Heparanase induces a differential loss of heparan sulphate domains in overt diabetic nephropathy

Aims/hypothesis Recent studies suggest that loss of heparan sulphate in the glomerular basement membrane (GBM) of the kidney with diabetic nephropathy is due to the increased production of heparanase, a heparan sulphate-degrading endoglycosidase. Our present study addresses whether heparan sulphate with different modifications is differentially reduced in the GBM and whether heparanase selectively cleaves heparan sulphate with different domain specificities. Methods The heparan sulphate content of renal biopsies (14 diabetic nephropathy, five normal) were analysed by immunofluorescence staining with four anti-heparan sulphate antibodies: JM403, a monoclonal antibody (mAb) recognising N-unsubstituted glucosamine residues; two phage display-derived single chain antibodies HS4C3 and EW3D10, defining sulphated heparan sulphate domains; and anti-K5 antibody, an mAb recognising unmodified heparan sulphate domains. Results We found that modified heparan sulphate domains (JM403, HS4C3 and EW3D10), but not unmodified domains (anti-K5) and agrin core protein were reduced in the GBM of kidneys from patients with diabetic nephropathy, compared with controls. Glomerular heparanase levels were increased in diabetic nephropathy kidneys and inversely correlated with the amounts of modified heparan sulphate domains. Increased heparanase production and loss of JM403 staining in the GBM correlated with the severity of proteinuria. Loss of modified heparan sulphate in the GBM as a result of degradation by heparanase was confirmed by heparan sulphate staining of heparanase-treated normal kidney biopsy specimens. Conclusions/interpretation Our data suggest that loss of modified heparan sulphate in the GBM is mediated by an increased heparanase presence and may play a role in the pathogenesis of diabetes-induced proteinuria. T. J. M. Wijnhoven and M. J. W. van den Hoven contributed equally to this study.     

Altered distribution of urinary glycosaminoglycans in diabetic subjects

We studied the influence of type 1 and type 2 diabetes mellitus with similar duration on the urinary excretion of total glycosaminoglycans and alteration of urinary glycosaminoglycan distribution pattern. Investigations were performed in the 24-hour urine samples of 31 children with type 1 diabetes mellitus, 36 adults with type 2 diabetes mellitus and in 30 age-matched controls for each group. We found that type 2 diabetes mellitus also induced an increased urinary excretion of total glycosaminoglycans and that both type 1 and type 2 diabetes alter the urinary distribution of heparan sulphate and dermatan sulphate. Observed changes correlate with duration of the disease. Microalbuminuria was detected in 9 of 36 type 2 adult diabetics (25%). The microalbuminic group had a significanlty higher heparan sulphate and/or dermatan sulphate excretion rate. To clarify whether an altered urinary distribution of heparan sulphate and dermatan sulphate may precede the development of microalbuminuria, it is necessary to performed a prospective study in which urinary glycosaminoglycans and microalbuminuria are measured year by year starting from the diagnosis of diabetes. Received: 23 August 2001 / Accepted in revised form: 3 April 2002     

肾小球基底膜中胶原的合成、降解与糖尿病肾病

糖尿病肾病是终末期肾病的主要原因,发病机制复杂.肾小球基底膜增厚及系膜区细胞外基质增生是主要的病理特点,胶原合成增多、降解减少是肾脏病理改变的主要原因.研究肾小球滤过屏障的生理功能与损伤机制,对深入了解糖尿病肾病的发生、发展尤为重要.     

Expression of glomerular extracellular matrix components in human diabetic nephropathy: decrease of heparan sulphate in the glomerular basement membrane

Summary Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against 1(IV)NC, 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-1(IV)NC no changes in GBM staining intensity were observed; with anti-3(IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions. No differences were observed for fibronectin although it was abundantly present in the mesangial area of biopsies from patients with diabetic nephropathy. In biopsies with mesangial expansion and in biopsies with diabetic nodules, we observed a decreased GBM (p = 0.001) HS side chain staining (JM403) without changes in HSPG-core protein staining (JM72,B31). The HS staining pattern regularly changed from a linear to a more granular and irregular pattern. In patients with a creatinine clearance of more than 15 ml/min, the intensity of GBM HS staining showed an inverse correlation with the rate of proteinuria (r = -0.85, p = 0.004), suggesting a functional relationship. The decreased HS staining in the GBM may reflect the potentially disrupted charge barrier in diabetic nephropathy.Abbreviations HS Heparan sulphate - GBM glomerular basement membrane - HSPG heparan sulphate proteoglycan - NC noncollagenous globular domain - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus     

生长因子与糖尿病肾病

糖尿病肾病是糖尿病常见的慢性并发症。目前生长因子的作用备受关注,主要包括结缔组织生长因子、转化生长因子β、生长激素/胰岛素样生长因子-1、血管内皮生长因子、血小板衍生因子等,它们通过引起肾小球基底膜的肥厚和细胞外基质沉积等,在糖尿病肾病的发展中起着关键作用,同时为探讨糖尿病肾病的治疗提供了新思路。     

炎性反应与糖尿病肾病

传统观点认为,代谢异常及血流动力学障碍是糖尿病肾病的主要原因.近来研究认为,天然免疫介导的慢性、低水平炎性反应在糖尿病肾病的发生及发展中起核心作用.细胞黏附分子、生长因子、趋化因子和促炎细胞因子在糖尿病患者肾组织的表达增多,且血清和尿中的细胞因子和黏附分子的水平与白蛋白尿相关.研究发现一些抗炎药物在糖尿病动物模型中有肾保护作用.因此深入了解糖尿病肾病的发病机制,并转化到临床开发相应的治疗策略,将可延缓甚至阻止糖尿病肾病的发生和发展.     

血清胱抑素C与糖尿病肾病

血清胱抑素C是一种半胱氨酸蛋白酶抑制剂,由机体所有有核细胞以恒定的速率产生,其相对分子质量小,可被肾小球自由滤过,在近端肾小管被代谢.血清胱抑素C作为一种内源性标志物,可以较早的反映肾小球滤过率的变化,故其在糖尿病肾病早期诊断中有重要的价值.     

糖尿病肾病并心肌损害的危险因素分析

目的 探讨糖尿病肾病（ＤＮ）并心肌损害的危险因素。方法 比较糖尿病肾病伴或不伴心肌损害的临床特征及其心肌损害的危险因素。结果 ＤＮ伴心肌损害组病程长（Ｐ〈０．０５）,空腹血糖及糖化血红蛋白高（Ｐ〈０．０１）,高血压发生率高（Ｐ〈０．０１）,且肾脏损害越重,死亡率越高,血ＢＵＮ水平和继发高血压与ＤＮ的心肌损害直接相关。ＤＮ与糖尿病性心肌病的发生时间无并行关系。结论 糖尿病病程长,血糖控制不佳,高血压     

Selective proteinuria in diabetic nephropathy in the rat is associated with a relative decrease in glomerular basement membrane heparan sulphate

Summary In the present study we investigated whether glomerular hyperfiltration and albuminuria in streptozotocin-induced diabetic nephropathy in male Wistar-Münich rats are associated with changes in the heparan sulphate content of the glomerular basement membrane. Rats with a diabetes mellitus duration of 8 months, treated with low doses of insulin, showed a significant increase in glomerular filtration rate (p<0.01) and effective renal plasma flow (p<0.05), without alterations in filtration fraction or mean arterial blood pressure. Diabetic rats developed progressive albuminuria (at 7 months, diabetic rats (D): 42±13 vs control rats (C): 0.5±0.2 mg/ 24 h, p<0.002) and a decrease of the selectivity index (clearance IgG/clearance albumin) of the proteinuria (at 7 months, D: 0.20±0.04 vs C: 0.39±0.17, p<0.05), suggesting loss of glomerular basement membrane charge. Light- and electron microscopy demonstrated a moderate increase of mesangial matrix and thickening of the glomerular basement membrane in the diabetic rats. Immunohistochemically an increase of laminin, collagen III and IV staining was observed in the mesangium and in the glomerular basement membrane, without alterations in glomerular basement membrane staining of heparan sulphate proteoglycan core protein or heparan sulphate. Giomerular basement membrane heparan sulphate content, quantitated in individual glomerular extracts by a new inhibition ELISA using a specific anti-glomerular basement membrane heparan sulphate monoclonal antibody (JM403), was not altered (median (range) D: 314 (152–941) vs C: 262 (244–467) ng heparan sulphate/mg glomerulus). However, the amount of glomerular 4-hydroxyproline, as a measure for collagen content, was significantly increased (D: 1665 (712–2014) vs C: 672 (515–1208) ng/mg glomerulus, p<0.01). Consequently, a significant decrease of the heparan sulphate/4-hydroxyproline ratio (D: 0.21 (0.14–1.16) vs C: 0.39 (0.30–0.47), p<0.05) was found. In summary, we demonstrate that in streptozotocin-diabetic rats glomerular hyperfiltration and a progressive, selective proteinuria are associated with a relative decrease of glomerular basement membrane heparan sulphate. Functionally, a diminished heparan sulphate-associated charge density within the glomerular basement membrane might explain the selective proteinuria in the diabetic rats.Abbreviations BW Body weight - ERPF effective renal plasma flow - GAG glycosaminoglycan - GBM glomerular basement membrane - GFR glomerular filtration rate - HS heparan sulphate - HSPG heparan sulphate proteoglycan - IDDM insulin-dependent diabetes mellitus - STZ streptozotocin     

糖尿病肾病诊断标志物研究进展

糖尿病肾病(DN)是造成终末期肾病的最常见原因,也是糖尿病患者发病率和死亡率显著增加的主要因素.早期诊断对DN的管理非常重要.尿微量白蛋白被认为是诊断早期DN的金标准,但具有一定的局限性.近年来发现了一些尿液中反映肾脏受损的新的标志物,包括肾小球损伤标志物(肾病蛋白、足细胞标记蛋白、肾母细胞瘤1基因),肾小管损伤标志物(中性粒细胞明胶酶相关载脂蛋白、肾脏损伤分子1)和炎性因子(肿瘤坏死因子受体)等.同时一些较新的方法如尿非编码RNA(微小RNA)及尿蛋白组学检测为诊断DN提供了新策略.     

IgA肾病伴肾小球基底膜虫蚀状改变的临床病理分析

目的了解IgA肾病伴肾小球基底膜(GBM)虫蚀状改变的临床及病理特点。方法通过电镜观察GBM的形态,将1997-01～2003-12间北京大学第一医院的756例原发性IgA肾病患者分为IgA肾病伴GBM虫蚀状改变组(共24例)及IgA肾病不伴GBM虫蚀状改变组(随机选取101例),观察并比较两组患者的临床及病理特点。结果GBM虫蚀状改变在原发性IgA肾病中的发生率为3.1%,患者临床上表现为更严重的镜下血尿(P=0.013)和更为多量的蛋白尿[(3.5±2.5)g/d对(2.1±2.4)g/d,P=0.011];病理上表现为更高的球性硬化比例(62.5%对49.5%,P=0.048)。结论本研究国内首次报告了原发性IgA肾病伴GBM虫蚀状改变的患者临床和病理改变均较重,但GBM虫蚀状改变对于IgA肾病患者的病程及预后的影响尚需扩大样本量和长期随访进一步探讨。     

Detailed glomerular ultrastructure in Japanese type 2 diabetic patients by the quick-freezing and deep-etching method

Mesangial expansion and glomerular basement membrane (GBM) thickening did not correlate with urinary albumin excretion (UAE) in type 2 diabetic patients in our previous studies; therefore, it was necessary to elucidate more detailed ultrastructural changes in the early stages of diabetic nephropathy (DN) in type 2 diabetic patients. The quick-freezing and deep-etching (QF–DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. The QF–DE method was applied to six type 2 diabetic patients without definable renal diseases other than DN. Four patients were normoalbuminuric (NA) and the other two were microalbuminuria (MA). Three control specimens were the normal parts from nephrectomies due to renal cell carcinomas. Electron microscopic morphometric analyses provided quantitative glomerular structural changes. Replica membranes were prepared by the QF–DE method, and diameters of mesh structures at the GBM and mesangial matrix (MM) were measured on electron micrographs as previously described. By the QF–DE method, both the GBM middle layer and MM were composed of polygonal meshwork structures. The mesh pores of the GBM and MM were more enlarged and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The mesh diameters of the GBM and MM in the diabetic patients were also larger than those of the controls. Such a mesh diameter of the GBM was well correlated with the amount of UAE, while the mesh diameter of MM showed a slight correlation with UAE. Although there were small number of subjects in the present study, the detailed ultrastructural changes in NA and MA type 2 diabetic patients, which had not been disclosed by conventional electron microscopy, were revealed by the QF–DE method. Increased mesh diameters of GBM might be related with the increase of UAE.     

79例糖尿病肾病五年随访分析

目的分析影响糖尿病肾病0DN)五年肾存活率的主要因素.方法调查了79例DN的临床、实验室及治疗有关资料,对各项临床参数进行单因素及多因素相关分析.结果蛋白尿程度较重0≥1.5g/24h),血肌酐升高0≥120umol/L),内生肌酐清除率下降0＜50ml/min)及血糖和血压控制不良对五年肾存活具有不良影响.结论严格控制血压,采取有效措施减轻肾脏受损程度,并及早控制血糖是提高肾存活率的关键.     

糖尿病肾病与颈动脉内膜中层厚度相关性研究

探讨糖尿病肾病的进展与大动脉硬化的关系.结果 发现糖尿病患者颈动脉内膜中层厚度 (CIMT)与尿白蛋白/肌酐显著正相关,与肾小球滤过率显著负相关.提示有微量白蛋白尿的糖尿病患者,CIMT的增厚预示其糖尿病临床肾病发生的风险增加.     

糖尿病肾病相关因素分析

目的探讨糖尿病肾病（DN）的相关因素,为DN的一级预防提供理论依据。方法选择2型糖尿病（T2DM）患者267例,其中DN患者102例（DN组）,单纯糖尿病无。肾病165例（NDN组）,采集血糖等临床资料,进行t检验、x2检验和Logistic回归分析。结果组间比较显示,DN组与NDN组的年龄、糖尿病（DM）病程、BMI、吸烟、高脂饮食、合并高血压、FPG、2hPG、0．5hPG、HbA1c、TC、TG、LDL-C和纤维蛋白原（Fib）差异有统计学意义（P〈0．05或P〈0．01）。多元Logistic回归分析中,DN的发生与DM病程、TG、HbA-C、FPG、2hPG和合并高血压有关（P〈0．05或P〈0．01）。结论TG、DM病程、HbA1C、FPG、2hPG和合并高血压是DN发生的独立相关因素。     

炎性因子与糖尿病肾病

糖尿病肾病是糖尿病的一个重要的微血管并发症，近年来已经成为导致。肾功能不全的一个主要原因。许多基础和临床研究证明多种炎性因子都参与糖尿病肾病的发生，其中包括趋化因子、黏附分子和前炎性细胞因子。这也为糖尿病肾病的治疗提供了新思路。     

NADPH氧化酶在糖尿病肾病中的作用

还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶是许多非吞噬细胞中活性氧簇（ROS）产生的主要来源。在高血糖、高血脂、血管紧张素Ⅱ及各种细胞因子、生长因子的作用下,NADPH氧化酶被激活,发生呼吸链级联反应,产生过量的ROS,参与氧化应激,并作为氧化应激的中心环节,通过形成晚期糖基化终末产物（AGE）、激活蛋白激酶C（PKC）、转录因子如核因子（NF）-κB等途径,改变肾脏血液动力学,影响细胞外基质的重构,激活细胞内信号转导等,促进糖尿病肾病的发生发展。调节NADPH氧化酶的活性以抑制肾脏的氧化应激,有望成为延缓肾功能损害的有效治疗措施。     

Hypotensive therapy reduces microvascular albumin leakage in insulin-dependent diabetic patients with nephropathy

The effect of hypotensive therapy on the transcapillary escape rate of albumin (TERalb) was studied in eight hypertensive insulin-dependent diabetic patients (mean age 29, range 19-42 years) with nephropathy and retinopathy. Transcapillary escape rate of albumin (initial disappearance of intravenously injected 125I-labelled human serum albumin), urinary albumin excretion rate (radial immunodiffusion), and glomerular filtrate rate (single bolus 51-Cr-EDTA technique) were measured. After hypotensive treatment (mean duration, 23 months, range 7-39 months) with combinations of metoprolol, hydralazine, and frusemide or thiazide diuretics, arterial blood pressure fell from 152/103 +/- 18/6 mmHg (mean +/- SD) to 133/81 +/- 12/10 mmHg (p less than 0.01), transcapillary escape rate of albumin from 10.2 +/- 1.8 to 8.1 +/- 1.8% of intravascular mass of albumin/h (p less than 0.01), albuminuria from 1803 (370-5066) micrograms/min to 940 (101-2676) micrograms/min (median and range, p less than 0.05), and glomerular filtration rate from 103 +/- 23 to 84 +/- 22 ml/min/1.73 m2 (p less than 0.01). Our study suggests that effective hypotensive treatment reduces the abnormally elevated albumin leakage characteristically found in insulin-dependent diabetic patients with clinical microangiopathy. This may be due to a reduction in the hydrostatic pressure in the microcirculation.     

Nephropathy in Type 1 Diabetes: the Role of Genetic Factors

Diabetic nephropathy affects up to 30% of all patients with Type 1 (insulin-dependent) diabetes and is associated with a high morbidity and mortality. A number of studies have suggested that, unlike retinopathy or neuropathy, the influence of hereditary factors on the development of nephropathy is strong. Much interest has focused on possible genetic markers indicating an increased risk for developing diabetic nephropathy. It is envisaged that patients with Type 1 diabetes may be screened at diagnosis for increased susceptibility to nephropathy and subsequently have intensified follow up and possibly even prophylactic therapy in order to prevent progression to nephropathy. Two groups of candidate genes have so far been of particular interest: those implicated in the aetiology of hypertension, and those involved in the metabolism of glomerular basement membrane proteins. This article aims to review the evidence suggesting a role for hereditary factors, possible genetic models, and the genetic loci thought to be involved.     

1059例2型糖尿病人糖尿病肾病患病率及其相关危险因素

目的：调查2型糖尿病病人糖尿病肾病患病率,并分析其相关危险因素,方法：对中山医院1997－2000年间收治的1059例糖尿病病人进行临床分析.结果：1053例2型糖尿病病人中糖尿病肾病的患病率为9．73％,终末期肾病的患病率为5．47％,高血压和以高甘油三酯,低HDL血症为特征的脂代谢紊乱是糖尿病肾病的重要危险因素。单次的HbAlc测定值与微量蛋白尿排泄率（UAER）水平间无显著相关,结论;本研究所得的糖尿病肾病患病率较国外报道稍低,而危险因素与文献报道相似,当前大多数高危患者尚未进入临床蛋白尿期时,正是积极开展糖尿病肾病早期防治的有利时机。