Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration

Abstract

Signal transduction and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although Stat3 deletion in tubular epithelial cells is known to protect mice from fibrosis, vFoxd1 cells remains unclear. Using Foxd1-mediated Stat3 knockout mice, CRISPR, and inhibitors of STAT3, we investigate its function. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury, whereas it is expanded to interstitial cells in fibrosis in mice and humans. Foxd1-mediated deletion of Stat3 protects mice from folic-acid- and aristolochic-acid-induced kidney fibrosis. Mechanistically, STAT3 upregulates the inflammation and differentiates pericytes into myofibroblasts. STAT3 activation increases migration and profibrotic signaling in genome-edited, pericyte-like cells. Conversely, blocking Stat3 inhibits detachment, migration, and profibrotic signaling. Furthermore, STAT3 binds to the Collagen1a1 promoter in mouse kidneys and cells. Together, our study identifies a previously unknown function of STAT3 that promotes kidney fibrosis and has therapeutic value in fibrosis.

Keywords: STAT3; acute kidney injury; chronic kidney disease; fibrosis; inflammation; macrophage infiltration; myofibroblasts transformation; pericytes; stromal cells.

Figure 1.. STAT3 is expressed in tubular epithelial cells in AKI and also in interstitial cells in CKD of human and mouse kidneys

(A) pSTAT3 immunostaining (red) in healthy (n=10), AKI (Acute Tubular Necrosis (ATN), n=5), and CKD (Diabetic Nephropathy (DN), n=10) paraffin sections of human kidneys. Na⁺ K⁺ ATPase (green) for tubular staining. DAPI staining (blue) for nuclei. Images were captured on a confocal microscope using 60X objective. Scale bar=10μm. Percentage of pSTAT3-positive tubular epithelial cells normalized to total nuclei (n=5–10). Percentage of pSTAT3-positive interstitial cells normalized to total nuclei (n=5–10). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (B) STAT3 immunostaining (green) in healthy and CKD paraffin sections of human kidneys (n=10). Images were captured on a confocal microscope using 60X objective. Scale bar=10 μm. White arrows show tubular and gray arrow show interstitial staining. Scale bar=10μm. Fold STAT3 intensity as compared to healthy kidneys plotted after normalization to number of total nuclei (n=10). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (C) Co-immunostaining for α-SMA (green) and pSTAT3 (red) staining on paraffin sections of human healthy and CKD kidneys (n=10). Arrows show double-positive cells. Scale bar=10μm. Images were captured on a confocal microscope using 60X objective. Fold of α-SMA and pSTAT3 double-positive cells were plotted as compared to healthy kidneys (n=10). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (D) α-SMA (green) and pSTAT3 (red) in normal (day 0) and folic acid-induced AKI (day 2) and fibrotic (day 14) paraffin kidney sections. Double-positive cells are shown by arrows (n=5). Scale bar=10μm. Quantitation of percentage of tubular pSTAT3-positive cells normalized to total nuclei (n=5). Quantitation of percentage of interstitial pSTAT3-positive cells normalized to total nuclei (n=5). Quantitation of percentage of α-SMA and pSTAT3-positive cells (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (E) Western blotting for pSTAT3 and STAT3 in normal (day 0) kidneys and folic acid (FA)-induced AKI (day 2) and fibrotic (day 14) kidneys. Quantitation of fold change in pSTAT3 compared to normal kidneys and normalized to GAPDH loading control (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (F) Western blotting for pSTAT3 and STAT3 in normal (day 0) kidneys and aristolochic acid (AA)-induced AKI (day 4) and fibrotic (day 14) kidneys. Quantitation of fold change in pSTAT3 compared to normal kidneys and normalized to GAPDH (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (G-I) Co-immunostaining for pSTAT3 (green) and NG2 (red) or PDGFRβ (red) or α-SMA (red) respectively, in mouse OCT kidney sections 14-days post-AA treatment (n=5). White arrow indicates double-positive cells. Scale bar=10μm. Images were captured on a wide field microscope using 60X objective.

Figure 2.. Foxd1 Cre-mediated depletion of Stat3 protects mice from folic acid and aristolochic acid-induced kidney fibrosis

(A-D) BUN and sCr estimation for kidney dysfunction in FA- (A and B) or AA- (C and D) induced kidney injury in control (Stat3^fl/fl) and Stat3 KO (Foxd1^GC Stat3^fl/fl) mice, 14-days post-treatment (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (E and F) Masson Trichrome Staining (MTS) and Interstitial Fibrosis and Tubular Atrophy (IFTA) scoring following 14-days post-FA- (E) or AA- (F) treatment in control and Stat3 KO mouse kidneys (n=5). Scale bar=10μm. Average IFTA scores were plotted for normal and Stat3 KO mice, 14-days post-FA- or AA-treatment (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (G and H) Co-immunostaining for LTL (green) and α-SMA (red) and its quantitation in kidneys at 14-days post-FA- (G) or AA- (H) induced kidney fibrosis in control and Stat3 KO mice. Scale bar=10 μm. Images were captured on a confocal microscope using 60X objective. Fold change in intensity for α-SMA normalized to cell number were plotted in comparison to control mice. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (I-P) TaqMan based gene expression analysis for Col1a1, Fn1, Acta2, and Havcr1 at 14-days post-treatment with FA (I-L) or AA (M-P) for control and Stat3 KO mouse kidneys (n=4). Fold changes were plotted in comparison to control mice at day 0. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (Q) Representative histology on PAS stain focusing on glomerulus from 14-days post-AA treated control and Stat3 KO mice. Scale bar=10μm. Fold of average glomerular size in comparison to control mice (n=4 mice). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (R) Co-immunostaining for podocin (green) and Na⁺ K⁺ ATPase (red) on kidney paraffin sections of 14-days post-AA treated control and Stat3 KO mice. Scale bar=10μm. Images were captured on a confocal microscope using 60X objective. Fold change in the podocin intensity normalized to number of glomeruli in comparison to control mice (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (S) Co-immunostaining for synaptopodin (green) and α-SMA (red) on kidney paraffin sections of 14-days post-AA treated control and Stat3 KO mice. Scale bar=10μm. Images were captured on a confocal microscope using 60X objective. Fold change in synaptopodin intensity normalized to number of glomeruli in comparison to control mice (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test.

Figure 3.. Foxd1 Cre-mediated depletion of Stat3 shows decreased macrophage infiltration, inflammation, decreased DNA damage signaling, and reduced cell proliferation in fibrotic kidneys

(A) Co-immunostaining for F4/80 (green) and Na⁺ K⁺ ATPase (red) on the paraffin sections of mouse kidneys at 14-days post-AA treatment. Scale bar=10μm. Fold change in the intensity of F4/80 normalized to nuclei and plotted in comparison to control kidneys at day 14 post-AA treatment (n=4). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (B) FACS sorting strategy for macrophages from control and Stat3 KO mouse kidneys at 14-days post-AA treatment. TaqMan base quantitative RT-PCR for (C) inflammatory mediators/M1 markers and (D) M2 markers from macrophages isolated from mouse kidneys at 14-days post-AA treatment. Fold changes were plotted in comparison to control mice at 14-days post-AA treatment (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (E) Co-immunostaining of F4/80 (green) and IL-10 (red) and IL-10 (red) alone on the paraffin sections of mouse kidneys 14-days post-AA treatment. Scale bar=10μm. Fold change in the intensity of IL-10 normalized to nuclei and plotted in comparison to control kidneys at day-14 post-AA treatment (n=4). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (F) Representative immunostaining for pH2AX (green) in the control and Stat3 KO mouse paraffin kidney sections at 14-days post-AA injection. Scale bar=10μm. Images were captured on a widefield immunofluorescence microscope using 60X objective. Quantitation of interstitial and tubular pH2AX positive cells (n=4). Fold changes were calculated by normalizing the number of pH2AX cells to number of nuclei and plotted in comparison to control mice at 14-days post-AA treatment. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (G) Co-immunostaining for PDGFRβ (green) and pH2AX (red) on the paraffin sections of control mouse kidneys at 14-days post-AA treatment. Scale bar=10μm. PDGFRβ and pH2AX double-positive cells are shown by white arrows. Number of PDGFRβ and pH2AX double-positive cells were counted and plotted (n=4). (H) Immunostaining for Ki67 (cyan) in the mouse kidneys 14-days post-AA injection. Images were captured on a widefield immunofluorescence microscope using 60X objective. Quantitation of interstitial and tubular Ki67 positive cells (n=4). Scale bar=10μm. Fold changes were calculated by normalizing the number of pH2AX cells to number of nuclei and plotted in comparison to control mice at 14-days post-AA treatment. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (I) Co-immunostaining for PDGFRβ (green) and Ki67 (red) on the paraffin sections of control mouse kidneys at 14-days post-AA treatment. Scale bar=10μm. PDGFRβ and Ki67 double-positive cells are shown by white arrows. Number of PDGFRβ and Ki67 double-positive cells were counted and plotted (n=4).

Figure 4.. IL-6-mediated STAT3 phosphorylation regulates proliferation, migration, and profibrotic signaling in pericyte-like 10T1/2 cells

(A) Representative western blots for pSTAT3 and STAT3 following treatment with IL-6 or IL-6 in combination with stattic in 10T1/2 cells. Fold changes were calculated after normalization to b-Actin and plotted in comparison to control (ctrl) cells (n = 3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (B) Nuclear translocation of STAT3 in 10T1/2 cells following IL-6 and or stattic treatment. Scale bar=10μm. Intensity of pSTAT3 were normalized to total number of cells and fold change in comparison to control cells were plotted (n = 3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. Proliferation of 10T1/2 cells following IL-6 and stattic treatments using BrdU incorporation assay in a (C) time- and (D) STAT3-dependent manner (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (E) Immunofluorescence staining for Ki67 with or without IL-6 and stattic treatments in 10T1/2 cells. Scale bar=10μm. Intensity of Ki67 were normalized to total number of cells and fold change in comparison to control cells were plotted (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (F) Migration of 10T1/2 cells following IL-6 and stattic treatment using scratch assay. Scale bar=50μm. Percent cell migration was calculated as compared to gap area in the control cells at 24 hours (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (G) Transwell migration of 10T1/2 cells treated with IL-6 or IL-6 in combination with stattic. Scale bar=50μm. Cell area normalized to cell number were calculated for CellMask stain (green) and fold change was calculated as compared to control cells (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (H-K) Immunostaining for (H) pSMAD2 (green), (I) Collagen 1 (green), (J) Fibronectin (green) and (K) α-SMA (green) following IL-6 alone or in combination with stattic on 10T1/2 cells. Scale bar=10μm. Intensity of pSMAD2, Collagen 1, Fibronectin, and α-SMA were normalized with number of cells and fold change were plotted in comparison to control cells (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test.

Figure 5.. STAT3 directly regulates attachment, spreading, migration, proliferation, and profibrotic signaling in 10T1/2 cells

(A) Scheme for STAT3 activation using synergistic activation mediators (SAM) and activation mutants (STAT3-C). (B-F) Immunostaining for (B) pSTAT3 (green), (C) pSMAD2 (green), (D) Collagen 1, (E) Fibronectin, and (F) α-SMA (green) on STAT3 activated 10T1/2 cells. Scale bar=10μm. Intensity of staining for pSTAT3, pSMAD2, Collagen 1, Fibronectin, and α-SMA were normalized with cell numbers and plotted in comparison to control (ctrl) cells (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (G) Transwell migration of 10T1/2 cells in STAT3-activated cells. Scale bar=50μm. Cell area normalized to cell number was calculated by CellMask and fold change was plotted in comparison to control cells (n=3). Total number of migrated cells were counted on the lower side of the polycarbonate membrane by counting the nuclei (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (H) Proliferation of Stat3 WT and KO 10T1/2 cells as assayed by BrdU incorporation assay for 5 days. Percent change was calculated in comparison to day-1 Stat3 WT cells (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (I) Adherence assay as shown by staining for F-actin (red) of Stat3 WT and Stat3 KO 10T1/2 cells at 1-hour after plating. Scale bar=10μm. Intensity of F-actin normalized to total cell number were calculated and fold change were calculated in comparison to Stat3 WT cells (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (J) Spreading assay as shown staining for F-actin (red) of Stat3 WT and Stat3 KO 10T1/2 cells at 3-hours after plating. Scale bar=10μm. Cell area of F-actin normalized to total cell number and fold change were calculated in comparison to Stat3 WT cells (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (K) Transwell migration assay as shown by staining for F-actin (red). Scale bar=10μm. Total number of migrated cells were calculated by counting the DAPI on the lower side of the polycarbonate membrane and fold change was calculated in comparison to Stat3 WT 10T1/2 cells. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (L) FACS sorting strategy for isolating pericytes from fibrotic mouse kidneys and its quantitation. (M) TaqMan based quantitative RT-PCR for profibrotic factors in the pericytes isolated from control and Stat3 KO mice at day-14 post-AA treatment (n=3). p-values are shown above the bars as determined by two-tailed unpaired T-Test.

Figure 6.. STAT3 binds Collagen1a1 promoter in mouse kidneys and in pericytes cell line to induce profibrotic signaling

(A and B) Analysis of previously published STAT3 ChIP sequencing data showing STAT3 binding mouse Collagen1a1 and Fibronectin 1 promoter regions. Log of fold enrichment are plotted for each replicate (n=2). (C) ChIP assay using anti-STAT3 antibody in normal and 14-days post-AA treatment in the mouse kidneys to detect STAT3 binding on Collagen1a1 promoter. Fold enrichments were normalized to input and were plotted in comparison to IgG at day 0 samples (n=3). Rabbit IgG and RNA polymerase (RP) antibodies are negative and positive controls. p-values are shown above the bars as determined by two-tailed unpaired T-Test. (D) ChIP assay using anti-STAT3 antibody in 10T1/2 cells with activated STAT3 to detect STAT3 binding on Collagen1a1 promoter. Fold enrichments were normalized to input and fold change were plotted in comparison to control cells (n=5). p-values are shown above the bars as determined by two-tailed unpaired T-Test. (E and F) Luciferase assay to detect STAT3 binding on STAT3 consensus sequence in (E) Stat3 KO and (F) STAT3-activated 10T1/2 cells. RLU values of firefly luciferase (STAT3 luciferase) was normalized to renilla luciferase (GAPDH luciferase) and fold changes were calculated in comparison to Stat3 WT or Ctrl cells in Figures 6E and 6F respectively. p-values are shown above the bars as determined by two-tailed unpaired T-Test.