| BackgroundDiabetic nephropathy(DN),a severe microvascular complication of diabetes mellitus,is becoming a major health problem worldwide.DN is a chronic metabolic disorder which involves a chronic capillary pathological change and is a leading cause for end-stage renal disease(ESRD).The underlying mechanisms of DN are complex and multifactorial,and yet the precise mechanisms are not fully known.Thus,a deeper understanding of the mechanisms involved in the pathogenesis of DN is needed in order to provide new effective therapeutic strategies to improve the treatment of renal diseases.Numerous evidences have pointed to the role of long term hyperglycemia-induced oxidative stress and inflammation state in the development of DN.Therefore,alleviating oxidative stress and inflammation state may be a novel prospective approach for DN.Our previous studies demonstrated that grape seed proanthocyanidin extracts(GSPE)exerts a variety of potent protective pharmacological effects on diabetic complications,especially on diabetic kidney.Grape seed procyanidin B2(GSPB2)is one of the main dimeric forms.We investigated the protective effects of GSPB2,a natural dietary antioxidant,on renal complications of db/db mice.In our team previous study,we resorted to a quantitative proteomic assay to get the differential renal protein profiles in GSPB2-treated and untreated diabetic mice.These differential protein profiles provided valuable insight into underlying mechanisms of GSPB2 protective effects on DN.Of all the differentially expressed proteins,an unexpected molecule mimecan showed a decrease in db/db mice versus db/m mice,and back-regulated by GSPB2 treatment.Mimecan is closely related to cardiovascular system,however it has not been found to be involved in the onset and development of DN.Therefore,we assessed the significance of mimecan as a marker to renal dysfunction and investigated whether GSPB2 could prevent renal injury through an anti-inflammatory mechanism in DN.It has been shown that the first intron of the mimecan gene has a binding sequence of nuclear factor kappa B(nuclear factor-kappaB,NF-kappa B).The NF-kappa B plays an important role in many physiological and pathological processes,such as immune response,inflammatory response,and so on.We hypothesized that NF-kappa B might be associated with the corresponding site of the mimecan gene,leading to impaired vascular endothelial cells and increasing the damage to diabetic kidney.On the basis of previous work,we proposed a hypothesis that GSPB2 might improve the inflammatory response of diabetic nephropathy by affecting the activity of the NF-kappa B signaling pathway.Therefore,we researched the expression of mimecan protein by western blot method,observed the NF-kappa B protein activity and investigated whether GSPB2 treatment of diabetic nephropathy was related with the NF-kappa B pathway.Objective1.To evaluate the effects of GSPB2 on renal functional and morphological changes in db/db mice.2.Among the primary protein expression detected by quantitative proteomic assay,mimecan,markedly lower-expressed protein in diabetic kidneys,is a hot spot which may play an important role in the pathogenesis of DN.3.We carried out western blot to confirm mimecan protein expression in db/db mice and in their nondiabetic db/m littermates.4.We further examined wheather GSPB2 affected NF-?B p65 activation by western blotting.5.The expression of fibrotic factors and inflammatory factors in kidney tissues of mice were observed by immunohistochemistry,RT-PCR and western blot.MethodsMale C57BLKS/J db/db(n = 24,7 weeks old)and db/m littermates(n = 12,7 weeks old)were caged at room temperature(22 ± 2 ?)and humidity levels of 50%± 5%under a 12 h light/dark cycle,with free access to tap water and regular diet for 1 week.After adaptation,age-matched db/m mice were assigned to a normal control group(CC,n = 12).Twenty-four db/db mice were divided into two groups at random:the vehicle-treated(normal saline solution)diabetic group(DM,n = 12)and GSPB2-treated diabetic group(DMT,n = 12)that was given daily with 30 mg/kg GSPB2 in normal saline solution orally.All animals were observed for ten weeks without any intervention of hypoglycemic therapy.At the end of the administration,all mice were kept in the metabolic cages to collect 24 h urine samples for measuring urinary albumin excretion.After that,all the animals fasted overnight were sacrificed.Fasting blood was sampled and the serum was stored at-80 ? for various laboratory test and quantification by competitive ELISA.Perfused kidneys were rapidly dissected and then frozen in liquid nitrogen until further analysis.All mice were weighed weekly for ten weeks.Renal tissues were stained with periodic acid-Sciff(PAS)and observed for histopathological changes by light microscopy.In our study,we resorted to western blot to get the differential mimecan protein expression in GSPB2-treated and untreated diabetic mice.To demonstrate the effects of GSPB2 on the DN,40? of nuclear extracts were performed western blot experiment.GAPDH was used as loading control.Each experiment was repeated at least three times.The expression of TGF-beta,collagen I(COL 1),alpha-smooth muscle actin(alpha-SMA)and inflammatory factors IL-6 and TNF-alpha in kidney tissues of mice were observed by immunohistochemistry,RT-PCR and western blot.Results1.General observationThroughout the experimental periods,db/m mice in CC group were in good condition,without any diabetic symptoms.The db/db mice in DM group were polydipsia,polyphagia and hyperdiuresis,with dirty furs and obesity.GSPB2 treated db/db mice looked abnormal,but better than the db/db mice.Body weights were consistently greater in db/db mice than in db/m mice(p<0.05).However,the increase of body weight in db/db mice was significantly attenuated from the second week after GSPB2 treatment(p<0.05).2.Effects of GSPB2 on fasting blood glucose(FBG),total cholesterol(TC),triglycerides(TG),advanced glycation end products(AGEs)and 24 h urinary albumin excretion rateAt the end of experiment,levels of FBG,TG,TC,AGEs and 24h urinary albumin excretion were remarkably elevated in db/db mice when compared to db/m mice.Treatment with GSPB2 to db/db mice induced a significant reduction in TG,TC,AGEs and 24 h urinary albumin excretion.However,GSPB2 treatment did not affect blood glucose levels in db/db mice.3.Effects of GSPB2 on renal histology of db/db miceThe renal section of db/m mice demonstrated normal architecture with normal glomeruli under light microscopy by PAS staining.However,db/db mice exhibited glomerular hypertrophy,mesangial expansion related to mesangial cell proliferation and matrix accumulation,along with visceral epithelial cells proliferation.These alterations in diabetic kidneys were ameliorated to near normalcy after treatments with GSPB2.4.Effects of GSPB2 on the mimecan expression in db/db miceWestern blot analysis showed that mimecan protein expression was significantly decreased in db/db mice compared to db/m mice,and nomalized by GSPB2 treatment,corresponding to the quantitative proteomic assay measurement.5.Effects of GSPB2 on the NF-?B in nuclear extracts of db/db miceThe expression of NF-?B p65 in nuclear extracts was markedly higher in the diabetic mice than in normal control(p<0.05),and was significantly suppressed by treatment with GSPB2,although the level of NF-?B p65 in GSPB2 treated group was still higher than in the control group(p<0.05).6.The expression of TGF-beta,COL I,alpha-SMA and inflammatory factors IL-6 and TNF-alpha in kidney tissues of DN mice were ascending by immunohistochemistry,RT-PCR and western blot.After treatment with GSPB2,the expression of those factors was reversed.Conclusion1.GSPB2 30mg/kg/day treatment significantly inhibit the obesity,decrease the TG,TC,AGEs level,improve renal function and alleviate renal histology injury in db/db mice.2.Mimecan contributed to the fibrosis pathogenesis of diabetic nephropathy.GSPB2 performed potent protective effect on DN by restoring rmimecan and inhibiting of NF-?B signaling pathway.Our research provides a new insight into the pathogenesis of diabetic nephropathy and indicates that targeting mimecan could be a novel strategy to retard the progression of DN.SignificanceIn this study,C57BLKs/J db/db mice were used as animal models of type 2 diabetic nephropathy.Our present study reported that GSPB2 treatment can significantly reduce body weight,decreases TG and TC lever,depress urine albumin excretion and decrease the serum AGEs levels in diabetic mice.These findings indicate oxidative stress(OS)is increased in the diabetic kidney and that GSPB2 can prevent renal damage associated with diabetes by attenuating the oxidative stress.A large number of researches show that oxidative stress and inflammation play a key role in the incidence of diabetic nephropathy.In diabetic nephropathy,oxidative stress can promote the secretion of inflammatory cytokines through different mechanisms.Similarly,inhibition of inflammatory response can significantly reduce oxidative stress in diabetic nephropathy.Oxidative stress and inflammation interact with each other to promote the development of DN,Our research group found mimecan protein expression level was reduced significantly in db/db mice kidneys,while restored after GSPB2 treatment in our western blot analysis,which was consistent with our previous quantitative proteomic analysis.Mimecan can promote the degradation of type I collagen and decrease the degree of fibrosis of skin or other tissues.Our analysis results showed that in diabetic nephropathy,reduced mimecan protein aggravated the deterioration of renal function,and caused the thickening of glomerular basement membrane.After GSPB2 treatment,the restored mimencan protein can increase the degradation of collagen I,reduced thickening of the glomerular basement membrane and plays an important role in protecting the kidney.Many studies have shown that inflammation is an important factor in the nosogenesis of diabetic nephropathy.Our study Indicates that the nuclear factor-kappa B(NF-?B)p65 level was elevated in the nulcler extract of diabetic mice whereas GSPB2 deactivated over-activation of NF-kB p65 in the kidney caused by hyperglycemia,suggesting that GSPB2 could inhibit the NF-kappa B mediated inflammatory response.In this study,NF-kappa B was found to be closely associated with fibrosis,and GSPB2 could effectively inhibit the inflammatory response to diabetes.We hypothesized that the reduced mimecan protein in renal tissue of diabetic mice may induce renal fibrosis by partly increasing the levels of NF-kappa B p65.The mimecan protein expression induced by GSPB2 is at least partly related to the NF-kappa p65 pathway.In conclusion,oxidative stress and inflammation are obvious in diabetic kidney.Mimecan can activate NF-kappa B signaling pathway,increased diabetic kidney inflammation,oxidation and fibrosis,however,GSPB2 may reduce inflammation and fibrosis in kidney of diabetic mice by inhibiting the excessive activation of the NF-kappa B signaling pathway in renal tissue.GSPB2 can improve oxidative stress,inhibit inflammatory response and fibrosis in diabetic nephropathy,which can prevent the development of diabetic nephropathy.BackgroundDiabetic nephropathy(DN)is the most common chronic microvascular complication of diabetes mellitus(DM)and is the leading cause of death in diabetic patients.Studies on the pathogenesis of diabetic nephropathy have previously focused on the accumulation of mesangial matrix and thickening of the glomerular basement membrane(GBM).However,intervention strategies for these factors are not effective in inhibiting the progression of diabetic nephropathy.Therefore,a more in-depth study of other mechanisms of the diabetic nephropathy pathogenesis is of great significance for finding new therapeutic targets.The most prominent clinical manifestation of diabetic nephropathy in the early stage is the increase in glomerular hyperfiltration and urinary albumin excretion.Abnormal changes in the glomerular filtration barrier are the key causes of these symptoms.In recent years,the changes in the structure and function of the glomerular filtration barrier,especially the role of podocytes in the development of diabetic nephropathy,have become the focus of attention and research.Podocyte is a small sac layer of epithelial cells attached to the outside of the glomerular basement membrane(GBM).The podocytes play an important role in maintaining the homeostasis of the glomerulus.It forms a glomerular filtration barrier together with glomerular endothelial cells and basement membrane.Podocyte injury is an early event in diabetic nephropathy that can lead to disruption of the filtration barrier,proteinuria,and ultimately renal failure.Podocyte injury is mainly due to the decrease in the density and number of podocytes.However,the mechanism of podocyte injury in diabetic nephropathy is not completely clear.Therefore,the research on the mechanism of podocyte apoptosis will provide theoretical basis for the occurrence and treatment of DN.MiRNAs are a class of endogenous,non-coding single-stranded small RNAs discovered in recent years that bind directly to the 3' untranslated region(3'-UTR)of their target genes and directly decrease mRNA level at the post-transcription.MiRNAs,which may negatively regulate the expression of target gene protein,plays an important role in the development and differentiation of organisms,organ formation,cell proliferation,apoptosis and even disease,and has become a research hot in recent years.At present,there are increasing evidences that abnormal expression of miRNA is closely related to the occurrence and development of DN.Many studies found that miRNAs in kidney tissue had their unique expression profiles,and the expression levels of miR-192,miR-194,miR-204,miR-215 and miR-216 were relatively higher than those in other tissues.Some researches had reported the role and mechanism of miR-192,miR-194,miR-215 and miR-216 in the development of diabetic nephropathy,but the function of miR-204 in diabetic nephropathy has not been reported.It has been found that miR-204 is associated with diabetes and diabetic retinopathy.However,the specific mechanism of its participation in diabetic nephropathy is still unclear and needs further study.Therefore,in this study we are going to analyze the expression and target of miR-204 in diabetic nephropathy,and to explore the mechanism of miR-204 in diabetic nephropathy.This study focused on the molecular mechanism of diabetic nephropathy,and used mouse models of diabetic nephropathy,diabetic nephropathy patients and high-glycemic mice podocytes as research vectors.Experimental methods,such as RT-PCR,western Blot,luciferase analysis,etc,were used to study the expression level of miR-204 in diabetic nephropathy and verify that its target is Bcl-2.Then the regulational mechanism of Bcl-2 by miR-204 in podocytes was discussed at the cellular level by MTT assay,flow cytometry and other methods.Finally,the effects of miR-204 on the diabetic nephropathy was explored by exogenous administration of miRNA-204 ASO overexpressing lentivirus in DN mouse models.The mechanism provides a theoretical basis for further research on the early specific diagnosis and treatment of diabetic nephropathy.Objective1.To analyze the expression of miR-204 in DN mice,DN patients and podocytes cultured under high glucose.2.To predict and verify the target gene of miR-204.The expression of target gene was detected in renal tissue of DN patients by RT-PCR.Then we transfected podocytes with miR-204 mimics/inhibitors to observe the mRNA and protein expression of target gene.3.Flow cytometry was used to study the effect of miR-204 on apoptosis of podocytes.The expression of apoptosis-related proteins in podocytes transfected with miR-204 mimics/inhibitors was observed by western blot.4.A mouse model of diabetic nephropathy was constructed to study the role of miR-204 in vivo.MethodKidney samples of 59 diabetic patients(31 were DN and 28 non-DN patients as controls)admitted between November 2015 and January 2017 were enrolled.The DBA/2 mice were intraperitoneal given by STZ with a daily dose of 50 mg/kg for 5 days at 10 weeks old and 15 weeks old to prepare a mouse model of diabetic nephropathy.Then the blood glucose,urea nitrogen,serum creatinine and 24-hour urine microalbumin were recorded.The podocytes were cultured in high glucose with glucose concentration of 30 mmol/L(HG group),and podocytes cultured in a medium with a glucose concentration of 5.6 mmol/L were set as normal control group(LG group).The expression level of miR-204 was detected by RT-PCR.TargetScan,PicTar and miRanda were used to predict the target gene of miR-204.The luciferase analysis was used to verify the target which had been predicted.MiR-204 mimics/inhibitors were used to up-regulate or down-regulate miR-204 level in podocytes,and the expression of Bcl-2 mRNA and protein was detected by RT-PCR and western blot.Podocytes cultured in high glucose medium were treated with miR-204 mimics/inhibitors,and the apoptosis of podocytes was detected by Annexin V-FITC/PI double staining.The expression level of Bcl-2 and caspase-3 protein by podocytes was investigated by western blot.We used intraperitoneal injections of STZ to prepare a mouse model of diabetic nephropathy just like before.After the mouse model was successfully established,miRNA-204 ASO overexpressing lentivirus was injected into the DN mouse model through the tail vein.We used PAS staining to study the morphological changes of the kidney,RT-PCR to detect the expression of miRNA-204 and western blot to detect the expression of Bcl-2 protein.Results1.The expression of miR-204 in kidney tissue of DN mice was significantly higher than that of normal mice,which was 1.64 times higher than that of normal mice.The expression of miR-204 in kidney tissue of patients with DN was significantly higher than that of non-DN diabetic patients,which was about 2.06 times that of non-DN.In podocytes cultured in high glucose,the expression level of miRNA-204 was about 2.2 times higher than that in the low glucose,and the difference was significant(p<0.05).2.The target mRNA of miR-204 was predicted and verified as the anti-apoptotic protein Bcl-2.3.The expression of Bcl-2 mRNA in renal tissues of patients with DN was significantly lower than that of non-DN diabetic patients,and the non-DN group was about 1.67 times that of DN.The expression of miR-204 was negatively correlated with the expression of Bcl-2 mRNA.4.Compared with the control group,up-regulation of miR-204 in podocytes significantly inhibited the expression of Bcl-2 mRNA and protein;conversely,the expression of Bcl-2 mRNA and protein was significantly increased after down-regulation of miR-204 expression(p<0.05).5.After podocytes cultured in high glucose medium were treated with miR-204 mimics/inhibitors,flow cytometry results showed that the apoptosis of podocytes in the scramble control group was comparable to that of the blank control group.Compared with the scramble control group,miR-204 mimics could induce podocyte apoptosis,while miR-204 inhibitors could protect podocytes from apoptosis(p<0.05).Western Blot results showed that compared with the control group,miR-204 inhibited Bcl-2 protein expression and increased caspase 3 protein expression.MiR-204 inhibitors had the opposite effect.6.After the DN mouse model was successfully established,renal histological sections stained with PAS were depicted under light microscope.Mesangial matrix hyperplasia,mesangial cell proliferation,glomeruli and renal tubular epithelial swelling were observed in DN mice.Scramble control group showed similar changes with DN group.MiR-204 ASO group showed no obvious change in proliferation of glomerular mesangial cells and in glomeruli swelling.After miRNA-204 ASO overexpressing lentivirus was injected into DN mouse modle,the expression of miRNA-204 was lower than that of the scramble control group and DN group by RT-PCR(p<0.05).Western blot results showed that the protein expression of Bcl-2 in the kidneys of DN mice was significantly decreased compared with blank control(p<0.05).However,the protein expression of Bcl-2 was significantly increased after microRNA-204 ASO overexpressing lentiviruses was injected(p<0.05).Lonclusion1.The expression of miR-204:The expression level of miR-204 in the kidney tissues of DN mouse model,kidney tissues of DN patients and podocytes cultured in high glucose was increased significantly,which preliminarily confirmed that miR-204 was involved in the pathogenesis of DN.2.Target prediction of miR-204 and validation:Bcl-2 was identified as the target gene of miR-204 by using on-line prediction software and luciferase reporter vectors.The expression level of Bcl-2 mRNA in renal tissue of DN patients was significantly lower than that of non-DN patients,and the expression level of miR-204 was negatively correlated with that of Bcl-2 mRNA.In podocytes,overexpression of miR-204 significantly inhibited the expression of Bcl-2 mRNA and protein,and down-regulation of miR-204 significantly increased the expression of Bcl-2 mRNA and protein.These results suggest that miR-204 might be involved in the development of DN through regulating Bcl-2 expression.3.Functional detection of miR-204:Annexin V-FITC/PI double staining showed that miR-204 promoted podocyte apoptosis.Western blotting showed that miR-204 mimics could inhibit the expression of Bcl-2 protein and increase the expression of caspase 3 protein;while miR-204 inhibitors had the opposite effect.The research showed that miR-204 promotes podocyte apoptosis by inhibiting Bcl-2 expression.4.Effects of miR-204 in DN mice:PAS staining results showed that the proliferation of glomerular mesangial cells and glomeruli swelling were significantly improved after miR-204 ASO overexpressing lentiviruses was injected.The expression of Bcl-2 protein was significantly increased in DN mice after injecting miR-204 ASO overexpressing lentivirus.These results suggested that miR-204 could promote podocyte apoptosis by targeting down-regulating the expression of anti-apoptotic protein Bcl-2 and could increase the risk of DN.SignificanceBased on the molecular mechanism of diabetic nephropathy,this study systematically found the expression level of miR-204 in kidney tissues of DN and the mechanism of miR-204 in the pathogenesis and development of diabetic nephropathy by inhibiting Bcl-2 pathway.This study explored the regulatory mechanism of miR-204 in the development of DN in vitro and in vivo,and predicted that miR-204 might be a potential biomarker and therapeutic target for DN.We successfully predicted and verified that Bcl-2 was the target gene of miR-204 and revealed the molecular mechanism of miR-204 in diabetic nephropathy on the cellular level.MiR-204 ASO overexpressing lentiviruses could specifically up-regulate the signal transduction of Bcl-2 in DN mice,and delay or even block the progression of diabetic nephropathy.In this study,we discovered that miR-204 could promote podocyte apoptosis and increase the risk of diabetic nephropathy by targeting the falling expression of anti-apoptotic protein Bcl-2.Therefore,miR-204 might be a potential therapeutic target for diabetic nephropathy. |
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