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Objective To analyze the correlation between the epithelial‐mesenchymal transition and the clinicopathologic features of IgA nephropathy .Methods A total of 168 patients diagnosed as IgA nephropathy by renal biopsy in Xinqiao hospital from Janu‐ary 2011 to December 2013 were divided into high expression of dual‐positive Snail and a‐SMA group (Group A ,117 cases) and low expression of dual‐positive Snail and a‐SMA group (Group B ,51 cases) according to results of immunohistochemical method .The clinical parameters (age ,gender ,course of disease ,BMI and chemical indicators) and renal pathology grade were compared by statis‐tical analysis .Results There were difference between group A and group B in the course of disease and BMI(P<0 .05) .There were differences between group A and group B in the incidences of creatinine ,blood urea nitrogen ,serum triglycerides and 24‐hour urine protein amount (P<0 .05) .The percentage of Lee′s grade Ⅰ ,Ⅱ ,Ⅲ in group B was significant ,while percentage of Lee′s gradeⅣ + Ⅴin group A was significant .The expression of Snail and a‐SMA in grade Ⅳ + Ⅴ was more than that in grade Ⅰ + Ⅱ (40 .2%vs .9 .4% ) ,and the difference between two groups was statistically significant (P< 0 .05) .Conclusion The expression of Snail and‐SMA were related with 24‐hour urine protein amount and kidney function in IgA nephropathy ;and Lee′s grade was severe in patients with high expression of Snail and‐SMA .
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Objective To construct and express a prokaryotic expression vector carrying the gene of FimH 1-156 that comprises human lysosome membrane protein 2 P41-49 gene ,and to express and purify the fusion protein .Methods FimH1-156 gene was cloned from plasmid pPKL241 by PCR ,and inserted into vector pET-28a(+ ) to obtain prokaryotic expression plasmid pET-28a-FimH . After transforming Escherichia coli BL21(DE3) with pET-28a-FimH ,fusion protein FimH1-156 was expressed under induction .The target fusion protein was purified ,and its antigenicity was detected through Western blot .Results The expressed recombinant pro-tein was purified ,the expression of protein was the highest when IPTG was 1 mmol/L and 4h after induction ,it was expressed as include body form ,and the expressed protein was identified to react with monoclonal antibodies 6 × His by Western blotting .Conclu-sion We cloned FimH1-156 fusion protein expressed genes successfully ,constructed prokaryotic expression vector ,and won the in-clusion body purification of FimH1-156 fusion protein .
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Objective To investigate the expression change of renal NLR family pyrin domain containing-3 protein(NALP3) in-flammasome in the nephrotic syndrome(NS) patients with focal segmental glomerulosclerosis(FSGS) and its relation with the tubu-lointerstitial pathogenic injury degree ,expression of inflammatory factors and clinical biochemical indexes .Methods Immunohisto-chemistry was used to detect the expressions of NALP3/ASC/caspase-1 and their downstream effector molecule IL-1β,IL-18 in re-nal tubular epithelial cells .The tubulointerstitial injury score and the activated macrophages F4/80 in renal interstitium of the FSGS patients and NS patiens were evaluated .The serum creatinine ,urea ,total protein ,albumin ,24 h urine protein and estimated glomer-ular filtration rate(eGFR) were observed .The correlation of tubulointerstitial injury with NALP3/ASC/caspase-1 ,IL-1β,IL-18 were respectively analyzed .Results The expression of NALP3/ASC/caspase-1 ,IL-1β,IL-18 in the renal tissue of the FSGS pa-tients was significantly increased compared with that in the control group (P<0 .01) .NALP3/ASC/capspase-1 expression was pos-itively correlated with the expression of IL-1β,IL-18(P< 0 .01) .NALP3/ASC/caspase-1 ,IL-1β,IL-18 expression was positively correlated with renal tubulointerstitial injury and the F4/80 expression intensity(P<0 .01) .NALP3/ASC/caspase-1 ,IL-1β,IL-18 was significantly positively correlated with 24 h urine protein and Scr ,and negatively correlated with the eGFR (P<0 .05) ,but had no obvious correlation with plasma urea ,plasma total protein and albumin concentrations .Conclusion The NALP3 inflammasome might participate in the pathogenic mechanism of FSGS through the activation of its downstream inflammatory factor of IL-1β,IL-18 ,the more higher its expression degree ,the more severe the renal tissue injury ,whether which could be served as the warning in-dex needs the further clinical verification .
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Objective To investigate the effects of p-cresol on human umbilical vein endothelial cells.Methods The effects of p-cresol on endothelial cell growth,cell cycle,cell morphological change and p21 protein were detected by the CCK-8 assay,flow cytometry assay,inverted microscope and Western blotting.Results P-cresol could inhibit the growth of human umbilical vein endothelial cells in dose-and time-dependent manners (all P ＜ 0.05).The human umbilical vein endothelial cells treated with p-cresol became elongated processes,cloudy cytoplasm,and irregular shapes.The p-cresol stopped human umbilical vein endothelial cells at cell cycle G1 and had no effect on cell apoptosis.The p-cresol could increase protein expression of p21 in a dose dependent manner (P ＜ 0.05).Conclusion P-cresol can increase protein expression of p21,induce cell cycle arrest at G1 stage and inhibit the proliferation of human umbilical vein endothelial cells.
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Objective To observe the effects of endoplasmic reticulum stress (ERS) on the activation of monocytes induced by high glucose and explore the underlying mechanism.Methods The monocyte cell line THP-1 was stimulated with high glucose,and then treated with molecular chaperone betaine.The levels of glucose regulation protein 78 (GRP78) and p-JNK,which were associated with ERS were detected by real-time PCR and Western blotting.The proliferation of the cell line was detected by MTT method.Transwell and immunofluorescence were applied to observe the chemotaxis and phenotype of cells respectively.Results The levels of GRP78 and p-JNK of THP-1 cells stimulated by high glucose were significantly increased compared with the normal control group (all P ＜ 0.05).The proliferation and chemotactic were also enhanced (all P ＜ 0.05).The number of cells in M1 phenotype was increased remarkably (P ＜ 0.05).All the indexes above could be rescued by betaine.Conclusion The activation of THP-1 cells can be induced by high glucose through ERS,while molecular chaperone betaine can reverse the activation.
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Objective To investigate the effect and significance of regulating endoplasmic reticulum stress on the expression of histone methyltransferases SET7/9 in the kidneys of db/db mice.Methods Db/db mice were randomly divided into two groups according to random number table method:diabetic nephropathy model group (DN group,n=18) and betaine treatment group (DN+B group,n =18),db/m mice were defined as normal control group (NC group,n =18).At the end of 4,8 and 12 weeks,the expression of GRP78,SET7/9,H3K4me2,and monocyte chemoattractant protein 1 (MCP-1) was determined by real-time fluorescence PCR and Western blotting.24-hour urinary protein excretion rate (UPER) and urine MCP-1 were measured by enzyme linked immunosorbent assay (ELISA).The dynamic changes of blood glucose(BG),serum creatinine (Scr),blood urea nitrogen (BUN) were tested by completely automatic biochemistry analyzer.The morphology of kidney was estimated by special staining of periodic acid-schiff (PAS).Results The levels of BG,BUN,UAER and MCP-1 were significantly higher in DN group than those in NC group (P ＜ 0.05),and were in time-dependent manner.Glomerular basement membrane thickening and mesangial cells proliferation began to emerge in DN group at the end of week 4 and mesangial matrix expansion was more obvious at the end of week 12.The mRNA and protein expression of GRP78 and SET7/9 were elevated significantly in DN group as compared to NC group.The H3K4me2 protein expression level was also increased in time-dependent manner.Compared with the DN group,in DN+B group glomerular lesions attenuated and the GRP78 and SET7/9 expression levels obviously decreased (P ＜ 0.05).Furthermore,the levels of BG,BUN,UPER,MCP-1,H3K4me2 in DN+B group were also reduced (P ＜ 0.05).Conclusion Endoplasmic reticulum stress may be the upstream mechanism of mediating the expression of SET7/9 in the kidneys of DN mice.
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Objective To investigate the correlation between plasma proteasome and endothelial dysfunction in patients with uremia. Methods Forty-five uremic patients who did not receive hemodialysis were defined as A group; seventy-five uremic patients who had received hemodialysis for 6 to 12 months were divided into sufficient hemodialysis group (44 cases,B group)and insufficient hemodialysis group (31 cases,C group).The primary disease of these patients was chronic glomerulonephritis.Fifteen healthy people were defined as healthy control group (D group).The diameter of radial artery lumen (DRL),intima-media thickness (IMT),intima-media area (IMA),endothelium-dependent or independent dilation (EDD or EID) of radial artery in right forearm were detected by diasonography.The levels of 20S proteasome,tumor necrosis factor α (TNF-α),C-reaction protein (CRP) and transforming growth factor β 1 (TGF-β1) of plasma and supernatant of cultured human umbilical veins endothelium (HUVEC) were determined by enzyme linked immunosorbent assay (ELISA).20S proteasome activity was analyzed by special substrate.Results Compared with D group,the level and activity of 20S proteasome,as well as TNF-α,CRP and TGF-β1 in A,B and C groups were significantly increased.Compared with A group,these plasma indices levels were significantly decreased in B group but strongly increased in C group.IMT and IMA were elevated,while DRL,EDD and EID were decreased significantly in A,B and C groups when compared with D group.These parameters were worse in C group than those in A and B groups.After co-culture of HUVEC with above mentioned human uremic serum,the level and activity of 20S proteasome and TNF-α were higher in A,B,C groups than that in D group.In A and C groups,there were negative correlations of EDD with the level or activity of 20S proteasome,TNF-α,CRP and TGF-β1,and there were positive correlations of 20S proteasome level or activity with TNF-α,CRP and TGF-β1. Conclusions 20S proteasome level and activity are significantly increased in uremic patients.There is a close correlation between 20S proteasome and endothelial dysfunction of radial artery.
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Objective To study the role of endoplasmic reticulum stress in phenotypic change of cultured human glomerular mesangial cells induced by high glucose.Methods Cultured human glomeruar mesangial cells were divided into three groups: control group,high glucose group and high glucose+ 4-phenylbutyric acid (4-PBA) group.Cell number of proliferation was assessed by MTT assay.Cell cycle was measured by flow cytometric analysis.Expression of α-SMA was assessed by immunohistochemistry and was observed by laser scanning confocal microscope.Involved mRNA and protein expression were measured by real-time PCR and Western blotting.Results (1)Cell number of proliferation and S transition proportion in high glucose group significantly increased than that in control group (P ＜ 0.05).High glucose could induce α-SMA expression significantly (P＜0.05).4-PBA could significantly inhibit human glomerular mesangial cells proliferation (P＜0.05),S transition arrest (P＜0.05) and expression of α-SMA (P＜0.05) induced by high glucose.(2) Compared with control group,high glucose could significantly increase the expression of glucose-regulated protein78(Grp78 ) mRNA and protein (P＜ 0.05),which could be inhibited by 4-PBA treatment (P＜0.05).(3)High glucose could induce the mRNA and protein expression of TGF-β1 and FN significantly,which could be inhibited by 4-PBA treatment (P＜0.05).Conclusion Endoplasmic reticulum stress plays an important role in phenotypic change of cultured human glomerular mesangial cells induced by high glucose.
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Infection control is important in quality management of blood purification center. Practice of Continuous Quality Improvement in infection control improves patients' living quality, and it is worth proceeding and generalization.
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Objective To investigate the effects of chronic renal failure rabbit serum on proliferation and nuclear factor kappa B (NF-κB) activation of rabbit arterial smooth muscle cells (ASMCs) and to explore the possible mechanism. Methods Rabbit model of chronic renal failure was established by the ligation of renal arterial branches. ASMCs were incubated in the media with various concentrations of chronic renal failure serum cultured in vitro. Cell proliferation was assessed by MTT. Cell apoptosis was detected by Hoechst33342 staining. NF-κB p65 nuclear translocation was analyzed by immunofluorescence. Expression of proliferating cell nuclear antigen (PCNA) and NF-κB p65 proteins in response to chronic renal failure serum in ASMCs was determined by Western blotting. Results Lower concentrations of chronic renal failure serum (≤ 10%) could significantly promot the proliferation of ASMCs in a dose- and time-dependent manner. Higher concentrations of chronic renal failure serum (＞10%) could significantly inhibit the proliferation and induce apoptosis of ASMCs compared to the normal control (P＜0.05). Under the stimulation of lower concentrations of chronic renal failure serum, the expression of PCNA and NF-κB p65 increased significantly compared to the normal control (P＜0.01), while decreased markedly under the stimulation of higher concentrations of chronic renal failure serum compared to the normal control (P＜0.01). Under the stimulation of 10% chronic renal failure serum, nuclear translocation of NF-κB p65 in ASMCs was found. Conclusions Different concentrations of chronic renal failure rabbit serum can effectively induce ASMCs proliferation or apoptosis. The mechanism of promoting proliferation may be mediated by activating NF-κB, which will be useful for the treatment of accelerated atherosclerosis in chronic renal failure.
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Objective To observe the stressor characteristic of patients with maintained hemodialy-sis (MHD). Methods The Chinese version of bemodialysis stressor seale(HSS) was adopted among 436 bemodialysis participants and the results underwent analysis. Results Among 436 patients psychological stressors> physiological stressers > social stressors, the most severe stressors included the requirement of long-term dialysis, the rest showed sequence from high to low: medical cost, concerning about the future,changes in family responsibility and decrease in sexuality. The slightest stressors included decreased ability to procreate, being limited in some styles of clothing, arterial and venous stick, reversal in family role with the children and changes in body appearance. Significant differences existed in patients with different age,gender, education background, working status and economic status, it manifested as follows: the more elder-ly, the more severity of stress, male patients had more severe stress than the female, patients with higher e-ducation background showed lower stress level, patients who kept working had lower level of stress, pa-tients who had better economic status showed lower stress level. Conclusions MHD patients often expe-rience high stress levels. Some measures should be taken to help MHD patients to cope better with stress.
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Objective To investigate the effects of the serum from chronic renal failure (CRF) rats on endoplasmic reticulum stress (ERS) and activation of activating transcription factor 4 (ATF4) of rat arterial smooth muscle cells (ASMCs) cultured in vitro and explore the possible mechanism. Methods The rat model of CRF was established by 5/6 nephrectomy. ASMCs were incubated in the media with 10% or 20% serum of CRF rats cultured in vitro. ATF4 nuclear translocation were analyzed by immunofluorescence. GRP78 (glucose regulated protein 78), p-PERK (pancreatic ER kinase) and ATF4 proteins in response to the CRF serum in ASMCs were determined by Western blot. The ATF4-DNA binding activity was analyzed by electrophoretic mobility shift assay (EMSA). Results The CRF serum could significantly promote the expression of GRP78 of ASMCs in dose-dependent manner. Under the stimulation of 20% CRF serum, nuclear translocation of ATF4 in ASMCs was found. Compared with control serum group, the expression of ATF4 and p-PERK of CRF serum group was increased significantly (P<0.01). The CRF serum could increase the ATF4-DNA binding activity. Conclusion The CRF rat serum can effectively induce ERS of ASMCs and activate the pathway of PERK-ATF4.
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Objective To compare the effect of hemodiafiltration(HDF) on the incidence rate of intradialytie hypotension. Methods We followed up eighteen patients with symptomatic hypotension history and divided them into group A and group B.Group A was treated with conventional hemodialysis(CHD) and HDF, Group B adopted HDF with low temperture hemodialysis. The number of hypotensive episodes, blood temperature during cardiopulmonary bypass, energy transfer rates were assessed. Results The incidence of symptomatic hypotension of patients was found lower in CHD (40%) than in HDF (6%),P<0.01. Energy transfer rates were founded higher in CHD than in HDF and low temperture hemodialysis, P<0.01. Conclusions HDF significantly reduced hemodialysis-related side effects and increased the tolerance ability of patients.
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Objective To investigate the activation of NF-κB and regulation by ubiquitin (Ub)-proteasome pathway in the aorta of rats with chronic renal failure(CRF).Methods The CRF rat model was established by right nephreetomy and left branch renal artery ligation.The CRF rats were were randomly divided into simple CRF group(n=20)and CRF+M used as control group(CON).The NF-κB and the Ub mRNA expression were detected by RT-PCR,and its protein expression was analyzed by immunohistochemistry method.The activity of NF-κBwas mesured by EMSA method.The concentration of IL-1 and TNF-α was detected by ELISA.Results Compared with the CON group,the concentration of serum IL-1 and TNF-α was increased significantly in CRF group [IL-1:(9.02±1.29) vs (2.74±0.96)mg/L,P＜0.01;TNF-α:(50.02±9.52) vs (14.04±1.29)mg/L,P＜0.01]at month 4 after operation.The mRNA expression of NF-κB and Ub in the aorta of CRF group was 1.38 and 1.29 times as that of CON group(P＜0.01).and the protein expression of NF-κB and Ub was 3.75 and 20.5 times as that of CON group(P＜0.01).Compared with the CON group,the activity of NF-κB in the aorta of rats of CRF group was elevated markedly at month 4 after operation(P＜0.01).All the indices were further increased at month 6 after operation.Compared with CRF group,the concentrations of serum IL-1and TNF-α were decreased significantly in CRF+M group[IL-1:(2.94±0.33)mg/L,P＜0.01;TNF-α:(12.80±2.12)mg/L,P＜0.01].The mRNA and protein expression of NF-κB and Ub were also decreased markedly(P＜0.01),and the activity of NF-κB was decreased significantly at month 4 to 6 after operation(P＜0.01).But the amount of ubiquitnative protein was increased significantly in the aorta of CRF+M group as compared to CRF group(P＜0.01). Conclusion The inflammatory signal pathway of ubquitin-proteasome-NF-κB pathway was activated in the aorta of CRF rats,and the proteasome was probablely an important pharmacological intervention target to regulate the activation of NF-κB.
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Objective To investigate the effects of resistin on mesangial cells proliferation induced by high glucose and subsequent change of p38MAPK signal pathway. Methods Human macrophrages were cultured and treated with adenovirus encoding for resistin (Ad-resistin) for 48 h and were then co-cultured with human mesangial cells stimulated by high glucose for another 48 h. Mesangial ceils were harvested and their proliferation was measured by 3H-TdR. Activator protein 1 (AP-1) was examined by immunocytochemistry and laminin of excellular matrix was observed with immuofluorescence. Protein levels of p38MAPK and TGF-β1 were measured by Western blot. Smad2 phosphatase activity was aslo detected by Western blot. Results The mRNA and protein levels of resistin were significantly higher in Ad-resistin treated macrophages than those in Ad treated cells (P<0.01). Over-expression of resistin up-regulated p38MAPK protein levels of human mesangial cells(P<0.05). Resistin also promoted the proliferation of mesangial cells (P<0.01) and the synthesis of laminin stimulated by high glucose. The expression of TGF-β1 and phosphorylation of Smad2 were up-regulated in the mesangial cells (P< 0.05). Conclusion Macrophage cytokine resistin may promote mesangial cells proliferation and abnormal accumulation of excellular matrix stimulated by high glucose via activating p38MAPK signal passway.
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72 h), the lowest increase of kidney weight ( P
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Objective To investigate the expression of indoleamine 2,3-dioxygenase(IDO)in renal tissues of crescent nephritis and its correlation with cell proliferation and pathologic tubulointerstitial lesion.Methods Immunohistochemistry was used to detect IDO expression in renal tissues of crescent nephritis and normal renal tissues.The correlation between IDO expression and the number of proliferating cell nuclear antigen(PCNA)positive cells or pathologic tubulointerstitial lesion were analyzed.Results IDO expression was found in the renal tubular epithelial cells of crescent nephritis,while that in the normal renal tissues was negative by immunohistochemical staining.The expression of IDO protein in tubular epithelial cells showed a significantly negative correlation with the number of PCNA positive cells or tubulointerstitial lesion of crescent nephritis.Conclusion IDO may participate in the pathogenesis of crescent nephritis by inhibiting the cell proliferation.
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<p><b>OBJECTIVE</b>To observe the ability of triple helix-forming oligonucleotides (TFOs) modified with manganese porphyrin to combine with and cleave HBV DNA fractions.</p><p><b>METHODS</b>TFO were modified with manganese porphyrin and acridines, and then reacted with the (32)P labeled HBV DNA fragments at 37 degrees C in vitro (pH 7.4). Electrophoretic mobility shift assays and DNase I footprinting tests were used to show the affinity and specificity of TFO to bind to target sequences. The ability of TFO to cleave HBV DNA fragments was tested by cleavage experiments.</p><p><b>RESULTS</b>TFO modified with manganese porphyrin and acridine could bind to the target sequence in a sequence-dependent manner, with a Kd value of 3.5 x 10(-7) mol/L and a relative affinity of 0.008. In the presence of potassium monopersulfate (KHSO(5)), TFO modified with manganese porphyrin and acridine could cleave the target sequence where the triplex DNA was formed.</p><p><b>CONCLUSION</b>In the presence of KHSO(5), TFO modified with manganese porphyrin and acridine could bind and cleave the target HBV-DNA in a sequence-dependent manner.</p>
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DNA , Pharmacology , DNA, Viral , Chemistry , Hepatitis B virus , Genetics , Manganese , Pharmacology , Metalloporphyrins , Pharmacology , Potassium Compounds , Pharmacology , Sulfates , PharmacologyABSTRACT
The authors analyzed the current problems about clinical postgraduate cultivation in the medical colleges or institutes or hospitals in China,and proposed some solving ideas for discussion,including raising the applicative qualification for enrolling postgraduate,reinforcing construction of the soft and hardware condition,guaranteeing enrolling number of postgraduate for key colleges and institutes.
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Objective To investigate the effect of bone morphogenetic protein-7 (BMP-7) on high glucose-induced expression of fibronectin (FN) and collagen Ⅳ (Col Ⅳ) and activity of nuclear factor-?? (NF-??) in human renal tubular epithelial cells. Methods Human renal tubular epithelial cells (HKCs) in culture were divided into 5 groups: normal glucose group with 5.5 mmol/L glucose (NG), high glucose group with 25 mmol/L D-glucose (HG), HG+100 ng/ml BMP-7, NG+100 ng/ml BMP-7 and high osmolality group with 25 mmol/L mannitol (HM). Thus HKCs were respectively stimulated with high glucose, high mannitol and BMP-7. Expression of collagen Ⅳ and fibronectin was determined by immunocytochemistry and enzyme linked immunosorbent assay (ELISA) and activity of nuclear factor-?? was assessed with electrophoretic mobility shift assay (EMSA). Results Compared with normal glucose, high glucose concentration up-regulated expression of FN and Col Ⅳ and activity of NF-?? (P0.05). With addition of BMP-7 in a concentration of 100ng/ml to the high glucose medium, expression of Col Ⅳ and FN was suppressed, and the activity of nuclear factor-?? was inhibited in human tubular epithelial cells (P