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     

T-Type calcium channel blockage ameliorates proteinuria and renal extracellular matrix accumulation in experimental diabetic rats

Calcium channels have been shown to play an important role in the pathogenesis of diabetic nephropathy. The purpose of this study is to investigate the effect of T-type calcium channel blockers in uninephrectomized diabetic rats. Two weeks after uninephrectomy, streptozotocin (65 mg/kg) was given to male Sprague-Dawley rats to induce diabetes mellitus. In the succeeding days, four groups of animals were randomized to receive 30 mg/kg mibefradil (Mib, T-type calcium channel blocker), 30 mg/kg amlodipine (Aml, L-type calcium channel blocker), 10 mg/kg cilazapril (Cil, angiotensin converting enzyme inhibitor) or 25 mg/kg Mib + 8 mg/kg Cil (M+C), which were delivered daily by gavage (n = 6-8 for each group). At the fourth week, 24-hour urine, blood and kidney samples were collected for examinations. Renal morphometric analyses and assay for type IV collagen and laminin were performed. Untreated diabetic rats (DM) exhibited overt hyperglycemia, proteinuria and albuminuria when compared with uninephrectomized (UNx) control (17.67 ±4.9 vs 2.97 ±0.72 μg/24 hours, p < 0.05), while Cil and M+C treated rats had significantly reduced urinary protein excretion (Cil: 5.53 ±1.45, M+C: 3.87 ±0.88 vs DM: 17.67 ±4.9 μg/24 hours; p < 0.05, respectively). Mibefradil alone also ameliorated albuminuria in DM rats (7.69 ±1.87 vs 17.67 ±4.9 μg/24 hours, p < 0.05). Histopathological study revealed significantly less glomerular matrix, laminin, type IV collagen and proximal tubule cells (PTC) hypertrophy in the Cil and M+C groups when compared with the DM group (Collagen IV: DM 7.3 ±0.65 vs Mib 4.82 ±0.78, Cil 4.11 ±0.74, M+C 2. 64 ±0.66, UNx 3.71 ±0.99, p < 0.05). Albuminuria, glomerular matrix accumulation and collagen IV deposition were not changed in the Aml treated group. There was, however, significantly lower creatinine clearance and laminin volume in the Aml group. In conclusion, our study demonstrated that T-type calcium channel blockage reduced albuminuria and renal extracellular matrix accumulation in experimental diabetic rats, suggesting an important role for T-type calcium channel in the progression of diabetic nephropathy. Additional benefit can be achieved by combination with an angiotensin converting enzyme inhibitor. The mechanism of action, however, requires further investigation.     

Glomerular structural and functional changes in a high-fat diet mouse model of early-stage Type 2 diabetes

Aims/hypothesis Type 2 diabetes often results in diabetic nephropathy, which is preceded by an elevated glomerular filtration rate (GFR). This study was designed to develop a mouse model of Type 2 diabetes and to elucidate the glomerular events in the early stages of diabetic nephropathy.Methods Four-week-old mice were fed a normal or high-fat (42% of total calories from fat) diet, and body weight, blood glucose, insulin, leptin, lipids and GFR were monitored from 9 to 21 weeks or longer after the feeding programme. Mesangial cell dedifferentiation was accessed by alpha-smooth muscle actin staining. Glomerular hypertrophy was determined using image analysis with haematoxylin–eosin staining. Matrix deposition was determined by type IV collagen staining.Results After 9 weeks, mice fed a high-fat diet weighed more than mice fed a normal diet (30.5±1.2 vs 22.3±0.5 g, p<0.05), and mice fed a high-fat diet were hyperinsulinaemic (283.9±69.7 vs 102.9±36.4 pmol/l, p<0.05), hyperglycaemic (8.0±0.6 vs 6.5±0.2 mmol/l, p<0.05) and their leptin levels were increased six-fold (1.48±0.45 vs 0.25±0.03 ng/ml, p<0.05). After 13 weeks, mice fed a high-fat diet showed hyperfiltration (GFR; 440±60 vs 210±10 µl/min, p<0.05). During the early stages of diabetic nephropathy, mesangial cell dedifferentiation was evident, shown by increased expression of alpha-smooth muscle actin in the glomeruli. After 9 weeks, mice fed a high-fat diet already demonstrated increased type IV collagen deposition. After 13 weeks, they developed enlarged glomerular tufts compared with those of their age-matched controls.Conclusions/interpretation The results of this study suggest that collagen IV deposition precedes the hyperfiltration and enlargement of glomeruli in early-stage diabetic nephropathy. Dedifferentiation of mesangial cells may be associated with collagen IV deposition.     

Genetic Variation of a Collagen IV α1-Chain Gene Polymorphism in Danish Insulin-dependent Diabetes Mellitus (IDDM) Patients: Lack of Association to Nephropathy and Proliferative Retinopathy

In insulin-dependent (Type 1) diabetes mellitus (IDDM) the development of nephropathy is partly due to genetic susceptibility. Previously one study has demonstrated a relationship between a HindIII restriction polymorphism of the collagen IV α1-chain gene and diabetic nephropathy. The aim of the present study was to evaluate such as association in a case–control study including 207 Danish IDDM patients: 116 with nephropathy (urinary albumin excretion rate (AER) > 300 mg 24 h⁻¹) and 91 without nephropathy (AER < 30 mg 24 h⁻¹). Using genomic DNA, HindIII restriction fragment length analysis revealed a bi allele polymorphism visualized by Southern hybridization with a cDNA probe recognizing the collagen IV α1-chain gene. No differences in genotype frequencies or allele frequencies were demonstrated comparing patients with and without nephropathy: p = 0.39 and p = 0.96, respectively. Neither were there any difference in genotype frequencies or allele frequencies when the patients were stratified according to the presence of proliferative retinopathy: p = 0.44 and p = 0.84, respectively. Pooling the diabetic groups revealed genotype frequencies and allele frequencies comparable to those found in 57 healthy unrelated Danish individuals. We conclude that in a Danish IDDM population a HindIII restriction polymorphism of the collagen IV α1-chain gene is not associated with diabetic nephropathy, diabetic retinopathy or with diabetes per se. © 1997 by John Wiley & Sons, Ltd.     

转化生长因子βI型受体在糖尿病大鼠肾皮质中的表达及苯那普利对其 …

目的 研究转化生长因子βⅠ型受体（ＴＧＦβＲⅠ）在糖尿病大鼠肾皮质中的表达及血管紧张素转换酶抑制剂苯那利普利对其调节作用。方法 纯种雄性成年Ｗｉｓｔａｒ大鼠随机分单侧肾切除组（Ｃ组）,糖尿病组（Ｄ组）和糖尿病苯那普利治疗组（ＤＢ组）,给药４周末观察各组血清、血肌酐水平、体重、肾重和肾组织蛋白含量及血浆、肾皮质、髓质血管紧张素转换酶（ＡＣＥ）活性的变化;分别采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）、Ｎ     

Suppression of transforming growth factor beta and vascular endothelial growth factor in diabetic nephropathy in rats by a novel advanced glycation end product inhibitor, OPB-9195

Aims/hypothesis. Advanced glycation end products (AGEs) participate in the pathogenesis of diabetic nephropathy. We reported earlier that OPB-9195, a synthetic thiazolidine derivative and novel inhibitor of advanced glycation, prevented progression of diabetic glomerulosclerosis by lowering serum concentrations of advanced glycation end products and reducing their deposition in the glomeruli. Here, we examined their contribution and that of growth factors, such as transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), to the progression of diabetic nephropathy. We also investigated the expression of type IV collagen in the kidneys of Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a Type II (non-insulin-dependent) diabetes mellitus model, after treatment with OPB-9195. Methods. Using northern blots and immunohistochemical techniques, we determined the renal expression of TGF-β and type IV collagen mRNAs and proteins in OLETF rats. We also examined OPB-9195's effects on renal expression of VEGF mRNA and protein. Results. Concomitant increases in TGF-β and type IV collagen expression were observed at each point in time in OLETF rats not given OPB-9195. In contrast, OPB-9195 treatment greatly suppressed the renal expression of TGF-β, VEGF and type IV collagen mRNAs and proteins to that seen in non-diabetic rats. Conclusion/interpretation. Since OPB-9195, an AGE-inhibitor, prevented the progression of diabetic nephropathy by blocking type IV collagen production and suppressing overproduction of two growth factors, TGF-β and VEGF, in diabetic rats, this compound warrants further investigation. [Diabetologia (1999) 42: 579–588] Received: 2 October 1998 and in final revised form: 28 December 1998     

Increased rat myocardial type VI collagen in diabetes mellitus and hypertension

Summary Diabetic cardiomyopathy, a condition characterized by the accumulation of carbohydrate-containing material surrounding the myocardial small blood vessels, has been studied in alloxan-diabetic normotensive and hypertensive rats. Immunochemical techniques were used to monitor several extracellular matrix constituents present in extracts of cardiac tissue, namely types I, IV and VI collagen, laminin and fibronectin, as well as myosin. These studies have indicated that after induction of diabetes, type VI collagen but none of the other matrix components studied, was significantly increased (from 2.29±0.04 mg/g in normal to 2.85±0.18 mg/g in diabetic ventricles, p<0.01). Hypertension, whether induced by the clipping of one renal artery or genetically determined (spontaneously hypertensive rats), resulted in a similar elevation in type VI collagen (2.71±0.12 mg/g, p<0.005 compared to normal rats). In the presence of diabetes plus hypertension the effect was not additive, the type VI collagen level being 2.93±0.15 (p<0.001 compared to normal rats). Basement membrane collagen (type IV) in the myocardium appeared to be unaffected by diabetes or hypertension and the myosin contents of the hearts of the four experimental groups were similar. Quantitative determinations indicate that compared to type IV collagen, laminin or fibronectin, type VI collagen represents the major periodic acid-Schiff-reactive extracellular constituent of the rat ventricle. Its preferential increase in the heart in diabetes may provide insight into the molecular mechanisms of the diabetic microvascular disease.     

Insertion/deletion polymorphism in the angiotensin-l-converting enzyme gene is associated with coronary heart disease in IDDM patients with diabetic nephropathy

Summary Insulin-dependent diabetic (IDDM) patients with diabetic nephropathy have a highly increased morbidity and mortality from coronary heart disease. An insertion (I) /deletion (D) polymorphism in the angiotensin-I-converting enzyme (ACE) gene has been shown to be associated with coronary heart disease. Therefore, we have investigated the role of this ACE/ID polymorphism in 198 IDDM patients with diabetic nephropathy and 190 normoalbuminuric IDDM patients. The prevalence of myocardial infarction and other coronary heart disease was significantly elevated in patients with nephropathy, 19 % (38/198) vs 8 % (15/190), p < 0.001. In the nephropathic group 12 of 63 (19 %), 23 of 95 (24 %), and 3 of 40 (7.5 %) patients with the DD, ID and II genotypes, respectively had a history of coronary heart disease, II vs DD and ID, p < 0.05 when compared to nephropathic patients without coronary heart disease. Multiple logistic regression analysis of the risk factors associated with coronary heart disease in univariate analysis revealed that the II genotype acts as an independent protective factor against coronary heart disease, odds ratio II/DD + ID 0.27 (95 % confidence interval 0.07–0.97, p < 0.05). There was no difference in genotype or allele frequency (D/I) between patients with and without nephropathy, 0.56/0.44 in both groups, but plasma ACE concentration was elevated in patients with nephropathy 609 (151–1504) ng/ml as compared to patients with normoalbuminuria, 428 (55–1630) ng/ml, p < 0.001. We suggest that ACE/ID polymorphism may influence the frequency of life-threatening cardiac complications in IDDM patients suffering from diabetic nephropathy, a condition characterized by increased plasma ACE concentration. [Diabetologia (1995) 38: 798–803] Received: 10 October 1994 and in revised form: 20 December 1994     

Increased urinary type IV collagen marks the development of glomerular pathology in diabetic d/db mice

The diabetic db/db mouse exhibits increased albumin excretion soon after the onset of obesity and hyperglycemia, and later manifests glomerular mesangial matrix expansion resembling that found in human diabetic nephropathy. Since the glomerular lesion in this rodent model of type 2 diabetes is associated with renal overexpression of mRNA encoding type IV collagen, we postulated that changes in the urinary excretion of collagen IV may reflect developing glomerular pathology. To explore this hypothesis, we monitored urinary collagen IV (measured by immunoassay) in db/db mice during the course of evolution of nephropathy. At age 8 weeks, collagen IV excretion was not different in diabetic compared to nondiabetic animals despite marked albuminuria, but was significantly increased in db/db compared to db/m mice at age 12 and 16 weeks. Serum levels of collagen IV did not significantly differ between normal versus diabetic mice at any age. Glomerular morphometry revealed mesangial matrix expansion at age 12 weeks, coincident with the rise in collagen IV excretion, which became more marked at age 16 weeks in association with reduced creatinine clearance and elevated serum creatinine. The findings suggest that increased urinary type IV collagen is a better indicator than albuminuria of developing glomerular matrix accumulation that results in compromised renal filtration function.     

Differential regulation of glomerular gelatinase B (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in obese Zucker rats

Summary The obese Zucker rat represents a model of obesity combined with insulin resistance and hyperlipidaemia, which over a period of several months develops spontaneous glomerulosclerosis. The present study addressed the question as to whether glomerular sclerosis was associated with alterations in the degradation of matrix components. In the early phase (up to 6 months) glomeruli from obese rats displayed increased total collagen content (+ 64 %) and decreased gelatinolytic activity (− 34 %) as compared to lean control animals. This decline in glomerular gelatinolytic activity was due to a reduction in gelatinase B [matrix metalloproteinase (MMP)-9]. Glomerular MMP-9 mRNA was reduced 4.6 ± 0.6-fold (n = 3; p < 0.05), MMP-9 protein was not detectable by Western blotting and MMP-9 activity was considerably suppressed in gelatin zymograms. MMP-2, in terms of mRNA expression and activity, was unchanged. Tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression, TIMP-1 protein (immunohistochemistry) and TIMP-1 activity (reverse zymography) were enhanced in glomeruli from obese rats, while TIMP-2 mRNA remained unchanged. Moreover, mRNA for the α₁ IV collagen chain was 2.1 ± 0.8-fold higher in glomeruli isolated from obese animals (n = 3; p < 0.05). These findings indicate that matrix expansion in glomeruli from obese Zucker rats is due to both enhanced synthesis of matrix components as well as reduced degradation by matrix metalloproteinases. Apparently the latter effect is based on a reduction in MMP-9 and up-regulation of its inhibitor TIMP-1. [Diabetologia (1997) 40: 1035–1043] Received: 16 January 1997 and in final revised form: 28 April, 1997     

Meta-analysis of association of insertion/deletion polymorphism of angiotensin I-converting enzyme gene with diabetic nephropathy and retinopathy

Summary An insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene has repeatedly been shown to be associated with ischaemic heart disease, but the association of this genetic marker with diabetic microangiopathy is controversial. To assess the association of the genotypes with the development of diabetic nephropathy or retinopathy, we performed a meta-analysis of data from the literature, using Mantel-Haenszel method followed by the Breslow-Day test for assessing homogeneity among data. In a total of 4773 diabetic patients from 18 studies with (n = 2495) and without (n = 2278) renal complications, the D allele was significantly associated with diabetic nephropathy (p < 0.0001) in a dominant model (summary odds ratio 1.32, 95 % confidence interval: 1.15 to 1.51). There was no significant evidence against homogeneity of the odds ratios (χ ² = 18.9, 20 df; p = 0.53). The association was significant both in non-insulin-dependent (p < 0.005) and in insulin-dependent diabetes mellitus (p < 0.05). Likewise, in a total of 2010 diabetic patients with (n = 1008) and without (n = 1002) retinopathy, there was no association of the I/D polymorphism with diabetic retinopathy. These data suggest that the ACE I/D polymorphism affects the risk for diabetic nephropathy, but not for diabetic retinopathy. [Diabetologia (1998) 41: 47–53] Received: 30 April 1997 and in revised form: 31 July 1997     

The presence of genetic hypertension stimulates early renal accumulation of fibronectin in experimental diabetes mellitus

Aims/hypothesis: To investigate the interaction between hypertension and diabetic nephropathy, we studied the renal accumulation of fibronectin in a genetic model of hypertension with streptozotocin-induced diabetes mellitus. Methods: Spontaneously hypertensive rats (SHR) and their genetically normotensive control Wistar Kyoto rats (WKY) were studied at 4 weeks of age. The rats were killed 20 days after the induction of diabetes mellitus. The renal accumulation of fibronectin was estimated using Western blot analysis as well as immunofluorescence technique and confocal microscopy. Results: Blood glucose concentrations were similar in diabetic SHR rats (27 ± 3.3 mmol/l) and WKY rats (25.5 ± 2.7 mmol/l). The systolic blood pressure was higher in both groups of SHR rats than in the control rats. The abundance of renal fibronectin as detected by Western blot analysis was (p < 0.05) higher in the diabetic SHR rats (41.4 ± 15.0 densitometric U, n = 8) than in the control rats, and no difference was observed between diabetic and control WKY rats (20.8 ± 6.2, n = 8) and (27.8 ± 17.2, n = 8). The mean peak intensity of fibronectin signal within the glomerulus as estimated by confocal microscopy was higher (p < 0.05) in the diabetic SHR rats (32.9 ± 3.5) than in control SHR rats (11.9 ± 5.7) or diabetic (7.4 ± 2.2) and control (15.2 ± 7.9) WKY rats. Conclusion/interpretation: In experimental diabetes the presence of genetic hypertension promotes earlier accumulation of renal fibronectin, a matrix protein implicated in renal glomerulosclerosis. [Diabetologia (2001) 44: 2088–2091] Received: 11 January 2001 and in revised form: 30 May 2001     

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     

Urinary mRNA expression of <Emphasis Type="Italic">ACE</Emphasis> and <Emphasis Type="Italic">ACE2</Emphasis> in human type 2 diabetic nephropathy

Aims/hypothesis The interplay of ACE and type 2 ACE (ACE2) has been recognised as playing an important role in the tissue renin–angiotensin system within the kidney. In the present study, we measured urinary mRNA expression of ACE and ACE2 in patients with type 2 diabetic nephropathy. Methods We studied 50 patients with diabetic nephropathy: 26 were being treated by ACE inhibitor (ACEI) alone (ACEI group), the other 24 by ACEI and angiotensin-receptor blocker (ARB) (ACEI+ARB group). mRNA expression of ACE and ACE2 was measured by real-time quantitative RT-PCR at 0 and 12 weeks. All patients were then followed for 56 weeks. Results Proteinuria correlated significantly with urinary ACE (r = 0.454, p = 0.001) and ACE2 expression (r = 0.651, p < 0.001). Urinary ACE2 expression correlated with estimated GFR (r = −0.289, p = 0.042). In the ACEI group, there was a significant inverse correlation between the rate of GFR decline and urinary ACE2 expression at baseline (r = −0.423, p = 0.031) as well as at 12 weeks (r = −0.395, p = 0.046). In contrast, there was no significant correlation between the rate of GFR decline and urinary ACE2 expression at baseline or at 12 weeks in the ACEI+ARB group. The rate of GFR decline did not correlate with the baseline urinary ACE expression of either group. Conclusion/interpretation There was a relationship between urinary mRNA expression of ACE2 and the degree of proteinuria. The physiological implication and possibility of clinical application of quantifying urinary ACE2 expression require further study.     

The effects of glucose-induced oxidative stress on growth and extracellular matrix gene expression of vascular smooth muscle cells

Summary Vascular smooth muscle cell (VSMC) dysfunction plays a role in diabetic macrovasculopathy and this may include abnormalities in growth characteristics and the extracellular matrix. As the actual mechanisms by which glucose induces VSMC dysfunction remain unclear, the aim of this study was to assess the potential role of glucose-induced oxidative stress. Porcine aortic VSMCs were cultured for 10 days in either 5 mmol/l normal glucose or 25 mmol/l D-glucose (high glucose). There was evidence of oxidative stress as indicated by a 50 % increase in intracellular malondialdehyde (p < 0.05), increased mRNA expression of CuZn superoxide dismutase and Mn superoxide dismutase (by 51 % and 37 % respectively, p < 0.01) and a 50 % decrease in glutathione in 25 mmol/l D-glucose (p < 0.001). Growth was increased by 25.0 % (p < 0.01). mRNA expression of extracellular matrix proteins (collagens I, III, IV and fibronectin) was not altered by high glucose in these experimental conditions. Repletion of glutathione with N-acetyl L-cysteine (1 mmol/l) in VSMC grown in high glucose was associated with reduction in malondialdehyde and restored growth to that of normal glucose. The water soluble analogue of vitamin E, Trolox (200 μmol/l), reduced malondialdehyde concentrations, but had no effect on glutathione depletion or the increased growth rate seen with high glucose. The addition of buthionine sulphoximine (10 μmol/l) to VSMC cultured in normal glucose reduced glutathione, increased malondialdehyde and increased growth to a similar extent as that found in high glucose alone. These results suggest that thiol status, rather than lipid peroxides, is a key factor in modulating VSMC growth and that mRNA expression of extracellular matrix proteins is not increased in VSMC under conditions of glucose-induced oxidative stress. [Diabetologia (1998) 41: 1210–1219] Received: 26 November 1997 and in revised form: 28 April 1998     

Aliskiren,a novel renin inhibitor,is renoprotective in a model of advanced diabetic nephropathy in rats

AIMS/HYPOTHESIS: Blockade of the renin-angiotensin system (RAS) with either ACE inhibitors or angiotensin receptor blocker is a key therapeutic strategy in slowing progression of diabetic nephropathy. Interruption of the RAS may also be achieved by blocking the activity of renin, the rate-limiting step in angiotensin II biosynthesis. However, it is not known whether drugs in this class also reduce the structural and functional manifestations of diabetic nephropathy. METHODS: Using diabetic transgenic (mRen-2)27 rats, a rodent model of advanced diabetic nephropathy, we compared the efficacy of the renin inhibitor, aliskiren (10 mg kg(-1) day(-1) by osmotic mini-pump), with the ACE inhibitor, perindopril (0.2 mg kg(-1) day(-1) in drinking water), over a 16 week period. RESULTS: Both perindopril and aliskiren reduced blood pressure, albuminuria and structural injury in experimental diabetic nephropathy, although not to the same extent. Aliskiren, at the dose used, did not reduce systemic blood pressure as much as perindopril, but both compounds were equally effective in reducing albuminuria and glomerulosclerosis in diabetic animals. The magnitude of interstitial fibrosis was attenuated to a greater degree by aliskiren than by perindopril. CONCLUSIONS/INTERPRETATION: These findings suggest that therapies aimed at different targets within the RAS may not have identical effects in attenuating structural injury in experimental diabetic nephropathy.     

Influence of hypertension on serum concentration of type IV collagen antigens in streptozotocin-diabetic and non-diabetic rats

Summary The serum concentration of 7S collagen was measured radioimmunologically as a marker of basement membrane type IV collagen synthesis in diabetic and nondiabetic rats with Goldblatt hypertension. In non-diabetic rats the 7S collagen level was significantly raised after induction of hypertension (51%; p<0.001), and showed a positive correlation with relative heart weight as an integral parameter of hypertension (r= 0.63; p<0.01). In diabetic rats, which displayed a 7S collagen concentration roughly 2.5 times as high as the metabolically normal animals, the 7S collagen level was 27% higher in the hypertensive animals (p<0.01). There was no correlation with blood pressure or heart weight, but only a positive correlation with blood glucose (r=0.51; p<0.05). The results indicate that haemodynamic alterations may alter basement membrane collagen metabolism. However, type IV collagen metabolism in diabetes is influenced to a greater extent by metabolic than by haemodynamic factors.     

Type IV collagen antigens in serum of diabetic rats: a marker for basement membrane collagen biosynthesis

Summary The nature of type IV collagen antigens in the serum of streptozotocin diabetic rats was studied using radioimmunoassays for the N-terminal (7S-collagen) and C-terminal domain of type IV collagen. Type IV collagen antigen crossreacting with antibodies to the C-terminal domain was elevated from 32.0±5.36 ng/ml (n=10) in serum of normal rats to 94.9±24.5 ng/ml (n=10,P<0.0001) in serum of streptozotocin diabetic rats and could be normalized to 40.1±8.30 ng/ml (n=18) by insulin treatment. Molecular sieve chromatography of serum demonstrated a high molecular weight fraction containing the C-terminal and N-terminal domains and smaller material containing only the N-terminal domain. Degradation of the high molecular weight material by collagenase indicates that it consists of intact collagen type IV. Its relative proportion increased from 42% to 54% 4 weeks after diabetes induction. Together with unaltered clearance rates of 7S collagen in normal and diabetic rats, the data suggest that the increase of collagen type IV antigens in diabetic states reflects increased synthesis of collagen type IV.     

Serum 7S domain of type IV collagen levels in essential hypertension and hypertensive type 2 diabetic patients

Metabolic alteration of Type IV collagen occurs in micro- or macrovascular basement membrane of diabetic patients. Hypertension, a risk factor for clinical progression of diabetic vascular disease, may influence this metabolic alteration. The object of this study was to evaluate the serum 7S domain of type IV collagen (7S-collagen) levels in patients with essential hypertension and in Type 2 diabetic patients with or without hypertension and to investigate the relationship between the type IV collagen metabolism and the arterial blood pressure. Serum 7S-collagen levels in 18 patients with essential hypertension were significantly higher than in 24 normal subjects (4.2 ± 0.5 vs 3.6 ± 0.4 ng ml⁻¹ p < 0.01). Serum 7S-collagen levels in 28 normotensive diabetic patients (4.2 ± 0.5 ng ml⁻¹) were significantly higher than in normal subjects (p < 0.01). The serum 7S-collagen levels were significantly higher in 22 diabetic patients with hypertension (4.8 ± 0.6 ng ml⁻¹) than in the other groups. There was a significant correlation between the serum 7S-collagen levels and the systolic blood pressure in cases with essential hypertension (r = 0.59, p < 0.001) and in all diabetic patients (r = 0.52, p < 0.001), suggesting that elevation of the systolic blood pressure may influence the type IV collagen metabolism of vascular basement membrane. We conclude that the metabolic alteration of basement membrane occurring in patients with diabetes mellitus may worsen in the presence of high systolic blood pressure. © 1997 by John Wiley & Sons, Ltd.