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Objective:To investigate the expression of integrin-linked kinase (ILK)/zinc finger transcription factor (Snail) signaling pathway in renal tubular epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN) and the effect of rhein.Methods:Healthy male Wistar rats of 8 weeks old were randomly divided into normal group, diabetic nephropathy group, rhein intervention group and valsartan intervention group, with 12 rats in each group. Streptozotocin (STZ) was used to induce the diabetic nephropathy model, then rhein intervention group and valsartan intervention group were given rhein 100 mg/(kg·d) and valsartan 30 mg/(kg·d), respectively. At the end of the 8th and 16th week, six rats of each group were killed, in situ lavage kidney, take out the kidney tissue of rats after fixed in wax block and slices. Renal tubular interstitial damage index and the relative area of interstitial collagen evaluated by hematoxylin-eosin (HE) and Masson staining respectively. The protein expression of E-cadherin, α-smooth muscle actin (α-SMA), ILK, Snail and matrix metalloproteinase-9 (MMP-2) in renal tubular epithelial cells were detected by immunohistochemistry.Results:Comparing to normal group, the renal tubular interstitial damage index and relative area of renal interstitial collagen of diabetic nephropathy rats were both increased. The expression of E-cadherin in renal tubular epithelial cells decreased and the expression of α-SMA significantly increased ( P<0.05). Comparing with diabetic nephropathy group, in rhein and valsartan intervention groups, the expression of E-cadherin in renal tubular epithelial cells increased, while the expression of α-SMA significantly decreased ( P<0.05). There was no significant difference in the above indexes between rhein intervention group and valsartan intervention group ( P>0.05). Compared to normal group, the expressions of ILK, Snail, MMP-2 increased progressively with the disease ( P<0.05). Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed significant decrease in expression of ILK, Snail, MMP-2 ( P<0.05). There was no significant difference in the above indexes between rhein intervention group and valsartan intervention group ( P>0.05). Conclusions:Rhein could inhibit EMT progression by down-regulating the expression of ILK/Snail signaling pathway in renal tubular epithelial cells of DN rats.
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Objective To investigate the possible regulating effect of integrin-linked kinase (ILK) towards matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 (MMP-9/TIMP-1) ratio in the process of transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial (HK-2) cells.Methods HK-2 cells were cultured and stimulated with 10 ng/ml TGF-β1.Specific ILK-small interfering RNA (ILK-siRNA) was used to inhibit ILK expression.The characteristic epithelial marker (E-cadherin) and mesenchymal marker (α-SMA) of EMT were examined by Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blot.The expressions of ILK,MMP-9,and TIMP-1 were also examined,to determine the regulating effect of ILK towards MMP-9/TIMP-1 ratio.Results In the HK-2 cells cultured with TGF-β1,the expression of E-cadherin decreased,and α-SMA expression increased;overexpression of ILK and an abnormal changing of MMP-9/TIMP-1 ratio were observed.ILK inhibition by ILK-siRNA could adjust MMP-9/TIMP-1 ratio to near normal.Meanwhile,the overexpressed ILK and α-SMA were decreased.Conclusions Our data indicates that ILK-siRNA successfully inhibits ILK expression,which regulates the MMP-9/TIMP-1 ratio in HK-2 cells.The inhibition of ILK expression suppresses TGF-β1-induced EMT partially.
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Objective To observe the expressions of c-Ski in renal tissue of diabetic nephropathy rats, and discuss its significance. Methods Wistar male rats were randomly assigned to 3 groups: Control group, diabetic group and treatment group. Rats were killed at the 16th week after experiment. Histopathologic changes,in renal tissue were observed and immunohistochemistry was performed to investigate the expression of TGF-β1, α-SMA and c-Ski. Results The expression of c-Ski had a negative correlation with TGF-β1 and α-SMA. Compared to diabetic group, the expression of c-Ski was significantly increased in treatment group. Conclusion c-Ski may be a protective factor of tu-bular epithelial-myofibroblast transformation.
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Objective To study the effect of rhein on the process of tubular epithelial-mesenchymat transformation in kidney of diabetic rats. Methods Wistar male rats were randomly assigned to 3 groups: Control group (N group, n=12),diabetic group(D group, n=12), rhein treatment group(R group, n=12).The rats of rhein treatment group were treated with daily intragastric administration of periment. The excretion of urinary protein and serum creatine were measured. Histological changes of renal tissue were observed by HE and MASSON stain. Immunohistochemistry was performed to investigate the expression of E-cadherin, α-SMA,FN and TGF-β1 in kidney. Results Compared with the control group, the tubulointerstitial injury and the accumulation of extraeellular matrix protein in diabetic models were obvious(P＜0.01).Compared with the control group, the expression of E-cadherin was decreased significantly and the expression of α-SMA,FN and TGF-β1 was increased significantly in diabetic group. E-cadherin was negatively correlated with TGF-β1(rs=-0.60,P＜0.05),α-SMA and FN was positively correlated with TGF-β1(rs=0.88,P＜0.05;rs:0.91,P＜0.01).In comparison with diabetic group,rhein could up-regulate the expression of E-cad and down-regulate the expression of α-SMA and FN in renal tubular epithelial cells(P＜0.01).Conclusion Rhein could protect kidney by ameliorating interstitial fibrosis in diabetic rats. The mechanism may be depend on down-regulating the expression of TGF-β1 and suppressing tubular epithelial-mesenchymal transformation.
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Objective To determine the association between asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, with atherosclerosis in patients with chronic kidney disease (CKD). Methods One hundred thirty-eight CKD patients were enrolled in this study. Serum levels of L-arginine, ADMA, and SDMA were measured by high-performance liquid chromatography (HPLC). Common carotid arteries intimae-medial thickness (CCA-IMT) ,cross-sectional calculated intimae-medial thickness(cIM area)and atherosclerotic plaque were detected by noninvasive high-resolution B-mode ultrasonography. Results Serum levels of ADMA and SDMA were significantly increased in CKD patients (n=138) compared with age matched healthy subjects (n=42,P＜0.01). ADMA and SDMA levels increased with the progression of renal dysfunction and were negatively related to creatinine clearance (Ccr) in pre-dialysis patients (r=-0.315,P＜0.05;r=-0.426,P＜0.01). Serum levels of ADMA and SDMA in dialysis patients (n=74)were significantly higher than those in pre-dialysis patients (P＜0.05). Patients with carotid artery plaques showed significantly higher levels of ADMA compared with those without plaques. Serum levels of ADMA closely correlated with the mean IMT (r=0.471, P＜0.01) and cIM area value (r=0.430, P＜0.01). These correlations remained significant even after adjusting GFR,age,gender ,and other risk factors for atherosclerosis in the multiple regression analysis. Conclusion Serum levels of ADMA increased with the progression of CKD and may play a role in the pathogenesis of accelerated atherosclerosis in CKD patients.