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. 2021 Sep 9;24(10):103112.
doi: 10.1016/j.isci.2021.103112. eCollection 2021 Oct 22.

The hepatocyte growth factor/c-met pathway is a key determinant of the fibrotic kidney local microenvironment

Haiyan Fu  1   2 , Yuan Gui  3 , Silvia Liu  1 , Yuanyuan Wang  1 , Sheldon Ira Bastacky  1 , Yi Qiao  4 , Rong Zhang  5 , Christopher Bonin  6 , Geneva Hargis  6 , Yanbao Yu  7 , Donald L Kreutzer  4 , Partha Sarathi Biswas  8 , Yanjiao Zhou  6 , Yanlin Wang  3 , Xiao-Jun Tian  5 , Youhua Liu  1 , Dong Zhou  3
Affiliations

The hepatocyte growth factor/c-met pathway is a key determinant of the fibrotic kidney local microenvironment

Haiyan Fu et al. iScience. .

Abstract

The kidney local microenvironment (KLM) plays a critical role in the pathogenesis of kidney fibrosis. However, the composition and regulation of a fibrotic KLM remain unclear. Through a multidisciplinary approach, we investigated the roles of the hepatocyte growth factor/c-met signaling pathway in regulating KLM formation in various chronic kidney disease (CKD) models. We performed a retrospective analysis of single-cell RNA sequencing data and determined that tubular epithelial cells and macrophages are two major cell populations in a fibrotic kidney. We then created a mathematical model that predicted loss of c-met in tubular cells would cause greater responses to injury than loss of c-met in macrophages. By generating c-met conditional knockout mice, we validated that loss of c-met influences epithelial plasticity, myofibroblast activation, and extracellular matrix synthesis/degradation, which ultimately determined the characteristics of the fibrotic KLM. Our findings open the possibility of designing effective therapeutic strategies to retard CKD.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

None
Graphical abstract
Figure 1
Figure 1
The characteristics of KLM in human diseased kidneys (A) Representative immunohistochemical staining images showing the patterns of α-SMA, vimentin, CD68, and CD14 expression in non-tumor normal human kidney (n = 3), and kidney biopsy specimens from patients diagnosed with focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN), and membrane nephropathy (MN). Five cases were employed per diagnosis. The images in the blue channel were used to identify the fibrotic area (blue outline). Arrows indicated positive staining. Scale bar, 50 μm. Quantitative data are presented (B–E). Data are represented as mean ± SEM. Statistical significance was assessed by one-way ANOVA, followed by the Student-Newman-Keuls test.
Figure 2
Figure 2
HGF/c-met expression in tubular epithelial cells and macrophages may mediate fibrotic KLM formation (A and B) Bulk RNA-seq data (Database: GSE125015) showing (A) normalized HGF and (B) c-met expression in whole male mouse kidneys subjected to sham surgery (control) or UUO after 5 and 10 days. Data are represented as mean ± SEM. Statistical significance was assessed by one-way ANOVA, followed by the Student-Newman-Keuls test. (C–H) Single-cell RNA-seq data (Database: GSE140023) were visualized by UMAP algorithm, where UMAP_1 and UMPA_2 represent the two reduced dimensions and each dot in the figure indicates one cell. (C) Kidney single cells clustered by sham control or 2 days and 7 days after UUO. At the single-cell level, (D) c-met expression was localized in specific subpopulations of kidney cells. Expression of Slc34a1, Umod, Tfcp2l1, and Cx3cr1 identified a subpopulation of kidney tubular cells (E-G) and macrophages (H).
Figure 3
Figure 3
Mathematical modeling predicts tubular epithelial cells and macrophages accelerate fibrotic KLM formation (A) Diagram of the cell-cell communication model for renal fibrosis. (B) Bifurcation diagram of ECM level with respect to macrophage activation rate (km0) (Left). The dependence of the fibrosis threshold (SN) on the levels of epithelial-HGF (E-HGF) and macrophage-HGF (M-HGF) (Right). (C) The dose-response curve of ECM levels with respect to the injury level under either E-HGF c-met−/− (orange line) or M-HGF c-met−/− (yellow line) knockdown (Left). The WT c-met+/+ (blue line) is shown as a control. The temporal dynamics of ECM level under normal, E-HGF knockdown, and M-HGF knockdown conditions (Right). The injury level is fixed as high. (D) The temporal dynamics of the system variables in the space of the injury levels under normal (c-met+/+), E-HGF knockdown, and M-HGF knockdown conditions. (E) The dependence of the repair and fibrosis scores on M-HGF (Left) and E-HGF (Right). (F) Phase diagram of repair and fibrosis scores in the space of M-HGF and E-HGF.
Figure 4
Figure 4
Tubular-specific ablation of c-met exacerbates fibroblast activation and ECM synthesis (A) Schematic diagram depicting the generation of tubular epithelial cell-specific c-met conditional knockout mice. (B) Mice genotyping analyses for the control mice used in this study (genotype: c-metfl/fl, lane 1), designated as Ksp-met+/+, while lane 2 demonstrates the genotyping of the tubular c-met knockout mice (genotype: c-metfl/fl, Cre), designated as Ksp-met−/−. (C) Immunofluorescence staining showing E-cadherin expression in the untreated Ksp-met+/+ and Ksp-met−/− mouse kidneys. Scale bar, 50 μm. (D–F) Diagrams representing the strategies for the surgeries or treatment in mice, IRI (10 days), FA (60 days), and UUO (7 days). (G–I) qPCR analyses revealed the mRNA abundance of FN, α-SMA, type I collagen, type IIIcollagen, or TGF-β in Ksp-met+/+ and Ksp-met−/− mice kidneys after IRI (G), FA (H), and UUO (I). n = 3–6. (J–O) Western blot analyses demonstrated FN and α-SMA protein expression in Ksp-met+/+ and Ksp-met−/− mice kidneys after IRI (J), FA (L), and UUO (N), and quantified data (K, M, and O). Dots indicate individual animals within each group. n = 3–4. (P–R) Representative micrographs for Masson's trichrome, Sirius red, α-SMA, and FN staining in kidneys from Ksp-met+/+ and Ksp-met−/− mice after IRI, FA, and UUO. n = 3–4. Scale bar, 50 μm. IRI, ischemia-reperfusion injury; FA, folic acid injection; UUO, unilateral ureteral obstruction. Data are represented as mean ± SEM. Statistical significance was assessed using a two-tailed Student's t-test or the Rank Sum Test if data failed in normality test or two-way ANOVA, followed by the Student-Newman-Keuls test.
Figure 5
Figure 5
Specific deletion of c-met in tubules influences epithelial plasticity towards kidney fibrosis (A–F) Western blot analyses demonstrated vimentin expression in Ksp-met+/+ and Ksp-met−/− mice kidneys after IRI (A), FA (C), and UUO (E). Quantitative data presented in B, D, and F, respectively. Dots indicate individual animals within each group. n = 3–4. Data are represented as mean ± SEM. Statistical significance was assessed using a two-tailed Student's t-test. (G) Immunohistochemical staining showing the distributions of vimentin in Ksp-met+/+ and Ksp-met−/− mice kidneys after IRI, FA, and UUO. Scale bar, 50 μm. (H) Representative images showing the induction of vimentin in diseased tubules after FA. Scale bar, 25 μm. (I and J) Immunofluorescence staining for vimentin expression in kidneys after FA. 3-D reconstruction images (J, J1–J4). Scale bar, 25 μm. (K) Representative micrographs showing the localization of td-Tomato + cells by co-staining with laminin and vimentin in Ksp-tdTom−/− mice treated with HGF inhibitor or vehicle at 10 days after IRI. Scale bar, 25 μm. (L) Pie chart of vimentin+/td-Tomato+ tubular cells in fibrotic kidneys after IRI. IRI, ischemia-reperfusion injury; FA, folic acid injection; UUO, unilateral ureteral obstruction.
Figure 6
Figure 6
Macrophage-specific deletion of c-met enhances fibrosis but has little effect on extracellular matrix (ECM) synthesis (A) Schematic diagram showing the generation of macrophage-specific c-met deletion mice. (B and C) qPCR analyses of FN, α-SMA, type I collagen, and type IIIcollagen mRNA in Lyz-met+/+ and Lyz-met−/− mice kidneys after IRI at 10 days and UUO at 7 (1 week [w]) and 14 days (2 w). n = 4–5. (D–G) Western blot analyses of FN, α-SMA, and TNC protein expression in Lyz-met+/+ and Lyz-met−/− mice kidneys after IRI (D) and UUO (F). Quantification of protein expression for IRI (E) and UUO (G). Dots indicate individual animals within each group. n = 3–5. (H and I) Representative micrographs for Sirius red, Masson's trichrome, and FN staining in Lyz-met+/+ and Lyz-met−/− mice kidneys after IRI and UUO, respectively. Scale bar, 50 μm. (J) Bar graph of collagen/non-collagen ratios in Lyz-met+/+ and Lyz-met−/− mice kidneys after IRI and UUO, respectively. n = 3–5. (K) qPCR analyses of MCP-1 mRNA in Lyz-met+/+ and Lyz-met−/− kidneys after IRI. n = 4. (L) qPCR analysis of MCP-1 mRNA in Lyz-met+/+ and Lyz-met−/− mice kidneys after UUO. n = 4. (M and N) Western blot analyses (M) and quantitative data (N) of MCP-1 protein expression in Lyz-met +/+ and Lyz-met −/− mice kidneys after IRI. Dots indicate individual animals within each group. n = 3. (O and P) Western blot analyses (O) and quantitative data (P) demonstrating MCP-1 protein expression in Lyz-met+/+ and Lyz-met−/− mice kidneys after UUO. Dots indicate individual animals within each group. n = 4. (Q and R) Representative images showing RANTES, MCP-1, and F4/80 expression in Lyz-met +/+ and Lyz-met−/− mice kidneys after UUO. Scale bar, 50 μm. (S and T) FACS analysis revealed no significant difference in macrophage numbers from Lyz-met+/+ and Lyz-met−/− mice fibrotic kidneys after IRI at 10 days. n = 6. Data are represented as mean ± SEM. Statistical significance was assessed using a two-tailed Student's t-test.
Figure 7
Figure 7
Macrophage loss of c-met repressed the capacity to degrade fibrotic ECM (A) The strategy of bone marrow-derived macrophage (BMDMs) isolation and culture. (B) Phase-contrast images of BMDM differentiation after stimulation with GM-CSF. (C) qRT-PCR analysis of tPA and uPA mRNA in BMDMs after incubation with HGF recombinant protein (20 ng/mL) for 1, 2, or 3 days. n = 3. (D) Western blot analyses demonstrating the induction of tPA in BMDMs after incubation with HGF recombinant protein (20 ng/mL). (E) Immunostaining showing HGF-induced tPA expression in macrophages. Cells were co-stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Scale bar, 25 μm. (F) RT-PCR analysis showed c-met gene expression in cultured c-met+/+ and c-met−/− BMDMs ex vivo. (G) Western blot analysis demonstrating reduced c-met phosphorylation after incubation with HGF recombinant protein in c-met−/− BMDMs. (H and I) qRT-PCR analysis of tPA and uPA mRNA in cultured c-met+/+ and c-met−/− BMDMs after incubation with HGF recombinant protein (20 ng/mL) for 1, 2, or 3 days. n = 4. (J and K) qPCR analysis revealing higher tPA and uPA mRNA abundance in Lyz-met+/+ than Lyz-met−/− kidneys after IRI and UUO. n = 3–5. (L–O) Western blot analyses (L, N) and quantitative data (M, O) demonstrating tPA protein abundance in Lyz-met +/+ and Lyz-met−/− mice kidneys after IRI and UUO. Dots indicate individual animals within each group. n = 3. (P–R) qRT-PCR analysis of tPA, and uPA mRNA in Ksp-met+/+ and Ksp-met−/− kidneys after IRI, FA, and UUO. n = 3–6. (S) Schematic diagram of our model for fibrotic KLM formation. Data are represented as mean ± SEM. Statistical significance was assessed using a two-tailed Student's t-test or the Rank Sum Test if data failed in normality test or one-way ANOVA, followed by the Student-Newman-Keuls test.
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