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Objective To explore the expression of miroRNA-21 (miRNA-21) in myocardial fibroblasts stimulated by indoxyl sulfate (IS) and its role on paracrine factors of myocardial fibroblasts.Methods Myocardial fibroblasts which derived from C57BL/6J mice were divided into control group and IS group,and their expressions of miRNA-21 were detected by real time PCR after 48 h.MiRNA-21 inhibitor transfection was applied to silence miRNA-21 expression.Myocardial fibroblasts were divided into creatinine (Scr) group (Scr treated for 48 h),Scr+IS group (Scr and 50 μmol/L IS treated for 48 h),Scr+miRNA-21 inhibitor group (miRNA-21 inhibitor treated for 24 h and then Scr treated for 48 h) and Scr+miRNA-21 inhibitor+ IS group (miRNA-21 inhibitor pretreated for 24 h and then Scr and IS treated for 48 h).Enzyme-linked immunosorbent assay was performed to evaluate the expressions of interleukin-1 (IL-1),interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α).Western blotting was applied to detect the expressions of transforming growth factor-β (TGF-β),matrix metalloproteinases 2 (MMP2),matrix metalloproteinases 9 (MMP9) and tissue inhibitor of metalloproteinases 1 (TIMP1).Results The expression of miRNA-21 was obviously increased in IS group than that in control group (P ＜ 0.01).Compared with those in Scr group,the expressions of IL-1,IL-6,TNF-α,TGF-β,MMP2 and MMP9 significantly increased (all P ＜ 0.05),while the expression of TIMP1 decreased (P ＜ 0.05).When the expression of miRNA-21 was inhibited,the expressions of IL-1,IL-6,TNF-α,TGF-β and MMP2 in Scr+miRNA-21 inhibitor+IS group significantly decreased than those in Scr+IS group (all P ＜0.05),and the expression of TIMP1 decreased further (P ＜ 0.05).Conclusions IS can promote the expression of miRNA-21 in myocardial fibroblasts.MiRNA-21 plays an important role in regulating inflammatory factors and pro-fibrogenic cytokines in myocardial fibroblasts.
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Objective To observe the quantity change of autophagosomes in podocytes and expressions of autophagy-related gene Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3) in different pathological stages of idiopathic membranous nephropathy (IMN),and to explore how autophagy is related to podocyte injury,the occurrence of proteinuria and the disease progression in IMN.Methods Clinical data of 26 patients who were diagnosed as IMN (14 IMN stage 1 and 12 IMN stage 2) admitted to Zhejiang Provincial people's Hospital from January 2013 to December 2014 were retrospectively analyzed.Normal renal tissue from 15 cases of kidney neoplasms with nephrectomy was collected as control.The changes of kidney tissue pathology were detected after PAS and PASM staining by light microscope.The autophagosomes of podocyte were detected by transmission electron microscopy.Expressions of Beclin-1 and LC3 protein were detected by immunohistochemistry.Expressions of LC3 and synaptopodin were detected by immunofluorescence.The correlation of autophagosomes and clinical pathologic factors in IMN patiens was analyzed.Results There were fewer autophagosomes of podocytes and lower expression of Beclin-1 and LC3 protein in IMN group than those in control group (P=0.034,P=0.011,P=0.013,respectively).Moreover,these effects were more obvious with the development of IMN.Compared with those in control group,autophagosomes,Beclin-1 and LC3 protien were reduced in IMN stage 2 group (P=0.009,P=0.030,P=0.015);the number of autophagosomes and the expressions of LC3 and Beclin-1 were decreased in IMN stage 1 group as well,however statistically insignificant (P=0.352,P=0.087,P=0.128);Comparisons between IMN stage 2 patients and IMN stage 1 patients shown significant difference in the number of autophagosomes (P=0.030),but no significant difference in expressions of Beclin-1 and LC3 (P=0.355,P=0.181).Autophagosomes number was not correlated with serum creatinine,serum urea nitrogen,24-hour urinary protein and eGFR (all P ＞ 0.05).The expressions of synaptopodin and LC3 protein were lower in IMN group than those in control group.Conclusion Autophagy may contribute to podocyte injury and the production of protein urine in IMN,and may be closely related to the progression of disease.
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Objective To investigate the clinical and pathological characteristics of IgA nephropathy (IgAN) in association with active tubular interstitial lesions. Methods 148 patients who were diagnosed as IgAN by renal biopsy and admitted to Zhejiang Provincial People's Hospital from March 2014 to December 2014 were enrolled. They were divided into IgAN with active tubular interstitial lesions group (IgAN?ATIL group, 23 patients) and IgAN without active tubular interstitial lesions group (control group, 125 patients). Clinical and pathological characteristics were retrospectively analyzed. Multivariate logistic regression analysis was used to analyze the influence factors. Results The prevalence of ATIL in 148 IgAN patients was 15.5%. IgAN?ATIL group showed an older average age and more higher proportion of medication history (antibiotics, diuretics, nonsteroidal anti ?inflammatory drugs, etc) than those in control group. There were significant differences in Alb, eGFR, Scr, BUN, 24?hour urinary protein quantity, urinary NGAL and urinary RBC count between two groups (P<0.05, respectively). A moderate of tubulointerstitial lesions of IgAN?ATIL group was shown, while the control group was mainly mild lesions. Multivariate logistic regression analysis showed that age, medication history and the urinary NGAL level were independent risk factors of IgAN ? ATIL. Conclusions IgAN patients with active tubular interstitial lesions had more severe clinical manifestations and chronic interstitial lesions. The age, medication history (antibiotics, diuretics, nonsteroidal anti?inflammatory drugs, etc) and the urinary NGAL level were independent risk factors of IgAN?ATIL.
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[ ABSTRACT] AIM:To investigate the effect of storage temperature in renal biopsy tissue frozen section on immu-nofluorescence results.METHODS:One hundred renal biopsy samples of Zhejiang Province People’ s Hospital from Janu-ary to June, 2015 were enrolled.Two sets of cutting slices were stored in -70 ℃ low temperature refrigerator as experi-mental group and in -12 ℃ freezing cryostat as control group.The immunoglobulins ( IgG, IgA and IgM) and comple-ments (C3, C4 and C1q) as well as fibrinogen were detected with direct immunofluorescence in the next day.The typeⅣcollagen a3 and a5 chains were also detected by indirect immunofluorescence, and the qualitative and semi-quantitative re-sults were observed under the fluorescence microscope.RESULTS:There were 16 cases of 3+~4+IgG in the experi-mental group, while the corresponding IgG was all 1+~2+in control group, significantly weaker than that in experimen-tal group.There were 99 cases of 3+~4+a3 and 3+~4+a5 in experimental group, white there were 92 cases of 1+~2+a3/a5 and 8 cases negative in control group.The positive intensity was decreased in control group with statistical difference between the 2 groups ( P<0.05) .There were 15 cases of 4+IgA, 19 cases of 3+IgA, 15 cases of 3+IgM, 11 cases of 2+IgM, 13 cases of 4+C3, and 12 cases of 3+C3 in experimental group.The control group were similar to the results of experimental group, and no difference between the 2 groups was observed.CONCLUSION:The immunoflu-orescence results of renal biopsy frozen sections are highly affected by the section storage temperature, which has greater in-fluence on the immunofluorescence positive intensity of IgG and typeⅣcollagen.The renal biopsy frozen section should be stored in -70 ℃low temperature refrigerator.
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Objective To investigate the clinical and pathological features of idiopathic membranous nephropathy (IMN) in young patients.Methods Clinical data of 20 young patients, 16 to 44 years, who were diagnosed as IMN admitted to the Zhejiang Provincial People's Hospital from January 2013 to December 2014 were retrospectively analyzed, comparing to 55 mid-aged patients who were diagnosed as IMN during the same period in the hospital.Clinical and pathological features of above mentioned patients were analyzed.Results Young patients with IMN accounted for 26.7％ of IMN patients.Compared to mid-aged patients, young patients with IMN had lower proportion of hypertension (P=0.003), lower blood glucose level (P=0.010), higher estimated glomerular filtration rate (eGFR) and low-density lipoprotein (LDL) (P=0.012, P=0.038), and lower levels of T3 and T4 (P=0.030, P=0.034).Furthermore, there were less sclerosis glomeruli (P＜ 0.001), hyaline change of arteriole (P=0.040) and arteriolar wall thickening (P ＜ 0.0001), lower positive ratios of IgA (P=0.008),and more without renal tubulointerstitial lesions (P=0.018) in young patients.There were no statistically significant differences between these two groups in other index.Conclusions Compared to mid-aged patients, young patients with IMN have better blood pressure and blood glucose level, higher glomerular filtration rate and LDL.Moreover, thyroid function is significantly affected, meanwhile the lesions of glomerular, interstitial and vascular are mild in young patients.
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Objective To investigate clinical and pathological features of idiopathic membranous nephropathy (IMN) accompanied by mesangial dense deposit.Methods Clinical data of 46 patients who were diagnosed as IMN accompanied by mesangial dense deposit admitted to Zhejiang Provincial People's Hospital from January 2013 to December 2014 were retrospectively analyzed.They were compared with those of 29 patients who were diagnosed as IMN without mesangial dense deposit during the same period in the hospital.Analysis of their clinical and pathological features was conducted.Results The IMN accompanied by mesangial dense deposit accounted for IMN 61.3％,and had more hyaline changes of arteriole (43.5％ vs 6.9％,P=0.001) and more obvious arteriolar wall thickening (78.2％ vs 51.7％,P=0.016) than IMN without mesangial dense deposit.Furthermore,the positive rate of IgA deposition in IMN accompanied by mesangial dense deposit was greatly higher than that in IMN without mesangial dense deposit (21.7％ vs 0,P=0.007).In other index,such as serum biochemical parameters,urine protein,glomerular lesion,tubulointerstitium pathological damage and other immunopathologic changes,no statistically significant differences were found between these two groups.Conclusions IMN patients accompanied by mesangial dense deposit have severe intrarenal artery lesions,and high positive rate of IgA deposition.