金属蛋白酶-2和金属蛋白酶组织抑制物-1与糖尿病肾病关系的实验研究

目的　观察链脲佐菌素 (STZ)实验性糖尿病大鼠肾脏基质金属蛋白酶 2 (MMP 2 )及金属蛋白酶组织抑制物 1(TIMP 1)蛋白的表达及功能、形态学改变 ,探讨MMP 2、TIMP 1在糖尿病肾病(DN)发生机制中的意义。　方法　 2 0只雄性Wistar大鼠随机分为糖尿病组和正常对照组 ,分别于第1、2、4、6、8周测定尿白蛋白排泄率 (UAER) ,第 9周用Western印迹方法检测MMP 2、TIMP 1蛋白表达水平 ,电镜观察肾小球基底膜厚度 (GBMT)。　结果　糖尿病组较正常组TIMP 1蛋白表达明显增加 ,MMP 2蛋白表达显著降低 (P <0 .0 1)。 4、6、8周UAER显著增加 ,GBMT明显增厚 (P <0 .0 1)。电镜发现糖尿病组肾小球基底膜弥慢性增厚 ,局部有系膜细胞插入和双轨征。　结论　持续高血糖可使大鼠肾脏MMP 2下降 ,TIMP 1增加。导致肾小球基底膜增厚 ,UAER增加。MMP 2、TIMP 1失衡可能对糖尿病肾病功能和形态学改变有重要意义。     

基质金属蛋白酶在类似人类2型糖尿病大鼠肾病中的研究

目的 动态研究基质金属蛋白酶－2（MMP－2）和其体内特异的抑制物－金属蛋白酶组织抑制剂－2（TIMP－2）在类似人类2型糖尿病大鼠肾组织的改变。方法 利用免疫组织化学观察Ⅳ型胶原在肾小球的表达,酶谱法测定肾皮质MMP－2的活性,蛋白印迹法检测肾皮质TIMP－2的含量,Northern Blot观察肾皮质MMP－2和TIMP－2的基因表达。结果 类似人类2型糖尿病大鼠肾小球Ⅳ型胶原成分表达增强,肾皮质MMP－2基因表达减弱（P＜0．01）;其活性进行性减少（P＜0．0001）,而TIMP－2基因表达（P＜0．01）及其含量逐渐增加（P＜0．01）。结论 类似人类2型糖尿病大鼠肾组织MMP－2活性进行性降低、TIMP－2含量增加,两者比例失衡为肾小球ECM成份沉积的重要原因之一,基质降解减弱也参与了2型糖尿病大鼠肾脏病变的发生和发展。     

虫草菌丝对糖尿病大鼠肾组织中基质金属蛋白酶2及其组织抑制因子2表达的影响

目的 观察虫草菌丝(CS)对糖尿病(DM)大鼠肾脏病理及肾组织中基质金属蛋白酶(MMP)-2和基质金属蛋白酶组织抑制因子(TIMP)-2表达的影响,探讨CS的肾脏保护作用及其可能机制.方法 应用链脲佐菌素(STZ)诱导DM大鼠模型,随机分为DM模型组、CS干预组1、2.同时设阴性对照组.干预组1成模后即予CS 5.0 g·kg-1·d-1进行干预,干预组2在成模4 w后开始予等量CS.8 w后测定各组血糖、血尿素氮(BUN)、血肌酐 (Scr)、24 h尿蛋白定量(Upro)等变化.光镜观察肾组织病理变化.用免疫组化法检测肾组织MMP-2、TIMP-2、Ⅳ胶原(Col Ⅳ)蛋白的表达.结果 与阴性对照组相比,其他3组大鼠血糖、BUN、Scr、Upro均显著上升(均P＜0.01);肾组织中MMP-2蛋白表达降低(P＜0.01),TIMP-2蛋白表达升高,Col Ⅳ表达亦增加(P＜0.01).与DM模型组相比,CS干预后,上述异常指标除血糖外均有明显改善(P＜0.05或P＜0.01),除血糖及BUN外干预组1优于干预组2(P＜0.05或P＜0.01).结论 CS对DM大鼠具有保护肾脏组织、改善肾脏功能的作用.其作用机制可能与上调肾组织MMP-2的表达和下调TIMP-2的表达,从而减轻肾小球细胞外基质沉积有关;且治疗越早,疗效越好.     

补脏通络方对糖尿病大鼠肾组织Ⅳ型胶原、转化生长因子-β表达及基质金属蛋白酶-9/金属蛋白酶抑制剂-1平衡的影响

目的观察补脏通络方对糖尿病(DM)大鼠肾组织Ⅳ型胶原(Ⅳ-C)、转化生长因子(TGF)-β表达及基质金属蛋白酶(MMP)-9/金属蛋白酶抑制剂(TIMP)-1平衡的影响。方法大鼠以脂肪乳灌胃21 d后一次性尾静脉注射链脲佐菌素(STZ)30 mg/kg造模,后予补脏通络方治疗。于第8周末取左肾,测算肾重指数;SABC-FITC免疫组织化学荧光法检测肾组织Ⅳ-C、TGF-β表达;免疫印迹法分析肾组织MMP-9、TIMP-1蛋白含量。结果病理组较正常组肾重指数增加(P<0.01),各治疗组较病理组肾重指数降低(P<0.01)。病理组肾小球体积增大,系膜区扩大病变经治疗后均有不同程度改善。病理组较正常组,肾组织Ⅳ-C、TGF-β、TIMP-1表达增高(P<0.01),MMP-9表达和MMP-9/TIMP-1比值下降(P<0.01);各治疗组较病理组,肾组织Ⅳ-C、TGF-β、TIMP-1表达下降(P<0.01),MMP-9表达和MMP-9/TIMP-1比值上升(P<0.01);中、高剂量组降低TIMP-1表达,升高MMP-9/TIMP-1比值的作用优于低剂量组(P<0.01)。结论下调肾组织TGF-β表达,调节MMP-9/TIMP-1平衡,促进胶原降解,可能是补脏通络方保护DM肾脏作用的机制之一。     

吡格列酮对糖尿病大鼠肾组织基质金属蛋白酶2和9表达的影响

免疫组化法研究吡格列酮对糖尿病大鼠肾脏基质金属蛋白酶 2、9(MMP 2、9)和Ⅳ型胶原 (C Ⅳ )表达的影响。结果提示 ,吡格列酮治疗可逆转糖尿病大鼠MMP 2的下降 ,降低C Ⅳ的表达 ,从而可能延缓肾纤维化的进展。     

L-158809对2型糖尿病大鼠模型肾脏保护的机制探讨

目的：观察血管紧张素Ⅱ（ATⅡ）受体拮抗剂L－158809对2型糖尿病大鼠模型肾脏的保护作用,并探讨其作用机制。方法：2型糖尿病大鼠在L－158809治疗16周后,测定肾组织ATⅡ含量,利用组织学检查观察肾组织结构的改变,免疫组化观察基质金属蛋白酶2（MMP－2）及其金属蛋白酶2组织抑制剂（TIMP－2）在肾小球的表达,酶谱法测定肾皮质MMP－2的活性,Western印迹检测肾皮质TIMP－2的含量,结果：L－158809治疗使糖尿病大鼠血浆和肾皮质ATⅡ浓度进一步升高,肾小球基质增生明显减轻,肾小球MMP－2染色强度和肾皮质MMP－2的活性增强,但不能使肾小球TIMP－2染色及肾皮质TIMP－2含量降至正常。结论：L－158809对类似人类2糖尿病大鼠肾脏病变有保护作用,对肾脏细胞外基质沉积的改善部分是通过促进基质转化而实现的。     

罗格列酮对糖尿病大鼠肾组织TGF-β1、MMP-2及Col-Ⅳ的影响

目的研究罗格列酮早期干预对STZ诱导的糖尿病大鼠肾皮质TGF-β1、MMP-2及Col-Ⅳ表达影响。方法40只雄性SD大鼠随机分为正常对照组(C组)、糖尿病组(DM组,链脲佐菌素60mg/kg腹腔注射)、糖尿病罗格列酮治疗组(DR组,4mg·kg-1·d-1罗格列酮无菌水混悬液灌胃),10w后观察大鼠HbA1c,肾功能、肾指数、尿微量白蛋白及肾病理学变化,透射电镜观察肾超微结构改变,免疫组化法观察肾皮质TGF-β1、MMP-2及Col-Ⅳ表达变化。结果DM组尿素氮、肾指数、尿微量白蛋白及肾皮质TGF-β1、Col-Ⅳ表达显著高于C组(P<0·01),DR组较DM组明显减低(P<0·01);同时观察到DM组、DR组MMP-2较C组减低(P<0·01),而DR组较DM组显著升高(P<0·01);透射电镜观察DM组肾小球基底膜增厚、肾小球系膜细胞增生,经罗格列酮治疗后的DR组上述改变明显减轻。结论罗格列酮对糖尿病大鼠具有肾脏保护作用,其部分机制是通过下调肾皮质中TGF-β1、Col-Ⅳ蛋白表达,上调MMP-2表达。     

基质金属蛋白酶/组织金属蛋白酶抑制物表达失衡在大鼠肾脏衰老过程中的意义

目的:探讨基质金属蛋白酶(MMPs)及其抑制物(TIMPs)在大鼠肾脏衰老过程中的作用。方法:选用3、12和24月龄大鼠，采用免疫组化技术分别检测基质金属蛋白酶—2、9(MMP—2、9)、组织金属蛋白酶抑制物—1、2(TIMP—1、2)、转化生长因子—β1(TGF—β1)等在不同月龄大鼠肾组织中的表达。结果TIMP—1、TIMP—2及TGF—β1主要表达在肾小球、肾小管、间质及血管，并随增龄表达增强(P＜O．01)；MMP—9、MMP—2主要表达在肾小管上皮细胞，随增龄表达无变化；TIMP—1与TGF—β1与肾小球硬化面积有相关性(r分别为O．751、O．771，P＜O．05)；TIMP—1与TIMP—2与小管间质纤维化面积有相关性(r分别为O．783、O．766，P＜O．O5)。结论:MMPs／TIMPs表达失衡在肾脏衰老过程中可能起重要作用。     

解聚复肾宁对糖尿病大鼠肾组织基质金属蛋白酶-9、金属蛋白酶组织抑制剂-1表达的影响

目的观察解聚复肾宁(JJFSN)对糖尿病(DM)大鼠肾组织基质金属蛋白酶-9(MMP-9)、金属蛋白酶组织抑制剂-1(TIMP-1)表达及肾脏保护作用机制。方法建立STZ诱导的DM SD大鼠模型,将成模DM大鼠随机分成4组:模型组、JJFSN组、厄贝沙坦组、JJFSN+厄贝沙坦组,同时设正常对照组。各组大鼠采用相应的干预措施处理12 w。检测各组大鼠第12周时肾重/体重、血糖、尿素氮、血肌酐、尿白蛋白排泄率(UAER),免疫组化法检测肾组织MMP-9、TIMP-1表达,透射电镜观察肾脏超微结构。结果模型组肾脏超微结构改变明显,血糖、UAER、尿素氮、血肌酐、肾重/体重显著增高。模型组大鼠肾组织TIMP-1的表达较正常对照组明显上调(P<0.01),JJFSN组、厄贝沙坦组、JJFSN+厄贝沙坦组肾组织TIMP-1表达明显下调(P<0.01),但仍高于正常对照组(P<0.01),JJFSN+厄贝沙坦组下调最明显(P<0.01)。DM模型组大鼠肾组织MMP-9的表达较正常对照组明显下调(P<0.01),解聚复肾宁组、厄贝沙坦组、解聚复肾宁+厄贝沙坦组肾组织MMP-9表达明显上调(P<0.01),但仍低于正常对照组(P<0.01),解聚复肾宁+厄贝沙坦组上调最明显(P<0.01)。结论 MMP-9、TIMP-l的表达变化与肾小球细胞外基质(ECM)降解减少相关,可能促进了糖尿病肾病(DN)的发生,JJFSN可能通过干预这种表达变化,减缓DN的发生和发展。     

重组人肝再生增强因子对5/6肾切除大鼠肾组织MMP-9、TIMP-1及Collgen-Ⅳ表达的影响

目的探讨重组人肝再生增强因子(rhALR)是否可以通过影响基质金属蛋白酶9(MMP-9)、基质金属蛋白酶组织抑制因子1(TIMP-1)及Ⅳ型胶原(Collgen-Ⅳ)的表达,来延缓肾小球硬化。方法雄性SD大鼠分为假手术组、对照组及rhALR组,以rhALR对5/6肾切除所致慢性肾衰竭大鼠进行干预。结果与假手术组比较,对照组肾小球硬化指数增大,Collgen-Ⅳ、TIMP-1表达增加,MMP-9表达减少(P均<0.05);外源性给予rhALR能降低TIMP-1表达,增加MMP-9表达,减轻Collgen-Ⅳ的沉积,改善肾小球硬化(P均<0.05)。结论 rhALR可通过上调MMP-9/TIMP-1比例减少Collgen-Ⅳ积聚而改善肾小球硬化,保护残肾功能。     

2型糖尿病大鼠心肌组织MMP-2、MMP-9、TIMP-1的表达与胶原代谢的关系

目的 了解2型糖尿病（T2DM）大鼠模型心肌组织基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）及其组织抑制物1（TIMP-1）的mRNA表达变化、蛋白水平及组织定位，探讨MMPs／TIMPs在T2DM心肌病变发生发展中的作用。方法 Masson染色观察心肌胶原含量变化；RT-PCR方法观察心肌MMP-、MMP-9和TIMP-1mRNA表达；免疫组化方法测定MMP-2、MMP-9、TIMP-1的蛋白表达和组织定位。结果 T2DM大鼠心肌胶原纤维明显增多；MMP-2mRNA表达明显下调，蛋白水平下降；MMP-9、TIMP-1的mRNA表达均增加，MMP-9、TIMP-1的蛋白水平也升高，但MMP-9／TIMP-1的比值下降。结论 T2DM大鼠心肌组织胶原聚集，心肌纤维化。MMPs／TIMP-1比例失衡可能与T2DM心肌病变的发生有关。     

Extracellular matrix in human diabetic nephropathy: reduced expression of heparan sulphate in skin basement membrane

Summary In diabetic nephropathy, expression of glycosaminoglycan side chains of heparan sulphate proteoglycan in the glomerular basement membrane is reduced proportionally to the degree of proteinuria. We performed a cross-sectional study to evaluate whether non-vascular basement membranes also show a decrease in heparan sulphate side chain staining in patients with diabetic nephropathy. We evaluated the skin basement membrane for extracellular matrix components in the following groups: control subjects (n = 16); patients with Type 1 diabetes and normoalbuminuria (n = 17), microalbuminuria (n = 7), and macroalbuminuria (n = 16); patients with Type 1 diabetes and diabetic nephropathy undergoing renal replacement therapy (n = 13); and non-diabetic patients undergoing renal replacement therapy (n = 21). The following antibodies were used for this immunohistochemical study: monoclonal antibodies against the heparan sulphate side chain (JM403) and core protein (JM72) of the glomerular heparan sulphate proteoglycan; polyclonal antibodies against the core protein (B31); polyclonal antibodies against collagen types I, III, and IV, fibronectin, and laminin; and monoclonal antibodies against the non-collagenous domain of α1(collagen IV) and α3(collagen IV), against transforming growth factor β(2G7), and against advanced glycosylation end products (4G9). Expression of heparan sulphate side chains was reduced in the skin basement membrane of patients with overt diabetic nephropathy, of those with Type 1 diabetes undergoing renal replacement therapy, and those with non-diabetic renal failure. Increased intensity of staining was found for collagen type I and advanced glycosylation end products in patients with diabetic nephropathy. Changes in the extracellular matrix of the skin basement membrane seem to be similar to those in the glomerular basement membrane. These findings support the suggestion that patients with diabetic nephropathy also have altered heparan sulphate and collagen staining in extrarenal basement membranes. However, patients with non-diabetic renal failure also had reduced expression of heparan sulphate in the skin basement membrane, suggesting that this finding is not specific for diabetic nephropathy. [Diabetologia (1998) 41: 791–798] Received: 13 November 1997 and in revised form: 10 February 1998     

Long-term overexpression of membrane type-1 matrix metalloproteinase and matrix metalloproteinase-2 in oleic acid-induced pancreatitis in rats

INTRODUCTION: The matrix metalloproteinase (MMP) plays important roles in extracellular matrix turnover. However, little is known about the roles of MMP-2 and type IV collagen, and the relationship between MMP-2 and membrane type-1 matrix metalloproteinase (MT1-MMP) during progressive destruction of acinar cells in pancreatitis. AIMS AND METHODOLOGY: To examine the serial changes in the expression and activity of MMP-2 and expression of MT1-MMP and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in rats after induction of pancreatitis by intraductal infusion of oleic acid, and to determine protein concentrations by Western blot analysis and localization of type IV collagen by immunostaining. RESULTS: Gelatinolytic activity of pro-and active MMP-2 and concentrations of MT1-MMP protein, as determined by zymography and Western blot analysis, respectively, increased significantly from 6 hours to day 42 after intraductal infusion of oleic acid. TIMP-2 mRNA expression was significantly higher than that at time 0 throughout the study period, and gelatinolytic activity of active MMP-2 increased from day 3 to day 42. In addition, immunoreactivity for type IV collagen was detected as a discontinuous line along the basement membranes of ducts, vessels, tubular complexes, and acinar cells. CONCLUSION: Our findings indicate that long-term increases of gelatinolytic activity of active MMP-2 cause continuous disorganization of type IV collagen in basement membranes during progressive atrophy of pancreatic gland in oleic acid-induced pancreatitis, and that MT1-MMP and TIMP-2 work as activating factors during proMMP-2 activation. Moreover, basement membranes disorganization in the sustentacula of acinar cells and duct epithelia seems to participate in continuous derangement of acinar cells and duct epithelium.     

The roles of MMP-2/TIMP-2 in extracellular matrix remodelling in the hearts of STZ-induced diabetic rats

OBJECTIVE: The present study was designed to examine the changes of MMP-2/TIMP-2 in the hearts of streptozotocin-induced diabetic rats and gain insight into their roles in extracellular matrix (ECM) remodelling on an experimental animal model of diabetic cardiomyopathy. METHODS AND RESULTS: Sixteen male Wistar rats were randomly divided into two groups: normal control and diabetic rats. Diabetes mellitus was induced in rats by streptozotocin injection. All rats were fed with standard chow and water ad libitum for 4 weeks. At 4 weeks diabetic rats were associated with a lower body weight (BW) and heart weight (HW) but a higher HW/BW. In the diabetic group, serum MMP-2 level had a tendency to increase but not significantly, while serum TIMP-2 significantly increased. Both the activity and expression of MMP-2 weakened in the hearts of diabetic rats. TIMP-2 gene expression in myocardium enhanced significantly. TIMP-2 protein level in diabetic heart was strengthened slightly but not significantly. VG staining showed a marked deposition of collagen in the diabetic group. Multivariate analysis revealed that total collagen content correlated negatively with the activity and gene expression of MMP-2 in the myocardium, and correlated positively with TIMP-1 mRNA expression. CONCLUSIONS: The decrease in MMP-2 activity and expression and increase in TIMP-2 gene expression in the myocardium of diabetic rats may lead to impairment of collagen degradation and contribute to the matrix deposition in diabetic myocardiopathy. The correlation between the serum level and cardiac expression of TIMP-2 in diabetic rats suggested that serum TIMP-2 level may be a viable marker for early diagnosis of diabetic myocardiopathy.     

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     

MMP-9/TIMP-1表达失衡与糖尿病大鼠皮肤"隐性损害"关系的初步探讨

目的 观察基质金属蛋白酶9(MMP-9)、组织金属蛋白酶抑制物1(TIMP-1)的表达和MMP-9/TIMP-1比值在糖尿病组和正常组大鼠皮肤表达的变化,并探讨其可能的作用.方法 采用链脲佐菌素制备糖尿病大鼠模型,6周后采用HE染色、免疫组织化学方法评估皮肤组织的组织学、细胞生物学行为的情况;通过逆转录-聚合酶链反应(RT-PCR)和Western印迹检测两组大鼠皮肤MMP-9、T1MP-1的表达情况.结果 与对照组比较,糖尿病组皮肤表皮厚度变薄,皮肤组织层次欠清晰、缺乏复层排列、明显萎缩,真皮纤维束排列紊乱、纤维间距增加、炎性细胞浸润.糖尿病组皮肤MMP-9的表达高于正常组,而TIMP-1的表达低于正常组,MMP-9/TIMP-1蛋白水平比值高于正常组.结论 糖尿病鼠皮肤在组织结构完整性未遭到破坏的情况下已经存在组织学、细胞生物学行为的改变,这种"隐性损害"可能与MMP-9/TIMP-1的平衡改变有关.     

Studies on glomerular basement membrane in experimental diabetes using lectin histochemistry in Wistar rats

Summary This study was designed to establish whether specific early changes in carbohydrate content of proteins in the glomerulus of the diabetic rat could be detected. Lectin staining of kidney sections from streptozotocin-induced diabetic rats were compared with similar sections from healthy and diabetic rats that were treated with insulin. Animal groups were killed 1 month, 3 months and 6 months after induction of diabetes. There were no differences in the staining of the glomerular basement membrane between control, insulin-treated and diabetic rats for the lectins concanavalin A, lotus tetragonolobus, soybean and kidney bean, with and without trypsinisation. Staining of the glomerulur basement membrane with wheat germ agglutinin after trypsinisation was significantly increased in the diabetic group when compared to both healthy and insulin-treated groups (p < 0.01). It was concluded that, in experimental diabetes mellitus in the rat, there is an accumulation of substances in the glomerular basement membrane and mesangium with an affinity for wheat germ agglutinin, most probably N-acetyl glucosamine, and this is partially prevented by insulin treatment.     

Alterations of glomerular basement membrane charge and structure in diabetic nephropathy

Summary We examined glomerular basement membrane anionic site distribution identified by cationic gold in seven patients with insulin-dependent and four patients with non-insulin-dependent diabetes mellitus, presenting a spectrum of clinical and glomerular changes. Anionic sites were investigated by pretreatment of tissue with glycosaminoglycan-degrading enzymes prior to cationic gold staining. The distribution of chondroitin sulphate proteoglycans — a previously unrecognized glomerular basement membrane component — and type IV collagen was examined by immunoelectron microscopy to identify structural changes in the basement membrane. Findings were compared with those of non-diabetic patients showing minor proteinuria and morphologically normal glomerular basement membranes. Two patients, originally diagnosed as having diabetic nephropathy were also examined at 19 weeks and 5 years after renal transplantation. Characteristic redistribution of type IV collagen and chondroitin sulphate proteoglycans was noted in thickened glomerular basement membrane segments (>400 nm) of diabetic patients and those with renal transplants. Extension of anionic sites deep into the glomerular basement membrane at pH 2.5, together with loss of interna sites at pH 5.8 is unique to diabetic nephropathy. Reduced charge density was apparent in some patients due to thickening of the glomerular basement membrane, although the number of anionic sites per unit length of membrane was actually increased. Thus, charge aberration in diabetic nephropathy is due to displacement rather than loss of anionic sites. Removal of more than 90% of these sites by heparitinase, confirms their association with heparan sulphate proteoglycans. Similar derangement of anionic sites in all patients with diabetic nephropathy irrespective of the degree of proteinuria, suggests that a heparan sulphate proteoglycan-related charge barrier plays a minor role in controlling permeability of the diabetic glomerular basement membrane.Abbreviations BSA Bovine serum albumin - CG cationic gold - CSPG chondroitin sulphate proteoglycans - GAG glycosaminoglycan - GBM glomerular basement membrane - HSPG heparan sulphate proteoglycans - LRE lamina rara externa - LRI lamina rara interna - PCI protein:creatinine index     

The role of matrix metalloproteinase 9 in the pathogenesis of chronic lymphocytic leukaemia

Matrix metalloproteinases (MMPs) are important for the pathogenesis and progression of different tumours. MMPs-2 and -9 are the principal MMPs produced by lymphocytes; these enzymes can degrade a number of matrix proteins but are the two main MMPs that digest type IV collagen, the major component of basement membranes. Therefore, these enzymes are potentially important for tissue invasion and remodelling by malignant lymphocytes. This study showed that chronic lymphocytic leukaemia (CLL) cells produce and secrete variable amounts of pro-MMP-9, but no MMP-2 or tissue inhibitor of metalloproteinase 1 (TIMP-1). The pro-enzyme was found in monomeric and dimeric forms and also complexed with lipocalin. Moreover, a small fraction of secreted monomer became associated with the cell surface and activated upon cell adhesion to insolubilized type IV collagen. High levels of intracellular MMP-9 were associated with advanced (stage C) disease and with poor patient survival. Immunohistochemical studies demonstrated that MMP-9 was associated with areas of tissue invasion and remodelling. The relatively specific MMP-9 inhibitors, Ro31-9790 (3 micromol/l) and TIMP-1, reduced CLL-cell migration through type IV collagen and through endothelial monolayers suggesting that the enzyme may also be important in malignant cell entry and egress to and from involved tissue. Our data raise the possibility that MMP-9 modulation may have therapeutic potential in advanced CLL.