Transgene-derived hepatocyte growth factor attenuates reactive renal fibrosis in aristolochic acid nephrotoxicity
Hirokazu Okada1,
Yusuke Watanabe1,
Tsutomu Inoue1,
Tatsuya Kobayashi1,
Yoshihiko Kanno1,
Goshi Shiota2,
Toshikazu Nakamura3,
Takeshi Sugaya4,
Akiyoshi Fukamizu4 and
Hiromichi Suzuki1
1Department of Nephrology, Saitama Medical College, Irumagun, 2Second Department of Internal Medicine, Tottori University School of Medicine, Yonago, 3Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Medical School, Suita and 4Center of Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Japan
Correspondence and offprint requests to: Hiromichi Suzuki, MD, PhD, Department of Nephrology, Saitama Medical College, 38 Morohongo, Moroyama-machi, Irumagun, Saitama 350-0495, Japan. Email: iromichi{at}saitama-med.ac.jp
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Abstract
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Background. Hepatocyte growth factor (HGF) has been demonstrated to attenuate acute tubular necrosis and interstitial fibrosis in a variety of rodent models of kidney disease. We investigated how HGF could affect chronic toxic nephropathy/interstitial fibrosis caused by aristolochic acid (AA).
Methods. Wild-type and HGF transgenic mice received daily intraperitoneal injections of AA for 14 days. At sacrifice, kidneys were removed and used for microscopy examination and in vitro studies. To determine the molecular mechanisms of anti-fibrotic effects of HGF, cultured murine tubular epithelial cells (mProx24) were employed.
Results. Significant tubular degeneration was observed in both the transgenic and the wild-type mice to the same degree after 2 weeks treatment with AA. Interstitial fibrosis subsequently developed in the wild-type mice 4 weeks after cessation of AA administration. However, the transgenic mice manifested less fibrotic changes. Decreased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased matrix metalloproteinase-9 activity could partially account for the attenuation of fibrogenesis in the transgenic mouse kidney. HGF at 10 and 100 ng/ml could block TIMP-1 gene expression in mProx24 induced by epidermal growth factor, but a decrease in the number of mProx24 via apoptosis induced by AA was blocked only by HGF at 100 ng/ml.
Conclusion. Circulating transgene-derived HGF (210 ng/ml) could not prevent tubular degeneration caused by AA, but attenuated interstitial fibrogenesis during tubular regeneration. These findings suggest a possible therapeutic efficacy for renal interstitial fibrosis following tubular degeneration even of low-dose HGF.
Keywords: aristolochic acid; hepatocyte growth factor; matrix metalloproteinase-9; renal fibrosis; tissue inhibitor of metalloproteinase-1
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Introduction
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Hepatocyte growth factor (HGF) was identified originally as a potent mitogen for mature hepatocytes, molecularly cloned, and recently shown to prevent apoptosis and accelerate regeneration of tubular parenchymal components, acting as a renotrophic factor [1]. Specifically, HGF attenuated progression of acute renal failure in a rodent model of ischaemic/toxic tubular necrosis [2,3], and prevented glomerulosclerosis and tubulointerstitial changes in rodent models of nephrotic syndrome [4] and obstructive nephropathy [5]. However, there have been no studies to date regarding long-term effects of HGF on toxic tubular degeneration/regeneration and subsequent reactive interstitial fibrosis.
In early 1993 in Belgium, a number of young women who had ingested slimming pills containing powdered Chinese herbs developed rapidly progressive renal failure [6]. This syndrome was once termed Chinese herbs nephropathy, characterized by extensive interstitial fibrosis without glomerular abnormalities. This was very similar to Balkan nephropathy, which often progressed steadily even after cessation of slimming pill ingestion [7]. In this latter case, the pills contained Aristolochia sp., from which aristolochic acid (AA) metabolites are derived. AA has been shown to be toxic to tubular epithelium and carcinogenic to urethral epithelium [8,9], and it formed DNA adducts in renal tissues of patients with Chinese herbs nephropathy [10]. The term Chinese herbs nephropathy has been criticized [11], and in this study we use the term aristolochic acid nephrotoxicity (AAN) instead.
In the present study, we developed a murine model of AAN, and investigated potential therapeutic effects of HGF on chronic tubulointerstitial damage, e.g. tubular epithelial degeneration and reactive interstitial fibrosis. To see the effects of HGF delivered constantly, we used AA in transgenic (tg) mice constitutively expressing HGF under the control of albumin regulatory sequences in the liver and secreting this factor into the peripheral circulation.
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Subjects and methods
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Transgenic mice
Mice of the TG.AH2 line, which was developed by means of microinjection of an albumin promoterHGF fusion gene and maintained by breeding to FVB inbred mice, were employed [12]. Heterozygous tg mice were routinely bred with wild-type FVB mice, and offspring heterozygous for the transgene were identified by means of standard polymerase chain reaction (PCR) methods using DNA extracted from tail biopsy specimens. The serum level of HGF was measured by the bioassay, and in the tg mice the HGF level was significantly higher than that in the wild-type mice (210 ng/ml vs an undetectable level) [12]. Such high serum levels of HGF were demonstrated to represent intrarenal HGF content [2,13]. In contrast to another HGF tg mouse line with the higher serum level of HGF [14], the induction of the transgene and the serum level of HGF in our tg mouse line have not been demonstrated to be pathogenic [12]. All animals received appropriate care in accordance with the institutional guidelines.
In vivo experimental design
Six- to 8-week-old male tg and wild-type mice were fed standard chow ad libitum and given free access to water. Pilot studies using wild-type mice established that daily intraperitoneal (i.p.) injections of 5.0 mg AA (Sigma, St Louis, MO) per kg of body weight for 2 weeks resulted in apparent tubular degeneration without significant lethality. Higher doses of AA tended to be lethal to the mice. Therefore, both groups (tg and wild-type mice) were given this daily dose. Control animals were injected daily with an equivalent volume of saline.
Fifteen experimental and 15 control animals in each group were used. After 2 weeks of daily i.p. injection of AA or saline, five experimental and five control animals in each group were sacrificed; the remaining mice were not treated further and were maintained for another 1 or 4 weeks prior to sacrifice.
At sacrifice, one kidney from each mouse was used for RNA extraction and zymography, and the other was divided into smaller pieces and fixed in 4% paraformaldehyde (PFA) or 4% glutaraldehyde (GA) overnight. Some of the PFA-fixed tissues were processed for paraffin blocks, and the rest were dehydrated and snap-frozen for immunofluorescence (IF) studies. The GA-fixed tissues were processed for electron microscopy according to the method described below.
In vitro experimental design
Cultured murine proximal tubular epithelial cells (mProx24) [15] were maintained in growth medium [Dulbeccos modified Eagles medium (DMEM) with 10% fetal calf serum (FCS)]. mProx24 were seeded in 6-well plates (1 x 105 cells/well), 4-chamber slides (3 x 104 cells/chamber) or 10 cm culture dishes (4 x 105 cells/dish) and kept overnight in growth medium. Medium was then changed to resting medium (DMEM with 0.5% FCS). After 12 h, human recombinant HGF (R&D Systems, Minneapolis, MN) was added to the medium at a final concentration of 10 and 100 ng/ml with or without subsequent supplementation with AA (6.0 µg/ml) 48 h later. After another 24 h of incubation, cells in the 6-well plates were collected. To determine the effects of HGF on cell survival, some of the cells were stained with 0.04% trypan blue for 5 min, and the cells excluding trypan blue were counted in a haemacytometer. To detect apoptosis in the rest of the cells, flow cytometry of annexin V staining was performed as described below. In addition, to detect apoptotic nuclei by another method, cells cultured in the 4-chamber slides were also used and processed according to the TUNEL staining method as described below. To determine the effects of HGF on the expression of pro- and anti-apoptotic proteins, cells cultured in 10 cm culture dishes were harvested for western blotting as described below. Also, in order to see the effects of HGF on the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA induced by another growth factor, cells treated with human recombinant epidermal growth factor (EGF) (R&D Systems) and HGF were harvested for RNase protection assay as described below.
Renal histopathology, immunohistochemistry and immunofluorescence studies
Sections were cut 4 µm in thickness from the paraffin blocks, and processed for haematoxylineosin (HE) and Massons trichrome (MT) staining. Collagenous, fibrotic areas stained in blue of 10 random cortical fields at 100x magnification were assessed quantitatively in each MT-stained section using a colour image computer analyser (Mac SCOPE Version 2.5, Mitani Corp., Fukui, Japan). All glomeruli and vessels were subtracted from a given field by manual tracing, yielding a target area of tubulointerstitium.
For the indirect immunoperoxidase method, after deparaffinization and rehydration, the sections were treated with proteinase K and boiled in citrate buffer under microwave for unmasking antigenicities. Endogenous biotin was blocked by a Biotin Blocking System (Dako Corp., Carpinteria, CA) and then they were immersed in 3% H2O2 in methanol for inhibition of endogenous peroxidase and flooded with 1% non-fat milk in phosphate-buffered saline (PBS) for inhibition of non-specific reactions. Goat anti-TIMP-1 (1:200, G-T Research Product, Minneapolis, MN) was applied as a primary antibody. The following immunoreaction was performed using a biotin-conjugated anti-goat IgG (1:500; Chemicon International, Temecula, CA) as a secondary antibody, a Vectastain ABC Standard Kit (Vector Laboratories, Burlingame, CA) and an AEC Standard Kit (Dako), according to the manufacturers instructions.
IF was performed on 4 µm cryostat sections. Fluorescein isothiocyanate (FITC)-conjugated mouse anti-
-smooth muscle actin (
SMA) (1:500; Sigma), rabbit anti-fibroblast-specific protein 1 (FSP1) (1:500) [16], anti-type I collagen (Col I) (1:400) [16] and anti-type III collagen (Col III) (1:400; Monosan, Uden, The Netherlands) were applied as primary antibodies. FITC-conjugated and rhodamine-conjugated anti-rabbit IgG (1:500; American Qualex, San Clement, CA, 1:500; Chemicon, respectively) were used as the secondary antibodies. The secondary antibodies used in this study had been isolated by immunoaffinity chromatography and absorbed for dual labelling. Control measures included omitting the primary antibody and substituting the primary antibody with normal IgG from the same animal.
Apoptosis assays
Flow cytometric analysis of annexin V staining was also employed for the detection of apoptosis. Annexin V detects phosphatidylserine externalized to the outer surface of the cell membrane in the early stage of apoptosis. FITCannexin V (PharMingen, San Diego, CA) was applied to determine the percentage of mProx24 undergoing apoptosis, according to the manufacturers instructions. The cells that were stained positive for annexin V were defined to be in the stage of apoptosis. For identification of apoptotic nuclei with TUNEL staining, DNA fragmentation within apoptotic cell nuclei was detected using the MEBSTAIN Apoptosis Kit Direct (Medical & Biological Laboratories, Nagoya, Japan) according to the manufacturers instructions. Briefly, 4 µm cryostat sections and mProx24 cultured in the 4-chamber slides, fixed with 4% PFA and permeabilized with 0.5% Tween-20 were incubated for 1 h at 37°C with FITCdUTP and terminal deoxynucleotidyl transferase, the latter linking FITCdUTP to 3'-OH fragmented ends of DNA.
Electron microscopy
The tissues fixed with 4% GA were post-fixed with osmium tetroxide, and then dehydrated through graded ethanol series followed by propylene oxide. These tissues were embedded in resin, and sectioned at 5070 nm with diamond knives. The ultrathin sections were stained with uranyl acetate and lead citrate, and examined using a transmission electron microscope.
RNase protection assay
Total RNA was extracted from the homogenates of whole kidneys and cultured cells with TRIzolTM (Gibco BRL, Grand Island, NY) according to the manufacturers instructions. RNase protection assay was performed as described previously [17]. The cRNA probes were generated from cDNA fragments encoding rat transforming growth factor-ß1 (TGF-ß1) (a generous gift from T. Yamamoto, Niigata University), rat HGF (GenBank accession No. E03330), rat TIMP-1 [17], mouse EGF (GenBank accession No. J00380) and mouse
1(I) procollagen (
1Col I) (GenBank accession No. MMU08020). The cDNA fragments of HGF, EGF and
1Col I were obtained in our laboratories by RTPCR according to the GenBank data. For quantifying the expression of mRNA, the autoradiograph bands were analysed by computerized densitometry using Mac SCOPE. Data are presented as a ratio of specific mRNA to GAPDH mRNA to equalize the quantity of RNA within each sample.
Gelatin zymography
To see matrix metalloproteinase (MMP) activities, gelatin zymography was performed with a Gelatin Zymography Kit (YU-68001; Yagai, Yamagata, Japan). Briefly, kidney tissues were homogenized in the extraction buffer (0.05 M TrisHCl pH 6.8, 2% SDS, 10% glycerol). The samples were then centrifuged for 5 min (14 000 g), and the supernatant was loaded onto the gel (15 µg/well) after protein concentration measurement using the Bradford assay (Bio-Rad, Hercules, CA). Human proMMP-2, MMP-2, proMMP-9 and MMP-9 were also loaded into the outer wells as standards. After electrophoresis, the gel was incubated overnight at 37°C in a solution containing 50 mM Tris pH 7.8 and 10 mM CaCl2, and then stained with 0.002% Coomassie blue. The sizes of lytic bands were measured using Mac SCOPE.
Western blotting
Western blotting was carried out as described previously using protein extracts from mProx24 [18]. Specific primary antibodies against Bcl-xL, Bcl-2 and Bax were obtained from Santa Cruz Biochemical (Santa Cruz, CA).
Serum biochemistry and urinary protein excretion
At sacrifice, blood samples were collected via the orbital vein. Serum creatinine and glutamicpyruvic transaminase levels were measured with an auto-analyser (Dry-Chem 3000, Fuji, Tokyo, Japan).
Statistical analysis
The values are presented as means ± SE. Statistical differences between the groups were evaluated by analyses of variance, followed by Duncans multiple range testing, with P < 0.05 used as the requirement for significance.
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Results
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In vivo experiments
Daily administration of AA for 2 weeks induced apparent tubular degeneration in HGF tg mice as well as in wild-type mice (Figure 1a and b). However, no significant fibrotic areas were found in either of them. Degenerated tubular epithelium, indicated by apoptotic and flattened epithelial cells, and disappearance of epithelial cells from the tubular structure, seemed to be mainly localized in the proximal and distal tubules in the cortex and the outer medulla, and in the medullary ray (Figure 1a and b). Administration of AA for 4 weeks also induced significant AAN, essentially to the same degree in both groups (data not shown). In the kidney with AAN of both mice at week 2, electron microscopy revealed detachment of tubular epithelial cells from the tubular structure (Figure 1c), and apoptotic epithelial cell nuclei were detected by TUNEL staining in the focal, restricted area (Figure 1d). Compared with the relevant control mice treated with saline, serum levels of creatinine were significantly increased in both tg and wild-type mice treated with AA (1.1 ± 0.3 mg/dl vs 0.3 ± 0.1 mg/dl in the tg mice, and 1.1 ± 0.3 mg/dl vs 0.3 ± 0.1 mg/dl in the wild-type mice; P < 0.05). No abnormalities were found in the serum levels of glutamicpyruvic transaminase of both mice. Therefore, the degree of tubulointerstitial changes of AAN evaluated in this study seemed substantially similar between tg and wild-type mice. After 4 weeks recovery time without AA administration, wild-type mice showed marked regeneration of tubular epithelium with moderate peritubular, interstitial fibrosis (Figure 2a and e) consisting of fibroblast-like cells (FbLCs) positive for FSP1 or
SMA, and accumulation of collagenous matrix such as Col I and Col III (data not shown). Although the tubules were regenerated similarly, tg mice had less fibrotic changes in the kidney than wild-type mice (Figure 2b and e), which was consistent with the finding that tubular expression of TIMP-1 in tg mice was suppressed compared with wild-type mice (Figure 2c and d). The serum levels of creatinine tended to be lower in tg mice compared with wild-type mice, but this difference failed to reach significance (0.4 ± 0.1 mg/dl vs 0.5 ± 0.1 mg/dl, respectively). In spite of 210 ng/ml of the circulating HGF derived from the transgene [12], the RNase protection assay revealed that mRNAs of
1Col I, TGF-ß1, and HGF per se were not altered in tg mice compared with wild-type mice throughout the study period (Figure 3a, b, d and f). In the case of EGF, the expression was enhanced in both mice 1 week after cessation of AA administration (Figure 3e and f), which might not only accelerate regeneration of tubules, but also promote fibrogenesis. In contrast, TIMP-1 mRNA was significantly downregulated in tg mice 1 week after cessation of AA administration (Figure 3c and f). The latter was consistent with the histopathological findings in tg mice compared with wild-type mice (Figure 2a and b). Gelatin zymography detected strong lytic bands representing MMP-9 activity and considerably smaller bands due to proMMP-9, proMMP-2 and MMP-2 activities (Figure 4a). After cessation of AA administration, renal MMP-9 activity was increased in tg mice compared with wild-type mice (Figure 4a and b), which was consistent with the lower TIMP-1 expression in tg mice (Figure 3e and f).

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Fig. 1. (a) AAN in wild-type mice at week 2 (HE stain, x200). (b) AAN in tg mice at week 2 (HE stain, x200). Focal disappearance of tubular epithelium in the kidney (arrows) was seen in both groups, whereas the glomerulus remained intact. (c) Detachment of a degenerated tubular epithelial cell (arrow) was observed in AAN in wild-type mice at week 2 (x2500). (d) Apoptotic cells within tubules of AAN in wild-type mice at week 2. TUNEL-positive nuclei were only seen focally (FITC, x400) There were no significant histopathological differences between the wild-type and tg mice at week 2.
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Fig. 2. Reactive interstitial fibrosis in AAN after cessation of AA administration. (a and b) Collagenous, fibrotic changes (in blue) were seen in the peritubular and perivascular interstitium in wild-type mice (a) and tg mice (b) (MT stain, x100). (c and d) TIMP-1 protein expression was suppressed in tubular epithelial cells in tg mice (d) compared with wild-type mice (c) (AEC, x100) (e) Quantification of collagenous, fibrotic areas (in blue) in the kidney shown in (a) and (b). Each value represents the fibrotic area as a fractional percentage of the tubulointerstitium. There were no significant fibrotic areas in either group at the start of AA administration (AAN D0). However, the fibrotic area was significantly increased in wild-type mice 4 weeks after cessation of AA administration compared with tg mice (REC D28).
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Fig. 4. MMP activities in the kidney at week 1 after cessation of AA. (a) Gelatin zymography showing renal proMMP-9, MMP-9, proMMP-2, and MMP-2 activities. The density of the lytic bands of MMP-9 were the most significant, and are arbitrarily expressed in (b). Renal MMP-9 activity was significantly increased in tg mice compared with wild-type mice. In contrast, there were no differences in other MMP activities between wild-type mice and tg mice. In (a), a representative gel selected from three separate experiments is shown, and the densitometric data in (b) were obtained from these three gels.
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In vitro experiments
HGF at a concentration of both of 10 and 100 ng/ml could promote the proliferation of subconfluent mProx24 (data not shown). However, a decrease in the number of mProx24 treated with AA could be blocked only by a 48 h pre-incubation with 100 ng/ml of HGF (data not shown). Preliminary experiments on co-incubation and pre-incubation for shorter periods with HGF failed to rescue mProx24 treated with AA. Treatment with AA significantly increased expression of a pro-apoptotic protein, Bax, in spite of the mildly increased expression of an anti-apoptotic protein, Bcl-xL, resulting in a decrease of the Bcl-xL/Bax ratio and induction of apoptosis in mProx24 (Figure 5a and b). One hundred but not 10 ng/ml of HGF could attenuate such a decrease in the Bcl-xL/Bax ratio in mProx24 treated with AA, thereby suppressing apoptosis (Figure 5a).

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Fig. 5. Anti-apoptotic effects of HGF on mProx24. (a) Fluorescence-activated cell sorting analyses of mProx24 labelled with annexin V (FITC; horizontal axis). Panels show the results of cells treated with AA alone or with pre-treatment with HGF at 10 and 100 ng/ml. Apoptotic cells are positive with annexin V, appearing in the right field. The percentage of the apoptotic cells in the control culture was 37.5 ± 5.6%, and 34.5 ± 5.2% in the culture pre-treated with 10 ng/ml HGF, but 14.4 ± 2.2% in the culture pre-treated with 100 ng/ml HGF (P < 0.05). Pre-treatment with 100 ng/ml HGF could significantly reduce the number of apoptotic cells, suggesting that HGF could prevent the decrease in the viable cell number of mProx24 treated with AA, at least partially, via its anti-apoptotic effects. (b) Expression of anti- and pro-apoptotic proteins in mProx24 treated with AA. One possible anti-apoptotic effect of HGF on mProx24 was found to result from the attenuation of a decrease in the Bcl-xL/Bax ratio.
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In mProx24, HGF treatment did not reduce the basal expression of TIMP-1 mRNA (data not shown), but lowered the expression of TIMP-1 mRNA induced by EGF in a dose-dependent manner (Figure 6a and b), presumably contributing to its anti-fibrogenic effects. These in vitro results were consistent with the in vivo findings (Figure 3c, e and f).


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Fig. 6. Anti-fibrotic effects of HGF on mProx24. (a) TIMP-1 gene expression induced by EGF in mProx24. HGF, even at 10 ng/ml, could significantly lower TIMP-1 mRNA expression of mProx24 treated with 10 ng/ml of EGF. (b) Densitometric analysis of (a). The blot is representative of three independent experiments, and the densitometric data were obtained from these three blots.
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Discussion
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In this study, we demonstrated that circulating HGF derived from the transgene could attenuate reactive renal fibrosis in a murine model of AAN. The resolution of toxic nephropathy after cessation of AA administration in wild-type mice was less complete, since there was more apparent peritubular, interstitial fibrosis compared with tg mice. The product of the proto-oncogene c-met is a receptor for HGF, but has never been detected in any interstitial FbLCs in vivo [1]. HGF itself can promote the transcription of the HGF gene and enhance HGF production [1]. Among known growth factors efficacious for tubular regeneration, e.g. EGF, fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF) and HGF, most of which are also mitogenic to interstitial FbLCs [19], only HGF is known to be more morphogenic to the tubular epithelium and less fibrogenic to the tubulointerstitium [1]. The present study suggests that HGF at 210 ng/ml in the serum is not enough to increase HGF expression, but enough to decrease the mRNA levels of some pro-fibrotic factors, e.g. TIMP-1, in the regenerating kidney, which was consistent with the increased activity of renal MMP-9 in tg mice. Dworkin et al. demonstrated that HGF can induce the expression of MMPs, and suppress the expression of TIMPs in tubular epithelial cells in vitro [20]. These anti-fibrotic effects of HGF are likely to be involved in the early phase of renoablation without significant renal fibrogenesis [20]. In the present model, HGF could suppress the expression of TIMP-1 not at 2 weeks of AA treatment, but 1 week after cessation of AA administration. Overall, organ fibrosis results from the balance between pro- and anti-fibrotic effects [19]. Although the exact mechanism remains unclear, the in vitro finding that HGF lowered the expression of TIMP-1 mRNA induced by EGF in mProx24 suggests that HGF could show its anti-fibrotic effects and counteract some pro-fibrotic factors active during the resolution phase, e.g. EGF.
In a series of experiments using recombinant HGF and rodent models of acute ischaemic/toxic tubular necrosis and urinary obstruction [2,3,5], HGF was demonstrated to be a strong inhibitor of tubular apoptosis and an accelerator of tubular regeneration, rescuing failing kidneys and preventing interstitial fibrosis. The reason why HGF derived from the transgene in the tg mice could not prevent the tubular apoptosis/degeneration in this model may be because of the relatively low serum level of transgene-derived HGF, which was 210 ng/ml in these tg mice [12]. On the other hand, serum levels of up to 100 ng/ml of injected recombinant HGF were required to treat experimental acute tubular injury [2]. Consistent with this, in the present study, 100 ng/ml but not 10 ng/ml of HGF prevented apoptosis of cultured tubular epithelial cells by AA.
This murine model of AAN did not support the development of significant interstitial fibrosis during 2 weeks of AA administration even in the wild-type mice. Daily administration of a high dose of AA for such a short period seems to account for the prominent tubular degeneration without interstitial fibrosis observed in this model. AAN in human (Chinese herbs nephropathy) is a unique disease which is characterized by prominent tubular atrophy and interstitial fibrosis in spite of the total absence of glomerulitis/glomerulosclerosis and massive proteinuria [6,7]. Although our model as used in this study is not identical to the human counterpart, it does seem to share some features. As mentioned above, a number of endogenous growth factors other than HGF are upregulated when degenerated tubular epithelium is being regenerated, and most of them are also mitogenic/fibrogenic for interstitial FbLCs. These facts may account for the comparative tubular regeneration and the reactive interstitial fibrosis observed in the wild-type mice during the resolution phase in this model. This also seems to be the case in AAN in humans because they developed progressive renal fibrosis even after cessation of taking pills containing AA [6,7]. Therefore, it is possible that chronic/repeated tubular injury resulting from habitual drinking of herbal tea containing AA most probably contributes to the progression of renal interstitial fibrosis via activation of pro-fibrotic growth factors in AAN in humans. Recently, human AAN-like disease has been reproduced in rabbits and rats treated with AA [21,22].
In conclusion, compared with the wild-type mice, resolution of AAN in the tg mice took place with fewer fibrotic changes. The serum level of HGF derived from the native gene and the transgene is thought to be insufficient to prevent direct tubular degeneration caused by AA, but high enough to attenuate reactive renal fibrosis during the resolution phase in AAN. This suggests an anti-fibrotic potential of HGF, even at low levels, for patients with chronic tubulointerstitial damage.
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Acknowledgments
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We are grateful to S. Yamada, J. Takahashi and K. Tanaka for their technical assistance. During this study, H.O. was supported by grants from the Ministry of Education, Science and Culture of Japan and the Uehara Memorial Science Foundation, and T.I. was a recipient of a grant from the Ochiai Memorial Foundation. A portion of this work was presented at the 35th Annual Meeting of the American Society of Nephrology in Philadelphia, Pennsylvania in 2002.
Conflict of interest statement. None declared.
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References
|
---|
- Matsumoto K, Nakamura T. Hepatocyte growth factor: renotrophic role and potential therapeutic for renal disease. Kidney Int 2001; 59: 20232038[CrossRef][ISI][Medline]
- Kawaida K, Matsumoto K, Shimazu H, Nakamura T. Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice. Proc Natl Acad Sci USA 1994; 91: 43574361[Abstract]
- Miller SB, Martin DR, Kissane J, Hammerman MR. Hepatocyte growth factor accelerates recovery from acute ischemic renal injury in rats. Am J Physiol 1994; 266: F129F134[ISI][Medline]
- Mizuno S, Kurosawa T, Matsumoto K et al. Hepatocyte growth factor prevents renal fibrosis and dysfunction in a mouse model of chronic renal disease. J Clin Invest 1998; 101: 18271834[Abstract/Free Full Text]
- Mizuno S, Matsumoto K, Nakamura T. Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy. Kidney Int 2001; 59: 13041314[CrossRef][ISI][Medline]
- Vanherweghem JL, Depierreux M, Tielemans C et al. Rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including Chinese herbs. Lancet 1993; 341: 387391[CrossRef][ISI][Medline]
- Cosyns JP, Jadoul M, Squifflet JP et al. Chinese herbs nephropathy: a clue to Balkan endemic nephropathy? Kidney Int 1994; 45: 16801688[ISI][Medline]
- Cosyns JP, Jadoul M, Squifflet JP, Wese F, De Strihou CY. Urothelial lesions in Chinese-herb nephropathy. Am J Kidney Dis 1999; 133: 10111017
- Nortier JL, Martinez MC, Schmeiser HH et al. Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi). N Engl J Med 2000; 342: 16861692[Abstract/Free Full Text]
- Stiborova M, Frei E, Breuer A, Bieler A, Schmeiser HH. Aristolactam I a metabolite of aristolochic acid I upon activation forms an adduct found in DNA of patients with Chinese herbs nephropathy. Exp Toxic Pathol 1999; 51: 421427[ISI][Medline]
- Chen HY, Ma BY, Grant A, Lampert N. Time to abandon the term Chinese herbs nephropathy. Kidney Int 2001; 60: 20392040
- Shiota G, Wang T, Nakamura T, Schmidt EV. Hepatocyte growth factor in transgemic mice: effects on hepatocyte growth, liver regeneration and gene expression. Hepatology 1994; 19: 962972[ISI][Medline]
- Igawa T, Matsumoto K, Kanda S, Saito Y, Nakamura T. Hepatocyte growth factor may function as a renotropic factor for regeneration in rats with acute renal injury. Am J Physiol 1993; 265: F61F69[ISI][Medline]
- Takayama H, LaRochelle WJ, Sabnis SG, Otsuka T, Merlino G. Renal tubular hyperplasia, polycystic disease, and glomerulosclerosis in transgenic mice overexpressing hepatocyte growth factor/scatter factor. Lab Invest 1997; 77: 131138[ISI][Medline]
- Takaya K, Koya D, Isono M et al. Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells. Am J Physiol 2003; 284: F1037F1045[ISI]
- Okada H, Ban S, Nagao S et al. Progressive renal fibrosis in murine polycystic kidney disease: an immunohistochemical observation. Kidney Int 2000; 58: 587597[CrossRef][ISI][Medline]
- Okada H, Inoue T, Kanno Y et al. Renal interstitial fibroblast-like cells in Goodpasture syndrome rats. Kidney Int 2001; 60: 597606[CrossRef][ISI][Medline]
- Okada H, Moriwaki K, Kalluri R et al. Osteopontin expressed by renal tubular epithelium mediates interstitial monocyte infiltration. Am J Physiol 2000; 278: F110F121[ISI]
- Eddy AA. Molecular basis of renal fibrosis. Pediatr Nephrol 2000; 15: 290301[CrossRef][ISI][Medline]
- Liu Y, Rajur K, Tolbert E, Dworkin LD. Endogenous hepatocyte growth factor ameliorates chronic renal injury by activating matrix degradation pathways. Kidney Int 2000; 58: 20282043[CrossRef][ISI][Medline]
- Cosyns JP, Dehoux JP, Guiot Y et al. Chronic aristolochic acid toxicity in rabbits: a model of Chinese herbs nephropathy? Kidney Int 2001; 59: 21642173[CrossRef][ISI][Medline]
- Debelle FD, Nortier JL, De Prez EG et al. Aristolochic acids induce chronic renal failure with interstitial fibrosis in salt-depleted rats. J Am Soc Nephrol 2002; 13: 431436[Abstract/Free Full Text]
Received for publication: 26. 9.02
Accepted in revised form: 9. 7.03