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Alport syndrome(AS)is a hereditary nephropathy associated with hematuria, proteinuria and progressive kidney failure.It is characterized by a defective glomerular basement membrane caused by mutations in type Ⅳ collagen genes COL4A3/A4/A5, which result in defective in the type Ⅳa3, a4 and a5 chains respectively.At present, there is no preventive or curative therapies for AS.Inhibitors of the renin angiotensin aldosterone system are routinely used to slow the progression of kidney disease and prolong life expectancy.Ramipril was found in retrospective studies to delay the onset of end stage renal disease(ESRD), supporting that early initiation of renin angiotensin aldosterone system blockade is very important.Advances in our understanding on the pathogenesis of AS has culminated in the development of innovative therapeutic approaches that are currently under investigation.This review will briefly outline novel therapeutic targets for the prevention of renal disease progression in AS.
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ObjectiveTo observe the modulatory effect of modified Zhenwutang on the interleukin-6 （IL-6）， matrix metallopeptidase-9（MMP-9）， type Ⅳ collagen（COL-Ⅳ） in rats with chronic renal failure （CRF） and to investigate the potential mechanism of its treatment of CRF. MethodFifty male SD rats were randomly divided into a modeling group of 40 rats and a normal group of 10 rats， and the modeling group was prepared by continuous adenine gavage for 12 weeks. After successful modelling， the modelling group was divided into the model group， the low dose （7.2 g·kg-1·d-1） group， the medium dose （14.4 g·kg-1·d-1） group， the high dose （28.8 g·kg-1·d-1） group and the Benadryl hydrochloride （10 mg·kg-1·d-1） group for gavage according to the random number table method， In the normal group and the model group， equal volume of distilled water was administered by gavage for 4 weeks. After the administration， the levels of blood creatinine （SCr）， blood urea nitrogen （BUN） and 24 h urine protein （24 h-UTP） were measured， the levels of serum IL-6 were measured by enzyme linked immunosorbent assay（ELISA）. Immunohistochemistry （IHC） was used to detect intercellular cell adhesion molecule-1 （ICAM-1）， IL-6， MMP-9， and other molecules in the rat kidney. The expression of ICAM-1 mRNA， IL-6 mRNA， MMP-9 mRNA and COL-Ⅳ mRNA in rat kidney tissues was measured by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expression levels of ICAM-1， IL-6， MMP-9 and COL-Ⅳ in rat kidney tissues were measured by Western blot. ResultCompared with the normal group， the levels of SCr， BUN and 24 h-UTP were significantly increased in the model group （P<0.01）； the serum IL-6 level was significantly increased （P<0.01）， the tubular lumen was dilated with atrophy， the tubular epithelial cells were necrotic， swollen and vacuolated， the interstitium was infiltrated by a large number of inflammatory cells and collagen fibers were deposited， the levels of IL-6， ICAM-1 and COL-Ⅳ were strongly positive in the tubular interstitium of the model group （P<0.01）， The levels of ICAM-1 mRNA， IL-6 mRNA and COL-Ⅳ mRNA were significantly increased （P<0.01） and MMP-9 mRNA was significantly decreased （P<0.05） in the model rats. ICAM-1， IL-6 and COL-Ⅳ protein expression was significantly increased （P<0.01） and MMP-9 protein expression was significantly increased （P<0.01） in the renal tissue， and MMP-9 protein expression was significantly decreased （P<0.01）. Compared with the model group， the 24 h-UTP， SCr and BUN levels of rats were significantly reduced after treatment with modified Zhenwutang （P<0.01）， the serum IL-6 level was significantly decreased （P<0.01）， the renal lesions of rats were significantly improved and collagen fiber deposition was reduced； the expression of IL-6， ICAM-1 and COL-Ⅳ in renal tubules and interstitium was weakened， and MMP-9 in ICAM-1 mRNA， IL-6 mRNA and COL-Ⅳ mRNA levels were significantly reduced （P<0.01） and MMP-9 mRNA levels were significantly increased （P<0.05）， ICAM-1， IL-6 and COL-Ⅳ protein expression was significantly reduced （P<0.01） and MMP-9 protein expression was significantly The expression of ICAM-1， IL-6 and COL-Ⅳ proteins was significantly decreased （P<0.01） and MMP-9 protein expression was significantly increased （P<0.01）. ConclusionModified Zhenwutang may regulate the IL-6/MMP-9/COL-Ⅳ signaling pathway， thereby reducing proteinuria， improving renal function， reducing renal pathological damage and delaying the progression of CRF interstitial fibrosis.
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ObjectiveTo observe the intervention of modified Sanrentang on the lipopolysaccharide-induced proliferation of rat glomerular mesangial cells， the phosphatidylinositol-3 kinase（PI3K）/protein kinase B（PKB/Akt）/nuclear factor kappa B（NF-κB） signaling pathway， and to investigate its mechanism in improving kidney inflammation in rats with immunoglobulin A nephropathy（IgAN）. MethodThe 18 rats were divided into 3 groups by serum pharmacology method： normal group， high-dose and low-dose （20.70，10.35 g·kg-1·d-1） groups with 6 rats in each group. Modified Sanrentang high- and low-dose groups were intragastric with the corresponding solution of modified Sanrentang， and normal group was intragastric with equal volume of distilled water. After 5 days of intragastric administration， blood samples were collected to prepare drug-containing serum. Rat mesangial （HBZY-1） were divided into five groups of normal group， LPS 10 mg·L-1 in the model group， benazepril（50 μmol·L-1）， modified Sanrentang high- and low-dose group. Preclude the use of methyl thiazolyl tetrazolium（MTT） method detect the proliferation activity of HBZY-1 cells， enzyme-linked immunosorbent assay（ELISA） was used to determine the content of each group type Ⅳ collagen（ColⅣ），Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） were used to detect protein and mRNA expression levels of PI3K/Akt/NF-κB signaling pathway. ResultAs compared with the normal group， MTT assay showed that exposure to LPS significantly enhanced the proliferative activity， the ColⅣ was increased significantly of HBZY-1 cells（P<0.01）， p-Akt， p-p65 was increased significantly （P<0.01）. Compared with the model group， the proliferation and ColⅣ of rat chronic glomerulonephritis cells induced by LPS by inhibiting PI3K/Akt/NF-κB signaling pathway（P<0.01）， and the phosphorylation of Akt was significantly inhibited（P<0.01）， the expression levels of NF-κB p65 was reduced in modified Sanrentang high-dose group（P<0.01）. ConclusionModified Sanrentang could inhibit cell proliferation and the content of ColⅣ in rat mesangial cells induced by LPS， and its mechanism might be related to suppression of PI3K/Akt signaling pathway.
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Objective To investigate the correlation between serum laminin (LN),type collagen (ⅣC),type procollagen N-terminal peptide (PⅢNP),hyaluronic acid (HA) and the severity as well as the prognosis of idiopathic pulmonary fibrosis (IPF).Methods From February 2015 to July 2016,160 IPF patients and 160 healthy subjects as controls were enrolled in this retrospective study.Serum LN,ⅣC,PⅢNP,and HA were analyzed in IPF patients and healthy controls.Pulmonary function test and chest high resolution computed tomography (HRCT) were carried out in IPF patients.Demographics and clinical characteristics,the percentage of forced vital capacity in the prediction value (FVC％pred),the percentage of diffusing capacity of the lung for carbon monoxide in the prediction value (DLCO％pred),and HRCTscore were collected.IPF patients were followed up.Results (1)There were no significant difference between two groups in age and sex ratio.The proportion of smoker in IPF patients was significantly higher than that in healthy controls(P ＜ 0.01).(2)Serum LN(P ＜ 0.01),ⅣC(P ＜ 0.01),PⅢNP(P ＜ 0.01),and HA(P ＜ 0.01) were significantly increased in the patients with IPF compared with the healthy controls.(3)Serum LN,ⅣC,PⅢNP,and HA of IPF patients positively correlated with HRCT score,all P ＜ 0.01,and negatively correlated with FVC％pred and DLCO％pred (all P ＜ 0.05).(4)Serum LN(P ＜ 0.01),ⅣC(P ＜ 0.05),PⅢNP(P ＜ 0.01),and HA(P ＜ 0.01) in acute exacerbation IPF patients were significantly higher than those in the stable IPF patients.(5)Serum LN(P ＜ 0.01),ⅣC(P ＜0.01),PⅢNP(P ＜ 0.01),and HA(P ＜ 0.01) in the survived patients were significantly lower than those inthe dead patients.Conclusions Serum LN,ⅣC,PⅢNP,and HA may reflect IPF prognosis and the severity of IPF.
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Objective To study the changes of serum markers of hepatic fibrosis in patients with fatty liver,and to provide clinical diagnosis and treatment for patients with fatty liver.Methods Three hundred and sixty-one patients with fatty liver were divided into light,medium and heavy three degrees according to the intrahepatic echo and structure.Serum markers of hepatic fibrosis-hyaluronic acid (HA),laminin (LN),procollagen Ⅲ (PC Ⅲ) and type Ⅳ collagen (C-Ⅳ) were examined by enzyme linked immunosorbent assay (ELISA),and the ordinal logistic regression model was used to analyze,regarding the grade of fatty liver as dependent variable.Results The levels of serum fibrosis markers HA,LN,PC Ⅲ and C-Ⅳ in patients with mild,moderate and severe fatty liver were (94.53 ± 16.21),(101.38 ± 20.42),(127.34 ± 26.54) μg/L,(107.25±22.63),(117.38±24.84),(136.62±32.27) μg/L,(110.27±23.15),(121.55±27.36),(138.62± 30.62) μg/L,(72.61 ± 15.46),(82.06 ± 18.28),(92.96 ± 21.35) μg/L respectively,there was significant difference in serum fibrosis markers among mild,moderate and severe fatty liver patients (F =675.719,398.771,303.960,840.570;P＜0.05),while the markers of patients with severe fatty liver were higher than normal.The ordinal logistic regression model showed that the serum fibrosis indexes of HA,LN,PC Ⅲ and C-Ⅳ had significant effects on the grading of fatty liver(P＜0.05),whose odds ratio were 1.322,1.229,1.899,3.935,that was the higher the HA,LN,PC Ⅲ,C-Ⅳ,the more severe fatty liver.Conclusion There were liver fibrosis trend in patients with severe fatty liver.Detection of serum markers of hepatic fibrosis can be used as an important basis for monitoring and diagnosis of fatty liver disease.
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Objective To investigate the effects of capparis spinosa total alkaloid on type collagen (ColⅣ - ),Ⅳmatrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in bleomycin-induced systemic sclerosis (SSc) mice; To explore the effective mechanism of capparis spinosa total alkaloid on fibrosis of SSc. Methods Totally 90 BALB/c mice were randomly divided into control group, model group, penicillamine group and capparis spinosa total alkaloid low-, medium- and high-dose group. Mice models with SSc were established by repeated local injections of bleomycin in mice back, except for the control group. Mice in medication groups received external application with capparis spinosa total alkaloid cream;mice in penicillamine group were given penicillamine for gavage; mice in the control and model group received external application without substance, one time a day, for 60 days. The contents of MMP-9, TIMP-1 and PAI-1 in serum and Col- in skin tissue were dⅣ etected respectively by ELISA after the last medication. Results Compared with the model group, the levels of MMP-9 and ratio of MMP-9/TIMP-1 markedly increased and the levels of Col-Ⅳand TIMP-1 markedly decreased in medium and high- dose of capparis spinosa total alkaloid group (P0.05). Conclusion Capparis spinosa total alkaloid is effective in treating fibrosis of SSc by adjusting imbalance of MMP-9/TIMP-1 and decreasing expression of Col-Ⅳ.
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To study the effects of forkhead transcription factor O1 (FoxO1) on the expression of type Ⅳ collagen and desmin in podocytes of diabetic rats.Streptozotocin-induced diabetic rats were divided into three groups:diabetic rats (DM group),rats transfected with blank lentiviral vectors (diabetes mellitus plus LV-pSC-GFP group,LV-NC group),and rats which were transfected with lentiviral vectors carrying constitutively active FoxO1 (diabetes mellitus plus LV-FoxO1-AAA group,LV-CA group).Rats which received an injection of diluent buffer served as normal control.At 2,4,and 8 weeks after transfection,the levels of urine albumin,blood glucose,blood urea nitrogen,and serum creatinine were measured.Realtime PCR and Western blotting were performed to measure the mRNA and protein levels of FoxO1,COL4A3,COL4A5,and desmin in the renal cortex.Moreover,light microscope and electron microscope were used to observe the structural changes in glomerulus and podocytes.Compared with LV-NC and DM group,in LV-CA group,there was a significant increase in the mRNA and protein levels of FoxO1,and a distinct decrease in the levels of urine albumin,blood urea nitrogen and serum creatinine of rats (except at the twoweek time point) (all P＜0.05),the mRNA and protein levels of COL4A3,COL4A5,and desmin were all decreased (all P＜0.05),and pathological changes in kidney were also improved.Upregulating the expression of FoxO1 by transfecting with constructed lentiviral vectors can definitely improve the abnormal expression of type Ⅳ collagen and desmin in podocytes of diabetic rats.
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Objective To investigate the influence of Yishen granule on the expression of type Ⅳ collagen,laminin (LN)and fibroneetin (FN)in diabetic nephropathy (DN) rats.Methods DN model rats were established by unilateral nephrectomy and intraperltoneal injection of STZ.The experimental rats were divided into six groups:normal group,DN model group,Yishen granule groups in low and high dose (8.1 g/kg、16.2 g/kg) and positive control medicine group with lotensin (0.0015 g/kg).After 16 weeks of treatment,kidney tissues were sampled and pathologically observed through Masson trichrome staining.The proteins of type Ⅳ collagen、LN and FN were determined through immunohistochemical methods.Results The type Ⅳ collagen in normal group,DN model group,Yishen granule groups in low and high dose,and positive control medicine group was (1521.22 ± 415.26),(1579.22 ± 343.26),(3402.00 ± 863.39),(2984.30 ± 674.53),(2959.15 ± 561.22),(2918.04±363.96); LN was (1968.04±522.17),(2004.52±417.19),(3299.04±665.78),(3116.89±540.10),(2932.63 ± 528.38),(2815.89 ± 798.58) ; FN was (2614.67 ± 533.82),(2742.63 ± 562.80),(3311.41 ± 529.29),(2993.44±548.66),(2953.30±535.74),(2897.41 ±505.84) respectively.The level of type Ⅳ collagen、LN and FN expression in Yishen granule group obviously decreased as compared with that in DN model group (P＜0.05).The mesangial cells,basement membrane thickening and endocapillary proliferation in glomerular filtration membrane were significantly alleviated in Yishen granule group.The pathology of kidney was obviously modulated by Yishen granule.Conclusion Yishen granule can significantly inhibits the expression of type Ⅳ collagen、LN and FN in DN rats,which may be the mechanisms for Yishen granule in protecting the DN rat's kidney.
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Objective To analyze the correlation of urinary angiotensinogen (AGT) with clinical index of kidney injury and intrarenal renin-angiotensin system (RAS) activity in chronic kidney disease (CKD) patients. Methods Urinary or plasma renin activity, AGT, angiotensin Ⅱ (Ang Ⅱ ), aldosterone were measured by RIA or ELISA in 129 CKD patients. Expression of intrarenal renin, AGT, Ang Ⅱ and angiotensinⅡ receptor was examined by immunohistochemistry staining (IHCS) in 73 CKD patients undergoing renal biopsy. Correlation of urinary AGT with other indexes was performed. Results Average urinary AGT in 129 CKD patients was (159.08 ± 125.18) μg/g Cr, Scr was (113.20± 105.05)μmol/L, and urinary AGT was positively correlated with Scr (r=0.51, P<0.01). Average estimated glomerular filtration rate (eGFR) was (58.52±27.15) ml·min-1·(1.73 m2)-1, which was negatively correlated with urinary AGT (r=-0.55, P<0.01). Average urinary protein was (2.03±2.65) g/24 h, which was positively correlated with urinary AGT (r=0.30, P<0.01). Average urinary Ang Ⅱ was (164.71 ±139.25) ng/g Cr, which was positively correlated with urinary AGT (r=0.20, P<0.05). Average urinary type Ⅳ collagen was (447.60± 800.66) μg/g Cr, which was positively correlated with urinary AGT (r=0.47, P<0.01). Average urinary soduim was (162.17±81.61) mmol/24 h, which was negatively correlated with urinary AGT (r=-0.20, P<0.05). Multiple regression analysis indicated that low eGFR (P<0.01), high Scr (P< 0.01), high urinary protein (P<0.05), high urinary Ang Ⅱ (P<0.05) and high urinary type Ⅲ collagen (P<0.01) were significantly correlated with high urinary AGT. In renal tissues of CKD patients, there was positive correlation of urinary AGT with positive IHCS area of AGT (r=0.45, P< 0.01), Ang Ⅱ (r=0.52, P<0.01) and angiotensin Ⅱ type 1 receptor (r =0.28, P <0.05). Conclusions Urinary AGT level may indicate the kidney injury severity, especially in chronic kidney injury, and may be used as a non-invasive marker of intrarenal Ang Ⅱ activity in CKD patients.
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Alport syndrome (AS) is a hereditary glomerular basement membrane disease characterized by hematuria, progressive renal hypofunction, usually with nervous deafness and ocular lesions, and its pathogenesis is the mutation in genes encoding the type Ⅳ collagen. Diagnosis of AS should combine with clinical manifestations, renal pathologic changes, immunofluorescence examination and gene diagnosis. At present, AS hasn t had any cure measure yet. As the pathogenesis of AS is much clearer in recent years, the researches of gene diagnosis and gene therapy have got some gratifying achievements.
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Objective To study the clinical significance of urinary type Ⅳ collagen(IVC)and UmAlb/UCr ratio in the diagnosis of early diabetic nephropathy. Methods We collected 52 cases of diabetes(group A)without DN(UAE ＜30 mg/24 h),35 cases of diabetes(group B)with early DN(UAE as 30-300 mg/24 h),and 50 cases of healthy controls. The differences of urine IVC,UmAlb/UCr were compared among group A,B and the control group. ROC curve was used for evaluating the use of urine IVC and UmAlb/UCr in the diagnosis of early DN. The correlation of urine IVC and UmAlb/UCr with UAE were investigated,and linear model curve were established. IVC in urine was detected by chemiluminescence,UmAlb was detected by immunoturbidimetric assay,UCr was detected by enzymatic. Statistical analysis were performed with SPSS13.0 statistical software. Results The urine IVC testing of group A,B and the control group were(2. 64 ± 0. 91),(3.91 ± 1.93)and(10. 08 ± 6. 50)μg/L,respectively. The UmAlb/UCr(mg/mmol)testing of group A,B and the control group were(1.50 ± 0. 40),(2. 58 ±2. 10)and(17.95 ± 13. 38)mg/mmol,respectively. Urine IVC and UmAlb/UCr were significant difference between group A,B and the control group(Ps ＜ 0. 01);the ROC area under the curve(AUCROC)of urine IVC and UmAlb/UCr were 0. 724,0. 945,the two indicators for early diagnosis of DN were significant(Ps ＜ 0. 01);The pearson correlation coefficients of the urine IVC and UmAlb/UCr were 0. 529,0. 919 ,respectively. They were positive and significant correlation(Ps ＜ 0. 01),On the basis of the correlation coefficient and linear model fitting curve with the UAE,the relationship of UmAlb/UCr with the UAE was better than that of IVC. Conclusions Urine IVC and UmAlb/UCr ratio,which significantly assists diagnosis in diabetic nephropathy ,can be used as a sensitive diagnostic indicator of diabetic nephropathy. Moreover,the relationship of UmAlb/UCr with the UAE is better than that with IVC.
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Objective To determine an early and noninvasive predictor of chronic kidney diseaase(CKD),by detecting the correlation between the urinary Col-Ⅳ concentration,the renal tissue and renal lesions in patients with CKD Col-Ⅳ expression.Methods 98 CKD patients proved by renal biopsy were selected and 76 healthy people were enrolled as control.Urinary and serum Col-Ⅳ concentration were examined by enzyme-linked immunosorbent assay(ELISA).Renal Col-Ⅳ expression was detected by immunohistochemistry.Renal injury grade was semiquantitative by CMIAS image analysis system.The association between urinary Col-Ⅳ level and renal Col-Ⅳexpression,as well as renal lesion was analyzed,meanwhile the proteinuria and glomerular filtration rate(GFR)were analysed simultaneously.Results Urinary Col-Ⅳ level in patients with CKD was significantly higher than that in control,however,there was no difference in blood Col-Ⅳ level.Expression of Col-Ⅳ could be found in the sclerosing glomeruli and tubulointerstitial tissue,and Col-Ⅳ expression degree was consistent with lesion degree.Urinary Col-Ⅳ level was correlated positively with renal Col-Ⅳ expression,as well as with density of glomerular basement membrane and interstitial damage.Elevated urinary Col-Ⅳ concentration was observed in patients with mild renal sclerosis prior to the change of GFR.No correlation between urinary Col-Ⅳ concentration and proteinuria was observed.Conclusions These results suggested urinary Col-Ⅳ concentration reflected renal Col-Ⅳ expression and renal sclerosis,and could predict the progression of CKD.
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Objective To explore the early diagnosis of Alport′s syndrome(AS).Methods Renal and skin biopsy was carried out in 5 patients who manifested with isolated hematuria and nephritic syndrome(NS).By using indirect immunofluorescence method,the expression of type Ⅳ collagen ? chains was detected on epidermal basement membrane(EBM) and glomerular basement membrane(GBM).Results ?_1 chains on EBM and GBM were expression in all patients,but ?_5 chains on EBM and ?_3,?_5 chains on GBM form 2 female patients were segmental expression.Thus the goal for early diagnosis was achieved.Conclusions ? chains for EBM type Ⅳ collagen and GBM type Ⅳ collegan should be investigated if condition permits for those patients with isolated hematuria,NS(steroid-resistant) and thinned GBM in electron microscopy.It can be useful for diagnosis and differenial diagnosis of AS.
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Aim To investigate the effects of fluvastatin on the cellular proliferation and the expression of connective tissue growth factor(CTGF)and type Ⅳ collagen(Col Ⅳ)in rat mesangial cells(MCs)induced by transforming growth factor-?1(TGF-?1).Methods MCs are divided into the following groups according to different factors:control group,TGF-?1 group,TGF-?1 plus fluvastatin(Flu,different concentrations)groups.The influence of Flu on rat proliferation of MCs was detected by MTT.The expression of CTGF was measured with RT-PCR and Western blot respectively.The secretion of Col Ⅳ protein was quantitated by ELISA.Results 5 ?g?L-1 TGF-?1 could stimulate proliferation of MCs and the expression of CTGF.Col Ⅳ in MCs significantly.Flu could inhibit proliferation of MCs and the expression of CTGF,Col Ⅳ in MCs induced by TGF-?1 in a dose-dependent manner:1 ?mol?L-1 Flu could suppress proliferation of MCs and downregulate the expression of CTGF,Col Ⅳ significantly,while 10 ?mol?L-1 Flu could just suppress the expression of CTGF protein significantly.Simultaneously exogenous CTGF could promote the expression of Col Ⅳ in a dose-dependent manner:2.5 ?g?L-1 CTGF could just upregulate the expression of Col Ⅳ.Conclusion Fluvastatin could inhibit proliferation of MCs and expression of CTGF-mediated Col Ⅳ in MCs.
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Objective To investigate the relation between the expression of vascular endothelial growth factor (VEGF) and type Ⅳ collagen and the renal lesion of diabetes mellitus in diabetic rats. Methods Wistar rats were divided at random into normal control group(NC group) and diabetic model group(DM group), each group containing 20 animals. The rats models of diabetic mellitus were induced by streptozotocin(STZ). At the 8th week after induction, the expression levels of VEGF and type IV collagen in the kidney tissues in the two groups of rats were detected using immunohistochemistry. At the same time, the renal function of the rats was measured. Results At the 8th week,the expression levels of VEGF and type IV collagen of kidney tissues in DM group was much higher than those in NC group(P
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Objective To study the relationship between levels of hyaluronic acid(HA),type Ⅳ collagen(ⅣC),Laminin (LN) in sera and hepatic fibrosis in patients with chronic hepatitis B.Methods 252 sera from patients with chronic hepatitis B were collected to measure hyaluronate,type Ⅳ collagen,laminin by radioimmunoassay,and sera HBV-DNA were also tested by quantitative fluorescence PCR respectively.Results Serum concentrations of HA,ⅣC and LN were remarkably increased with the progression of the disease,and reached the highest levels in chronic severe hepatitis and liver cirrhosis(P
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Objective To investigate the effect of baicalain on high expression of ECM and TGF-?1 in proximal tubular epithelial cells cultured in high glucose concentration. Methods LLC-PK1 cells were divided into six groups: (1)normal glucose group(NG, 5. 5 mmol/L D-glucose), (2)high glucose group(HG, 25 mmol/L D-glucose), (3) HG + PKC inhibitor(10?mol/L chelerythrine chloride), (4)HG + baicalein(50 ?mol/L), (5) HG + baicalein(100 ?mol/L), (6) HG + baicalein (200 ?mol/L) . PKC activity was detected. Expression of ColⅣ, FN and TGF-?1 was examined by in situ hybridization and immunocytochemistry(ABC) techniques. Results At concentrations of 100 ?mol/L and 200 ?mol/L, baicalein decreased membranous PKC activity in LLC-PK1 cells by 42% and 68% , respectively ( P
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Objective To screen the mutations in 30 exons of COL4A5 gene from X-linked Alport syndrome. Methods 20 Chinese X-linked Alport syndrome patients from 16 families were examined. Genomic DNA was extracted from peripheral leukocyte and exon-specific primers were designed for 30 exons(1-25、31、 32、41、 50、51) . Polymerase chain reaction amplification was performed and followed bydegenerating gel gradient electrophoresis (DGGE) analysis. All abnormal migration bands were sequenced and one hundred normal persons selected as control. Results Four abnormal bands were detected, which were all point mutations and predicted to be functionally pathogenic, in four unrelated patients. One patienthad a nonsense mutation in exon 1 (Glu 22 Term); one had a missense mutation in exon 3l(Gly852Val).Two patients carried splicing mutations in intron 1 and 25 respectively(283+1G→T、2150 + 1G→T) .Conclusions X-linked Alport syndrome is caused by various kinds of COL4A5 gene mutations without any hotspot. Paralleled with the significance of exon mutations, intron mutations also play a critical role in the pathogenesis. Furthermore, these four pathogenic mutations have never been reported in the genebank and showed good correlation with clinical manifestations.
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Objective To investigate the roles of activin A(ACT-A) and follistatin(FS) in renal interstitial fibrosis.Methods Renal interstitial fibroblasts were isolated from SD rat kidney and primarily cultured.The cells were divided into A group,F group and A+F group,and then each group was further divided into 5 subgroups according to their culture media being added with ACT-A(0,0.3,3,30,or 100 ng/ml),FS(0,0.3,3,30,or 100 ng/ml),and ACT-A(30 ng/ml) plus FS(0,0.3,3,30,or 100 ng/ml) respectively.Levels of type Ⅳ collagen(Ⅳ-C) and hydroxyproline(HYP) in the supernatants of cultured fibroblasts were measured by ELISA assay.Results Concentrations of Ⅳ-C and HYP in cultured rat renal interstitial fibroblasts were in a dose-dependent manner with ACT-A treatment.FS had no effect on these concentrations,but it inhibited the effects of ACT-A on Ⅳ-C and HYP in the supernatants of cultured fibroblasts in a dose-dependent manner.Conclusion ACT-A might be involved in occurrence and development of renal fibrosis by affecting renal interstitial fibroblasts.Exogenous FS could suppress the effects of ACT-A on the cultured renal fibroblasts.