Transforming growth factor β-1 stimulates profibrotic epithelial signaling to activate pericyte-myofibroblast transition in obstructive kidney fibrosis

Abstract

Pericytes have been identified as the major source of precursors of scar-producing myofibroblasts during kidney fibrosis. The underlying mechanisms triggering pericyte-myofibroblast transition are poorly understood. Transforming growth factor β-1 (TGF-β1) is well recognized as a pluripotent cytokine that drives organ fibrosis. We investigated the role of TGF-β1 in inducing profibrotic signaling from epithelial cells to activate pericyte-myofibroblast transition. Increased expression of TGF-β1 was detected predominantly in injured epithelium after unilateral ureteral obstruction, whereas downstream signaling from the TGF-β1 receptor increased in both injured epithelium and pericytes. In mice with ureteral obstruction that were treated with the pan anti-TGF-β antibody (1D11) or TGF-β receptor type I inhibitor (SB431542), kidney pericyte-myofibroblast transition was blunted. The consequence was marked attenuation of fibrosis. In addition, epithelial cell cycle G2/M arrest and production of profibrotic cytokines were both attenuated. Although TGF-β1 alone did not trigger pericyte proliferation in vitro, it robustly induced α smooth muscle actin (α-SMA). In cultured kidney epithelial cells, TGF-β1 stimulated G2/M arrest and production of profibrotic cytokines that had the capacity to stimulate proliferation and transition of pericytes to myofibroblasts. In conclusion, this study identified a novel link between injured epithelium and pericyte-myofibroblast transition through TGF-β1 during kidney fibrosis.

Figure 1

Activation of TGF-β1 signaling during obstructive kidney fibrosis. A: qPCR time course of whole-kidney TGFB1 gene transcript after UUO surgery. Expression levels were normalized by GAPDH. B: Western blot of whole kidney after UUO surgery for p-Smad2, Coll-GFP, α-SMA, and GAPDH in Coll-GFP transgenic mice. C: Confocal micrographs show Coll-GFP⁺ pericytes in normal control kidney (CON) and Coll-GFP⁺ myofibroblasts with α-SMA expression. In control kidney, α-SMA is expressed only in arterial vascular smooth muscle cells (a). D: Confocal micrographs show p-Smad2 expression in both tubular epithelial cells (arrowheads) and Coll-GFP⁺ cells (arrows) of day-4 UUO kidney, but not in control kidney. Tubular epithelial cells are indicated by the letter T. E: Quantification of cell numbers with positive nuclear p-Smad2 staining. F: Immunofluorescence micrographs show primary cultured kidney pericytes colabeled with α-SMA. G: qPCR of gene transcripts of α-SMA of primary kidney pericyte culture in the presence and absence of TGF-β1 and SB431542. Blots are representative of three independent experiments with similar results. Data are expressed as means ± SEM. n = 5 per time point (A) or 3 per group (G). ***P < 0.001 versus normal kidney at day 0 (A) or as indicated by brackets (E and G).

Figure 2

Blocking TGF-β1 signaling inhibited pericyte-myofibroblast transition. A and B: Blocking TGF-β1 signaling by pan anti–TGF-β antibody 1D11 (5 mg/kg every other day) (A) or type I TGF-β receptor (TGF-βRI) small-molecule inhibitor SB431542 (5 mg/kg every day) (B) inhibited expression of p-Smad2, Smad2, and α-SMA expression in UUO kidneys. 13C4 was administered as isotype control antibody. Lane C, control; Lane U, UUO kidney at day 4. C and D: Picrosirius Red-stained kidney sections for interstitial fibrillar collagens (red) in mice treated with control antibody13C4 or anti–TGF-β antibody 1D11 (C) or treated with vehicle (VEH) or SB431542 (D) for 10 days after UUO surgery, with morphometric quantification of fibrillar collagen from whole sagittal kidney sections. E and F: qPCR analysis showed that increased expression of collagen I(α1) (Col1α1) and transcripts of α-SMA in UUO kidney were inhibited by either 1D11 antibody (E) or SB431542 (F). G and H: Immunofluorescence detection of Coll-GFP⁺ cells in control and day-4 UUO kidneys treated with 13C4, 1D11, and SB431542 and in control kidney (G), with quantification of Coll-GFP⁺ cells (H). I and J: Confocal micrographs show Coll-GFP⁺ cells colabeled with myofibroblast marker α-SMA (Coll-GFP+α-SMA–cells are indicated by arrows, I), with quantification of the percentage of Coll-GFP⁺ cells with α-SMA expression (J). K and L: Fluorescence-activated cell sorting quantified the percentage of α-SMA⁺ cells in Coll-GFP⁺PDGFR-α⁺ cells (K) and the mean peak fluorescence of α-SMA in Coll-GFP⁺PDGFR-α⁺ cells (L) of control and UUO kidneys from mice treated with 13C4, 1D11, or SB431542. Blots (A and B) are representative of six mice per group. Data are expressed as means ± SEM. n = 6 per group (C-F, H, J); n = 3 per group (K, L). *P < 0.05, **P < 0.01. Scale bars: 25 μm (C, D, G); 20 μm (I).

Figure 3

TGF-β1 signaling stimulated cell proliferation of Coll-GFP⁺ pericytes in vivo, but not in vitro. A and B: Blocking TGF-β1 signaling by 1D11 decreased proliferation of Coll-GFP⁺ myofibroblasts in UUO kidneys. Immunofluorescence micrographs show Coll-GFP⁺ cells (arrows) colabeled with the cell proliferation marker PCNA in control or day-4 UUO kidneys (A), with quantification of the percentage of PCNA⁺ cells in all Coll-GFP⁺ cells (B). Renal tubules are indicated by the letter T. C and D: Platelet-derived growth factor (PDGF-BB), but not TGF-β1, stimulated proliferation of primary cultured kidney pericytes. Cell cycle profiles were determined by flow cytometric analysis in serum-starved cells without (Con) or with TGF-β1, PDGF-BB stimulation for 24 hours (C) or at different time points, from 8 to 48 hours (D). Data are expressed as means ± SEM. n = 6 per group (A, B); n = 3 per group (C, D). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar = 25 μm.

Figure 4

Blocking TGF-β1 signaling inhibits profibrotic phenotype of injured tubular epithelial cells. A: qPCR analysis showed that increased expression of TGFB1 and PDGFB gene transcripts in day-4 UUO kidney was inhibited by either 1D11 antibody or SB431542. B: qPCR analysis of PTECs purified from control and day-4 UUO kidneys using FACS showed that blocking TGF-β1 signaling inhibited the increased transcripts of TGF-β1 and PDGFB in UUO-injured PTECs. Data are expressed as means ± SEM. n = 6 per group (A); n = 3 per group (B). *P < 0.05, **P < 0.01.

Figure 5

Blocking TGF-β1 signaling prevents G2/M arrest of tubular epithelial cells. A: Confocal micrographs show tubular epithelial cells in cell cycle (staining with pan-cell cycle marker Ki-67-specific antibody) and in G2/M phase [staining with phosphorylation-specific antibody against histone H3 with Ser10 phosphorylation (p-H3)]. The p-H3 staining shows chromatin patterns depending on the cells in respective G2 and M phases of the cell cycle. Basement membrane nidogen staining was used to identify the tubules. Ki-67⁺p-H3⁺ tubular epithelial cells are indicated by arrows. B and C: Blocking TGF-β1 signaling by either 1D11 or SB431542 decreased tubular epithelial cells entering cell cycle (B) and the proportion of tubular epithelial cells in G2/M phase (C). Data are expressed as means ± SEM. n = 6 per group. *P < 0.05. Scale bar = 20 μm.

Figure 6

TGF-β1 stimulated profibrotic epithelial signaling to pericytes. A–C: TGF-β1 arrested nonsynchronizing PTECs in cell cycle G2/M phase. D: TGF-β1 induced profibrotic phenotype of PTECs with increased transcripts of TGF-β1 and PDGFB. E: Conditioned medium from TGF-β1–treated PTECs (TGF-β1-PTEC) increased cell number in primary kidney pericyte culture. White bars, Con-PTEC; black bars, TGF-β1-PTEC. F: Conditioned medium from TGF-β1-PTEC increased cell proliferation and transcripts of Col1a1 and α-SMA in primary kidney pericyte cultures, which were blocked by anti–PDGFR-β antibody and anti–TGF-β antibody, respectively. G: TGF-β1 increased Smad2 phosphorylation and p21 expression, but decreased p27, all of which were reversed by the TGF-βRI inhibitor SB431542. H: TGF-βRI inhibitor SB431542, but not pan c-jun NH₂-terminal kinase (JNK) inhibitor SP600125 and p38 inhibitor SB203580, reversed cell cycle G2/M arrest of TGF-β1–treated PTECs. I: SB431542 and SP600125, but not SB203580, decreased transcripts of PDGFB in TGF-β1–treated PTECs. J: TGF-β1 induced phosphorylation of p38 (p-p38) and JNK (p-JNK), but not extracellular regulated kinase (p-ERK). K: Silencing p21 reversed cell cycle G2/M arrest of TGF-β1–treated PTECs. The control was nontargeting siRNA. Data are expressed as means ± SEM. Quantification was from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 7

Schematic of TGF-β1 stimulated profibrotic epithelial signaling to pericytes during fibrotic kidney injury. Fibrotic injury induced TGF-β1 production of tubular epithelial cells. TGF-β1 then induced G2/M cell cycle arrest and profibrotic phenotype through up-regulation of p21 and activation of the JNK pathway, respectively. TGF-β1 and PDGF subsequently stimulated pericyte-myofibroblast transition through differentiation and proliferation, respectively.