Volume 84, Issue 6, Pages 1129-1144 (December 2013) The microRNA miR-433 promotes renal fibrosis by amplifying the TGF-β/Smad3-Azin1 pathway  Rong Li, Arthur C.K. Chung, Yuan Dong, Weiqin Yang, Xiang Zhong, Hui Y. Lan  Kidney International  Volume 84, Issue 6, Pages 1129-1144 (December 2013) DOI: 10.1038/ki.2013.272 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 MiR-433 is an important mediator of TGF-β-induced renal fibrosis. (a) Real-time PCR analysis demonstrates that renal miR-433 is elevated in UUO kidneys, and deletion of Smad3 in mice blocks miR-433 expression in UUO kidneys. (b) Real-time PCR analysis demonstrates that renal miR-433 is elevated in anti-GBM glomerulonephritis (day 14) in WT mice, and deletion of Smad3 in mice blocks the elevation of renal miR-433. (c) Real-time PCR analysis demonstrates that renal miR-433 is also elevated in diseased kidneys at 4 weeks (4W) after injection of adriamycin. (d) Real-time PCR shows that TGF-β1 (2ng/ml), not the control, induces miR-433 expression in a time-dependent manner. (e) Real-time PCR demonstrates that TGF-β1 induces miR-433 expression at 12h in a dose-dependent manner, being significant at 2.0ng/ml. The induction of miR-433 expression by TGF-β1 is inhibited by a neutralizing TGF-β1 antibody (A-T-Ab), but not by an isotype control antibody (Ctl-Ab). (f) Real-time PCR analysis shows that miR-433 expression is suppressed in TECs stably expressed with miR-433 shRNA, and miR-433 expression is enhanced in TECs stably expressed with miR-433 expression plasmid. (g) Representative western blots demonstrate that knockdown (KD) of miR-433 (miR-433 KD) in TECs suppressed protein expression of collagen I, fibronectin, and α-SMA induced by TGF-β1. (h) Real-time PCR analysis shows that TECs stably expressed with miR-433 shRNA plasmid suppress TGF-β1-induced expression of miR-433, collagen I, fibronectin, and α-SMA. (i) Representative western blots demonstrate that overexpression (OE) of miR-433 (miR-433 OE) in TECs enhances protein expression of collagen I, fibronectin, and α-SMA induced by TGF-β1. (j) Real-time PCR analysis shows that TECs stably expressed with miR-433 expression plasmid enhance TGF-β1-induced expression of miR-433, collagen I, fibronectin, and α-SMA. Each bar represents the mean±s.e.m. for at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 compared with time 0 (or dosage 0), or with normal WT mice; #P<0.05, ##P<0.01, ###P<0.001 compared with control, or normal TEC, or WT mice with treatment, or as indicated. Col I, collagen I; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GBM, glomerular basement membrane; shRNA, small hairpin RNA; α-SMA, α-smooth muscle actin; TEC, tubular epithelial cell; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction; WT, wild type. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Delivery of miR-433 shRNA plasmids attenuates renal fibrosis in mice. (a) Schematic diagram of the experimental approach. (b) Real-time PCR results show that TGF-β1 expression in ligated kidneys increases at day 7 after UUO but reduces after gene transfer of miR-433 shRNA plasmids. (c) Real-time PCR results show that miR-433 expression in ligated kidneys increases at day 7 (7d) after UUO but reduces after gene transfer of miR-433 shRNA plasmids. (d) In situ hybridization shows that miR-433 is upregulated in the UUO kidney, and its expression is remarkably reduced in the UUO kidney with ultrasound-mediated miR-433 gene therapy. (e) Histology, Masson’s trichrome staining, and immunohistochemistry. Results from histology and immunohistochemistry demonstrate that renal fibrosis is ameliorated in mice treated with shRNA plasmids (KD), as evidenced by reduction of the expression of collagen I in day 7 UUO kidneys. Masson’s trichrome staining reveals that suppression of miR-433 significantly inhibits collagen matrix deposition in the obstructed kidney. In contrast, Azin1 is downregulated in day 7 UUO kidneys, but partially restored after treatment with miR-433 shRNA plasmid. Each bar represents the mean±s.e.m. for at least eight mice. **P<0.01, ***P<0.001 compared with normal mice; ##P<0.01, ###P<0.001 as indicated. Azin1, antizyme inhibitor 1; Col I, collagen I; Ctl, control plasmid; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KD, miR-433 knockdown plasmid; PAS, periodic acid–Schiff; Scramble, Scramble microRNA; shRNA, small hairpin RNA; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction. Original magnification × 200. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Delivery of miR-433 shRNA plasmids reduces the expression of fibrotic markers in ligated kidneys. (a) Real-time PCR, (b) representative western blots, and (c) quantitative analysis of western blots. Expression of collagen I, fibronectin, and α-SMA in ligated kidneys increases at day 7 (7d) after UUO but reduces after gene transfer of miR-433 shRNA plasmid. In contrast, Azin1 is downregulated in day 7 UUO kidneys, but it is partially restored after treatment with miR-433 shRNA plasmid. Each bar represents the mean±s.e.m. for at least eight mice. **P<0.01, ***P<0.001 compared with normal mice; #P<0.05, ##P<0.01, ###P<0.001 as indicated. Azin1, antizyme inhibitor 1; Col I, collagen I; Ctl, control plasmid; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KD, miR-433 knockdown plasmid; shRNA, small hairpin RNA; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Delivery of miR-433 shRNA plasmids holds the progression of renal fibrosis in mice. (a) Schematic diagram of the experimental approach. (b) Real-time PCR results show that TGF-β1 expression in ligated kidneys increases at day 10 after UUO but reduces after gene transfer of miR-433 shRNA plasmid. (c) Real-time PCR results show that miR-433 expression in ligated kidneys increases at day 10 (10d) after UUO but reduces after gene transfer of miR-433 shRNA plasmids. (d) Histology, Masson’s trichrome staining, and immunohistochemistry. Results from histology and immunohistochemistry demonstrate that renal fibrosis is ameliorated in mice treated with miR-433 shRNA as evidenced by the reduction of the expression of collagen I, fibronectin, and α-SMA in day 10 UUO kidneys. Masson’s trichrome staining reveals that suppression of miR-433 significantly reduces collagen matrix deposition in day 10 UUO kidneys. In contrast, Azin1 is downregulated in day 10 UUO kidneys but partially restored after treatment with miR-433 shRNA plasmid. Each bar represents the mean±s.e.m. for at least eight mice. **P<0.01, ***P<0.001 compared with normal mice; ##P<0.01, ###P<0.001 as indicated; †††P<0.001 as indicated. Azin1, antizyme inhibitor 1; Col I, collagen I; Ctl, control plasmid; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KD, miR-433 knockdown plasmid; PAS, periodic acid–Schiff; shRNA, small hairpin RNA; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction. Original magnification × 200. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Delivery of miR-433 shRNA plasmids reduces the expression of fibrotic markers in ligated kidneys. (a) Real-time PCR, (b) representative western blots, and (c) quantitative analysis of western blots. Expression of collagen I, fibronectin, and α-SMA in ligated kidneys increases at day 10 (10d) after UUO but reduces after gene transfer of miR-433 shRNA plasmid. In contrast, Azin1 is downregulated in day 10 UUO kidneys, but it is partially restored after treatment with miR-433 shRNA plasmid. Each bar represents the mean±s.e.m. for at least eight mice. *P<0.05, **P<0.01, ***P<0.001 compared with normal mice; #P<0.05, ##P<0.01, ###P<0.001 as indicated; ††P<0.01 as indicated. Azin1, antizyme inhibitor 1; Col I, collagen I; Ctl, control plasmid; Fn, fibronectin; KD, miR-433 knockdown plasmid; shRNA, small hairpin RNA; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Differential mediation of Smads on miR-433 expression during renal fibrosis. (a) Real-time PCR analysis of miR-433 expression. (ai) Overexpression of Smad7 in TECs inhibits the induction of miR-433 by TGF-β1. (aii) Delivery of Smad7 overexpression plasmids into UUO kidneys abolishes the expression of miR-433. (aiii) Disruption of Smad7 gene in mice promotes the expression of miR-433 at day 7 after UUO. (b) Real-time PCR analysis of miR-433 expression. (bi) Real-time PCR analysis demonstrates that knockdown of Smad3 in TECs blocks TGF-β-induced miR-433 expression, whereas Smad2 knockdown promotes miR-433 expression. (bii) MiR-433 expression is enhanced in Smad2 knockout (KO) UUO kidneys. (ci) Sequence analysis by rVista 2.0 program shows the degree of sequence conservation of miR-433 promoter and reveals two conserved SBSs located at -1845 to -582bp from the miR-433 mature sequence in human, rat, and mouse genomes. (cii) DNA sequence alignments of two potential Smad-binding sites (SBS1 and SBS2). Bold and underlined sequences indicate the location of SBSs and italics indicate the mismatches. (d) Schematic representation of mouse miR-433 promoter reporter constructs (left) and analysis of their reporter activities (middle, right). (i) After treatment with TGF-β1 for 24h, firefly luciferase (Luc) reporter activities are induced when the constructs contain the SBS2. (ii) Mutation of the SBS2 abolishes the TGF-β1-induced luciferase reporter activities. (e) ChIP assays for Smad3 were performed with chromatin from TEC and MEF cells treated with TGF-β1. Precipitated DNA was amplified with oligonucleotides spanning regions of SBSs. Total inputs are indicated. Each bar represents the mean±s.e.m. for at least three independent experiments or at least five mice. *P<0.05, **P<0.01, ***P<0.001 compared with either time 0, or in the absence of TGF-β1, or with normal mice; #P<0.05, ##P<0.01, ###P<0.001 compared with the TGF-β1-treated samples of normal TECs, with the kidneys from WT-UUO, or with delivery of Smad7 (S7) plasmids, or with ff-UUO, or as indicated. Ab, antibody; ChIP, chromatin immunoprecipitation; Ctl, control plasmids; Cre, cause recombination; Dox, doxycycline; EV, empty vector; ff, flox/flox; FL, full length of the promoter of miR-433 (-2840 to -65bp); IgG, immunoglobulin G; MEF, mouse embryonic fibroblast; Mut, mutated; SBS, Smad-binding site; TEC, tubular epithelial cell; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction WT, wild type. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Azin1 may be a target of miR-433 during renal fibrosis. (a) Schematic diagrams of numbers of target genes obtained from four microRNA target prediction programs (TargetScan, miRDB, miRanda, and Diana Lab) and a list of five potential targets. (b) Representative western blots and (c) real-time PCR assays show that Azin1 expression in TECs is reduced after TGF-β1 treatment. (d) Knockdown (KD) of miR-433 in TECs restores Azin1 expression, but overexpression (OE) of miR-433 further aggravates TGF-β1-suppression of Azin1 at both RNA and protein levels. (e) Western blot assays demonstrate that renal expression of fibrotic markers, such as collagen I, α-SMA, and fibronectin, increased in ligated kidneys at 7 days (7d) after UUO (UUO 7d) compared with sham kidneys (Sham), whereas Azin1 expression was greatly reduced. (f) Real-time PCR assays demonstrate the negative correlation between renal miR-433 and Azin1 expression. (g) Immunohistochemical assays shows that Azin1 expression is reduced in ligated kidneys at 7 days after UUO compared with sham kidneys (Sham). (h) DNA sequence alignments of miR-433-binding sites in five mammalian Azin1 3′-untranslated regions (UTRs). Bold and underlined sequences indicate the location of the miR-433-binding site. The lower panel indicates the sequence alignments of miR-433 with human Azin1 3′-UTR. (i) Schematic representation of mouse Azin1 3′-UTR reporter constructs (left) and analysis of their reporter activities (right). Firefly luciferase (Luc) reporter activities are abolished when the constructs contain the miR-433-binding site in miR-433 OE TECs. Mutation of the miR-433-binding site restores the luciferase reporter activities. Each bar represents the mean±s.e.m. for at least three independent experiments or at least five mice. *P<0.05, **P<0.01, ***P<0.001 compared with either time 0, with normal TECs, or with normal mice; #P<0.05, ##P<0.01, ###P<0.001 compared with the TGF-β1-treated samples of normal TECs, or with the sham kidneys, as indicated. Azin1, antizyme inhibitor 1; Col I, collagen I; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ODC, ornithine decarboxylase; α-SMA, α-smooth muscle actin; TEC, tubular epithelial cell; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Azin1 has a protective role in renal fibrosis. (a) Real-time PCR analysis shows that transient transfection with an Azin1 overexpression (OE) plasmid in TECs suppresses TGF-β1-induced expression of collagen I, fibronectin, and α-SMA. (b) Real-time PCR analysis shows that transient transfection with a small interfering RNA (siRNA) against Azin1 in TECs enhances TGF-β1-induced expression of collagen I, fibronectin, and α-SMA. (c) Representative western blots demonstrate that overexpression of Azin1 in TECs attenuates the expression of fibrotic markers after TGF-β1 treatment. In contrast, knockdown (KD) of Azin1 in TECs demonstrates opposite expression patterns of these markers. In addition, overexpression of Azin1 restores the ODC renal expression. (d) Representative results and quantification of thin-layer chromatography (TLC) assay demonstrate that TGF-β1 treatment suppresses the cellular levels of spermidine and spermine. Overexpression of miR-433 promotes TGF-β1-caused reduction in spermidine and spermine, but overexpression of Azin1 suppresses this reduction. (e) Representative results and quantification of TLC assay demonstrate that renal levels of spermidine and spermine are reduced in UUO kidneys, but the knockdown of miR-433 partially restores the levels of cellular polyamines. Each bar represents the mean±s.e.m. for at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 compared with either time 0; #P<0.05, ##P<0.01, ###P<0.001 compared with the TGF-β1-treated samples of normal TECs. Azin1, antizyme inhibitor 1; Col I, collagen I; Ctl, control plasmid; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KD, miR-433 knockdown plasmid; ODC, ornithine decarboxylase; α-SMA, α-smooth muscle actin; TEC, tubular epithelial cell; TGF-β1, transforming growth factor-β1; UUO, unilateral ureteral obstruction. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 9 Suppression of antizyme reduces TGF-β1-induced expression of fibrotic markers, and alteration of Azin1 expression affects TGF-β signaling. (a) Representative western blots demonstrate that treatment with antizyme small interfering RNA (siRNA) in TECs attenuates TGF-β1-induced expression of collagen I, fibronectin, and α-SMA, and restores the ODC expression. (b) Representative results and quantification of the thin-layer chromatography (TLC) assay show that treatment with antizyme siRNA in TECs restores cellular levels of spermidine and spermine. (c) Representative western blots demonstrate that overexpression of Azin1 in TECs attenuates the expression of TGF-β1, TβRI, and phosphorylation of Smad3 after TGF-β1 treatment, but knockdown (KD) of Azin1 in TECs reverses the pattern. (d) Schematic diagram of how miR-433 forms a positive feedback loop to amplify TGF-β signaling by targeting Azin1. Each bar represents the mean±s.e.m. for at least three independent experiments. **P<0.01 compared with treatment of negative control siRNA in the absence of TGF-β1; #P<0.05 as indicated. Azin1, antizyme inhibitor 1; Col I, collagen I; Ctl, control plasmid; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ODC, ornithine decarboxylase; α-SMA, α-smooth muscle actin; TEC, tubular epithelial cell; TGF-β1, transforming growth factor-β1; TβRI, TGF-β receptor type I; TβRII, TGF-β receptor type II. Kidney International 2013 84, 1129-1144DOI: (10.1038/ki.2013.272) Copyright © 2013 International Society of Nephrology Terms and Conditions