The motor protein Myo1c regulates transforming growth factor-β-signaling and fibrosis in podocytes

Abstract

Transforming growth factor-β (TGF-β) is known to play a critical role in the pathogenesis of many progressive podocyte diseases. However, the molecular mechanisms regulating TGF-β signaling in podocytes remain unclear. Using a podocyte-specific myosin (Myo)1c knockout, we demonstrate whether Myo1c is critical for TGF-β-signaling in podocyte disease pathogenesis. Specifically, podocyte-specific Myo1c knockout mice were resistant to fibrotic injury induced by Adriamycin or nephrotoxic serum. Further, loss of Myo1c also protected from injury in the TGF-β-dependent unilateral ureteral obstruction mouse model of renal interstitial fibrosis. Mechanistic analyses showed that loss of Myo1c significantly blunted TGF-β signaling through downregulation of canonical and non-canonical TGF-β pathways. Interestingly, nuclear rather than the cytoplasmic Myo1c was found to play a central role in controlling TGF-β signaling through transcriptional regulation. Differential expression analysis of nuclear Myo1c-associated gene promoters showed that nuclear Myo1c targeted the TGF-β responsive gene growth differentiation factor (GDF)-15 and directly bound to the GDF-15 promoter. Importantly, GDF15 was found to be involved in podocyte pathogenesis, where GDF15 was upregulated in glomeruli of patients with focal segmental glomerulosclerosis. Thus, Myo1c-mediated regulation of TGF-β-responsive genes is central to the pathogenesis of podocyte injury. Hence, inhibiting this process may have clinical application in treating podocytopathies.

Keywords: TGF-beta; fibrosis; focal segmental glomerulosclerosis; glomerulonephritis; glomerulus; podocyte.

Figure 1:. Construction of podocyte-specific Myo1c null mice:

(A) Schematic diagram of Myo1c targeting vector shows deletion of Myo1c Exons 5–13 following cre recombination. (B) Podocyte-specific deletion of Myo1c was confirmed by staining of paraffin-embedded mouse kidney sections from Myo1c^fl/fl (control) and Myo1c^fl/flpod- Cre^Tg/+ mice using Neph1 (green) and Myo1c (Red) antibodies and DAPI (Blue). The images were collected using an confocal microscope. Immunofluorescence staining confirm deletion of Myo1c protein in podocytes (marked with arrows). Scale bars: 20μm (C) Podocyte-specific deletion of Myo1c was confirmed by staining with NM1 (green) and synaptopodin (Red) antibodies and DAPI (Blue). The images were collected using confocal microscopy. Scale bars: 20μm. (D) Myo1c^fl/flpod-Cre^Tg/+ or Myo1c^+/+pod-Cre^Tg/+ mice were crossed with ROSA^mT/mG mice to generate GFP expressing podocytes, while all the other cell types remained red (Texas-red). (E) The green podocytes were separated by FACS sorting (~40000–50000 cells were obtained from glomeruli isolated from kidneys of 3 mice each). (F) qPCR analysis of isolated podocytes showed more than 90% reduction in cytoplasmic and nuclear Myo1c expression.

Figure 2:. Podocyte specific Myo1c deletion protects mice from adriamycin induced podocytopathy:

(A). Experimental timeline of adriamycin injection and urine collections. (B). Adriamycin sensitivity was tested in Myo1c^fl/fl and Myo1c^fl/flpod-Cre^Tg/+ mice on adriamycin sensitive C57BL/6N background. The SDS-PAGE analysis of 3 and 4 weeks post adriamycin injection urine samples showed heavy albuminuria in Myo1c^fl/fl mice but not in Myo1c^fl/flpod-Cre^Tg/+ mice. (C) The albumin/creatinine analysis further showed significant reduction in adriamycin-induced albuminuria in Myo1c^fl/flpod-Cre^Tg/+ mice. ***P≤0.001, **P≤0.01 vs. Myo1c^fl/fl, 2-way ANOVA (Sidak’s multiple comparison test). Myo1c^fl/fl, n=6 mice and Myo1c^fl/flpod-Cre^Tg/+, n=9 mice. Data presented in means±SEM. (D) Adriamycin-induced damages were assessed by TEM analysis of kidney sections, which showed areas of flattening with significant shortening and fusion of podocyte foot processes and loss of slit diaphragm in control Myo1c^fl/fl mice, whereas the podocytes of Myo1c^fl/flpod-Cre^Tg/+ or saline treated control mice were largely healthy. Scale bars: 1μm (Left panel); 500nm (Right panel). (E) Quantitative analysis of electron micrographs showed significant reduction in the number of slit-diaphragm/μm in adriamycin treated Myo1c^fl/fl mice but not in Myo1c^fl/flpod-Cre^Tg/+ or saline treated control mice. *P≤0.05 vs Myo1c^fl/fl (Saline) and Myo1c^fl/flpod-Cre^Tg/+ (Adriamycin), one-way ANOVA (Kruskal-Wallis test), Multiple scoring of each animal was first averaged and the average value of each animal was used for constructing graphs and statistical analysis. n=5 mice in each group, The bar graphs represent mean±SEM. (F) SEM analysis showed flattening of podocytes in adriamycin treated Myo1c^fl/fl. Scale bars: 2μm (Left panel); 1μm (Right panel).

Figure 3:. Podocyte-specific Myo1c deletion protects mice from NTS-induced podocytopathy:

(A) Experimental timeline of NTS injection and urine collections. (B) SDS-PAGE and (C) albumin creatinine analysis and showed albuminuria was significantly reduced in Myo1c^fl/flpod-Cre^Tg/+ mice, when compared to the Myo1c^fl/fl mice. *P≤0.05 vs Myo1c^fl/fl (NTS), one-way ANOVA (Kruskal-Wallis test), n=6 mice per group. Data presented in means±SEM. (D-F) The microscopic analysis (SEM and TEM) of kidney sections showed significant podocytes flattening and increased foot process fusion with loss of slit diaphragm in NTS treated Myo1c^fl/fl mice but not in Myo1c^fl/flpod- Cre^Tg/+ or sheep IgG treated mice. (E) Quantitative analysis of TEM images showed numbers of slit-diaphragm per micrometer of GBM were reduced in NTS treated Myo1c^fl/fl mice. *P≤0.05 vs Myo1c^fl/fl (NTS), one-way ANOVA (Kruskal-Wallis test), Multiple scoring of each animal was first averaged and the average value of each animal was used for constructing graphs and statistical analysis. n=5 mice in each group. Data presented in mean±SEM. Scale bars: 800nm (D, Left panel); 200nm (D, Right panel); 1 μm (F).

Figure 4:. Podocyte-specific Myo1c null mice were protected from adriamycin induced renal fibrosis:

Glomerular fibrosis in adriamycin treated Myo1c^fl/fl and Myo1c^fl/flpod-Cre^Tg/+ mice was assessed using Masson’s trichrome and Sirius red stainings. (A) Increased Masson’s trichrome staining was noted in the glomeruli of Myo1c^fl/fl mice. Scale bars: left panel, 100μm; middle panel 50μm and right panel 20μm. (B) Fibrotic area assessment from the Masson’s trichrome stained glomeruli of Myo1c^fl/fl mice showed ~20% fibrosis, whereas the Myo1c^fl/flpod-Cre^Tg/+ showed ~6% fibrosis. *P≤0.05 vs Myo1c^fl/fl (Adriamycin), one-way ANOVA (Kruskal-Wallis test), n=6 mice in each group using manual outlining method. Data presented in mean±SEM. (C) Similar to Masson’s trichrome, the Sirius red staining was also elevated in Myo1c^fl/fl mice glomeruli. Scale bars: left panel, 50μm and right panel 20μm. (D) Quantitative assessment using five glomeruli from six mice in each group showed significantly increased fibrotic areas in Myo1c^fl/fl mice, when compared to Myo1c^fl/flpod-Cre^Tg/+ mice. </P/> *P≤0.05 vs Myo1c^fl/fl (Adriamycin), one-way ANOVA (Kruskal-Wallis test), n=6 mice in each group. Data presented in means±SEM. (E) Immunostaining of kidney sections using α-SMA antibody and DAPI (blue) showed increased α-SMA expression in Myo1c^fl/fl mice in response to injury. Scale bars: 20μm (F) Quantitative analysis of immunofluorescence images showed that injury-induced α-SMA expression was elevated in Myo1c^fl/fl mice when compared to Myo1c^fl/flpod-Cre^Tg/+ mice. *P≤0.05 vs Myo1c^fl/fl (Adriamycin), one-way ANOVA (Kruskal-Wallis test), n=6 mice in each group. Data presented in mean±SEM.

Figure 5:. Podocyte-specific Myo1c deletion attenuates post-UUO renal fibrosis:

(A) Experimental timeline of UUO procedure (upper panel) and representative image of non-obstructed right kidney (left panel) and obstructed left kidney (right panel). (B) Representative macrophotographs (increasing magnification) of Masson-stained kidney sections from sham and day 7 post-UUO procedure performed on, Myo1c^fl/fl and Myo1c^fl/flpod-Cre^Tg/+ mice are shown (left panel). Scale bars: left panel, 200μm; middle panel 100μm and right panel 50μm. n=6 mice in each group. (C) Representative images of stained kidney sections show increased Sirius red staining in Myo1c^fl/fl mice as compared to the Myo1c^fl/flpod-Cre^Tg/+ and sham operated mice. Images were collected at 40x magnification and enlarged glomerular regions are presented in the right panel. Scale bars: left panel 50μm and right panel 20μm. (D) Reduction in total renal area (% area, normalized with sham) post UUO procedure was quantitatively assessed and showed ~30% reduction in Myo1c^fl/fl, whereas in Myo1c^fl/flpod-Cre^Tg/+ mice ~20% reduction was noted. ***P=0.0001, *P<0.05, vs Sham, one-way ANOVA (Kruskal-Wallis test). Data presented in mean±SEM. n=6 mice in each group. (E) Quantitative analysis of interstitial fibrosis using Masson’s trichrome staining showed that fibrosis increased by ~6% in Myo1c^fl/fl renal tissue, whereas in Myo1c^fl/flpod-Cre^Tg/+ mice only ~2% increase was observed. *P≤0.05, Myo1c^fl/fl (UUO) vs Myo1c^fl/flpod-Cre^Tg/+ (UUO), one-way ANOVA (Kruskal-Wallis test). Data presented as mean±SEM. n=6 mice in each group. (F) Representative confocal images from Myo1c^fl/fl and Myo1c^fl/flpod-Cre^Tg/+ mice immunostained with α-SMA (Red) and Neph1 (Green) antibodies and DAPI (Blue) are shown. Scale bars: 20μm. (G) Bar graphs represent mean fluorescent intensity of α- SMA staining. *P≤0.05, Myo1c^fl/fl (UUO) vs Myo1c^fl/flpod-Cre^Tg/+ (UUO), one-way ANOVA (Kruskal-Wallis test). Data presented in mean±SEM. n=6 mice in each group.

Figure 6:. TGF-β induced signaling is blunted in Myo1c-KD podocytes:

(A and B) The signaling components of canonical and non-canonical TGF-β signaling pathways in control and Myo1c-KD podocytes were screened using western blotting. Quantitative analysis showed reduced phosphorylation of SMAD3, P38 and ERK1/2 proteins and reduced expression of α-SMA proteins in Myo1c-KD podocytes. All experiments were performed in triplicate. Data are presented in mean±SEM and p-values were calculated using one way ANOVA (Kruskal-Wallis test). (^aP≤0.05, control-KD vs Myo1c-KD; ^bbP≤0.001, ^bP≤0.05, control-KD TGFβ stimulated vs Myo1c-KD TGFβ stimulated; **P≤0.001, *P≤0.05, control-KD vs control-KD TGFβ stimulated). n=3 experimental replicates. (C) GFP-Myo1c over-expression was confirmed using western blot analysis. (D-E) TGF-β stimulation of in GFP-Myo1c over-expressed podocytes induced the expression of p-SMAD2, p-SMAD3 and p-ERK2/3. All experiments were performed in triplicate. Data are presented in mean±SEM and p-values were calculated using Mann-Whitney test (nonparametric test). *P≤0.05, TGFβ- vs TGFβ+.

Figure 7:. Genes involved in TGF-β signaling were DE in Myo1c-KD podocytes.

(A) The heat map from RNA-Seq data shows DEGs in Myo1c-KD podocytes in response to TGF-β +/− stimulation. (B) GO enrichment analysis showed multiple cellular events (presented as histograms) that were enriched in response to TGF-β stimulation of Myo1c-KD podocytes. P≤0.05, n=3 experimental replicates. (C) qPCR analysis showed upregulation of Myo1c and nuclear myosins isoforms NM(16) and NM(35) in response to TGF-β stimulation in a time dependent manner. *P≤0.05 vs untreated, one way ANOVA (Kruskal-Wallis test). n=3 experimental replicates. (D) Comparative qPCR analysis showed significant upregulation of Myo1c and nuclear myosin NM1(N16) in glomeruli isolated from adriamycin injured kidney. Data are presented as mean±SEM. *P≤0.05 vs. Myo1c^fl/fl, Mann-Whitney test (nonparametric test). n=3 mice glomeruli per group. (E) Myo1c-KD was performed using gene-specific shRNA and validated using qPCR. The qPCR analysis showed that expression of all Myo1c isoforms including, Myo1c, NM1(N16) and NM1(N35) was significantly reduced when compared to the control-KD cells. *P≤0.05 vs control-KD, Mann-Whitney test (nonparametric test). Data presented in mean±SEM. n=3 experimental repeats.

Figure 8:. NM1 associates with TGF-β responsive proteins GDF15 and JunB that are DE in Myo1c-KD podocytes.

(A) The heat map shows genes that associate with Myo1c from the Chip-Seq analysis and were DE upon stimulatiuon of podocytes with TGF-β. (B) qPCR analysis was performed of the genes that were upregulated upon TGF-β stimulation but were downregulated in Myo1c-KD podocytes. All experiments were performed in triplicate. Data are presented in mean±SEM and p-values were calculated using one way ANOVA (Kruskal-Wallis test). (^aaaP≤0.0001, ^aP≤0.05, control KD vs Myo1c-KD; ^bbbP≤0.0001, ^bP≤0.05, control-KD TGFβ stimulated vs Myo1c-KD TGFβ stimulated; **P≤0.001, *P≤0.05, control-KD vs control-KD TGFβ stimulated; #P≤0.05, Myo1c-KD vs Myo1c-KD TGFβ stimulated). n=3 experimental replicates. (C-D) The protein expression of GDF-15 and JUNB was evaluated by western blotting using JunB and GDF-15 antibodies (C) and quantitatively assessed by densitometric analysis of the blots (D). All experiments were performed in triplicate. Data are presented in mean±SEM and p-values were calculated using one way ANOVA (Kruskal-Wallis test). (*P≤0.05, control-KD vs control-KD TGFβ stimulated). (E) qPCR analysis to evaluate the expression of genes (that associate with NM1) in response to NTS and protamine sulphate-induced injuries in cultured human podocytes. All experiments were performed in triplicate. Data are presented in mean±SEM and p-values were calculated using one way ANOVA (Kruskal-Wallis test).*P≤0.05, compare to untreated. (F) Parafilm embedded sections of kidneys from control and FSGS patients were immunostained with ZO1 (Green) and GDF-15 (Red) antibodies and DAPI (Blue). Expression of GDF- 15 was significantly upregulated in FSGS patients glomerulus. Scale bars: 20μm. (G) NM1 directly targets GDF-15 in podocytes. ChIP-qPCR analyses for NM1 occupancy of GDF-15 promoter in podocytes. Human podocytes were subjected to ChIP using anti- NM1 antibody/IgG antibody followed by qPCR of GDF-15 promoter regions. Schematic of the three sets of GDF-15 primers from the GDF-15 promoter are presented (upper panel). The bar-graph shows NM1 enriched region in the GDF15 promoter (F1/R1), which is proximal to the transcription initiation site (lower panel). Three independent experiments were performed with similar results. *P<0.05 Mann-Whitney test (nonparametric test) 1F/1R vs IgG. Data are presented as mean±SEM.

Figure 9:. Schematic description for the role of Myo1c in podocyte pathogenesis:

Stimulation of TGF-β receptor either by injury or TGF-β induces canonical and non-canonical signaling cascades, along with NM1, which leads to the activation of TGF-β responsive genes including GDF15 in a NM1 dependent fashion. Since GDF15 can also act as a TGF-β receptor ligand, it can further regulate TGF-β signaling and fibrogenesis. Whether and how NM1 collaborates with Smads and non-Smads during TGF-β-induced EMT, needs further investigation.