Both in patients with IDDM (1) and in healthy control subjects (2,3), increased urinary albumin excretion rate (AER) is associated with high relative morbidity and mortality. In IDDM patients, genetic susceptibility factors are most likely contributing to an increased AER (4,5) resulting in a cumulative incidence of nephropathy (AER >300 mg/24 h) of -30% (6). So far, candidate genes, proposed as susceptibility markers linked to abnormal albuminuria, have been identified because of the knowledge of pathophysiological events related to diabetic nephropathy. Heparan sulfate proteoglycan (HSPG, i.e., Perlecan) constitutes an integrated part of the glomerular basement membrane. It consists of a central core protein to which anionie sulfated polysaccharide chains (heparan sulfate [HS]) are linked (7), thus contributing to the negative charge of the glomerular filtration barrier and thereby indirectly to the composition of the glomerular filtration product (8,9). In IDDM patients, it has been demonstrated that increased AER is reduced by the administration of heparin (10) most likely because of a stimulating effect on the HS synthesis (11).

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 Hind III restriction polymorphism of the collagen IV alpha 1-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, Hind III restriction fragment length analysis revealed a bi allele polymorphism visualized by Southern hybridization with a cDNA probe recognizing the collagen IV alpha 1-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 Hind III restriction polymorphism of the collagen IV alpha 1-chain gene is not associated with diabetic nephropathy, diabetic retinopathy or with diabetes per se.

The aim of this follow-up study was to assess whether slightly elevated urinary albumin excretion, i.e., microalbuminuria, precedes development of atherosclerotic vascular disease in IDDM. Out of 259 IDDM-patients 30 developed vascular disease during 2,457 person-years. Microalbuminuria was significantly predictive of vascular disease (hazard ratio (95% confidence interval) 1.06 (1.02-1.18) per 5 mg/24 hours increase in urinary albumin excretion; p = 0.002). The predictive effect was independent of age, sex, blood pressure, tobacco smoking, serum concentrations of total-cholesterol, HDL-cholesterol, sialic acid, and von Willebrand factor, and of haemoglobin A1c, insulin dose, diabetes duration, and diabetic nephropathy (hazard ratio (95% confidence interval) 1.04 (1.01-1.08) per 5 mg/24 hours increase in urinary albumin excretion; p = 0.03). It is concluded that slightly elevated urinary albumin excretion is an independent predictor of atherosclerotic vascular disease in insulin-dependent diabetes mellitus.

In IDDM patients, an increased permeability of the glomerular capillaries has been associated with a general loss of negatively charged heparan sulfate proteo-glycans (HSPGs) within basement membranes (BMs). In the present study, we used immunohistochemical staining to quantify heparan sulfate (HS), HSPG core protein, and collagen IV in capillary basement membranes of skeletal muscle biopsies taken from 9 healthy control subjects (C) and 20 IDDM patients: 7 with normal albumin excretion rate (<30 mg/24 h) (D0), 5 with incipient nephropathy (albumin excretion rate 30–300 mg/24 h) (Dx), and 8 with clinical nephropathy (albumin excretion rate >300 mg/24 h) (D2). In the capillaries, staining was measured by a scanning and integrating microspectrophotometer. A significant difference in the absorbance of HS was found among the four subgroups (means ± SD): 0.477 ± 0.082 (C), 0.627 ± 0.031 (D0), 0.542 ± 0.098 (D1, and 0.371 ± 0.118 (D2) (P = 0.006). Similarly, an overall significant difference in the absorbance of collagen IV was demonstrated (means ± SD): 0.836 ± 0.111 (C), 0.838 ± 0.300 (D0), 0.970 ± 0.173 (D1), and 0.512 ± 0.248 (D2) (P = 0.02). No statistical difference in the absorbance of core protein was demonstrated among the groups. Within the diabetic groups, HS was inversely correlated to albumin-uria (r = −0.76, P = 0.003) and albuminuria corrected for creatinine clearance (r = −0.69, P = 0.008). Because, in IDDM patients with albuminuria, alterations of the content of HS and collagen IV within the capillary BM have been demonstrated immunohistochemically, not only in the glomerular filtration barrier, but also in the skeletal muscle capillary BM, we suggest that these changes reflect universal quantitative or qualitative alterations within the capillary filtration barrier.

OBJECTIVE The high risk for cardiovascular disease in IDDM patients with nephropathy may be mediated by abnormal function of the vascular wall. We investigated whether heparin was able to modulate markers of vascular wall and hemostatic function in patients with incipient nephropathy. RESEARCH DESIGN AND METHODS Thirty-five IDDM patients with incipient nephropathy were randomized to treatment with placebo, unfractioned heparin, or low molecular weight heparin in a double-blind trial. The treatment was given as 1 h of conventional intravenous high-dose treatment and in a conventional subcutaneous low-dose regime for 3 months. Transcapillary escape rate of albumin and plasma levels of von Willebrand factor, fibrinogen, prothrombin fragment 1 + 2, thrombin-antithrombin III complexes, tissue type plasminogen activator, tissue plasminogen activator inhibitor type 1, total cholesterol, HDL cholesterol, and triglycerides were measured before and after treatment. Of the patients, 31 completed the study. RESULTS We found no significant effect of heparin on markers of vascular wall and hemostatic function by any of the treatments. CONCLUSIONS Treatment with high- or low-dose heparin induced no modulation of markers of vascular wall or hemostatic function in IDDM patients with incipient diabetic nephropathy.

OBJECTIVE To investigate the long-term course of glomerular filtration rate (GFR) in IDDM patients with microalbuminuria in order to identify patients with stable or declining kidney function over a 5-year study. RESEARCH DESIGN AND METHODS Forty normotensive (129 ± 11/80 ± 8 mmHg) IDDM patients with persistent microalbuminuria (mean urinary albumin excretion [UAE] 84 mg/24 h [range 30–300]) were followed prospectively for 5 years of clinical examinations that included the measurement of GFR (51Cr-labeled EDTA clearance) at least once a year. The mean GFR at baseline was 120 ± 18 ml · min−1 · 1.73 m−2. RESULTS Using multiple regression analysis, the rate of decline in GFR was independently correlated to onset of diabetic nephropathy (P < 0.001) and systolic blood pressure (sBP) at baseline (P < 0.05). Increase in UAE was correlated to the mean HbA1c during the observation period. Out of 40 patients, 14 progressed to diabetic nephropathy (UAE > 300 mg/24 h). These patients had a significant reduction in GFR (mean −2.2 ± 3.8 ml · min−1 · year−1; P = 0.05), while GFR remained stable in the remaining 26 patients with nonprogressive microalbuminuria (change in GFR 0.5 ± 2.1 ml · min−1 · year−1; NS). The difference in the rate of decline of GFR was significant (mean 2.7 ml · min−1 · year−1; P < 0.05). CONCLUSIONS Normotensive IDDM patients with nonprogressive microalbuminuria have a stable GFR. Progression of UAE to diabetic nephropathy heralds a progressive loss of kidney function. Efforts should be made to prevent the progression from microalbuminuria to diabetic nephropathy in every IDDM patient with microalbuminuria.

In patients with insulin-dependent diabetes mellitus (IDDM), albuminuria reflects widespread vascular dysfunction. Albuminuria has been associated to defects of heparan sulfate proteo- glycan (HSPG) within the extracellular matrix. Our hypothesis is that loss of HSPG in vascular walls reduces the HSPG-bound lipoprotein- lipase activity (LPLA), thereby causing elevated levels of plasma triglyceride (TG) seen in IDDM patients with albuminuria. The aim of the present study was to evaluate whether LPLA in muscle capillaries could be related to TG in IDDM patients with and without albuminuria. This is a cross-sectional study including ten healthy control subjects (group C), nine patients with IDDM and urinary albumin excretion rate (AER) of 30 mg/24 h or less (group D0) and 20 patients with IDDM and AER greater than 30 mg/24 h (group DA). Muscle LPLA, plasma TG, total cholesterol, high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), and very-low-density lipoprotein cholesterol (VLDL) were measured. Between groups no difference in total cholesterol, TG, VLDL, and LDL was found. In patients with albuminuria, LPLA was reduced compared to controls, however, the difference between the groups was not statistically significant [median (range)] 35.9 mU/g (20.4–103) versus 44.6 mU/g (28.2–57.2) and 40.9 mU/g (21.7–53.5) in group DA, C, and D0, respectively, p = 0.76. AER was not correlated to LPLA. An overall negative correlation between TG and LPLA was found; r = −0.33, p = 0.04, supported by an overall significant positive correlation between LPLA and HDL; r = 0.32, p = 0.045. We conclude that, in insulin-dependent diabetes mellitus, skeletal muscle lipoprotein-lipase activity is associated with plasma triglyceride, while an association between lipoprotein-lipase activity and urinary albumin excretion is questionable.

The effects of heparin and aminoguanidine on glomerular basement membrane thickening were studied in streptozotocin diabetic Sprague-Dawley rats. A placebo-treated group and a non-diabetic group served as controls. All diabetic rats remained severely hyperglycaemic (23 mmol/l) throughout the 8-month study period. At the end of this time relative kidney weight was significantly increased in diabetic control rats (4.9 +/- 0.5 g/kg b.w.) compared with non-diabetic rats (3.3 +/- 0.3 g/kg). This increase was not affected by the intervention treatments. Glomerular basement membrane thickness increased 32% in diabetic control rats (240 +/- 24 nm) compared with non-diabetic rats (182 +/- 20 nm). This increase was prevented by s.c. treatment with both unfractionated and low molecular weight heparins, while basement membrane thickness was the same in animals treated with oral heparins and aminoguanidine and untreated diabetic rats. Macroscopic malignant kidney tumours were seen in three aminoguanidine-treated rats. In conclusion, subcutaneously administered heparin prevents diabetes-induced glomerular basement membrane thickening.

OBJECTIVE Elevated concentrations of serum sialic acid, a potent cardiovascular risk factor in the general population, have been found in patients with IDDM and microalbuminuria. We investigated whether a coincidence exists between the increase of sialic acid concentrations and albuminuria in the transition from normoalbuminuria to microalbuminuria. Furthermore, the predictability of increased sialic acid as well as von Willebrand factor (vWF) and total and HDL cholesterol concentrations in development of persistent microalbuminuria in IDDM was investigated. RESEARCH DESIGN AND METHODS This 10-year prospective study was carried out in a cohort of 209 IDDM patients with normoalbuminuria at baseline. RESULTS Of the cohort, 198 patients completed the follow-up period and 27 developed persistent microalbuminuria (urinary albumin excretion rate [UAER] ≥ 30 mg/24 h). A coincident increase of UAER and serum sialic acid concentration was seen before persistent microalbuminuria was diagnosed. Elevation of serum sialic acid concentrations in those who later developed microalbuminuria occurred 3 years before the diagnosis of persistent microalbuminuria. Baseline serum sialic acid concentrations were significantly higher in the group of patients who later developed microalbuminuria than in the group who remained normoalbuminuric (2.02 ± 0.41 vs. 1.85 ± 0.31 mmol/l [means ± SD], P < 0.05). Baseline serum sialic acid concentration correlated significantly with HbA1c, UAER, blood pressure, total cholesterol, HDL cholesterol, and vWF and was significantly predictive for development of microalbuminuria (hazards ratio [95% CI], 3.1 [1.2–8.1]; P = 0.02) after adjustments for sex, duration of diabetes, smoking, blood pressure, vWF, total cholesterol, and HDL cholesterol. Adjustment for the effects of HbA1c and UAER, however, canceled out the predictive effect of serum sialic acid. CONCLUSIONS UAER and serum sialic acid concentration increase coincidentally before the onset of persistent microalbuminuria. An increased serum sialic acid concentration is predictive for the onset of microalbuminuria independent of age, sex, diabetes duration, smoking, blood pressure, vWF, and total HDL cholesterol.

Objective-To examine whether slightly elevated urinary albumin excretion precedes development of atherosclerotic vascular disease in patients with insulin dependent diabetes independently of conventional atherogenic risk factors and of diabetic nephropathy.Design-Cohort study with 11 year follow up.Setting-Diabetes centre in Denmark.Subjects-259 patients aged 19-51 with insulin dependent diabetes of 6-34 years' duration and without atherosclerotic vascular disease or diabetic nephropathy at baseline.Main outcome measures-Baseline variables: urinary albumin excretion, blood pressure, smoking habits, and serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor. End point: atherosclerotic vascular disease assessed by death certificates, mailed questionnaires, and hospital records.Results-Thirty patients developed atherosclerotic vascular disease during follow up of 2457 person years. Elevated urinary albumin excretion was significantly predictive of atherosclerotic vascular disease (hazard ratio 1.06 (95% confidence interval 1.02 to 1.18) per 5 mg increase in 24 hour urinary albumin excretion, P=0.002). Predictive effect was independent of age; sex; blood pressure; smoking; serum concentrations of total cholesterol, high density lipoprotein cholesterol, sialic acid, and von Willebrand factor; level of haemoglobin A(1c); insulin dose; duration of diabetes; and diabetic nephropathy (hazard ratio 1.04 (1.01 to 1.08) per 5 mg increase in 24 hour urinary albumin excretion, P=0.03).Conclusion-Slightly elevated urinary albumin excretion independently predicted atherosclerotic vascular disease in patients with insulin dependent diabetes.

The effects of heparin and aminoguanidine on glomerular basement membrane thickening were studied in streptozotocin diabetic Sprague-Dawley rats. A placebo-treated group and a non-diabetic group served as controls. All diabetic rats remained severely hyperglycaemic (23 mmol/l) throughout the 8-month study period. At the end of this time relative kidney weight was significantly increased in diabetic control rats (4.9±0.5 g/kg b.w.) compared with non-diabetic rats (3.3±0.3 g/kg). This increase was not affected by the intervention treatments. Glomerular basement membrane thickness increased 32% in diabetic control rats (240±24 nm) compared with non-diabetic rats (182±20 nm). This increase was prevented by s.c. treatment with both unfractionated and low molecular weight heparins, while basement membrane thickness was the same in animals treated with oral heparins and aminoguanidine and untreated diabetic rats. Macroscopic malignant kidney tumours were seen in three aminoguanidine-treated rats. In conclusion, subcutaneously administered heparin prevents diabetes-induced glomerular basement membrane thickening.

Patients with insulin-dependent diabetes mellitus (IDDM) and albuminuria are at high risk for severe micro- and macrovascular complications. Diabetic vascular complications are characterized by structural alterations of extracellular matrix (ECM) components in glomeruli and large vessel walls, namely, accumulation of collagen IV, collagen VI and fibronectin and relative decrease of heparan sulphate proteoglycan (HSPG). We hypothesize that the defect remodelling of ECM contributing to nephropathy and macrovascular disease is induced by overproduction of transforming growth factor-beta (TGF-β). Recent reports indicate that hyperglycaemia, increased intraglomerular pressure, and glycated proteins potentially induce overproduction of TGF-β in diabetes. TGF-β stimulates production of ECM components such as collagen IV, fibronectin, proteoglycans (decorin and biglycan) without increasing HSPG. TGF-β overproduction leads to glomerulosclerosis and TGF-β is a causal factor in myointimal hyperplasia after balloon injury of carotid artery. It mediates angiotensin II modulator effect on smooth muscle cell growth. These findings may indicate TGF-β overproduction to be a common pathogenetic step explaining the well-known association between micro- and macrovascular complications in diabetic patients. TGF-β antagonists, such as decorin, betaglycan, and possibly also heparin, might be potential candidates for future therapy to prevent diabetic vascular disease.

The pathophysiologic mechanism behind microalbuminuria, a potential atherosclerotic risk factor, was explored by measuring fractional clearances of four endogenous plasma proteins of different size and electric charge (albumin, beta 2-microglobulin, immunoglobulin G, and immunoglobulin G4). Twenty-eight clinically healthy individuals with microalbuminuria, defined as a urinary albumin excretion of 6.6-150 micrograms min-1, and 60 matched control subjects were studied. Fractional immunoglobulin G clearance was higher (geometric means (95% confidence intervals)) 3.0 (2.3-3.9) x 10(-6), n = 28, vs. 2.1 (1.8-2.4) x 10(-6), n = 60; P = 0.02), whereas the ratio immunoglobulin G clearance/immunoglobulin G4 clearance was lower (geometric means (95% confidence intervals)) 1.8 (1.4-2.2), n = 28, vs. 2.3 (2.0-2.5), n = 60; P = 0.03) in microalbuminuric than in normoalbuminuric individuals. Fractional beta 2-microglobulin clearance was similar in the two groups. Since total IgG and the IgG4 subclass are of similar size and configuration but electrically neutral and negative, respectively; these findings indicate that microalbuminuria is associated with decreased size- and charge-selectivity of the glomerular vessel wall. Hypothetically, such alterations may reflect generalized vascular abnormalities linking microalbuminuria to atherogenesis.

The urinary albumin excretion rate (AER) in a subgroup of patients with insulin-dependent diabetes mellitus (IDDM) steadily increases. In these patients a concomitant reduction of the glomerular charge selectivity index (SI) has been demonstrated. The aim of the present study was to evaluate whether diurnal variation in AER could be related to a diurnal variation in SI and/or a diurnal blood pressure variation. Thirty-three patients with IDDM, 27 with normal albumin excretion (AER < 20 micrograms/min; group D(o)) and six with incipient nephropathy (AER from 20 to 200 micrograms/min; group DA), were studied. AER and SI (renal clearance ratio of total-IgG/IgG4) were measured in three different urine collecting periods: period A (8:00 a.m. to 12:00 a.m.), period B (12:00 a.m. to bedtime) and period C (bedtime to 8:00 a.m.). A significant increase in SI was seen during the nighttime: period A, 1.6 (0.2 to 3.8; mean, range); period B, 1.7 (0.3 to 3.0); and period C, 2.0 (0.2 to 4.0); P = 0.01. Corresponding to this observation, an overall significant decrease in AER was found: period A, 10 (3 to 137) micrograms/min (median, range); period B, 8 (3 to 84) micrograms/min; and period C, 5 (0 to 78) micrograms/min; P < 0.001. In all three sampling periods a negative correlation was found between AER and SI. When group D(o) was analyzed alone, the results were similar. Diurnal variation in blood pressure was significantly positively correlated with AER in group DA, but was not correlated to variation in AER in D(o). We suggest that in normoalbuminuric IDDM patients diurnal variation in AER is related to diurnal variation in SI.

Abstract. Objectives. Elevated serum sialic acid concentration is a strong predictor of cardiovascular mortality in non-diabetic subjects. Because patients with insulin-dependent diabetes mellitus (IDDM) and albuminuria have a highly increased cardiovascular morbidity and mortality, we hypothesized that IDDM patients with albuminuria would have an increased concentration of serum sialic acid. Design. Cross-sectional study. Setting. Outpatient clinic at Steno Diabetes Centre, Gentofte, Denmark. Subjects. Twenty-six non-diabetic controls and 74 IDDM patients with normoalbuminuria (urinary albumin excretion [UAE] < 30 mg 24 h −1 ; n = 37), incipient nephropathy (UAE 30–300 mg 24 h −1 ; n = 20) and clinical nephropathy (UAE > 300 mg 24 h −1 ; n = 17), matched for sex, age and body mass index (BMI). Main outcome measures. Serum sialic acid concentration, concurrent fasting blood glucose, glycated haemoglobin (HbA1c), serum creatinine, plasma fibrinogen and erythrocyte sedimentation rate. Results. Normoalbuminuric patients had a higher serum sialic acid concentration (mmol L −1 ) than non-diabetic controls (1.83 ± 0.24 vs. 1.67 ± 0.26; P < 0.02). Serum sialic acid concentration was further increased in patients with incipient nephropathy (2.02 ± 0.37; P < 0.03) and in patients with clinical nephropathy (2.13 ± 0.33; P < 0.002) compared with normoalbuminuric IDDM patients. Serum sialic acid correlated strongly with plasma fibrinogen ( r = 0.78; P < 0.0001) and erythrocyte sedimentation rate ( r = 0.62; P < 0.0001). In a multiple regression analysis including UAE, retinopathy status, fasting blood glucose, HbA1c, mean blood pressure, serum creatinine, age, BMI, duration and smoking, UAE and fasting blood glucose were the independent variables which correlated significantly with serum sialic acid concentration ( P < 0.0001 and P < 0.05, respectively). Conclusion. Serum sialic acid is elevated in IDDM especially in albuminuric patients. Whether elevated serum sialic acid is predictive for early diabetic nephropathy and cardiovascular disease in IDDM has to be shown in the future.

The purpose of this study was to describe the clinical course in patients followed right from the onset of microalbuminuria to the development of diabetic nephropathy. A 10‐year prospective follow‐up of 209 consecutive normotensive insulin‐dependent diabetic patients with normal urinary albumin excretion (UAE <30 mg 24 h−1), age 34 (18–50) years and duration of diabetes 17 (10–30) years was performed. Twenty‐four‐hour urinary albumin excretion was measured every 4 months, glycated haemoglobin and supine blood pressure was measured annually. Two‐hundred (96%) patients completed 10 (range 5–10) years follow‐up. Twenty‐nine (15%) patients developed persistent microalbuminuria (UAE 30–300 mg 24 h−1). Eight of these have progressed to nephropathy and one had died of diabetic nephropathy. Multiple stepwise logistic regression analysis demonstrated baseline urinary albumin excretion (p = 0.0016) and glycated haemoglobin (p = 0.0014) but not blood pressure as predictors of development of microalbuminuria within the following 10 years. The median annual increase in urinary albumin excretion was 27 (range 17–65)% in the 29 patients developing microalbuminuria. The median duration from onset of microalbuminuria to development of nephropathy was 7 years. The prevalence of patients receiving antihypertensive treatment (BP > 140/90 mmHg) increased from 10% at onset of microalbuminuria to 45% 4 years after onset of microalbuminuria. The prevalence of patients with proliferative retinopathy increased from 7% at onset of microalbuminuria to 28% 4 years after onset of microalbuminuria. The incidence of persistent microalbuminuria in normotensive insulin‐dependent diabetic patients is 2% per year, and development of persistent microalbuminuria is a strong predictor of overt nephropathy. Development of hypertension is frequent in the early course of microalbuminuria and treatment modalities for normotensive patients with microalbuminuria are urgently needed.