Fig 2: The MET receptor is not required for HGF-induced NEPHRIN and NEPH1 phosphorylation. Recombinant HGF (20 ng/ml) was added to HEK293 cells overexpressing (A) NEPH1-FLAG or (B) NEPHRIN-FLAG in the presence or absence of 100 nM Crizotinib, a MET inhibitor. Phosphorylation of NEPH1 and NEPHRIN was unchanged by the presence of Crizotinib. Data are presented as mean ± SEM, and p-values were calculated using the Kruskal–Wallis one-way analysis of variance. C, D, NEPH1-FLAG or NEPHRIN-FLAG were overexpressed in HEK293 cells with stable shRNA-mediated knockdown (KD) of the MET receptor. Recombinant HGF (20 ng/ml) was added to these cells, and NEPHRIN and NEPH1 phosphorylation in the cell lysate showed no change in phosphorylation following MET knockdown (KD). Data are presented as mean ± SEM, and p-values were calculated using the Kruskal–Wallis one-way analysis of variance. E, using CRISPR-Cas9, stable MET knockout HEK293 cells were generated and transfected with NEPHRIN- and NEPH1-expressing plasmids. Red fluorescent protein (RFP) is a marker for transfection and confirms the stable knockout of MET post puromycin selection. Data are presented as mean ± SEM, and p-values were calculated using a two-tailed Student’s t test. The scale bar represents 25 µm. F and G, using HGF-overexpressing cells grown on the Transwell filter and HEK293 MET knockout cells expressing NEPH1 or NEPHRIN growing on the plastic at the bottom of well (first panel, schematic), we demonstrate that NEPH1 and NEPHRIN phosphorylation occurs following complete loss of the MET receptor. Data are presented as mean ± SEM, and p-values were calculated using a two-tailed Student’s t test. ns, nonsignificant, *p = 0.05, **p = 0.01, ****p = 0.0001. All experiments were performed in triplicate and repeated three times with similar results, and representative images of the results are presented in the figure.

Fig 3: HGF treatment repairs podocytes/nephrocytes in a NEPH1- and NEPHRIN-dependent fashion.A and B, cultured human podocytes were treated with protamine sulfate (PS), and actin cytoskeleton (green) disorganization was visualized by phalloidin staining. To induce recovery, HGF (50 ng/ml) was added to the PS-treated podocytes. The addition of NEPH1 inhibitory peptide blocked HGF-induced recovery. KD of NEPH1 also prevented HGF-induced recovery. Ten cells per experimental condition were evaluated from three experimental replicates. The scale bar represents 25 µm. Data are presented as mean ± SEM, and p-values were calculated using Tukey's multiple comparisons test (one-way ANOVA). C and D, Sns (Drosophila ortholog of NEPHRIN) staining of Drosophila nephrocytes treated with PS in the presence or absence of HGF and NEPHRIN or NEPH1 peptides. Decreased Sns staining was noted in PS-treated nephrocytes, which was rescued by treatment with HGF. Addition of HGF-interacting NEPH1 Peptide-1 and NEPHRIN Peptide-1, but not NEPHRIN Peptide-2, blocked the rescue by HGF. The scale bar represents 5 µm; scale bar for insets represents 1 µm. For quantification, seven nephrocytes from three flies for each condition were analyzed. Data are presented as mean ± SEM, and p-values were calculated using the Tukey's multiple comparisons test (one-way ANOVA). ns, nonsignificant, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001.

Fig 4: NEPH1/NEPHRIN augments HGF-mediated recovery in protamine sulfate (PS)-induced injury in MET knockout podocytes.A and B, using CRIPR-Cas9 MET knockout podocytes were generated. Red fluorescent protein (RFP) is a marker for transfection and confirms the stable knock out of MET post puromycin selection. Western blot for the MET knockout and control podocyte cell lysates. C and D, MET knockout podocytes were transfected with NEPHRIN- and NEPH1-expressing plasmids and then treated with PS and the actin cytoskeletal (green) disorganization was visualized by phalloidin staining. To induce recovery, HGF (50 ng/ml) was added to the PS-treated podocytes. Podocytes transfected with NEPHRIN and NEPH1 showed significantly improved recovery with HGF. Ten cells per experimental condition were evaluated from three experimental trials. The scale bar represents 25 µm. Data are presented as mean ± SEM, and p-values were calculated using the Kruskal–Wallis test one-way analysis of variance. ns, nonsignificant, *p = 0.05, ** p = 0.01, ***p = 0.001.

Fig 5: The HGF-binding site for MET does not overlap with the HGF-binding sites for NEPH1 and NEPHRIN.A, schematic for the competitive ELISA. HGF was immobilized on the wells of an ELISA plate, and its binding to NEPH1, NEPHRIN, and MET alone and in combination with NEPH1 peptide-1 and NEPHRIN peptide-1 was analyzed. B, quantification of the data. All comparisons are with NEPH1 or NEPHRIN alone. Data are presented as mean ± SEM, and p-values were calculated using a two-tailed Student’s t test. *p = 0.05, **p = 0.01, ***p = 0.001, ****p < 0.0001.