Alteration in the distribution of type IV collagen in glomerular basal laminae in diabetic rats as revealed by immunocytochemistry and morphometrical approach

Summary The gomerular basal laminae of normoglycaemic and long-term streptozotocin-induced hyperglycaemic rats was studied by morphometrical and immunocytochemical approaches. Using the orthogonal intercept method, we have confirmed that in long-term diabetes, the thickness of the glomerular basal laminae increases significantly. Applying the high resolution protein A-gold immunocytochemical technique, type IV collagen was localized in the glomerular basal laminae. In tissues from normoglycaemic animals the labell ing was present over the central lamina densa. However, the labelling obtained over the thickened glomerular basal laminae of the hyperglycaemic animals was restricted to the subendothelial site of the lamina. Thus major alteration in the distributioa of type IV collagen occurs during the development of diabetic microangiopathy in hyperglycaemic animals.     

Increased vascular permeability in pancreas of diabetic rats: detection with high resolution protein A-gold cytochemistry

Summary The role of the pancreatic microcirculation in the pathogenesis of Type 1 (insulin-dependent) diabetes mellitus remains poorly understood. Herein, a method is described for the ultrastructural investigation of the integrity of the pancreatic microvasculature. The method consists of histochemical detection and isolation of the islets followed by albumin and protein A-gold immunocytochemistry, whereby the distribution of endogenous albumin is used as a marker of endothelial integrity. This technique, applied to the study of spontaneously diabetic rats, reveals a selective increase in permeability of islet capillaries and post-capillary venules at the onset of diabetes, while acinar capillaries and arterioles remain intact. At 50 days of age, before the onset of diabetes, the microvasculature of diabetes-prone rats shows no alterations in permeability to albumin. When used in conjunction with morphometric analyses, this methodological approach may be useful for further studies in pathologic or experimental conditions involving the pancreatic microvasculature.     

伊贝沙坦对大鼠糖尿病模型肾脏肥大和肾小球毛细血管基底膜厚度的影响

目的 观察伊贝沙坦对大鼠糖尿病模型肾脏肥大和肾小球毛细血管基底膜厚度的影响。方法 将SD大鼠分为糖尿病肾病组(A组)、伊贝沙坦治疗组(B组)、健康对照组(C组),A和B组大鼠制成糖尿病模型,B组予以50mg／kg伊贝沙坦灌胃。观察第4、8、12周大鼠的血糖、体重、尿白蛋白、24h尿蛋白的改变及第12周时的肌酐清除率(Ccr)、肾重、肾脏肥大指数、肾组织总蛋白含量、肾小球面积和体积、肾小球毛细血管基底膜(GBM)厚度的改变。通过免疫组化观察肾结缔组织生长因子(CTGF)和转化生长因子(TGF)-βl的表达。结果 A组和B组大鼠血糖较C组明显升高且维持在一个较高水平(P＜0．01)。A组大鼠的体重较C组明显下降,B组有所增加(P＜0．05)。随时间的推移(第4、8、12周),A组大鼠尿蛋白、尿白蛋白逐渐增加,B组明显减少(P＜0．01)。A组大鼠Ccr较C组显著升高(P＜0．01),B组Ccr明显下降(P＜0．05)。至第12周时,A组大鼠肾脏重量、肾脏肥大指数、肾组织总蛋白含量、肾小球面积和体积均较C组明显增加(P＜0．01),B组均较A组降低(P＜0．01,P＜0．05)。免疫组化半定量分析显示,A组大鼠CTGF与TGF-βl的表达均高于C组(P＜0．01),B组明显低于A组(P＜0．01,P＜0．05)。A组大鼠GBM较C组明显增厚(P＜0．01),B组较A组明显变薄(P＜0．01）。肾小球CTGF、TGF—βl的表达与肾脏体积呈正相关(r＝0．83,r＝0．83;P＜0．05)。结论 早期应用伊贝沙坦可抑制糖尿病大鼠早期肾脏肥大和CTGF表达等。     

ACE2对糖尿病大鼠肾小球足细胞的影响及厄贝沙坦干预作用的研究

目的 探讨糖尿病大鼠肾组织局部血管紧张素转换酶(ACE)2对肾小球足细胞的影响及厄贝沙坦的干预作用.方法 56只Wistar大鼠经链脲佐菌素诱导糖尿病模型,死亡9只,其余随机分为糖尿病组(n=11)、30 mg/(kg·d)肼屈嗪干预组(n=9)、25 mg/(kg·d)厄贝沙坦干预组(n=9)、50 mg/(kg·d)厄贝沙坦干预组(n=9)、200 mg/(kg·d)厄贝沙坦干预组(n=9),同时设正常对照组(n=7).于造模后第4周开始予肼屈嗪、厄贝沙坦干预,第12周观察24h尿微量白蛋白排泄率(UAER),采用免疫组化技术检测肾组织ACE2的分布和表达情况,免疫组化SP法检测肾小球WT1表达情况,Image.Pro Plus 6.0彩色图像分析系统半定量检测足细胞密度.结果 正常对照组UAER低于糖尿病组和药物干预组[包括肼屈嗪干预组、25 mg/(kg·d)厄贝沙坦干预组、50 mg/(kg·d)厄贝沙坦干预组、200 mg/(kg·d)厄贝沙坦干预组](P＜0.001);药物干预组UAER低于糖尿病组(P＜0.001);所有厄贝沙坦干预组UAER较肼屈嗪干预组显著降低(P ＜0.05);50 mg/(kg·d)厄贝沙坦干预组、200 mg/(kg·d)厄贝沙坦干预组UAER较25 mg/(kg·d)厄贝沙坦干预组明显降低(P＜0.001).肾组织ACE2表达与肾小球足细胞密度呈正相关(r=0.381,P＜0.001),糖尿病组ACE2表达较正常对照组明显降低(P＜0.05),而所有厄贝沙坦干预组显著高于糖尿病组和肼屈嗪干预组(P ＜0.001),厄贝沙坦干预组、肼屈嗪干预组足细胞密度显著高于糖尿病组(P ＜0.001);200 mg/(kg·d)厄贝沙坦干预组高于肼屈嗪干预组、25 mg/(kg·d)厄贝沙坦干预组、50 mg/(kg·d)厄贝沙坦干预组、糖尿病组.结论 肾组织ACE2减少可导致足细胞密度降低,进而引起肾脏损害;而厄贝沙坦可上调肾组织ACE2的表达,增加足细胞密度,发挥降压外的肾脏保护作用,且呈现剂量依赖性.     

糖尿病患者尿白蛋白与肌酐比值与肾动态显像肾小球滤过率的相关性研究

目的探讨尿白蛋白与肌酐比值（UACR）和肾小球滤过率（GFR）联合诊断在糖尿病肾脏病变评估中的临床意义。方法分析294例住院糖尿病患者的资料,行99mTC-DTPA肾小球滤过率和UACR检查。结果正常白蛋白尿、微量白蛋白尿及大量白蛋白尿患者中分别有9．6％（15／157）、25．3％（23／91）、45．7％（21／46）出现了GFR的下降;以UACR为标准诊断糖尿病患者肾损害的灵敏度、特异度分别为88．9％、59．3％,以GFR为标准其灵敏度、特异度分别为80．6％、88．8％,而联合UACR与GFR诊断其灵敏度、特异度分别为94．4％、54．3％;结论UACR正常的糖尿病患者已有部分出现肾功能下降,联合GFR有助于更好地评价糖尿病患者的肾脏病变情况。     

Micro vascular permeability to albumin and glomerular filtration rate in diabetic and normal children

Summary In order to examine the permeability of microvessels in diabetic children, the glomerular filtration rate, urinary excretion rates of albumin and ₂-microglobulin, intravascular mass of albumin, and transcapillary escape rate of albumin were studied in 26 diabetic children without clinical signs of microangiopathy (age: 7–14 years; duration of disease: 3–14 years). Similar measurements were made in 28 healthy school children (age: 8–14 years). Mean glomerular filtration rate in the diabetic children was higher than in the normal children (138 versus 109 ml/min per 1.73 m², p<0.01). Urinary excretion rates of albumin and ₂-microglobulin did not differ in diabetics. Mean intravascular albumin mass in the diabetic girls (1.64 g/kg body weight) was lower (p<0.01) than in the diabetic boys (1.89 g/kg body weight) and also lower (p<0.02) than in the normal girls (1.94 g/kg body weight). Mean transcapillary escape rate of albumin in the twenty diabetics with duration of diabetes less than 10 years (7.14%/h) was lower (p<0.01) than that in normal children (8.90%/h); the escape rate showed a positive correlation with duration of diabetes (r=0.47; p<0.02). Thus glomerular filtration rate in diabetic children is elevated to the same extent as in adult short-term juvenile diabetics while the permeability of the glomerular membrane to macromolecules is normal. Interpretation of the results on intravascular albumin mass and transcapillary escape rate of albumin requires further investigation.     

链脲佐菌素所致糖尿病大鼠骨髓中存在胰岛样细胞

本研究证实糖尿病大鼠骨髓中存在表达胰岛素、C肽、胰高血糖素、生长抑素和胰淀粉样多肽的细胞簇，并检测到胰岛发育和功能相关基因的表达，这些胰岛样细胞可能是骨髓中的成体干细胞转分化而来。     

Effect of thalidomide and rosiglitazone on the prevention of diabetic retinopathy in streptozotocin-induced diabetic rats

Aims/hypothesis Our aim was to compare the therapeutic effect of thalidomide and rosiglitazone on the prevention of diabetic retinopathy in streptozotocin-induced diabetic rats.Methods Male Holtzman rats of 6 to 8 weeks of age and weighing 170±30 g were randomly divided into four groups: control (n=13), untreated diabetic (n=17) and diabetic rats treated with thalidomide (200 mg kg^–1 day^–1) (n=8) or rosiglitazone (1 mg kg^–1 day^–1) (n=22) for 3 months. Diabetes was induced by streptozotocin with the rats having a body weight of 70 mg/kg. After treatment, vascular endothelial growth factor (VEGF) concentrations in ocular fluid were compared between the different groups, and retinal capillary basement membrane thickness was measured by electron microscopy.Results Higher VEGF concentrations in ocular fluid and thicker basement membranes were observed in untreated diabetic rats compared to the control rats. Similar VEGF concentrations and basement membrane thickness were observed for the thalidomide-treated group compared with the control group, whereas no difference in these parameters was observed between the rosiglitazone-treated rats and the control or untreated diabetic rats.Conclusions/interpretation Our findings confirm the association between VEGF concentrations and diabetic retinopathy as suggested by other investigators. Thalidomide, but not rosiglitazone, was associated with the inhibition of basement membrane thickening and the blockade of the increase of VEGF in ocular fluid, thus representing a potential therapeutic drug for the prevention of diabetic retinopathy.Abbreviations T Thalidomide - R rosiglitazone - VEGF vascular endothelial growth factor - BM basement membrane - BMT basement membrane thickness An erratum to this article can be found at     

Immunocytochemical studies of pancreatic acinar cells in normal and streptozotocin-induced diabetic rats

Summary Amylase and chymotrypsinogen in pancreatic tissue from normal and diabetic rats were revealed by immunocytochemistry and analyzed biochemically. In acinar cells of control animals, both enzymes were localized with high resolution in the rough endoplasmic reticulum, Golgi apparatus, immature and mature secretory granules. Quantitative evaluations of the intensities of labelings have demonstrated, for both enzymes, the presence of an increasing gradient which followed precisely their secretory pathway. This gradient reflects the normal processing of both proteins through secretion. In streptozotocin-induced diabetic animals, labeling for amylase in acinar cells was markedly reduced (remaining about 11% of the normal values). The gradient along the secretory pathway was abolished, indicating an alteration in the processing and secretion of amylase. On the other hand, labeling for chymotrypsinogen was significantly increased (to 170%p < 0.0005), and its processing remained normal. In insulin-treated diabetic animals, immunolabeling for amylase was restored and the gradient re-established, indicating a normalization of the secretion. Labeling for chymotrypsinogen was reduced towards normal values. These results were found to be in agreement with those obtained by biochemical approaches and demonstrate that, in the diabetic condition, secretion of amylase is selectively impaired.     

糖尿病大鼠骨骼肌的早期超微结构改变

链脲佐菌素复制的糖尿病大鼠在第4周和对照组相比，出现骨骼肌线粒体数目增加，线粒体变性、肿胀和排列紊乱。     

Effects of early insulin treatment on ultrastructural changes of glomeruli in diabetic rats revealed by the quick-freezing and deep-etching method

Summary The three-dimensional ultrastructure of glomerular basement membrane (GBM) and mesangial matrix (MM) at an early stage of streptozotocin (STZ)-induced diabetes mellitus in rats was examined by the quick-freezing and deep-etching method. In diabetic rats, the GBM inner layer was diffusely enlarged and the meshwork structure not only in the GBM middle layer but also in the MM became markedly irregular due to the rupture of fine fibrils. This irregularity and enlargement of the mesh pores in diabetic rats developed during the experimental period and was significantly different from results in control rats. Insulin treatment from 1 week after STZ injection had significant effects in preventing the ultrastructural changes in the GBM and MM. It is suggested that early insulin treatment has significant effects in preventing size barrier disturbance of GBM and MM in STZ-induced diabetes.Abbreviations GBM Glomerular basement membrane - MM mesangial matrix - STZ streptozotocin - QF-DE quick-freezing and deep-etching - LDM longest dimension of meshpores     

Increased glomerular permeability to albumin induced by exercise in diabetic subjects

Summary The urinary excretion of albumin was measured in insulin-dependent diabetics under ordinary conditions of life and in response to exercise. Possible mechanisms of exercise induced albuminuria in diabetics were also investigated. Under ordinary conditions of life the insulin-treated diabetics, as a group, had a higher mean urinary albumin excretion than normal controls; however, half of the diabetics had albumin excretion rates within the control range. A given exercise load (600 kpm/min for 20 min) produced an exaggerated albumin excretion in diabetics, particularly evident in the post-exercise period. The elevated urinary albumin excretion was due to an increased transglomerular passage of albumin, not to reduced tubular reabsorption. The increase was not associated with differences in blood pressure or urine flow between controls and diabetics. This exercise test has proved to be a suitable provocation test to unmask abnormalities in the glomerular handling of albumin that might not be recognisable at rest.Presented in part at the 12th Annual Meeting of the European Association for the Study of Diabetes, Helsinki, 1976     

Effect of octreotide and insulin on manifest renal and glomerular hypertrophy and urinary albumin excretion in long-term experimental diabetes in rats

Summary Treatment of diabetic rats with octreotide can inhibit early diabetic renal hypertrophy. Octreotide administration for 6 months from the day of diabetes induction inhibits renal hypertrophy and diminishes increase in urinary albumin excretion. To investigate the effect of octreotide on manifest diabetic renal changes, octreotide treatment was given for 3 weeks after an untreated diabetic period of 3 or 6 months. In addition, following 6 months of diabetes, a group of diabetic rats was treated with insulin for 3 weeks. Renal and glomerular hypertrophy, and increased urinary albumin excretion were observed in diabetic rats compared to non-diabetic control rats from 3 months and throughout the study period. Octreotide treatment did not affect body weight, food intake, blood glucose or serum fructosamine levels. We observed no effect of octreotide treatment on renal and glomerular hypertrophy or urinary albumin excretion compared to placebotreated diabetic rats. Insulin treatment for 3 weeks after 6 months of untreated diabetes normalized blood glucose and serum fructosamine levels, and furthermore renal hypertrophy was significantly diminished compared to the placebo-treated diabetic rats. However, insulin treatment had no effect on glomerular hypertrophy or urinary albumin excretion. In conclusion, octreotide treatment for 3 weeks following an untreated diabetic period of 3 or 6 months is unable to reduce the increased renal and glomerular volume or urinary albumin excretion. However, insulin treatment for 3 weeks with induction of euglycaemia diminishes the renal hypertrophy but has no effect on glomerular volume or urinary albumin excretion.Abbreviations UAE Urinary albumin excretion - IGF-I insulin-like growth factor I - IGFBP insulin-like growth factor binding protein - STZ streptozotocin - TGV total glomerular volume - BW body weight - GH growth hormone - RPF renal plasma flow     

Role of curcumin in the prevention of cholinergic mediated cortical dysfunctions in streptozotocin-induced diabetic rats

Diabetes exacerbates neuronal injury mediated through neurotransmitters deregulation in cerebral cortex. Our study analyzed the neuroprotective effect of curcumin to prevent cortical dysfunction associated with diabetes. Our study revealed decreased gene expression of muscarinic M1, insulin receptor, SOD, choline acetyl transferase and increased gene expression of muscarinic M3, α7-nicotinic acetylcholine receptor, acetylcholine esterase and GLUT3 in cerebral cortex of diabetic rats. Curcumin and insulin treatment reversed this altered parameters to near control. Immunohistochemistry studies of muscarinic M1 and M3 confirmed the gene expression at protein level. Decreased novel arm entry of diabetic rats in Y-maze test, improved in treatment group. These results suggest that cholinergic dysfunction, impaired glucose transport and oxidative stress contributes to learning and memory deficits in diabetes and further suggest that antioxidant curcumin has potential therapeutic role in preventing and/or delaying the diabetic complications associated with brain.     

Clinical significance of P-glycoprotein expression in acute leukaemia as analysed by immunocytochemistry

Abstract: Multidrug resistance, mediated by the overexpression of an energy-dependent transport protein, P-glycoprotein, has been one of the major targets of interest in solving the mechanisms of clinical drug resistance of malignant cells. To evaluate the correlation between P-glycoprotein overexpression and the response to chemotherapy, we analysed cytospin preparations of gradient-separated blood or bone marrow mononuclear cells from 79 patients with acute leukaemia by means of the P-glycoprotein-directed monoclonal antibody JSB-1 and immunocytochemistry using the alkaline phosphatase-antialkaline phosphatase technique. P-glycoprotein expression was detected in all disease phases of acute leukaemia. Thirteen out of 51 patients at diagnosis, 10/29 patients in relapse or during residual disease and 8/27 patients in remission overexpressed P-glycoprotein. Seven out of the 8 positive remission samples were collected between the cycles of consolidation treatment. Our results suggest that increased P-glycoprotein expression in samples collected between the cycles of consolidation treatment during remission may be induced in normal leukocytes by cytotoxic drug treatment, infections, or by some physiological mechanisms related to the disease. Patients older than 45 years of age were significantly more often P-glycoprotein-positive (11/25) at diagnosis than younger patients (2/26). P-glycoprotein expression at diagnosis was significantly correlated with a low remission rate after the first cycle of induction therapy. Of 34 P-glycoprotein-negative patients, 25 achieved remission after the first cycle as compared to 4/12 of the P-glycoprotein-positive patients. Our results indicate that the method used is specific and sensitive enough for the analysis of P-glycoprotein expression and that the expression at initial presentation is inversely correlated with the outcome of induction therapy.     

Effect of rosuvastatin on capillary filtration of albumin and blood pressure in rats with streptozotocin-induced diabetes

An increase in capillary filtration of albumin (CFA) is well demonstrated in diabetes. Statins may exert a protective effect against endothelial dysfunction. The aim of this study was to test whether rosuvastatin may prevent the increase in peripheral CFA in diabetic rats and the role of blood pressure lowering. Rats with streptozotocin-induced diabetes were randomized to receive either rosuvastatin 20 mg/kg/d (group R) or both rosuvastatin 20 mg/kg/d and mevalonate 20 mg/kg/d (group RM) or no treatment (group U). CFA index was measured on a limb by a non-invasive isotopic test using technetium-labelled albumin, at three time points: at mean age of 3 months, before treatment; at 5 and 8 months, i.e. after 2 and 5 months of treatment. At 3 months, interstitial albumin retention (AR) was markedly increased in the 3 groups. From 3 to 5 months, AR increased significantly in group U, decreased in group R and in group RM. At 5 and 8 months, AR was significantly lower in groups R and RM than in group U. Systolic blood pressure (SBP) was measured at 8 months and was significantly lower in group R than in group U and RM. At 8 months, serum cholesterol levels were not different between the three groups whereas triglycerides were significantly lower in groups R and RM than in group U. In conclusion, in diabetic rats rosuvastatin prevents the increase in peripheral CFA and induces a decrease in blood pressure. The beneficial effect of rosuvastatin on endothelial function does not seem to result from blood pressure reduction nor lipid lowering effects.     

C-type natriuretic-peptide-potentiated relaxation response of gastric smooth muscle in streptozotocin-induced diabetic rats

AIM： To study the sensitivity of gastric smooth muscle to C-type natriuretic peptide （CNP） in streptozotocin（STZ）-induced diabetic rats.METHODS： The spontaneous contraction of a gastric smooth muscle strip was recorded by using physiological methods in rats. The expressions of CNP and natriuretic peptide receptor-B （NPR-B） in gastrictissue were examined by using immunohistochemistry techniques in the diabetic rat.RESULTS： At 4 wk after injection of STZ and vehicle,the frequency of spontaneous contraction of gastric smooth muscle was significantly reduced in diabeticrats, and the frequency was decreased from 3.10 ±0.14 cycle/min in controls to 2.23± 0.13 cycle/min（n = 8, P 〈 0.01）. However, the amplitude of spontaneous contraction was not significant different from the normal rat. CNP significantly inhibited spontaneous contraction of gastric smooth musclein normal and diabetic rats, but the inhibitory effect was significantly potentiated in the diabetic rats.The amplitudes of spontaneous contraction were suppressed by 75.15% ± 0.71% and 58.92% ±1.32% while the frequencies were decreased by 53.33% ±2.03% and 26.95% ± 2.82% in diabetic and normal rats, respectively （n = 8, P 〈 0.01）. The expression ofCNP in gastric tissue was not changed in diabetic rats,however the expression of NPR-B was significantlyincreased in diabetic rats, and the staining indexes of NPR-B were 30.67± 1.59 and 17.63 ± 1.49 in diabeticand normal rat, respectively （n = 8, P 〈 0.01）.CONCLUSION： The results suggest that CNP inducedan inhibitory effect on spontaneous contraction ofgastric smooth muscle, potentiated in diabetic ratvia up-regulation of the natriuretic peptides-NPR-B-particulate guanylyl cyclase-cyclic GMP signal pathway.