Volume 21, Issue 2, Pages 388-398 (February 2013) Smad7 suppresses renal fibrosis via altering expression of TGF-β/Smad3-regulated microRNAs  Arthur C.K. Chung, Yuan Dong, Weiqin Yang, Xiang Zhong, Rong Li, Hui Y. Lan  Molecular Therapy  Volume 21, Issue 2, Pages 388-398 (February 2013) DOI: 10.1038/mt.2012.251 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Delivery of Smad7 overexpression plasmids attenuates renal fibrosis and the expression levels of fibrotic markers in mice. (a) Histology and immunohistochemistry. (b) Quantitative analysis of immunohistochemical staining. (c) Representative western blots. Expression of collagen I, α-SMA, and phosphorylated Smad3 (P-Smad3) in ligated kidneys increases at day 7 after unilateral ureteral obstruction (UUO) but reduces after gene transfer of Smad7 overexpression plasmids (S7 plasmid). Note that the expression levels of total Smad3 (T-Smad3) are not affected during fibrosis. Owing to the cross-reactivity of the antibody, the lower bands are total Smad3 while the upper bands are total Smad2 (T-Smad2). Each bar represents the mean + SEM for at least eight mice. **P < 0.01, ***P < 0.001 as compared with normal mice; ##P < 0.01, ###P < 0.001 as compared with UUO mice with control plasmids (Ctl plasmid). α-SMA, α-smooth muscle actin. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Smad7 overexpression attenuates the expression levels of fibrotic markers and alters the expression levels of fibrosis-related microRNAs. (a) Real-time PCR of expression of fibrotic markers in the unilateral ureteral obstruction (UUO) model. Expression of collagen I, fibronectin (Fn), and α-smooth muscle actin (α-SMA) in ligated kidneys increases after UUO but reduces after gene transfer of Smad7 overexpression plasmids (S7 plasmid). (b) Real-time PCR of expression of renal expression of miR-29b, miR-192, and miR-21 in the UUO model. (c–e) The combination of immunohistochemical staining with the anti-Smad7 antibody (red) and in situ hybridization with FITC-miR-29b, miR-192, and miR-21 (green). At Day7 of UUO, expression levels of miR-192 and miR-21 are elevated and miR-29b expression is reduced. During UUO, endogenous Smad7 expression is reduced in ligated kidneys. Smad7 overexpression restores miR-29b expression but abolishes expression of miR-21 and miR-192. No signal was observed when a scramble microRNA probe was used (data not shown). Each bar represents the mean + SEM for at least eight mice. ***P < 0.001 as compared with normal mice; ##P < 0.01, ###P < 0.001 as compared with UUO mice with control plasmids (Ctl plasmid). Ctl: control plasmid; S7: Smad7 overexpression plasmid. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Overexpression of Smad7 reduces transforming growth factor-β1 (TGF-β1)-induced expression of fibrotic markers and alters expression levels of miR-29b, miR-21, and miR-192 in rat mesangial cells (MCs) and tubular epithelial cells (TECs). (a) Representative western blots of expression of fibrotic markers in TECs at 24 hour after treatment with TGF-β1. (b) Representative western blots of expression of fibrotic markers in MCs at 24 hour after treatment with TGF-β1. (c) Real-time PCR analysis of expression of fibrotic markers in TECs. (d) Real-time PCR analysis of expression of fibrotic markers in MCs. (e) Real-time PCR analysis of microRNA expression in TECs. (f) Real-time PCR analysis of microRNA expression in MCs. Overexpression of Smad7 in MCs and TECs suppresses mRNA and protein expression of collagen I (Col I), fibronectin (Fn), and α-smooth muscle actin (α-SMA) induced by TGF-β1 after Dox treatment. MCs and TECs transfected with Smad7 overexpression plasmids restores miR-29b expression, and inhibits the induction of miR-21 and miR-192 by TGF-β1. Each bar represents the mean + SEM for at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with time 0; #P < 0.05, ##P < 0.01, ###P < 0.001 as compared with without Dox treatment. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Overexpression of Smad7 restores miR-29b expression, and suppresses the induction of miR-21 and miR-192 after advanced glycated end products (AGE) and angiotensin II (AngII) treatments in rat mesangial cells (MCs) and tubular epithelial cells (TECs). (a) Real-time PCR analysis of microRNA expression in TECs after treatment of AGE (33 µg/ml). (b) Real-time PCR analysis of microRNA expression in MCs after treatment of AGE (33 µg/ml). (c) Real-time PCR analysis of microRNA expression in TECs after treatment of AngII (1.0 µmol/l). (d) Real-time PCR analysis of microRNA expression in MCs after treatment of AngII (1.0 µmol/l). Treatment with Ang II and AGE in MCs and TECs induce expression of miR-21 and miR-192 but suppress miR-29b. In MCs and TECs transfected with Smad7 overexpression plasmids restores miR-29b expression, and inhibits the induction of miR-21 and miR-192 by AGE and AngII. No effect is observed when BSA control or no AngII is used in the absence of Dox. BSA, bovine serum albumin (33 µg/ml) as a control of AGE treatment without Dox treatment. No AngII, absence of AngII as a control of AngII treatment without Dox treatment. Each bar represents the mean + SEM for at least 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with time 0; #P < 0.05, ##P < 0.01, ###P < 0.001 as compared with no Dox treatment. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Disruption of Smad7 gene in mice promotes renal fibrosis by increasing renal collagen I deposition and altering the expression of miR-21 and miR-29b at day 7 after unilateral ureteral obstruction (UUO). (a) Immunohistochemical analysis of collagen I. (i) A normal kidney from an untreated wild-type (WT) mouse, (ii) a normal kidney from an untreated Smad7ΔE1 mouse, (iii) a UUO kidney from a WT mouse, (iv) a UUO kidney from a Smad7ΔE1 mouse. (b) Real-time PCR results of miR-21 and miR-29b expression in Smad7 WT/ΔE1 kidneys with or without UUO at day 7. Note that both untreated WT and Smad7ΔE1 mice show little accumulation of collagen I within the interstitium (i and ii), but severe renal fibrosis with an abundant collagen I accumulation within the tubulointerstitium is developed in WT mice with UUO (iii), which is significantly enhanced in Smad7ΔE1 mice (iv). The slides were counterstained with hematoxylin for the nuclei. Each bar represents the mean + SEM for a group of eight mice. **P < 0.01, ***P < 0.001 as compared with the normal mice. ##P < 0.01 as compared with the WT mice with UUO. Magnification ×400. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Delivery of miR-192 mimic re-induces transforming growth factor-β1 (TGF-β1)-induced expression of fibrotic markers in Smad7 overexpressing tubular epithelial cells (TECs). (a) Real-time PCR analysis of miR-192 in TECs at 24 hours after TGF-β1 treatment (i) with all samples (ii) without miR-192 mimic. (b) Representative western blots of fibrotic markers. (c) Quantitative analysis of western blots. (d) Real-time PCR analysis of fibrotic markers. Note that (a-i) TECs transfected with miR-192 mimics contain an excessive amount of the mature form of miR-192. (a-ii) TECs transfected with Smad7 overexpression plasmids show a reduction of TGF-β1-induced miR-192 expression. After treatment with TGF-β1 for 24 hours, overexpression of Smad7 in TECs does not totally suppress TGF-β1-induced expression of collagen I (Col I), fibronectin (Fn), and α-SMA after the delivery of miR-192 mimic. Each bar represents the mean + SEM for at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with time 0; #P < 0.05, ##P < 0.01, ###P < 0.001 as compared with no delivery of miR-192 mimic. ††P < 0.01, †††P < 0.001 as compared with no Dox treatment. SCR, scramble microRNA; α-SMA, α-smooth muscle actin. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Delivery of miR-21 mimic re-induces transforming growth factor-β1 (TGF-β1)-induced expression of fibrotic markers in Smad7 overexpressing tubular epithelial cells (TECs). (a) Real-time PCR analysis of miR-21 in TECs at 24 hours after TGF-β1 treatment (i) with all samples (ii) without miR-21 mimic only. (b) Representative western blots of fibrotic markers. (c) Quantitative analysis of western blots. (d) Real-time PCR analysis of fibrotic markers. Note that (a-i) TECs transfected with miR-21 mimics contain an excessive amount of the mature form of miR-21. (a-ii) TECs transfected with Smad7 overexpression plasmids show a reduction of TGF-β1-induced miR-21 expression. After treatment with TGF-β1 for 24 hours, overexpression of Smad7 in TECs partly suppresses TGF-β1-induced expression of collagen I (Col I), fibronectin (Fn), and α-SMA after the delivery of miR-21 mimic. Each bar represents the mean + SEM for at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with time 0; #P < 0.05, ##P < 0.01, ###P < 0.001 as compared with no delivery of miR-21 mimic. ††P < 0.01, †††P < 0.001 as compared with no Dox treatment. SCR: scramble microRNA; α-SMA, α-smooth muscle actin. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Delivery of anti-miR-29b re-induces transforming growth factor-β1 (TGF-β1)-induced expression of fibrotic markers in Smad7 overexpressing tubular epithelial cells (TECs). (a) Real-time PCR analysis of miR-29b in TECs at 24 hours after TGF-β1 treatment (i) with all samples (ii) without anti-miR-29b only. (b) Representative western blots of fibrotic markers. (c) Quantitative analysis of western blots. (d) Real-time PCR analysis of fibrotic markers. Note that (a–i) TECs transfected with anti-miR-29b suppresses the expression of the mature form of miR-29b. TECs transfected with Smad7 overexpression plasmids show a restoration of TGF-β1-suppresed miR-29b expression. After treatment with TGF-β1 for 24 hours, overexpression of Smad7 in TECs partly suppresses TGF-β1-induced expression of collagen I (Col I), fibronectin (Fn), and α-SMA after the delivery of anti-miR-29b. Each bar represents the mean + SEM for at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with time 0; #P < 0.05, ##P < 0.01, ###P < 0.001 as compared with no delivery of miR-21 mimic. ††P < 0.01, †††P < 0.001 as compared with no Dox treatment. SCR: scramble microRNA; α-SMA, α-smooth muscle actin. Molecular Therapy 2013 21, 388-398DOI: (10.1038/mt.2012.251) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions