Objective     To investigate the effect of fluvastatin（FLV）on the expression of β1 integrin in puromycin aminonucleoside (PAN)?treated podocytes and its mechanism.    Methods    Cultured human podocytes were divided into PAN, different concentrations of fluvastatin（1×10-8 to 1×10-5 mol/L），SOD, H2O2 groups respectively. Expressions of β1 integrin and reactive oxygen species (ROS) in podocytes were detected by Western blotting and DCFHDA (2’7’?Dichlorofluoresecein 3’6’?diacetate) respectively. The viability of podocyte was determined by MTT colorimetry.    Results    PAN and H2O2 significantly decreased the expression of β1 integrin and increased the synthesis of ROS in podocytes (P<0.05 respectively). Lower concentration fluvastatin or SOD treatment up?regulated β1 integrin and down?regulated ROS of podocytes induced by PAN (P<0.05 respectively). MTT revealed that lower podocyte viability was found in higher concentration fluvastatin, PAN and H2O2 groups. Lower concentration fluvastatin and SOD could protect podocytes against PAN.    Conclusion    Fluvastatin attenuates the injury of podocyte induced by PAN and increases the expression of β1 integrin, whose mechanism may be associated with the inhibition of the ROS activity.
 

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.
  

Objective     To explore the effect of Sunset Abelmoschus on podocyte injury in adriamycin?induced nephropathy rats.    Methods    Fifty male SD rats were randomly divided into five groups: sham operation group (n=10), model group (n=10), Sunset Abelmoschus low dose group (0.5 g•kg-1•d-1 n=10), middle dose group (1.0  g•kg-1•d-1, n=10) and high dose group (2.0 g•kg-1•d-1, n=10). Unilateral nephrectomy combined repeated adriamycin injection were performed to establish adriamycin?induced nephropathy models. The rats were administered with the corresponding dose of Sunset Abelmoschus during the experiment period. Urinary protein, urinary N?acetyl glucose aminotransferase (NAG), serum albumin, serum creatinine and blood lipid were measured before operation and 2, 4, 6, 8 weeks after operation. The rats were sacrificed on week 8 for the renal histological examination, including light microscope and electron microscope. Expression of nephrin was examined by immunofluorescence assays.    Results    As compared to model group, urinary protein and NAG significantly decreases in Sunset Abelmoschus groups in each time point, especially in high dose group (P<0.01), meanwhile the serum albumin increased and the disturbance of lipid metabolism was improved in Sunset Abelmoschus groups (P<0.05). Compared with sham group, Scr increased significantly in model group and Sunset Abelmoschus groups at the 4th week. At the 8th week, Scr in high dose group was lower than that in model group (P<0.05), and the ratio of glomerular globe sclerosis and segmental sclerosis, tubulointerstitial damage reduced in Sunset Abelmoschus groups, especially in high dose group. The podocyte damage and the extent of foot process fusion were improved in Sunset Abelmoschus groups compared with model group. Expression of nephrin increased in Sunset Abelmoschus groups than that in model group.    Conclusion    Sunset Abelmoschus can ameliorate proteinuria and renal tissue damage of adriamycin?induced nephropathy rats, whose mechanism may be associated with the improvement of podocyte injury.

Objective     To evaluate the efficacy, safety and tolerance of continuous erythropoietin receptor activator (CERA) once every 2 weeks intravenous injection on anemia correction in dialysis patients compared to Epoetin?β (EPO?β) administration.    Methods    An open?label, randomized, parallel, active?control and multi?center clinical trial was performed. All the hemodialysis or peritoneal dialysis patients with chronic renal anemia who had not  been treated with erythropoiesis?stimulating agents (ESAs) for at least 8 weeks before entering the treatment phase were randomized (1∶1) to receive either CERA once every 2 weeks intravenous administration (CERA group, n=132) or intravenous EPO?β three times weekly (EPO group, n=133) for 24 weeks including 16?week correction period and 8?week efficacy evaluation period. At week 25, the patients who reached the target Hb (defined as Hb≥110 g/L and increase in Hb≥10 g/L from baseline without red blood cell transfusion during the 24 weeks after the first dose) were kept on CERA or EPO?β treatment regimen for the subsequent 28 weeks to evaluate the long?term safety and tolerability. The starting dose of CERA was 0.4 μg/kg. Two primary endpoints were (1) the Hb response rate during the first 24 weeks; and (2)the mean change in Hb between the baseline and the evaluation periods (week 17 to week 24).     Results    Totally 232 patients (87.5%) completed the first 24?week treatment and 198 patients (74.7%) completed the whole study treatment (52 weeks). The response rate in CERA group during the first 24 weeks was 87.12%[95% CI（80.2% to 92.3%）]. Since the lower limit of the 95%CI was greater than 60% (P＜0.01), CERA once every 2 weeks intravenous administration was considered as effective in correction of renal anemia. The difference between CERA group and EPO group in mean change of Hb from evaluation periods to baseline in the per?protocol (PP) population was -4.7 g/L [95%CI (-7.38 g/L to -1.92 g/L)]. Since the lower limit of 95%CI was greater than the pre?defined non?inferiority margin -7.5 g/L (P=0.0205), CERA was considered as non?inferior to EPO in the maintenance of Hb after anemia correction. The Hb level remained stable during the subsequent 28?week extension period in both CERA and EPO groups. During the whole study period, the overall safety findings were similar in CERA and EPO groups, 50.0% and 54.6% of patients experienced at least one adverse event (AE) respectively. The findings from AEs were in accordance with the characteristics of the studied population.    Conclusions    Intravenous CERA once every 2 weeks is safe and effective for correcting anemia in dialysis patients. Treatment with CERA once every 2 weeks is also non?inferior to 3 times weekly EPO in maintaining the Hb level after the correction. In general, long?term intravenous administration of CERA is well tolerated by dialysis patients with chronic renal anemia. 

Objective    To evaluate the functional magnetic resonance (MR) imaging in the assessment of renal involvement and pathological changes in patients with lupus nephritis (LN).    Methods   Seventeen patients with LN and 10 healthy controls underwent coronal echo?planar diffusion?weighted (DW) MR imaging and blood oxygen level dependent (BOLD) MR imaging of the kidneys with a single breath?hold time of 16 s. The apparent diffusion coefficient (ADC) and R2* value of the kidneys were calculated with high b values (b=500 s/mm2). The correlation between the renal injury variables and the ADCs or R2* values was evaluated.    Results    The mean ADC value of kidneys in patients with LN was (2.43±0.24)×10-3 mm2/s, the mean R2* values of the renal cortex and medulla were (11.72±2.35)/s and (13.07±2.35)/s respectively, which were all significantly lower than those in volunteers (P=0.045，P=0.048 and P=0.001, respectively). In the patients with LN, the mean ADC values were positively correlated with estimated glomerular filtration rate (eGFR) (r=0.558, P<0.05). There was a negative correlation between the ADC values of the right kidneys and pathological chronic indexes (r=-0.493, P<0.05). Moreover, the R2*values of the renal medulla were negatively correlated with 24 hours proteinuria, serum creatinine, pathological active indexes. The patients were assigned to group A (class Ⅲ, Ⅳ, Ⅴ, n=8) and group B (class Ⅴ+Ⅲ and Ⅴ+Ⅳ, n=9). The tubulointerstitial lesions in group B were more severe than those in group A, while the mean ADC values and R2* values of the renal cortex in group B were lower as compared to group A.    Conclusion     DW MR imaging and BOLD MR imaging may be used to non?invasively monitor the disease activity and evaluate the efficacy in lupus nephritis.  
   

Objective    To detect the α1?antitrypsin (AAT) concentration in urine samples of children with primary nephrotic syndrome (PNS) before initiation of glucocorticoid treatment, in order to verify whether it could predict the response to glucocorticoid?based therapy.    Methods    Forty?three children diagnosed as PNS initially were chosen as subjects, namely steroid?sensitive nephrotic syndrome (SSNS) and steroid?resistant nephrotic syndrome (SRNS) depending on reaction to glucocorticoid therapy four weeks later, and 15 healthy children serving as normal control. The mid stream of the first morning urine samples were collected from children before taking glucocorticoid. ELISA kit was used to quantify the urinary AAT concentration which was revised by urine creatinine further. The data of urine AAT/Cr were expressed as median with interquartile range. Data analysis was performed using the SPSS 17.0.    Results    AAT was absent in urine samples of normal healthy children, and there were no statistic differences of the AAT concentrations in urine between children with SSNS and SRNS [(30.4±4.5) mg/L vs (31.8±4.6) mg/L，t=-1.0, P=0.33]. The level of urine AAT/Cr in children with SRNS was higher than that in children with SSNS [0.049(0.028?0.073) vs 0.028(0.022?0.036), Z=2.4, P=0.02]. Among the laboratory parameters of the two subgroups before taking glucocortiod, the levels of platelet, blood white cell count, serum globulin, urine white cell count, urine red cell count, urine IgG and urine α1?microglobulin were significantly different (P<0.05). Three parameters that included urine AAT/Cr (OR=6.81×1028, P=0.005), serum globulin (OR=1.69, P=0.01) and urine α1?microglobulin (OR=1.05, P=0.009) further entered the logistic regression model to predict the SRNS independently. The ROC curve based on the level of the urine AAT/Cr was constructed, and the area under the curve (AUC) was 0.72. When the cutoff value of urine AAT/Cr was 0.035, the sensitivity and specificity of the urine AAT/Cr prediction were 68% and 75% respectively (Youden’s index 0.43) . The AUC that based on the logistic regression model which included urine AAT/Cr,  serum globulin and urine α1?microglobulin was improved to 0.94, and the sensitivity and specificity of the model prediction were 95% and 83% respectively (Youden’s index 0.78). There was no significant difference of the urine AAT/Cr level among the different pathological types of the children undergoing renal biopsy.   Conclusions    There are no statistic differences of the AAT concentrations in urine between children with SSNS and SRNS. The level of urine AAT/Cr is significantly higher in the SRNS than that in the SSNS which can be as a candidate biomarker to predict the response to glucocorticoid?based therapy. It has a better prediction efficacy based on the model which includes urine AAT/Cr, serum globulin and urine α1?microglobulin.

Objective    To investigate the incidence and influencing factors of aldosterone breakthrough during therapy with angiotensin II receptor blockers (ARB) alone, or combined with angiotensin?converting enzyme inhibitors（ACEI） in Chinese patients with non?diabetic nephropathy.   Methods    A total of 144 patients with non?diabetic nephropathy were treated with ARB or combination therapy of ACEI and ARB for a mean follow?up period of 12 months. Aldosterone breakthrough  was determined according to the change of plasma aldosterone concentration before and after treatment during 6?month and 12?month ACEI/ARB treatment.    Results     In 6 months, aldosterone breakthrough occurred in 21 patients, corresponding to 14.58%, while in 12 months, occurred in 39 patients, corresponding to 27.08%. Although the overall urinary protein excretion (UPE) decreased after treatment in both groups (P<0.05), non?breakthrough group had a more remarkable reduction in UPE (P<0.05). Univariate Logistic regression demonstrated that risk factors of aldosterone breakthrough included pre?treatment values of UPE (OR=3.643, P=0.073) and eGFR（OR=0.980, P=0.025）. Multivariate Logistic model revealed pre?treatment values of eGFR was positively associated with aldosterone breakthrough (OR=0.980, P=0.025).    Conclusions    The incidence of the aldosterone breakthrough increases with duration of treatment. The patients with aldosterone breathrough have higher level of UPE, and enhanced decline in eGFR. Pre?treatment value of eGFR is independent risk factor of aldosterone breakthrough.

Objective    To study the relationship between the medial artery calcification and expression of core?binding factor alpha 1 (Cbfα?1) and collagen Ⅱ (ColⅡ) in chronic kidney disease(CKD) stage 5 patients.     Methods    Pieces of radial arteries were taken from 40 patients with CKD stage 5 during internal arteriovenous fistula operation. Ten patients with subtotal gastrectomy and normal renal function were chosen as control. The vessels were examined for calcification by von Kossa stain and for the presence of Cbfα?1 and ColⅡ by immunohistochemistry. According to von Kossa stain, CKD stage 5 patients were divided into no calcification group, mild?moderate calcification group and severe calcification group. Other related factors including serum calcium，phosphate, intact parathyroid hormone (iPTH), C?reactive protein (CRP), triglyceride(TG), cholesterol(TC) and low?density lipoproteins(LDL) were also detected.    Results    Seventeen (42.5%) of CKD Stage 5 patients showed vascular calcification, while calcification was not found in controls. Most calcification occurred in medial layer．Positive immunohistochemical staining of core?binding factor and ColⅡ was found in the smooth muscular cell plasma of medial layer in the vessels with calcification. However, above positive staining was also observed in 78.3% of no calcification group. But there was little staining in control group. Positive staining score of  Cbfα?1 and ColⅡ in severe calcification group was significantly higher than that in no calcification group. Same findings were obtained in mild?moderate calcification group, but the difference between them was not statistically significant. CRP and Ca×P were positively correlated with staining  score of Cbfα?1 and ColⅡ. Serum phosphate was positively correlated with Cbfα?1 (r=0.786, P<0.01) and ColⅡ (r=0.785, P<0.01) respectively．   Conclusions     42.5% of CKD stage 5 patients in our group shows vascular calcification, which occurrs mainly in medial layer. High expression of     Cbfα?1 and ColⅡ can be observed in vascular calcification of radial arteries, which is earlier than vascular histological changes. Cbfα?1 and ColⅡ may be involved in the development of vascular calcification.

Objective     To explore the mechanism of protecting cells from hypoxia/reoxygenation (H/R) injury by constructing specific small interference RNA (siRNA) to inhibit NALP3 expression in rat renal tubular epithelial cells (NRK?52E).    Methods    (1) To establish the H/R injury model of NRK?52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 1 h and then with reoxygenation for 1 h, 2 h, 4 h, 8 h, 16 h and 24 h. The activity of lactae dehydrogenase (LDH) in the culture medium, cell count and cell viability, the expression of NALP3 were determined by biochemical method, trypan blue exclusion and Western blotting. (2) The siRNA was transfected into NRK?52E. The irrespective siRNA transfected group was used as control. NALP3 expression was examined by Western blotting. (3) The cells were divided into 4 groups: control group, H/R group, irrespective siRNA transfected group and NALP3?siRNA transfected group. To establish the H/R injury model of NRK?52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 1 h and then with reoxygenation for 4 h. And the expression of NALP3 was determined by Western blotting. (4)Cellular apoptosis was examined by Annexin V/PI staining and flow cytometry. NF?κB DNA binding activity, IκB?α, Bcl?2 and Bax expression were examined by EMSA and Western blotting.    Results    (1)Compared with the control group, the activity of LDH significantly increased, cell count and cell viability significantly decreased (all P<0.05). The expression of NALP3 significantly increased and peaked at 4 h after H/R. (2)The specific siRNA could efficiently inhibit NALP3 expression in NRK?52E. Compared with the irrespective siRNA transfected group, the protein expression of NALP3 was significantly down?regulated in NALP3 siRNA transfected group (P<0.05). (3)After hypoxia 1 h and reoxygenation 4 h, the activity of LDH and the expression of NALP3 increased. Compared with the irrespective siRNA transfected group, LDH concentration in media and the expression of NALP3 significantly decreased in NALP3?siRNA transfected group. (4)After hypoxia 1 h and reoxygenation 4 h, NF?κB DNA binding activity was increased, IκB?α phosphorylation and degradation, Bcl?2 and Bax were significantly up?regulated. However, compared with the irrespective siRNA transfected group , NF?κB DNA binding activity, IκB?α degradation and Bax/Bcl?2 were significantly decreased (P<0.05) in NALP3?siRNA transfected group. At the same time, the ratio of apoptosis was significantly increased in three groups than that in control. Compared with the irrespective siRNA transfected group, the ratio of apoptosis in NALP3?siRNA transfected group was significantly decreased (P<0.05).    Conclusions    H/R induces the expression of NALP3 in NRK?52E. The synthesized siRNA can inhibit the expression of NALP3 and protect NRK?52E from hypoxia/reoxygenation injury. The mechanism may be via inhibiting the activation of NF?κB, modulating expression of Bcl?2 and Bax, as well as decreasing cell apoptosis.

Objective     To investigate the effect of estrogen on regression of vascular calcification in rats induced by vitamin D3 plus nicotine.    Methods    Ninety?six female SD rats were divided randomly into control group (n=24) and calcification group (n=72). Vascular calcification of 72 rats was induced by vitamin D3 and nicotine (VDN). On the day 1, the VDN group rats were injected with vitamin D3(300 000 U/kg, i.m), and were intragastric administrated with nicotine (25 mg/kg), after 9 hours, another dosage of nicotine was given again. After 4 weeks, the VDN group rats were subdivided randomly into 4 groups: VDN group(n=16), Sham operation group (n=16), ovariotomy group (n=16), estrogen group(after ovariotomy, 17β ?estrogen was subcutaneously injected, 50 μg•kg-1•d-1, n=16).    Results    After 4 weeks，the VDN group showed obvious vascular calcification, and calcium content of the vessel wall was significantly higher than that of control group (P<0.01). Extensive calcification was witnessed on the aortic tunica media of the VDN group. After 12 and 8 weeks, the calcium content of the vessel wall in each subdivided groups was significantly lower than that at 4 weeks point(P<0.01), and the lowest calcinm content was in estrogen group, meanwhile the reduction of previously accumulated arterial calcium precipitate in each group was different.    Conclusions    It is a reversible process that vascular calcification induced by vitamin D plus nicotine in rats. Estrogen can promote the regression of vascular calcification.

Objective    To investigate the effect of histone acetylation change on the transforming growth factor β1 (TGF?β1)?associated plasminogen activator inhibitor 1 (PAI?1) regulation in mesangial cells (MCs).    Methods    MCs were transfected with histone acetyltransferase (HAT) CREB?binding protein(CBP), P300 or histone deacetylase (HDAC) 1, 3, 5 expression vectors, followed by luciferase assay, real?time PCR and Western blotting to see the change of PAI?1 gene's transcriptional activity, mRNA and protein in response to TGF?β1. HDAC inhibitor trichostatin A(TSA) was used and TGF?β1?Smad signaling activity was detected also in this experiment.    Results     TGF?β1 enhanced PAI?1 transcriptional activity and mRNA expression (P＜0.05). Over?expression of CBP or P300 significantly increased TGF?β1?associated PAI?1 transcriptional activity, mRNA and protein expression (P＜0.05). On the contrary, over?expression of HDAC1, 3, 5 or dominant negative CBP or dominant negative P300 obviously reduced PAI?1 gene's expression induced by TGF?β1 (P＜0.05). Change of HAT/HDAC did not affect TGF?β1?associated Smad2/3 phosphorylation. TSA enhanced TGF?β1 induced PAI?1 gene's regulation markedly, but did not change TGF?β1?Smad2/3 signaling activity.    Conclusion     Change of histone acetylation can affect TGF?β1 associated PAI?1 gene's regulation in MCs.
  

Objective    To explore the effect and mechanism of fluvastatin on the expression of fibronectin(FN) in human peritoneal mesothelial cells (HPMCs) induced by high?glucose peritoneal dialysate (HGPDS).    Methods    Cultured HPMCs were randomly divided into control, HGPDS, HGPDS plus GSK650394 10-5 mol/L (the competitive inhibitor of SGK1), different concentrations of fluvastatin, fluvastatin 10-6 mol/L and GSK650394 10-5 mol/L alone. The morphology change of HPMC was observed by light microscopy. The cellular viability was detected by MTT colorimetry. The mRNA and protein expressions of serum and glucocorticoid?inducible kinase 1 (SGK1) and FN were detected by RT?PCR, Western blotting or ELISA.    Results    After incubation with HGPDS, the cell morphology changed from typical cobblestone?like appearance to fibroblast?like appearance, and the cell viability was inhibited significantly (P<0.05). Fluvastatin 10?6mol/L and GSK650394 could improved the cell morphology and the cell viability injured by HGPDS (P<0.05). Compared with the normal control group, the mRNA and protein expressions of SGK1 and FN increased significantly in HPMC treated with HGPDS(P<0.05). GSK650394 significantly decreased the high expression of SGK1 and FN (P<0.05), also the fluvastatin had same effects as GSK650394 in dose?dependent manner (P<0.05).      Conclusions    High?glucose peritoneal dialysate can increase FN expression in human peritoneal mesothelial cells, which can be attenuated by fluvastatin. The protective role of fluvastatin in HPMC may be partially achieved through the signal pathway of SGK1.

Objective    To investigate the effects of triptolide on proliferation, apoptosis and the changes of Ski, Smad3, Smad7 and collagen type Ι (ColΙ) in cultured rat mesangial cells induced by transforming growth factor (TGF)?β1.    Methods    Cultured HBZY?1 rat mesangial cells were divided into 5 groups: (1)normal control group; (2)TGF?β1 group (10 μg/L); (3)-(5)triptolide (0.4, 2, 10 μg/L)+TGF?β1 (10 μg/L) groups. The cell proliferation was detected by MTT. Apoptosis of mesangial cells was detected by TUNEL assay. The expressions of Ski, Smad3, Smad7 mRNA were examined by real?time quantitative PCR. The expressions of Ski, Smad3, Smad7 and ColΙ protein were detected by Western blotting. The localizations of Ski and Smad3 protein were detected by laser confocal fluorescence microscope.    Results    Compared with the normal control, TGF?β1 (10 μg/L) significantly stimulated mesangial cells proliferation, while decreased apoptosis. The mRNA and protein expressions of Ski, Smad7, Smad3 and ColΙ protein expression in TGF?β1 group were increased (P＞0.05). In comparison with TGF?β1 group, triptolide could significantly inhibit TGF?β1?induced mesangial cells proliferation in dose?dependent manner, and promote the apoptosis of mesangial cells. In TGF?β1 group, mRNA and protein expresscons of Ski and Smad7 were increased (P<0.05), Smad3 mRNA and protein were decreased (P＞0.05), and ColΙ protein was decreased (P<0.01). In comparison with TGF?β1 group, fluorescence intensity of Ski, Smad3 proteins was significantly increased in cytoplasm, while decreased in nucleus.    Conclusions    Triptolide can inhibit TGF?β1?induced mesangial cells proliferation through regulating the expressions of Ski, Smad7 mRNA and protein, inhibiting Ski. Smad7 translocation to the nucleus, and down?regulating Smad3 mRNA and protein expression. Triptolide can promote apoptosis of mesangial cells.