BACKGROUND Diabetic nephropathy(DN)is a leading cause of chronic kidney disease and endstage renal disease,and is a significant global healthcare burden.Although proximal tubular epithelial cells(PTECs)and podocytes a...BACKGROUND Diabetic nephropathy(DN)is a leading cause of chronic kidney disease and endstage renal disease,and is a significant global healthcare burden.Although proximal tubular epithelial cells(PTECs)and podocytes are involved in DN progression,the specific molecular interactions between these cells are not well understood.AIM To elucidate the role of interleukin-6(IL-6)/Rab5 signaling in mediating crosstalk between PTECs and podocytes,and to evaluate the protective effects of nicotinamide mononucleotide(NMN)against DN progression.METHODS We utilized in vitro and in vivo models to investigate the pathogenesis of DN.In vitro,human PTECs and murine podocytes were cultured under high-glucose conditions,and IL-6 neutralizing antibodies or NMN treatments were applied.Podocyte injury was assessed by measurements of nephrin endocytosis,Rab5 activity,cytoskeletal organization,cell adhesion,and cell-spreading assays.In vivo,DN was induced in mice using streptozotocin,and mice then received NMN,insulin,or both treatments over an 8-week period.Renal tissues were analyzed histologically,ultrastructurally,and immunochemically,and urinary albumin excretion was measured to assess renal function.Statistical analyses were conducted using one-way ANOVA and Tukey's test.RESULTS High-glucose conditions induced the epithelial-mesenchymal transition(EMT)in PTECs,increased IL-6 secretion,and activated Rab5 signaling in podocytes,leading to increased nephrin endocytosis and podocyte injury.Blocking IL-6 significantly attenuated these effects.NMN treatment of diabetic mice markedly reduced podocyte injury,glomerular hypertrophy,foot-process effacement,and urinary albumin excretion.Mechanistically,NMN suppressed the EMT and IL-6 secretion by PTECs,inhibited Rab5 activation in podocytes,and prevented nephrin endocytosis,thereby preserving the cytoskeletal integrity and function of podocytes.CONCLUSION Our findings reveal a novel pathogenic mechanism of DN in which IL-6 released from glucose-stressed PTECs activates Rab5 signaling in podocytes,followed by nephrin endocytosis and structural injury of podocytes.Importantly,NMN treatment effectively disrupted this pathological pathway of intercellular communication,and provided significant protection against DN progression.These results suggest that NMN supplementation and targeting the IL-6/Rab5 signaling axis has promise as a therapeutic strategy for managing DN.展开更多
OBJECTIVE: To observe the effects and mechanisms of Bushenhuoxue on desmin and nephrin expression in mice podocytes, and to investigate its effects on wt1 expression in Wilms' tumor.METHODS: Adriamycin(ADR) was us...OBJECTIVE: To observe the effects and mechanisms of Bushenhuoxue on desmin and nephrin expression in mice podocytes, and to investigate its effects on wt1 expression in Wilms' tumor.METHODS: Adriamycin(ADR) was used to induce focal segmental glomerulous sclerosis(FSGS) in mice. Bushenhuoxue was used to treat FSGS for 6 weeks. We measured body mass and right renal mass, and determined serum albumin(ALB) levels,protein content in urine, and urinary protein and albumin creatinine ratio(UACR). Changes in renal tissue morphology were evaluated by microscopy.wt1 and nephrin expression in podocytes were detected using immunofluorescence. Expression levels of desmin, wt1 and nephrin m RNAs in renal tissue were determined using reverse transcription polymerase chain reaction assays.RESULTS: Protein levels in urine and UACR were significantly increased in FSGS model mice compared with Bushenhuoxue-treated and control mice.Body mass and ALB levels were decreased in FSGS mice compared with control and Bushenhuoxue-treated mice. Expression of the wt1 protein was observed in control mice. Compared with controls,wt1 expression levels were reduced in Bushenhuoxue-treated mice, and to a greater extent in FSGS mice. Nephrin protein expression was widespread in FSGS mice, and significantly reduced in control and Bushenhuoxue mice. Expression levels of wt1 and nephrin m RNAs in FSGS mice were lower compared with those in control and Bushenhuoxue-treated mice. Desmin m RNA levels in FSGS mice were reduced compared with those in control and Bushenhuoxue-treated mice.CONCLUSION: Bushenhuoxue ameliorated albuminuria in FSGS mice; this was possibly related to the up-regulation of wt1 and nephrin, and down-regulation of desmin.展开更多
To investigate the effects of albumin on the production of matrix metalloproteinases-2 （MMP-2） and matrix metalloproteinases-9 （MMP-9）in podocytes. Podocytes were treated with bovine serum albumin （BSA） at the c...To investigate the effects of albumin on the production of matrix metalloproteinases-2 （MMP-2） and matrix metalloproteinases-9 （MMP-9）in podocytes. Podocytes were treated with bovine serum albumin （BSA） at the concentration of 0.1, 0.5, 1, 2 g/L, respectively. Conditioned media were harvested 12, 24, 48 and 72 h after the treatment. The expression of MMP-2 and MMP-9 was assayed by gelatin zymography, RT-PCR and Western blotting analysis. Our results showed that in comparison with the control group, BSA increased the expression of MMP-2 and MMP-9 mRNA and protein in a doseand time-dependent manner （P〈0.05）. Meanwhile, the enzymatic activities of MMP-2 and MMP-9 in the culture supernatants of podocytes were also increased （P〈0.05）. It is concluded that albumin up-regulated the expression of MMP-2 and MMP-9 at gene and protein levels in a time-and dose-dependent manner.展开更多
Objective:Previous studies have found that Qidi Tangshen granules(QDTS),a combination therapy of supplementing essence(Tianjing,TJ)and unblocking the collaterals(Tongluo,TL),can reduce kidney damage in db/db mice.This...Objective:Previous studies have found that Qidi Tangshen granules(QDTS),a combination therapy of supplementing essence(Tianjing,TJ)and unblocking the collaterals(Tongluo,TL),can reduce kidney damage in db/db mice.This study aimed to explore the effect of QDTS and their separate prescriptions on podocytes in mice with diabetic nephropathy.Methods:The db/db mice were used in this experiment as an animal model,while wild-type C57BL/6J mice were used as normal controls.At the age of 12 weeks,the db/db mice were randomly divided into 5 groups(db/db,db/dbþvalsartan,db/dbþQDTS,db/dbþTJ and db/dbþTL).The urine albumin excretion ratio(UAE)was measured by enzyme-linked immunosorbent assay before and after the intervention.The ultrastructure of the kidney podocytes was observed by transmission electron microscopy.The protein expression levels of nephrin and desmin were detected by immunohistochemistry.Results:QDTS and their separate prescriptions significantly decreased the UAE and attenuated the renal pathological injury.QDTS and their separate prescriptions also reduced the fusion rate of the foot processes and increased the expression of nephrin protein.In contrast,QDTS and their separate prescriptions(TJ and TL)reduced the expression level of desmin protein.Conclusion:QDTS and their separate prescriptions might reduce diabetes-induced renal injury by reducing podocyte damage.The therapeutic effect of QDTS was more pronounced than TJ and TL.展开更多
OBJECTIVE:To investigate the effects of Tongluo Digui decoction on renal injury and streptozotocin-induced podocyte autophagy in diabetic rats.METHODS:Male Sprague-Dawley rats were randomly divided into six groups:nor...OBJECTIVE:To investigate the effects of Tongluo Digui decoction on renal injury and streptozotocin-induced podocyte autophagy in diabetic rats.METHODS:Male Sprague-Dawley rats were randomly divided into six groups:normal,model,Tongluo Digui decoction(high,medium,and low dose)and valsartan.Streptozotocin was injected intraperitoneally to replicate the diabetic animal model.After 8 weeks,proteinuria was evaluated to establish the diabetic nephropathy model.Treatments were administered daily via the intragastric route.At 16 weeks after gavage,we determined 24 h urine protein concentration,and blood glucose,serum creatinine,and urea nitrogen concentrations.Then,rats were sacrificed,and kidneys were harvested and stained with periodic acid-Schiff to evaluate the pathological changes in glomeruli,including glomerular podocytes by transmission electron microscopy.Western blot analysis was used to determine the expression of nephrin,podocin,p62,beclin-1,LC3Ⅱ/Ⅰ,and p-m TOR/m TOR protein in kidney tissues.RESULTS:Compared with the model group,Tongluo Digui decoction was associated with decreases in 24 h urine protein concentration,and blood glucose,hemoglobin A1 c,serum creatinine,urea nitrogen concentrations,total serum protein and albumin.Concurrently,mesangial mesenteric broadening and fusion of foot processes were reduced,the glomerular basement membrane was not significantly thickened,and the number of podocytes and the number of autophagosomes in the podocytes was increased.Further,expression of nephrin,podocin,LC3Ⅱ,and beclin-1 protein in kidney tissue was up-regulated,while expression of p62 protein was down-regulated and m TOR phosphorylation was inhibited.CONCLUSION:Tongluo Digui decoction may inhibit the progression of diabetic nephropathy by inhibiting m TOR phosphorylation,thereby increasing autophagy to protect podocytes and reducing proteinuria.展开更多
Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes ...Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.展开更多
Objective:Diabetic nephropathy is one of the most important microvascular complications of diabetes,which mainly refers to glomerular capillary sclerosis.Podocytes are an important part of glomerular capillaries.Previ...Objective:Diabetic nephropathy is one of the most important microvascular complications of diabetes,which mainly refers to glomerular capillary sclerosis.Podocytes are an important part of glomerular capillaries.Previous clinical and basic studies have shown that fibrosis is the main factor of diabetic nephropathy.This study aimed to assess the protective mechanism of glycyrrhizic acid(GA)on glomerular podocytes induced by high glucose as we hypothesized that GA may have antifibrotic and anti-inflammatory effects on podocytes through regulation of the adenosine 5'-monophosphate-activated protein kinase(AMPK)/sucrose nonfermenting AMPK-related kinase(SNARK)signaling pathway.Methods:SNARK siRNA was used to transfect podocytes.Real-time quantitative polymerase chain reaction and immunofluorescence staining assays were used for molecular and pathological analysis.The expression levels of key pathway proteins(including TGF-β1,α-SMA,SITR1,AMPKα,LKB1,PGC-1α,NF-κB,IL-6,and TNF-α)were verified by Western blotting.The expression of inflammatory factors in podocytes was detected by ELISA.Results:We demonstrated that GA decreased the expression of podocyte fibrosis signaling pathway-related factors by upregulating the AMPK pathway and its related factors.However,after transfection of podocytes with SNARK siRNA,there was an increased expression of fibrosis-related factors and inflammation-related factors.Conclusion:GA can protect podocytes and alleviate fibrosis and inflammation induced by high glucose,which is related to the AMPK signaling pathway.Meanwhile,knockdown of SNARK protein can inhibit the AMPK signaling pathway,aggravate fibrosis,and increase inflammation.展开更多
The role of B7-1 in podocyte injury has received increasing attention.The aim of this study was to investigate whether losartan protects podocytes of patients with diabetic kidney disease(DKD)by regulating B7-1 and th...The role of B7-1 in podocyte injury has received increasing attention.The aim of this study was to investigate whether losartan protects podocytes of patients with diabetic kidney disease(DKD)by regulating B7-1 and the underlying mechanisms.Rats with streptozotocin-induced DKD were treated with losartan for 8 weeks.Biochemical changes in blood and urine were analyzed.Kidneys were isolated for electron microscopy,immunofluorescence,real-time quantitative PCR(RT-PCR),and Western blot analysis.Immortalized mouse podocyte cells were cultured in normal or high glucose medium in the presence or absence of losartan for 48 h,and then the cells were collected for immunofluorescence,PCR,Western blotting and monolayer permeability detection.The phosphatidylinositol 3-kinase(PI3K)110a subunit and angiotensin II type 1 receptor(AT1R)plasmids were transfected into podocytes,respectively,and then Western blotting was performed to assess the expression of B7-1 protein.The results showed that losartan ameliorated podocyte structure and function in the rat model of DKD,and reduced the expression of B7-1 protein.Overexpression of PI3K 110a subunit in podocytes attenuated the inhibitory effect of losartan on B7-1 expression in high glucose-stimulated podocytes.The expression of B7-1 was significantly increased by overexpression of ATI R and significantly reduced by blocking PI3K 110a subunit.We conclude that losartan protects podocytes against high glucose-induced injury by inhibiting AT1R-mediated B7-1 expression.This effect is dependent on the AT1R-PI3K 110a subunit pathway.展开更多
Objective To explore the influence of Linggui Zhugan Decoction(LGZGD) on high glucose induced podocyte autophagy.Methods LGZGD containing serum was prepared by intragastric administation of 4.2 g/kg(low dose), 8.4 g/k...Objective To explore the influence of Linggui Zhugan Decoction(LGZGD) on high glucose induced podocyte autophagy.Methods LGZGD containing serum was prepared by intragastric administation of 4.2 g/kg(low dose), 8.4 g/kg(medium dose), and 12.6 g/kg(high dose) LGZGD into SD rats respectively. MPC5 and AB8/13 podocyte cells were treated with 60 mmol/L glucose to establish diabetic nephropathy podocyte model in vitro. Both podocytes were divided into control group, high glucose group, low dose LGZGD group, medium dose LGZGD group, and high dose LGZGD group, respectively. For the three LGZGD groups, before LGZGD intervention, podocytes were treated with 60 mmol/L glucose for 3 days. After treated with LGZGD containing serum, cells were collected to analyze cell migration using Transwell assay, proliferation using CCK8, apoptosis and cell cycle using flow cytometry, autophagosome formation using transmission electron microscopy, and expression levels of Beclin-1, Atg5, LC3II/I, and P62 proteins using Western blot.Results Compared with the control group, the proliferation and migration of MPC5 and AB8/13 cells in the high glucose group slightly decreased, whereas these parameters restored after intervention with low and medium concentrations of LGZGD, with the medium dose LGZGD having the better effect(P < 0.05). Flow cytometry showed that the medium dose LGZGD group had a significantly lower apoptosis rate(P < 0.05) and higher survival rate(P > 0.05) compared to the high dose LGZGD group. High glucose arrested podocytes in G1 phase, whereas LGZGD shifted podocytes from being predominant in G1 phase to G2 phase. High dose LGZGD significanly reduced high glucose-increased autophagosome formation in both podocytes(P < 0.05). Western blot analysis showed that Beclin-1, Atg5, LC3II/I, and P62 expressions were increased in MPC5 cells treated with high glucose and reversed after adminstration of low and medium doses of LGZGD(P < 0.05).Conclusion LGZGD reduced apoptosis and enhanced autophagy in high glucose treated podocytes via regulating Beclin-1/LC3II/I/Atg5 expression.展开更多
Glomerular tuft immune reactive Ezrin surface area (EzA) and fraction of EzA to total glomerular tuft area significantly increased, indicating podocyte growth, rounding and altered cytoskeletal interactions at 1 week ...Glomerular tuft immune reactive Ezrin surface area (EzA) and fraction of EzA to total glomerular tuft area significantly increased, indicating podocyte growth, rounding and altered cytoskeletal interactions at 1 week of STZ diabetes. Podocyte number per glomerulus (WT1+ nuclei) did not change indicating no detachment, but density decreased due to tuft hypertrophy. Treatment with PLZ or Insulin for one week, prevented increase in proteinuria and hyperglycemia but not the decrease in podocyte density. PLZ but not Insulin prevented increase in ezrin positive area in glomeruli and per podocyte. In podocytes in culture neither 25 mM glucose with or without PLZ (2.5 or 25 uM) altered Ezrin expression measured in western blots. In summary, the Ezrin positive glomerular surface area increase seen after 1 week of STZ diabetes, reflects altered podocyte morphology and cytoskeletal interactions, prevented by PLZ but not by insulin. Ezrin area increase preceded podocyte detachment and in podocytes in culture is not associated with increases in podocyte Ezrin protein expression. It is a likely precursor of shape changes in podocytes and of alterd interactions with basement membrane that contribute to detachment and thickening. Glomerular capillary tuft hypertrophy and reduced podocyte density persisted despite PLZ or insulin treatments, independently of levels of glycemia and of proteinuria.展开更多
Accumulation of plasma advanced oxidation protein products(AOPPs) promotes progression of proteinuria and glomerulo-sclerosis.To investigate the molecular basis of AOPPs-induced proteinuria,normal Sprague-Dawley rats ...Accumulation of plasma advanced oxidation protein products(AOPPs) promotes progression of proteinuria and glomerulo-sclerosis.To investigate the molecular basis of AOPPs-induced proteinuria,normal Sprague-Dawley rats were treated with AOPPs-modified rat serum albumin.The expression of glomerular podocyte slit diaphragm(PSD)-associated proteins,nephrin and podocin,was significantly decreased coincident with the onset of albuminuria in rats treated with AOPPs.Chronic inhibi-tion of NADPH oxidase by apocynin prevented down-regulation of nephrin and podocin and decreased albuminuria in AOPPs-challenged rats.This suggested that accumulation of AOPPs promotes proteinuria,possibly via down-regulating the expression of PSD-associated proteins.展开更多
Background:Nephrotic syndrome is the most common cause of kidney disease in children,but its pathogenesis remains unclear.This article reviews the novel aspects of the mechanisms underlying massive proteinuria in mini...Background:Nephrotic syndrome is the most common cause of kidney disease in children,but its pathogenesis remains unclear.This article reviews the novel aspects of the mechanisms underlying massive proteinuria in minimal-change disease,which is the most common form of childhood nephrotic syndrome.Data sources:This article integrates the findings of a PubMed database search for English language articles published in the past 40 years(from September 1974 to February 2014)using the key words"pathogenesis","minimal change nephrotic syndrome"or"idiopathic ne phrotic syndrome".Results:Unknown humoral factors associated with T-cell dysfunction have been thought to play an important role in the pathogenesis of minimal-change disease.However,recent findings are changing this paradigm,i.e,visceral glomerular epithelial cells(podocytes)may be involved via expression of molecules such as CD80 and angiopoietin-like 4.Conclusions:Recent evidence suggests that minimal-change disease results from interactions between humoral factors and dysfunctional podocytes.In addition to immunosuppressant drugs that target lymphocytes,a biological agent such as an antibody against the abnormal molecule(S)expressed by podocytes may provide novel drug treatment for minimal-change disease.展开更多
Glomerular podocytes are highly specialized epithelial cells and play an essential role in establishing the selective permeability of the glomerular filtration barrier of kidney.Maintaining the viability and structura...Glomerular podocytes are highly specialized epithelial cells and play an essential role in establishing the selective permeability of the glomerular filtration barrier of kidney.Maintaining the viability and structural integrity of podocytes is critical to the clinical management of glomerular diseases,which requires a thorough understanding of podocyte cell biology.As mature podocytes lose proliferative capacity,a conditionally SV40 mutant tsA58-immortalized mouse podocyte line(designated as tsPC)was established from the Immortomouse over 20 years ago.However,the utility of the tsPC cells is hampered by the practical inconvenience of culturing these cells.In this study,we establish a user-friendly and reversibly-immortalized mouse podocyte line(designated as imPOD),on the basis of the tsPC cells by stably expressing the wildtype SV40 T-antigen,which is flanked with FRT sites.We show the imPOD cells exhibit long-term high proliferative activity,which can be effectively reversed by FLP recombinase.The imPOD cells express most podocyte-related markers,including WT-1,Nephrin,Tubulin and Vinculin,but not differentiation marker Synaptopodin.The imPOD cells do not form tumor-like masses in vivo.We further demonstrate that TGFb1 induces a podocyte injury-like response in the FLP-reverted imPOD cells by suppressing the expression of slit diaphragm-associated proteins P-Cadherin and ZO-1 and upregulating the expression of mesenchymal markers,a-SMA,Vimentin and Nestin,as well as fibrogenic factors CTGF and Col1a1.Collectively,our results strongly demonstrate that the newly engineered im-POD cells should be a valuable tool to study podocyte biology both under normal and under pathological conditions.展开更多
The underlying molecular changes that result in minimal change disease(ne-phrotic syndrome)require an in-depth analysis.Current molecular studies have shown the involvement of zinc fingers and homeobox transcriptional...The underlying molecular changes that result in minimal change disease(ne-phrotic syndrome)require an in-depth analysis.Current molecular studies have shown the involvement of zinc fingers and homeobox transcriptional factors in its pathogenesis.The application of therapeutic drugs relies on understanding the cascade of molecular events to determine their efficacy in managing the clinical condition.展开更多
Objectives:Podocytes undergo epithelial-mesenchymal transition(EMT)and ferroptosis in response to hyperglycemic stimulation.This is considered an important early event in the development and progression of diabetic ne...Objectives:Podocytes undergo epithelial-mesenchymal transition(EMT)and ferroptosis in response to hyperglycemic stimulation.This is considered an important early event in the development and progression of diabetic nephropathy(DN).Rhein is the main active anthraquinone derivative in several common traditional herbal medicines.This study aimed to investigate the protective effects of Rhein on podocyte ferroptosis and EMT.Methods:The mouse glomerular podocyte cell line MPC5 was stimulated with high glucose(HG),Rhein,and the ferroptosis inhibitor ferrostatin-1(Fer-1).Mechanistic investigations employed plasmids to overexpress and knockdown Sirtuin-1(SIRT1),solute carrier family 7 member 11(SLC7A11),or p53 and measure ferroptosis-or EMT-related indicators.Results:In the HG-injured podocytes,Rhein enhanced cell viability,reduced malondialdehyde(MDA),ferrous iron(Fe2+),and reactive oxygen species(ROS)levels,increased glutathione(GSH)production,accompanied by the restoration of ferroptosis-and EMT-associated indicator expressions.Mechanistically,Rhein induced SIRT1 and SLC7A11 expression and attenuated p53 expression.SIRT1 knockdown upregulated p53 and downregulated SLC7A11,thereby abolishing the protective effects of Rhein against podocyte ferroptosis and EMT.However,the effects of SIRT1 overexpression were reversed by SLC7A11 knockdown.Conclusion:Rhein activated the SIRT1/p53/SLC7A11 axis to protect podocytes against ferroptosis and EMT.This suggests that Rhein has a potential therapeutic effect on DN patients associated with podocyte injury,and targeting SIRT1/p53/SLC7A11 may represent an innovative therapeutic strategy for DN patients.展开更多
OBJECTIVE:To investigate the therapeutic action and mechanism of the Qizhi Jiangtang capsule(芪蛭降糖胶囊,QZJT)on diabetic kidney disease(DKD)treatment.METHODS:This experiment used db/db mice and podocytes(MPC5)to dev...OBJECTIVE:To investigate the therapeutic action and mechanism of the Qizhi Jiangtang capsule(芪蛭降糖胶囊,QZJT)on diabetic kidney disease(DKD)treatment.METHODS:This experiment used db/db mice and podocytes(MPC5)to develop DKD model.Evaluation of the effect of the QZJT on db/db mice by testing urine and blood biochemical parameters(24-h urinary albumin,serum creatinine,blood urine nitrogen),pathological kidney injury,and podocyte integrity.Moreover,autophagosomes in podocytes of DKD mice and cultured podocytes were detected using electron microscopy.Additionally,Western blotting was applied to detect the expression of podocyte marker protein(podocin),autophagy-associated proteins,and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)signaling pathway changes in vivo and in vitro.RESULTS:QZJT significantly reduced urine protein,blood nitrogen urea,and serum creatinine and showed histological restoration of renal tissues.QZJT also significantly improved the down-regulation of podocin and foot fusion and effacement in db/db mice.QZJT increased autophagic vesicles in mice and cultured podocytes.QZJT also upregulated microtubuleassociated protein 1 light chain 3-II(LC3-II)/(LC3-I)and Beclin-1 and downregulated phosphorylated-PI3K(pPI3K),p-AKT,and p-mTOR in db/db mice and MPC5 cells.However,autophagy inhibitor 3-methyladenine partially alleviated the above effects in MPC5 cells.CONCLUSIONS:These results showed that the QZJT can enhance podocyte autophagy and ameliorate podocyte injury in DKD by inhibiting the PI3K/AKT/mTOR signaling pathway.展开更多
Objective： To observe the podocyte injury in diabetic nephropathy （DN） patients by identifying the urinary podocytes and the situation of detached podocytes in glomeruli and to demonstrate the correlation between p...Objective： To observe the podocyte injury in diabetic nephropathy （DN） patients by identifying the urinary podocytes and the situation of detached podocytes in glomeruli and to demonstrate the correlation between podocyte excretion and proteinuria, blood glucose, serum creatinine in different phases in DN patients. Methods： Urinary podocytes and the podocalyxin （PCX） expression state of podocytes in glomeruli were identified and observed by indirect immunofluorescent method. The DN patients were divided into three groups according to the volume of proteinuria, namely small, medium and large volume proteinuria groups. The podocytes in the urine of every group were calculated. The DN patients were divided into five groups according to the chronic kidney disease （CKD） phases, then the positive podocytes in urine were calculated. Meanwhile, the 24-hour protein in urine, fasting blood glucose （FBG） and the serum creatinine of DN patients were tested. The correlations among the proteinuria, serum creatinine, FBG and the number of positive podocytes in the urine of DN patients were statistically analyzed. Results： Urinary positive podocytes were found in 88% of the patients with DN, whereas podocytes were found in 0% of patients with minimal changed disease （MCD） and healthy cases. The expression of PCX was absent in DN patients. In contrast, PCX was expressed integrally in MCD patients. The positive podocytes was 1.49±0.95/ml in small-volume proteinuria group, 2.15±0.70/ml in the medium-volume proteinuria group, and 3.48±1.27/ml in the large-volume proteinuria group. There was no significant difference between the small- and medium- volume proteinuria groups, and there were significant differences between other groups （P〈0.05）. The positive podocyte number tended to increase as proteinuria was increased. By Pearson analysis, the correlation between podocyte number and proteinuria was podocytes in urine from different groups of DN patients, CKD pc I sitive statistically. The difference of the number of positive -V group was significant statistically. The correlation between serum creatinine of CKD Ⅰ -Ⅲ group and positive podocytes in urine was positive statistically. The correlation between serumcreatinine of CKD Ⅳ- Ⅴ group and positive podocytes in urine was not significant statistically. The correlation between FBG and positive podocytes in urine was not significant either. Conclusion： The mechanism of the podocyte injury in DN patients is present. The podocyte injury in DN may positively correlate to proteinuria and serum creatinine of CKD Ⅰ -ⅢDN patients, but not to the FBG and serum creatinine of CKD Ⅳ-Ⅴ patients.展开更多
BACKGROUND A number of recent studies indicate a transformation in the natural course of chronic kidney disease(CKD)in type 2 diabetes(T2D)patients:an increasing prevalence of declined renal function without proceedin...BACKGROUND A number of recent studies indicate a transformation in the natural course of chronic kidney disease(CKD)in type 2 diabetes(T2D)patients:an increasing prevalence of declined renal function without proceeding to the accompanying elevation of albuminuria.It has been suggested that albuminuric and nonalbuminuric CKD patterns could be different in their phenotypes and pathogenic mechanisms.AIM To identify the risk factors and biomarkers of albuminuric and non-albuminuric patterns of CKD in patients with T2D.METHODS Three hundred sixty patients with T2D duration≥10 years were included in this observational cross-sectional study.The associations of a panel of demographic and clinical characteristics,complications,comorbidities,and metabolic and hematology parameters with albuminuric and non-albuminuric CKD patterns were analyzed.The urinary excretion of nephrin and podocin,two podocytespecific markers,and WAP-four-disulfide core domain protein 2(WFDC-2),a marker of tubulointerstitial fibrosis,was determined by ELISA in comparison with healthy controls.RESULTS Non-albuminuric CKD was associated with age≥65 years(P=0.0001),female sex(P=0.04),diabetes duration≥15 years(P=0.0009),and the use of diuretics(P=0.0005).Male sex(P=0.01),smoking(P=0.01),waist-to-hip ratio>1.0(P=0.01)and hemoglobin A1c(HbA1c)>8.0%(P=0.005)were risk factors for elevated albuminuria not accompanied by a decrease in estimated glomerular filtration rate(eGFR).Duration of diabetes≥15 years and the use of calcium channel blockers were risk factors for albuminuria with decreased eGFR(both P=0.01).In multivariate logistic regression analysis,age,HbA1c,female sex and diuretics were significant predictors for reduced eGFR,while waist-to-hip ratio,HbA1c and male sex were associated with elevated urinary albumin-to-creatinine ratio(UACR).Excretion of nephrin and podocin was increased in patients with albuminuria,regardless of decline in renal function(P<0.001),correlating positively with UACR.The urinary excretion of WFDC-2 was markedly higher in men than in women(P<0.000001).Men with T2D demonstrated increased WFDC-2 levels independently of the CKD pattern(all P<0.05).In T2D women,WFDC-2 excretion was increased in those with reduced renal function(P≤0.01),correlating negatively with eGFR.CONCLUSION The data provide further evidence that albuminuric and non-albuminuric CKD phenotypes correspond to different pathways of diabetic kidney disease progression.展开更多
Costimulatory pathways(Cluster of differentiation 28,tumor necrosis factor-related,adhesion and T Cell Ig-and mucin-domain molecules) regulating the interactions between receptors on the T cells andtheir ligands expre...Costimulatory pathways(Cluster of differentiation 28,tumor necrosis factor-related,adhesion and T Cell Ig-and mucin-domain molecules) regulating the interactions between receptors on the T cells andtheir ligands expressed on several cell types,have a key role in controlling many immunological and non immunological processes.Indeed,accumulating evidence indicate that these molecules are involved in the pathogenesis of numerous conditions,such as allograft rejection,atherosclerosis,rheumatoid arthritis,psoriasis and renal diseases,including glomerulonephritis.Primary or secondary(i.e.,associated with infections,drugs or systemic diseases,such as systemic lupus erythematosus,diabetes,etc.) glomerulonephritis represent a group of heterogeneous diseases with different pathogenic mechanisms.Since costimulatory molecules,in particular CD80 and CD40,have been found to be expressed on podocytes in the course of different experimental and clinical glomerulonephritis,costimulation has been thought as a new therapeutic target for patients with glomerular diseases.However,although experimental data suggested that the blockade of costimulatory pathways is effective and safe in the prevention and treatment of glomerular diseases,clinical trials reported contrasting results.So,at this moment,there is not a strong evidence for the general use of costimulatory blockade as an alternative treatment strategy in patients with primary or secondary glomerulonephritis.Here,we critically discuss the current data and the main issues regarding the development of this innovative therapeutic approach.展开更多
基金Supported by Hubei Provincial Natural Science Foundation,No.2023AFB732and Scientific Research Project of Hubei Provincial Health Commission,No.WJ2023M053.
文摘BACKGROUND Diabetic nephropathy(DN)is a leading cause of chronic kidney disease and endstage renal disease,and is a significant global healthcare burden.Although proximal tubular epithelial cells(PTECs)and podocytes are involved in DN progression,the specific molecular interactions between these cells are not well understood.AIM To elucidate the role of interleukin-6(IL-6)/Rab5 signaling in mediating crosstalk between PTECs and podocytes,and to evaluate the protective effects of nicotinamide mononucleotide(NMN)against DN progression.METHODS We utilized in vitro and in vivo models to investigate the pathogenesis of DN.In vitro,human PTECs and murine podocytes were cultured under high-glucose conditions,and IL-6 neutralizing antibodies or NMN treatments were applied.Podocyte injury was assessed by measurements of nephrin endocytosis,Rab5 activity,cytoskeletal organization,cell adhesion,and cell-spreading assays.In vivo,DN was induced in mice using streptozotocin,and mice then received NMN,insulin,or both treatments over an 8-week period.Renal tissues were analyzed histologically,ultrastructurally,and immunochemically,and urinary albumin excretion was measured to assess renal function.Statistical analyses were conducted using one-way ANOVA and Tukey's test.RESULTS High-glucose conditions induced the epithelial-mesenchymal transition(EMT)in PTECs,increased IL-6 secretion,and activated Rab5 signaling in podocytes,leading to increased nephrin endocytosis and podocyte injury.Blocking IL-6 significantly attenuated these effects.NMN treatment of diabetic mice markedly reduced podocyte injury,glomerular hypertrophy,foot-process effacement,and urinary albumin excretion.Mechanistically,NMN suppressed the EMT and IL-6 secretion by PTECs,inhibited Rab5 activation in podocytes,and prevented nephrin endocytosis,thereby preserving the cytoskeletal integrity and function of podocytes.CONCLUSION Our findings reveal a novel pathogenic mechanism of DN in which IL-6 released from glucose-stressed PTECs activates Rab5 signaling in podocytes,followed by nephrin endocytosis and structural injury of podocytes.Importantly,NMN treatment effectively disrupted this pathological pathway of intercellular communication,and provided significant protection against DN progression.These results suggest that NMN supplementation and targeting the IL-6/Rab5 signaling axis has promise as a therapeutic strategy for managing DN.
基金Supported by Protective Effect of Extract of Fructus Schisandrae Chinensis on Fibrosis of Diabetic Nephropathy among C57BL/6 Mice of National Natural Science Foundation of China(No.81150012)
文摘OBJECTIVE: To observe the effects and mechanisms of Bushenhuoxue on desmin and nephrin expression in mice podocytes, and to investigate its effects on wt1 expression in Wilms' tumor.METHODS: Adriamycin(ADR) was used to induce focal segmental glomerulous sclerosis(FSGS) in mice. Bushenhuoxue was used to treat FSGS for 6 weeks. We measured body mass and right renal mass, and determined serum albumin(ALB) levels,protein content in urine, and urinary protein and albumin creatinine ratio(UACR). Changes in renal tissue morphology were evaluated by microscopy.wt1 and nephrin expression in podocytes were detected using immunofluorescence. Expression levels of desmin, wt1 and nephrin m RNAs in renal tissue were determined using reverse transcription polymerase chain reaction assays.RESULTS: Protein levels in urine and UACR were significantly increased in FSGS model mice compared with Bushenhuoxue-treated and control mice.Body mass and ALB levels were decreased in FSGS mice compared with control and Bushenhuoxue-treated mice. Expression of the wt1 protein was observed in control mice. Compared with controls,wt1 expression levels were reduced in Bushenhuoxue-treated mice, and to a greater extent in FSGS mice. Nephrin protein expression was widespread in FSGS mice, and significantly reduced in control and Bushenhuoxue mice. Expression levels of wt1 and nephrin m RNAs in FSGS mice were lower compared with those in control and Bushenhuoxue-treated mice. Desmin m RNA levels in FSGS mice were reduced compared with those in control and Bushenhuoxue-treated mice.CONCLUSION: Bushenhuoxue ameliorated albuminuria in FSGS mice; this was possibly related to the up-regulation of wt1 and nephrin, and down-regulation of desmin.
基金supported by grants from the National Natural Sciences Foundation of China (No 30500245, 30871174)the Science Research Foundation of Health Department of Hubei Province (No NX200510)
文摘To investigate the effects of albumin on the production of matrix metalloproteinases-2 （MMP-2） and matrix metalloproteinases-9 （MMP-9）in podocytes. Podocytes were treated with bovine serum albumin （BSA） at the concentration of 0.1, 0.5, 1, 2 g/L, respectively. Conditioned media were harvested 12, 24, 48 and 72 h after the treatment. The expression of MMP-2 and MMP-9 was assayed by gelatin zymography, RT-PCR and Western blotting analysis. Our results showed that in comparison with the control group, BSA increased the expression of MMP-2 and MMP-9 mRNA and protein in a doseand time-dependent manner （P〈0.05）. Meanwhile, the enzymatic activities of MMP-2 and MMP-9 in the culture supernatants of podocytes were also increased （P〈0.05）. It is concluded that albumin up-regulated the expression of MMP-2 and MMP-9 at gene and protein levels in a time-and dose-dependent manner.
基金This study was supported by the National Natural Science Foundation of China program(81774273 and 82004275)Beijing Municipal Science and Technology Commission(Z161100001816003).
文摘Objective:Previous studies have found that Qidi Tangshen granules(QDTS),a combination therapy of supplementing essence(Tianjing,TJ)and unblocking the collaterals(Tongluo,TL),can reduce kidney damage in db/db mice.This study aimed to explore the effect of QDTS and their separate prescriptions on podocytes in mice with diabetic nephropathy.Methods:The db/db mice were used in this experiment as an animal model,while wild-type C57BL/6J mice were used as normal controls.At the age of 12 weeks,the db/db mice were randomly divided into 5 groups(db/db,db/dbþvalsartan,db/dbþQDTS,db/dbþTJ and db/dbþTL).The urine albumin excretion ratio(UAE)was measured by enzyme-linked immunosorbent assay before and after the intervention.The ultrastructure of the kidney podocytes was observed by transmission electron microscopy.The protein expression levels of nephrin and desmin were detected by immunohistochemistry.Results:QDTS and their separate prescriptions significantly decreased the UAE and attenuated the renal pathological injury.QDTS and their separate prescriptions also reduced the fusion rate of the foot processes and increased the expression of nephrin protein.In contrast,QDTS and their separate prescriptions(TJ and TL)reduced the expression level of desmin protein.Conclusion:QDTS and their separate prescriptions might reduce diabetes-induced renal injury by reducing podocyte damage.The therapeutic effect of QDTS was more pronounced than TJ and TL.
基金Supported by the funds of Key Scientific Research Project of Colleges and Universities in Henan Province in 2017(Explore the Mechanism of Huoxue Tongluo Decoction in Preventing and Treating Diabetic Nephropathy from the Regulation of Podocyte Autophagy by Mtor Signal Pathway,No.17A360003)the Research Project of National TCM Clinical Research Base(Project Number:2019jdzx068+9 种基金Project Name:Study on the Effect of Tongluo Digui Decoction on Circadian Rhythm of Diabetic Nephropathy Based on Lncrna-mrna Chip Technology)the Special Research Project of TCM In Henan Province(Project Number:2019zybj17Project Name:Study on the Mechanism of Tongluo Digui Decoction in the Treatment of Diabetic Kidney Disease by Regulating the Pyroptosis of Podocyte through Autophagy-related Molecular Pathways)Henan Province Key R&D and Promotion Special(Science and Technology)Project(Project Number:202102310505Project Name:Study on the Mechanism Of Tongluo Digui Decoction in the Treatment of Diabetic Nephropathy Based on Autophagy Regulating Renal Tubular Epithelial Cell Pyroptosis)Henan Province Key R&D and Promotion Special(Science and Technology)Project(Project Number:202102310171Project Name:to Explore the Mechanism of Tongluo Digui Decoction on Improving Non-dipper Blood Pressure In Diabetic Nephropathy Based on Circadian Rhythm)a Sub-station Project of the Inheritance Studio of Famous Old Chinese Medicine Experts Across the CountryTCM Top Talent Training Project of Henan ProvinceNational TCM Innovational Core Talent Training Project。
文摘OBJECTIVE:To investigate the effects of Tongluo Digui decoction on renal injury and streptozotocin-induced podocyte autophagy in diabetic rats.METHODS:Male Sprague-Dawley rats were randomly divided into six groups:normal,model,Tongluo Digui decoction(high,medium,and low dose)and valsartan.Streptozotocin was injected intraperitoneally to replicate the diabetic animal model.After 8 weeks,proteinuria was evaluated to establish the diabetic nephropathy model.Treatments were administered daily via the intragastric route.At 16 weeks after gavage,we determined 24 h urine protein concentration,and blood glucose,serum creatinine,and urea nitrogen concentrations.Then,rats were sacrificed,and kidneys were harvested and stained with periodic acid-Schiff to evaluate the pathological changes in glomeruli,including glomerular podocytes by transmission electron microscopy.Western blot analysis was used to determine the expression of nephrin,podocin,p62,beclin-1,LC3Ⅱ/Ⅰ,and p-m TOR/m TOR protein in kidney tissues.RESULTS:Compared with the model group,Tongluo Digui decoction was associated with decreases in 24 h urine protein concentration,and blood glucose,hemoglobin A1 c,serum creatinine,urea nitrogen concentrations,total serum protein and albumin.Concurrently,mesangial mesenteric broadening and fusion of foot processes were reduced,the glomerular basement membrane was not significantly thickened,and the number of podocytes and the number of autophagosomes in the podocytes was increased.Further,expression of nephrin,podocin,LC3Ⅱ,and beclin-1 protein in kidney tissue was up-regulated,while expression of p62 protein was down-regulated and m TOR phosphorylation was inhibited.CONCLUSION:Tongluo Digui decoction may inhibit the progression of diabetic nephropathy by inhibiting m TOR phosphorylation,thereby increasing autophagy to protect podocytes and reducing proteinuria.
基金supported by grants from the NationalNatural Science Foundation of China(No.30500245 and No.30871174)the Science Research Foundation of HealthDepartment of Hubei Province(No.NX200510)
文摘Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.
基金supported by the Natural Science Foundation of Ningxia Province(No.2021AAC03296).
文摘Objective:Diabetic nephropathy is one of the most important microvascular complications of diabetes,which mainly refers to glomerular capillary sclerosis.Podocytes are an important part of glomerular capillaries.Previous clinical and basic studies have shown that fibrosis is the main factor of diabetic nephropathy.This study aimed to assess the protective mechanism of glycyrrhizic acid(GA)on glomerular podocytes induced by high glucose as we hypothesized that GA may have antifibrotic and anti-inflammatory effects on podocytes through regulation of the adenosine 5'-monophosphate-activated protein kinase(AMPK)/sucrose nonfermenting AMPK-related kinase(SNARK)signaling pathway.Methods:SNARK siRNA was used to transfect podocytes.Real-time quantitative polymerase chain reaction and immunofluorescence staining assays were used for molecular and pathological analysis.The expression levels of key pathway proteins(including TGF-β1,α-SMA,SITR1,AMPKα,LKB1,PGC-1α,NF-κB,IL-6,and TNF-α)were verified by Western blotting.The expression of inflammatory factors in podocytes was detected by ELISA.Results:We demonstrated that GA decreased the expression of podocyte fibrosis signaling pathway-related factors by upregulating the AMPK pathway and its related factors.However,after transfection of podocytes with SNARK siRNA,there was an increased expression of fibrosis-related factors and inflammation-related factors.Conclusion:GA can protect podocytes and alleviate fibrosis and inflammation induced by high glucose,which is related to the AMPK signaling pathway.Meanwhile,knockdown of SNARK protein can inhibit the AMPK signaling pathway,aggravate fibrosis,and increase inflammation.
基金the National Natural Science Foundation of China(No.81400333).
文摘The role of B7-1 in podocyte injury has received increasing attention.The aim of this study was to investigate whether losartan protects podocytes of patients with diabetic kidney disease(DKD)by regulating B7-1 and the underlying mechanisms.Rats with streptozotocin-induced DKD were treated with losartan for 8 weeks.Biochemical changes in blood and urine were analyzed.Kidneys were isolated for electron microscopy,immunofluorescence,real-time quantitative PCR(RT-PCR),and Western blot analysis.Immortalized mouse podocyte cells were cultured in normal or high glucose medium in the presence or absence of losartan for 48 h,and then the cells were collected for immunofluorescence,PCR,Western blotting and monolayer permeability detection.The phosphatidylinositol 3-kinase(PI3K)110a subunit and angiotensin II type 1 receptor(AT1R)plasmids were transfected into podocytes,respectively,and then Western blotting was performed to assess the expression of B7-1 protein.The results showed that losartan ameliorated podocyte structure and function in the rat model of DKD,and reduced the expression of B7-1 protein.Overexpression of PI3K 110a subunit in podocytes attenuated the inhibitory effect of losartan on B7-1 expression in high glucose-stimulated podocytes.The expression of B7-1 was significantly increased by overexpression of ATI R and significantly reduced by blocking PI3K 110a subunit.We conclude that losartan protects podocytes against high glucose-induced injury by inhibiting AT1R-mediated B7-1 expression.This effect is dependent on the AT1R-PI3K 110a subunit pathway.
基金supported by Guangdong Bureau of Traditional Chinese Medicine (20211082)
文摘Objective To explore the influence of Linggui Zhugan Decoction(LGZGD) on high glucose induced podocyte autophagy.Methods LGZGD containing serum was prepared by intragastric administation of 4.2 g/kg(low dose), 8.4 g/kg(medium dose), and 12.6 g/kg(high dose) LGZGD into SD rats respectively. MPC5 and AB8/13 podocyte cells were treated with 60 mmol/L glucose to establish diabetic nephropathy podocyte model in vitro. Both podocytes were divided into control group, high glucose group, low dose LGZGD group, medium dose LGZGD group, and high dose LGZGD group, respectively. For the three LGZGD groups, before LGZGD intervention, podocytes were treated with 60 mmol/L glucose for 3 days. After treated with LGZGD containing serum, cells were collected to analyze cell migration using Transwell assay, proliferation using CCK8, apoptosis and cell cycle using flow cytometry, autophagosome formation using transmission electron microscopy, and expression levels of Beclin-1, Atg5, LC3II/I, and P62 proteins using Western blot.Results Compared with the control group, the proliferation and migration of MPC5 and AB8/13 cells in the high glucose group slightly decreased, whereas these parameters restored after intervention with low and medium concentrations of LGZGD, with the medium dose LGZGD having the better effect(P < 0.05). Flow cytometry showed that the medium dose LGZGD group had a significantly lower apoptosis rate(P < 0.05) and higher survival rate(P > 0.05) compared to the high dose LGZGD group. High glucose arrested podocytes in G1 phase, whereas LGZGD shifted podocytes from being predominant in G1 phase to G2 phase. High dose LGZGD significanly reduced high glucose-increased autophagosome formation in both podocytes(P < 0.05). Western blot analysis showed that Beclin-1, Atg5, LC3II/I, and P62 expressions were increased in MPC5 cells treated with high glucose and reversed after adminstration of low and medium doses of LGZGD(P < 0.05).Conclusion LGZGD reduced apoptosis and enhanced autophagy in high glucose treated podocytes via regulating Beclin-1/LC3II/I/Atg5 expression.
文摘Glomerular tuft immune reactive Ezrin surface area (EzA) and fraction of EzA to total glomerular tuft area significantly increased, indicating podocyte growth, rounding and altered cytoskeletal interactions at 1 week of STZ diabetes. Podocyte number per glomerulus (WT1+ nuclei) did not change indicating no detachment, but density decreased due to tuft hypertrophy. Treatment with PLZ or Insulin for one week, prevented increase in proteinuria and hyperglycemia but not the decrease in podocyte density. PLZ but not Insulin prevented increase in ezrin positive area in glomeruli and per podocyte. In podocytes in culture neither 25 mM glucose with or without PLZ (2.5 or 25 uM) altered Ezrin expression measured in western blots. In summary, the Ezrin positive glomerular surface area increase seen after 1 week of STZ diabetes, reflects altered podocyte morphology and cytoskeletal interactions, prevented by PLZ but not by insulin. Ezrin area increase preceded podocyte detachment and in podocytes in culture is not associated with increases in podocyte Ezrin protein expression. It is a likely precursor of shape changes in podocytes and of alterd interactions with basement membrane that contribute to detachment and thickening. Glomerular capillary tuft hypertrophy and reduced podocyte density persisted despite PLZ or insulin treatments, independently of levels of glycemia and of proteinuria.
基金supported by the National Natural Science Foundation of China (Grant Nos 30830056 and 30830056)the National Basic Research Program of China (Grant No 2006CB503904) to Dr HOU FanFan+1 种基金the National Natural Science Foundation of China (Grant No 30971382)the Natural Science Foundation of Guangdong Province (Grant No 06024402) to Dr LIANG Min
文摘Accumulation of plasma advanced oxidation protein products(AOPPs) promotes progression of proteinuria and glomerulo-sclerosis.To investigate the molecular basis of AOPPs-induced proteinuria,normal Sprague-Dawley rats were treated with AOPPs-modified rat serum albumin.The expression of glomerular podocyte slit diaphragm(PSD)-associated proteins,nephrin and podocin,was significantly decreased coincident with the onset of albuminuria in rats treated with AOPPs.Chronic inhibi-tion of NADPH oxidase by apocynin prevented down-regulation of nephrin and podocin and decreased albuminuria in AOPPs-challenged rats.This suggested that accumulation of AOPPs promotes proteinuria,possibly via down-regulating the expression of PSD-associated proteins.
文摘Background:Nephrotic syndrome is the most common cause of kidney disease in children,but its pathogenesis remains unclear.This article reviews the novel aspects of the mechanisms underlying massive proteinuria in minimal-change disease,which is the most common form of childhood nephrotic syndrome.Data sources:This article integrates the findings of a PubMed database search for English language articles published in the past 40 years(from September 1974 to February 2014)using the key words"pathogenesis","minimal change nephrotic syndrome"or"idiopathic ne phrotic syndrome".Results:Unknown humoral factors associated with T-cell dysfunction have been thought to play an important role in the pathogenesis of minimal-change disease.However,recent findings are changing this paradigm,i.e,visceral glomerular epithelial cells(podocytes)may be involved via expression of molecules such as CD80 and angiopoietin-like 4.Conclusions:Recent evidence suggests that minimal-change disease results from interactions between humoral factors and dysfunctional podocytes.In addition to immunosuppressant drugs that target lymphocytes,a biological agent such as an antibody against the abnormal molecule(S)expressed by podocytes may provide novel drug treatment for minimal-change disease.
基金The reported work was supported in part by research grants from the National Institutes of Health(CA226303 to TCH)the National Key Research and Development Program of China(2016YFC1000803 and 2011CB707906 to TCH)+1 种基金This project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430.
文摘Glomerular podocytes are highly specialized epithelial cells and play an essential role in establishing the selective permeability of the glomerular filtration barrier of kidney.Maintaining the viability and structural integrity of podocytes is critical to the clinical management of glomerular diseases,which requires a thorough understanding of podocyte cell biology.As mature podocytes lose proliferative capacity,a conditionally SV40 mutant tsA58-immortalized mouse podocyte line(designated as tsPC)was established from the Immortomouse over 20 years ago.However,the utility of the tsPC cells is hampered by the practical inconvenience of culturing these cells.In this study,we establish a user-friendly and reversibly-immortalized mouse podocyte line(designated as imPOD),on the basis of the tsPC cells by stably expressing the wildtype SV40 T-antigen,which is flanked with FRT sites.We show the imPOD cells exhibit long-term high proliferative activity,which can be effectively reversed by FLP recombinase.The imPOD cells express most podocyte-related markers,including WT-1,Nephrin,Tubulin and Vinculin,but not differentiation marker Synaptopodin.The imPOD cells do not form tumor-like masses in vivo.We further demonstrate that TGFb1 induces a podocyte injury-like response in the FLP-reverted imPOD cells by suppressing the expression of slit diaphragm-associated proteins P-Cadherin and ZO-1 and upregulating the expression of mesenchymal markers,a-SMA,Vimentin and Nestin,as well as fibrogenic factors CTGF and Col1a1.Collectively,our results strongly demonstrate that the newly engineered im-POD cells should be a valuable tool to study podocyte biology both under normal and under pathological conditions.
文摘The underlying molecular changes that result in minimal change disease(ne-phrotic syndrome)require an in-depth analysis.Current molecular studies have shown the involvement of zinc fingers and homeobox transcriptional factors in its pathogenesis.The application of therapeutic drugs relies on understanding the cascade of molecular events to determine their efficacy in managing the clinical condition.
基金supported by the Hunan Province Natural Science Foundation Medical Industry Joint Fund(No.2024JJ9421)Clinical Medical Technology Innovation Guide Project of Hunan Province(No.2021SK51418).
文摘Objectives:Podocytes undergo epithelial-mesenchymal transition(EMT)and ferroptosis in response to hyperglycemic stimulation.This is considered an important early event in the development and progression of diabetic nephropathy(DN).Rhein is the main active anthraquinone derivative in several common traditional herbal medicines.This study aimed to investigate the protective effects of Rhein on podocyte ferroptosis and EMT.Methods:The mouse glomerular podocyte cell line MPC5 was stimulated with high glucose(HG),Rhein,and the ferroptosis inhibitor ferrostatin-1(Fer-1).Mechanistic investigations employed plasmids to overexpress and knockdown Sirtuin-1(SIRT1),solute carrier family 7 member 11(SLC7A11),or p53 and measure ferroptosis-or EMT-related indicators.Results:In the HG-injured podocytes,Rhein enhanced cell viability,reduced malondialdehyde(MDA),ferrous iron(Fe2+),and reactive oxygen species(ROS)levels,increased glutathione(GSH)production,accompanied by the restoration of ferroptosis-and EMT-associated indicator expressions.Mechanistically,Rhein induced SIRT1 and SLC7A11 expression and attenuated p53 expression.SIRT1 knockdown upregulated p53 and downregulated SLC7A11,thereby abolishing the protective effects of Rhein against podocyte ferroptosis and EMT.However,the effects of SIRT1 overexpression were reversed by SLC7A11 knockdown.Conclusion:Rhein activated the SIRT1/p53/SLC7A11 axis to protect podocytes against ferroptosis and EMT.This suggests that Rhein has a potential therapeutic effect on DN patients associated with podocyte injury,and targeting SIRT1/p53/SLC7A11 may represent an innovative therapeutic strategy for DN patients.
基金National Natural Science Foundation of China Project:Experimental Research on Podocyte Autophagy of Diabetic Nephropathy Regulated by Qizhi Jiangtang Capusul(No.81874440)Natural Science Foundation of Shandong Province Project:Curcumin Ameliorates Diabetic Nephropathy via Regulating the Intestinal Barrier-Inflammation“cross-talk”(ZR2020QH063)。
文摘OBJECTIVE:To investigate the therapeutic action and mechanism of the Qizhi Jiangtang capsule(芪蛭降糖胶囊,QZJT)on diabetic kidney disease(DKD)treatment.METHODS:This experiment used db/db mice and podocytes(MPC5)to develop DKD model.Evaluation of the effect of the QZJT on db/db mice by testing urine and blood biochemical parameters(24-h urinary albumin,serum creatinine,blood urine nitrogen),pathological kidney injury,and podocyte integrity.Moreover,autophagosomes in podocytes of DKD mice and cultured podocytes were detected using electron microscopy.Additionally,Western blotting was applied to detect the expression of podocyte marker protein(podocin),autophagy-associated proteins,and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)signaling pathway changes in vivo and in vitro.RESULTS:QZJT significantly reduced urine protein,blood nitrogen urea,and serum creatinine and showed histological restoration of renal tissues.QZJT also significantly improved the down-regulation of podocin and foot fusion and effacement in db/db mice.QZJT increased autophagic vesicles in mice and cultured podocytes.QZJT also upregulated microtubuleassociated protein 1 light chain 3-II(LC3-II)/(LC3-I)and Beclin-1 and downregulated phosphorylated-PI3K(pPI3K),p-AKT,and p-mTOR in db/db mice and MPC5 cells.However,autophagy inhibitor 3-methyladenine partially alleviated the above effects in MPC5 cells.CONCLUSIONS:These results showed that the QZJT can enhance podocyte autophagy and ameliorate podocyte injury in DKD by inhibiting the PI3K/AKT/mTOR signaling pathway.
文摘Objective： To observe the podocyte injury in diabetic nephropathy （DN） patients by identifying the urinary podocytes and the situation of detached podocytes in glomeruli and to demonstrate the correlation between podocyte excretion and proteinuria, blood glucose, serum creatinine in different phases in DN patients. Methods： Urinary podocytes and the podocalyxin （PCX） expression state of podocytes in glomeruli were identified and observed by indirect immunofluorescent method. The DN patients were divided into three groups according to the volume of proteinuria, namely small, medium and large volume proteinuria groups. The podocytes in the urine of every group were calculated. The DN patients were divided into five groups according to the chronic kidney disease （CKD） phases, then the positive podocytes in urine were calculated. Meanwhile, the 24-hour protein in urine, fasting blood glucose （FBG） and the serum creatinine of DN patients were tested. The correlations among the proteinuria, serum creatinine, FBG and the number of positive podocytes in the urine of DN patients were statistically analyzed. Results： Urinary positive podocytes were found in 88% of the patients with DN, whereas podocytes were found in 0% of patients with minimal changed disease （MCD） and healthy cases. The expression of PCX was absent in DN patients. In contrast, PCX was expressed integrally in MCD patients. The positive podocytes was 1.49±0.95/ml in small-volume proteinuria group, 2.15±0.70/ml in the medium-volume proteinuria group, and 3.48±1.27/ml in the large-volume proteinuria group. There was no significant difference between the small- and medium- volume proteinuria groups, and there were significant differences between other groups （P〈0.05）. The positive podocyte number tended to increase as proteinuria was increased. By Pearson analysis, the correlation between podocyte number and proteinuria was podocytes in urine from different groups of DN patients, CKD pc I sitive statistically. The difference of the number of positive -V group was significant statistically. The correlation between serum creatinine of CKD Ⅰ -Ⅲ group and positive podocytes in urine was positive statistically. The correlation between serumcreatinine of CKD Ⅳ- Ⅴ group and positive podocytes in urine was not significant statistically. The correlation between FBG and positive podocytes in urine was not significant either. Conclusion： The mechanism of the podocyte injury in DN patients is present. The podocyte injury in DN may positively correlate to proteinuria and serum creatinine of CKD Ⅰ -ⅢDN patients, but not to the FBG and serum creatinine of CKD Ⅳ-Ⅴ patients.
文摘BACKGROUND A number of recent studies indicate a transformation in the natural course of chronic kidney disease(CKD)in type 2 diabetes(T2D)patients:an increasing prevalence of declined renal function without proceeding to the accompanying elevation of albuminuria.It has been suggested that albuminuric and nonalbuminuric CKD patterns could be different in their phenotypes and pathogenic mechanisms.AIM To identify the risk factors and biomarkers of albuminuric and non-albuminuric patterns of CKD in patients with T2D.METHODS Three hundred sixty patients with T2D duration≥10 years were included in this observational cross-sectional study.The associations of a panel of demographic and clinical characteristics,complications,comorbidities,and metabolic and hematology parameters with albuminuric and non-albuminuric CKD patterns were analyzed.The urinary excretion of nephrin and podocin,two podocytespecific markers,and WAP-four-disulfide core domain protein 2(WFDC-2),a marker of tubulointerstitial fibrosis,was determined by ELISA in comparison with healthy controls.RESULTS Non-albuminuric CKD was associated with age≥65 years(P=0.0001),female sex(P=0.04),diabetes duration≥15 years(P=0.0009),and the use of diuretics(P=0.0005).Male sex(P=0.01),smoking(P=0.01),waist-to-hip ratio>1.0(P=0.01)and hemoglobin A1c(HbA1c)>8.0%(P=0.005)were risk factors for elevated albuminuria not accompanied by a decrease in estimated glomerular filtration rate(eGFR).Duration of diabetes≥15 years and the use of calcium channel blockers were risk factors for albuminuria with decreased eGFR(both P=0.01).In multivariate logistic regression analysis,age,HbA1c,female sex and diuretics were significant predictors for reduced eGFR,while waist-to-hip ratio,HbA1c and male sex were associated with elevated urinary albumin-to-creatinine ratio(UACR).Excretion of nephrin and podocin was increased in patients with albuminuria,regardless of decline in renal function(P<0.001),correlating positively with UACR.The urinary excretion of WFDC-2 was markedly higher in men than in women(P<0.000001).Men with T2D demonstrated increased WFDC-2 levels independently of the CKD pattern(all P<0.05).In T2D women,WFDC-2 excretion was increased in those with reduced renal function(P≤0.01),correlating negatively with eGFR.CONCLUSION The data provide further evidence that albuminuric and non-albuminuric CKD phenotypes correspond to different pathways of diabetic kidney disease progression.
文摘Costimulatory pathways(Cluster of differentiation 28,tumor necrosis factor-related,adhesion and T Cell Ig-and mucin-domain molecules) regulating the interactions between receptors on the T cells andtheir ligands expressed on several cell types,have a key role in controlling many immunological and non immunological processes.Indeed,accumulating evidence indicate that these molecules are involved in the pathogenesis of numerous conditions,such as allograft rejection,atherosclerosis,rheumatoid arthritis,psoriasis and renal diseases,including glomerulonephritis.Primary or secondary(i.e.,associated with infections,drugs or systemic diseases,such as systemic lupus erythematosus,diabetes,etc.) glomerulonephritis represent a group of heterogeneous diseases with different pathogenic mechanisms.Since costimulatory molecules,in particular CD80 and CD40,have been found to be expressed on podocytes in the course of different experimental and clinical glomerulonephritis,costimulation has been thought as a new therapeutic target for patients with glomerular diseases.However,although experimental data suggested that the blockade of costimulatory pathways is effective and safe in the prevention and treatment of glomerular diseases,clinical trials reported contrasting results.So,at this moment,there is not a strong evidence for the general use of costimulatory blockade as an alternative treatment strategy in patients with primary or secondary glomerulonephritis.Here,we critically discuss the current data and the main issues regarding the development of this innovative therapeutic approach.
