Inhibition of TGF-β1-receptor posttranslational core fucosylation attenuates rat renal interstitial fibrosis

basic research

http://www.kidney-international.org & 2013 International Society of Nephrology

see commentary on page 11

Inhibition of TGF-b1-receptor posttranslational core fucosylation attenuates rat renal interstitial fibrosis Nan Shen1, Hongli Lin1, Taihua Wu2, Dapeng Wang1, Weidong Wang1, Hua Xie1, Jianing Zhang3 and Zhe Feng4 1

Department of Nephrology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China; 2Central Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China; 3Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian, Liaoning, China and 4Department of Nephrology, State Key Laboratory of Kidney Disease, Beijing, China

The profibrotic cytokine transforming growth factor-b1 (TGF-b1) causes renal fibrosis by binding to receptors at the cell surface; however, it is not clear which of the TGF-b superfamily receptors correlates with renal fibrosis. To resolve this, we quantified TGF-b superfamily receptor expression in the kidneys of rats with unilateral ureteral obstruction using a real-time PCR gene array. Expression of activin receptor-like kinase (ALK)-5, ALK7, and TGF-b receptor II (TGF-bRII) mRNA increased significantly, while ALK6 mRNA expression was significantly decreased in the obstructed rat kidney. Core fucosylation is essential for the proper function of both TGF-bRII and ALK5 in cultured human renal proximal tubular epithelial cells in vitro. Therefore, we targeted posttranslational core fucosylation, regulated by a-1,6 fucosyltransferase (FUT8), by adenoviral-mediated knockdown of FUT8 mRNA in vivo and measured TGF-bRII and ALK5 expression and the progression of renal fibrosis. Despite long-term obstruction injury, inhibition of TGF-bRII and ALK5 of core fucosylation ameliorated the progression of renal fibrosis, an effect independent of TGF-bRII and ALK5 expression. Thus, the regulation of TGF-b1-receptor core fucosylation may provide a novel potential therapeutic strategy for treating renal fibrosis. Kidney International (2013) 84, 64–77; doi:10.1038/ki.2013.82; published online 13 March 2013 KEYWORDS: glycosylation; obstructive nephropathy; receptors; renal fibrosis; transforming growth factor-beta; unilateral urethral obstruction

Correspondence: Hongli Lin, Department of Nephrology, The First Affiliated Hospital of Dalian Medical University, No. 222 Zhongshan Road, Dalian 116011, China. E-mail: [email protected] or Taihua Wu, Central Laboratory, The First Affiliated Hospital of Dalian Medical University, No. 222 Zhongshan Road, Dalian 116011, China. E-mail: [email protected] Received 10 September 2012; revised 29 December 2012; accepted 10 January 2013; published online 13 March 2013 64

Renal fibrosis is an inevitable consequence of many chronic kidney diseases. Among the diverse causative factors, transforming growth factor-b1 (TGF-b1) can strongly induce renal fibrosis.1–3 TGF-b1 is a multifunctional cytokine that evokes diverse cellular responses,4–9 including fibrosis, by binding to cell surface receptors.10 TGF-b superfamily receptors include seven type I receptors (activin receptor-like kinases 1 to 7 (ALK1–7), five type II receptors (TGF-b receptor II (TGF-bRII), activin type II receptor, activin type IIB receptor, bone morphogenetic protein receptor type II, and anti-Mu¨llerian hormone type II receptor), and two type III receptors (Betaglycan and Endoglin) in mammals.11,12 TGF-b1 signals through receptor complexes consisting of type I (TGF-bRIs) and type II receptors (TGF-bRIIs), and the activated receptors phosphorylate and activate the Smad proteins, which form transcriptional complexes that control the expression of a number of target genes with various functions.13 Therefore, we hypothesized that some of the receptors may have important roles during renal fibrosis, and that these receptors might be novel therapeutic targets for renal fibrosis. However, the subtype-specific expression of the diverse range of TGF-b superfamily receptors during renal fibrosis remains to be determined. Currently, inhibition of protein expression is a common strategy to abolish the function of proteins in certain diseases. However, data increasingly indicate that posttranslational modifications mediate direct and definitive regulation of protein function, which can be independent of the protein expression levels in some pathophysiological processes.14,15 Therefore, such modifications may be a critical regulator of protein function. Glycosylation is a crucial posttranslational modification that has profound effects on the regulation of various physiological processes, including cell growth, differentiation, and migration,16,17 all of which are involved in kidney disease. The TGF-b superfamily receptors TGF-bRII and TGF-bRI (ALK5) are both glycoproteins.18 Our recent study showed that diminishing the core fucosylation of TGF-bRII and ALK5, which is catalyzed by a-1,6 fucosyltransferase (FUT8), blocked renal tubular epithelial–mesenchymal transition in Kidney International (2013) 84, 64–77

N Shen et al.: TGF-b1-R fucosylation affects renal fibrosis

cultured human renal proximal tubular epithelial cells in vitro.19 However, to date, the blockade of protein function in vivo by inhibiting core fucosylation has not yet been reported in kidney disease. In this study, we screened and identified the TGF-b superfamily receptors that correlated with renal fibrosis induced by unilateral ureteral obstruction (UUO). We observed that TGF-bRII and TGF-bRI (ALK5) were the critical TGF-b1 receptors for this process. We also investigated the role of core fucosylation on the expression and function of these TGF-b1 receptors in vivo during renal fibrosis. Our results suggest that core fucosylation has a crucial role in TGF-b1R function, and the blockade of core fucosylation successfully abolished the activation of TGF-b/Smad signaling and attenuated renal fibrosis induced by UUO. Targeting the posttranslational modifications of key proteins may provide a novel and effective strategy for the treatment of fibrotic renal disorders. RESULTS The TGF-b superfamily receptors TGF-bRII, TGF-bRI (ALK5), ALK6, and ALK7 correlate with renal fibrosis in UUO rats

To examine the expression levels of the TGF-b superfamily receptors in the UUO kidney, we compared the gene expression profiles of kidneys from the UUO7d and Control groups using an RT2Profiler Array. Among the 23 genes examined, significant differential expression of six genes was detected compared with the control group; in the UUO7d group, ALK5, ALK7, TGF-bRII, Smad2, and Smad3 were significantly upregulated, whereas ALK6 was significantly downregulated. Significant differential expression of the other 17 genes was not detected (Figure 1a). Compared with the Control group, ALK5, ALK7, and TGF-bRII mRNA increased progressively in the UUO group from 3 days after surgery (Po0.05), reaching peak levels at 21 days after surgery (Po0.01). The expression of ALK6 mRNA decreased progressively in the UUO group from 3 days after surgery (Po0.05), reaching the lowest level at 21 days after surgery (Po0.01; Figure 1b). Western blot analysis confirmed these observations at the protein level (Figure 1c and d). We investigated the activation of the TGF-b/Smad2/3 pathway in the UUO kidney by quantifying Smad2/3 phosphorylation. Western blot analysis indicated that Smad2/3 and p-Smad2/3 expression increased progressively in the UUO group, was significantly increased 3 days after surgery (Po0.05), and reached peak levels at 21 days after surgery (Po0.01 vs. Control group; Figure 1c and d). Core fucosylation levels are elevated in the rat kidney during UUO

An increasing number of studies indicate that posttranslational modifications crucially regulate protein function, and posttranslational modifications may represent potential targets for the treatment of some diseases. Therefore, we examined the core fucosylation levels in the UUO kidney by fluorescent Lens culinaris agglutinin–fluorescein complex Kidney International (2013) 84, 64–77

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(LCA-FITC). We observed low levels of core fucosylation in the normal kidney; however, these levels markedly and progressively increased following surgery in the UUO group (Figure 2a and b). Construction of the FUT8-knockdown UUO rat model

FUT8 is the unique fucosyltransferase responsible for core fucosylation.20,21 To investigate the function of core fucosylation in UUO-induced renal fibrosis, we created a FUT8-knockdown UUO rat model. We constructed a FUT8 short hairpin RNA (shRNA) recombinant adenovirus vector (Ad-FUT8shRNA) and control adenovirus encoding green fluorescent protein (Ad-GFP), amplified and purified these adenoviruses from HEK-293 cells, and performed adenoviral transfection through rat tail vein injection (Figure 3). Exogenous FUT8shRNA was expressed in the kidney 1 day after infection and was maintained at similar levels for 21 days (Figure 4a and b). We investigated the effects of Ad-FUT8shRNA on endogenous FUT8 expression in the UUO kidney. Real-Time reverse transcriptase–PCR (RT–PCR) and western blot analysis (Figure 4c–e) showed that the endogenous FUT8 expression levels obviously increased in the UUO kidney; however, these levels were significantly downregulated at the mRNA and protein levels in both the ShamFUT8shRNA and UUOFUT8shRNA groups. These data indicated successful generation of a FUT8-knockdown UUO rat model. FUT8shRNA suppresses core fucosylation in the UUO kidney

We investigated whether core fucosylation could be reduced by FUT8shRNA in the UUO kidney using rhodamine-labeled Lens culinaris agglutinin (LCA-TRITC). As shown in Figure 5a and b, FUT8shRNA effectively inhibited core fucosylation in the Control group (ControlFUT8shRNA þ rats). FUT8shRNA also prevented an increase in core fucosylation after surgery in the kidney of the UUO FUT8shRNA group as compared with the progressive increase observed after surgery in the UUO group. TGF-bRII and ALK5 are modified by core fucosylation

Our data indicated that TGF-bRII, TGF-bRI (ALK5), ALK6, and ALK7 are the key TGF-b superfamily receptors involved in the renal fibrosis in UUO rats. We hypothesized that targeting of these receptors could provide a possible therapeutic target interference point for renal fibrosis. Recently, we reduced the core fucosylation of TGF-bRII and TGF-bRI (ALK5) in cultured human renal proximal tubular epithelial cells by transient transfection of FUT8shRNA, which successfully reversed the renal tubular epithelial–mesenchymal transition in vitro.19 Therefore, in this study, we investigated whether TGF-bRII and ALK5 are modified by core fucosylation in the kidney. Core fucose bands were detected after immunoprecipitation with TGFbRII and ALK5 antibodies, demonstrating that TGF-bRII and ALK5 are modified by core fucosylation (Figure 6a and b). Next, we determined the spatial relationship of core fucose 65

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Figure 1 | Relative mRNA and protein expression levels of the transforming growth factor-b1 (TGF-b1) receptors and Smad family in the unilateral ureteral obstruction (UUO) rat kidneys. (a) Representative RT2Profiler PCR Array analysis of the TGF-b 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-b receptor II (TGF-bRII) expression in the UUO kidneys; *Po0.05 and **Po0.01 versus Control or Sham groups. ALK, activin receptor-like kinase; d, day. (c) Representative western blotting and (d) quantification of ALK5, ALK6, ALK7, TGF-bRII, 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 ¼ 6 per group). b-Actin was used as an internal control; *Po0.05 and **Po0.01 versus Control or Sham groups.

with TGF-bRII and ALK5 using double fluorescent labeling. The expression of core fucose epitopes completely overlapped with that of both TGF-bRII and ALK5 epitopes in a significant number of tubular epithelial cells, confirming that both TGF-bRII and ALK5 are modified by core fucosylation in the UUO kidney (Figure 6c–f). FUT8shRNA inhibits core fucosylation of TGF-bRII and ALK5 in the UUO kidney without affecting protein expression levels

We measured the protein expression levels of TGF-bRII and ALK5, and observed that TGF-bRII and ALK5 were upregulated with increased core fucosylation in UUO kidneys 66

(Figure 6a and b). This effect was markedly suppressed by FUT8shRNA; however, FUT8shRNA had no effect on the expression levels of TGF-bRII and ALK5 (Figure 6a and b). Double fluorescent labeling confirmed that the core fucosylation of TGF-bRII and ALK5 markedly and progressively increased after surgery in the UUO group; however, this effect was prevented by FUT8shRNA (Figure 6c–f). Inhibiting TGF-bRII and ALK5 core fucosylation suppresses Smad2/3 phosphorylation in the UUO kidney

To explore the effects of core fucosylation on the functionality of TGF-bRII and ALK5, we investigated whether Kidney International (2013) 84, 64–77

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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 ¼ 5 per group); *Po0.05, **Po0.01 versus Control group.

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Figure 3 | Schematic outline of the construction of recombinant adenovirus. Three short hairpin RNAs (shRNAs; FUT8shRNA1, 50 -GTGGTCATTTGGTTCGAGA-30 ; FUT8shRNA2, 50 -GCGAATGGCTGAGTCTCTA-30 ; FUT8shRNA3, 50 -GGCTGGAAACCATTGGGAT-30 ) 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, a-1,6 fucosyltransferase; GFP, green fluorescent protein. Kidney International (2013) 84, 64–77

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Figure 4 | FUT8shRNA effectively inhibits endogenous a-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 ¼ 5 per group); *Po0.05 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; *Po0.05, **Po0.01 versus Control, Sham, or ShamGFP groups; #Po0.05, ##Po0.01 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 ¼ 6 per group). b-Actin was used as an internal control; *Po0.05, **Po0.01 versus Control, Sham, or ShamGFP groups; #Po0.05, ##Po0.01 versus UUO group. FUT8, a-1,6 fucosyltransferase; shRNA, short hairpin RNA.

inhibiting core fucosylation of TGF-bRII and ALK5 could abolish the activation of the TGF-b/Smad2/3 pathway. Western blot and immunofluorescence analysis indicated that p-Smad2/3 was expressed mostly in tubule epithelium, rather than in the interstitial cells, and the expression increased progressively after surgery in the UUO group; however, the abolition of ALK5 and TGF-bRII core fucosylation using FUT8shRNA effectively suppressed this increase after surgery in the UUOFUT8shRNA group (Figure 7a–d). Blocking TGF-bRII and ALK5 core fucosylation inhibits extracellular matrix protein expression in the UUO kidney

Extracellular matrix accumulation is critically involved in UUO-induced renal fibrosis. Therefore, we examined extra68

cellular matrix–associated protein markers to determine the effects of reduced TGF-bRII and ALK5 core fucosylation on renal fibrosis. Western blot analysis demonstrated that the expression of extracellular matrix proteins (collagen I, collagen III, fibronectin, and tissue inhibitor of metalloproteinase 1 (TIMP-1)) were markedly elevated after surgery in the UUO group, consistent with an increase in core fucose staining. Transfection of FUT8shRNA markedly prevented UUO-induced changes in extracellular matrix protein expression (Figure 8a and b). Furthermore, inhibition of core fucosylation using FUT8shRNA reversed UUO-induced expression of the inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1; Figure 8c and d). Kidney International (2013) 84, 64–77

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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, a-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 ¼ 5 per group); *Po0.05, **Po0.01 versus Control group.

Blocking TGF-bRII and ALK5 core fucosylation ameliorates UUO-induced renal fibrosis

Finally, we examined the effects of FUT8shRNA on pathological changes in the renal interstitium and tubules in paraffin-embedded tissue sections using periodic acid–Schiff, periodic acid–Schiff-methenamine silver, and Masson’s trichrome staining. On day 7 after UUO, the kidneys of untreated rats (FUT8shRNA  ) were characterized by widespread renal tubulointerstitial damage and fibrosis. In comparison, FUT8shRNA þ rats exhibited markedly decreased tubulointerstitial damage and fibrosis at 7 days after UUO, and these differences were more evident at day 14 after UUO (Figure 9a and b). DISCUSSION

This study has provided novel insights into the pathogenesis of renal fibrosis in obstructive nephropathy by probing the role of the posttranslational modification, core fucosylation, in kidney disease for the first time. We found that core fucose was expressed at low levels in the normal rat kidney, and markedly increased in rat UUO kidneys, which was closely associated with the overexpression of extracellular matrix components, including collagen I, collagen III, fibronectin, and TIMP-1. In addition, we demonstrated that core fucosylation of ALK5 and TGF-bRII was essential for their function and subsequent activation of TGF-b/Smad signaling. Furthermore, the downregulation of core fucosylation by transfection of Ad-FUT8shRNA inhibited extracellular matrix molecule overexpression and ameliorated renal fibrosis in UUO rats because of the inactivation of the TGF-b/Smad pathway. Our findings further elucidate the mechanisms of renal fibrosis Kidney International (2013) 84, 64–77

and suggest that core fucosylation may be exploited as a novel therapeutic target for the treatment of renal fibrosis. TGF-b1 has long been considered to be a key mediator of renal fibrosis in both experimental and human kidney disease.1–3 TGF-b1 is a highly pleiotropic cytokine4–6 that exerts a broad range of anti-inflammatory and immunosuppressive effects.6–7 Knockout of TGF-b1 in mice results in autoimmunity and early death due to multiorgan inflammatory disease.8–9 In this study, we confirmed that the expression of ALK5, ALK7, and TGF-bRII increased during the progression of renal fibrosis induced by UUO, whereas ALK6 decreased (Figure 1). This result indicates that the activation of TGF-b and activin signaling and interference with bone morphogenetic protein signaling may occur during renal fibrosis in UUO kidneys. Several studies have previously demonstrated that the diversity of TGF-b1 functions relies on its different receptors.22,23 Recently, aberrant protein glycosylation, rather than altered protein expression levels, has been associated with tumor development.24 For example, a-fetoprotein (AFP) is a well-known glycoprotein that is commonly used to detect hepatocellular carcinoma. The increased expression of AFP provides limited clinical information and is only suggestive of prognosis in hepatocellular carcinoma because of low specificity (B50%); however, the aberrantly glycosylated AFP-L3 isoform is particularly useful for the early identification of aggressive hepatocellular carcinoma, with a specificity of 96%.25 Interestingly, the majority of the US Food and Drug Administration–approved cancer biomarkers are glycoproteins, including AFP, prostate cancer antigen, carcinoembryonic antigen, and Her-2/neu,26 and 69

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Figure 6 | Transforming growth factor-b receptor II (TGF-bRII) 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-bRII and core fucose, or ALK5 and core fucose in unilateral ureteral obstruction (UUO) kidneys infected with green fluorescent protein (GFP) or FUT8shRNA. TGF-bRII 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 ¼ 6 per group). b-Actin was used as an internal control; *Po0.05, **Po0.01 versus Control, Sham, or ShamGFP groups; #Po0.05, ##Po0.01 versus UUO group. FUT8, a-1,6 fucosyltransferase; shRNA, short hairpin RNA. (c) Representative images of double fluorescent labeling for TGF-bRII 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-bRII and core fucose or (f) ALK5 and core fucose. Results are expressed as the mean±s.e.m. of five independent experiments (n ¼ 5 per group); *Po0.05, **Po0.01 versus Control group, #Po0.05, ##Po0.01 versus UUO group.

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UUO7d UUO14d UUO21d – –

+ –

– –

+ –

– –

25

Relative protein expression

Control

+ –

Smad2/3 p-Smad2/3 β-Actin

Smad2/3

**** ** ** ** *

10

** *

5

0 FUT8shRNA GFP

Control

– –

– –

– +

+ –

Sham

– –

+ –

UUO3d

UUO7d +

#

##

#

– –

+ –

UUO7d

– –

+ –

UUO14d

## – –

+ –

UUO21d

UUO14d

–

+

+

–

p-Smad2/3

–

** **

15

Control

FUT8shRNA

p-Smad2/3

20

x,y20 um

x,y20 um

x,y20 um

Fluorescence intensity

10

x,y20 um

p-Smad2/3

x,y20 um

x,y20 um

**

7.5

* 5 ##

# 2.5

0 FUT8shRNA

–

+ Control

–

+ UUO7d

–

+

UUO14d

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 ¼ 6 per group). b-Actin was used as an internal control; *Po0.05, **Po0.01 versus Control, Sham, or ShamGFP groups; #Po0.05, ##Po0.01 versus UUO group. FUT8, a-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 ¼ 5 in each group); *Po0.05, **Po0.01 versus Control group, #Po0.05, ##Po0.01 versus UUO group.

posttranslational glycosylation is becoming an important, attractive biomarker and a treatment target in cancer.27–30 Increased expression of fucosyltransferases is found in various tumor cells and has been correlated with aspects of tumor progression such as metastasis and cell adhesion. Thus, fucosyltransferase inhibitors are potentially useful as antitumor agents.31 In contrast to studies in cancer, studies on the posttranslational glycosylation of proteins in kidney disease are almost nonexistent. In fact, the diverse range of glycan structures has specific functions in eukaryotic cells. Altered cellular glycosylation is associated with increased cell proliferation, migration, and apoptosis,16,17,32 all of which are involved in kidney disease. Our previous studies confirmed that blocking the core fucosylation of TGF-bRII and ALK5, a major mechanism of glycosylation, inactivated TGF-b/ Kidney International (2013) 84, 64–77

Smad2/3 signaling and reversed the renal tubular epithelial–mesenchymal transition in cultured human renal proximal tubular epithelial cells in vitro.19 In this study, we investigated the role of TGF-bRII and ALK5 core fucosylation in vivo. We used a UUO fibrosis model, in which TGF-b/Smad2/3 signaling is the key profibrotic pathway. We found that core fucose was present at low levels in the renal epithelial cells of the normal rat kidney and was elevated significantly in the UUO kidney (Figure 2). We also observed that the level of core fucosylation increased with the levels of TGF-b receptors (Figure 1b–d), suggesting that core fucosylation may have a pathological role in renal fibrosis. Wang et al.33 demonstrated that TGF-bRII was regulated by core fucosylation, which was dependent on FUT8, a fucosyltransferase that specifically catalyzes the 71

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N Shen et al.: TGF-b1-R fucosylation affects renal fibrosis

Sham

Control FUT8shRNA GFP

– –

– –

UUO3d

– +

+ –

– –

+ –

UUO7d – –

UUO21d

UUO14d

+ –

– –

+ –

– –

+ –

Collagen I Collagen III Fibronectin TIMP-1 β-Actin

– –

Control

– +

+ –

Sham

– –

+ –

**

##

#

4

20

**

*

8

– –

##

+ –

– –

##

+ –

– –

+ –

*

15

*

10 ##

##

5

Control

– –

– +

+ –

Sham

– –

+ –

# – –

+ –

– –

#

+ –

– –

+ –

# ##

7 # – –

Control

– +

+ –

– –

Sham

##

+ –

– –

– –

+ –

– –

+ –

UUO3d UUO7d UUO14d UUO21d

TIMP-1

** **

10

* *

##

#

#

5

##

0 FUT8shRNA – GFP –

– –

Control

– +

+ –

– –

Sham

+ –

– –

+ –

– –

+ –

– –

+ –

UUO3d UUO7d UUO14d UUO21d

UUO7d +

UUO14d +

–

+

–

Core fucose

40

*

30 20

##

## 10

0 FUT8shRNA

–

+

Control

72

MCP-1

**

–

+

UUO7d

–

+

UUO14d

Positive stain area/ tubulointerstitium

Positive stain area/ tubulointerstitium

MCP-1

Core fucose

–

+ –

15

UUO3d UUO7d UUO14d UUO21d Control

FUT8shRNA

*

14

20

*

**

*

0 FUT8shRNA – GFP –

**

**

Collagen III

21

UUO3d UUO7d UUO14d UUO21d

Fibronectin

0 FUT8shRNA – GFP –

Relative protein expression

12

0 FUT8shRNA – GFP –

Relative protein expression

28

**

Collagen I

Relative protein expression

Relative protein expression

16

40

**

*

30

#

20

##

10

0 FUT8shRNA

–

+

Control

–

+

UUO7d

–

+

UUO14d

Kidney International (2013) 84, 64–77

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N Shen et al.: TGF-b1-R fucosylation affects renal fibrosis

introduction of fucose to position 6 of the initial N-acetyl glucosamine residue of the N-glycan core to produce ‘core fucose.’20,21 Wang et al.33 showed that FUT8-knockout mice induced severe growth retardation, early death during postnatal development, and emphysema-like changes in the lung due to the absence of core fucosylation of TGF-bRII.34 To further elucidate the role of core fucosylation in the UUO kidney, we designed, synthesized, and fluorescently labeled a FUT8shRNA adenovirus (Figure 3) FUT8shRNA, which was expressed continually for 21 days in adenovirus-infected rats (Figure 4a and b), resulting in marked knockdown of Control FUT8shRNA

endogenous FUT8 expression (Figure 4c–e) and leading to the inhibition of core fucosylation in the UUO kidney (Figure 5). In accordance with previous research,13,19,35 this study confirmed that TGF-bRII and ALK5 are critical receptors for the profibrotic function of TGF-b1. Both TGF-bRII and ALK5 were modified by core fucosylation in the kidney, with protein expression levels obviously upregulated in the UUO kidney. Interestingly, FUT8shRNA suppressed core fucosylation of TGF-bRII and ALK5, but had no effect on their protein expression levels (Figure 6a–f). These data UUO7d

+

–

+

–

+

Masson staining

PAM staining

PAS staining

–

UUO14d

**

10

* #

5

0 FUT8shRNA

##

–

+

Control

–

+

UUO7d

–

+

UUO14d

PAM

24

*

16

##

#

8

0 FUT8shRNA

–

+

Control

–

+

UUO7d

–

Masson

32

**

+

UUO14d

Fibrotic area (%)

32

PAS

15

Fibrotic area (%)

Fibrotic area (%)

20

**

24

*

16

#

##

8

0 FUT8shRNA

–

+

Control

–

+

UUO7d

–

+

UUO14d

Figure 9 | FUT8shRNA blocks tubular–interstitial injury and fibrosis. (a) Representative periodic acid–Schiff (PAS), periodic acid–Schiffmethenamine 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, a-1,6 fucosyltransferase shRNA, short hairpin RNA. Results are expressed as the mean±s.e.m. of five independent experiments (n ¼ 5 per group). *Po0.05, **Po0.01 versus Control group; # Po0.05, ##Po0.01 versus UUO group.

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 ¼ 6 per group). b-Actin was used as an internal control; *Po0.05, **Po0.01 versus Control, Sham, or ShamGFP groups; #Po0.05, ##Po0.01 versus UUO group. FUT8, a-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 ¼ 5 per group). *Po0.05, **Po0.01 versus Control group; #Po0.05, ##Po0.01 versus UUO group. Kidney International (2013) 84, 64–77

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indicate that the effects of fucosylation of TGF-bRII and ALK5 on renal fibrosis are independent of their protein expression levels. As Smad2/3 phosphorylation is a marker of TGF-b/Smad2/3 signaling activation,11 we quantified Smad2/ 3 phosphorylation to investigate whether core fucosylation regulates the TGF-b/Smad2/3 signaling pathway. Suppression of TGF-bRII and ALK5 core fucosylation effectively decreased Smad2/3 phosphorylation in the UUO kidney (Figure 7). In addition, using immunostaining, we showed that the upregulation of TGF-bRII, ALK5, and p-Smad2/3 expression occurs mainly in tubule epithelium, rather than in the interstitial cells, consistent with prior research on the UUO kidneys. Recent research suggests that tubule cell apoptosis, autophagy, and atrophy are major features of pathology in UUO,36 raising the possibility that severe tubule pathology and tubule-located TGF-b/Smad2/3 signaling are closely related. Importantly, we investigated whether the inhibition of the core fucosylation of TGF-bRII and ALK5 could effectively prevent renal fibrosis, even in the presence of high levels of TGF-bRII and ALK5 protein expression. Therefore, we investigated the key event of renal fibrosis: extracellular matrix accumulation.37 This process is characterized by increased expression of collagen, fibronectin, and TIMP-1.38 During renal fibrosis, increased expressions of extracellular matrix components (collagen I, collagen III, fibronectin, and TIMP-1) and the inflammatory marker MCP-1 were observed, which was successfully reversed by inhibition of core fucosylation mediated by transfection of FUT8shRNA (Figure 8). Renal fibrosis is characterized by widespread renal tubulointerstitial damage and fibrosis. After induction of UUO, rats transfected with FUT8shRNA exhibited markedly decreased tubulointerstitial damage and fibrosis, and these differences increased with time after surgery (Figure 9). These findings confirm that UUO rats underwent significant tubular atrophy during renal fibrosis, which was notably reduced by the inhibition of core fucosylation using FUT8shRNA. Although the inhibition of TGF-b1 expression was shown to effectively attenuate organ fibrosis 420 years ago,39,40 this strategy has not yet been used in clinical practice because of the potential for blockade of all the biological functions of TGF-b1. However, inhibition of TGF-b1/Smad signaling by suppression of TGF-b receptor expression using a soluble TGF-bRII extracellular domain-Fc fusion protein,41 ALK5 inhibitor,42–45 can effectively prevent the development of organ fibrosis. All this evidence, together with the findings of our study, indicates that the TGF-b1 receptors might be potential targets for the development of novel therapeutic agents for renal fibrosis. Furthermore, it is likely that this approach will affect TGF-b1 biologic functions to a lesser extent than direct inhibition of TGF-b1. However, complete deletion of FUT8 might also impair the actions of several other ligand–receptor systems and produce detrimental side effects,33,34 thus carrying similar risks such as inflammation, abnormal immunity, lung 74

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damage, and cancer, associated with the inhibition of TGF-b signaling.8–9 Our experimental conditions were not suitable for determining the effect of fucosylation on TGF-b receptors and ligands binding through direct or indirect mechanisms. However, this study provides the basis of the further investigations required to elucidate details of the mechanisms of this process, such as the indirect effects of other signaling pathways and key inflammatory mediators. In summary, this study demonstrates that inhibition of TGF-bRII and ALK5 core fucosylation successfully suppressed the profibrotic function of these key TGF-b1 receptors, inhibited the activation of TGF-b/Smad signaling, and prevented renal fibrosis in the UUO kidney. Our results suggest that posttranslational modification of key proteins by core fucosylation could provide an attractive novel approach for the treatment of renal fibrotic disease. MATERIALS AND METHODS Care and use of laboratory animals Animal experiments were conducted in accordance with the regulations set by the institutional committee for the care and use of laboratory animals, and approved by local authorities. Adult male Wistar rats (250–300 g) were housed with a 12 h light/dark cycle, and were provided free access to food and water. Experimental UUO animal model and in vivo infection Animals were anesthetized using an intraperitoneal injection of freshly prepared chloral hydrate. A midline incision was made in the abdominal wall, and the left ureter was isolated and ligated using a 4.0 silk suture at two points along its length. The abdominal wound was closed with silk suture and the animals were returned to their cages. Six groups of rats (n ¼ 24 per group) were treated as follows. (1) Control group: received tail vein injection of phosphate-buffered saline (PBS) twice a week from the day of surgery; (2) Sham group: ureters were exposed and manipulated but not ligated (sham surgery) and received tail vein injection of PBS twice a week; (3) ShamGFP group: underwent sham surgery and single tail vein injection of 1106 plaque-forming units (PFUs) of Ad-GFP (Genepharma, Wuhan, China); (4) ShamFUT8shRNA group: underwent sham surgery and single tail vein injection of 1106 PFUs of Ad-FUT8shRNA (Genepharma); (5) UUO group: underwent unilateral ureteral ligation and received tail vein injection of PBS twice a week; and (6) UUOFUT8shRNA group: underwent unilateral ureteral ligation and received a single tail vein injection of 1106 PFUs of Ad-FUT8ShRNA. Rats were killed on days 1, 3, 7, 14, and 21 (n ¼ 6 at each time point) and the kidneys were harvested for morphological and biochemical studies. Construction of recombinant adenoviruses encoding rat FUT8shRNA Chemically synthesized FUT8shRNAs were designed to target the FUT8 gene. The shRNA sequences were identified using BLAST and the rat genome database to assess possible crossreactivity. Three shRNAs (FUT8shRNA1, 50 -GTGGTCATTTGGTTCGAGA-30 ; FUT8shRNA2, 50 -GCGAATGGCTGAGTCTCTA-30 ; and FUT8shRNA3, 50 -GGCTGGAAACCATTGGGAT-30 ) were synthesized and connected. The FUT8shRNA1 þ 2 þ 3 encoding three rat FUT8shRNAs was cloned into the multicloning site of the AdTrack-CMV shuttle vector containing a GFP reporter gene. FUT8shRNA was Kidney International (2013) 84, 64–77

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cloned using BamHI and HindIII restriction sites between the cytomegalovirus (CMV) promoter and the polyadenylation (polyA) site. The resultant plasmid was linearized by PmeI digestion and subsequently cotransformed into Escherichia coli BJ5183 cells with the AdEasy-1 adenoviral plasmid that contains all sequences of adenovirus serotype Ad5 except nucleotides encompassing the E1 and E3 genes. Recombinant bacteria were selected for kanamycin resistance, and recombination was confirmed by PacI digestion. Finally, the linearized recombinant plasmid was transiently transfected into the packaging HEK293 cells (ATCC, cat. no. CRC-1573, Manassas, VA) that supply the E1 proteins necessary to generate adenovirus, using Lipofectamine 2000 (Invitrogen, Gaithersburg, MD) according to the manufacturer’s protocol. The recombinant Ad-GFP was constructed similarly; the protocol was facilitated because the GFP gene was integrated into the pAdTrack-CMV. Recombinant adenoviruses were released from 293 infected cells by freeze-thawing three times and detected each time by confocal microscopy (Leica Microsystems, Bannockburn, IL), real-time RT–PCR, and western blotting, as described to confirm FUT8shRNA expression. Real-time PCR gene array Total RNA was extracted from the kidney tissue of the Control and UUO7d group using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Total RNA (2 mg) was reverse transcribed using SuperScript II and random hexamers (Invitrogen). The cDNA (20 ng) was added to each well of a PCR array to simultaneously examine the mRNA expression levels of 23 genes (TGF-b Superfamily Receptors and Smads arrays, RT2Profiler PCR Array; SuperArrays Biosciences, Bethesda, MD), according to the manufacturer’s instructions. The expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the DDCt method; fold changes in expression were calculated for each gene as the difference in gene expression between the UUO7d and Control groups. Positive values indicated upregulation and negative values indicated downregulation. Real-time RT–PCR analysis Total RNA was isolated from each sample and first-strand cDNA synthesis was performed using the SYBR PrimerScript RT–PCR Kit (Takara, Otsu, Shiga, Japan) according to the manufacturer’s protocol. The relative expression levels of ALK5, ALK6, ALK7, TGF-bRII, and FUT8 mRNA were examined by real-time PCR using the primers described in Table 1 on a LightCycler (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The PCR products were amplified at 30 s at 95 1C, followed by 40 cycles of 5 s at 95 1C, 20 s at 60 1C, and 30 s at 72 1C, and then assessed using melting curve analysis. The levels of gene expression were calculated using the DDCt method after normalization to GAPDH; all samples were analyzed in triplicate. Detection of fucosylation The levels of core fucosylation in the kidney sections were analyzed using LCA-FITC (Vector Laboratories, Burlingham, CA). Frozen kidney sections (4 mm) were fixed in 4% paraformaldehyde, blocked using 3% (w/v) goat serum for 1 h, incubated with LCA-FITC (1:1000) for 30 min at 25 1C, washed three times with 0.01 M PBS, counterstained with 1 mg/ml 4’,6-diamidino-2-phenylindole for 1 min, and then mounted in an anti-fade medium for examination by fluorescence microscopy. Kidney International (2013) 84, 64–77

Table 1 | RT–PCR primer sequences Gene/primer

Direction

Sequence (50 to 30 )

ALK5

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

GCTGACATCTATGCAATGGGCTTA AGGCAACTGGTAGTCTTCGTGGA ATTGCCCATCGGGACTTGAA GGTGGAATGTCAACCTCATTTGTG AAACATAGTGACCGTGGCTGGAA CTCGGTGAGCAATAGCAGGCTTA CTCGGTGAGCAATAGCAGGCTTA GGGCCATGTATCTCGCTGTTC GCTACCGATGACCCTGCTTTG CCGATTGTGTAATCCAGCTGACC GCACCGTCAAGGCTGAGAAC TGGTGAAGACGCCAGTGGA

ALK6 ALK7 TGF-bRII FUT8 GAPDH

Abbreviations: ALK, activin receptor-like kinase; FUT8, a-1,6 fucosyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT–PCR, reverse transcriptase–PCR; TGF-bRII, transforming growth factor-b receptor II.

Immunoprecipitation Kidney tissues were homogenized in cold RIPA lysis buffer (Laiwen, Jiangsu, China), incubated on ice for 1 h, centrifuged at 12,000 g for 20 min at 4 1C, and then the supernatants were transferred to a clean tube. Lysates were precleared with Protein G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA), and then 500 mg tissue lysate was incubated with 2 mg of anti-TGF-bRII or anti-ALK5 antibodies at 4 1C under rotary agitation for 4 h. Protein–antibody complexes were collected by adding 20 ml of Protein G PLUS-Agarose to each sample, and the mixtures were incubated at 4 1C under rotary agitation overnight. Beads were washed three times and equal amounts of protein were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) for lectin blotting. The primary antibody was omitted in negative controls. Lectin blotting Polyvinylidene difluoride membranes were blocked for 1 h with 100 ml of 5% bovine serum albumin (w/v) at 25 1C with gentle agitation, and then incubated for 2 h at 25 1C in blocking solution containing 1 mg/ml LCA-Biotin (Vector Labs), which preferentially recognizes Fuc-1,6GlcNAc. The membranes were washed with PBS containing Tween-20 six times for 10 min and an ECL kit (Amersham, Pittsburgh, PA) was used to visualize the lectin-reactive proteins. Western blotting Kidney tissues were prepared as described for immunoprecipitation. The protein concentration of the whole lysates was determined using BCA protein assay kits (Pierce Biotechnology, Rockford, IL). Protein samples were heated to 100 1C for 5 min, and 20 ml aliquots were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies against TGF-bRII, ALK5, ALK6, ALK7, Smad2/3, p-Smad2/3, FUT8, TIMP-1, fibronectin, collagen-1, and collagen-3 (1:200; Santa Cruz Biotechnology) in 10 ml dilution buffer (1 Tris-buffered saline, 0.1% Tween-20 with 5% bovine serum albumin), with agitation overnight at 4 1C. Membranes were then incubated with the appropriate horseradish peroxidase–conjugated secondary antibody (1:5000; Zhongshan Biotechnology, Beijing, China) in 10 ml dilution buffer with agitation for 1 h at 25 1C. Bands were detected using an ECL kit (Amersham) and protein expression was quantified using Labworks Image Analysis software (UVP, Upland, CA). 75

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Immunofluorescent analysis of p-Smad2/3 Cryostat sections (4 mm thick) fixed in cold acetone were incubated with rabbit anti-p-Smad2/3 for 1 h, and then with FITC-labeled anti-rabbit IgG (Biosource, Camarillo, CA) for 1 h at 25 1C in the dark. After washing three times in PBS for 3 min, sections were counterstained with 1 mg/ml 40 ,6-diamidino-2-phenylindole for 1 min and mounted in anti-fade medium. Slides were viewed under a Leica TCS-SL confocal microscope (Bannockburn, IL). Staining intensity was analyzed using image analysis software (GraphPad Software, San Diego, CA).

N Shen et al.: TGF-b1-R fucosylation affects renal fibrosis

3. 4. 5.

6. 7. 8. 9.

Double fluorescent labeling of TGF-bRII or ALK5 and core fucose Frozen kidney sections (4 mm) were incubated overnight with primary anti-TGF-bRII antibody (1:100) or anti-ALK5 antibody (1:100) and LCA-TRITC (1:1000; Vector Laboratories), and then incubated with FITC-goat anti-rabbit antibody (1:200; Zhongshan Biotechnology) for 30 min at 25 1C in the dark. After washing three times in PBS for 3 min, the sections were counterstained and mounted as previously described. The slides were viewed and staining intensity was analyzed as previously described. Histology and immunohistochemistry For histological analysis, kidney tissues were fixed in 4% buffered paraformaldehyde and embedded in paraffin. Sections (3 mm thick) were prepared and stained with periodic acid–Schiff, periodic acid–Schiff-methenamine silver, and Masson’s trichrome. For immunohistochemical analysis, the sections were deparaffinized, endogenous peroxidase activity was quenched in 3% H2O2 for 10 min, the sections were washed in PBS, incubated with LCA-Biotin (Vector Labs) or anti-MCP-1 antibodies (Santa Cruz Biotechnology) for 1 h, and then incubated with the appropriate biotinylated secondary antibodies, followed by treatment with avidin–biotin coupling (ABC) reagent (Vector Laboratories) as recommended by the manufacturer. Color development was achieved with the substrate diaminobenzidine. Slides were then counterstained with Mayer’s hematoxylin and mounted in glycerol jelly. Renal fibrotic areas were quantified by morphometric analysis using a light microscope (Carl Zeiss,Thornwood, NY). Quantification of fibrotic areas and positive areas of immunostaining for core fucose and MCP-1 for each antibody in the renal fibrotic regions (brown color) were evaluated by computer-based morphometric analysis (Olympus, Tokyo, Japan).

10.

11. 12.

13.

14. 15.

16.

17. 18.

19.

20. 21.

22. 23.

Statistical analysis Data are expressed as the mean±s.d. values. Statistical analysis was performed using analysis of variance with Tukey’s test post hoc analysis. Statistical significance was set at Po0.05. DISCLOSURE

All the authors declared no competing interests.

24.

25.

26. 27.

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China (NSFC) grant 81070564 (to HL), National Basic Research Program of China grant 2011CB944000 (to HL), and National Natural Science Foundation of China (NSFC) grant 30871125 (to TW).

28.

29. 30.

REFERENCES 1. 2.

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