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Hepatocyte growth factor counteracts transforming growth factor-ß₁, through attenuation of connective tissue growth factor induction, and prevents renal fibrogenesis in 5/6 nephrectomized mice¹

TSUTOMU INOUE, HIROKAZU OKADA, TATSUYA KOBAYASHI, YUSUKE WATANABE, YOSHIHIKO KANNO, JEFFREY B. KOPP^*, TAKASHI NISHIDA^#, MASAHARU TAKIGAWA^#, MUNEHISA UENO^##, TOSHIKAZU NAKAMURA and HIROMICHI SUZUKI²

Departments of Nephrology and
^## Urology, Saitama Medical College, Saitama;
^# Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine and Dentistry, Okayama;
Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Suita, Japan; and
^* Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, Maryland, USA

²Correspondence: Department of Nephrology, Saitama Medical College, 38 Morohongo, Moroyama-machi, Irumagun, Saitama 350–0495, Japan. E-mail address:iromichi{at}saitama-med.ac.jp

SPECIFIC AIMS

The aim of this study was to investigate the mechanisms that underlie the anti-fibrotic effects of hepatocyte growth factor (HGF) on fibrosis in a variety of organs, especially its action against transforming growth factor ß₁ (TGF-ß₁).

PRINCIPAL FINDINGS

1. Expression of fibrosis-related genes in wild-type (WT) and TGF-ß₁ transgenic (TG) mice with 5/6 nephrectomy (Nx)
Five-wk-old male TGF-ß₁ TG mice and WT mice were used. Alb/TGF-ß₁ TG mice express the TGF-ß₁ transgene exclusively in the liver. At 8 to 24 h after the Nx, there were significant increases in all the mRNAs examined, i.e., TGF-ß₁, HGF, connective tissue growth factor (CTGF), and 1(I) procollagen (COLI), in both mice. Expression of those mRNAs gradually decreased to basal levels by wk 2–4. However, in the remnant kidneys of NxWT mice, expression of TGF-ß₁ and HGF mRNAs started to increase again. In NxTG mice, although HGF gene expression was similar to that of NxWT mice (Fig. 1 B),the level of TGF-ß₁ expression was significantly higher (Fig. 1A ). Whereas the expression of CTGF and COLI mRNAs remained at basal levels in NxWT mice, it increased significantly in the remnant kidneys of NxTG mice at wk 8 (Fig. 1C, D ), remaining increased until wk 12. The kidney tissues of NxTG mice at wk 12 (Fig. 1E-b ), in contrast to the NxTG mice on day 0 (Fig. 1E-a ) and the NxWT mice at wk 12, showed prominent interstitial fibrosis. Immunohistochemical and in situ hybridization analyses revealed that in the remnant kidneys of NxTG mice, tubular epithelial cells and glomerular epithelial cells, expressed CTGF, possibly in response to TGF-ß₁ (Fig. 1F ).

View larger version (43K): [in this window] [in a new window]   Figure 1. Quantification of expression of mRNAs of fibrosis-related molecules in the remnant kidneys of NxWT mice and NxTG mice. Expression levels of all the molecules examined in the remnant kidneys were higher in NxTG mice on day 0 (A–D). Although HGF expression in NxTG mice was similar to that in NxWT mice (B), endogenous TGF-ß1 expression in the remnant kidney was higher in NxTG mice (A). The expression of CTGF and COLI mRNAs increased significantly in the remnant kidney of NxTG mice at wk 8 (C, D). *P < 0.05 vs. NxTG as the control, #P < 0.05 vs. NxWT at each point. Light microscopic finding of the remnant kidney tissues from NxTG mice (E). The interstitium had no significant alternations on day 0 (E-a). In contrast, at wk 12, the kidney tissues showed interstitial expansion and increased extracellular matrix accumulation (E-b) (H&E staining, MO: 20x). Immunohistochemical analysis using anti-CTGF antibody and in situ hybridization using CTGF antisense cRNA probes revealed that in the remnant kidney of NxTG mice, the tubular epithelial cells as well as glomerular epithelial cells expressed CTGF, possibly in response to TGF-ß1 (F-a, c). No CTGF protein expression was detected in NxWT mice (F-b). Sense probes yielded no signals in NxTG mice (F-d).

2. Cell culture
Murine renal proximal tubular epithelial cells (PTEC) were cocultured with murine renal fibroblasts (TFB). CTGF gene expression increased significantly in PTEC in response to TGF-ß₁. COLI synthesis also increased in TFB after TGF-ß₁ treatment. The COLI synthesis in TFB was reduced significantly after treatment with anti-CTGF neutralizing antibody, showing that COLI synthesis in TFB was mediated at least partially by CTGF secreted by PTEC. The increase in COLI synthesis after TGF-ß₁ treatment was inhibited by HGF pretreatment through attenuation of CTGF induction in PTEC. This result was consistent with the finding that coadministration of CTGF rescued TGF-ß₁ induction of COLI synthesis in TFB pretreated with HGF.

3. Effects of recombinant deleted HGF (dHGF) administration in TG mice with 5/6 Nx
To examine the effects of HGF on renal fibrosis, TG mice were given twice daily s.c. injections of dHGF (5.0 mg·kg^-1·day^-1) or an identical volume of PBS from wk 2 to 6 after the Nx. dHGF administration significantly reduced expression of the CTGF gene in the remnant kidneys of the NxTG+dHGF group (Fig. 2A ), resulting in a decrease in COLI expression in the remnant kidneys (Fig. 2B ). Quantitative analysis demonstrated that the interstitial fibrosis area was markedly narrowed in the NxTG+dHGF group (Fig. 2C, D ). The survival rate of the NxTG mice treated with dHGF was improved significantly (Fig. 2E ), perhaps because of the attenuated renal fibrogenesis.

View larger version (61K): [in this window] [in a new window]   Figure 2. Quantification of CTGF mRNA in the remnant kidney of NxTG+PBS mice and NxTG+dHGF mice. Expression of CTGF mRNA was reduced significantly in the remnant kidney of NxTG+dHGF mice after dHGF administration (at wk 6 after the Nx) (A). HGF treatment reduced CTGF expression significantly, resulting in a decrease in COLI expression in the remnant kidney of NxTG+dHGF mice (B). *P < 0.05 vs. NxTG+PBS mice. Histology of the remnant kidney tissues of NxTG+PBS mice and NxTG+dHGF mice (C). The remnant kidneys at wk 2 (before dHGF administration) showed slightly widened interstitium (C-a, b). At wk 6, the remnant kidney tissues of NxTG+PBS mice showed tubular atrophy, interstitial expansion, and increased matrix accumulation (C-c, d); in the remnant kidney of NxTG+dHGF mice, however, such alterations were attenuated significantly (C-e, f) (upper row; H&E staining, MO: 20x, lower row; MT staining, MO: 4x). Quantitative analysis of interstitial fibrosis by light microscopy (D). The area of fibrosis in blue in the MT-stained sections was measured quantitatively using a computer-assisted image analyzer, revealing that dHGF administration prevented interstitial fibrogenesis significantly. #P < 0.05 vs. NxTG+PBS mice, P < 0.05 vs. NxTG mice at wk 2. Survival of the 5/6 NxTG mice in the NxTG+PBS and NxTG+dHGF groups (E). Life table analyses are presented as Kaplan-Meier plots. Survival rate was improved significantly in the 5/6 NxTG mice treated with dHGF.

CONCLUSIONS

TGF-ß₁ is a well-characterized, profibrogenic cytokine whose expression is markedly up-regulated in most organ fibrosis. CTGF is one of the downstream mediators of TGF-ß₁, modulating fibroblast cell growth and extracellular matrix production. CTGF gene expression is induced strongly by TGF-ß₁ but not by other growth factors. HGF is a pleiotropic protein that has mitogenic, motogenic, and morphogenic effects on the epithelial cells of some organs. In the normal kidney, HGF is expressed mainly in interstitial cells, endothelial cells, and macrophages whereas its receptor, c-Met, is present on the surface of proximal tubular epithelial cells. It was recently been reported that HGF administration can prevent renal fibrogenesis in spontaneously nephrotic mice, in which HGF was assumed to counteract against TGF-ß₁.

As the principal producers of TGF-ß₁, interstitial fibroblasts and macrophages have been demonstrated to be important in renal fibrogenesis. HGF cannot inhibit directly the production of TGF-ß₁ by those cells because they do not express c-Met. To investigate the interaction between TGF-ß₁ and HGF at the late stage in remnant kidneys, we focused on changes in the expression of CTGF mRNA. Removal of 5/6 kidney induced advanced renal fibrosis reproducibly in TG mice by wk 12 after the Nx. The relative potency of TGF-ß₁ in the remnant kidney was supposed to predominate over endogenous HGF, resulting in the up-regulation of CTGF gene expression, followed by an increase in COLI gene expression. We demonstrated CTGF expression in the tubular epithelial cells in the remnant kidneys of NxTG mice. Therefore, proximal tubule injury likely results in adjacent interstitial fibrosis through paracrine mechanisms. Based on these findings, we proposed the hypothesis that the reciprocal balance between the effects of TGF-ß₁ and HGF within the tubular epithelial cells may determine the CTGF expression level and overall renal fibrogenesis.

In in vitro experiments, PTEC expressed the CTGF gene, which was induced significantly by TGF-ß₁. We showed that such a stimulatory effect of TGF-ß₁ on PTEC was reduced by HGF pretreatment. Counteractions between TGF-ß₁ and HGF within PTEC can affect COLI expression in TFB by regulating CTGF induction in PTEC. In in vivo experiments, daily injections of dHGF decreased the expression of CTGF and COLI mRNAs and markedly narrowed the area of interstitial fibrosis in the remnant kidney of NxTG mice, improving their survival rate. TGF-ß₁ gene expression was not down-regulated in the remnant kidney after dHGF administration, and the plasma level of TGF-ß₁ remained high in the NxTG+dHGF mice. Therefore, exogenous HGF attenuated the COLI expression in the remnant kidneys not via a reduction in TGF-ß₁ gene expression, but by a blockade of CTGF induction.

No data have been reported concerning the interplay of TGF-ß₁ and HGF. TGF-ß signals are transmitted through the phosphorylation of Smad proteins by receptor serine/threonine kinase, leading to nuclear accumulation and DNA binding of Smad proteins. We demonstrated that phosphorylation of Smad3 induced by TGF-ß₁ in PTEC was not affected by HGF pretreatment, suggesting that an interplay of these growth factors likely exists at subsequent downstream levels. TGF-ß induces a member of the AP-1 family c-jun, and interaction of c-Jun with Smad3 results in a reduction of the Smad3/DNA interaction, effectively a negative feedback mechanism counteracting Smad-driven gene trans-activation. In rat hepatocytes, HGF through transmembrane receptor tyrosine kinases increased the expression of c-jun mRNA in a concentration-dependent manner, so we assume that c-jun gene expression induced by HGF pretreatment leads to inhibition of the transcription of the CTGF gene via a Smad3/c–Jun interaction.

In conclusion, the profibrotic effects of TGF-ß₁ were exerted through expression of the CTGF gene in tubular epithelial cells; HGF can block such CTGF induction, resulting in the anti-fibrotic effects (Fig. 3 ). CTGF, rather than TGF-ß₁, likely serves as a specific target for selective intervention in the process involved in connective tissue formation during wound repair or tissue fibrogenesis, because the biological actions of TGF-ß₁ are complex and affect a number of different cell types.

View larger version (25K): [in this window] [in a new window]   Figure 3. The long-term overproduction of TGF-ß1 is a key event leading to the pathology of fibrotic disorders. TGF-ß1 induces potently growth arrest and apoptosis of epithelial cells, stimulates extracellular matrix deposition, inhibits matrix degradation and induces its own production by target cells. CTGF, induced by TGF-ß1 in epithelial cells, has an important role in fibrogenesis. HGF counteracts the action of TGF-ß1 in epithelial cells and prevents tubular atrophy and interstitial fibrosis. The anti-fibrotic effect of HGF is exerted through CTGF expression inhibition in epithelial cells. These findings mean that the reciprocal balance between TGF-ß1 and HGF is associated closely with the pathogenesis of renal fibrosis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0442fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0442fje

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