Clinical, Pathological and Genetic Analysis of Alport Syndrome in Children / 罕见病研究

  Objective  To explore the phenotype-genotype correlation of Alport syndrome in children.  Methods  Retrospectively analyze the clinical and pathological features of 55 patients with Alport syndrome with COL4A mutations detected by second-generation sequencing, who were treated at Beijing Children's Hospital from January 2016 to December 2020.  Results  A total of 55 children with Alport syndrome were included. All cases had hematuria, including 31 cases (56.4%) with gross hematuria and 24 cases (43.6%) with microscopic hematuria. A total of 39 (70.9%) patients also had proteinuria. Extrarenal manifestations were pres- ent in 12 patients (21.8%). 36(65.4%) patients had a family history of Alport syndrome. 32 patients underwent pathological examination and 23 of them had the specific pathological changes of Alport syndrome. In 55 cases, 36 (65.4%) were diagnosed as X-linked Alport syndrome, 5(9.1%) were diagnosed as autosomal recessive Alport syndrome, 10(18.2%) were diagnosed as autosomal dominate Alport syndrome, and 4(7.3%) were diagnosed as digenic Alport syndrome. Missense mutations in COL4A genes accounted for 62.5%, 67.5% of missense mutations resulted in glycine substitution. There were statistical significances in proteinuria degree and hearing loss between male and female patients with XLAS (P < 0.05) as well as statistical significance in the degree of proteinuria between autosomal recessive Alport syndrome and autosomal dominate Alport syndrome (P=0.044), and there was critical statistical significance in the age of onset. There was statistical significance in hearing loss between children with renal impairment and children with normal renal function (P=0.001).  Conclusions  Most of the pathogenic variants in COL4A genes that cause Alport syndrome result in glycine substitutions. The degree of proteinuria and hearing loss of males with XLAS were greater than those of females. The degree of proteinuria in autosomal recessive Alport syndrome was greater than that of children with autosomal dominate Alport syndrome, and the age of onset was earlier than that of autosomal dominate Alport syndrome. Renal manifestation was more severe in children with hearing loss. The early clinical manifestations of Alport syndrome are diverse and pathological manifestations may be atypical. The application of next-generation sequencing can reduce misdiagnosises of Alport syndrome.

Clinical significances of the synchronous detection of mucin 1 and collagen Ⅳ in breast cancer by using quantum dots immunofluorescence imaging technique / 肿瘤研究与临床

Objective@#To investigate the relationship between the expression of mucin 1 and collagen Ⅳ and the clinical characteristics in invasive breast cancer by using the quantum dots immunofluorescence imaging technique.@*Methods@#The expressions of mucin 1 and collagen Ⅳ were detected simultaneously by quantum dots immunofluorescence imaging technique in 94 cases of breast cancer from July 2007 to July 2010 in Affiliated Hospital of Guilin Medical University. The correlation of mucin 1 and collagen Ⅳ quantitative parameters with clinicopathological features and the prognosis were also analyzed.@*Results@#The positive rates of mucin 1 in human breast cancer tissues marked by quantum dots immunofluorescence imaging technique and immunohistochemistry were 73.4 % (69/94) and 69.1 % (65/94), the positive rates of collagen Ⅳ were 53.2 % (50/94) and 47.9 % (45/94), and the differences were not statistically significant (both P > 0.05) Quantum dots immunofluorescence imaging technique was consistent with conventional immunohistochemistry (IHC) in detecting the expressions of mucin 1 and collagen Ⅳ in human breast cancer tissues (κ = 0.763, P = 0.000; κ=0.759, P = 0.000). The expression of mucin 1 was negatively correlated with collagen Ⅳ (r = -0.883, P < 0.01). The expressions of mucin 1 and collagen Ⅳ were respectively associated with tumor size (F = 3.683, P = 0.029; F = 4.922, P = 0.009), histological grading (F = 3.529, P = 0.033; F = 3.912, P = 0.023), lymph node metastasis (t = -4.868, P = 0.000; t = 3.868, P = 0.000), pathological stage (t = -8.337, P = 0.000; t = 5.962, P = 0.000) and 5-year disease free survival rate (both P = 0.000), and the differences were statistically significant (all P < 0.05).@*Conclusion@#The co-detection of mucin 1 and collagen Ⅳ by using quantum dots immunofluorescence imaging technique provides direct evidence determining the biologic behaviors of breast cancer and evaluating the prognosis.

Emodin alleviated kidney damage in high fat diet induced obese mice / 中华内分泌代谢杂志

Objective To explore the protective effect of emodin on kidney damage in high-fat diet-induced obese mice. Methods Male C57bl/ 6 mice were divided into groups according to diet and treatment. Collagen Ⅳ(Col4), phosphatidylinositide 3-kinase(PI3K), phosphorylated phosphoinositol 3-kinase(p-PI3K), protein kinase (Akt), and phosphorylated protein kinase(p-Akt) were measured by Western blotting method. Results Col4 was increased, while p-PI3K and p-Akt were decreased in kidney tissue in high-fat diet-induced obese mice. However, there was lower level of Col4, but higher levels of p-PI3K and p-Akt in kidneys. Conclusion Kidney damage of high-fat diet-induced obese mice seems to be alleviated by emodin via improving insulin sensitivity.

Effects of centellaasiatica granule on the expression of Smad 2/3, Smad 7 and collagen Ⅳ in the mesangial cells stably expressed TGF-β1 / 中国应用生理学杂志

OBJECTIVES@#Stably expressed transforming growth factor -beta 1(TGF-β1)MCs were obtained and the effects of centellaasiatica (CA) granule on the expressions of Smad 2/3, Smad 7 and collagen Ⅳ and the level of Smad 2/3 phosphorylation were observed.@*METHODS@#Lipofectin method was used to transfect TGF-β1 vector into MC, and the stably expressed TGF-β1 cell lines were selected by G418. The cells were divided into three groups. Control group:normal MC + RPMI 1640 + 10% normal rat serum; TGF-β1 group:stably expressed TGF-β1 MC + RPMI 1640 + 10% normal rat serum; CA group:stably expressed TGF-β1 MC + RPMI 1640 + 10% rat serum containing high CA. The experiments were repeated for five times. The contents of TGF-β1 and collagen Ⅳ in the culture medium were detected with ELISA, the expressions of mRNA and protein of TGF-β1, Smad 2/3, Smad 7 and the level of Smad 2/3 phosphorylation were detected by using real time quantitative polymerase chain reaction and Western blot.@*RESULTS@#The contents of TGF-β1 and collagen Ⅳ in the culture medium of stably-expressed TGF-β1 MC were increased significantly, and the CA could reverse the effects of TGF-β1. The expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation were increased significantly in TGF-β1 transfected MC, and CA could dramatically reduce the expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation. The high expression of TGF-β1 decreased the expression of Smad 7 mRNA and protein, and the CA could antagonize the effect of mRNA expression.@*CONCLUSIONS@#The MCs stably-expressed TGF-β1 can activate the TGF-β1/Smad signal pathway and increase the expression of collagen Ⅳ. CA can decrease the occurrence of diabetic nephropathy(DN) by reducing the production of collagen Ⅳ through inhibiting the TGF-β1/Smad signal pathway.

Advances in Alport syndrome / 国际儿科学杂志

Alport syndrome is a rare genetic disorder of specialized basement membranes in the kidney,ear,and eye.Eighty percent of patients have X-linked disease caused by mutations in the COL4A5 gene;autosomal forms of the other Alport syndrome are caused by mutations in COI4A3 and COL4A4 genes.These mutations result in progressive irregular thickening,thinning,and splitting of the GBM,leading to end stage renal failure.During the past two decades,research into this rare disease has focused on the effects of mutations in collagen type Ⅳ α chains and the role of changes in podocytes and the glomerular basement membrane that lead to early kidney fibrosis.In this review,we discuss the latest basic and clinical research on Alport syndrome,focusing on the roles of podocyte pathology and the extracellular matrix.

Expression of FLi-1, h-caldesmon and collagen Ⅳ in glomus tumor: a clinicopathologic study / 临床与实验病理学杂志

Purpose To explore the expression of FLi-1,h-caldesmon and collagen Ⅳ in glomus tumor and possible relationship between different histological types and clinical-pathological characteristics.Methods Immunohistochemistry of EnVision method was used to detect the expression of FLi-1,h-caldesmon and collagen Ⅳ in 35 cases of glomus tumor under microscope,and the relationship between different histological types and clinical-pathological characteristics was analyzed.Results The pathological morphology of tumor cells was round or polygonal,patchy distribution in blood vessels or arranged in a ring around the blood vessels.The tumor cell boundary was clear and the shape was regular,gradual transition between tumor cells and spindle smooth muscle cells was sometimes visible.The positive rate of FLi-1,h-caldesmon and collagen Ⅳ in 35 cases of glomus tumor were 58.6％,97.1％ and 100％ respectively.Different histological types of glomus tumors had no correlation with the sex of patient and the size of tumor,but had correlation with the age of patient.Vimentin and SMA were positive for glomus tumors by immunohistochemistry in 35 cases.CD34 was positive in 2 cases.Desmin positive was found in 1 case.EMA,S-100,CgA,CD68 and CD99 were negative.Conclusion h-caldesmon and collagen Ⅳ are the markers for glomus tumor diagnosis,and FLi-1 can serve as a good auxiliary reference marker.

The relationship of expression of zinc, matrix metalloproteinases-9 and collagen Ⅳ levels and premature rupture of membranes at term / 中国综合临床

Objective To investigate the changes of zinc in maternal plasma and MMP-9,collagen Ⅳ levels in serum and matrix metalloproteinases-9 (MMP-9) level in amniotic fluid in patients with premature rupture of membranes at term(TPROM).Methods Thirty cases who were diagnosed as PROM at term during Nov.2012 to Mar.2013 were enrolled as case group,and 30 cases delivered during the same time without PROM were enrolled as control group.Maternal blood and ammiotic fluid were collected from all the cases.The level of zinc in maternal plasma was measured by atomic absorption method and the levels of MMP-9 in serum and amniotic fluid were detected by enzyme linked immunosorbent assay(ELISA),while the level of collagen Ⅳ in serum was measured by up-conversion luminescence method.The relationship among them was analyzed.Results Compared to control group,there were statistically significant difference between TPROM and control groups in terms of the level of zinc,collagen Ⅳ,MMP-9 in serum and MMP-9 in amniotic fluid (zinc:(109.10 ± 16.07) μmol/L vs.(90.54 ± 10.99) μmol/L; t =-5.22,P ＜ 0.001 ; collagen Ⅳ:(56.86 ±41.26) μg/L vs.(88.61 ±44.87) μg/L;t =2.852,P =0.006;MMP-9 in serum:(1 463.25 ±483.6) μg/L vs.(1 196.9 ± 357.43) μg/L,t =-2.426,P =0.018 ; MMP-9 in amniotic fluid:(125.48 ± 67.18) μg/L vs.(72.64 ± 60.74) μg/L,t =-2.873,P =0.006).Zinc level in maternal plasma and collagen Ⅳ in serum had a negative relationship in TPROM (r =-0.261,P =0.044).Zinc level in maternal plasma and MMP-9 level in serum had a positive relationship in TPROM (r =0.274,P =0.034).MMP-9 levels in serum and amniotic fluid had a positive relationship in TPROM (r =0.264,P =0.047).There were no significant relationship between zinc level in maternal plasma,MMP-9 level in amniotic fluid,collagen Ⅳ and MMP-9 levels in serum,collagen Ⅳ in serum and MMP-9 in amniotic fluid (r =0.215,-0.172,-0.172 ; P ＞ 0.05).Conclusion The level of zinc in maternal plasma and increase of MMP-9 in serum and amniotic fluid of women and decrease of the level of collagen Ⅳ in serum are related to the occurrence of TPROM.

Observation of extracellular matrix damage in the kidney of rats with multiple organ dysfunction syndrome / 中华急诊医学杂志

ObjectiveTo determine the levels of type Ⅳ collagen and matrix metalloproteinase-9 ( MMP-9 ) in the serum and kidney of rats with the multiple organ dysfunction syndrome ( MODS ) and investigate the mechanism of extracellular matrix damage in renal failure of MODS.MethodsForty adult male Sprague- Dawley (SD) rats were randomly (ramdom number) divided into two groups,namely the normal control group ( n =8 ) and the MODS model group ( n =32 ).The rats of model group were further divided into four sub-groups as per different intervals,6 h,12 h,24 h and 48 h,after modeling (n =8 in each).The animal models of MODS were established by two hits,the left eyeball of each model rat was removed to bleed to 2 ml bloocd/100 g body weight and lipopolysaccharide ( LPS,5 mg/kg) was injected into intraperitoneal cavity of model rats four hours later. The same volume of saline instead was injected intraperitoneally into rats of control group.All rats were sacrificed at different intervals.Creatinine (Cr)and blood urea nitrogen (BUN) were determined by using a Hitachi Automatic Biochemical Analyzer.The histological changes in renal tissue were observed under light microscope and transmission electronic microscope.The levels of serum type Ⅳ collagen and MMP - 9 protein were measured by using enzyme linked immunosorbent assay (ELISA).Type Ⅳ collagen and MMP- 9 protein levels in renal tissue were detected by western blot.One - way ANOVA was used for comparison among multiple groups.Results Compared with the control group,Cr and BUN were significantly higher in MODS group ( P ＜0.05 ).There were no histopathological changes in kidney of rats in control group,and the renal injury was serious in rats with MODS.The remarkable edema of basement membrane and defect of collagen fibers in renal tissue were observed in MODS group.Compared with the control group,the levels of MMP-9 in serum increased 6-48 hours after modeling and peaking at interval of 12 hours after modeling ( P ＜ 0.05 ).The protein levels of type Ⅳ collagen increased not significantly in 6 h group and 12 h group in comparison with control group (P ＞ 0.05 ),while those in 24 h group and 48 h group significantly increased ( P ＜ 0.01 ).The levels of MMP -9 in renal tissue of rats with MODS increased in 6 h group and 12 h group (P＜0.05),peaked in 24 h group ( P ＜ 0.05 ),and decreased in 48 hours.However,the level of Ⅳ collagen in serum of rats with MODS decreased significantly 6-48 hours after modeling (P ＜ 0.05 ) Conclusions The injury of extracellular matrix is an important factor to the kidney damage in MODS and it may he used for early diagnose and as a treatment target for kidney injury in MODS.

Pigment epithelium-derived factor inhibits high glucose-stimulated transforming growth factor β1,fibronectin, and collagen Ⅳ expressions in human mesangial cells / 中华内分泌代谢杂志

The expressions of transforming growth factor β1 (TGF-β1), fibronectin (FN), and collagen Ⅳ in human mesangial cells were augmented with increased concentrations of glucose.Pigment epithelium-derived factor (PEDF) at concentrations of 40-160 nmol/L significantly inhibited TGF-β1 expression in a dose-dependent manner(P＜0.01).PEDF at concentrations of 5-40 nmol/L significantly decreased FN and collagen Ⅳ expressions in a dose-dependent manner(P＜0.01) ,suggesting that PEDF may play a salutary role in diabetic nephropathy by its antifibrogenic activity.

Expression and significance of CD147 and collagen Ⅳ in gastric carcinoma / 肿瘤研究与临床

Objective To study the expression of CD147 and collagen Ⅳ protein in gastric carcinoma.Methods Immunohistochemical assay was used to detect the expression of CD147 and collagen Ⅳ protein in 119 cases of gastric carcinoma, 20 cases of normal gastric tissues, and the clinical data was analyzed.Results In 119 cases of gastric carcinoma, the positive expression of CD147 protein was 83.2 %(99/119), the positive expression of collagen Ⅳ protein was 31.9 %(38/119). The expression of CD147 and collagen Ⅳprotein had relationship with metastasis of lymph nodes,depth of invasion and clinical phases (P ＜0.05). There was negative significant correlation between the expression of CD147 and collagen Ⅳ protein in gastric carcinoma (P＜0.01). Conclusion The expressions of CD147 and collagen Ⅳ protein may be used as one of the marks to study gastric carcinoma.

Effects of Electroacupuncture on Serum Indexes of Rats with Hepatic Fibrosis / 针灸推拿医学（英文版）

Objective: To observe the effects of electroacupuncture on the histopathological changes and serum indexes of rats' liver, and to investigate the mechanism of electroacupuncture in treating hepatic fibrosis of rats. Methods: The rat model of hepatic fibrosis was induced with carbon tetrachloride. Then, the rats were divided into electroacupuncture group, medicine group, and model group. The collagen of liver, and serum hyaluronic acid (HA), laminin (LN), procollagen (PⅢNP), and collagen Ⅳ (C-Ⅳ) were detected with morphologic methods and radioimmunoassay. Results: Compared with normal rats, the collagen was hyperplasia in the liver tissue, and the contents of the serum HA, LN, and PⅢ NP were higher in the model group. The collagen area of liver tissue, and the contents of the serum HA, and LN were lower in rats treated with electroacupuncture than model rats. The contents of serum of PⅢ NP, and C-Ⅳ changed little. Conclusion: Electroacupuncture had some effects of prevention and treatment, and the mechanism may relate to the effects of electroacupuncture in protecting liver cells, inhibiting synthesis of extracellular matrix, and promoting the breakup of collagen.

Effects of Calcium Dobesilate on Glomerulus TIMP1 and Collagen Ⅳ of Diabetic Rats / 华中科技大学学报（医学）（英德文版）

To observe the effects of calcium dobesilate on the expression of glomerular tissue inhibitor of metalloproteinase 1 (TIMP1), collagen Ⅳ, and ultrastructure of glomerular basement membrane in diabetic rats, rats model of diabetes was established by unilateral nephrectomy and intraperitoneal injection of 1% STZ (55 mg/kg), and rats were administered calcium dobesilate 100 mg/kg (DD group) or distilled water (DM group) respectively. 12 weeks later, the changes in the renal ultrastructure and creatinine clearance rate (Ccr) were examined in each group. The expression of glomerular TIMP1 and collagen Ⅳ were studied by immunohistochemical staining. Our results showed that after 12 weeks, the Ccr in DD group increased and was significantly higher than that in DM group. Electron microscopy showed that thickness of glomerular capillary basement membrane (GBM) in Group DD was less than that of DM group. No hyperplasia of collagen fibers was found, and the distance between the holes of endothelial cells in DD group was not as even as that in the normal group, but more even than that of DM group, and podocyte processes was still in order. Immunohistochemical staining of glomeruli showed that expression of TIMP1 and collagen Ⅳ in DD group were significantly less than those of DM group DM. It is concluded that calcium dobesilate can improve diabetic nephropathy by inhibiting the over- accumulation of collagen Ⅳ and calcium dobesilate may also contribute to diabetes by inhibiting the expression of TIMP1.

DISTRIBUTION OF COLLAGEN Ⅳ IN KIDNEY OF IGA NEPHROPATHY AND ITS CORRELATION WITH RENAL PATHOLOGY / 解放军医学杂志

The distribution of collagen Ⅳ (CⅣ) and its correlation with renal histological lesions were investigated in 107 cases with IgA nephropathy. Strong positive staining of CⅣ and Fn was found in glumerruli with crescents, segmental and global sclerosis, as well as in the tubular basement membrane and interstitium. The extent of CⅣ deposit was correlated well with tubular atrophy, interstitium fibrosis, infiltration of cells and renal pathologic changes. The results show that the increment of CⅣ and Fn in glomeruli is associated with the development of crescents, glomerular sclerosis. The inflammation of interstitium may stimulate renal tubular cells to produce extravagent amounts of CⅣ, which might further result in tubular atrophy and fibrosis.

Study on the methods of qualitative diagnosis of laryngeal displasia and early carcinoma / 吉林大学学报(医学版)

Objective:To study the methods of early qualitative diagnosis of laryngeal displasia and earlycarcinoma. Methods:We investigated displasia (34 cases),squamous cell carcimoa (19 cases) and vocalcord polyps (17 cases) of larynx which were embeded in paraffin by PCR－SSCP analysis of p53 gene,im－munochemi stain of p53 protein and Ⅳ collagen,cytometer (determining fraction of cell cycle) and AgNORstain. Results :Eleven of 34 cases of displasia were all positive in the indexes. Seven of 11 cases were alsopositive in pathology,but others were negative. Five of 34 cases of displasia were all negative in the index-es,2 of 5 cases were positive and 1 of 5 cases were negative,other two cases did not have following-up re-sults in pathology. Conclusion :The results suggest that it is the combination of the morphological indexeswith biological indexe that is more scientific and more accurate in quatitative diagnosis of premalignantchange.

Effects of breviscapine on protein expression of c-fos,c-jun in glomerular mesangial cells cultured under high glucose conditions / 中国药理学通报

AIM To explore effects of protein kinase C (PKC) inhibitor breviscapine on protein expression of c fos?c jun and collagen Ⅳ(C Ⅳ) production in rat glomerular mesangial cells (GMC) cultured under high glucose conditions. METHODS Rat GMCs were cultured under normal glucose, high glucose or high glucose and breviscapine. After 24 h, 48 h or 1 wk, protein expression of c fos?c jun, C Ⅳ and PKC activity were measured. RESULTS Increased protein expression of c fos?c jun, C Ⅳ and PKC activity were observed under high glucose compared to normal glucose. All the above mentioned effects of high glucose were inhibited completely or partly by breviscapine. CONCLUSION High glucose may increase protein expression of c fos?c jun and C Ⅳ production mediated by PKC activation in cultured GMCs. PKC inhibitor breviscapine may ameliorate these abnormalities efficiently.

Effect of TIMP-1 on expression of FN and type Ⅳ collagen in rat glomerular mesangial cells / 中华肾脏病杂志

ObjectⅣe To investigate the effect of sense and antisense TIMP-1 on the expression of FN and type Ⅳ collagen of rat glomerular mesangial cells in vitro. Methods Two recombinant retroviral vectors, pLXIN-TIMP-1 (PLT)encoding sense TIMP-1 and pLXIN-ATIMP-1 (PLA)encoding antisense TIMP-1 were constructed by using DNA recombining techniques. These two vectors were then introduced into the PA317 packaging cell line with lipofectin DOTAP. The high-titer retroviral supematants containing sense or antisense TIMP-1 were used to infect rat glomerular mesangial cells. PCR and Northern blot were used to detect the integration and expression of human TIMP-1. Northern blot and ELISA were employed to investigate the expression of endogenous TIMP-1, FN, and Ⅳ collagen. Results Both sense and antisense TIMP-1 were successfully integrated into rat mesangial cells and expressed the sense and antisense ITMP-1 RNA. Overexpression of TIMP-1 induced by sense TIMP-1 caused upregulation of FN and Ⅳ type collagen in protein level, in contrast, supprssion of TIMP-1 expression by antisense TIMP-1 caused downregulation of FN and Ⅳ type collagen protein; but neither sense nor antisense TIMP-1 infection had effects on the RNA level of FN and Ⅳ type collagen. Conclusion TIMP-1 suppresses the degradation of ECM in rat glomerular mesangial cells and antisense TIMP-1 may be available for renal fibrosis in the future.

A method of assessing the thickness of myocardial basement membranes / 中国药理学通报

Aim To observe the thickness changes of cardiomyocyte and microvascular basement membranes(BM)and the expression of collagen Ⅳ in Gk rats of different months and STZ-induced diabetic rats,and to explore the method that operated easily and could assess the thickness of myocardial BM.Methods GK rats of the fourth,fifth,sixth,seventh months,Streptozotocin induced diabetes rats of fifth months and normal Wister rats were respectively divided into different groups with ev eight rats in each group.The myocardial section was stained by methenamine silver and immunohistochemistry was performed for collagen Ⅳ.Left ventricular endocardium was selected as measuring view.Through computer image analysis,the ratio of BM positive staining density(BMPSD)and positive staining density of collagen Ⅳ(CPSD)were calculated separately in all myocardium.Take the ratio to assess the thickness of myocardial BM and the expression of collagen Ⅳ.Results The glycemia,BMPSD and CPSD of the two diabetes rats were higher than those of the normal rats,having highly remarkable difference(P0.05).Conclusion By methenamine silver staining for myocardial section,calculating BMPSD through computer image analysis,can be an index to assess the thickness of myocardial BM.