Role For Wnt/β-catenin Signaling Pathway And Integrin-linked Kinase In Mediating Tubulointerstitial Fibrogenesis In Diabetic Kidney

Objectives: Diabetic nephropathy(DN) is one of the most common complications of diabetes, which is histologically characterized by hypertrophy of glumeruli, increased thickness of basement membrane, overaccumulation of extracellular matrix, glomerulosclerosis, tubular atrophy, interstitial fibrosis and so on. Much more researches have focused on glomerular lesions, little emphasized the crucial role of tubulointerstitial injury in diabetic kidney in the past.It has recently been demonstrated that the development of tubulointerstitial fibrosis is more closely correlated with a progressive decline in renal function compared to glomerularsclerosis. Profibrotic switches in the phenotype of epithelial cells and epithelial–mesenchymal transition (EMT) can be induced by high glucose . Transdifferentiated epithelial cells become reprogrammed to secrete and excessively accumulate extracellular matrix which promote tubulointerstitial fibrosis in diabetic kidney. It is widely recognized that tubulointerstitial injury serves as an important mediator of chronic renal failure and predictor of outcome in patients with diabetic nephropathy.Wnt/β-catenin signalling pathway plays an important role in mammalian developmental processes, tumorigenesis, and organic fibrosis by regulating a variety of cellular processes including cell proliferation, differentiation, survival and apoptosis.Wnt/β-catenin signalling pathway regulates apoptosis of mesangial cells induced by high glucose,and the pathway is activated after unilateral ureteral obstruction, suggesting that the Wnt/β-catenin signalling pathway is likely implicated in the pathogenesis of chronic kidney desease.Integrin-linkedkinase (ILK) is one of the most important downstream effector of TGF-β1 in mediating renal fibrosis.Oversxpression of ILK aggravate extracellular matrix deposition and tubulointerstitial fibrosis in chronic kidney desease , diabetic nephropathy and mouse models of unilateral ureteral obstruction. Activation of the downstream components of the Wnt signaling pathway, namely inhibition of glycogen synthase kinase 3β(GSK -3β)activity and nuclear accumulation ofβ-catenin, has also been achieved through overexpression of ILK. Cross talk between ILK and Wnt signalling pathway likely contribute to the onset and progression of tubulointerstitial fibrosis.Role for Wnt/β-catenin signaling pathway in mediating tubulointerstitial fibrogenesis in diabetic kidney and tubular epithelial–mesenchymal transition induced by high glucose remains to be unknown. Although ILK has been known to regulate the effector activity of Wnt signaling, direct interplay between ILK and the Wnt pathway in diabetic nephropathy has remained to be clarified.In the present study, using STZ-induced diabetic model and HKC cells treated with high glucose ,we investigated the expression of Wnt/β-catenin signaling pathway and ILK and the relationship between them in early stage of DN and transdifferentiated tubular epithelial cells. Furthermore, we investigated the role of ILK in modulating the components expression of Wnt signaling in HKC cells by silencing ILK gene via specific ILK shRNA. At last, the expression of Wnt/β-catenin pathway was examined in renel tissues from patients with diabetic nephropathy to elucidate the potential role of this signaling pathway for tubulointerstitial fibrosis in diabetic kidney,in order to provide experimental and clinical evidences for explaining the mechanism of the development of tubulointerstitial disease in diabetic nephropathy and seeking new treatment target.Methods:1 Preparation of diabetic model and examination of ILK,Wnt4,GSK -3β,β-catenin protein and mRNA expressionMale Wistar rats were randomly divided into two groups after unilateral nephrectomy:control group(n=18), diabetic group(n=18). The rats of diabetic group were injected intraperitoneally with 65 mg/kg body weight STZ in 0.1mol/L sodium citrate solution (pH 4.5), and the rats in the control group were injected with 0.1mol/L sodium citrate solution. Diabetic model was considered to be successful when the blood glucose was≥16.7mmol/L and the urine glucose was +++~++++ after 48 hours of the injection. Six rats from each group were sacrificed respectively at weeks 4, 8 and 12 after STZ injection. Partial renal tissues were fixed in 4% formaldehyde for light microscopic and immunohistochemical staining. Partial renal cortices were used to extracte RNA and total and nuclear protein . The expressions of ILK,Wnt4,β-catenin andα-SMA in renal tissues were evaluated by immunohistochemistry . Western blot was used to examine the expression of ILK,Wnt4,GSK-3β,P-GSK -3β, total and nuclearβ-catenin . The mRNA levels of Wnt4 andβ-catenin were measured by reverse transcription and polymerase chain reaction (RT-PCR).2 Cell culture and examination of ILK,Wnt4,GSK -3β,β-catenin protein and mRNA expressionHuman kidney proximal tubular epithelial cell line (HKC) cells were incubated with serum-free DMEM for 24 hours to synchronize the cell growth,then the cells were divided into three groups:NG group (media containing 5.5mM glucose); mannitol control group (media containing 5.5mM glucose and 24.5mM mannitol); HG group (media containing 30mM glucose). The cells were collected to extracte total RNA and protein at 12, 24, 48 and 72 hours after incubation. The expression of Wnt4,β-catenin, E-cadherin andα-SMA were examined by immunocytochemistry. Western-blot was also used to detect the expression of ILK,Wnt4,GSK-3β,P-GSK-3β, total and nuclearβ-catenin proteins. The mRNA levels of Wnt4,GSK-3βandβ-catenin were examined by RT-PCR .3 Transfection of ILKsiRNA into HKC cells and examination of ILK, GSK -3β,β-catenin protein and mRNA expressionThree small interfering RNA (siRNA) targeting human ILK gene sequences were synthesized to silence ILK gene expression.HKC cells were divided into six groups: NG group, HG group ,HK control group(a vector containing the non-specific siRNA was designed as negative control), pGenesil-1.1-ILK-1siRNA group, pGenesil-1.1-ILK-2siRNA group and pGenesil-1.1-ILK-3 siRNA group. Transient transfection of HKC cells was carried out using Lipofectamine 2000 according to the manufacturer's instruction. Six hours after transfection, the medium of NG group was replaced by normal glucose and the medium of other groups was replaced by high glucose DMEM medium (30mM glucose) with 10% FBS .The cells were incubated for an additional 48h. Fluorescent microscopy was used to examine GFP expression . Then ILK protein and RNA expression was examined by Western-blot and RT-PCR. Immunocytochemistry was used to observe the expression of P-GSK-3βandβ-catenin. The expression of GSK-3β,P-GSK-3β, total and nuclearβ-catenin ,E-cadherin andα-SMA proteins was examined by Western-blot.4 Patients and observation of Wnt4,P-GSK -3βandβ-catenin expressionThirty three patients diagnosed as diabetic nephropathy by renal biopsy and clinical data from October 2006 to October 2008 at the Second Hospital of Hebei Medical University were included in this study.There were 18 male and 15 female patients whose ages were from 28 to 70(48.7±10.3)years old.The cases of lupus nephritis,Henoch-Schonlein nephritis, HBV associated glomerulonephritis, thyropathy associated glomerulonephritis,renal damage induced by rheumatoid arthritis and cancer associated nephropathy were excluded.Those cases complicated with acute renal tubular necrosis,acute or chronic interstitial nephritis were also excluded. The patients were divided into two groups according to the degrees of tubulointerstitial fibrosis: mild /moderate group (n=20)and advanced group(n=13). The renal tissues without evidence of renal disease (n=10) obtained from distant portions of kidneys surgically excised because of the presence of a localized neoplasm were used as control. Clinical data were collected including age, duration of diabetes, plasma albumin,urine protein excretion, serum creatinine and so on. Tubulointerstitial expression of Wnt4,P-GSK-3βandβ-catenin was assessed by immunhistochemical staining.Results:1 Protein and mRNA expression of ILK,Wnt4,GSK-3β,β-catenin in the renal tissues of diabetic rats①Immunohistochemical positive staining of ILK and Wnt4 was observed in renal tubular epithelial cytoplasim whileα-SMA is restricted to the vascular smooth muscle cells in control group. They were all remarkably increased in diabetic group (P<0.05).In control group,β-catenin showed a basolateral localization within the proximal tubules,but was observed in cytoplasm and nuclei of tubular epithelial and renal interstitial cells in diabetic group (P<0.05).②From the results of Western blot analysis, the diabetic rats showed increased expression of ILK ,Wnt4,P-GSK-3βand nuclearβ-catenin in the kidney from week 4, and maintained at a higher level until week 12 (P<0.05). There is no significant difference of GSK-3βand totalβ-catenin protein expression between the groups (P>0.05).③The mRNA levels of Wnt4 in the kidney of diabetic rats increased compared with control group (P<0. 05). No significant difference ofβ-catenin mRNA expression was found between different groups(P> 0. 05).2 The expression of ILK,Wnt4,GSK-3β,β-catenin in HKC cells incubated in HG①Immunocytochemical staining showed that Wnt4 weakly expressed in HKC cytoplasm in NG group and mannitol control group while the expression enhanced after HG stimulation.β-catenin was mainly anchored at the membrane and faintly expressed in cytoplasm of HKC cells in control group . In HG group ectopic expression ofβ-catenin was found in cytoplasm and translocated into nuclei of HKC cells (P<0.05 or P<0.01). In HG group the expression of E-cadherin decreased compared with control groups. The expression ofα-SMA was opposite to that of E-cadherin(P<0.05).②Western blot analysis indicated that the expression of Wnt4,p-GSK-3β,nuclearβ-catenin and ILK were increased in HKC cells of HG group(P<0.05). Wnt4,p-GSK-3βand nuclearβ-catenin expression in HKC reached the peak at 24h and that of ILK peaked at 48h after HG stimulation(P<0.01). No significant difference of GSK-3βand totalβ-catenin protein expression was observed among the three groups (P>0.05).③The mRNA levels of Wnt4 was up-regulated in HG group compared with control group (P<0.05). There was no significant difference of GSK-3βandβ-catenin mRNA expression found among different groups(P>0.05).3 The expression of ILK,GSK-3βandβ-catenin in HKC cells transfected with ILKsiRNA①pGenesil-1.1-ILK-1siRNA, pGenesil-1.1-ILK-2siRNA and pGenesil-1.1-ILK-3 siRNA were transfected into HKC cells to silence ILK expression. At 48 hours after transfection, green fluorescent protein(GFP)was observed in about 60%-70% HKC cells, showing that the transfection is successful.②The ILK mRNA and protein were examined using the methods of RT-PCR and Western blot. Compared with HG and non-specific siRNA transfected control group(HK), the ILK mRNA was significantly inhibited by 36.47%,25.11% and 43.15% respectively in groups transfected with ILKsiRNA(P<0.05).Correspondingly,protein expression of ILK was inhibited by 41.59% , 29.78% and 56.12% respectively(P<0.05).③Immunocytochemical staining showed that P-GSK-3βas well as cytosolic and nuclearβ-catenin expression remarkbly reduced in HKC cells transfected with ILK siRNA compared with that of HG and non-specific siRNA transfected control group(P<0.05).④Western blot showed ILK siRNA suppressed phosphorylation of GSK-3βandβ-catenin translocation into nuclei, whereas the level of P-GSK-3βand nuclearβ-catenin remained increased in HG and non-specific siRNA transfected control group(P<0.05). No significant difference of GSK-3βand totalβ-catenin protein expression was observed among different groups (P>0.05). Downregulation of E-cadherin and upregulation ofα-SMA induced by HG were recovered to a certain degree in HG+ILK-3siRNA group(P<0.05).4 Pathological changes and the expression of Wnt4,P-GSK-3,β-catenin in renal tissues of patients with diabetic nephropathy①pathological changes including glomerular enlargement, mesangial matrix expansion, increase of glomerular and tubular basement membrane in thickeness, focal tubular epithelial vacuolar degeneration and atrophy as well as mild interstitial fibrosis were observed in the patients of mild/morderate group. Progressive glomerulosclerosis with the presence of Kimmelstiel–Wilson lesions, global sclerosis and diffused tubulointerstitial fibrosis could be found in the patients of advanced group.②Immunohistochemical staining displayed that Wnt4 and p-GSK-3βweakly expressed in renal tubular epithelial cytoplasim in control group. The expression remarkably increased in mild/morderate DN group,whereas decreased in advanced DN group(P<0.05). In control kidneys,β-catenin was found to be localized basolaterally within the proximal tubules. Increase of cytosolic and nuclearβ-catenin in both tubular epithelial and interstitial cells were observed in mild/morderate DN group. However, in advanced DN group cytosolic and nuclearβ-catenin is downregulated,and is barely detectable in severe tubulointerstitial lesions (P<0.05).③The expression of Wnt4, p-GSK-3β, nuclearβ-catenin had a positive correlation with urine protein excretion, and a negative correlation with estimated glomerular filtration rate(eGFR) (P<0.05) .Conclusions:1 Wnt/β-catenin signaling pathway activity is up-regulated in the early stage of DN and in the HKC cells incubated in high glucose medium,and the up-regulation is correlated with tubular epithelial–mesenchymal transition, suggesting that the pathway may be involved in the pathogenesis of tubulointerstitial fibrogenesis of DN.2 The expression of ILK is also increased in the early stage of DN and in the HKC cells incubated in high glucose medium, and correlated with tubular epithelial–mesenchymal transition. ILKsiRNA transfection into HKC cells successfully silence ILK expression and reverse tubular epithelial–mesenchymal transition by downregulating the expression of P-GSK-3βand nuclearβ-catenin.Thus,the effect of ILK to modulate the activity of downstream componants in Wnt/β-catenin signaling pathway may be involved in the tubular epithelial–mesenchymal transition3 The expression of Wnt/β-catenin signaling pathway is increased in the patients of mild/morderate DN group,but decreased in the patients of advanced DN group.These changes of the pathway may be contributable to the onset and progression of tubulointerstitial fibrosis of human diabetic nephropathy.