Volume 85, Issue 2, Pages 352-361 (January 2014) Transforming growth factor-β1-mediated renal fibrosis is dependent on the regulation of transforming growth factor receptor 1 expression by let-7b  Bo Wang, Jay C. Jha, Shinji Hagiwara, Aaron D. McClelland, Karin Jandeleit-Dahm, Merlin C. Thomas, Mark E. Cooper, Phillip Kantharidis  Kidney International  Volume 85, Issue 2, Pages 352-361 (January 2014) DOI: 10.1038/ki.2013.372 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Transforming growth factor-β1 (TGF-β1) induces profibrotic changes and reduces let-7b expression in renal cells. (a) NRK52E cells were cultured in high-glucose medium in the presence of TGF-β1 (10ng/ml, 3 days), after which the expression of let-7b was reduced (*P<0.05 compared with control). (b) The expression of several genes was significantly elevated by TGF-β1, as measured by real-time quantitative polymerase chain reaction (rt QPCR), including collagen (Col) I and IV, plasminogen activator inhibitor-1 (PAI-1), fibronectin (Fibr), and TGF-β1 receptor 1 (TGFBR1), whereas E-cadherin (E-cad) was significantly decreased (*P<0.05 compared with control). Let-7b transfection significantly decreased collagen I, PAI-1, Fibr, and TGFBR1 expression (*P<0.05 compared with control transfected cells). Transfection with let-7b also attenuated the effect of TGF-β1 on collagen I, collagen IV, PAI-1, and TGFBR1 (#P<0.05 compared with TGF-β1 treatment alone). (c) Primary mouse mesangial cells (MCs) were cultured for 3 days in high-glucose medium with TGF-β1 (10ng/ml), resulting in reduced let-7b expression (*P<0.05 compared with control). (d) TGF-β1 also significantly elevated the level of collagen I, III, IV, and fibronectin mRNA in primary mouse mesangial cells, as well as TGFBR1 (*P<0.05 compared with control cells). (e) Western blot analysis of let-7b-transfected NRK52E cells revealed a significant reduction in TGFBR1 protein levels (*P<0.02 compared with control). (f) Similarly, collagen I protein levels were significantly reduced by let-7b transfection (*P<0.05 compared with control transfected). LNA, locked nucleic acid. Kidney International 2014 85, 352-361DOI: (10.1038/ki.2013.372) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 The 3′-UTR of transforming growth factor-β1 receptor 1 (TGFBR1) is regulated by let-7b. NRK52E cells were transfected with TGFBR1 3′-UTR-luciferase-reporter plasmids (1μg), β-galactosidase plasmid (0.2μg), and either miR-C (100nmol/l) or let-7b (100nmol/l), and cells were analyzed for β-galactosidase and luciferase activity after 3 days. The 5′ and 3′ constructs of the 3′-UTR of TGFBR1 represent the two regions of the UTR-containing let-7b sites. (a) Sequence of the 5′ and 3′ regions of the 3′-UTR of TGFBR1 containing the let-7b binding site and the mutant sequence used for the mutant 3′-UTR constructs. The sequence in bold represents the nucleotides involved in binding to the seed sequence in the 3'UTR of TGFBR1. (b) TGF-β1 significantly increased luciferase activity in cells containing both of the 3′-UTR regions of TGFBR1 (*P<0.05 compared with control transfected cells). In each case, let-7b prevented this increase and even reduced luciferase activity below control transfected cells (#P<0.05 compared with control transfected cells). (c) Similar experiments were carried out using luciferase constructs with TGFBR1 3′-UTRs containing mutated 5′ and 3′ let-7b binding sites. Let-7b had no effect in these constructs where the let-7b binding sites were mutated. Kidney International 2014 85, 352-361DOI: (10.1038/ki.2013.372) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Let-7b regulates transforming growth factor-β1 (TGF-β1) signaling. (a) NRK52E cells were cotransfected with the p(CAGA12) SMAD3 activity reporter construct, a β-galactosidase construct, and let-7b. After 4h, cells were treated with TGF-β1 and harvested 3 days later. TGF-β1 resulted in increased SMAD3 activity with miR-C (*P<0.01 compared with control (Cont)), which was strongly inhibited by let-7b (#P<0.01 compared with miR-C with TGF-β1). (b) In a separate experiment, SB431542 also strongly inhibited SMAD3 activity (*P<0.001 compared with control; #P<0.005 compared with TGF-β1-treated cells). (c) TGF-β1 treatment also increased plasminogen activator inhibitor-1 (PAI-1 expression) (*P<0.005 compared with miR-C control), whereas let-7b transfection reduced the expression of PAI-1 in untreated cells and attenuated the induction of PAI-1 in TGF-β1-treated cells (#P<0.05 compared with miR-C control and TGF-β1 treatment alone). (d) The expression of PAI-1 was strongly inhibited by SB431542 in NRK52E cells in the presence and absence of TGF-β1 (*P<0.01 compared with control; #P<0.001 compared with TGF-β1-treated cells). (e) Treatment of cells with the TGFBR1 blocker, SB431542, resulted in decreased expression of collagen I and IV (Col I and Col IV), as well as fibronectin (Fibr) and TGF-β1 receptor 1 (TGFBR1) (*P<0.05 compared with untreated cells), even in the presence of TGF-β1 in the case of collagen I and IV and fibronectin (*P<0.01 compared with control; #P<0.05 compared with TGF-β1 treatment). (f) Western blot analysis demonstrated a significant decrease in collagen I protein expression by SB431542 (*P<0.01 compared with control cells). (g) TGFBR1 RNA is efficiently knocked down in transfected NRK52E cells by three small interfering RNAs (siRNAs) designed against TGFBR1 and used at a concentration of 10nmol/l (*P<0.02 compared with control). (h) NRK52E cells were transfected with the combination of the three siRNAs shown in g at a concentration of 10nmol/l each and then treated with TGF-β1. Knockdown of TGFBR1 attenuated the expression of Col I, fibronectin, and PAI-1, even in the presence of TGF-β1 (*P<0.05 compared with control; #P<0.05 compared with TGF-β1 treatment alone). Kidney International 2014 85, 352-361DOI: (10.1038/ki.2013.372) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Inhibition of let-7b increases the expression of transforming growth factor-β1 receptor 1 (TGFBR1) in proximal tubular cells. (a) NRK52E cells were transfected with locked nucleic acid (LNA)-let-7b, and 4h later they were treated with TGF-β1 for 3 days. TGF-β1 treatment increased TGFBR1 expression (*P<0.05 compared with untreated control), which was further increased by LNA-let-7b transfection (#P<0.05 compared with miR-C+TGF-β1 treatment). (b) Western blot analysis of cells transfected with LNA-let-7b demonstrated significantly increased TGFBR1 protein levels (*P<0.01 compared with control). (c) NRK52E cells transfected with both LNA-let-7b and the luciferase reporter with the region of the TGFBR1 3′-UTR containing the 5′ let-7b binding site demonstrated that LNA-let-7b can further increase TGFBR1 levels above that of TGF-β1 alone (*P<0.01 compared with control; #P<0.05 compared with LNA-C and TGF-β1 treatment). (d) Experiments similar to that in c but using the luciferase reporter with the region of the TGFBR1 3′-UTR containing the 3′ let-7b binding site demonstrated that LNA-let-7b can increase TGFBR1 levels above that of control (*P<0.01 compared with control), as well as that of TGF-β1 (#P<0.05 compared with LNA-C and TGF-β1 treatment). (e) NRK52E cells were cotransfected with the p(CAGA)12 SMAD3 activity reporter construct, a β-galactosidase construct, and LNA-let-7b. After 4h, cells were treated with TGF-β1 and harvested 3 days later. TGF-β1 predictably resulted in increased SMAD3 activity in LNA-C-transfected cells (*P<0.01 compared with control). LNA-let-7b further enhanced SMAD3 activity compared with TGF-β1 treatment alone (#P<0.05). (f) Ectopic expression of LNA-let-7b further increased plasminogen activator inhibitor-1 (PAI-1) expression compared with TGF-β1-treated and LNA-C-transfected proximal tubular cells (*P<0.005 compared with control; #P<0.001 compared with TGF-β1-treated and LNA-C-transfected cells). (g) LNA-let-7b transfection of NRK52E cells did not alter the baseline expression of collagen I and IV, fibronectin, or SMAD7 gene expression, but was able to enhance the effect of TGF-β1 on the expression of these genes (*P<0.05 compared with untreated LNA-C controls; #P<0.05 compared with TGF-β1-treated and LNA-C-transfected cells). Kidney International 2014 85, 352-361DOI: (10.1038/ki.2013.372) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Changes in let-7b expression in experimental renal fibrosis. (a) Immunohistochemical analysis of kidneys from control and 10-week diabetic apolipoprotein E (apoE) mice (n=8 per group) showing increased fibronectin (Fibr) and collagen IV (Col IV) expression (magnification × 40). (b) Quantification of the staining in a is shown. (c) Total RNA was extracted from the kidney cortex of these animals. The relative expression of let-7b was decreased in the kidney cortex of 10-week diabetic apoE-/- mice (*P<0.05 compared with control mice). (d) Gene expression analysis in the kidney cortex of these animals revealed significantly increased levels of collagen I, III, and IV, α-smooth muscle actin (α-SMA), vimentin (Vim), and fibronectin in diabetic animals (P<0.05 compared with control). The expression of the profibrotic factors connective tissue growth factor (CTGF) and TGF-β1 was also elevated, as was the receptor for TGF-β1, TGF-β1 receptor 1 (TGFBR1) (*P<0.05 compared with control). (e) The expression of collagen I, fibronectin, and TGFBR1 genes was increased in mRNA from the isolated renal proximal tubular fraction of 10-week streptozotocin (STZ)-diabetic apoE knockout (KO) mice (*P<0.05 compared with control mice). (f) Stained sections of the tubulointerstitium in 20-week control and STZ-diabetic apoE KO mice reveals an increase in tubulointerstitial area (TIA) (magnification × 20) in the diabetic mouse kidney, which is quantified (g) (*P<0.05 compared with control). (h) The expression of let-7b is significantly reduced in the kidney cortex of 20-week STZ-diabetic apoE KO mice (*P<0.05 compared with control). (i) Expression of let-7b was significantly reduced in the kidney cortex of mice with adenine-induced renal fibrosis (*P<0.01 compared with control mice). (j) TGFBR1 mRNA was significantly increased in the kidneys of mice with adenine-induced renal fibrosis (*P<0.05 compared with control mice). Kidney International 2014 85, 352-361DOI: (10.1038/ki.2013.372) Copyright © 2014 International Society of Nephrology Terms and Conditions