Delayed administration of hepatocyte growth factor reducesrenal fibrosis in obs - 《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 13:32 |只看该作者 |倒序浏览 |打印

作者：JunweiYang and YouhuaLiu作者单位：Division of Cellular and Molecular Pathology, Department ofPathology, University of Pittsburgh School of Medicine, Pittsburgh,Pennsylvania 15261 6 l2 X, E& U$ j+ a
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      【摘要】
   Hepatocyte growth factor(HGF) is a renotropic protein that elicits antifibrogenic activity bypreventing the activation of matrix-producing myofibroblast cells inanimal models of chronic renal diseases. However, whether a delayedadministration of HGF can still attenuate renal fibrosis remainsuncertain. In this study, we examined the therapeutic potential ofexogenous HGF on an established renal interstitial fibrosis induced byunilateral ureteral obstruction (UUO). Three days after UUO, theobstructed kidneys displayed interstitial fibrotic lesions withcharacteristic features of an established renal fibrosis, as manifestedby myofibroblast activation, fibronectin overexpression, interstitialmatrix deposition, and transforming growth factor- 1 upregulation.Beginning at this time point, administration of recombinant HGF intomice by intravenous injections for 11 days markedly suppressed theprogression of renal interstitial fibrosis. HGF significantlysuppressed renal -smooth muscle actin expression, total kidneycollagen contents, interstitial matrix components, such as fibronectin,and renal expression of transforming growth factor- 1 and its type Ireceptor. Compared with the starting point (3 days after UUO), HGFtreatment largely blunted the progression of myofibroblast accumulation and collagen deposition but did not reverse it. Delayed administration of HGF also suppressed the myofibroblastic transdifferentiation fromtubular epithelial cells in vitro, as demonstrated by a decline in -smooth muscle actin and fibronectin expression. These results suggest that exogenous HGF exhibits potent therapeutic effects onretarding the progression of an established renal fibrosis. 7 i/ f( |* T' P) Y
      【关键词】 cmet renal fibrosis ureteral obstruction smooth muscleactin myofibroblast( }' s6 t9 x- j5 @  t
   INTRODUCTION. y" X. H7 d& d2 r! ~& W
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END-STAGE RENAL DISEASE (ESRD) presently affects a large number of patientsworldwide, and the incidence of affected patients is increasing at arate of 6-8% annually ( 4, 33 ). Once patients reachESRD, mortality on dialysis is high. These data highlight theimportance and urgency of developing effective therapeutic strategiesfor the treatment of ESRD. Importantly, most patients with chronicrenal disease (CRD) are identified well before they reach end-stagerenal failure; however, no presently available treatment is effectivein halting the progressive loss of renal function.( N) M' _3 _! V; P, X! a

Several pieces of evidence suggest that hepatocyte growth factor (HGF)is an excellent candidate for therapeutic use in the treatment of CRDs( 20, 26, 40 ). HGF and c-met expression are increased, atleast in the initial stage, in a variety of experimental animal modelsof CRD ( 21, 23, 28 ). Similarly, serum HGF levels areelevated in patients with end-stage renal failure ( 24 ).Both of these findings suggest that HGF may be part of thetissue-healing response. Moreover, administration of either recombinantHGF protein or its gene markedly prevents renal myofibroblastactivation and interstitial fibrosis in numerous animal models of CRD( 2, 27, 29, 42 ). Conversely, blockage of endogenous HGFsignaling by a neutralizing antibody promotes the onset and progressionof renal tissue fibrosis and kidney dysfunction in two distinct modelsof CRD ( 21, 28 ). Recently, we have demonstrated that HGFdramatically inhibits the activation of matrix-producing myofibroblastsby blocking epithelial-to-mesenchymal transition under pathologicalconditions ( 44 ). This suggests that HGF is capableof precisely targeting a critical event during renal fibrogenesis.
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Despite several studies demonstrating an apparent efficacy of HGFin preventing renal fibrogenesis, it remains unclear whether HGF has atherapeutic role in established renal fibrosis. This unansweredquestion obviously has its profound clinical relevance, because mostpatients diagnosed with CRD already have different degrees of fibroticlesions in their kidneys. In addition, contradicting data exist as tothe role of HGF in progressive renal diseases ( 19, 41 ).For instance, it is reported that administration of HGF intogenetically obese db diabetic mice actually reduces creatinineclearance and increases microalbuminuria ( 19 ).Clearly, more studies are needed in animal models to evaluate thetherapeutic efficacy of exogenous HGF in an established renal fibrosisbefore clinical use of HGF can be considered for patients with chronic renal insufficiency./ H% Q) @1 ~  o% t6 n3 y2 j

In this study, we have examined the ability of HGF to suppressthe progression of established renal fibrotic lesions in a mouse modelinduced by unilateral ureteral obstruction (UUO). Our results suggestthat delayed administration of exogenous HGF is also effective inretarding the progression of an established renal fibrosis.4 w% Q1 a* T7 i6 \- C7 \

MATERIALS AND METHODS- T: H  Z; z7 X5 H; R& x% _9 n8 {

Animal model. Male CD-1 mice (18-22 g) were purchased from Harlan-SpragueDawley (Indianapolis, IN). They were housed in the animal facilities ofthe University of Pittsburgh Medical Center with free access to foodand water. UUO was performed with an established procedure ( 6, 11, 42 ). Briefly, under general anesthesia, complete ureteralobstruction was performed by the double ligation of the left ureterwith 4-0 silk after a midline abdominal incision. Sham-operatedmice had their ureters exposed and manipulated but not ligated. Inpreliminary studies to establish the course of renal fibrogenesis inthis model, four groups of mice ( n = 4) were killed at days 1, 3, 7, and 14,respectively, after ureteral obstruction. One group of sham-operatedmice ( n = 4) was killed at day 3 after surgery.
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After preliminary studies on the kinetics of renal interstitialfibrosis after UUO in mice, four groups of animals were used in theexperiments (Fig. 1 ). Groups 1 and 2 ( n = 4) were sham-operated orUUO mice killed at 3 days after surgery. Eight additional mice underwent complete UUO. Three days later, these mice ( n = 4) were randomly assigned into an HGF treatment ( group 3 )or vehicle control ( group 4 ). Recombinant human HGF protein(kindly provided by Genentech, South San Francisco, CA) wasadministrated by intravenous injections via tail vein at 400 µg/kgbody wt every 12 h for 11 days. Control mice were injected withthe same volume of vehicle (PBS) in an identical manner. Mice werekilled at day 14 after initial UUO, and the kidneys wereremoved. One part of the kidneys was fixed in 10% phosphate-bufferedformalin for histological studies after being embedded in paraffin.Another part was immediately frozen in Tissue-Tek OCT compound forcryosection. The remaining kidneys were snap-frozen in liquid nitrogenand stored at 80°C for protein extractions.
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Fig. 1. Experimental design. Mice were assigned into 4 groups,with 4 animals/group ( n = 4). Groups 1 and 2 : sham-operated or unilateral ureteral obstruction (UUO)mice were killed at 3 days after surgery. Three days after UUO, micereceived either hepatocyte growth factor (HGF) injections ( group3 ) or vehicle ( group 4 ) for 11 days and were killed at14 days after initial surgery. Arrows, 2 injections separated by 12 h/day.
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Western blot analysis. Western blot analysis was carried out essentially according to theprocedures described previously ( 22 ). Briefly, the kidney was homogenized in RIPA lysis buffer (1% Nonidet P-40, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 0.5% sodium deoxycholate, 1 mMsodium orthovanadate, 2 µg/ml aprotinin, 2 µg/ml antipain, and 2 µg/ml leupeptin in PBS) on ice, and the supernatants were collectedafter centrifugation at 13,000 g at 4°C for 20 min. Protein concentration was determined by using a bicinconinic acid protein assay kit (Sigma, St. Louis, MO), and tissue lysates were mixedwith an equal amount of 2× SDS loading buffer (100 mMTris · HCl, 4% SDS, 20% glycerol, and 0.2%bromophenol blue). Samples were heated at 100°C for 5-10 minbefore loading and separated on precasted 10 or 5% SDS-polyacrylamidegels (Bio-Rad, Hercules, CA). The proteins were electrotransferred to anitrocellulose membrane (Amersham, Arlington Heights, IL) in transferbuffer containing 48 mM Tris · HCl, 39 mMglycine, 0.037% SDS, and 20% methanol at 4°C for 1 h.Nonspecific binding to the membrane was blocked for 1 h at roomtemperature with 5% Carnation nonfat milk in Tris-buffered salinebuffer (20 mM Tris · HCl, 150 mM NaCl, and 0.1%Tween 20). The membranes were then incubated for 16 h at 4°Cwith various primary antibodies in blocking buffer containing 5% milk.The mouse monoclonal anti- -smooth muscle actin ( -SMA) antibody(clone 1A4) was purchased from Sigma. The anti-fibronectin antibody wasobtained from Transduction Laboratories (Lexington, KY). The polyclonalantibodies against transforming growth factor- [TGF-; TGF- type I receptor (sc-398) and actin (sc-1616)] were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA). After being washedextensively three times, the membranes were then incubated withhorseradish peroxidase-conjugated secondary antibody (Bio-Rad) for1 h at room temperature in 5% nonfat milk. The signals werevisualized by an enhanced chemiluminescence system (ECL, Amersham).Quantitation was performed by measuring the intensity of thehybridization signals with the Image analysis program (National Institutes of Health).9 E8 |4 x9 K8 b9 N# i0 B6 v

Histology and immunohistochemical staining. Tissue sections from the mice were prepared at 4-µm thickness by aroutine procedure. Sections were stained with hematoxylin/eosin forgeneral histology. Immunohistochemical staining for -SMA wasperformed by using the Vector MOM immunodetection kit (Vector Laboratories, Burlingame, CA), according to the procedures reported previously ( 44 ). To evaluate the extent of collagendeposition, Masson-Trichrome staining was performed according to thestandard protocol by using a reagent kit purchased from Sigma.
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Immunofluorescence microscopy. Kidney cryosections were prepared and fixed for 5 min in PBS containing3% paraformaldehyde. After being blocked with 1% normal donkey serumin PBS for 30 min, the sections were incubated with primary antibodiesagainst -SMA, fibronectin, and collagen I, respectively, in PBScontaining 1% BSA overnight at 4 o C. Sections were thenincubated for 1 h with fluorescein-conjugated secondaryantibodies, at a dilution of 1:200 in PBS containing 1% BSA, beforebeing washed extensively with PBS. As a negative control, the primaryantibody was replaced with nonimmune IgG and no staining occurred. Fordouble staining of -SMA and proximal tubular marker, the slides werestained with fluorescein-conjugated lectin from Tetragonolobuspurpureas (Sigma) after first being stained with -SMA( 44 ). Slides were mounted with antifade mounting media(Vector Laboratories) and viewed with an Eclipse E600 Epi-fluorescence microscope equipped with a digital camera (Nikon, Melville, NY).0 R) ]) H8 a1 P3 `4 p* h/ A
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Biochemical measurement of total kidney collagen content. For quantitative measurement of collagen deposition in the kidney,total tissue collagen content was determined by biochemical analysis ofthe hydroxyproline in the hydrolysates extracted from kidney samples,according to established procedures ( 16, 31 ). Briefly,accurately weighed portions of the obstructed kidneys were homogenizedin distilled H 2 O. The homogenates were hydrolyzed in 10 NHCl by incubation at 110°C for 18 h. The hydrolysates were driedby speed vacuum centrifugation over 3-5 h and redissolved in abuffer containing 0.2 M citric acid, 0.2 M glacial acetic acid, 0.4 Msodium acetate, and 0.85 M sodium hydroxide, pH 6.0. Hydroxyprolineconcentrations in the hydrolysates were chemically measured accordingto the techniques previously described ( 16, 31 ). Totalcollagen was calculated on the assumption that collagen contains 12.7%hydroxyproline by weight. The results of total tissue collagen contentwere expressed as micrograms of collagen per milligram of kidney weight.
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Determination of tissue TGF- 1 levels by enzyme-linkedimmunosorbent assay. To measure renal TGF- 1 levels, kidneys from mice were homogenized inthe extraction buffer containing 20 mMTris · HCl, pH 7.5, 2 M NaCl, 0.1% Tween 80, 1 mM ethylenediamine tetraacetate, and 1 mM phenylmethylsulfonylfluoride, and the supernatant was recovered after centrifugation at19,000 g for 20 min at 4°C. Kidney tissue TGF- 1 levelwas determined by using the commercial Quantikine TGF- 1 ELISA kit inaccordance with the protocol specified by the manufacturer (R&DSystems, Minneapolis, MN). This kit measures the abundance of activeTGF- 1 protein that binds to its soluble type II receptor precoatedonto a microplate. Total protein levels were determined by using abicinconinic acid protein assay kit as described above. Theconcentration of TGF- 1 in kidneys was expressed as picograms permilligram of total protein.

Cell culture and treatment. Human proximal tubular epithelial HKC cells (clone 8) were provided byDr. L. Racusen (Johns Hopkins University, Baltimore, MD) and maintainedin DMEM/F-12 medium supplemented with 10% FBS (Life Technologies,Grand Island, NY), as described previously ( 44 ). The HKCcells were seeded on six-well culture plates to 60-70% confluencein complete medium containing 10% FBS for 16 h and then changedto serum-free medium after being washed twice with medium. The cellswere pretreated with recombinant human TGF- 1 (R&D Systems) at theconcentration of 1 ng/ml for 48 h. Thereafter, the medium waschanged, and the cells were continuously incubated with either TGF- 1(1 ng/ml) alone or in combination with 50 ng/ml of recombinant humanHGF (Genentech) for an additional 24 and 48 h, respectively. Thecell lysates were subjected to Western blot analysis. For controlexperiments, the cells were incubated with vehicle (PBS).8 m: Y8 |0 w  N( j4 o; c
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Statistical analysis. Animals were randomly assigned to control and treatment groups. Dataare expressed as means ± SE. Statistical analysis of the data wasperformed by using SigmaStat software (Jandel Scientific, San Rafael,CA). Comparison among groups was made with one-way ANOVA followed bythe Student-Newman-Keuls test. A P value of significant.

RESULTS/ C# E! X( O* A6 h

Mice underwent complete UUO to generate a model of renalinterstitial fibrosis characterized by myofibroblast activation, tubular atrophy, and interstitial matrix deposition ( 5, 17 ). Figure 2 shows the timecourse of fibronectin expression in the obstructed kidneys after thesurgical operation. A more than 20-fold induction of fibronectinexpression was observed in the obstructed kidneys as early as 1 dayafter UUO. Although the renal fibronectin level increased progressivelyin accordance with the duration of ureteral obstruction, 3 days ofobstruction appeared sufficient to induce marked accumulation offibronectin and renal fibrosis in the obstructed kidneys. Furtherobstruction beyond this time point only resulted in a moderate increasein fibronectin expression (Fig. 2 ). Consistently, 3 days of obstructionbegan to induce significant morphological lesions and overexpression ofTGF- 1 in the obstructed kidneys (Fig. 2 ). An approximately ninefold induction of the active TGF- 1 protein was detected in the kidneys at3 days after ureteral obstruction (Fig. 2 ). These alterations arereminiscent of the characteristic features of an established renalfibrosis.
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Fig. 2. Early onset of renal fibrotic lesions in obstructivenephropathy. A and B : time-dependent induction offibronectin (FN) expression in kidneys after ureteral obstruction. Miceunderwent UUO or sham operation for various durations (days) asindicated. A : representative Western blot analysisdemonstrates an early induction of fibronectin expression in theobstructed kidneys after UUO. The blot was reprobed with actin toconfirm equal loading. Two individual animals are represented at eachtime point. B : graphic presentation of the relativeabundance of renal fibronectin after normalization with actin. Values(fold-induction relative to sham control) are means ± SE of 4 animals/group ( n = 4). * P C ) or UUO ( D ).Morphological injury manifested by tubular dilation and interstitialexpansion was evident in the obstructed kidneys at 3 days after UUO.Bar = 20 µm. E : transforming growth factor (TGF)- 1protein levels determined by ELISA in the kidneys at 3 days after UUOor sham operation. Values are means ± SE of 4 animals/group( n = 4). * P7 ?' I/ v. b) ^7 h1 Q

Figure 3 demonstrates the activation of -SMA expression in the obstructed kidneys at 3 days after ureteralobstruction. Quantitative determination of Western blot analysisrevealed that an ~17-fold induction of -SMA was found in theobstructed kidneys at as early as 3 days, suggesting a dramaticactivation of myofibroblasts, the principal cells known to beresponsible for accumulation and deposition of the interstitial matrix( 9, 34, 36 ). This activation of renal myofibroblasts wasindependently confirmed by immunohistochemical staining for -SMA(Fig. 3, C and D ). Of note, -SMA-positivecells were largely confined in the interstitial compartment at thisstage.+ C3 X7 K4 d9 N( R. ?

Fig. 3. Induction of -smooth muscle actin ( -SMA) expression in theobstructed kidneys. A : Western blot analysis shows thelevels of -SMA protein in the obstructed kidneys at 3 days after UUOor sham operation. Numbers ( 1-4 ) indicate 4 individual animals/group. B : graphic presentation of therelative abundance of renal -SMA after normalization with actin.Values (fold-induction relative to sham control) are means ± SEof 4 animals/group ( n = 4). * P -SMA in the kidneys at 3 days after either shamoperation ( C ) or UUO ( D ). Bar = 20 µm.! d3 j7 g: p$ G: m. e  J
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After demonstrating that significant renal fibrosis was established at3 days after UUO (Figs. 2 and 3 ), we next began to administerrecombinant human HGF into mice at this time point to evaluate itstherapeutic effects on established renal fibrosis. Figure 4 shows the -SMA levels in theobstructed kidneys after intravenous injection of human HGF at 400 µg/kg body wt every 12 h for 11 days. Compared with the vehiclecontrol, exogenous HGF markedly suppressed 70%in the obstructed kidneys. Immunoflurorescence staining alsodemonstrated that the number of -SMA-positive cells was dramaticallydecreased (Fig. 4, C and D ). Of interest, doublestaining for -SMA (red) and proximal tubular marker (green) revealedthat in the obstructed kidneys of the control group, few cells retainedthe tubular marker, whereas -SMA-positive cells were present in awidespread fashion. This suggests that at this advanced stage (14 days)of obstructive nephropathy, renal tubules already lost theircharacteristic epithelial marker, presumably due toepithelial-to-myofibroblast transition (EMT). Consistent with this,cells at the transitional stage with both -SMA and tubular marker(yellow) were observable. However, administration of exogenous HGFlargely preserved tubular marker and tubular structural integrity inthe diseased kidneys (Fig. 4 D ), possibly by blocking tubularEMT as seen in the diseased kidneys.
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Fig. 4. Exogenous HGF inhibits -SMA expression in theobstructed kidneys. A : Western blot analysis shows thelevels of -SMA protein in the obstructed kidneys after injections ofeither HGF or vehicle. The same blot was reprobed with actin to ensureequal loading. Numbers ( 1-4 ) indicate 4 individualanimals/group. Top : nonspecific band is mostly likelycontributed by the heavy chain of IgG. B : graphicpresentation of the relative abundance of renal -SMA afternormalization with actin. Values are means ± SE of 4 animals/group ( n = 4). * P -SMA (red) andproximal tubular marker, lectin from Tetragonolobuspurpureas (green), in the obstructed kidneys after injections ofeither HGF ( D ) or vehicle ( C ). Arrowhead, cellcoexpressing -SMA and tubular marker. Bar = 20 µm. E and F : direct comparison of the -SMA levelsin the kidneys of 4 groups of animals illustrated in Fig. 1.Representative Western blot analysis ( E ) and graphicalpresentation ( F ) show -SMA abundance in various groups.* P P

Direct comparison of intrarenal -SMA levels among different groupsis also presented in Fig. 4. Delayed administration of HGF starting at day 3 after UUO almost completely blunted the progressiveaccumulation of -SMA protein but did not reverse it. Quantitativedetermination revealed that similar levels of -SMA were observed inthe obstructed kidneys between the starting point ( day 3 ofUUO) and the end point ( day 14 of UUO) of HGF treatment.+ x& P3 w; A* ?. D

Along with the suppression of myofibroblastic activation, delayedadministration of exogenous HGF also ameliorated the morphological lesions and extracellular matrix deposition seen in the obstructed kidneys. Figure 5, A and B, shows representative micrographs of the obstructedkidneys receiving either vehicle or HGF protein. Kidneys with ureteralobstruction for 14 days displayed severe morphological lesionscharacterized by tubular dilation with epithelial atrophy, interstitialexpansion with collagen accumulation and deposition (Fig. 5 A ). The interstitial space was clearly widened withsome degree of hypercellularity, while the glomerular structure wasgenerally well preserved. By contrast, kidneys with injections ofexogenous HGF exhibited obvious attenuation of these morphological lesions with less fibrosis in the interstitium (Fig. 5 B ).
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Fig. 5. HGF ameliorates the accumulation and deposition of total collagenand fibronectin in the obstructed kidneys. Representative micrographsshow collagen deposition in the control ( A ) and HGF-treated( B ) groups by Masson-Trichrome staining. Bar = 20 µm. C : total kidney collagen contents in 4 groups of animalsillustrated in Fig. 1. Values are means ± SE of 4 animals/group( n = 4). * P P D : Westernblot analysis shows the levels of fibronectin in the obstructed kidneysafter injections of either HGF or vehicle. The same blot was reprobedwith actin. Numbers ( 1-4 ) indicate 4 individualanimals/group. E : graphic presentation of the relativeabundance of renal fibronectin after normalization with actin. Valuesare means ± SE of 4 animals/ group ( n = 4).* P1 f# ^- K5 G3 P: [, R: M- t. H
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Figure 5 C shows total kidney collagen contents from varioussamples determined by a quantitative, biochemical assay. Delayed administration of exogenous HGF markedly reduced total collagen deposition in the obstructed kidneys at day 14 afterpersistent injury. Direct comparison of collagen levels among differentgroups indicated that HGF largely blocked the progressive accumulation of renal total collagen after continuous, complete obstruction, although it did not reverse it. Accordingly, the expression of collagenI, a major component of the interstitial matrix, was also markedlysuppressed by HGF in the obstructed kidneys, as demonstrated byimmunofluorescence staining (data not shown). In addition, comparedwith that in the control group, exogenous HGF injections resulted insignificant reduction of fibronectin expression in the kidneys. Morethan 48% repression of the fibronectin protein abundance was detectedby Western blot analysis of whole kidney lysates (Fig. 5, D and E ). Similar results were obtained by using an indirectimmunofluorescence staining for fibronectin in the kidneys (not shown).
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We further examined the expression of TGF- 1 and its type I receptorin the obstructed kidneys. As shown in Fig. 6, ureteral obstruction markedly inducedthe expression of TGF- 1 in the diseased kidneys, as detected by aspecific ELISA. Administration of exogenous HGF considerably inhibitedthe induction of TGF- 1 expression (Fig. 6 ). Moreover, Western blotanalysis exhibited that exogenous HGF also significantly downregulatedthe expression of TGF- type I receptor in the obstructed kidneys(Fig. 6, B and C ).
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Fig. 6. HGF suppresses the expression of TGF- 1 and its type Ireceptor in the obstructed kidneys. A : TGF- 1 proteinlevels determined by ELISA in the obstructed kidneys after injectionsof either HGF or vehicle. Values are means ± SE of 4 animals/group ( n = 4). * P B : Western blot analysis shows thelevels of TGF- type I receptor (T RI) protein in the obstructedkidneys after injections of either HGF or vehicle. The same blot wasreprobed with actin. Numbers ( 1-4 ) indicate 4 individual animals/group. C : graphic presentation of therelative abundance of renal TGF- type I receptor after normalizationwith actin. Values are means ± SE of 4 animals/group( n = 4). * P
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In an effort to provide mechanistic insight into understanding theactions of HGF on an established renal fibrosis, we investigated theeffects of HGF on the phenotypes of newly transdifferentiated tubularepithelial cells by using an in vitro culture system. A treatmentscheme was designed to mimic in vivo situations as described in thisstudy (Figs. 1 and 7 A ). Asshown in Fig. 7 B, preincubation of renal proximal tubularepithelial cells (HKC) with TGF- 1 for 48 h inducedmyofibroblastic transdifferentiation, as evidenced by de novo -SMAexpression as well as an induced fibronectin production. After thispretreatment, which is analogous to the onset of renal fibrosis in vivo( day 3 after UUO), HGF was added to the cultures with freshmedium in the continuous presence of TGF- 1. As shown in Fig. 7 B, delayed administration of exogenous HGF suppressed -SMA expression in HKC cells. In fact, the abundance of -SMA inHKC cells after HGF treatment was below the preexisting level( lane 5 vs. lane 2, Fig. 7 B ). Accordingly, delayed administration of HGF also inhibited fibronectin expression in TGF- 1-pretreated HKC cells. These results suggest that delayed administration of HGF effectively suppresses, and possiblyreverses, myofibroblastic transition of tubular epithelial cells.* l/ v& {% Y- q! F

Fig. 7. Delayed administration of HGF suppresses TGF- 1-induced -SMA and fibronectin expression in tubular epithelial cells invitro. A : diagram depicts treatment scheme. The in vitroexperiments were designed to mimic HGF treatment scheme illustrated inFig. 1. B : Western blot analysis shows the levels of -SMAand fibronectin protein in renal tubular epithelial cells (HKC) aftervarious treatments.' s) n: |( k9 T" S- |* p/ \
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DISCUSSION. g* _! ]: k1 @' K+ R# H3 ^
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When patients are diagnosed with chronic renal insufficiency,their kidneys may already display different degrees of renal fibrosisunder most circumstances. Hence, a key to an effective therapy forpatients with CRD is to develop a strategy that blocks the progressionof an established renal fibrosis and dysfunction in a clinical setting.The purpose of this study was to test whether exogenous HGF, which hasbeen shown to be preventive in retarding the onset of renal fibrosisand kidney dysfunction ( 27, 29, 42 ), also has therapeuticeffects to ameliorate an established renal fibrosis. Our resultsdemonstrate that HGF markedly suppresses renal myofibroblast activationand attenuates renal interstitial matrix deposition in the obstructedkidneys when given at 3 days after complete ureteral obstruction, atime point when significant renal fibrosis has clearly emerged. Theseobservations suggest that the supplement of HGF not only may preventthe onset and progression of CRD as previously reported ( 27, 29, 42 ) but also may exhibit therapeutic effects on the diseasedkidneys where tissue fibrosis is already established.9 T( ~, j& r8 M5 N) t& S

Although the pathological mechanism underlying chronic obstructivenephropathy is not completely elucidated, the fibrogenic processclearly plays a critical role in ultimately leading to permanent lossof the normal structural and functional integrity of the kidney( 8, 18, 35 ). One of the intriguing observations in thisstudy is that renal fibrogenesis initiates at a very early stage afterureteral obstruction and progresses rapidly in our mouse model. Unlikemost forms of CRD models, such as remnant kidneys and diabeticnephropathy, that often take months to establish fibrotic lesions,obstructive nephropathy induced by complete, continuous ureteralobstruction perhaps is an exceptionally aggressive form of interstitialfibrogenesis. In accordance with this, an ~80-fold induction infibronectin, 17-fold in -SMA, and 9-fold in TGF- 1 expression areobserved in the obstructed kidneys at 3 days after complete obstructioncompared with the sham control group (Figs. 2 and 3 ). The kidneysobstructed for such a short duration display the characteristicfeatures of chronic renal interstitial fibrosis often seen in diseasedkidneys under various pathological conditions. Consistent with this, anearlier study shows that a significant upregulation of TGF- 1 and itstype I receptor expression takes place in the kidneys as early as 1 day after obstruction ( 43 ). Thus major hallmarks forinterstitial fibrotic lesions are already established in the obstructedkidneys at day 3 after complete obstruction. This notion isfurther supported by the fact that relief of obstruction after a shortduration (days) does not completely reverse renal interstitial lesions ( 7, 30 ).
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Although the therapeutic efficacy of exogenous HGF on established renalfibrosis is discernible, the mechanism underlying the therapeutic roleof HGF remains largely unsolved. One clue for a potential mechanismcomes from the observation that exogenous HGF inhibited the expressionof both TGF- 1 and its specific type I receptor in the obstructedkidneys in vivo (Fig. 9). In light of the role of aberrant expressionof TGF- 1 in the pathogenesis of many forms of CRDs ( 3, 25, 39 ), it is plausible to speculate that the inhibition of renalfibrosis may be mediated, at least in part, by the suppression ofTGF- 1 axis expression in the obstructed kidneys. TGF- 1 is awell-documented profibrogenic factor that plays a determinant role inthe pathological accumulation of extracellular matrix in normal tissuesafter various injurious insults ( 1 ). Studies indicate thatoverexpression of TGF- 1 in transgenic mice induced CRD withincreased expression of fibrotic matrix protein. Conversely,suppression of TGF- 1 signaling either by truncated, soluble receptoror by an antisense approach significantly retards the progression ofrenal interstitial fibrosis in animals ( 13, 14 ). Wepreviously demonstrated that the upregulation of both TGF- 1 and itstype I receptor is an early event preceding the onset of significantrenal fibrosis in the obstructed kidneys ( 43 ), supportinga causal relationship between overexpression of TGF- 1 axis and thedevelopment of renal interstitial fibrosis. Altogether, inhibition ofTGF- 1 axis expression by exogenous HGF in vivo may play an importantrole in mediating its antifibrotic actions. However, it remains unclearwhether the decreased TGF- axis expression is due to a direct effectof HGF or a secondary consequence resulting from the antifibroticaction of HGF. Preliminary studies in our laboratory suggest that HGFdoes not directly inhibit TGF- 1 gene expression in cultured renaltubular cells (data not shown), implying that the antifibrogenicactivities of HGF are probably mediated by a mechanism involvingblockade of TGF- 1 signaling rather than via a direct inhibition ofits expression.7 ]& X2 e2 X% o% L+ f+ V
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The observation that HGF specifically blocks the activation of renalmyofibroblasts in vivo may provide significant insights into thecellular mechanism underlying the amelioration of renal fibroticlesions. Renal interstitial myofibroblast cells are -SMA-positive, activated matrix-producing cells responsible for relentlessaccumulation and deposition of extracellular matrix in the interstitialcompartments of diseased kidneys ( 12, 34, 37 ). Becausemyofibroblast cells are not present in normal kidneys, their originunder pathological conditions is uncertain. They are often presumed toderive from local resident interstitial fibroblasts. However, emergingevidence suggests that these cells may also come from tubularepithelial cells via a process known as epithelial-to-mesenchymaltransition ( 10, 15, 32, 38, 45 ). It has been shownthat TGF- 1 plays an essential role in initiating EMT that leads tothe accumulation of matrix-producing myofibroblast cells ( 10, 43 ). Earlier studies from our laboratory show that HGFdramatically prevents myofibroblast activation from tubular epithelialcells triggered by TGF- 1 ( 44 ). Using an in vitro cellculture model system that recapitulates the in vivo treatment scheme,we show herein that delayed administration of HGF also suppresses, andpossibly reverses, TGF- 1-induced -SMA expression in tubularepithelial cells (Fig. 7 ). Such dramatic blockage of tubular EMT by HGFwill undoubtedly lead to preservation of epithelial cell phenotypes andtubular structural integrity, thereby limiting the continuous suppliesto the myofibroblast pool in obstructed kidneys after persistentinjury. In view of the efficacy of HGF administrated in a delayedfashion on blocking tubular EMT and renal fibrosis in obstructivenephropathy, it is reasonable to speculate that a supplement of HGF mayhold promise as a novel therapeutic strategy for halting theprogression of an established renal interstitial fibrosis in patients.
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ACKNOWLEDGEMENTS) b( J$ n" g7 ?

This work was supported by National Institute of Diabetes andDigestive and Kidney Diseases Grants DK-02611, DK-54922, and DK-61408(Y. Liu) and a postdoctoral fellowship from the American HeartAssociation Pennsylvania-Delaware Affiliate (J. Yang)." O5 j8 D. w0 M4 n: Z5 V) }) H
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