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. 2015 Aug 3;125(8):2935-51.
doi: 10.1172/JCI74929. Epub 2015 Jul 20.

Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis

Rafael Kramann, Susanne V Fleig, Rebekka K Schneider, Steven L Fabian, Derek P DiRocco, Omar Maarouf, Janewit Wongboonsin, Yoichiro Ikeda, Dirk Heckl, Steven L Chang, Helmut G Rennke, Sushrut S Waikar, Benjamin D Humphreys

Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis

Rafael Kramann et al. J Clin Invest. .

Abstract

Chronic kidney disease is characterized by interstitial fibrosis and proliferation of scar-secreting myofibroblasts, ultimately leading to end-stage renal disease. The hedgehog (Hh) pathway transcriptional effectors GLI1 and GLI2 are expressed in myofibroblast progenitors; however, the role of these effectors during fibrogenesis is poorly understood. Here, we demonstrated that GLI2, but not GLI1, drives myofibroblast cell-cycle progression in cultured mesenchymal stem cell-like progenitors. In animals exposed to unilateral ureteral obstruction, Hh pathway suppression by expression of the GLI3 repressor in GLI1+ myofibroblast progenitors limited kidney fibrosis. Myofibroblast-specific deletion of Gli2, but not Gli1, also limited kidney fibrosis, and induction of myofibroblast-specific cell-cycle arrest mediated this inhibition. Pharmacologic targeting of this pathway with darinaparsin, an arsenical in clinical trials, reduced fibrosis through reduction of GLI2 protein levels and subsequent cell-cycle arrest in myofibroblasts. GLI2 overexpression rescued the cell-cycle effect of darinaparsin in vitro. While darinaparsin ameliorated fibrosis in WT and Gli1-KO mice, it was not effective in conditional Gli2-KO mice, supporting GLI2 as a direct darinaparsin target. The GLI inhibitor GANT61 also reduced fibrosis in mice. Finally, GLI1 and GLI2 were upregulated in the kidneys of patients with high-grade fibrosis. Together, these data indicate that GLI inhibition has potential as a therapeutic strategy to limit myofibroblast proliferation in kidney fibrosis.

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Figures

Figure 8
Figure 8. Upregulation of GLI in human kidney fibrosis.
(A and B) Human kidney tissue was obtained from tumor nephrectomy specimens from 10 patients and scored by an experienced kidney pathologist for the degree of interstitial fibrosis. (A) Representative images of trichrome and periodic acid-Schiff–stained (PAS-stained) sections of specimens assigned to the high-grade versus low-grade fibrosis groups. (B) Four specimens were assigned on the basis of fibrosis severity to the high-grade fibrosis group (interstitial fibrosis >40%) and 6 specimens to the low-grade fibrosis group (interstitial fibrosis <20%). Clinical data on the patients are provided in Supplemental Table 3. (C) qRT-PCR analysis indicated significantly higher mRNA expression levels of the fibrotic readouts COL1A1, FN, and ACTA2 in the high-grade fibrosis group compared with levels in the low-grade fibrosis group, as expected. (D) qRT-PCR analysis indicated a significant upregulation of GLI1, GLI2, and PTCH1 mRNA levels in the high-grade fibrosis group compared with that detected in the low-grade fibrosis group. P values were calculated by t test. Data represent the mean �� SEM. Scale bars: 100 μm.
Figure 7
Figure 7. Targeting of GLI proteins by GANT61 ameliorates renal fibrosis following UUO.
(A) To test whether specific inhibition of GLI ameliorates renal fibrosis, 8- to 10-week-old male WT mice on a C57BL/6J background were subjected to UUO surgery and treated on the indicated days (arrows) with either GANT61 (n = 5) or vehicle (ethanol/corn oil, 1:4; n = 6) (50 mg/kg BW, s.c.). (B and C) Trichrome staining and scoring for interstitial fibrosis in UUO kidneys revealed significantly less fibrosis in GANT61-treated mice. (D–F) Determination of mRNA levels revealed significantly lower expression levels of the fibrotic readouts Col1a1 (D), Acta2 (E), and fibronectin (Fn) (F) in the UUO kidneys of GANT61-treated animals when compared with those of the vehicle-treated animals. (G–L) Representative Western blots and quantification by IOD for fibronectin, α-SMA, GLI1, and GLI2 from whole UUO kidney lysates, demonstrating reduced GLI1 and GLI2 protein expression levels as well as reduced fibrotic readouts. (M and N) GANT61 treatment resulted in a significant reduction in Gli1 and Ptch1 mRNA levels following UUO. *P < 0.05, **P < 0.01, and ***P < 0.001, by t test. Data represent the mean ± SEM. Scale bars: 100 μm.
Figure 6
Figure 6. Darinaparsin induces myofibroblast-specific cell-cycle arrest, but does not affect the cell cycle of tubular epithelial cells.
Eight- to ten-week-old WT male mice on a C57BL/6J background were treated with darinaparsin (50 mg/kg, n = 6) or vehicle (n = 6) starting 2 days before UUO surgery and sacrificed on day 3 after surgery. BrDU was injected (100 mg/kg) 3 hours prior to sacrifice. (A) Representative images of kidney sections from UUO and noninjured CLKs costained for Ki67, BrdU, and α-SMA, demonstrating reduced proliferation of interstitial myofibroblasts in UUO kidneys of the darinaparsin-treated group compared with the vehicle-treated group. (Quantification of Ki67+ cells is shown in Supplemental Figure 14.) (B–D) Costaining of sections from noninjured CLKs and UUO kidneys for BrDU (cells in S phase), p-H3 (G2/M phase), and α-SMA allowed quantification of the cell-cycle stages for interstitial myofibroblasts (α-SMA+) and tubular epithelial cells. Darinaparsin treatment resulted in a specific G0/G1 cell-cycle arrest of interstitial myofibroblasts in UUO kidneys (C), whereas the cell-cycle distribution of tubular epithelial cells was not effected (D). (A similar analysis was performed in mice treated from day 2 until day 10 after UUO and is shown in Supplemental Figures 15 and 16). (E and F) Representative Western blots and quantification by IOD of whole UUO kidney lysates for the cyclin-dependent kinase inhibitor p21/CIP1 and p-Rb (Western blots for noninjured CLKs are shown in Supplemental Figure 16). *P < 0.05 by 1-way ANOVA, followed by Bonferroni’s post-hoc test (C); **P < 0.01 by t test (F). Data represent the mean ± SEM. Scale bars: 60 μm.
Figure 5
Figure 5. Darinaparsin treatment ameliorates renal interstitial fibrosis after UUO and AKI-to-CKD progression after severe IRI.
(A) WT mice (8- to 10-week-old males on a C57BL/6J background) were treated with darinaparsin (50 mg/kg, n = 9) or vehicle (normal saline, n = 7), as indicated, underwent UUO surgery and were sacrificed on day 10 after surgery. (B) Representative Western blots of whole UUO kidney lysates for GLI1 and GLI2 (Western blots for noninjured CLKs and quantification by IOD are shown in Supplemental Figure 10). (C and D) Representative trichrome-stained and α-SMA–immunostained UUO kidneys. (E and F) Quantification of interstitial fibrosis and α-SMA+ surface area. (G) Representative Western blot of whole UUO kidney lysates for fibronectin and α-SMA (Western blots for noninjured CLKs and quantification by IOD are shown in Supplemental Figure 11). (H) WT mice (n = 19 male 8- to 10-week-old mice on a C57BL background) underwent severe bilateral IRI and were randomized, on the basis of their day-1 and day-7 BUN levels (Supplemental Figure 12), to darinaparsin or vehicle treatment. (I) BUN measurement at randomization (day 7) and after 14 or 21 days of treatment (BUN levels at baseline and on days 1 and 14, and BW and creatinine data are shown in Supplemental Figure 12). (J) Representative images of α-SMA–immunostained kidneys after IRI. (K) Quantification of interstitial fibrosis (n = 11, darinaparsin; n = 8, vehicle). (L) Relative mRNA expression for Col1a1 and Acta2 (n = 11, darinaparsin; n = 8, vehicle). *P < 0.05,**P < 0.01, and ***P < 0.0 01 versus vehicle-treated mice, by t test. Data represent the mean ± SEM. Scale bars: 50 μm.
Figure 4
Figure 4. Darinaparsin reduces GLI protein levels and induces cell-cycle arrest in vitro, while overexpression of GLI2 rescues this cell-cycle effect of darinaparsin.
(A and B) Darinaparsin induced G0/G1 cell-cycle arrest in the mouse MSC-like, pericyte-like 10T1/2 cell line (BrDU, S phase; 7AAD, 7-aminoactinomycin, DNA content). (C) Representative Western blots of whole-cell lysate from 10T1/2 cells treated with darinaparsin or vehicle in the presence or absence of Shh. Quantification of Western blot analysis for p21 and p-Rb (3 biological replicates) is shown in Supplemental Figure 8. (D and E) Quantification by IOD showing reduced GLI1 and GLI2 protein levels after treatment with with darinaparsin (data are from 2 pooled experiments, with a total of 3 biological replicates). (F and G) qRT-PCR analysis of mRNA expression of Gli1 and Gli2 in 10T1/2 cells after treatment with vehicle or darinaparsin in the presence or absence of Shh (n = 3 replicates). (H and I) Retroviral expression of GLI2 rescued the cell-cycle effect of darinaparsin and drove proliferation of 10T1/2 cells (n = 3 biological replicates; gating is shown in Supplemental Figure 2). (J) 293T cells were transfected with full-length GLI2-myc, cotransfected with GFP-myc as a control protein, and treated with darinaparsin or vehicle. GST agarose beads were added to the cell lysate to bind the glutathione moiety of darinaparsin. The immunoblot for myc suggests that in the presence of darinaparsin, GST is able to pull down GLI2 (myc), indicating darinaparsin binding to GLI2 (IP + DAR). Importantly, the GFP-myc control protein was not detectable, indicating the specificity of the IP. *P < 0.05, **P < 0.01, and ***P < 0.001, by t test (B, D, and F) and by 2-way ANOVA followed by Bonferroni’s post-hoc test (I). Data represent the mean ± SEM. CV, control virus; DAR, darinaparsin; Veh, vehicle.
Figure 3
Figure 3. Conditional KO of Gli2 or overexpression of the GLI3 repressor in GLI1+ cells induces myofibroblast-specific cell-cycle arrest.
(A) WT littermates, Gli1-KO, Gli2-KO, Gli1/2-KO, and Gli3T mice (n = 5/group, 3 males and 2 females/group; mice were on a 129S-C57BL/6J mixed background and were 8–10 weeks of age) received tamoxifen, underwent UUO surgery, and were euthanized on day 3 following surgery. BrdU was administered 3 hours before sacrifice. Representative images of UUO kidneys after costaining for BrdU (S phase), p-H3 (G2/M phase), and α-SMA/myofibroblasts (representative images and quantification of Ki67+ cells are shown in Supplemental Figures 6 and 7). Scale bars: 50 μm, 25 μm (insets). (B) Cell counting and quantification of myofibroblast cell cycle (S phase = α-SMA+/BrdU+; G2/M phase = α-SMA+/p-H3+; G0/G1 phase = α-SMA+ – α-SMA+/BrdU+ – α-SMA+/p-H3+). (C) Cell counting and quantification of tubular epithelial cell cycle (S phase = tubular epithelial cells [TE] – TE/BrdU+; G2/M phase = TE/p-H3+; G0/G1 phase = TE – TE/BrdU+ – TE/p-H3+). (D) Western blot of whole UUO kidney lysates for p-Rb and p21. (E and F) Quantification of Western blots for p-Rb and p21 by IOD. Representative Western blots of noninjured CLKs are shown in Supplemental Figure 7C. *P < 0.05 and **P < 0.001 versus WT; #P < 0.05 and ##P < 0.001 versus Gli1-KO, by 1-way ANOVA followed by Bonferroni’s post-hoc test. Data represent the mean ± SEM. hpf, high-power field.
Figure 2
Figure 2. Conditional KO of Gli2 or overexpression of the GLI3 repressor in GLI1+ cells ameliorates kidney fibrosis following UUO.
(A) WT littermates (n = 9, 5 males), Gli1-KO (Gli1CreERt2/CreERt2 Gli2+/+; n = 5, 3 males), conditional Gli2-KO (Gli1CreERt2/+ Gli2fl/fl; n = 12, 7 males), Gli1/2-KO (Gli1CreERt2/CreERt2 Gli2fl/fl; n = 9, 5 males), or Gli3T (Gli1CreERt2/+ Rosa26-Gli3T; n = 6, 4 males) mice were injected with tamoxifen, underwent UUO surgery as indicated, and were sacrificed 10 days after UUO. All Tg mice underwent surgery at 8 to 10 weeks of age and can be considered as being on a mixed C57BL/6J and 129S background. WT controls were littermates of the Tg mice used in the experiments. (B and C) qRT-PCR analysis of Gli1 and Gli2 mRNA levels in whole-kidney lysates from conditional-KO experiments. (D) Western blots for GLI1 effector proteins and fibrotic readouts fibronectin and α-SMA in UUO kidneys. Representative Western blots for the CLKs and quantification by integrated OD (IOD) are presented in Supplemental Figure 4. (E) qRT-PCR analysis of mRNA expression of fibrotic readouts α-SMA (Acta2) and collagenIαI (Col1a1). (F) Representative images of trichrome- or α-SMA–stained UUO kidneys on day 10 after surgery. (G and H) Quantification of interstitial fibrosis and α-SMA+ surface area. *P < 0.05, **P < 0.01, and ***P < 0.001 versus WT and #P < 0.05, ##P < 0.01, and ###P < 0.001 versus Gli1-KO, by 1-way ANOVA followed by Bonferroni’s post-hoc test. Data represent the mean ± SEM.
Figure 1
Figure 1. Lowering GLI2, but not GLI1, levels by RNAi induces cell-cycle arrest of MSC-like cells in vitro.
(A and B) mRNA and protein levels of GLI1 and GLI2 in 10T1/2 cells treated with siRNA, as indicated. Shh was added to the medium to increase GLI protein levels for better detection (n = 3 biological replicates). Quantification of Western blot data from 3 replicates is shown in Supplemental Figure 1. (C and D) Representative flow cytometric cell-cycle plots and quantification of 10T1/2 cells after siRNA-mediated knockdown of GLI1, or GLI2, or both GLI1 and GLI2, compared with scrambled siRNA (n = 3 replicates). (E) Representative Western blot of cell-cycle regulators at the G1 restriction point p-Rb and p21 following knockdown of GLI1, GLI2, or GLI1/2. Quantification of Western blot data from 3 replicates is shown in Supplemental Figure 1. (F–I) Overexpression of GLI2 by retroviral delivery activates the Hh pathway, with increased downstream expression of GLI1 and PTCH1. *P < 0.05, **P < 0.01, and ***P < 0.001, by t test (A) or 1-way ANOVA followed by Bonferroni’ post-hoc test (D). Data represent the mean ± SEM.
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