Volume 84, Issue 1, Pages 64-77 (July 2013) Inhibition of TGF-β1-receptor posttranslational core fucosylation attenuates rat renal interstitial fibrosis  Nan Shen, Hongli Lin, Taihua Wu, Dapeng Wang, Weidong Wang, Hua Xie, Jianing Zhang, Zhe Feng  Kidney International  Volume 84, Issue 1, Pages 64-77 (July 2013) DOI: 10.1038/ki.2013.82 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Relative mRNA and protein expression levels of the transforming growth factor-β1 (TGF-β1) receptors and Smad family in the unilateral ureteral obstruction (UUO) rat kidneys. (a) Representative RT2Profiler PCR Array analysis of the TGF-β superfamily receptors and Smad family expression levels in UUO kidneys. Rats were killed 7 days after UUO. RNA from three different normal rat kidneys and UUO rat kidneys was analyzed. (b) Representative real-time reverse transcriptase–PCR (RT–PCR) analysis of ALK5, ALK6, ALK7, and TGF-β receptor II (TGF-βRII) expression in the UUO kidneys; *P<0.05 and **P<0.01 versus Control or Sham groups. ALK, activin receptor-like kinase; d, day. (c) Representative western blotting and (d) quantification of ALK5, ALK6, ALK7, TGF-βRII, Smad2/3, and p-Smad2/3 expression in the UUO kidneys. Results are expressed as the mean±s.e.m. of three independent experiments (n=6 per group). β-Actin was used as an internal control; *P<0.05 and **P<0.01 versus Control or Sham groups. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Core fucose levels are elevated in the unilateral ureteral obstruction (UUO) rat kidneys. (a) Representative images and (b) densitometric quantification of Lens culinaris agglutinin–fluorescein complex (LCA-FITC) staining of core fucose in UUO kidneys. Original magnification × 400. Data are the mean±s.e.m. of five independent measurements (n=5 per group); *P<0.05, **P<0.01 versus Control group. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Schematic outline of the construction of recombinant adenovirus. Three short hairpin RNAs (shRNAs; FUT8shRNA1, 5′-GTGGTCATTTGGTTCGAGA-3′; FUT8shRNA2, 5′-GCGAATGGCTGAGTCTCTA-3′; FUT8shRNA3, 5′-GGCTGGAAACCATTGGGAT-3′) were synthesized and cloned in reverse direction into the pAdTrack-CMV shuttle vector. The resultant plasmid was linearized by PmeI digestion and cotransformed into Escherichia coli BJ5183 strain cells with the pAdEasy-1 adenoviral plasmid. Recombinant bacteria were selected by kanamycin resistance, and recombination was confirmed by PacI digestion. Finally, the linearized recombinant plasmid was transiently transfected into the packaging 293 cells. Recombinant adenoviruses were typically generated within 7 days. The ‘left arm’ and ‘right arm’ represent the regions of homologous recombination between the adenoviral backbone vector and the shuttle vector. FUT8, α-1,6 fucosyltransferase; GFP, green fluorescent protein. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 FUT8shRNA effectively inhibits endogenous α-1,6 fucosyltransferase (FUT8) expression. (a) Representative photomicrographs and (b) densitometric quantification of green fluorescent protein (GFP)–tagged FUT8shRNA adenovirus infection in the rat kidneys after tail vein injection; original magnification × 400. d, day. Results are expressed as the mean±s.e.m. of five independent experiments (n=5 per group); *P<0.05 versus tail vein injection 0 d (0 day) group. (c) Representative real-time reverse transcriptase–PCR (RT–PCR) analysis of FUT8 mRNA expression in the unilateral ureteral obstruction (UUO) kidneys infected with GFP or FUT8shRNA; *P<0.05, **P<0.01 versus Control, Sham, or ShamGFP groups; #P<0.05, ##P<0.01 versus UUO group. (d) Representative western blot analysis and (e) quantification of FUT8 protein expression in UUO kidneys infected with GFP or FUT8shRNA. Results are expressed as the mean±s.e.m. of three independent experiments (n=6 per group). β-Actin was used as an internal control; *P<0.05, **P<0.01 versus Control, Sham, or ShamGFP groups; #P<0.05, ##P<0.01 versus UUO group. FUT8, α-1,6 fucosyltransferase; shRNA, short hairpin RNA. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 FUT8shRNA suppresses core fucosylation in unilateral ureteral obstruction (UUO) rat kidneys. (a) Representative photomicrographs of rhodamine-labeled Lens culinaris agglutinin (LCA-TRITC) staining for core fucosylation in control kidneys (Control) and UUO kidneys infected with FUT8shRNA; original magnification × 400. FUT8, α-1,6 fucosyltransferase; shRNA, short hairpin RNA. (b) Densitometric quantification of core fucosylation levels. Data are the mean±s.e.m. of five independent experiments (n=5 per group); *P<0.05, **P<0.01 versus Control group. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Transforming growth factor-β receptor II (TGF-βRII) and activin receptor-like kinase 5 (ALK5) are regulated by core fucosylation, which can be inhibited by FUT8shRNA. (a) Representative western and lectin blot analysis and (b) quantification of the expression levels of TGF-βRII and core fucose, or ALK5 and core fucose in unilateral ureteral obstruction (UUO) kidneys infected with green fluorescent protein (GFP) or FUT8shRNA. TGF-βRII and ALK5 were immunoprecipitated from tissue lysates and then subjected to electrophoresis (12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)). After electroblotting, blots were probed by Lens culinaris agglutinin (LCA)–Biotin. Results are expressed as the mean±s.e.m. of three independent experiments (n=6 per group). β-Actin was used as an internal control; *P<0.05, **P<0.01 versus Control, Sham, or ShamGFP groups; #P<0.05, ##P<0.01 versus UUO group. FUT8, α-1,6 fucosyltransferase; shRNA, short hairpin RNA. (c) Representative images of double fluorescent labeling for TGF-βRII and core fucose or (e) ALK5 and core fucose in UUO kidneys infected with FUT8shRNA. A significant number of tubular epithelial cells showed colocalization of both molecules; original magnification × 400. (d) Densitometric quantification of the expression levels of TGF-βRII and core fucose or (f) ALK5 and core fucose. Results are expressed as the mean±s.e.m. of five independent experiments (n=5 per group); *P<0.05, **P<0.01 versus Control group, #P<0.05, ##P<0.01 versus UUO group. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 FUT8shRNA suppresses phosphorylation of Smad2/3. (a) Representative western blot analysis and (b) quantification of Smad2/3 and p-Smad2/3 protein expression in unilateral ureteral obstruction (UUO) kidneys infected with green fluorescent protein (GFP) or FUT8shRNA. Results are expressed as the mean±s.e.m. of three independent experiments (n=6 per group). β-Actin was used as an internal control; *P<0.05, **P<0.01 versus Control, Sham, or ShamGFP groups; #P<0.05, ##P<0.01 versus UUO group. FUT8, α-1,6 fucosyltransferase; shRNA, short hairpin RNA. (c) Representative images of immunofluorescent analysis and (d) densitometric quantification of p-Smad2/3 expression in the UUO kidneys infected with FUT8shRNA. P-Smad2/3 was mainly localized in tubule epithelium and, to a lesser extent, in the interstitial cells; original magnification × 400. Results are expressed as the mean±s.e.m. of five independent experiments (n=5 in each group); *P<0.05, **P<0.01 versus Control group, #P<0.05, ##P<0.01 versus UUO group. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Effects of FUT8shRNA on the expression of core fucose, collagen I, collagen III, fibronectin, tissue inhibitor of metalloproteinase 1 (TIMP-1), and monocyte chemoattractant protein-1 (MCP-1) in the unilateral ureteral obstruction (UUO) kidney. (a) Representative western blot analysis and (b) quantification of collagen I, collagen III, fibronectin, and TIMP-1 protein expression levels in the UUO kidneys infected with adenovirus-encoding green fluorescent protein (GFP) or FUT8shRNA. Results are expressed as the mean±s.e.m. of three independent experiments (n=6 per group). β-Actin was used as an internal control; *P<0.05, **P<0.01 versus Control, Sham, or ShamGFP groups; #P<0.05, ##P<0.01 versus UUO group. FUT8, α-1,6 fucosyltransferase; shRNA, short hairpin RNA. (c) Representative images of immunohistochemical staining for core fucose, MCP-1, and (d) computer-based morphometric analysis of kidney fibrosis in the UUO kidneys infected with FUT8shRNA; original magnification × 400. Results are expressed as the mean±s.e.m. of five independent experiments (n=5 per group). *P<0.05, **P<0.01 versus Control group; #P<0.05, ##P<0.01 versus UUO group. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 9 FUT8shRNA blocks tubular–interstitial injury and fibrosis. (a) Representative periodic acid–Schiff (PAS), periodic acid–Schiff-methenamine silver (PAM), Masson’s trichrome staining, and (b) computer-based morphometric analysis of kidney fibrosis in the unilateral ureteral obstruction (UUO) kidneys infected with FUT8shRNA; original magnification × 200. FUT8, α-1,6 fucosyltransferase shRNA, short hairpin RNA. Results are expressed as the mean±s.e.m. of five independent experiments (n=5 per group). *P<0.05, **P<0.01 versus Control group; #P<0.05, ##P<0.01 versus UUO group. Kidney International 2013 84, 64-77DOI: (10.1038/ki.2013.82) Copyright © 2013 International Society of Nephrology Terms and Conditions