| BackgroundDiabetes mellitus (DM) is a noncommunicable chronic disease worldwide. Its prevalence has increased significantly in recent decades. It negatively affects a patient’s quality of life and social environment, and poses a burden on national health care budgets. Diabetic kidney disease (DKD) is a major microvascular complication of DM and the main cause of end-stage renal disease. It has been recognized as the leading cause of renal disease worldwide, occurring in 30%-40% of patients with DM.Although DKD was traditionally considered as a glomerular disease, it is now widely accepted that the deterioration rate of renal function correlates best with the degree of renal tubulointerstitial fibrosis (TIF). Recent studies suggested that proximal tubular changes were evident even in early stage. Thus, it is important to explore how to delay tubulointerstitial fibrosis to improve the prognosis of the patients.Although the precise mechanisms mediating the pathogenesis of TIF remain unclear, a growing body of evidence indicates that apoptosis of renal tubular cells combined with epithelial-mesenchymal transition is the major cause. The term "EMT"is used preferentially to describe the conversion of terminally differentiated epithelial into cells with a mesenchymal phenotype. It is the claasical characteristic of renal tubulointerstitial fibrosis in DKD. A wide range of animal studies have established TGF-β1 as the predominant pathogenic factor that drives glomerular and tubulointerstitial fibrosis. It induces renal fibrosis through several mechanisms: by causing cell loss through apoptosis; by acting directly on fibroblasts and other cell types to induce extracellular matrix (ECM) synthesis and reduce ECM degradation;and by inducing the transition of various cell types into fibroblast-type cells that are capable of depositing ECM in injured kidney. TGF-β1 can induce cultured tubular epithelial cells to differentiate into cells with a distinct myofibroblast morphology(including altered polarity, actin microfi laments and dense bodies) and marked upregulation of collagen production. These changes are predominately mediated by Smad signaling. Specifically, TGF-β stimulation promotes Smad2 and Smad3 phosphorylation, Smad complex formation. and nuclear accumulation that ultimately result in the induction of a profibrotic gene expression program. In addition,activation of Smad2/3 has been observed in both experimental and human diabetic kidney, implicating that activation of the renal TGF-P system plays a critical role in the pathogenesis of DKD.The Chinese medicine Astragalus is the root of the legumous herb Mongolia Astragalus and Astragalus membranaceus. Astragaloside IV (ASI) is the main active component of astragalus,and it has been shown to have broad application prospects in acute kidney injury, membranous nephropathy and DKD. However, few research concentrated on renal tubulointerstitial fibrosis in DKD.ObjectiveTo investigate ASI effect on TGF-β/Smad pathway in kidney tissue of diabetic KKAy mice and high glucose-induced tubular epithelial cells (NRK-52E) to reveal its potential impact on renal interstitial fibrosis and provide basis for its clinical application.Methods1. Mice were purchased and given 2 weeks adaptive feed. KKAy mice were fed with high-fat diet to 14 weeks old. Diabetic model was determined according to the random blood glucose level > 13.9 mmol/L. KKAy mice with similar blood glucose level were randomly equally divided into model group and drug group. ASI at 40mg/(kg·d) was given to the drug group through intragastric administration. 10 C57BL/6J mice were selected as normal control with normal diet. The mice in the normal control and model group received normal saline at 40 mg/ (kg·d).2. Mice general status was observed daily. The body weight were recorded every week throughout the experiment. Blood glucose and creatinine were detected at different time points. Urine creatinine and microalbuminuria were also tested to calculate ACR.3. The mice were sacrificed at 24 weeks old and the kidney tissue samples were collected. Renal tissue was received conventional HE staining and Masson staining,and observed under the light microscope.4. Renal tissue TGF-β1 Smad2/3, and α-SMA expression were detected by immunohistochemistry according to the SABC kit manual.5. We examined the effect of ASI on cell viability to determine whether the inhibitory effect of ASI was related to the cytotoxicity induced by this compound.Viability of NRK-52E cells was determined by cell counting kit-8 (CCK-8) assay.6. We detected apoptotic rate by flow cytometric analysis. Cells were treated with increasing concentrations of ASI (20, 40, 80 and 100μg/ml) for 24 h in high glucose and we also stimulated cells in ASI 100μg/ml with high glucose for various lengths of time. The apoptosis rate was detected according to the manufacturer’s instructions of the Annexin V-FTIC Apoptosis Detection Kit.7. Cells were treated with increasing concentrations of ASI (20, 40, 80 and 100μg/ml) for 24h in high glucose. The mRNA and protein expression of TGF-β1,α-SMA. Smad2. Smad3 were detected by real-time PCR and Western Blotting.respectively.8. All statistical analyses were performed using SPSS 19.0 software. Numerical data were presented as means and standard deviation. Differences between multiple groups were analyzed by t-test or one-way ANOVA and LSD test. P < 0.05 was considered as significant difference, while P < 0.01 as remarkable difference.Results1. Mice in control group showed sensitive response and smooth hair. Mice in model group exhibited weak mental state, slow movement, polydipsia and polyuria,insensitive response, and rough hair. The symptom became worse gradually following the age. Mice in ASI group presented better mental state and response sensitivity than the model group, but worse than the control. At each observation time points, mice in model and ASI group were heavier than control group (P < 0.01). Compared to model group, weights of mice in model group were growing more slowly (P < 0.05).2. ASI impact on blood glucose, creatinine and urine ACR: At each observation time points, KKAy mice showed significantly higher fasting blood glucose and urine ACR level than the control (P < 0.01). Compared to model group, mice in ASI group showed lower glucose and urine ACR (P < 0.05, P < 0.01, respectively). Blood creatinine showed no difference among three groups .3. ASI impacts on kidney tissue histomorphology: 24 weeks mice in control showed clear glomerular and renal tubular structure without interstitial fibrosis. Mice in model group exhibited increased glomerular mesangial matrix, significantly proliferated mesangial cells, renal tubular epithelial cell cytoplasm vacuoles degeneration, and increased renal interstitial inflammatory cells. Renal tubular cytoplasm vacuoles were relatively fewer in the mice from the ASI group with no significant fibrosis.4. ASI impacts on TGF-β1 Smad2/3, and a-SMA expression on kidney tissue:TGF-β1 expressed weak in the control, while it expressed strongly in the renal tubular epithelial cell cytoplasm in model and ASI group (P < 0.01,P< 0.05, respectively).Compared with the model group. TGF-β1 decreased obviously in the ASI group (P <0.01) . Cytoplasm α-SMA expression increased in model group and ASI group.Specially, it significantly reduced in ASI group compared with the model group (P <0.01). There was less Smad2/3 in the renal tubular and glomerular cell nucleus in the control group. It increased markedly in the model group and ASI group (P < 0.01, P <0.05, respectively), while it declined in the ASI group compared to model group (P <0.01).5. Effects of ASI on cell viability: Cells were treated with ASI at concentrations of 0, 10, 20, 40, 80 and 100 μg/ml for 24 h. The results showed that ASI at the above concentrations had no inhibitory effect on cell viability.6. Effects of ASI on cell apoptosis: Compared to control group, treatment with high glucose (25 mmol/L) significantly increased cell apoptosis (P < 0.01). However,ASI inhibited high glucose-induced cell apoptosis in a dose-dependent manner, with a maximal inhibitory effect achieved at 100 μg/ml. The significant inhibition caused by ASI was observed at 8h after the start of pretreatment and increased in a time-dependent manner.7. Effects of ASI on mRNA expression of TGF-β1 a-SMA, Smad2, Smad3 : The mRNA expression of Smad2, TGF-β1, a-SMA and Smad3 was higher in HG and HG+ASI 20 group as compared to the control group (P < 0.01, P < 0.01, P < 0.01 and P < 0.05, respectively). Besides, the mRNA expression of a-SMA in HG+ASI 40 was also upregulated as compared to the control group (P < 0.01). The mRNA expression of TGF-β1, Smad3,a-SMA and Smad2 was lower in HG+ASI 80 and HG+ASI 100 group as compared to HG group (P < 0.01 , P < 0.05, P < 0.05 and P < 0.05,respectively). Whereas TGF-β1 expression in HG+ASI 40 and Smad2 in HG+ASI 20 was also decreased (P < 0.01 and P < 0.05, respectively).8. Effects of ASI on protein expression: The expression of Smad2, p-Smad2.Smad3, p-Smad3 and α-SMA was higher in HG,HG+ASI 20 and HG+ASI 40 group as compared to the control group (P < 0.01, respectively), and p-Smad2 and p-Smad3 were also increased in HG+ASI 80 group (P < 0.01, respectively). Whereas TGF-β1 was upregulated in HG, HG+ASI 20 group as compared to the control group (P <0.01. respectively). All indexes were downregulated in HG+ASI 80 and HG+ASI 100 group as compared to HG group (P < 0.05, respectively), and TGF-β1 was also decreased in HG+ASI 40 group (P < 0.05).Conclusions1. ASI alleviated high glucose, weight and urine ACR in diabetic KKAy mice.but didn’t affect blood creatitine.2. ASI ameliorated TIF in diabetic KKAy mice by down-regulating TGF-β/Smad signaling pathway.3. ASI inhibited NRK-52E cells apoptosis induced by high glucose in dose-and time-dependent manners.4. ASI reduced high glucose induced expression of TGF-β1. α-SMA. Smad2,Smad3 both at the mRNA and protein level in NRK-52E cells, thus alleviated EMT and delayed TIF. |
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