Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

Rapamycin markedly slows disease progression in a rat model of passive Heymann nephritis

AIM：To determine the effect of rapamycin on the progression of passive Heymann nephritis (PHN), and whether autophagy is involved in this process. METHODS：Male Sprague-Dawley rats (n=24) were randomly divided into 3 groups: control group, PHN group and rapamycin treatment group. The rat PHN model was induced by injection of anti-Fx1A serum through penile vein, and all rats were sacrificed on day 21. Automatic biochemical analyzer was used to detect 24 h urine protein, blood urea nitrogen and serum creatinine. Renal damage was observed through per-iodic acid-silver methenamine staining. The number of podocyte was estimated by Weibel-Gomez method. The glomerular deposition of C5b-9, the expression of caspase-3 and expression of autophagy marker LC3 in glomeruli were examined by immunofluorescence staining, immunohistochemical staining and Western blotting, respectively. RESULTS：Rapamycin significantly reduced proteinuria in the PHN rats (P<0.05), while the renal functions in 3 groups were normal, without significant difference. Although rapamycin limited weight gain in the rats, the health of the rats during drug treatment was not affected. Rapamycin retarded glomerular basement membrane thickening in the PHN rats. Rapamycin significantly reduced the podocyte deletion by preventing podocyte apoptosis. Rapamycin enhanced the level of autophagy of glomerular inherent cells. CONCLUSION：In the disease process of PHN, appropriate strength of autophagy plays a protective role. Rapamycin appropriately enhances autophagy and prevents podocyte apoptosis, thus reducing nephropathy and proteinuria. This may be one of the important mechanisms of rapamycin to slow down the progress of PHN.     

Autophagy protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting C/EBP homologous protein expression

AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: The RAW264.7 macrophages were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid (PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of lactate dehydrogenase (LDH) in the medium and caspase-3 in the cells were determined by detection kits. The protein levels of beclin-1 (a molecular marker of autophagy), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein (CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis) were examined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope.RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability, and dramatic elevation in LDH leakage, cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autophagy inducer). ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap. Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL. Moreover, PBA (endoplasmic reticulum stress inhibitor) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granulation of LC3. CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages, and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression.     

Effect of irbesartan on nephrin expression in the podocyte of early diabetic nephropathy rats

AIM: To investigate the expression of the nephrin in podocyte of the diabetic nephropathy(DN) rats and the mechanism of irbesartan-induced renal protection.METHODS: The DN model was established by a single injection of streptozotocin(STZ),and DN rats were randomly divided into 2 groups: model group and irbesartan treatment group.In addition,the normal rats served as a normal control group. All the rats were received daily gavage respectively for 8 weeks. The urinary protein quality in 24 hours,body weight(BW),kidney weight (KW),KW/BW,glucemia,urea nitrogen,creatinine,total cholesterol, triacylglycerol were detected with correlative methods and the pathological changes of kidney were also detected with optic microscope and transmission electron microscope.The expression of nephrin in podocyte were detected by immunohistochemistry. RESULTS: In DN rats, irbesartan reduced the urinary protein quality in 24 hours (P<0.01) and alleviated the damage of kidney. Meanwhile，the expression of nephrin was declined remarkably in podocytes in irbesartan treatment group compared with model group（P<0.05）.CONCLUSION: Irbesartan might protect kidney against STZ-induced injury via decreasing the expression of nephrin in podocytes.     

Hydrogen inhibits C/EBP homologous protein-mediated macrophage apoptosis by activating autophagy

AIM: To investigate the protective effect of hydrogen (H₂) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H₂-saturated medium was added to murine RAW264.7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine (3-MA) and 3 μmol/L rapamycin (Rap) for 1 h, and then treated with ox-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI staining, respectively. The activity of lactate dehydrogenase (LDH) in medium was detected. The protein levels of beclin-1 (a molecular marker of autophagy) and C/EBP homologous protein (CHOP, a key signaling component of endoplasmic reticulum stress-associaed apoptosis pathway) were determined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope. RESULTS: Hydrogen attenuated the reduction of cell viability, LDH leakage, apoptosis and CHOP upregulation induced by ox-LDL. Hydrogen promoted ox-LDL-induced autophagy in macrophages as assessed by beclin-1 upregulation, and LC3 granulation, and this promotion effect of hydrogen was inhibited by 3-MA (an autophagy inhibitor) and further enhanced by Rap (an autophagy inducer). Moreover, the inhibitory effect of hydrogen on ox-LDL-induced macrophage apoptosis, reduction of cell viability and CHOP upregulation were also blocked by 3-MA and enhanced by Rap. Similar results were obtained in human THP-1-derived macrophages, as assessed by the inhibition of ox-LDL-induced apoptosis and CHOP upregulation, and the promotion of beclin-1 expression by hydrogen. CONCLUSION: Hydrogen may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression, and the upstream mechanism may partially involved in the activation of autophagy.     

Effects of hydrogen sulfide on high glucose-induced injury of mouse glomerular podocytes

AIM: To investigate the effect of hydrogen sulfide (H2S) on high glucose （HG）-induced injury of the mouse podocyte cell line MPC5. METHODS: The cultured MPC5 cells were randomly divided into 4 groups: HG group, normal glucose (NG) group, NG+DL-propargylglycine (PPG) group, and HG+NaHS group. After treated for a certain time, the cells were collected for further detection. The expression of zonula occludens-2 (ZO-2), nephrin, β-catenin and cystathionine γ-lyase (CSE) was determined by Western blotting. RESULTS: High glucose significantly reduced the expression of nephrin, ZO-2 and CSE (P<0.05), while the level of β-catenin was elevated obviously (P<0.05), all in a time-dependent manner. NG+PPG inhibited the levels of ZO-2 and nephrin significantly (P<0.05), and increased the level of β-catenin (P<0.05), all in a PPG concentration-dependent manner. HG+NaHS induced a more significant increase in the levels of ZO-2 and nephrin as compared with HG group (P<0.01), whereas a severe reduction of β-catenin in HG+NaHS group was observed as compared with HG group. Compared with NG group, the expression of ZO-2 and nephrin was decreased obviously, and the level of β-catenin was increased in HG+NaHS group. CONCLUSION: Down-regulation of CSE contributes to hyperglycemia-induced podocyte injury. Exogenous H2S protects against hyperglycemia-induced podocyte injury, possibly through up-regulation of ZO-2 and subsequent suppression of Wnt/β-catenin pathway.     

Role of autophagy in inhibition of human lung cancer PC9 cell proliferation by ursolic acid

AIM: To investigate the role of autophagy in inhibition of human lung cancer PC9 cell proliferation by ursolic acid (UA) as well as the underlying mechanism. METHODS: MTT assay and Trypan blue exclusion test were performed to analyze the effect of UA on the proliferation of PC9 cells. The PC9 cells were treated with UA, and autophagy was observed under fluorescence microscope through acridine orange staining. The expression of autophagy-associated proteins LC3 and ATG5 in the PC9 cells were detected by Western blot. The effect of UA, 3-methyladenine (3-MA) 3-MA or their combination on the cell viability was measured by MTT assay. RESULTS: The viability of PC9 cells was significantly inhibited by UA (P<0.05 or P<0.01). The number of bright red fluorescence positive cells was significantly increased after treatment with UA. The protein expression of LC3-Ⅱ and ATG5 was significantly up-regulated compared with control group (P<0.01). Furthermore, combination of UA and 3-MA resulted in a substantial decrease in cell viability compared with using UA alone (P<0.01). CONCLUSION: UA inhibits the proliferation and induces the autophagy of the PC9 cells, in which autophagy plays a protective role. The inhibition of autophagy significantly promotes the death of the PC9 cells induced by UA.     

Astrocyte activity and autophagy after radiation-induced brain injury in rats

AIM: To investigate the activity of astrocytes and autophagy-related changes after radiation-induced brain injury (RBI) in rats.METHODS: A total of 36 Sprague-Dawley rats, weighing 180~200 g, were trained for 4 d in the Morris water maze. They were randomly divided into sham group, model group and 3-methyladenine (3-MA) group. The rats in model group and 3-MA group were given single whole-brain X-ray irradiation at a dose of 20 Gy after intraperitoneal anesthesia. After the irradiation was completed, the rats in model group was given 5 μL of NaCl into the lateral ventricle, and the rats in 3-MA group was injected with 3-MA at 600 nmol into the lateral ventricle. After 8 weeks of feeding, Morris water maze was used for measuring the learning and memory abilities. The brain tissues were taken and HE staining was used to observe the pathological changes of the hippocampus. The protein level of GFAP was determined by immunohistochemistry and Western blot for evaluating astrocyte activity. Dual fluorescence staining of GFAP and LC3 was performed for evaluating the changes of autophagy in the astrocytes. The protein level of cleaved caspase-3 detected by Western blot and TUNEL staining in the ipsilateral hippocampus were used to evaluate the apoptosis. The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were examined by ELISA to assess the inflammatory response in the hippocampus.RESULTS: Radiation inhibited astrocyte activity, activated autophagy in astrocytes, and aggravated brain damage. 3-MA promoted the activation of astrocytes and promoted the repair of brain tissue damage.CONCLUSION: The injury of rat hippocampus after radiation is obvious, and the number of astrocytes is significantly reduced. 3-MA significantly attenuates the damage. This finding may provide a new approach for the treatment of radiation-induced brain injury.     

Role of nephrin in preventing angiotensinⅡ-induced cytoskeletal rearrangement in glomerular podocytes

AIM: To investigate the role of nephrin, a slit diaphragm-associated protein, in angiotensinⅡ (AngⅡ)-induced cytoskeleton rearrangement in podocytes. METHODS: Immortalized mouse podocytes were exposed to AngⅡ (10^-8 mol/L) with or without AngⅡ receptor antagonist lorsatan and Akt inhibitor LY294002. FITC-conjugated phalloidin was used to stain F-actin, and semi-quantitative system with cortical F-actin score (CFS) was introduced to analyze the degree of actin cytoskeleton arrangement. The expression of nephrin was assessed by quantitative real-time RT-PCR,RT-PCR and Western blotting. Undifferentiated podocytes were transfected with pcDNA3.1-mNPHS1 plasmid containing the full length of nephrin. The stably transfected cell line was generated by G418 selection. Phosphorylation level of Akt was assessed by Western blotting, and F-actin distribution was further evaluated in transfected cells exposed to AngⅡ or not. RESULTS: Cytoskeletal rearrangements including cortical F-actin ring formation and stress fiber attenuation were observed in Ang II-and LY294002-stimulated podocytes. Pretreatment with losartan significantly prevented Ang II-induced actin cytoskeleton reorganization. The mRNA and protein levels of nephrin and phosphorylation of Akt were obviously decreased in the podocytes exposed to Ang II, which were dramatically reversed by pcDNA3.1-mNPHS1 transfection. Transfection of pcDNA3.1- mNPHS1 induced the formation of short filopodia and partially prevented AngⅡ-induced F-actin remodeling. CONCLUSION: PI3K/Akt signaling is a common downstream pathway of nephrin and Ang Ⅱ. Nephrin is able to stabilize AngⅡ-induced cytoskeletal rearrangement via PI3K/Akt signaling pathway.     

Hypoxic preconditioning alleviates oxygen-glucose deprivation in PC12 cells:the involvement of autophagy

AIM: To examined the effects of hypoxic preconditioning(HPC) on oxygen-glucose deprivation(OGD)-induced PC12 cells, and to investigate its possible mechanisms of autophagy.METHODS: Cultured PC12 cells were randomly divided into control group, HPC group, 3-methyladenine(3-MA) group, HPC+OGD group, 3-MA+HPC+OGD group and OGD group. CCK-8 assay was used to detect the cell viability. The caspase-3 activity was also tested. TUNEL staining and flow cytometry were used to detect the cell apoptosis. The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot.RESULTS: Compared with control group, the viability of PC12 cells was significantly reduced, and the activity of caspase-3 was significantly increased in OGD group. Compared with 3-MA+ HPC+OGD group and OGD group, the viability of PC12 cells was significantly increased, and the activity of caspase-3 was significantly reduced in HPC+OGD group(P<0.05). The PC12 cell injury was apparent after OGD with a great increase in the apoptotic rate(P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in HPC+OGD group(P<0.05). Compared with control group, the protein level of cleaved caspase-3 was significantly increased in OGD group(P<0.05). Compared with OGD group, the protein level of cleaved caspase-3 was significantly decreased, and the levels of LC3-2 and beclin-1 were significantly increased in HPC+OGD group(P<0.05).CONCLUSION: OGD decreases cell survival and induces apoptosis.Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC12 cells from OGD induced injury.     

Effects of astragaloside IV on autophagy in rats with cerebral ischemia/reperfusion injury

AIM: To investigate the effects of astragaloside IV (AS-IV) on autophagy in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral ischemia/reperfusion of rat left middle cerebral artery occlusion (MCAO) was induced by suture method. Male SD rats (n=70) were randomly divided into sham operation group, I/R group, solvent control group, AS-IV group, AS-IV+autophagy inhibitor (3-methyladenine, 3-MA) group, 3-MA group and autophagy activator (rapamycin, Rapa) group. Except for sham operation group, the rats in other groups were subjected to ischemia for 2 h and reperfusion for 24 h. The rats with successful modeling were selected according to Zea Longa scoring criteria. The volume of cerebral infarction was measured by TTC staining. The morphological changes of nerve cells in the rats were observed with Nissl staining. The phenomenon of autophagy was observed under transmission electron microscope. The protein expression of beclin-1 and LC3-Ⅱ was determined by Western blot. RESULTS: No neurological deficit in sham operation group was observed, and the cerebral infarction was not found. Compared with sham operation group, obvious cerebral infarction was observed, the Nissl bodies were small in size and number and stained light, typical autophagosomes were observed, and the protein expression of beclin-1 and LC3-Ⅱ was increased in I/R group (P<0.05). Compared with I/R group, the volume of cerebral infarction was decreased obviously, neurological deficit restored significantly, and the number of autophagosomes and the protein expression of beclin-1 and LC3-Ⅱ were increased in AS-IV group and Rapa group (P<0.05). However, no significant difference between solvent control group and I/R group was observed (P>0.05). Compared with AS-IV group, the neurological deficit was serious, the volume of cerebral infarction and the number of autophagosomes were increased, while the expression of beclin-1 and LC3-Ⅱ was decreased in AS-IV+3-MA group and 3-MA group (P<0.05). CONCLUSION: Astragaloside IV may play an important role in atte-nuating cerebral ischemia/reperfusion injury by activating autophagy.     

miR-181a regulates CSE-induced autophagy dysfunction and releases of pro-inflammatory factors in NR8383 alveolar macrophages

AIM:To investigate the effect of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced autophagy disorder and releases of pro-inflammatory factors in NR8383 rat alveolar macrophages. METHODS:The NR8383 cells were treatment with 5%,10% and 20% CSE. The release levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-8 were measured by ELISA. The level of miR-181a was detected by RT-qPCR. The numbers of autophagosomes were observed by Cyto-ID staining. The expression levels of LC3-Ⅱ, beclin-1 and p62 were determined by Western blot. NR8383 cells were pretreated with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin (Rapa) before treatment with 20% CSE, and the release levels of TNF-α, IL-6 and IL-8 were measured by ELISA. Furthermore, NR8383 cells were transfected with miR-181a mimic or miR-181a inhibitor before treatment with 20% CSE, and the release levels of TNF-α, IL-6 and IL-8, and the expression of LC3-Ⅱ, beclin-1 and p62 were detected by ELISA and Western blot, respectively. RESULTS:CSE increased release levels of pro-inflammatory factors and autophagy disorder in a concentration-dependent manner in the NR8383 cells (P<0.05). 3-MA increased CSE-induced releases of pro-inflammatory factors. However, Rapa partially reversed CSE-induced releases of pro-inflammatory factors. Additionally, miR-181a mimic inhibited CSE-induced releases of pro-inflammatory factors and promoted autophagy. However, miR-181a inhibitor increased CSE-induced releases of pro-inflammatory factors and autophagy disorder. CONCLUSION:miR-181a regulates CSE-induced releases of pro-inflammatory factor in the NR8383 cells, which may be related to the regulatory role of miR-181a in autophagy disorder.     

Analysis of glomerular complement C5b-9 deposits and synthesis of NO,TNFα in the model of the rats with anti-thymocyte serum nephritis

AIM: To explore the localization and semi-quantification of the glomerular complement C5b-9 complexes and synthesis of some inflammatory mediators or cytokines such as nitric oxide (NO) and tumor necrosis factor α(TNFα) in the rats with anti-thymocyte serum nephritis(ATSN). METHODS: The animal model of rat ATSN was reproduced by a single intravenous injection of anti-thymocyte serum (ATS). Then, the deposits of glomerular C5b-9 complexes were localized and quantified by immunohistochemical staining and microscopic image scanning separately. And the glomerular mesangial cells (MC) surrounded by C5b-9 complexes were counted under microscope. In addition, the expression of glomerular MC inducible NO synthase(iNOS) mRNA and excretion of urinary NO metabolite (NO^-₂／NO^-₃) and TNF α in the rats with ATSN were detected. RESULTS: The MC in the rats with ATSN emerged necrosis followed by a rapid proliferation. In the early time of MC injury, the C5b-9 complexes were mainly seen in glomerular mesangium and MC surface. But with the progression of ATSN, the MC enclosed by C5b-9 appeared gradual decrease. Moreover, the expression of MC iNOS mRNA in early stage of ATSN obviously increased and the excretion of urinary NO^-₂／NO^-₃ and TNF α also significantly increased. However, the changes of parameters mentioned above in ATSN proliferative stage (after 7 days) alleviated gradually. CONCLUSION: The secondary lysis of MC has relation to the deposition of C5b-9 complexes and synthesis and release of NO and TNF α in rats with ATSN.     

Effect of autophagy on necroptosis of renal tubular epithelial cells in subtotal nephrectomy rats

AIM: To explore whether autophagy is involved in the excessive death of renal tubular epithelial cells in subtotal nephrectomy(SNx) rats and the relationship between autophagy and necroptosis in the kidney of SNx rats. METHODS: Male Sprague-Dawley rats were randomly assigned to control group(n=6) and SNx group(n=42). The rats in SNx group were subjected to SNx. Sham surgery was performed in the rats in control group. The rats in SNx group were divided into subgroups at 0, 4, 8 and 12 weeks(n=6) and the other rats in SNx group were divided into SNx+vehicle group, SNx+necrostatin-1(Nec-1) group and SNx+3-methyladenine(3-MA) group. The expression of RIP1, RIP3, LC3 and beclin-1 at mRNA and protein levels was measured at 0, 4, 8 and 12 weeks by qPCR and immunohistochemistry. The effects of Nec-1 or 3-MA on the protein expression of LC3-I, LC3-II and beclin-1, and production of reactive oxygen species(ROS) in the rat kidney were determined by Western blot and DCFH-DA staining. The death of renal tubular epithelial cells in the SNx rats was observed by TUNEL staining and electron microscopy. Finally, the effects of Nec-1 and 3-MA on blood urea nitrogen(BUN), serum creatinine(SCr) and the pathological changes of the renal tissues were analyzed. RESULTS: The highest mRNA and protein levels of RIP1, RIP3, LC3 and beclin-1 appeared at the 8th week after SNx(P<0.01). Compared with the rats in SNx+vehicle group, the protein over-expression of LC3-II/I and beclin-1, renal tubular epithelial cells with typical morphological features of necroptotic cell death and TUNEL-positive renal tubular cells were decreased in the SNx rats treated with Nec-1 and 3-MA(P<0.01), but 3-MA did not reduce the increased concentration of ROS. In addition, treatment with Nec-1 and 3-MA obviously reduced BUN, SCr(P<0.05), glomerulosclerosis index and tubulointerstitial injury score(P<0.01). CONCLUSION: Autophagy participates in the excessive death of renal tubular epithelial cells in SNx rats. Inhibition of autograph prevents necroptotic cell death of renal tubular cells, and alleviates chronic renal injury in SNx rats.     

Role of connective tissue growth factor in high glucose-induced epithelial-mesenchymal transition in podocytes

AIM: To investigate the role of connective tissue growth factor (CTGF) in high glucose-induced epithelial-mesenchymal transition (EMT) in podocytes. METHODS: The differentiated podocytes cultured under different conditions for 24 h at 37 ℃ were divided into 4 groups: control (5 mmol/L glucose solution), 5 mmol/L glucose solution supplemented with 50 μg/L CTGF, high glucose (30 mmol/L glucose solution) and high glucose supplemented with CTGF (50 μg/L). The morphology of cultured podocytes was observed under phase-contrast microscope. To study the relevant markers of EMT, the mRNA and protein expression was analyzed by real-time RT-PCR and Western blotting, respectively. In addition, the effect of CTGF inhibition with anti-CTGF antibody on high glucose-induced EMT in podocytes was investigated. RESULTS: High glucose induced phenotypic transition in the podocytes from an arborization morphology to a cobblestone morphology. After addition of CTGF to high glucose culture, the podocytes developed a feature of spindle. Under high glucose condition, podocytes underwent EMT, as the epithelial marker (nephrin) was decreased and the mesenchymal marker (desmin) was increased. Exogenous CTGF showed the effect of synergy on high glucose-induced EMT. Moreover, high glucose-induced EMT in podocytes was prevented by CTGF inhibition with anti-CTGF antibody. CONCLUSION: CTGF is involved in high glucose-induced EMT in podocytes. Inhibition of CTGF might prevent podocytes from injury in diabetes.     

Role of autophagy inhibitor 3-methyladenine in the injury of U251 cells induced by H2O2

AIM: To investigate the role of autophagy inhibitor 3-methyladenine(3-MA) in the injury of U251 glioma cells induced by H₂O₂. METHODS: The following groups in this study were set up: control group, 10 mmol/L 3-MA group, 1 mmol/L H₂O₂ group and 1 mmol/L H₂O₂ +10 mmol/L 3-MA group. The viability of U251 cells in each group was detected by MTT assay. Autophagic vacuoles in the cells were observed by staining with MDC. The cells were stained with Hoechst 33342 to determine the chromatin condensation. Cell apoptotic ratio was measured by flow cytometry analysis. RESULTS: Compared with control group, no effect of 3-MA on the viability of U251 cells was observed. In H₂O₂ group, the cell viability decreased and cell apoptotic ratio increased.The autophagic vacuoles and nuclear chromatin condensation in the cells were also detected. Compared with H₂O₂ group, addition of 3-MA inhibited the increase in autophagic vacuoles but exacerbated the apoptosis. CONCLUSION: Autophagy inhibitor 3-MA inhibits autophagy partially, but exacerbates apoptosis in U251 cells, indicating that autophagy exerts protective effect in the process of injury in U251 cells induced by H₂O₂.     

Mechanism of Sos2 inhibiting NFATc1-mediated podocyte injury in diabetic nephropathy

AIM: To observe the effect of high glucose (HG) stimulation on the expression of guanine nucleotide exchange factor Sos2 (Son of Sevenless homolog 2) in mouse podocytes, and to explore the role of Sos2 in HG-induced podocyte damage and its possible molecular mechanisms. METHODS: The expression of Sos2 in the podocytes of diabetic nephropathy patients was observed by immunofluorescence staining and laser confocal microscopy. In vitro, the Sos2 expression at mRNA and protein levels in immortalized podocytes with HG (30 mmol/L glucose) stimulation for 48 h was determined by the methods of RT-PCR, Western blot and immunofluorescence. Using Western blot, immunofluorescence and wound-healing assay, the expression of podocin, the translocation of NFATc1 into the nucleus and the podocyte migration with or without Sos2 silencing or overexpression were analyzed. The expression of downstream target genes for NFATc1 was detected by RT-PCR. RESULTS: The expression of Sos2 was significantly decreased in the podocytes of diabetic nephropathy patients and in vitro cultured podocytes with HG stimulation (P<0.05). When Sos2 was silenced, the expression of podocin was significantly decreased, the migration ability of podocytes was increased, and the translocation of NFATc1 into the nucleus was increased (P<0.05). In contrast, after overexpression of Sos2 in the podocytes with HG stimulation, the podocin expression level was obviously higher, and the podocyte migration ability and the translocation of NFATc1 into the nucleus were decreased (P<0.05).CONCLUSION: Sos2 may attenuate the diabetic nephropathy-induced podocyte injury by inhibiting NFATc1.     

Effect of antiserum against complement C5b-9 complexes on nitric oxide(NO) synthesis and pathologic changes in renal tissue of ATSN rats

AIM: To explore the effect of antiserum against rat complement C5b-9 complexes on nitric oxide synthesis and pathologic changes in renal tissue of rats with mesangioproliferative glomerulonephritis. METHEDS: The rat model of mesangioproliferative glomerulonephritis,namely,anti-thymocyte serum nephritis(ATSN) was established and the rats with ATSN were treated with antiserum against complement C5b-9 complexes. Some parameters related to NO and pathologic changes of the rats were observed.RESULTS: Anti-C5b-9 serum not only reduced the expression of inducible NO synthase(iNOS) mRNA in renal tissue and urinary content of nitrate/nitrite(NO₃^-/ NO₂^-) of rats with ATSN, but also reduced renal pathologic changes. L-N G-nitro arginine methylester (L-NAME) also reduced the contents of urinary NO₃^-/NO₂^- and the renal pathologic changes of rats with ATSN. CONCLUSION: The antiserum against rat complement C5b-9 complexes inhibited glomerular NO synthesis and glomerular mesangial cell proliferation of rats with ATSN.     

Role of microRNA-126-5p in myocardial injury induced by doxorubicin

AIM: To observe the expression of microRNA-126-5p during myocardial injury and its role in myocardial cell injury induced by adriamycin (also called doxorubicin, DOX). METHODS: The BALB/c mouse model of DOX-induced acute and chronic myocardial injury was established via intraperitoneal injection of DOX. HE staining was applied to observe the morphological changes of myocardial tissues. Lactate dehydrogenase (LDH) in serum was detected and PowerLab system was used to detect the influence of DOX on the changes of ±dp/dt_max. The expression of microRNA-126-5p in injured myocardial tissues and the H9c2 cells exposed to DOX was detected by real-time PCR. Gain-and loss-of-function experiments were conducted to detect the role of microRNA-126-5p in H9c2 cells treated with DOX on LDH release and caspase-3 activation. RESULTS: In acute and chronic DOX myocardial damage models in mice, HE staining showed disarranged myocardial fibers, dissolved myofibril and inflammatory cell infiltration. Higher serum LDH level and lower ±dp/dt_max in DOX-treated mice than those in normal mice were found. Compared with the normal mice, the expression level of microRNA-126-5p was significant increased in the myocardium with DOX-induced injury. Similarly, the expression level of microRNA-126-5p was significant increased in the H9c2 cells treated with DOX. In addition, over-expression of microRNA-126-5p decreased cell viability and promoted apoptosis, while microRNA-126-5p ablation promoted the viability and inhibited the apoptosis of H9c2 cells. CONCLUSION: The microRNA-126-5p expression is up-regulated in myocardial injury induced by DOX, and microRNA-126-5p inhibits cell viability and promotes apoptosis induced by DOX.     

Autophagy is activated during glutamic acid-induced cortical neuron injury

AIM: To investigate whether autophagy is activated during glutamic acid-induced neuron injury and the possible neuroprotective effect of 3-methyl adenine(3-MA) (an autophagy inhibitor).METHODS: Glutamic acid or 3-MA was added to the medium of cultured cortical neurons. Cell viability was measured by MTT assay. The formation of autophagosome was observed under transmission electron microscope. The marker protein light chain 3(LC3) for autophagy was detected by immunofluorescence assay and visualized under laser confocal microscope.RESULTS: The cell viability declined during glutamic acid treatment and the autophagosomes were increased. LC3, the marker protein of autophagy, also significantly increased. The autophagy level was lowered by 3-MA, and cell viability was increased.CONCLUSION: The results suggest that autophagy is activated during glutamic acid treatment and inhibition of autophagy may have neuroprotective effect. The autophagy inhibitor 3-MA may be a potential neuroprotective agent.