Attenuation of high glucose induced podocyte injury by ursolic acid up-regulating autophagy level

Objective To investigate the effects of ursolic acid (UA) on autophagy and podocyte injury induced by high glucose. Methods Conditionally immortalized murine podocyte were cultured in high glucose, the effect of PI3K inhibitor LY294002 and ursolic acid treatment were observed. The miR-21 expression was detected using RT-qPCR. The activation of PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related protein and podocyte marker protein were determined by Western blot. Immunofluorescence staining showed the expression of podocyte marker protein and endogenous accumulation of LC3. Autophagosomes were observed using electron microscopy. Results Compared with normal control group,the cells exposed to high glucose condition showed down-regulated synaptopodin, podocin and nephrin expression (P＜0.01), up-regulated miR-21 expression (P＜0.01), down-regulated PTEN expression (P＜0.01), up-regulated p85-P13K, phospho(p)-Akt, p-mTOR,p62/SQSTMI, expression and down-regulated LC3II and Beclin1 expression (all P＜0.01). Ursolic acid and LY294002 promoted synaptopodin, podocin and nephrin expression (all P＜0.01), up-regulated LC3II, Beclin1 expression and down-regulated p62/SQSTM1 expression (all P＜0.01), down-regulated p85-PI3K, p-Akt, p-mTOR expression (all P＜0.01). However, LY294002 did not affect the expression of miR-21 and PTEN. Ursolic acid inhibited miR-21 expression and upregulated PTEN level. Conclusions The podocyte injury is associated with defective autophagy level under high glucose condition. Ursolic acid could reduce podocyte injury by increasing autophagy level via inhibition of miR-21 expression and PTEN/Akt/mTOR pathway.     

Oxidized LDL leads to podocyte injury through PTEN/SR-A pathway

Objective To evaluate the effect of oxidized LDL (Ox-LDL) on scavenger receptor A (SR-A) expression in mouse podocytes and to explore the possible mechanism. Methods The conditionally immortalized mouse podocyte cell line was cultured in vitro and exposed to Ox-LDL of different concentrations for 24 h, or 20 mg/L Ox-LDL for different hours. Cell cholesterol accumulation was examined. Real-time quantity PCR and Western blotting were used to analyze the expression level of PTEN, SR-A and nephrin. Podocytes were incubated with DiI labeled Ox-LDL for 4 h and immunofluorescence was used to analyze lipid uptaking. To further confirm the relationship between PTEN and SR-A, PTEN inhibitor bpv (hOpic) [dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (V) ], PTEN siRNA and PTEN adenovirus (adPTEN) were used to dual-directional regulate PTEN expression, so as to observe the change of SR-A, nephrin, cell cholesterol accumulation and lipid uptake. Results SR-A was expressed on mouse podocyte and mediated podocyte lipid uptake. Compared with control group, Ox-LDL increased cell cholesterol accumulation, and up-regulated SR-A expression along with inhibited expression of PTEN and nephrin (P＜0.05), which were correlate with dose and exposure time of Ox-LDL. Expression of PTEN significantly inhibits the expression level of SR-A and lipid uptake induced by Ox-LDL (P＜0.05), thereby decreasing cell cholesterol accumulation, but up-regulating nephrin level (P＜0.05). However, down-regulation of PTEN could cause opposite effect. Conclusion Ox-LDL up-regulates SR-A through decreasing the expression of PTEN, and contributes to podocyte injury.     

Tacrolimus protects podocytes by up-regulating autophagy in type 2 diabetic model rats

Objective To assess the effects of tacrolimus (FK506) on podocyte in type 2 diabetic model rats and to explore the potential mechanism. Methods The model rats were fed with high fat and high sugar food and combining with a low-dose of streptozotocin (STZ). They were then randomly divided into a diabetic mellitus group (DM group) and a FK506 group. A normal control group (NC group) was also set. The rats in FK506 group were given with 0.5 mg?kg-1?d-1 FK506 for 8 weeks. The biochemical parameters were measured. The changes of renal pathology and ultrastructure of podocyte were observed by the light and electron microscopy. The expression of nephrin and LC3-Ⅱ was determined by immunohistochemistry and Western blotting. Results (1) Compared with those in NC group, KW/BW, systolic blood pressure (SBP), fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), urinary albumin excretion rate (UAE) and creatinine clearance rate (Ccr) in DM group were significantly increased (all P＜0.05). And the KW/BW, UAE and Ccr were decreased in FK506 group compared to those in DM group (all P＜0.05), while other parameters had no significant difference (all P＞0.05). (2) Compared with those in NC group, the glomerular volume, mesangial cell proliferation and accumulation of mesangial matrix were increased, and the foot process became disorder and fusion in DM group, while these changes were significantly reduced in FK506 group. (3) Compared with that in NC group, the expression of nephrin and LC3-Ⅱ was decreased in DM group (all P＜0.05), and both of parameters were higher in FK506 group than those in DM group (all P＜0.05). Conclusion FK506 may enhance podocyte autophagy in type 2 diabetic model rats and attenuate podocyte injury.     

Early autophagy activation inhibits podocytes from apoptosis induced by aldosterone

Objective To explore the protection of early autophagy activation on podocyte injury induced by aldosterone. Methods In vitro cultured mouse podocyte clones (MPC5) were treated with aldosterone for 6, 12, 24, 48 h respectively. Apoptosis of podocytes was detected by Annexin V combined with flow cytometry. After 24 h treatment with aldosterone, the existence of apoptotic body and autophagosome was observed by electron microscopy. The protein expressions of LC3, caspase?3 and nephrin were examined by Western blotting. The mRNA expression of Beclin?1 was detected by real?time PCR. Results The induction of apoptosis and autophagy by aldosterone in podocytes was in time?dependent mannner. After 24 h treatment with aldosterone, the apoptosis was increased by 26.5% (P＜0.05) and the expression of nephrin was decreased by 28.0% (P＜0.05) compared to control group. Aldosterone remarkably induced the expression of Beclin?1 at 6 h and promoted the transformation of LC3?Ⅰto LC3?Ⅱ at 12 h (P＜0.05). Compared to simple aldosterone treatment, the apoptosis rate of podocyte was increased by 39.0%（P＜0.05）and the expression of nephrin was declined by 19.5%（P＜0.05) after 3?methyladenine (3?MA) pre?treatment. Conclusions Aldosterone can induce autophagy and apoptosis in podocytes. Autophagy occurs earlier (12 h) than apoptosis (24 h). The occurrence of autophagy can inhibit the apoptosis,so the autophagy pathway may be a new research topic of glomerular disease treatment.     

Bufalin alleviates adriamycin-induced podocyte injury by up-regulating the expression of vitamin D receptor

Objective To investigate the role of vitamin D receptor (VDR) in the protection of bufalin on podocyte injury induced by adriamycin (ADR). Methods (1) In vitro: the toxic effect of different concentrations of bufalin (10-9, 10-8, 10-7, 10-6 mol/L) on podocyte was evaluated by lactate dehydrogenase (LDH) test;Annexin V-FITC and RT-PCR were utilized for podocyte apoptosis and VDR mRNA level respectively. Western blotting was used to analyze the protein expression of VDR and nephrin. SiRNA intervene was also applied to evaluate the role of VDR in bufalin's protective effect on podocyte injury induced by ADR. (2) In vitro: 24 SD rats were randomly divided into three groups: control group, ADR group and ADR+bufalin group. TUNEL assay was applied to detect the apoptosis of podocytes in the kidney. Immunofluorescence and transmission electron microscope (TEM) were applied to analyze the expression of VDR and the ultrastructure of the glomerulus. Results Bufalin concentration lower than 10-7 mol/L had no toxicity on normal podocyte. Bufalin reduced the urinary protein excretion (P＜0.05), alleviated the removal of podocyte foot processes and attenuated the changes in nephrin expression in the glomerulus of the adriamycin (ADR) rats (P＜0.05). Bufalin notably inhibited the down-regulation of VDR in protein levels on the glomerulus of the ADR rats. Additionally, bufalin inhibited the down-regulation of VDR in both mRNA levels and protein levels (P＜0.05）, nephrin protein expression (P＜0.05), and apoptosis induced by ADR in cultured podocytes. Additionally, VDR specific siRNA intervene abolished the protective effect of bufalin in ADR-induced podocyte injury. Conclusion Bufalin can alleviate ADR-induced podocyte injury via enhancing VDR expression.     

The effect of acteoside on puromycin nephropathy and podocyte injury model

Objective To explore the effect of acteoside (one of the ingredients of Total Glycosides Extracted from Rehmannia capsules) on treatment of proteinuria and protection of podocytes. Methods In this study, puromycin nephropathy rat model was successfully established. After detecting the degree of proteinuria, the expression of podocyte injury markers and the degree of podocyte foot process fusion were investigated by electron microscope. In addition, puromycin treated podocyte injury model was also successfully established in vitro. Podocyte viability, migration, cytoskeleton and injury marker were detected. Results In vivo study showed that acteoside could effectively reduce proteinuria (P＜0.05), restore the expression of podocyte injury markers such as nephrin and synaptopodin (all P＜0.05), and alleviate the degree of podocyte foot process fusion. In vitro study showed that acteoside could effectively restored podocyte viability (P＜0.05), reduce abnormal migration ability (P＜0.05), protecte cytoskeleton and restore the expression of podocyte injury marker nephrin (P＜0.05). Conclusions This study confirms that acteoside can reduce the degree of proteinuria in puromycin nephropathy rat model in vivo and alleviate the degree of podocyte injury in vitro as well as enrich the molecular mechanism of Total Glycosides Extracted from Rehmannia capsules in treatment of proteinuria.     

Effects of B7-1 on the cytoskeleton rearrangement in podocytes induced by angiotensin Ⅱ

Objective To observe the effect of costimulatory molecule B7-1 on cytoskeleton rearrangement in mouse podocytes induced by angiotensinⅡ(AngⅡ), and to study the underlying molecular mechanism of B7-1 in the pathological changes of podocytes. Methods All cultivation of conditionally immortalized mouse podocytes (MPC) in vitro were divided into the following groups: normal control group, CTLA-4 group, AngⅡgroup (10-6 mmol/L 12 h, 24 h; 10-8 mmol/L 12 h, 24 h) and CTLA-4 with AngⅡgroup. Transfect B7-1 RNA interference fragment (siRNA) to the mature podocytes, and then restimulated by AngⅡ(10-6 mmol/L 12 h), the change of podocyte cytoskeleton after AngⅡ stimulation were observed. The expression of B7-1 in each group was assayed by flow cytometry and Western blotting. The nephrin and p-nephrin protein levels in the four groups were also analyzed by Western blotting. At the same time, the podocyte cytoskeleton distribution as indicated by F-actin was observed by fluorescence microscopy. Results Flow cytometry and Western blotting showed that B7-1 was not expressed in the normal control group. AngⅡshowed a concentration and time dependent induction of B7-1 expression in mouse podocytes (P＜0.05). Western blotting indicated that AngⅡinduced B7-1 protein expression (P＜0.05). Expression of nephrin and p-nephrin was significantly down-regulated by AngⅡ(P＜0.05). Compared with the normal control group, the expression of podocyte protein nephrin and p-nephrin in Ang II stimulation group was significantly reduced (P＜0.05). Using FITC phalloidin fluorescence staining showed that CTLA-4+AngⅡ stimulation group cytoskeleton rearrangement was improved significantly and F-actin recombinant score (mCFS) decreased compared with AngⅡ group (P＜0.05), suggesting that AngⅡ led to the disorder of the podocytes cytoskeleton and the destruction of the cytoskeleton of podocytes by AngⅡ could be improved after B7-1 blocking. Compared with the AngⅡ stimulation, transfection of B7-1 siRNA+AngⅡ stimulation group improved F-actin cytoskeletal rearrangement, and mCFS also decreased significantly (P＜0.05), suggesting that transfection of B7-1 siRNA might improve the damage of AngⅡ on podocytes cytoskeleton. Conclusions B7-1 participates in the process of cytoskeleton reconstruction and plays an important role in the pathological changes of podocytes.     

Role of ABCA1 in angiotensinⅡ-induced cholesterol accumulation in podocytes

Objective To investigate the effects of angiotensinⅡ(AngⅡ) on the expression of ATP-binding cassette transporter A1(ABCA1) in AngⅡ-infused rat model and cultured human podocytes, and to explore the role of ABCA1 in AngⅡ-induced cholesterol accumulation of podocytes. Methods Twelve Wistar rats were randomly subjected to normal saline infusion, or AngⅡ infusion at 400 ng?kg-1?min-1 (via subcutaneous osmotic minipumps) for 8 weeks. The expression of glomerular ABCA1 was analyzed by Western blotting and real-time fluorescent quantitative PCR. In vitro, conditionally immortalized human podocytes were divided into normal group, AngⅡ group, AngⅡ+scrambled siRNA group, AngⅡ+ABCA1 siRNA group. The expression of podocyte ABCA1 was assessed by immunofluorescence, Western blotting and real-time fluorescent quantitative PCR. Oil Red O staining was used to observe the lipid droplets in podocytes and cholesterol efflux assay kit was used to measure the cholesterol efflux rate of podocytes. Fluorescein isothiocyanate (FITC)-conjugated phalloidin was used to observe the podocyte cytoskeleton. Results In vivo, compared with normal group, the protein and mRNA expression of glomerular ABCA1 in AngⅡ-infused rats were decreased (P＜0.05). In vitro, ABCA1 was distributed in the cytomembrane of podocytes, and compared with normal group, AngⅡtreatment down-regulated the expression of ABCA1 (P＜0.05). Increased lipid droplets, decreased cholesterol efflux and cytoskeletal rearrangement were observed in AngⅡ-treated podocytes. When compared to AngⅡ group, podocytes stimulated by AngⅡand then transfected with ABCA1 siRNA had lower expression level of ABCA1 mRNA and protein (all P＜0.05). More lipid droplets and lower cholesterol efflux rate could be observed in AngⅡ+ABCA1 siRNA group (P＜0.05). Conclusion The reduced expression of ABCA1 may be involved in AngⅡ-induced cholesterol accumulation in podocytes.     

Podocyte-specific knock-in of PTEN protects kidney against high fat diet

Objective To evaluate the effect of over-expression of phosphatase and tensin homolog does on chromosome ten (PTEN) in podocytes on kidney under high fat diet (HFD) in vivo and clarify the mechanism how PTEN regulates scavenger receptor A (SR-A) expression exposed to oxidized low density lipoprotein (ox-LDL) in podocytes in vitro. Methods The podocyte-specific PTEN knock-in (PPKI) mice were fed with HFD to establish mouse model of lipid-induced renal injury. Mice were divided into four groups: ND+Ctrl group, ND+PPKI group, HFD+Ctrl group and HFD+PPKI group. After 24 weeks of dietary intervention, all mice were tested for clinical and biochemical parameters, including serum creatinine (Scr) as well as urine albumin excretion rate (UAER); renal lipid content was measured by oil red O staining and cholesterol quantitative analysis; the pathological changes of glomeruli were observed by PAS staining and electron microscope. Podocyte injury was induced by ox-LDL in vitro. Western blotting was used to detect the changes of SR-A expression induced by ox-LDL after YAP-siRNA interfering (si-YAP), as well as YAP phosphorylation induced by ox-LDL after interfering by PTEN-siRNA (si-PTEN) and PTEN phosphatase inhibitor (Bpv-PTEN), and over-expressing by recombinant adenovirus (ad-PTEN). Results Compared with ND+Ctrl group, HFD+Ctrl group significantly aggravated the levels of Scr and UAER, the expression of SR-A in podocytes, renal lipid content, mesangial matrix expansion, effacement of podocyte foot processes, and incrassation of glomerular basement membrane (all P＜0.05). Conversely, compared with HFD+Ctrl group, HFD+PPKI group obviously alleviated the above lipid-induced renal damage (all P＜0.05). In vitro, the expression of SR-A in podocytes was up-regulated when stimulated with ox-LDL (P＜0.05), and the knockout of YAP significantly down-regulated the expression of SR-A induced by ox-LDL (P＜0.05). Exposed to ox-LDL, the expression of p-YAP increased in podocytes (P＜0.05); over-expression of PTEN inhibited p-YAP up-regulation induced by ox-LDL (P＜0.05), while either knockdown of PTEN or inhibition of PTEN phosphatase activity displayed opposite effect (all P＜0.05). Conclusions Over-expression of PTEN in podocytes protected the kidney against damage from HFD in vivo and PTEN might suppress SR-A mediated lipid uptake via dephosphorylating p-YAP to prevent podocyte injury from ox-LDL.     

Disruption of low-density lipoprotein receptor pathway induced by inflammation contributes to podocyte injury in diabetic nephropathy

Objective To investigate the effects of low density lipoprotein receptor (LDLr) pathway on podocyte injury in diabetic nephropathy (DN) under inflammatory stress. Methods Male db/db mice and db/m mice were randomly divided into four groups (8 mice in each group): db/m group (control), casein injected db/m group (db/m＋casein), db/db group (db/db), and casein injected db/db group (db/db＋casein). An inflamed model of DN was established according to our previous study. 24-hour urinary protein was measured every week. The plasma lipid profile was detected by clinical biochemistry assay. Podocyte changes were evaluated by electron microscope and immunofluorescent staining. Lipid accumulation in the kidney was evaluated by oil red O staining and intracellular cholesterol quantitative assay. The protein expression of Wilm's tumor-1 (WT-1), nephrin, α-smooth muscle actin (α-SMA), and molecules correlated with LDLr pathway were examined by immunohistochemical staining or Western blotting. The colocalized protein expression of LDLr with WT-1 was examined by immunofluorescent staining and laser confocal microscopy. Results There were no differences in plasma levels of LDL and HDLamong four groups. Compared with db/db group, the db/db+casein group showed markedly increased 24-hour urinary protein, more significant podocyte foot process effacement and podocyte damage, increased lipid droplet accumulation in kidneys, increased protein expressions of LDLr, SCAP and SREBP-2 in kidneys (all P＜0.05). Interestingly, increased LDLr protein expression in kidneys of db/db mice was negatively correlated with decreased nephrin protein expression (r＝-0.855, P＜0.01) and positively correlated with increased α-SMA protein expression (r＝0.768, P＜0.01). Conclusions The disruption of LDLr pathway induced by inflammation contributes to podocyte injuries in diabetic nephropathy.     

Effects of bone marrow-derived mesenchymal stem cells on glomerular podocyte injured by lipopolysaccharide

Objective To observe the effects of bone marrow-derived mesenchymal stem cells (BMSC) on glomerular podocyte injured by lipopolysaccharide (LPS) and the expression of related protein. Methods Podocytes are divided into control group, BMSC group, LPS group and LPS plus BMSC group. After 24 hours of intervention, observing each experimental group podocyte form under inverted phase contrast microscope;detecting the expressions of mRNA and protein of nephrin, CD2AP, synaptopodin, and TRPC6 by RT-PCR and Western-blot. Results Compared with control group, expressions of nephrin, CD2AP, and synaptopodin in LPS group decreased (P＜0.05) while that of TRPC6 increased (P＜0.05); compared with LPS group, expressions of nephrin, CD2AP, and synaptopodin in LPS+MSC group increased (P＜0.05) while that of TRPC6 decreased (P＜0.05). Conclusion BMSC may relieve LPS-induced podocyte injury.     

Protective effect ofACTH4-10 on adriamycin-induced podocyte injury

Objective To observe the influence of adrenocorticotropic hormone (ACTH4-10) in the changes of podocyte proliferation, apoptosis and expression of nephrin and podocin on adriamycin(ADR) -induced podocyte injury and investigate the protective effect ofACTH4-10. Methods All podocytes were randomly divided into following groups: normal control, ADR-induced group andACTH4-10 intervention group (low, middle and high concentration). Normal control group was not treated, ADR-induced group was induced to set the model of podocyte injury by ADR (1 μmol/L) for 24 hours andACTH4-10 intervention groups were intervened by 1 μg/L, 10 μg/L and 100 μg/LACTH4-10 for 1 hours respectively, prior to setting the model of podocyte injury. Cell counting kit (CCK-8) was used to detect the multiplication of podocytes and TUNEL apoptosis detection kit was used to detect podocyte apoptosis. Real-time PCR and Western blotting were used to examine the expression of nephrin and podocin. Results Compared with control group, podocyte proliferation and expression of nephrin and podocin was decreased significantly in ADR-induced group (P＜0.05), meanwhile podocyte apoptosis was increased obviously (38.14% vs 5.12%). Compared with ADR-induced group, podocyte proliferation and expression of nephrin and podocin was increased generally with concentration ofACTH4-10. Although podocyte apoptosis rates (20.45%, 17.39%, 11.02%) were increased inACTH4-10 intervention group (low, middle and high concentration) while comparing with normal control group, podocyte apoptosis decreased obviously while comparing with ADR-induced group. ConclusionsACTH4-10 can stabilize the expression of nephrin and podocin on slid diaphragm, and has the protective effect on podocyte injury induced by ADR, while the effect depends on the concentration ofACTH4-10.     

法舒地尔对血管紧张素Ⅱ诱导足细胞骨架重构的干预作用

目的 观察ROCK抑制剂法舒地尔对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导小鼠足细胞骨架重构的影响,探讨法舒地尔在足细胞病变中的保护机制.方法 体外采用AngⅡ(]0-7 mol/L)致足细胞损伤模型.不同浓度法舒地尔(10-8、10-7、10-6mol/L)与足细胞预孵育30 min或60 min后,再加入含AngⅡ(10-7 mol/L)的培养基,继续作用24 h.异硫氰酸荧光素(FITC)-鬼笔环肽荧光染色和Western印迹法分析足细胞骨架相关蛋白F-actin和synaptopodin 的变化,并进一步观察细胞内调节骨架装配的Rho-ROCK信号通路的活性,即采用Western印迹法检测Rho激酶1(ROCK-1)及磷酸化肌球蛋白磷酸酯酶靶点亚单位1(MYPT1)表达,代表ROCK的表达及活性.结果 与对照组相比,AngⅡ可明显降低足细胞synaptopodin的表达(P＜0.05),使F-actin重构,而法舒地尔预处理可减轻AngⅡ介导的synaptopodin的下调(P＜0.05)和F-actin的重构.法舒地尔可下调AngⅡ诱导的足细胞ROCK-1 (P＜ 0.05)及MYPT1蛋白表达(P＜0.05).结论 法舒地尔可稳定足细胞的细胞骨架,拮抗AngⅡ对足细胞的损伤.法舒地尔的上述作用可能与细胞内Rho-ROCK信号通路有关.     

Role of autophagy in high glucose-induced podocyte lipid droplet accumulation

Objective To investigate the role of autophagy in high glucose-induced podocyte lipid droplet metabolism. Methods (1) Cultured, conditionally immortalized human podocytes (HPC) were divided into normal control group, high glucose group and mannitol group. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Protein level of SREBP-1 was analyzed by Western blotting. (2) HPC were cultured and divided into normal control group, high glucose group, high glucose+3-methyladenine (3-MA) group, and mannitol group. Acridine orange staining was used to observe the formation of autophagosomes. Western blotting was used to detect the protein levels of beclin-1 and LC3-II/LC3-I. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Western blotting was used to analyze the expression of SREBP-1. Results (1) Compared with the normal control group, the lipid accumulation in the high glucose group was increased and the lipid metabolism related molecule SREBP-1 was up-regulated (P＜0.05); There was no significant difference between the normal control group and the mannitol group in lipid accumulation (P＞0.05). (2) Compared with the normal group, the number of autophagosomes was increased and autophagy-related proteins beclin-1 and LC3-II/LC3-I were up-regulated in high glucose group (all P＜0.05). After intervened with 3-methyladenine, a significant decrease in autophagosomes was observed; Protein levels of autophagy-related proteins beclin-1 and LC3-II/LC3-I were decreased (all P＜0.05); The lipid droplets in the high glucose+3-MA group was decreased and lipid metabolism related molecule SREBP-1 was down-regulated (all P＜0.05). Conclusion Autophagy may be involved in the process of high-glucose-induced podocyte lipid accumulation by affecting SREBP-1 expression, and inhibition of autophagy can alleviate the high-glucose-induced podocyte lipid accumulation.     

1,25(OH)2D3 ameliorates high glucose-induced podocyte injury via PI3K/p-Akt signalling pathway

Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced podocyte injury and its signal transduction mechanism. Methods Differentiated mouse podocytes were exposed to normal glucose, high glucose, and different concentrations of 1,25(OH)2D3 or LY294002 (a selective PI3K inhibitor) for 24 h. PCR and immunofluorescent staining were used to detect nephrin, podocin, and desmin. Western blotting was used to detect protein expression of nephrin, podocin, desmin, PI3K, Akt and p-Akt. Results Compared with high glucose group, 1,25(OH)2D3 (100 nmol/L and 1000 nmol/L) significantly up-regulated the expression of podocin and nephrin in podocytes induced by high glucose (P＜0.05). Meanwhile, 1,25(OH)2D3 (100 nmol/L) significantly reduced the expression of desmin (P＜0.05). PI3K and p-Akt were obviously reduced in high glucose group. In the presence of 1,25(OH)2D3, the trends were reversed. However the above effects of 1,25(OH)2D3 were abolished when p-Akt was blocked by the PI3K inhibitor LY294002. Conclusions 1,25 (OH)2D3 can inhibit high glucose-induced podocyte injury through PI3K/p-Akt signaling pathway.     

nephrin通过PI3K-Akt途径抑制血管紧张素Ⅱ诱导的足细胞凋亡

目的 研究nephrin在血管紧张素Ⅱ（AngⅡ）诱导足细胞凋亡中的作用,以及可能的分子机制。 方法 体外培养永生化小鼠足细胞（MPC）,以不同浓度AngⅡ处理MPC和10-8 mol/L AngⅡ刺激不同时间,用流式细胞仪检测细胞凋亡率;实时定量PCR、免疫荧光和Western印迹法检测nephrin的表达和分布;Western印迹法检测Akt磷酸化水平。脂质体法转染pcDNA3.1-mNPHS1质粒,G418筛选稳定转染细胞系。用Akt抑制剂LY294002或与AngⅡ共孵育刺激MPC和pcDNA3.1-mNPHS1转染细胞,检测Akt磷酸化水平和细胞凋亡率。 结果 （1）AngⅡ以剂量和时间依赖方式诱导MPC凋亡。AngⅡ受体拮抗药氯沙坦与AngⅡ共孵育18 h,显著降低AngⅡ单独刺激的足细胞凋亡率（P < 0.05）。（2）10-8 mol/L AngⅡ刺激12 h后,nephrin mRNA和蛋白较对照组显著降低,24 h nephrin mRNA约为正常对照的50%（P < 0.05）。正常足细胞nephrin主要分布于核膜周围的胞质和细胞膜,随刺激时间延长,胞膜和胞质nephrin表达逐渐降低。（3）10-8 mol/L AngⅡ刺激15 min后,Akt磷酸化显著降低,约为正常对照细胞的50%（P < 0.01）。（4）AngⅡ与LY294002共孵育12 h后,细胞凋亡率显著高于AngⅡ和LY294002单独刺激（均P < 0.05）。（5）pcDNA3.1-mNPHS1稳定转染显著上调足细胞Akt磷酸化水平（P < 0.05）,抑制 AngⅡ诱导的细胞凋亡（P < 0.05）。 结论 AngⅡ通过AngⅡ受体诱导小鼠足细胞凋亡,抑制足细胞nephrin表达。nephrin通过PI3K-Akt信号通路调节足细胞存活状态。     

EP1 receptor mediates Adriamycin-induced podocyte injury through activating p38 MAPK

Objective To investigate the effect of prostaglandin E2 receptor 1 (EP1) on Adriamycin (ADR)-induced glomerular podocytes injury and its possible mechanism. Methods (1) In vivo experiments: 6-8 weeks old male Balb/c mice were randomly divided into four groups: Control group; ADR group; EP1 agonist 17-phenyl PGE2+ADR group; EP1 antagonist SC-19220+ADR group. The mouse model of nephrotic syndrome was induced by injection of ADR (10 mg/kg) into tail vein, and then EP1 agonist (1 μg/g) and antagonist (25 μg/g) were administered respectively. Six weeks later, all mice were sacrificed and urine, blood and kidney tissues were collected. Detecting urine protein, blood chemistry, changes of renal pathology and podocyte-related proteins, electron microscopy changes of podocytes. (2) In vitro experiments: Podocytes were cultured in vitro and divided into different groups: Control group; ADR group (0.2 μmol/L); EP1 agonist (0.1, 1, 10 μmol/L)+ADR (0.2 μmol/L) group; antagonist (0.1, 0.5, 1 μmol/L)+ADR (0.2 μmol/L) group. The proliferation of podocytes was measured by CCK-8. Expression of PGE2 in podocytes was detected by ELISA. Indirect immunofluorescence was used to determine the localization of podocyte-related proteins nephrin, podocin and CD2AP. Expression of nephrin, podocin, CD2AP, COX2 in podocytes was detected by Western blotting and Real-time quantitative PCR. p38 MAPK or phospho-p38 MAPK was measured by Western blotting as well. Flow cytometry was used to detect cell apoptosis. Results (1) In vivo experiments: Compared with control group, obvious proteinuria, blood biochemical changes and renal pathological changes were observed in ADR group, proteinuria, blood biochemical and renal pathological changes were more serious in mice dealt with agonist, while antagonist could reduce ADR-induced injury (all P＜0.05). Results of immunohistochemistry showed that the expression of podocyte-related proteins nephrin, podocin and CD2AP in ADR group were significantly lower than those in control group, and EP1 agonist could further inhibit expression of these proteins, while antagonist could reverse this inhibitory effects (P＜0.05). Electron microscopic results showed that mice in ADR group appeared foot enlargement and fusion, and the agonist group further aggravated the injury, while antagonist intervention could inhibit the injury of podocytes. (2) In vitro experiments: Compared with control group, expression of PGE2 and COX2 were increased; mRNA and protein expression of nephrin, podocin, CD2AP were decreased, p38 MAPK activity and podocytes apoptosis were increased in ADR group (P＜0.05); Agonist could aggravate podocytes damage (P＜0.05), while Antagonist could down-regulate the expression of PGE2 and COX2, promote the expression of nephrin, podocin and CD2AP, and inhibit the activity of p38 MAPK and podocytes apoptosis (P＜0.05). The addition of p38 MAPK inhibitor(10 μmol/L) could reduce the inhibitory effect of EP1 agonist on the expression of podocyte-related proteins nephrin, podocin and CD2AP (P＜0.05). Conclusions EP1 receptor may activate the p38 MAPK signaling pathway to inhibit podocytes-related proteins nephrin, podocin and CD2AP, as well as mediate the ADR induced podocyte injury. Inhibition of EP1 receptor however have a protective effect.     

Autophagy alleviate podocytes injury induced by contrast media via oxidative stress

Objective To evaluate the effects of autophagy on oxidative stress induced by contrast media in podocytes. Methods The differentiated mouse podocytes were exposed to contrast media (Iopromide, 50 mg/L)、rapamycin (Rap, autophagy enhancer, 1 ng/L), 3-methyladenine (3-MA, autophagy inhibitor, 2 mmol/L) for 2 hours. The expression of autophagy protein LC3-Ⅱand Beclin-1 as well as oxidative stress-related proteins Catalase, MnSOD were detected by Western blot. The formations of autophagy were observed by MDC staining, and the levels of reactive oxygen species (ROS) by CM-H2DCFDA staining. Cell activity was evaluated by CCK8 assay. Results Both the levels of oxidative stress and autophagy in podocytes increased when stimulated by contrast media, the expression of LC3-Ⅱand Beclin-1 were enhanced, Catalase and MnSOD were inhibited (all P＜0.05). Rapamycin increased the expression of Catalase, MnSOD and cell activity of podocytes, reduced the generation of ROS (all P＜0.05), but in Rap group, cell activity showed no significant difference (P＞0.05). 3-MA decreased the expression of Catalase、 MnSOD and inhibited the cell activity of podocyte, increased the generation of ROS (all P＜0.05). Conclusion Autophagy protects podocyte from contrast media by the means of reducing oxidative stress.     

血管紧张素Ⅱ对足细胞nephrin 磷酸化水平的影响

目的 通过建立血管紧张素Ⅱ( AngⅡ)输注大鼠模型及体外足细胞培养,观察AngⅡ刺激对足细胞nephrin磷酸化水平的影响.方法 30只SPF级Wistar大鼠皮下埋置渗透性微泵,随机分为AngⅡ组(AngⅡ400 ng· kg-1·min-1,n=12)、替米沙坦(Tel)组(AngⅡ+Tel 3 mg· kg-1·d-1,n=12)和正常对照组(生理盐水代替AngⅡ,n=6),于实验0、7、14、21、28 d测量大鼠尾动脉收缩压,收集24 h尿液,检测尿白蛋白.分别在14、28 d处死动物,收集血液标本,检测血肌酐;收集肾脏标本,透射电镜观察肾小球足细胞形态,放免法检测血浆及肾组织AngⅡ水平;Western印迹检测nephrin表达及其磷酸化水平.体外培养小鼠永生化足细胞,AngⅡ (10-6 mol/L)刺激不同时间,并行洛沙坦(10-5 mol/L)干预,Western印迹法检测足细胞nephrin 表达及其磷酸化水平.异硫氰酸荧光素( FITC)-鬼笔毒环肽(phalloidin)染色标记足细胞F-actin,用外周F-actin环评分系统(CFS)半定量分析足细胞骨架运动.结果 (1)与正常对照组同时间点相比,AngⅡ组大鼠自7d起尾动脉收缩压开始升高(P＜0.05),随着作用时间的延长,血压持续升高.AngⅡ输注7d大鼠开始出现蛋白尿,并持续增加.各组大鼠血肌酐、尿肌酐及内生肌酐清除率无明显变化.AngⅡ输注大鼠血浆及肾组织AngⅡ浓度明显增高(均P＜0.05).(2)与正常对照组相比,AngⅡ输注组大鼠肾小球足细胞nephrin表达明显减少,其磷酸化水平亦显著下降(P＜0.05).(3)与正常对照组相比,AngⅡ刺激早期(3～6 h),足细胞nephrin磷酸化表达即明显下调(P＜0.05),刺激12～24 h维持低水平表达.(4)AngⅡ刺激后,F-actin排列紊乱,逐渐向细胞外周分布形成F-actin环,CFS评分显著高于正常对照组(P＜0.05).结论 正常状态下足细胞nephrin维持一定水平磷酸化状态,AngⅡ可以诱导足细胞nephrin磷酸化水平下调.nephrin磷酸化水平改变可能是AngⅡ诱导足细胞骨架重排、足突融合的重要分子机制.