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AIM:Bone morphogenetic protein 7(BMP7)reduces the expression of Yes-related protein 1(YAP1)by down-regulating Ajuba level and decreasing extracellular matrix(ECM)deposition.This study aimed to inves-tigate the influence of these factors on modifying the degree of renal fibrosis in rats with diabetic nephropathy.METH-ODS:Eighteen Sprague-Dawley(SD)rats were randomly divided into three groups:the normal control(NC)group,the diabetes mellitus(DM)group,and the DM group treated with BMP7 overexpressing adeno-associated virus(DM+rAAV-BMP7).Each group consisted of six rats.Diabetic kidney disease(DKD)was established in the DM and DM+rAAV-BMP7 groups by injecting 55 mg/kg streptozotocin(STZ)via the tail vein.NRK-52E cells were divided into three groups:the normal glucose(NG)group,the high glucose(HG)group,and the high glucose group treated with recombinant hu-man BMP7(HG+rhBMP7)group.Pathological changes in renal tissues were observed using hematoxylin and eosin(HE)and Sirius red staining.Immunohistochemical staining was performed to examine the expression sites of Ajuba and YAP1 in the renal cortex.Western blot analysis was conducted to determine the expression levels of BMP7,Ajuba,YAP1,colla-gen type Ⅲ(Col-Ⅲ),and fibronectin(FN)in the rat renal cortex and NRK-52E cells.RT-qPCR was used to measure the mRNA levels of Ajuba and YAP1 in the rat renal cortex.RESULTS:Biochemical indices revealed significantly ele-vated levels of blood glucose,serum creatinine,triglycerides,total cholesterol,and 24-hour urinary protein in the DM group compared to the NC group(P<0.05).In the DM+rAAV-BMP7 group,the levels of serum creatinine,24-hour uri-nary protein,triglycerides,and total cholesterol were lower than those in the DM group(P<0.05).Pathological staining demonstrated that the renal interstitium of the DM group exhibited inflammatory cell infiltration,fibrous tissue,collagen fi-ber deposition,disordered renal tubule arrangement,atrophy,and vacuolar degeneration,which were ameliorated in the DM+rAAV-BMP7 group.Immunohistochemistry revealed that Ajuba and YAP1 were mainly expressed in the cytoplasm and nucleus,with high expression in the cytoplasm of the DM group,which was significantly decreased in the DM+rAAV-BMP7 group.Western blot results indicated that the protein levels of FN,Col-Ⅲ,Ajuba,and YAP1 were up-regulated in the DM and the HG groups(P<0.05),but significantly down-regulated in the DM+rAAV-BMP7 group(P<0.05).RT-qP-CR results demonstrated that the mRNA levels of Ajuba and YAP1 were higher in the DM group and significantly lower in the DM+rAAV-BMP7 group(P<0.05).CONCLUSION:The overexpression of BMP7 can ameliorate renal fibrosis in rats with DKD.This effect is likely mediated by the down-regulation of Ajuba,reduction of YAP1 expression,and subse-quent inhibition of ECM deposition.
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Objective:To investigate the lipidomics differences of CD4+T immune cells in diabetic kidney disease(DKD)mice,and screen out the differential metabolites with biological significance.Methods:CD4(L3T4)MicroBeads immunomagnetic beads were used to isolate CD4+T immune cells from spleen of BKS.Cg-Dock7m+/+Leprdb/J mice with spontaneous DKD;the purity of CD4+T cells were identified by flow cytometry.The non-targeted lipidomics of CD4+T cells were detected by LC-MS/MS,and the differ-ential metabolites were analyzed.Results:A total of 463 metabolites were detected by LC-MS.PCA and OPLS-DA analysis showed that the metabolic components were significantly separated;twenty-four differential metabolites were screened out.KEGG and enrich-ment analysis showed that the differential metabolites involved in the disorder of glycerol phospholipid metabolism.Conclusion:Phos-pholipid metabolism of CD4+T cells is closely related to the occurrence of DKD.Phospholipid metabolism targeting DKD CD4+T cells in DKD may be a new direction of DKD treatment.
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Objective To investigate the effect of Dapagliflozin on high glucose-induced podocyte proliferation and apoptosis through p38 mitogen-activated protein kinase(p38 MAPK)pathway.Methods Human glomerular podocytes(HGPC)were divided into control(Con)group,low/medium/high D-glucose(Glu 10,Glu 20,Glu 30)group,high glucose(HG)group,low/medium/high concentration Dapagliflozin(HG+Dap 12.5,HG+Dap 25,HG+Dap 50)group,Dapagliflozin(HG+Dap)group,inhibitor(HG+ SB 203580)group,Dapagliflozin + inhibitor(HG+Dap+SB 203580)group and Dapagliflozin + activator(HG+Dap+C16-PAF)group.After 24 hours of intervention,the cell viability,proliferation rate,apoptosis rate and levels of related factors were tested.Results Compared with Con group,IL-1β,TNF-α,apoptosis rate,Caspase-3 mRNA and protein expression,p53,p-p38 MAPK protein expression were increased(P＜0.05),while cell proliferation rate,Cyclin D1 mRNA and protein expression were decreased in HG group(P＜0.05).Compared with HG group,the proliferation rate,Cyclin D1 mRNA and protein expression were increased(P＜0.05),while IL-1β,TNF-α,apoptosis rate,Caspase-3 mRNA and protein expression,p53,p-p38 MAPK protein expression were decreased in the HG+Dap and HG+SB 203580 groups(P＜0.05).Compared with HG+Dap group,cell proliferation rate,Cyclin D1 mRNA and protein expression were increased(P＜0.05),while IL-1β,TNF-α,apoptosis rate,Caspase-3 mRNA and protein expression,p53,p-p38 MAPK protein expression were decreased in HG+Dap+SB 203580 group(P＜0.05).In HG+Dap+C16-PAF group,IL-1β,TNF-α,apoptosis rate,Caspase-3 mRNA and protein expression,p53,p-p38 MAPK protein expression were increased(P＜0.05),while cell proliferation rate,Cyclin D1 mRNA and protein expression were decreased(P＜0.05).Conclusion Dagagliflozin can promote HGPC proliferation and inhibit apoptosis and inflammation in high D-glucose environment,and its mechanism may be related to the inhibition of p38 MAPK pathway signal transduction.
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Objective To investigate the effect of miR-130a targeting phosphase and tensin homology deleted on chromosome ten(PTEN)/phosphoinositide 3 kinase(PI3K)/protein kinase B(AKT)pathway on renal tissuecell apoptosis in diabetic kidney disease(DKD)rats.Methods The DKD rat model was constructed by feeding high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin(STZ).72 rats were divided into normal control group(NC),DKD model group(DKD),miR-130a agonist negative control group(NC agomir),and miR-130a agonist group(miR-130a agomir),miR-130a agomir+ PTEN overexpression negative control group(miR-130a agomir+pcDNA),and miR-130a agomir+ PCDNA-PTEN overexpression group(miR-130a Agomir + PCDNA-PTEN),with12 rats in each group.Urinary microalbumin kit was used to detect 24 h urine albumin(UAlb).Fasting blood glucose(FBG),serum creatinine(Scr)and blood urea nitrogen(BUN)were detected by automatic biochemical analyzer.Pathological changes of renal tissue were detected by HE staining.The levels of serum IL-6 and TNF-α were detected by ELISA.The apoptosis of renal tissue was detected by TUNEL staining.The expression of miR-130a was detected by qRT-PCR,and the expression of B-cell lymphoma-2-associated X protein(Bax),B-cell lymphoma-2(Bcl-2)and PTEN/PI3K/AKT pathway were detected by Western blot.Dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-130a and PTEN.Results Compared with DKD and NC agomir groups,24 h UAlb,FPG,Scr,BUN,IL-6,TNF-α,renal cell apoptosis rate,Bax protein expression and PTEN protein expression in miR-130a agomir group were decreased(P＜0.05).The expressions of miR-130a,Bcl-2,p-Akt/AKT protein were increased(P＜0.05).Compared with miR-130a agomir group,24 h UAlb,FPG,Scr,BUN,IL-6,TNF-α,renal cell apoptosis rate,Bax protein expression and PTEN protein expression were increased in miR-130a agomir+pcDNA-PTEN group(P＜0.05).The expression of Bcl-2,p-Akt/AKT protein decreased(P＜0.05).Conclusion Overexpression of miR-130a may inhibit renal cell apoptosis in DKD rats by down-regulating PTEN to activate PI3K/AKT pathway.
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Objective To investigate the protective effect and mechanism of oleanolic acid(OA)on kidneys in rats with type 2 diabetes mellitus(T2DM).Methods A total of 35 Sprague-Dawley(SD)rats were enrolled in this study.25 SD rats were randomly selected to establish T2DM model,after modeling,20 rats remained and divided into T2DM group(n=6),low-dose oleanolic acid group(LOA,n=6)and high-dose oleanolic acid group(HOA,n=8).And ten rats were selected as normal control group(NC,n=10).The biochemical indicators,24 h urine volume and 24 h urinary microalbumin(UAlb)were compared among the four groups.Renal lipid deposition was evaluated by Oil red O staining.The protein expressions of Adenosine 5'-monophosphate-activated protein kinase(AMPK),p-AMPK and peroxisome proliferator-activated receptor γ coactivator-1 α(PGC-1 α)were detected by Western blot.Results Compared with the NC group,the levels of 24 h urine volume,fasting blood glucose(FPG),serum total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C),serum creatinine(Scr),serum uric acid(SUA)and 24 hUAlb were increased(P<0.05),while the body weight,high density lipoprotein cholesterol(HDL-C),p-AMPK and PGC-1α were decrease in the T2DM group(P<0.05).Compared with the T2DM group,the expressions of HDL-C,p-AMPK and PGC-1α were increased(P<0.05),while the levels of 24 h urine volume,FPG,TG,TC and LDL-C,Scr,SUA and 24 hUAlb were decreased in the LOA and HOA groups(P<0.05).Compared with LOA group,the expressions of HDL-C,p-AMPK and PGC-1α were increased(P<0.05),while the levels of 24 h urine volume,FPG,TG,TC,LDL-C,Scr,SUA and 24 h UAlb were decreased in the HOA group(P<0.05).Compared with the NC group,a large number of red-stained lipid droplets were deposited in the renal tubular epithelial cells in the T2DM group.Compared with the T2DM group,the lipid droplet deposition was reduced in the LOA and HOA groups,and the improvement was more significant in the HOA group.Conclusion OA can alleviate renal injury in T2DM rats,which may be linked to activation of AMPK/PGC-1α pathway.
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Diabetic kidney disease(DKD)is a common microvascular complication of diabetes mellitus.Exosomes play a key role in mesangial cell proliferation,podocyte apoptosis,and epithelial mesenchymal transformation induced by renal tubular epithelial cells,which are important links in the pathogenesis of DKD.Based on the correlation of abnormal function of exosomes of DKD with theory of"disperse essence by spleen qi"and the theory of"kidney collaterals stasis"in TCM,this article reviewed the research progress and regulation mechanism of TCM in regulating exosomes to intervene in diabetic kidney disease,providing reference for related research.
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Objective:To analyze the level of interleukin-36(IL-36) family cytokines in peripheral blood, and explore the regulatory role of recombinant human IL-36α in monocyte function in patients with diabetic kidney disease(DKD).Methods:A total of 41 type 2 diabetes mellitus(T2DM) patients, 36 DKD patients, and 20 controls were consecutively enrolled. Plasma and peripheral blood mononuclear cells(PBMCs) were isolated. Enzyme-linked immunosorbent assay(ELISA) was used to measure plasma levels of IL-36α, IL-36β, IL-36γ, and IL-36 receptor antagonist(IL-36Ra). PBMCs were sorted, and real-time quantitative PCR was performed to detect the mRNA expression of IL-36 receptor subunits in monocytes. Monocytes were stimulated with recombinant IL-36α, and levels of cytotoxic molecules and cytokines in the culture supernatant were measured. Flow cytometry was used to assess the expressions of programmed death receptor-1(PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA-4). Co-culture of monocytes with Vero cells was performed to evaluate monocyte cytotoxicity.Results:Plasma levels of IL-36α and IL-36β in the T2DM and DKD groups were significantly higher than those in the control group. The DKD group also showed higher plasma levels of IL-36α compared to the T2DM group( P<0.05). There were no significant differences in IL-36γ and IL-36Ra levels among the three groups( P>0.05). The mRNA expression of IL-36 receptor subunits in monocytes was comparable among the three groups( P>0.05). The DKD group had higher level of tumor necrosis factor-alpha(TNF-α) compared to the control and T2DM groups( P<0.05). The levels of PD-1 and CTLA-4 were lower in the DKD group than those in the control and T2DM groups( P<0.05). The proportion of monocyte-induced Vero cell death was significantly higher in the DKD group compared to the control and T2DM groups( P<0.05). After stimulation with recombinant human IL-36α, monocytes from DKD patients showed a significant increase in the secretion of granzyme B and TNF-α( P<0.05), as well as an increased proportion of monocyte-induced Vero cell death( P=0.024). Conclusion:In DKD patients, elevated IL-36α and granzyme B levels in monocytes enhance monocyte function.
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Objective:To explore the influencing factors of hemodialysis (HD) initiation in non-diabetic kidney disease (NDKD) patients with predialysis arteriovenous fistula (AVF) creation.Methods:This was a single-center prospective cohort study. The NDKD patients undergoing predialysis AVF creation were enrolled at the First Affiliated Hospital of Xi'an Jiaotong University from 2015 to 2018. According to the estimated glomerular filtration rate (eGFR, the Chronic Kidney Disease Epidemiology Collaboration equation) and age, patients were divided into different subgroups, eGFR: group 1 [eGFR<10 ml·min -1·(1.73 m 2) -1], group 2 [ eGFR between 10 to 15 ml·min -1·(1.73 m 2) -1], and group 3 [eGFR > 15 ml·min -1·(1.73 m 2) -1]; age: age ≥65 years group and age <65 years group. The primary outcome was defined as the initiation of HD within 1 year after AVF surgery. The second outcome was the use of AVF access at the time of HD initiation. Cox proportional hazard regression was performed to identify which demographic and clinical factors were associated with the initiation of HD after AVF surgery. Logistic regression analysis was performed to investigate factors associated with AVF use at the initiation of HD. Results:A total of 220 patients were enrolled, with age of (48.1±16.2) years, of which 143(65.0%) were males. Overall, the clinical parameters of eGFR, cystatin C, serum albumin, 24h-Urine protein, serum phosphorus were as follows respectively, 7.7 (6.6,9.2) ml·min -1·(1.73 m 2) -1, (3.93±1.12) mg/L, (36.0±4.0) g/L, (2.22±1.36) g, (1.71±0.53) mmol/L. The proportion of patients initiating HD within 6 months ( Fisher=6.832, P=0.020) and the level of hemoglobin ( F=3.112, P=0.047) were higher in group 3 compared to the other two eGFR groups. While the median time interval between AVF creation and HD initiation ( H=6.295, P=0.043) was shorter in group 1. In age <65 years group, the level of serum albumin ( t=2.076, P=0.039), triglyceride ( t=1.995, P=0.048) were higher compared with age ≥65 years group; interestingly, the proportion of patients initiated HD within 3 months ( χ2=4.033, P=0.045) and 6 months ( χ2=5.012, P=0.025) were lower in age <65 years group. The median time interval between AVF creation and HD initiation among these patients was 84 (49,174) days. The patients initiating HD within 3 months, 6 months, and 1 year after AVF creation were 112 (50.9%), 152 (69.1%), and 202 (91.8%), respectively. Multivariate Cox regression analysis indicated that higher cystatin C level ( HR=1.283, 95% CI 1.121-1.469, P<0.001) was associated with earlier HD initiation within 1 year of AVF surgery in NDKD patients. AVF usage was accomplished in 64.3% of patients who initiated HD within 90 days, the ratio was 100.0% in those initiated HD between 91 to 180 days, and 88.0% in those ≥181 days after AVF surgery. No factor was independently associated with AVF use at HD initiation identified by multivariate logistic regression analyses in patients with NDKD. Conclusion:Serum cystatin C level is associated with HD initiation within 1 year of the predialysis AVF creation in NDKD patients.
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[Objective]To observe the effect of Xiaoyu Xiezhuo Drink on podocyte apoptosis and hypoxia inducible factor 1 alpha(HIF1α)expression in db/db diabetic kidney disease(DKD)model mice.[Methods]Six db/m mice were chosen as negative control group,eighteen db/db mice were chosen and divided into DKD model group,low dosage Xiaoyu Xiezhuo Drink group,high dosage Xiaoyu Xiezhuo Drink group,with six mice in each group.After gastric irrigation for twelve weeks,the urine was collected to test the levels of protein,β2-microglobulin(β2-MG),albumin/creatinine ratio(Acr),β2-microglobulin/creatinine ratio(β2-MG/Ucr);blood was collected to test the level of triglyceride(TG),total cholesterol(TC),albumin(Alb),blood urea nitrogen(BUN),serum creatinine(Scr);the kidney tissue was collected to observe the pathological change by light and electron microscope,and to test HIF1α,nephrin mRNA by Real-time polymerase chain reaction(RT-PCR).[Results]Compared with negative control group,the levels of urine protein,β2-MG,Acr,β2-MG/Ucr,serum TG,TC,BUN,Scr were increased(P＜0.01),serum Alb was decreased(P＜0.01);glomerular volume increased,capillary loops lobed,mesangial cells and matrix hyperplasia,interstitial inflammation and fibrosis increased,foot process fusion increased,basement membrane thickened;podocyte apoptosis was increased;expression of HIF1α mRNA was elevated(P＜0.01),and nephrin mRNA was descended in kidney tissue of DKD model group(P＜0.01).Compared with DKD model group,the level of urine protein,β2-MG,Acr,β2-MG/Ucr,serum TG,TC,BUN,Scr were decreased(P＜0.01),serum Alb was incresed(P＜0.01);the pathological changes of the kidney was improved;the apoptosis of podocyte was reduced;the expression of HIF1α mRNA was decreased(P＜0.01),and nephrin mRNA was incresed(P＜0.01)in the kidney tissue of varied dosage Xiaoyu Xiezhuo Drink groups.There was no statistical significance in the level of urine protein,β2-MG,Acr,β2-MG/Ucr,serum TG,TC,BUN,Scr,Alb,podocyte apoptosis,and HIF1α,nephrin mRNA in the kidney tissue between different dosage Xiaoyu Xiezhuo Drink groups(P＞0.05).[Conclusion]Xiaoyu Xiezhuo Drink could improve urinary protein,renal function,renal structure lesion and podocyte apoptosis in DKD mice,which perhaps by regulating the expression of HIF1α.
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ObjectiveTo investigate the impact of yang deficiency syndrome on the progression to end-point events of diabetic kidney disease （DKD）. MethodsA retrospective study among patients with stage Ⅳ DKD admitted to Dongzhimen Hospital of Beijing University of Chinese Medicine from September 1st， 2016 to September 30th， 2021 was conducted. Data on the patients' general information， clinical indicators including duration of diabetes， duration of proteinuria， history of smoking and drinking， hemoglobin （HGB）， fasting blood glucose （FBG）， albumin （ALB）， serum creatinine （Scr）， urea nitrogen （BUN）， uric acid （UA）， cholesterol （TC） ， triglycerides （TG）， low-density lipoprotein （LDL）， 24-hour urine protein quantification （24h-UTP） and estimated glomerular filtration rate （eGFR）， and TCM syndromes including symptoms， tongue and pulse， and syndrome scores were collected. The patients were divided into exposure group （yang-deficiency group） and non-exposure group （non-yang-deficiency group）. The general information， clinical indicators and incidence rates of end-point events were compared， and the impact of yang deficiency syndrome on the end-point events of stage Ⅳ DKD was analyzed. Survival analysis was performed using Kaplan-Meier method， and multivariate Cox proportional risk models were used to identify independent predictors of end-point events. ResultsA total of 160 patients with stage Ⅳ DKD were included in the study， including 43 cases of yang deficiency syndrome and 117 cases of non-yang deficiency syndrome. Compared to those in the non-yang deficiency group， the waist circumference， BUN and the incidence of end-point events in the yang deficiency group were significantly higher （P＜0.05 or P＜0.01）. Spearman correlation analysis showed that yang deficiency syndrome was positively correlated with incidence of end-point events of stage Ⅳ DKD （r = 0.167， P = 0.035）. Furthermore， 24h-UTP and BUN levels were also positively correlated with end-point events in stage Ⅳ DKD patients （P＜0.01）， while ALB and HGB levels were negatively correlated （P＜0.01）. Kaplan-Meier survival curves showed that yang deficiency syndrome was associated with an increased risk of end-point events （Log Rank P = 0.011）. Moreover， 24h-UTP levels ≥3500 mg， BUN level ≥8 mmol/L， ALB level ＜30 g and HGB level ＜11 g were all associated with the increase of the risk of end-point events （P＜0.05 or P＜0.01）. Multivariate Cox regression analysis showed that yang deficiency syndrome was an independent risk factor for patients with stage Ⅳ DKD to progress into end-point events （HR = 2.36， 1.32 to 4.21； P = 0.004）， as well as 24h-UTP ≥ 3500 mg， BUN ≥ 8 mmol/L， HGB＜11 g and ALB＜30 g （P＜0.05 or P＜0.01）. ConclusionsFor stage Ⅳ DKD， patients with yang deficiency syndrome are more likely to have end-point events， which is an independent risk factor for the progression into end-point events.
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Diabetic kidney disease （DKD） is a chronic kidney disease caused by diabetes， influenced by genetic and environmental factors and their interaction. It is the primary cause of chronic kidney disease and end-stage renal disease. Recent studies have found， as a natural isoquinoline alkaloid， berberine （BBR） has hypoglycemic， hypolipidemic， antioxidant， anti-inflammatory and anti-fibrotic properties， thus protects against kidney injury in DKD. The mechanisms of action of BBR may involve improving glucolipid metabolism， reducing oxidative stress， alleviating inflammatory responses， mitigating renal fibrosis， regulating DNA methylation， promoting mitochondrial function and modulating the gut microbiota to enhance gut metabolism and clearance. This article systematically reviews the current status of research on the mechanisms of BBR in the treatment of DKD and provides reference for future clinical application of BBR in the treatment of DKD.
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OBJECTIVE To investigate the effects of Taohong siwu decoction modified granules on podocyte epithelial- mesenchymal-transition （EMT） and renal fibrosis in diabetic kidney disease （DKD） model rats. METHODS Eight rats were selected as normal group （ordinary feed）； the remaining rats were given a high-glucose and high-fat diet combined with intraperitoneal injection of streptozotocin （35 mg/kg） to induce the DKD model. Model rats were randomly divided into model group， irbesartan group [positive control， 13.5 mg/（kg·d）] and modified Taohong siwu decoction group [6.48 g/（kg·d）]， with 8 rats in each group. All groups were given relevant medicine intragastrically， once a day， for 16 consecutive weeks. Twenty-four- hour urinary total protein （24 h UTP） was detected at the end of the 4th， 8th， 12th and 16th week of administration. After the last medication， the body mass， water intake， food intake， urine output， the levels of fasting blood glucose， serum creatinine （Scr） and blood urea nitrogen （BUN） as well as mRNA and protein expressions of P-cadherin， nephrin， α -smooth muscle actin （α-SMA）， Wilms’ tumor gene 1 （WT1）， transforming growth factor-β1（ TGF-β1） and type Ⅳ collagen （Col-Ⅳ） in renal tissue were determined. The pathological and morphological changes in renal tissue were observed and the thickness of the glomerular basement membrane was determined. RESULTS Compared with the model group， 24 h UTP of rats was significantly decreased in modified Taohong siwu decoction group since the 8th weekend （P＜0.05）； the body weight of rats increased significantly， but the amount of water intake and urine decreased significantly； Scr and BUN level， mRNA expression of α-SMA， mRNA and protein expressions of TGF-β1 and Col-Ⅳ were significantly reduced， while the mRNA expressions of P-cadherin， nephrin and WT1 were increased significantly （P＜0.05）； the protein deposition of α-SMA was reduced， protein depositions of P-cadherin， nephrin and WT1 were increased； the pathological damage and fibrosis of renal tissue were relieved； the thickness of glomerular basement membrane was decreased significantly （P＜0.05）. CONCLUSIONS Taohong siwu decoction modified granules can inhibit the EMT of podocyte in DKD model rats， and alleviate renal pathological damage and podocyte damage， thus protecting renal function， and delaying the process of renal fibrosis.
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OBJECTIVE To investigate the effects of Taohong siwu decoction modified granules on podocyte epithelial- mesenchymal-transition （EMT） and renal fibrosis in diabetic kidney disease （DKD） model rats. METHODS Eight rats were selected as normal group （ordinary feed）； the remaining rats were given a high-glucose and high-fat diet combined with intraperitoneal injection of streptozotocin （35 mg/kg） to induce the DKD model. Model rats were randomly divided into model group， irbesartan group [positive control， 13.5 mg/（kg·d）] and modified Taohong siwu decoction group [6.48 g/（kg·d）]， with 8 rats in each group. All groups were given relevant medicine intragastrically， once a day， for 16 consecutive weeks. Twenty-four- hour urinary total protein （24 h UTP） was detected at the end of the 4th， 8th， 12th and 16th week of administration. After the last medication， the body mass， water intake， food intake， urine output， the levels of fasting blood glucose， serum creatinine （Scr） and blood urea nitrogen （BUN） as well as mRNA and protein expressions of P-cadherin， nephrin， α -smooth muscle actin （α-SMA）， Wilms’ tumor gene 1 （WT1）， transforming growth factor-β1（ TGF-β1） and type Ⅳ collagen （Col-Ⅳ） in renal tissue were determined. The pathological and morphological changes in renal tissue were observed and the thickness of the glomerular basement membrane was determined. RESULTS Compared with the model group， 24 h UTP of rats was significantly decreased in modified Taohong siwu decoction group since the 8th weekend （P＜0.05）； the body weight of rats increased significantly， but the amount of water intake and urine decreased significantly； Scr and BUN level， mRNA expression of α-SMA， mRNA and protein expressions of TGF-β1 and Col-Ⅳ were significantly reduced， while the mRNA expressions of P-cadherin， nephrin and WT1 were increased significantly （P＜0.05）； the protein deposition of α-SMA was reduced， protein depositions of P-cadherin， nephrin and WT1 were increased； the pathological damage and fibrosis of renal tissue were relieved； the thickness of glomerular basement membrane was decreased significantly （P＜0.05）. CONCLUSIONS Taohong siwu decoction modified granules can inhibit the EMT of podocyte in DKD model rats， and alleviate renal pathological damage and podocyte damage， thus protecting renal function， and delaying the process of renal fibrosis.
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ObjectiveTo observe the protective effect of Didang Xianxiong decoction on the kidneys of diabetic rats， its regulation on the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， and its influence on podocyte apoptosis and explore the mechanism of Didang Xianxiong decoction in improving diabetic nephropathy. MethodThe diabetic model was established by a single intraperitoneal injection of streptozotocin （STZ） solution of 55 mg·kg-1. The successfully replicated model rats were randomly divided into the model group， Didang Xianxiong decoction group （8.10 g·kg-1）， Xiao Xianxiongtang group （4.05 g·kg-1）， Didangtang group （4.05 g·kg-1）， and alagebrium （ALT-711） group （3 mg·kg-1）， with six rats in each group. In addition， six rats were included in the blank group. After continuous administration for eight weeks， hematoxylin-eosin （HE） staining was used to observe the pathological changes in rats' kidney tissue. Masson staining was used to observe the degree of collagen deposition. Periodic acid-Schiff （PAS） staining was used to observe basement membrane lesions， and immunohistochemistry was used to detect the expression of phosphorylation （p）-PI3K and p-Akt proteins in rats' kidney tissue. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） method was used to detect podocyte apoptosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of PI3K and Akt in rats' kidney tissue. Western blot was used to detect the protein expression of PI3K， p-PI3K， Akt， p-Akt， B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， phosphorylation glycogen synthase kinase-3β （p-GSK-3β）， and Caspase-3 in the kidney tissue. ResultCompared with the normal group， the model group had compensatory expansion of glomeruli， proliferation of mesangial cells， a large amount of collagen deposition in the mesangial stroma， thickening of the basement membrane， decreased mRNA expression of PI3K and Akt， and inhibition of PI3K and Akt protein phosphorylation （P<0.01）. It also underwent enhanced apoptotic signaling， decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）， and increased expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）. Compared with the model group， Didang Xianxiong decoction significantly improved kidney tissue pathology， increased mRNA expression of PI3K and Akt （P<0.01）， significantly up-regulated phosphorylation levels of PI3K and Akt proteins （P<0.01） and Bcl-2 expression （P<0.01）， downregulated the expression of Bax， p-GSK-3β， and Caspase-3 （P<0.01）， and weakened podocyte apoptotic signaling. ConclusionDidang Xianxiong decoction may promote the activation of the PI3K/Akt signaling pathway， inhibit podocyte apoptosis， and thus slow down the progression of diabetic nephropathy.
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Objective To investigate the distribution of traditional Chinese medicine(TCM)syndrome types in diabetic kidney disease(DKD),and to explore the correlation between TCM syndrome types and laboratory indices,so as to provide an objective basis for the TCM syndrome differentiation and treatment of DKD.Methods Syndrome differentiation was carried out in the 157 patients with DKD at stages Ⅲ and Ⅳ,and then the distribution of the syndromes of deficiency in the origin and the syndromes of excess in the superficiality was explored.The levels of 24-hour urinary total protein(24hUTP),serum creatinine(Scr),blood urea nitrogen(UREA),plasma albumin(Alb),total cholesterol(TC),and triglyceride(TG)of the patients were detected,and then the relationship between the TCM syndrome types and the biochemical indexes was analyzed.Results(1)The distribution of the syndromes of deficiency in the origin in DKD patients at different stages showed that DKD patients at stage Ⅲ were mainly differentiated as yin deficiency and dryness-heat syndrome[58.57％(41/70)],qi and yin deficiency syndrome[28.57％(20/70)],yin and yang deficiency syndrome[10.00％(7/70)],and spleen and kidney qi deficiency syndrome[2.86％(2/70)];DKD patients at stage Ⅳ were mainly differentiated as yin deficiency and dryness-heat syndrome[40.23％(35/87)],qi and yin deficiency syndrome[29.89％(29/87)],spleen and kidney qi deficiency syndrome[18.39％(16/87)],and yin and yang deficiency syndrome[11.49％(10/87)].The differences in the distribution of the syndromes of deficiency in the origin among the DKD patients at different stages were statistically significant(P<0.05).However,with the progression of the disease,DKD patients at different stages in general showed a trend of the decrease in the proportion of yin deficiency and dryness-heat syndrome while the increase in the proportions of qi and yin deficiency syndrome,spleen and kidney qi deficiency syndrome,and yin and yang deficiency syndrome.(2)The distribution of the syndromes of excess in the superficiality in DKD patients at different stages showed that DKD patients at stage Ⅲ were mainly differentiated as damp-heat syndrome[54.29％(38/70)],phlegm-stasis syndrome[27.14％(19/70)],blood-stasis syndrome[10.00％(7/70)],and cold-damp syndrome[8.57％(6/70)];DKD patients at stage Ⅳ were mainly differentiated as damp-heat syndrome[44.83％(39/87)],phlegm-stasis syndrome[35.63％(31/87)],cold-damp syndrome[14.94％(13/87)],and blood-stasis syndrome[4.60％(4/87)].There were no significant differences in the distribution of the syndromes of excess in the superficiality among the DKD patients at different stages(P>0.05).(3)The analysis of relationship between TCM syndrome type and biochemical indexes showed that Scr and UREA levels of DKD patients with spleen and kidney qi deficiency syndrome were significantly higher than those of patients with yin deficiency and dryness-heat syndrome,and the differences were statistically significant(P<0.05);Scr and 24hUTP levels of DKD patients with cold-damp syndrome were significantly higher than those of patients with damp-heat syndrome,and the differences were statistically significant(P<0.05).Conclusion DKD patients at stages Ⅲ and Ⅳ are all predominantly suffering from yin deficiency and dryness-heat syndrome,and with the progression of the disease,the syndrome of yin deficiency and dryness-heat develops into qi and yin deficiency syndrome,spleen and kidney qi deficiency syndrome,and yin and yang deficiency syndrome sequentially.Pathogenic dampness and blood stasis are the main pathogenic factors of DKD.And Scr,UREA,and 24hUTP are correlated with the TCM syndrome types of DKD,which will be helpful for the differentiation of TCM syndrome types of DKD.
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Hypoxia-inducible factor-1α(HIF-1α)is a nuclear transcription factor.Under high glucose and hypoxia conditions,the expression of HIF-1α is elevated,result in the increased expression of its downstream target genes VEGF,HO-1 and BNIP3,which affect angiogenesis,extra cellular matrix deposition,iron metabolism,and mito-phagy,participating in the occurrence and development of diabetic kidney disease(DKD).In addition,HIF-1α promotes inflammation and renal fibrosis by affecting the production of cytokines in DKD.
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Objective To explore the regulatory role of fat mass and obesity-associated protein(FTO)and serine-threonine kinase protein kinase D2(PRKD2)in progression of diabetic kidney disease(DKD)and its regulatory mechanisms.Methods DKD model in vitro was constructed by podocytes(MPC5 cells)treated with high glucose(HG,35 mmol/L glucose)for 24 h.HG-induced MPC5 cells were transfected with FTO overexpression vector(pcDNA-FTO)and PRKD2 overexpression vector(pcDNA-PRKD2),or empty vector.The overexpression efficiency of FTO and PRKD2 were detected with RT-qPCR.MeRIP was used to detect the N6-methyladenosine(m6A)modification level of PRKD2 mRNA.The activity of Caspase-3 and the secretion of IL-6,TNF-α and monocyte chemotactic protein-1(MCP-1)were detected by ELISA.Cell apoptosis rate was analyzed by flow cytometry.The protein levels of FTO and PRKD2,as well as the key proteins in SIRT1/HIF-1α pathway,were evaluated by Western blot.Pearson analysis was used to analyze the correlation between FTO levels and PRKD2 levels.Results Compared with the control group without HG-induction,the protein expression of FTO(0.51±0.04 vs 1.00±0.03)and PRKD2(0.45±0.03 vs 1.01±0.04)was significantly down-regulated in HG-induced podocytes,and the differences were statistically significant(t=13.17,16.76,all P＜0.001).FTO protein levels were positively correlated with PRKD2 protein levels in HG-induced podocytes(r2=0.705 1,P＜0.001).Compared with the vector group,the m6A levels of PRKD2 mRNA(0.56±0.09 vs 1.01±0.13)in the pcDNA-FTO group were decreased,and the mRNA levels of PRKD2(3.16±0.14 vs 1.03±0.02)were increased,with significant differences(t=51.37,11.82,all P＜0.001).Compared with the control group(IL-6:512.76±61.85 pg/ml,TNF-α:28.17±2.83 pg/ml,MCP-1:157.31±17.69 pg/ml)and the vector group(IL-6:498.41±87.51 pg/ml,TNF-α:26.35±5.47 pg/ml,MCP-1:165.52±16.87 pg/ml),the secretion of IL-6(301.86±21.85 pg/ml),TNF-α(11.06±4.12 pg/ml)and MCP-1(81.45±9.03 pg/ml)were significantly decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=7.51,10.47,61.97,all P＜0.01).Compared with the control group(caspase-3 activity:689.65±79.5 U/L,cell apoptosis:22.31%±2.69%)and the vector group(Caspase-3 activity:715.91±113.58 U/L,cell apoptosis:21.07%±3.28%),Caspase-3 activity(437.64±104.76 U/L)and the rate of apoptosis(8.41%±3.15%)were significantly decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=2.35,79.13,all P＜0.01).Compared with the control group(SIRT1:1.01±0.05,HIF-1α:1.03±0.07)and the vector group(SIRT1:0.97±0.05,HIF-1α:1.02±0.03),SIRT1 protein levels(3.51±0.15)were increased and HIF-1α protein levels(0.37±0.07)were decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=31.54,8.31,all P＜0.01).Conclusion FTO-mediated and m6A-modified PRKD2 suppresses inflammation and apoptosis in HG-induced podocytes through the SIRT1/HIF-1 pathway.
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ObjectiveTo explore the underlying mechanism by which the Chinese medicine compound Yitangkang granule（YTK） treats diabetic kidney disease （DKD） by observing its effects on podocyte autophagy through the regulation of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/forkhead transcription factor O1 （FoxO1） signaling pathway mediated by silent information regulator 1 （SIRT1） via advanced glycation end products （AGE）/receptor for AGE （RAGE） axis. MethodNinety-six 8-week-old healthy male SPF-grade Wistar rats were selected and randomly divided into blank control group （B）， model control group， high-dose YTK （40 g·kg-1）， medium-dose YTK （20 g·kg-1）， low-dose YTK （10 g·kg-1）， and Western medicine control （20 mg·kg-1 losartan） groups. The DKD rat model was established by high-fat diet feeding combined with intraperitoneal injection of streptozotocin. After successful modeling， the rats in each group received the corresponding treatments for eight weeks. The levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， and catalase （CAT） were measured according to the instructions of the respective assay kits. Hematoxylin and eosin （HE） staining was used to observe pathological changes in kidney tissues. Immunohistochemistry was employed to detect the average optical density values of α-smooth muscle actin （α-SMA）， fibronectin （FN）， desmin， and nephrin. Western blot analysis was used to measure the expression levels of PI3K， phosphorylated PI3K （p-PI3K）， Akt， phosphorylated Akt （p-Akt）， RAGE， SIRT1， Caspase-3， and FoxO1 proteins in kidney tissues of DKD rats. ResultCompared with the blank control group， the model group showed significantly lower levels of SOD， GSH-Px， and CAT， and significantly higher levels of MDA （P<0.01）. The rats exhibited severe kidney damage. The positive expression of podocyte marker proteins α-SMA， FN， and desmin increased significantly， while nephrin and podocin significantly decreased （P<0.01）. The expression levels of PI3K， p-PI3K， Akt， p-Akt， RAGE， and Caspase-3 proteins were significantly elevated， while SIRT1 and FoxO1 protein levels were significantly reduced （P<0.01）. Compared with the model control group， rats in the YTK treatment groups showed significantly higher levels of SOD， GSH-Px， and CAT， and significantly lower levels of MDA in serum （P<0.01）. The degree of kidney damage was reduced to varying extents. The average optical density values of podocyte marker proteins α-SMA， FN， and desmin were significantly decreased， while nephrin and podocin significantly increased （P<0.01）. The expression levels of PI3K， p-PI3K， Akt， p-Akt， RAGE， and Caspase-3 in kidney tissues were significantly reduced， while SIRT1 and FoxO1 expression levels significantly increased （P<0.01）. The Chinese medicine groups demonstrated a clear dose-response trend. ConclusionYTK may alleviate kidney pathological damage， reduce proteinuria， and protect kidney function in DKD rats， thereby delaying the progression of DKD by improving podocyte autophagy through the AGE-RAGE axis-mediated SIRT1 regulation of the PI3K/Akt/FoxO1 signaling pathway. Additionally， a dose-response relationship was observed in the Chinese medicine groups.
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Diabetic kidney disease(DKD)is one of the most serious microvascular complications of diabetes and a common cause of end-stage renal disease.Early kidney injury lacks typical clinical symptoms and its onset is insidiously.The common clinical indicators of kidney injury are urine microalbumin,glomerular filtration rate,etc.Due to their lack of sensitivity and specificity,it is easy to cause missed diagnosis,misdiagnosis,and delay the disease.Therefore,it is of great significance to find more convenient,stable,and less invasive detection indicators for the early diagnosis of DKD.This paper reviews the research progress of biological indicators related to glomerular and tubular injury,oxidative stress,inflammatory response,microRNA and proteomics,which provides a certain reference value for the early diagnosis of clinical DKD.
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ObjectiveTo investigate the effect and mechanism of Zhenwutang on renal oxidative damage in the mouse model of diabetic kidney disease with the syndrome of spleen-kidney Yang deficiency via the nuclear factor erythroid 2-related factor-2 （Nrf2）/heme oxygenase-1 （HO-1）/glutathione peroxidase 4 （GPX4） signaling pathway. MethodTwenty-five 7-week-old SPF-grade male db/m mice and 95 7-week-old SPF-grade male db/db mice were adaptively fed for a week. A blank group was set with the db/m mice without treatment， and the other mice were administrated with Rhei Radix et Rhizoma decoction and hydrocortisone for the modeling of diabetic kidney disease with the syndrome of spleen-kidney Yang deficiency. The modeled mice were randomized into the model， irbesartan （25 mg·kg-1）， and high-， medium-， low-dose （33.8， 16.9， 8.45 g·kg-1） Zhenwutang groups （n=15） and administrated with corresponding drugs for 8 weeks. The survival status of mice was observed， and the traditional Chinese medicine （TCM） syndrome score was recorded. The indicators related to spleen-kidney Yang deficiency， fasting blood glucose （FBG）， and renal function indicators were determined. Hematoxylin-eosin staining was employed to observe the histopathological changes of the renal tissue in each group. Biochemical kits were used to determine the oxidative stress-related indicators in the renal tissue. Real-time polymerase chain reaction and Western blotting were employed to determine the mRNA and protein levels， respectively， of Nrf2， HO-1， glutamate-cysteine ligase catalytic subunit （GCLC）， and GPX4 in the renal tissue of mice in each group. ResultCompared with the blank group， the modeling increased the TCM syndrome score （P<0.05）， elevated the estradiol （E2） and FBG levels （P<0.05）， lowered the testosterone （T）， triiodothyronine （T3）， and tetraiodothyronine （T4） levels （P<0.05）， and weakened the renal function （P<0.05）. In addition， the modeling led to glomerular hypertrophy and glomerular mesangial and basal thickening， decreased the catalase （CAT） activity， total antioxidant capacity （T-AOC）， and glutathione （GSH） content （P<0.05）， increased the malondialdehyde （MDA） content （P<0.05）， and down-regulated the mRNA and protein levels of Nrf2， HO-1， GCLC， and GPX4 in the renal tissue （P<0.05）. Compared with the model group， high and medium doses of Zhenwutang decreased the TCM syndrome score and E2 content （P<0.05）， increased the T， T3， and T4 content （P<0.05）， improved the renal function （P<0.05）， alleviated the pathological changes in the renal tissue， increased CAT， T-AOC， and GSH （P<0.05）， reduced MDA （P<0.05）， and up-regulated the mRNA and protein levels of Nrf2， HO-1， GCLC， and GPX4 in the renal tissue （P<0.05）. ConclusionZhenwutang can improve the general state and renal function and reduce the oxidative damage and pathological changes in the renal tissue of db/db mice with spleen-kidney Yang deficiency by regulating the Nrf2/HO-1/GPX4 signaling pathway.