Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea.
R&D Center for Innovative Medicines, Helixmith Co., Ltd., Seoul, Korea.
Department of Internal Medicine, Seoul National University Boramae Medical Center, Seoul, Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.
Department of Medical Science, Seoul National University College of Medicine, Seoul, Korea.
Kidney Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Kidney Research Institute, Seoul National University College of Medicine, Seoul, Korea. ysh5794@gmail.com.
Seoul National University Biomedical Research Institute, Seoul, Korea. ysh5794@gmail.com.
Department of Internal Medicine, Seoul National University Boramae Medical Center, Seoul, Korea. nephrolee@gmail.com.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. nephrolee@gmail.com.
Abstract
Hepatocyte growth factor (HGF) and its receptor, cMet, activate biological pathways necessary for repair and regeneration following kidney injury. Because HGF is a highly unstable molecule in its biologically active form, we asked whether a monoclonal antibody (Ab) that displays full agonist activity at the receptor could protect the kidney from fibrosis. We attempted to determine whether the cMet agonistic Ab might reduce fibrosis, the final common pathway for chronic kidney diseases (CKD). A mouse model of kidney fibrosis disease induced by unilateral ureteral obstruction was introduced and subsequently validated with primary cultured human proximal tubular epithelial cells (PTECs). In kidney biopsy specimens from patients with CKD, cMet immunohistochemistry staining showed a remarkable increase compared with patients with normal renal functions. cMet Ab treatment significantly increased the levels of phospho-cMet and abrogated the protein expression of fibrosis markers such as fibronectin, collagen 1, and αSMA as well as Bax2, which is a marker of apoptosis triggered by recombinant TGF-β1 in PTECs. Remarkably, injections of cMet Ab significantly prevented kidney fibrosis in obstructed kidneys as quantified by Masson trichrome staining. Consistent with these data, cMet Ab treatment decreased the expression of fibrosis markers, such as collagen1 and αSMA, whereas the expression of E-cadherin, which is a cell-cell adhesion molecule, was restored. In conclusion, cMet-mediated signaling may play a considerable role in kidney fibrosis. Additionally, the cMet agonistic Ab may be a valuable substitute for HGF because it is more easily available in a biologically active, stable, and purified form.
Figures
Figure 1cMet expression in normal human kidney tissue. (A) Representative images of IHC staining for cMet in the glomerulus and tubulo-interstitium from normal human kidney tissue. Original magnification: X400 (Left), X200 (Right). Scale bar, 100 µm. (B) Representative confocal microscopy images of human kidney biopsy samples from human normal kidney tissues; the samples were costained for cMet (green), aquaporin-1 (red) and DAPI (blue). Original magnification: X200. (C) Representative images of phospho-cMet expression in IgAN patients. Original magnification: X200. Scale bar, 100 µm. Symbols represent individual data points, horizontal bars indicate the mean, and error bars indicate SEM (*p < 0.05, ***p < 0.001).Figure 2Effect of cMet agonistic antibody on human umbilical vein endothelial cells (HUVECs). (A) cMet Ab-mediated activation of the cMet receptor. (B) Representative image of the HUVEC migration assay. (C) Quantification of (B). (D) Analyzing the biological activity of the cMet Ab in A549 cells. (E) cMet Ab-mediated activation of the cMet receptor. (F) Representative image of cell proliferation assay in A549 cells.Figure 3Injection of cMet Agonistic antibody in mice did not produce changes in the immune cell profile. After C57BL/6J wild-type mice were treated with cMet agonistic Ab (10 mg/kg, 20 mg/kg) or control IgG, their peripheral blood (Figure A-D) or spleens (Figure E-H) were harvested 2 hours later. Also we injected cMet agonistic Ab or control IgG under the same schedule as UUO experiment and their peripheral blood/spleens were harvested 7 days later. The different immune cell populations were enumerated by flow cytometry or an automated hematology analyzer. In the spleen cell suspensions, cMet treatment did not lead to obvious changes in the percentages of the CD3+ and CD25+ regulatory T cell populations (A,E), CD3+CCD44+ effector T cell populations (B,F), CD3+CCD25+CD44+ effector T cell populations (C,G), or CD11b+Gr-1+ myeloid (nonlymphoid) cell populations (D,H) compared with those in the control IgG treatment group. (I) Pro-inflammatory cytokines expression (IL-6, TNFα, IFNγ) didn’t show difference between the spleen cells of control IgG or cMet Ab injected mice.Figure 4cMet agonistic Ab and kidney fibrosis in a unilateral ureteral obstruction (UUO) mouse model. (A) Representative images of kidney sections stained with Masson’s trichrome stain in IgG control- or cMet Ab-treated mice at day 14 after UUO. Original magnification: X20. Scale bar, 200 µm. Symbols represent individual data points, horizontal bars indicate the mean, and error bars indicate SEM. (B) cMet expression in proximal tubular cells in n IgG control- or cMet Ab-treated mice at day 14 after UUO. DAPI (blue), cMet (green), and aquaporin-1 (red).Original magnification: X400. Scale bar, 50 µm. (C,D) The expression levels of fibronectin and collagen 1 were increased and those of E-cadherin were decreased in the cMet group, and treatment with the cMet agonistic Ab prevented fibrosis as indicated by western blot. (E) Quantitative RT-PCR analyses of the indicated mRNA transcript showed similar expression patterns as those observed by western blot. The combined data represent at least 2 independent experiments with similar results. Symbols represent individual data points, horizontal bars indicate the mean, and error bars indicate SEM (*p < 0.05, **p < 0.01, ***p < 0.001).Figure 5Effect of the cMet agonistic antibody on primary cultured human proximal tubular epithelial cells (PTECs). (A) Representative western blot analysis of primary cultured human PTECs after treatment with rHGF (10 ng/ml) or cMet Ab (250, 500 ng/ml) for various time periods. (B) The expression of phosphorylated cMet was increased at 1 hour and 3 hours after treatment with rHGF or cMet Ab.Figure 6Effect of cMet agonistic antibody on kidney fibrosis and wound healing in primary cultured human proximal tubular epithelial cells (PTECs) in vitro. (A) Representative confocal images of primary cultured human PTECs costained for DAPI (blue), fibronectin (green), and collagen IV (red). Fibrosis was induced with TGFβ, and treatment with the cMet agonistic Ab (500 ng/ml) indicated improvements in fibrosis. Original magnification: X200. (B,C) In the presence or absence of either rHGF (10, 20 ng/ml) or cMet Ab (250, 500, 1000 ng/ml) for 30 minutes, rTGFβ-stimulated PTECs exhibited significantly increased protein levels of fibronectin, αSMA and Bax2. (D,E) The migratory capacity of PTECs was assessed using a scratch wound healing assay and observing cell movement at 0 and 10 hours after scratching. There was a significant difference in the migratory potential of cells in the high-dose cMet Ab group compared to that in the control groups. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus control group.
