting. Valuesrepresent the relative abundance of phosphorylated ERK vs. total ERKprotein. The value obtained at 10 min after HGF treatment was taken as1.0. Values are means ± SD of 3 independent experiments.* P P

Suppression of mesangial cell proliferation, glomerular ERKphosphorylation, and -SMA expansion in anti-Thy 1.1 glomerulonephritis by HGF. Based on the finding that HGF inhibited the proliferation of activatedmesangial cells stimulated by PDGF in vitro, we considered it importantto determine whether administration of HGF would exert negativeregulatory functions in the expansion of mesangial cells duringprogression of anti-Thy 1.1 nephritis. For this purpose, recombinanthuman HGF was repeatedly administrated later than 2 days afterinjection of anti-Thy 1.1 antibody (Fig. 5 A ). Because disruption ofglomerular construction occurs during the first 2 days after anti-Thy1.1 treatment ( 11 ), treatment with HGF later than 2 daysafter anti-Thy 1.1 treatment excludes the possibility that HGF wouldaffect the onset of nephritis itself in this model. Compared withcontrol saline-treated rats, the mean number of total cells perglomerulus significantly decreased on days 4 and 8 in HGF-treated rats (Fig. 5 B ). To observewhether a decrease in the number of cells in glomeruli after HGFtreatment was due to a decrease in the number of proliferating cells,cells undergoing DNA synthesis were determined based on BrdUincorporation and subsequent immunohistochemistry (Fig. 6 A ). The number ofBrdU-positive cells per glomerulus in HGF-treated rats decreased to74.7% on day 4 and 58.0% on day 8, comparedwith findings in saline-treated rats. On the other hand, apoptoticcell death in activated mesangial cells was proposed to be a cellclearance mechanism in anti-Thy 1.1 nephritis, thereby contributing tothe resolution of glomerular hypercellularity ( 2 ).However, during our present observation, the number of terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick-endlabeling-positive apoptotic cells was few, and there was not asignificant change with HGF treatment (data not shown).

Fig. 5. Experimental protocol for administration of HGF into ratswith anti-Thy 1.1 glomerulonephritis and change in glomerular cell no.by HGF treatment. A : recombinant human HGF (HGF) or the samevolume of saline was given subcutaneously every 12 h from 48 h after anti-Thy 1.1 treatment. Analysis was made on days 4 and 8. B : change in the total no. of cells perglomerulus on days 4 and 8 after anti-Thy 1.1 treatment. Hematoxylin-eosin staining was used. Magnification: ×200.Values are means ± SD ( n = 6). * P6 n4 g4 ^5 v7 f! `

Fig. 6. Effect of HGF on proliferation of glomerular cells andexpansion of -SMA. HGF was administrated to rats with anti-Thy 1.1 glomerulonephritis, as described in Fig. 5 A. A :distribution of 5-bromo-2'-deoxyuridine (BrdU)-positive cells andchange in the no. of BrdU-positive cells per glomerulus by HGFtreatment at days 4 and 8 after anti-Thy 1.1 treatment. Micrographs indicate localization of BrdU-positive cells inglomeruli on day 4. B : distribution of cellspositive for BrdU and -SMA and change in the no. of BrdU-positivecells with HGF treatment in activated mesangial cells and other cellsin glomeruli at day 4. Note the distribution of cellsstained for BrdU (brown) and -SMA (red). Expression of BrdU and -SMA was detected using double immunohistochemistry. Originalmagnifications: ×200. C : tissues were analyzed for -SMAexpression on days 4 and 8, and expression of -SMA was evaluated using the scoring method described by Floege etal. ( 13 ). A - C : values aremeans ± SD ( n = 6). * P D : densitometric analysis of glomerular ERK phosphorylationas detected by Western blot analysis. Values represent the relativeabundance of phosphorylated ERK vs. total ERK protein. The valueobtained in normal glomerular lysate was taken as 1.0. Values aremeans ± SD ( n = 3/group). * P

We then asked whether a decrease in the proliferating glomerular cellswith HGF treatment is related to changes in proliferation of activatedmesangial cells. When the numbers of proliferating activated mesangialcells and nonmesangial cells were determined by double staining for -SMA and BrdU, the number of -SMA-positive mesangial cellsundergoing DNA synthesis decreased on day 4 to 52.0% of thecontrol value seen with HGF treatment (Fig. 6 B ). Incontrast, HGF treatment had no statistically significant effect on theproliferation of nonmesangial cells. Similarly, accompanied by adecrease in the number of proliferating activated mesangial cells,expansion of -SMA-positive cells in glomeruli, as evaluated byimmunohistochemical staining and its scoring, was suppressed by HGFtreatment on both days 4 and 8 (Fig. 6 C ). To further confirm the suppressive effect of HGF onactivated mesangial cell proliferation, we analyzed the activationstatus of ERK in glomerular lysates (Fig. 6 D ). GlomerularERK was phosphorylated/activated in rats with anti-Thy 1.1 treatment on day 4, whereas glomerular ERK activation was significantlyattenuated by HGF treatment. The attenuation of glomerular ERKactivation by HGF may possibly be involved in the suppressive effect ofHGF on activated mesangial cell proliferation. Taken together, theseresults indicate that HGF treatment preferentially suppressedproliferation of activated mesangial cells but not of nonmesangialcells during development of anti-Thy 1.1 nephritis.6 Z! ?: d# G. {- a2 S
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When we analyzed expression of the PDGF-B chain mRNA and PDGF-BBprotein levels in glomeruli on day 4 after disease induction with real-time quantitative RT-PCR and ELISA, respectively, HGF treatment did not significantly change either PDGF-B mRNA expression orPDGF-BB levels in glomeruli (Table 1 ).Thus HGF suppressed PDGF-induced mesangial cell proliferation withoutaffecting PDGF expression in glomeruli.7 V) h( G% g' g0 Q7 O! c! m8 K2 ]

Table 1. Changes in PDGF mRNA expression and PDGF-BB protein level after HGFtreatment in rats with anti-Thy 1.1 glomerulonephritis. s7 W9 O3 v3 t

DISCUSSION

In the kidney, activation of glomerular mesangial cells inresponse to glomerular damage and proliferation of these cells arethought to be risk factors for the progression of glomerular nephritisto irreversible glomerular scarring and play an important role in thepathogenesis of a variety of glomerular diseases ( 12 ). Therefore, understanding regulatory mechanisms governing proliferation of mesangial cells is important in designing effective treatments forglomerular nephritis and gaining a better understanding of pathophysiological aspects. In this study, we demonstrated for thefirst time that the c-Met receptor was induced in mesangial cells onstimulus toward activated mesangial cells and that HGF suppressedPDGF-induced proliferation of activated mesangial cells both in vivoand in vitro.
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During successive cultivation, mesangial cells spontaneously undergo aphenotypic change into activated, myofibroblast-like cells( 8 ). Previous reports showed that mesangial cells express the c-Met receptor in vitro, but the effects of HGF on cultured mesangial cells and in vivo expression of the c-Met receptor in mesangial cells were controversial ( 19, 21, 37 ). In the present study, we found that mesangial cells barely expressed the c-Metreceptor in the normal glomerulus, but after anti-Thy 1.1 treatment thec-Met receptor was induced in -SMA-positive activated mesangialcells. Thus in glomerular mesangial cells, expression of the c-Metreceptor is inducible, when mesangial cells are activated to become -SMA-positive myofibroblast-like cells. Although mechanisms forinduction of the c-Met receptor during activation of mesangial cellsremain to be addressed, Liu et al. ( 24 ) reported that incultured mesangial cells the c-Met receptor was upregulated by severalgrowth factors, including PDGF. Therefore, the increased expression ofgrowth factors such as PDGF in this model ( 15 ) may bepartially responsible for c-Met receptor induction in this model.. A3 V  B( {. m2 y
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Infusion of PDGF into the kidney or transfection of the PDGF gene inthe kidney selectively induced mesangial cell proliferation ( 10, 16 ). Conversely, neutralization of PDGF resulted in the suppression of mesangial cell proliferation ( 18, 33 ).Similarly, it was reported that glomerular PDGF and PDGF receptorexpression are increased in patients with various forms ofglomerulonephritis ( 12 ). Thus considering the closeinvolvement of PDGF in mesangioproliferative glomerulonephritis, weinvestigated a potential role for HGF, focusing on mesangial cellproliferation in combination with a PDGF stimulus. Other studies haveshown that HGF alone had no apparent effect on rat, mouse, or humanmesangial cell proliferation ( 21, 37 ), or that it isweakly mitogenic ( 19 ). In the present study, HGF alone hadno apparent effect on cultured mesangial cells, but HGF did suppressthe proliferation of activated mesangial cells promoted by PDGF.Because HGF did not alter PDGF expression in cultured mesangial cells,we studied the activation of ERK, an intracellular event closelyassociated with mesangial cell proliferation. Recent reportsdemonstrated that ERK is a mediator of the proliferative response inmesangioproliferative glomerulonephritis ( 4 ) and thatinhibition of ERK in the proliferative phase of anti-Thy 1.1 nephritisprevented mesangial cell proliferation ( 5 ). Importantly,we found that ERK was differently regulated after stimulation of cellswith PDGF, HGF, or both combined. The earlier inactivation of ERK bythe simultaneous addition of PDGF and HGF seems to explain thesuppressive effect of HGF on PDGF-dependent growth in activatedmesangial cells. Our study clearly showed the counteractive interactionof two different growth factors and their receptors, either of whichhas the tyrosine kinase receptor.

Consistent with in vitro findings on the counteractive effects of HGFon PDGF-induced proliferation of activated mesangial cells,administration of HGF to rats with anti-Thy 1.1 nephritis suppressedglomerular ERK phosphorylation and the proliferation of activatedmesangial cells, and these events were associated with a reduction inglomerular expansion of -SMA expression. Because HGF did notsignificantly change the glomerular expression of PDGF, theintracellular counteraction of HGF against PDGF-dependent mesangialcell proliferation seems to be a predominant mechanism by which HGFsuppressed expansion of activated mesangial cells inmesangioproliferative glomerulonephritis. Because endogenous glomerularHGF expression was decreased, rather than increased, after anti-Thy 1.1 treatment, decreased glomerular HGF expression might to some extent andin part allow for the proliferation of activated mesangial cells.5 J" Z8 C7 \' m

Ostendorf et al. ( 33 ) showed that the transient inhibitionof PDGF-B chain by a specific aptamer during the mesangioproliferative phase in the irreversible glomerulosclerosis model diminished proliferation of activated mesangial cells and that this resulted inalmost complete prevention of the development of renal failure andglomerular as well as tubulointerstitial scarring. Their results provide a strong argument against concerns that inhibition of overshooting mesangial cell growth after injury might lead to theinhibition of healing and thus exacerbate glomerular damage. Areduction in early glomerular proliferation may be important for anysequent reduction of glomerular scarring and renal failure. Whetherantiproliferative effects of HGF on activated mesangial cells areassociated with the prevention of glomerulosclerosis remains to beaddressed, using a model for glomerulosclerosis.

Previous approaches using distinct animal models of tissue fibrosisprovided evidence that HGF has antifibrogenic actions on tissuefibrosis, including liver cirrhosis ( 26, 34 ), renal tubulointerstitial fibrosis ( 29 ), and lung fibrosis( 36 ). Based on the notions that the c-Met receptor ispreferentially expressed in epithelial cells and endothelial cellsunder physiological conditions and that transforming growth factor- (TGF- ) plays a role in tissue fibrosis, these studies addressedmechanisms involved in antifibrotic actions of HGF, focusing on celldivision and antiapoptosis in epithelial cells, expression ofTGF-, and proteases involved in the breakdown of ECM ( 23, 26, 29, 34, 36 ). However, little attention was directed to thepotential role of HGF to directly attenuate stromal cell expansion. Our original findings here are that HGF exerts antiproliferative actions, directly targeting stromal myofibroblast-like cells. Our observations provide a better understanding of the pathogenic mechanisms of as wellas therapeutic approaches to fibrotic disorders, from the aspect oftissue remodeling regulated by growth factor networks.
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ACKNOWLEDGEMENTS  s1 g+ n! b, H! b7 v2 K! {. p
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We are grateful to M. Ohara for comments and language assistance.
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