Prevalence of left ventricular hypertrophy in Type I diabetic patients with diabetic nephropathy

Summary The increased mortality of patients with diabetic nephropathy is mainly due to cardiovascular disease and end stage renal failure. Left ventricular hypertrophy is an independent risk factor for myocardial ischaemia and sudden death. The aim of our cross-sectional study was to evaluate left ventricular structure and function in Type I (insulin-dependent) diabetic patients with diabetic nephropathy. M-mode and Doppler echocardiography were done on 105 Type I diabetic patients with diabetic nephropathy [61 men, age (means ± SD) 44 ± 9 years, and albuminuria [median(range)] 567(10–8188) mg/24 h, serum creatinine 109 (53–558) μmol/l], and 140 Type I diabetic patients with persistent normoalbuminuria [79 men, 47 ± 10 years, urinary albumin excretion rate 8 (0–30) mg/24 h, and serum creatinine 81 (55–121) μmol/l]. Patients with and without nephropathy were comparable with respect to sex, body mass index, and duration of diabetes. Arterial blood pressure was slightly higher in patients with nephropathy: 140/79 ± 17/9 mm Hg vs 134/78 ± 15/8 mm Hg, p < 0.01, and the majority of proteinuric patients received antihypertensive drugs, 84 vs 17 %, respectively, p < 0.001. Left ventricular mass index was increased in the nephropathic group (means ± SD) 100.6 ± 23.9 g/m² compared with the normoalbuminuric group 91.4 ± 21.9 g/m², p = 0.002. Left ventricular hypertrophy was found more often in patients with nephropathy 23 (14–31)% compared with patients with normoalbuminuria 9 (5–14)%, p < 0.005. Diastolic function, assessed by the ratio between the peak diastolic velocity and the peak atrial systolic velocity (E/A ratio) and isovolumic relaxation time, was reduced in patients with vs without nephropathy: 1.17 ± 0.29 vs 1.34 ± 0.32, and 81.7 ± 16.5 vs 74.6 ± 14.5, p < 0.001 and p = 0.002, respectively. Systolic function was about the same and normal in both groups. Our study suggests that an increase in left ventricular mass index and a decrease in diastolic function occurs early in the course of diabetic nephropathy. [Diabetologia (1999) 42: 76–80] Received: 16 April 1998 and in final revised form: 5 August 1998     

Renal kallikrein in diabetic patients with hypertension accompanied by nephropathy

Summary We measured the 24-h excretion of urinary kallikrein in 27 patients with Type 2 (non-insulin-dependent) diabetes and in 10 normal control subjects. Mean (± SD) kallikrein excretion in diabetic patients with nephropathy (6.2±2.4 naphthyl units (NU)/day,n=13) was significantly lower than in control subjects (12.8±3.4NU/day,p<0.01) and in diabetic patients without nephropathy (9.4±3.4NU/day,n=14,p<0.05). Kallikrein excretion in hypertensive diabetic patients with nephropathy (5.1±1.6 NU/day,n=8) was significantly lower (p<0.05) than in normotensive patients with nephropathy (8.3±2.1 NU/day,n=5). There were no significant differences in kallikrein excretion rate (24-h excretion of urinary kallikrein/24-h creatinine clearance) among control subjects (9.9±4.3 NU/ml), diabetic patients with (9.0±3.2 NU/ml) and without (9.3±3.5 NU/ml) nephropathy. However, kallikrein excretion rate in hypertensive diabetic patients with nephropathy (7.7±3.3 NU/ml) was significantly lower (p<0.05) than in normotensive diabetic patients with nephropathy (11.8 ±2.0 NU/ml,n=10). Respective basal and post-stimulated (with intravenous furosemide 40 mg plus 60 min ambulation) plasma aldosterone concentrations measured in control subjects and in hypertensive diabetic patients with nephropathy were similar and increased to the same extent in the 2 groups (5.5±3.2 versus 5.3±3.2 and 9.3±2.6 versus 10.5±3.4 ng/ml), although the respective plasma renin activity tended to be lower in diabetic patients than in control subjects (0.7±0.6 versus 1.3±0.9 and 1.8±1.8 versus 3.0±2.6 ng⁻¹ · ml⁻¹ · h⁻¹). The results indicate that urinary kallikrein excretion is decreased in hypertensive diabetic patients with nephropathy, and that the decrease might not be attributable to an altered renin-aldosterone system.     

Macro-microangiopathy and endothelial dysfunction in NIDDM patients with and without diabetic nephropathy

Summary The Steno hypothesis suggests that albuminuria reflects widespread vascular damage (proliferative retinopathy and severe macroangiopathy) due to a generalized vascular (endothelial) dysfunction. We assessed this concept in NIDDM (non-insulin-dependent diabetic) patients with (13 female/39 male, age 60 ± 7 years, group 1) and without (12 female/41 male, age 61 ± 7 years, group 2) diabetic nephropathy compared to matched non-diabetic subjects (7 female/15 male, age 58 ± 8 years, group 3). A 12-lead ECG was recorded and coded blindly using the Minnesota Rating Scale; the World Health Organization cardiovascular questionnaire was used to assess past and present evidence of myocardial infarction, angina pectoris, stroke, and peripheral vascular disease (digital systolic blood pressure determination). The degree of diabetic retinopathy was scored from fundus photography. The following variables were measured: transcapillary escape rate of albumin (initial disappearance of intravenously injected ¹²⁵I-labelled human serum albumin), plasma concentrations of prorenin (radioimmunoassay) and serum concentrations of von Willebrand factor (enzyme-linked immunoadsorbent assay). Prevalence of ischaemic heart disease (ECG reading) (49/20/5)% and peripheral vascular disease as indicated by reduced systolic blood pressure on big toe (69/30/14)% was significantly higher in group 1 vs group 2 (p < 0.01) and in group 2 vs group 3 (p < 0.01), respectively. The prevalence and severity of retinopathy was higher in group 1 vs 2 (p < 0.01). Transcapillary escape rate of albumin (%/h) was elevated in group 1 and 2 as compared to control subjects: 7.9 (4.3–13.7); 7.4 (3.7–16.4) vs 6.0 (3.4–8.7), (p < 0.005), respectively. Plasma prorenin activity (IU/ml) was raised in group 1 and group 2 as compared to group 3: 272 (59–2405); 192 (18–813), and 85 (28–246), p < 0.001, respectively. Serum von Willebrand factor (IU/ml)was elevated in group 1 as compared to group 2 and 3: 2.07 (0.83–4.34); 1.60 (0.30–2.99) and 1.50 (1.00–2.38), p < 0.001, respectively. Our study demonstrated that NIDDM patients with and without albuminuria had increased transcapillary escape of albumin and raised prorenin activity, whereas only those with albuminuria had increased von Willebrand factor. Patients with NIDDM may have abnormal endothelial function in the absence of albuminuria. [Diabetologia (1996) 39: 1590–1597]     

Size- and charge selectivity of glomerular filtration in Type 1 (insulin-dependent) diabetic patients with and without albuminuria

Summary Albuminuria is the first clinical event in the development of diabetic nephropathy. We assessed glomerular charge- and size selectivity in 51 patients with Type 1 (insulin-dependent) diabetes mellitus of juvenile onset and 11 healthy individuals. Patients were allocated to five groups. The urinary albumin excretion rate was normal in group D1; 30–100 mg/24 h in group D2; 101–300 mg/24 h in group D3 and greater than 300 mg/24 h in groups D4 and D5. Group D5 had elevated serum creatinine (above 110 mol/l). Glomerular filtration rate and renal plasma flow were determined by constant infusion techniques and tubular protein reabsorption by excretion of 2-microglobulin. Charge selectivity was estimated from the IgG/IgG4 selectivity index. Size selectivity was measured by dextran clearance. Dextran was measured by refractive index detection after fractionation (2 Å fractions in the range 26–64 Å) by size exclusion chromatography. IgG/IgG4 selectivity index was significantly decreased in patients with albuminuria (p<0.001). The drop in IgG/IgG4 selectivity index was found in patients with minimal albuminuria (D2) and was not accompanied by any changes in tubular function or glomerular haemodynamics. Size selectivity was significantly altered only in patients with the most advanced nephropathy (D5) as reflected by an increase in the clearance of 62 Å dextran (p<0.04). We conclude that loss of glomerular charge selectivity precedes or accompanies the formation of new glomerular macromolecular pathways in the development of diabetic nephropathy.     

Renal functional reserve in IDDM patients

Summary The aim of this study was to determine whether renal functional reserve (RFR) is altered in insulin-dependent diabetic (IDDM) patients according to the stage of diabetic nephropathy. RFR was examined in 33 IDDM patients in similar glycaemic and metabolic control and compared to 12 healthy control subjects, during eight 1 h clearance periods prior to, during and after a 3-h stimulation by amino acid infusion (4.5 mg · kg⁻¹· min⁻¹). RFR was calculated as the difference between stimulated and baseline glomerular filtration rates (GFR). In 14 early normotensive diabetic patients with normal urinary albumin excretion, mean baseline GFR (133 ± 3 ml · min⁻¹· 1.73 m⁻²) was higher whereas RFR (10 ± 4 ml · min⁻¹· 1.73 m⁻²) was lower (p < 0.05) than in control subjects (113 ± 4 and 28 ± 2 ml · min⁻¹· 1.73 m⁻², respectively). In 10 normotensive patients who had lived with IDDM for 16 years and who had microalbuminuria, baseline GFR and RFR (109 ± 7 and 24 ± 6 ml · min⁻¹· 1.73 m⁻², respectively) were similar to those in control subjects. In 9 patients who had suffered IDDM for 23 years and had developed macroalbuminuria and hypertension, baseline GFR (78 ± 8 ml · min⁻¹· 1.73 m⁻²) was lower than in control subjects (p < 0.05) and RFR (8 ± 4 ml · min⁻¹· 1.73 m⁻²) was not significant. In addition, renal vascular resistance decreased significantly during infusion (p < 0.05) in microalbuminuric normotensive patients as well as in control subjects (by 9 ± 4 and 11 ± 4 mm Hg · l⁻¹· min⁻¹· 1.73 m⁻², respectively) but not in normoalbuminuric normotensive or macroalbuminuric hypertensive patients. These results indicate that microalbuminuric normotensive patients retain a normal RFR, whereas RFR is reduced or suppressed at two opposite stages of the disease: in normoalbuminuric normotensive patients with a high GFR and in macroalbuminuric hypertensive patients with a decreased GFR. This dissimilar impairment reveals permanent glomerular hyperfiltration in both early IDDM without nephropathy and IDDM with overt diabetic nephropathy, but not in IDDM with incipient nephropathy. [Diabetologia (1998) 41: 86–93] Received: 12 February 1997 and in final revised form: 28 August 1997     

Effects of dietary protein restriction on albumin and fibrinogen synthesis in macroalbuminuric type 2 diabetic patients

Aims/hypothesis Diabetic nephropathy is associated with hypoalbuminaemia and hyperfibrinogenaemia. A low-protein diet has been recommended in patients with diabetic nephropathy, but its effects on albumin and fibrinogen synthesis are unknown. Methods We compared the effects of a normal (NPD; 1.38 ± 0.08 g kg⁻¹ day⁻¹) or low (LPD; 0.81 ± 0.04 g kg⁻¹ day⁻¹) -protein diet on endogenous leucine flux (ELF), albumin and fibrinogen synthesis (l-[5,5,5,-²H₃]leucine infusion), and markers of inflammation in nine type 2 diabetic patients with macroalbuminuria. Six healthy participants on NPD served as control participants. Results In comparison with healthy participants, type 2 diabetic patients on an NPD had similar ELF, reduced serum albumin (38 ± 1.1 vs 42 ± 0.8 g/l; p < 0.05), similar fractional synthesis rates (FSR) and absolute synthesis rates (ASR) of albumin, and both increased plasma fibrinogen concentration [10.7 ± 0.6 vs 7.2 ± 0.5 μmol/l (3.64 ± 0.22 vs 2.45 ± 0.18 g/l); p < 0.05] and fibrinogen ASR [11.03 ± 1.17 vs 6.0 ± 1.8 μmol 1.73 m⁻² day⁻¹ (3.7 ± 0.4 vs 1.9 ± 0.3 g 1.73 m⁻² day⁻¹); p < 0.01]. After LPD, type 2 diabetic patients had the following changes in comparison with NPD: reduced proteinuria (2.74 ± 0.4 vs 4.51 ± 0.8 g/day; p < 0.05), ELF (1.93 ± 0.08 vs 2.11 ± 0.08 μmol kg⁻¹ min⁻¹; p < 0.05) and total fibrinogen pool; increased serum albumin (42 ± 1 vs 38 ± 1 g/l; p < 0.01) and albumin ASR (14.1 ± 1 vs 9.9 ± 1 g 1.73 m⁻² day⁻¹; p < 0.05); and reduced plasma IL-6 levels, which were correlated with albumin ASR (r = −0.749; p < 0.05). Conclusions/interpretation LPD in type 2 diabetic patients with diabetic nephropathy reduces low-grade inflammatory state, proteinuria, albuminuria, whole-body proteolysis and ASR of fibrinogen, while increasing albumin FSR, ASR and serum concentration. ISRCTN ID no: CCT-NAPN-16911     

Short-term treatment with ramipril normalizes renal haemodynamics and the natriuretic response to a sodium load in type 1 diabetic patients with early nephropathy

The objective of the present study was to determine whether acute inhibition of angiotensin converting enzyme (ACE) normalizes intrarenal sodium handling, renal haemodynamics and renal dopamine output in response to an i.v. NaCl infusion in type 1 diabetic patients with early nephropathy. Nine diabetic patients (aged 28±3 years) with elevated urinary albumin excretion (173±39 mg ⋅ min^–1) were studied. The effects of a 2-hour NaCl infusion (12.5 ml ⋅ kg^–1⋅ h^–1) on para-amino hippuric acid (PAH), inulin, lithium and sodium clearances as well as the urinary dopamine excretion were studied before and after 2 days of acute ACE inhibition. Fifteen healthy subjects (aged 34±1 years) served as controls. The results showed that 2 days of ACE inhibition improved the natriuretic response significantly (P<0.05) within the first 2 h following an i.v. NaCl load due to a normalization of the proximal tubular sodium handling. In control subjects urinary dopamine output increased by 14% (P<0.01) following i.v. NaCl infusion, whereas a blunted increase was seen in the diabetic patients, which tended to normalize following inhibition of ACE. In conclusion, this study demonstrates that patients with type 1 diabetes and early nephropathy display abnormalities in renal haemodynamics, natriuresis and urinary dopamine mobilization in response to a sodium load, which can be reversed by short-term inhibition of ACE. Received: 27 September 1996 / Accepted in revised form: 2 December 1996     

Plasma homocysteine is related to albumin excretion rate in patients with diabetes mellitus: a new link between diabetic nephropathy and cardiovascular disease?

Summary The high risk of cardiovascular disease in patients with diabetes mellitus, particularly in those with nephropathy, is not completely explained by classical risk factors. A high plasma homocysteine concentration is an independent risk factor for cardiovascular disease but information on its association with diabetes is limited. Fasting homocysteine concentrations were measured in the plasma of 165 diabetic patients (75 with insulin-dependent [IDDM]; 90 with non-insulin-dependent diabetes [NIDDM]) and 56 non-diabetic control subjects. Other measurements included the prevalence of diabetic complications, glycaemic control, lipid and lipoprotein levels, vitamin status and renal function tests. Patients with NIDDM had higher homocysteine levels than control subjects, whereas IDDM patients did not (9.2 ± 4.5 vs 7.7 ± 2 μmol/l, p < 0.01; and 7.0 ± 3 vs 7.4 ± 2 μmol/l, NS). Univariate correlations and multiple regression analysis showed albumin excretion rate to be the parameter with the strongest independent association with homocysteine. Patients with both types of diabetes and nephropathy had higher plasma homocysteine concentrations than those without nephropathy. Increases of homocysteine in plasma were related to increases in the severity of the nephropathy. Fasting hyperhomocysteinaemia was considered as the mean of the plasma homocysteine for all control subjects (7.5 ± 2.1 μmol/l) + 2 SD (cut-off =11.7 μmol/l). Nephropathy was present in 80 % of diabetic patients with fasting hyperhomocysteinaemia. In conclusion, increases in fasting homocysteine in diabetic patients are associated with increased albumin excretion rate, especially in those with NIDDM, thus providing a potential new link between microalbuminuria, diabetic nephropathy and cardiovascular disease. [Diabetologia (1998) 41: 684–693] Received: 4 August 1997 and in final revised form: 4 February 1998     

Attenuated neutrophil respiratory burst following acute hypoglycaemia in diabetic patients and normal subjects

The effects of insulin-induced hypoglycaemia on the neutrophil respiratory burst were investigated in six patients with type 1 diabetes and six non-diabetic control subjects. Plasma glucose reached similar nadirs in control subjects (0.9±0.1 mmol l^–1; mean±SEM) and diabetic patients (1.2±0.2 mmol l^–1) (NS). The resting neutrophil respiratory burst was similar in control subjects (1.26±0.15 mV) and diabetic patients (1.03±0.18 mV) (NS). The neutrophil respiratory burst fell following hypoglycaemia in control subjects and diabetic patients to 0.38±0.05 mV (P<0.001) and 0.54±0.09 mV (P<0.05), respectively. This fall was significantly greater in control subjects (ANOVA; P<0.001). Resting neutrophil counts were not significantly different in control subjects (3.2±0.3×10⁹ l^–1) and diabetic patients (6.1±1.5×10⁹ l^–1). Following hypoglycaemia, neutrophil numbers increased in control subjects and diabetic patients to 11.5±1.4×10⁹ l^–1 (P<0.01) and 9.7±1.7×10⁹ l^–1 (P<0.05), respectively. This increase was significantly greater in control subjects (ANOVA; P<0.001). These results suggest that the neutrophil respiratory burst is suppressed in response to hypoglycaemia and that this phenomenon is more pronounced in non-diabetic subjects. Received: 28 March 1997 / Accepted in revised form: 31 July 1997     

Thiol group modulation of sodium-lithium countertransport kinetics in diabetic nephropathy

Summary Abnormal erythrocyte sodium-lithium countertransport (Na-Li CT) activity, traditionally measured at a single sodium concentration of 140 mmol · l^–1 (V₁₄₀), may represent an inherited risk marker for diabetic nephropathy. The membrane defect underlying this association is poorly understood, though modulation by key protein thiol groups appears to be important in essential hypertension. To improve understanding of this abnormality, Na-Li CT kinetics in untreated erythrocytes and after thiol group alkylation with N-ethylmaleimide were investigated in 18 subjects with diabetic nephropathy, 20 normoalbuminuric insulin-dependent diabetic (IDDM) subjects and 18 non-diabetic individuals. Using the traditional assay, V₁₄₀ was similar in subjects with diabetic nephropathy compared to IDDM control subjects vs 0.311 (0.152–0.475) (0.247 (0.111–0.498) mmol Li · h^–1· l erythrocytes^–1). Kinetic parameters were abnormal in subjects with diabetic nephropathy compared with diabetic and non-diabetic control subjects, with both V_max (maximal Na-Li CT activity) (0.454 (0.257–0.963) vs 0.338 (0.183–0.972) vs 0.332 (0.213–0.603) mmol Li · h^–1· l erythrocytes^–1, p < 0.05), and V_max/K_m(So) ratio, reflecting ion association (6.03 (2.3–9.6) vs 4.73 (2.0–10.4) vs 4.48 (1.5–7.1), p < 0.01), significantly higher. N-ethylmaleimide decreased K_m(So) and V_max abolishing differences in V_max/K_m(So) ratio between groups (2.45 (1.18–4.21) vs 2.23 (0.96–4.3) vs 2.44 (1.4–3.7), but enhancing the differences in V_max (0.186 (0.090–0.315) vs 0.120 (0.051–0.256) vs 0.128 (0.080–0.206) mmol Li · h^–1· l erythrocytes^–1, p < 0.0001). Of subjects with diabetic nephropathy, 78 % were outside the 75th percentile of the non-diabetic control subjects when V_max and V_max/K_m(So) ratio were combined, compared to 20 % of the normoalbuminuric control subjects. We conclude that the traditional assay, V₁₄₀, is poor at detecting individuals with diabetic nephropathy. Study of the kinetic parameters of the transporter, including thiol group modulation, suggests that increased ion association, V_max/K_m(So) ratio may represent the inherited defect and improves identification of subjects with diabetic nephropathy. [Diabetologia (1997) 40: 1079–1084] Received: 17 January 1997 and in revised form: 5 May 1997     

The course of kidney function in Type 2 (non-insulin-dependent) diabetic patients with diabetic nephropathy

Summary We evaluated the impact of some putative progression promoters on kidney function in albuminuric Type 2 (non-insulin-dependent) diabetic patients with biopsyproven diabetic glomerulosclerosis. Twenty-six patients (1 female) with a mean age of 52 (standard error 2) years and a known mean duration of diabetes of 9 (1) years were followed-up prospectively for a mean of 5.2 (range 1.0–7.0) years. Twenty-one patients received antihypertensive treatment. During the observation period the glomerular filtration rate decreased from 83 (24–146) to 58 (2–145) ml·min⁻¹·1.73 m⁻² (mean (range)) (p<0.001). The mean rate of decline in glomerular filtration rate was 5.7 (−3.5 to 22.0) ml/min per year. Albuminuria increased from 1.2 (0.3–7.2) to 2.3 (0.4–8.0) g/24 h (geometric mean (range)) (p<0.001). Arterial blood pressure remained unchanged: 162/93 (SE 4/3) and 161/89 (4/2) mm Hg. Univariate analysis showed the rate of decline in glomerular filtration rate to correlate with systolic blood pressure (r=0.71,p<0.001), mean blood pressure (r=0.56,p<0.005), albuminuria (r=0.58,p<0.005) and the initial glomerular filtration rate (r=−0.49,p<0.02). The rate of decline in glomerular filtration rate did not correlate significantly with dietary protein intake, total cholesterol, high-density lipoprotein cholesterol or HbA_1c. Three patients died from uraemia and four patients died from cardiovascular disease. Two patients required renal replacement therapy at the end of the observation period. Our prospective observational study revealed that one-fifth of the patients developed end-stage renal failure during the 5-year observation period. The decline in glomerular filtration rate varied considerably between patients. Increase in arterial blood pressure to a hypertensive level is an early feature of diabetic nephropathy. Elevated systolic blood pressure accelerates the progression of diabetic nephropathy in Type 2 diabetic patients.     

Glomerular hyperfiltration in microalbuminuric NIDDM patients

Summary Glomerular hyperfiltration and microalbuminuria are both regarded as risk factors for the development of diabetic nephropathy in insulin-dependent diabetic patients. Information on glomerular hyperfiltration is scarse in microalbuminuric non-insulin-dependent diabetic (NIDDM) patients. Therefore, we performed a cross-sectional study of glomerular filtration rate (single i. v. bolus injection of ⁵¹Cr-EDTA, plasma clearance for 4 h) in 158 microalbuminuric NIDDM patients compared to 39 normoalbuminuric NIDDM patients and 20 non-diabetic control subjects. The groups were well-matched with regard to sex, age and body mass index. The uncorrected (ml/min) and the adjusted (ml · min^–1· 1.73 m^–2) glomerular filtration rate were both clearly elevated in the microalbuminuric patients: 139 ± 29 and 117 ± 24 as compared to 115 ± 19 and 99 ± 15; 111 ± 23 and 98 ± 21 in normoalbuminuric NIDDM patients and control subjects, respectively (p < 0.001). The glomerular filtration rate (ml · min^–1· 1.73 m^–2) in NIDDM patients who had never received antihypertensive treatment was also clearly elevated in the microalbuminuric patients (n = 96): 119 ± 22 as compared to 100 ± 14 and 98 ± 21 in normoalbuminuric NIDDM patients (n = 27) and control subjects (n = 20), respectively (p < 0.001). Glomerular hyperfiltration (elevation above mean glomerular filtration rate plus 2 SD in normoalbuminuric NIDDM patients) was demonstrated in 37 (95 % confidence interval 30–45)% of the microalbuminuric patients. Multiple regression analysis revealed that HbA_{1 c}, 24-h urinary sodium excretion, age and known duration of diabetes were correlated with glomerular filtration rate in microalbuminuric NIDDM patients (r ² = 0.21, p < 0.01). Our cross-sectional study indicates that NIDDM patients at high risk of developing diabetic nephropathy are also characterized by an additional putative risk factor for progression, glomerular hyperfiltration. [Diabetologia (1996) 39: 1584–1589]     

Structural basis of diabetic nephropathy in microalbuminuric NIDDM patients: a light microscopy study

Summary The objective of the study was to evaluate early structural changes occurring in patients with non-insulin-dependent diabetes mellitus (NIDDM) and microalbuminuria by light microscopy. Basal renal biopsy was performed in patients who were subsequently randomized to different antihypertensive treatments. Fourteen NIDDM patients aged 36–65 years (duration of diabetes 9 ± 7 years) with microalbuminuria (mean urinary albumin excretion 66 ± 49 μg/min) underwent percutaneous renal biopsy. Control biopsies were obtained from five patients of similar age undergoing nephrectomy for renal neoplasia with normal renal function and no history of renal disease. Control and diabetic biopsies were processed by light microscopy and stained with haematoxylin and eosin, periodic acid Schiff, Masson's trichrome and silver methenamine. The percentage of globally sclerotic glomeruli was evaluated. Glomerular volume was determined using perimeter analysis. A semiquantitative assessment (range 0 to 3 +) was made of mesangial sclerosis, interstitial fibrosis, tubular atrophy, arteriosclerosis and arteriolar hyalinosis. Glomerular volume was significantly increased in diabetic as compared to control glomeruli (3.2 ± 8 vs 1.8 ± 7, p < 0.01). Mesangial sclerosis (0.9 vs 0, p < 0.0001) and arteriolar hyalinosis (0.91 vs 0.2, p < 0.022) were significantly higher in diabetic compared to control subjects. No significant differences between diabetic and control subjects were found in the percentage of globally sclerotic glomeruli or in the extent of interstitial fibrosis, tubular atrophy and arteriosclerosis. Thus NIDDM patients with microalbuminuria show histological findings consistent with diabetic nephropathy characterized by glomerular hypertrophy, mesangial sclerosis and arteriolar hyalinosis. However, the renal histological changes are mild and appear less marked than in insulin-dependent diabetic patients. [Diabetologia (1996) 39: 1625–1628].     

Plasma concentration of C-reactive protein is increased in Type I diabetic patients without clinical macroangiopathy and correlates with markers of endothelial dysfunction: evidence for chronic inflammation

Summary Moderately increased plasma concentrations of C-reactive protein are associated with an increased risk of cardiovascular disease. C-reactive protein, its relation to a low degree of inflammatory activation and its association with activation of the endothelium have not been systematically investigated in Type I (insulin-dependent) diabetes mellitus. C-reactive protein concentrations were measured in 40 non-smoking patients with Type I diabetes without symptoms of macrovascular disease and in healthy control subjects, and in a second group of Type I diabetic patients (n = 60) with normo- (n = 20), micro- (n = 20) or macroalbuminuria (n = 20). Differences in glycosylation of α₁-acid glycoprotein were assayed by crossed affinity immunoelectrophoresis. Activation of the endothelium was measured with plasma concentrations of endothelial cell markers. The median plasma concentration of C-reactive protein was higher in Type I diabetic patients compared with healthy control subjects [1.20 (0.06–21.64) vs 0.51 (0.04–9.44) mg/l; p < 0.02]. The Type I diabetic subjects had a significantly increased relative amount of fucosylated α₁-acid glycoprotein (79 ± 12 % vs 69 ± 14 % in the healthy control subjects; p < 0.005), indicating a chronic hepatic inflammatory response. In the Type I diabetic group, log(C-reactive protein) correlated significantly with von Willebrand factor (r = 0.439, p < 0.005) and vascular cell adhesion molecule-1 (r = 0.384, p < 0.02), but not with sE-selectin (r = 0.008, p = 0.96). In the second group of Type I diabetic patients, increased urinary albumin excretion was associated with a significant increase of von Willebrand factor (p < 0.0005) and C-reactive protein (p = 0.003), which were strongly correlated (r = 0.53, p < 0.0005). Plasma concentrations of C-reactive protein were higher in Type I diabetic patients without (clinical) macroangiopathy than in control subjects, probably due to a chronic hepatic inflammatory response. The correlation of C-reactive protein with markers of endothelial dysfunction suggests a relation between activation of the endothelium and chronic inflammation. [Diabetologia (1999) 42: 351–357] Received: 4 September 1998 and in final revised form: 24 November 1998     

Altered properties of the fibrin gel structure in patients with IDDM

Summary High plasma fibrinogen levels are associated with vascular complications in the general population. Fibrin, the structural element in a clot, is derived from fibrinogen by activation of thrombin. An abnormal fibrin gel structure has been demonstrated in patients with myocardial infarction and in diabetic patients during poor metabolic control. In the present study the properties of fibrin gel structure were investigated in 20 patients with insulin-dependent diabetes mellitus (IDDM): 10 patients without (age: 30 ± 8; diabetes duration: 7 ± 6 years), and 10 patients (age: 44 ± 7; diabetes duration: 27 ± 9 years) with microangiopathy. Fifteen healthy subjects served as controls (age: 40 ± 8 years). The glycosylated haemoglobin level (HbA_1c) was elevated (p < 0.001) in the patients: 6.5 ± 1.5 % in diabetic patients without, and 7.1 ± 1.0 % in diabetic patients with microangiopathy. C-reactive protein and plasma fibrinogen were similar as compared to healthy control subjects. The properties of the fibrin gel structure; i. e. the permeability coefficient (Ks) and the fibre mass length ratio (μ) formed in recalcified plasma on addition of thrombin were investigated. Ks was decreased in the diabetic patients, with (6.5 ± 2.0 cm²; p < 0.01) and without microangiopathy (6.5 ± 2.7 cm²; p < 0.05), as compared to healthy subjects (10.0 ± 3.4 cm²), while μ was not significantly (p = 0.14) altered. The results indicate a lower fibrin gel porosity in patients with IDDM, despite normal plasma fibrinogen and irrespective of microangiopathy. The abnormal fibrin gel structure may be due to an increased glycosylation of the fibrin (-ogen) molecule caused by long-term hyperglycaemia and may be of importance for the development of angiopathy in diabetic patients. [Diabetologia (1996) 39: 1519–1523] Received: 7 May 1996 and in revised form: 9 September 1996     

Uptake mechanisms for ascorbate and dehydroascorbate in lymphoblasts from diabetic nephropathy and hypertensive patients

Summary In diabetic nephropathy and hypertension, a major cause of mortality is from cardiovascular disease. Since low levels of antioxidants such as vitamin C have been associated with such complications, we have examined the uptake mechanisms for ascorbic acid (AA) and dehydroascorbic acid (DHA) in lymphoblasts from normal control subjects (CON), normoalbuminuric insulin-dependent diabetic (IDDM) patients (DCON), patients with IDDM and nephropathy (DN) and hypertensive patients (HT) using mass assays of uptake and measuring AA using high-performance liquid chromatography. Precautions were taken to prevent oxidation of AA and to take into account the instability of DHA in buffers. DHA uptake was the major mechanism in all four groups of subjects, and the V_max (maximal uptake rate) was significantly lower in the DN cells (24.7 ± 1.0 nmol [95 % confidence intervals CI 22.5, 26.3] 10⁶ cells^–1 h^–1) compared to CON and DCON cells (33.9 ± 2.1 [95 % CI 29.4, 38.4] and 37.0 ± 2.2 [95 % CI 32.2, 41.8] nmol 10⁶ cells^–1 h^–1, respectively, p < 0.001 for both). DHA V_max was also lower in the HT group (23.2 ± 1.1 [95 % CI 20.7, 25.7] nmol 10⁶ cells^–1 h^–1) compared to the CON group (p < 0.001). There were no significant differences in the K_m or passive membrane permeability for DHA or the AA uptake. DHA uptake showed a negative correlation to systolic blood pressure (r _s = –0.49, p < 0.001). These findings suggest that impaired DHA uptake may be one component of the phenotype expressed by DN cells that may persist in culture. Impaired DHA uptake in vivo, especially in the presence of hyperglycaemia, leads to impaired regeneration of AA and depletion of anti-oxidant defences, exposing such individuals to increased risk of cardiovascular disease. [Diabetologia (1998) 41: 435–442] Received: 10 July 1997 and in final revised form: 24 November 1997     

Association of diabetic neuropathy with Na/K ATPase gene polymorphism

Summary Diabetes mellitus induces a decrease in Na/K ATpase activity in man and animals, and this decrease plays a role in the development of diabetic neuropathy. Na/K ATPase is encoded by various genes, of which the ATP1 A1 gene is expressed predominantly in peripheral nerves and in erythrocytes. To investigate whether a polymorphism in the Na/K ATPase genes could explain the predisposition of some patients with insulin-dependent diabetes mellitus (IDDM) to develop polyneuropathy, a restriction fragment length polymorphism (RFLP) of the ATP1 A1 gene was studied together with erythrocyte Na/K ATPase activity in 81 Caucasian patients with more than 10 years' duration of IDDM. Associations with diabetic neuropathy, retinopathy and nephropathy were sought. Digestion of the first intron of the ATP1 A1 gene by the Bgl II restriction enzyme revealed a dimorphic allelism. Frequency of the restricted allele was 0.18 in this selected series (however, it was 0.10 in representative samples of IDDM patients and of normal subjects in our area). Mean erythrocyte Na/K ATPase activity was lower in diabetic patients than in 42 control subjects (292 ± 10, vs 402 ± 13 nmol Pi · mg protein^{− 1}· h^{− 1}, p < 0.0001) and was not related to HbA_1c value or to diabetes duration. It was lower in the group of the 28 patients bearing the restricted allele (241 ± 10 vs 319 ± 11 nmol Pi · mg protein^{− 1}· h^{− 1}, p < 0.0001). Neuropathy was absent in 50 patients, mild in 15 and severe in 16. When classified accordingly the three groups of patients did not differ with respect to sex, age and duration of diabetes. The respective frequency of the restricted allele among the groups was 10, 73 and 81 %, (p < 0.0001) and mean erythrocyte Na/K ATPase activity was respectively: 322 ± 10.7 nmol Pi · mg protein^{− 1}· h^{− 1}, 268 ± 15 and 229 ± 17, (p < 0.001). A borderline association between renal status or retinal status and repartition of polymorphism and a borderline correlation between renal status and Na/K ATPase activity were found, but significance disappeared after checking for the presence or absence of neuropathy. IDDM patients bearing the ATP1 A1 variant detected by Bgl II RFLP are much more frequently affected by neuropathy (relative risk 6.5, with 95 % CI 3.3–13). Identification of this risk factor may help to prevent this complication. It is suggested that the restricted allele is in linkage disequilibrium with a genomic mutation allowing diabetes to induce a greater impairment of Na/K ATPase activity which could in turn favour the development of neuropathy. [Diabetologia (1997) 40: 506–511] Received: 26 August 1996 and in revised form: 28 January 1997     

Glomerular hypertension as one cause of albuminuria in Type II diabetic patients

Aims/hypothesis. Results from animal models of glomerular hypertension have suggested that this disorder is one cause of albuminuria in diabetic nephropathy. We evaluated this hypothesis clinically. Methods. The subjects were 20 patients with Type II (non-insulin-dependent) diabetes mellitus but without uraemia or hypertension: 8 had normoalbuminuria and 12 had albuminuria ( ≥ 20 μg/min). In the 2-week study, patients were on a diet with ordinary amounts of sodium for 1 week and on a sodium-restricted diet for 1 week. Urinary excretion of sodium and albumin and the systemic blood pressure were measured daily. Intrarenal haemodynamics, in terms of the glomerular pressure and resistance of afferent and efferent arterioles, were calculated from renal clearance, the plasma total protein concentration, and the pressure-natriuresis relation. In 8 of the 12 patients with albuminuria, an angiotensin-converting enzyme inhibitor, cilazapril, was given orally (2 mg/day) and the 2-week study was repeated. Results. In patients with albuminuria, resistance of efferent arterioles and the glomerular pressure were higher than in patients with normoalbuminuria (glomerular pressure, 53 ± 5 vs 43 ± 5 mmHg, means ± SD, p < 0.001). Urinary excretion of albumin correlated (n = 20, r = 0.675, p < 0.001) with the glomerular pressure but not with systemic pressure. The increased glomerular pressure and the albuminuria were decreased by cilazapril but systemic pressure was not. Conclusion/interpretation. These findings are consistent with the hypothesis that glomerular hypertension is present in Type II diabetic patients with early nephropathy and can cause albuminuria. [Diabetologia (1999) 42: 999–1005] Received: 25 January 1999 and in revised form: 15 March 1999     

Angiotensin converting enzyme inhibiting therapy is associated with lower vitreous vascular endothelial growth factor concentrations in patients with proliferative diabetic retinopathy

Aims/hypothesis: Vascular endothelial growth factor (VEGF) is thought to be instrumental in the progression of diabetic retinopathy. Indications exist that the renin-angiotensin system is involved in VEGF overexpression. We assessed the vitreous VEGF concentrations in patients and related them to anti-hypertensive treatment, with special interest in the use of ACE-inhibitors. Methods: Samples of vitreous fluid (10–80 μl) were obtained from 39 patients both with Type I (insulin-dependent) and Type II (non-insulin-dependent) diabetes mellitus and 11 non-diabetic patients undergoing intra-ocular surgery. The VEGF-A concentrations were assessed by immunoassay. Results: Control patients and patients without proliferative diabetic retinopathy (n = 8) had low and comparable VEGF concentrations (medians < 50 pg/ml). In contrast, patients with proliferative diabetic retinopathy (n = 31) had high vitreous VEGF concentrations (median 1134 pg/ml), which showed a negative correlation with the use of ACE inhibiting medication (Spearman rank-R = – 0.54; p = 0.002, n = 13). Diastolic and systolic blood pressure did not differ significantly between the two subgroups with proliferative diabetic retinopathy, i. e. those patients receiving ACE-inhibition (medians 88/160 mm Hg, respectively) and the others (90/160). For the mostly used ACE-inhibitor in the proliferative diabetic retinopathy group, i. e. enalapril (n = 8), a linear dose-effect relation was observed (–20 ± 4 pg · ml^–1· mg^–1· day^–1; p = 0.024; coefficient ± SEM). Conclusion/interpretation: Treatment with ACE-inhibitors attenuates retinal overexpression of VEGF-A in patients with proliferative diabetic retinopathy, probably by interference with a local effect of angiotensin II. [Diabetologia (2002) 45: 203–209] Received: 25 June 2001 and in revised form: 25 October 2001     

Baroreflex sensitivity is depressed in microalbuminuric Type I diabetic patients at rest and during sympathetic manoeuvres

Abstract Aims/hypothesis. To evaluate baroreflex sensitivity (BRS) in microalbuminuric and normoalbuminuric Type I (insulin-dependent) diabetic patients without autonomic neuropathy and in healthy control subjects. Methods. Microalbuminuric Type I diabetic patients (n = 15) were matched for age, sex, body mass index (BMI) and smoking habits with 15 normoalbuminuric patients and with 15 healthy control subjects. All subjects had a blood pressure less than 160/95 mmHg, a BMI less than 30 kg/m² and normal autonomic function on standard tests. Blood pressure and heart rate were measured non-invasively (Finapres) at rest and during sympathetic activation (handgrip, mental stress, standing). The baroreflex sensitivity was defined as the mean gain between blood pressure variability and heart rate variability in the 0.07–0.15 Hz frequency band. Results. Resting baroreflex sensitivity was decreased in the microalbuminuric patients (3.5 ± 0.4 ms/mmHg) compared with the normoalbuminuric patients and the healthy subjects (7.6 ± 1.6 and 9.5 ± 1.1 ms/mmHg, respectively, p < 0.001). The sympathetic tests reduced baroreflex sensitivity similarly in the groups without changing the between group differences. Conclusion/interpretation. Baroreflex sensitivity is reduced in Type I diabetic patients with microalbuminuria but without autonomic neuropathy. A prospective study should indicate whether this early abnormality in cardiovascular reflex function is a risk factor of cardiovascular mortality in these patients. [Diabetologia (1999) 42: 1345–1349] Received: 20 May 1999 and in revised form: 8 July 1999