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Aims/hypothesis: Methylglyoxal (MGO) is a major precursor for advanced glycation end-products (AGEs), which are thought to play a role in vascular complications in diabetes. Known MGO-arginine-derived AGEs are 5-hydro-5-methylimidazolone (MG-H1), argpyrimidine and tetrahydropyrimidine (THP). We studied THP in relation to type 1 diabetes, endothelial dysfunction, low-grade inflammation, vascular complications and atherosclerosis., Methods: We raised and characterised a monoclonal antibody against MGO-derived THP. We measured plasma THP with a competitive ELISA in two cohort studies: study A (198 individuals with type 1 diabetes and 197 controls); study B (individuals with type 1 diabetes, 175 with normoalbuminuria and 198 with macroalbuminuria [>300 mg/24 h]). We measured plasma markers of endothelial dysfunction and low-grade inflammation, and evaluated the presence of THP and N (ε)-(carboxymethyl)lysine (CML) in atherosclerotic arteries., Results: THP was higher in individuals with type 1 diabetes than in those without (median [interquartile range] 115.5 U/μl [102.4-133.2] and 109.8 U/μl [91.8-122.3], respectively; p = 0.03). THP was associated with plasma soluble vascular cell adhesion molecule 1 in both study A (standardised β = 0.48 [95% CI 0.38, 0.58]; p < 0.001) and study B (standardised β = 0.31 [95% CI 0.23, 0.40]; p < 0.001), and with secreted phospholipase A2 (standardised β = 0.26 [95% CI 0.17, 0.36]; p < 0.001) in study B. We found no association of THP with micro- or macro-vascular complications. Both THP and CML were detected in atherosclerotic arteries., Conclusions/interpretation: Our results suggest that MGO-derived THP may reflect endothelial dysfunction among individuals with and without type 1 diabetes, and therefore may potentially play a role in the development of atherosclerosis and vascular disease.

Aims/hypothesis: Parental type 2 diabetes mellitus increases the risk of diabetic nephropathy in offspring with type 1 diabetes mellitus. Several single nucleotide polymorphisms (SNPs) that predispose to type 2 diabetes mellitus have recently been identified. It is, however, not known whether such SNPs also confer susceptibility to diabetic nephropathy in patients with type 1 diabetes mellitus., Methods: We genotyped nine SNPs associated with type 2 diabetes mellitus in genome-wide association studies in the Finnish population, and tested for their association with diabetic nephropathy as well as with severe retinopathy and cardiovascular disease in 2,963 patients with type 1 diabetes mellitus. Replication of significant SNPs was sought in 2,980 patients from three other cohorts., Results: In the discovery cohort, rs10811661 near gene CDKN2A/B was associated with diabetic nephropathy. The association remained after robust Bonferroni correction for the total number of tests performed in this study (OR 1.33 [95% CI 1.14, 1.56], p = 0.00045, p (36tests) = 0.016). In the meta-analysis, the combined result for diabetic nephropathy was significant, with a fixed effects p value of 0.011 (OR 1.15 [95% CI 1.02, 1.29]). The association was particularly strong when patients with end-stage renal disease were compared with controls (OR 1.35 [95% CI 1.13, 1.60], p = 0.00038). The same SNP was also associated with severe retinopathy (OR 1.37 [95% CI 1.10, 1.69] p = 0.0040), but the association did not remain after Bonferroni correction (p (36tests) = 0.14). None of the other selected SNPs was associated with nephropathy, severe retinopathy or cardiovascular disease., Conclusions/interpretation: A SNP predisposing to type 2 diabetes mellitus, rs10811661 near CDKN2A/B, is associated with diabetic nephropathy in patients with type 1 diabetes mellitus.

Aims/hypothesis: This study aimed to investigate the associations of plasma levels of the pro-inflammatory cytokine high-mobility group box 1 (HMGB1) with incident cardiovascular disease (CVD) and all-cause mortality in patients with type 1 diabetes., Methods: We prospectively followed 165 individuals with diabetic nephropathy and 168 individuals with persistent normoalbuminuria who were free of CVD at study entry and in whom levels of HMGB1 and other cardiovascular risk factors were measured at baseline., Results: During the course of follow-up (median, 12.3 years [interquartile range, 7.8-12.5]), 80 patients died, 82 suffered a fatal (n = 46) and/or non-fatal (n = 53) CVD event. After adjustment for age, sex, case-control status and other risk factors, patients with higher levels of log(e) HMGB1 had a higher incidence of fatal and non-fatal CVD and all-cause mortality: HR 1.55 (95% CI 0.94, 2.48) and HR 1.86 (95% CI 1.18, 2.93), respectively. Further adjustments for differences in markers of low-grade inflammation, endothelial and renal dysfunction and arterial stiffness did not attenuate these associations because plasma levels of HMGB1 were not independently associated with these variables., Conclusions/interpretation: In patients with type 1 diabetes, higher levels of plasma HMGB1 are independently associated with a higher risk of all-cause mortality and, to a lesser extent, with a higher incidence of CVD. Larger studies are needed to ascertain more definitely the role of HMGB1 in the development of vascular complications in diabetes.

Aims/hypothesis: The teratogenic consequences of angiotensin-converting enzyme inhibitors angiotensin receptor blockers (ARBs) during the second and third trimesters of pregnancy are well described. However, the consequences of exposure during the first trimester are unclear, especially in diabetes. We report the experience from DIRECT (DIabetic REtinopathy and Candesartan Trials), three placebo-controlled studies designed to examine the effects of an ARB, candesartan, on diabetic retinopathy., Methods: Over 4 years or longer, 178 normotensive women with type 1 diabetes (86 randomised to candesartan, 32 mg once daily, and 92 assigned to placebo) became pregnant (total of 208 pregnancies)., Results: More than half of patients were exposed to candesartan or placebo prior to or in early pregnancy, but all discontinued it at an estimated 8 weeks from the last menstrual period. Full-term pregnancies (51 vs 50), premature deliveries (21 vs 27), spontaneous miscarriages (12 vs 15), elective terminations (15 vs 14) and other outcomes (1 vs 2) were similar in the candesartan and placebo groups. There were two stillbirths and two 'sick babies' in the candesartan group, and one stillbirth, eight 'sick babies' and one cardiac malformation in the placebo group., Conclusions/interpretation: The risk for fetal consequences of ARBs in type 1 diabetes may not be high if exposure is clearly limited to the first trimester. Long-term studies in fertile women can be conducted with ARBs during pregnancy, provided investigators diligently stop their administration upon planning or detection of pregnancy., Trial Registration: ClinicalTrials.gov DIRECT-Prevent 1 NCT00252733; DIRECT-Protect 1 NCT00252720; DIRECT-Protect 2 NCT00252694., Funding: The study was funded jointly by AstraZeneca and Takeda.

Aims/hypothesis: To assess the effect of an angiotensin receptor blocker (ARB) on serum potassium and the effect of a serum potassium change on renal outcomes in patients with type 2 diabetes and nephropathy., Methods: We performed a post hoc analysis in patients with type 2 diabetes participating in the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) study. Renal outcomes were defined as a composite of doubling of serum creatinine or end-stage renal disease., Results: At month 6, 259 (38.4%) and 73 (10.8%) patients in the losartan group and 151 (22.8%) and 34 (5.1%) patients in the placebo group had serum potassium ≥5.0 mmol/l and ≥5.5 mmol/l, (p < 0.001), respectively. Losartan was an independent predictor for serum potassium ≥5.0 mmol/l at month 6 (OR 2.8; 95% CI 2.0-3.9). Serum potassium at month 6 ≥ 5.0 mmol/l was in turn associated with increased risk for renal events (HR 1.22; 95% CI 1.00-1.50), independent of other risk factors. Adjustment of the overall treatment effects for serum potassium augmented losartan's renoprotective effect from 21% (6-34%) to 35% (20-48%), suggesting that the renoprotective effects of losartan are offset by its effect on serum potassium., Conclusions/interpretation: In this study, we found that treatment with the ARB losartan is associated with a high risk of increased serum potassium levels, which is in turn associated with an increased risk of renal outcomes in patients with diabetes and nephropathy. Whether additional management of high serum potassium would further increase the renal protective properties of losartan is an important clinical question.

Aims/hypothesis: Homozygosity for a five leucine repeat (5L-5L) in the carnosinase gene (CNDP1) has been found to be cross-sectionally associated with a low frequency of diabetic nephropathy (DN), mainly in type 2 diabetes. We prospectively investigated in patients with type 1 diabetes whether: (1) 5L-5L is associated with mortality; (2) there is an interaction of 5L-5L with DN or sex for prediction of mortality; and (3) 5L-5L is associated with progression to end-stage renal disease (ESRD)., Methods: In this prospective study in white European patients with type 1 diabetes, individuals with DN were defined by persistent albuminuria ≥ 300 mg/24 h. Controls without nephropathy were defined by persistent (>15 years) normoalbuminuria < 30 mg/24 h. Leucine repeats were assessed with a fluorescent DNA analysis system. Onset of ESRD was defined by need to start chronic dialysis or kidney transplantation., Results: The study involved 916 patients with DN and 1,170 controls. During follow-up for 8.8 years, 107 patients (14%) with 5L-5L died compared with 182 patients (13.8%) with other genotypes (p = 0.99). There was no significant interaction of 5L-5L with DN for prediction of mortality (p = 0.57), but a trend towards interaction with sex (p = 0.08). In patients with DN, HR for ESRD in 5L-5L vs other genotypes was not constant over time, with increased risk for 5L-5L beyond 8 years of follow-up (p = 0.03)., Conclusions/interpretation: CNDP1 polymorphism was not associated with mortality, and nor was there an interaction of this polymorphism with DN for prediction of mortality in patients with type 1 diabetes. CNDP1 polymorphism predicts progression to ESRD in patients with DN, but only late after baseline measurements.

Aims/hypothesis: Endothelial progenitor cells (EPC) augment vascular repair and neovascularisation. Patients with type 2 diabetes have reduced EPC and increased risk of cardiovascular disease (CVD), which is reduced by multifactorial intervention. Our aim, therefore, was to evaluate in type 2 diabetic patients whether the numbers of EPC derived from peripheral blood mononuclear cells is influenced by a multifactorial treatment strategy., Methods: We enrolled 28 patients newly referred for initiation of multifactorial treatment, which consisted of improving glycaemic, lipid and blood pressure control, as well as antithrombotic therapy and lifestyle modification. EPC count was assessed by in vitro cultures at baseline and after 90 days of treatment. After 7 days in culture, we identified EPC by fluorescent staining of attached cells. Patients were treated with metformin, aspirin, statins and angiotensin II receptor blockers, and divided accordingly into groups of mono-, dual-, triple- or quadruple therapy., Results: After 90 days of treatment, glycaemic control improved and total cholesterol decreased. Multifactorial intervention for 90 days significantly increased EPC count in cultures by 35% (from 105 [SE 8] to 140 [11] cells per field [p = 0.002]). The change in EPC among patients with quadruple therapy was higher (63%) than in untreated patients (-32%, p = 0.043)., Conclusions/interpretation: Numbers of EPC derived from peripheral blood mononuclear cells increased significantly after multifactorial intervention in type 2 diabetic patients. It remains to be shown whether these changes contribute to the beneficial effects of multifactorial intervention on diabetic micro- and macroangiopathy.

Aim: The optimal antiproteinuric dose of aliskiren is unknown. This study compared the effect of placebo and increasing doses of aliskiren on urinary albumin excretion rate (UAER)., Methods: The trial was a double-blind crossover design. Twenty-six patients with type 2 diabetes mellitus, hypertension and albuminuria were randomised to 2-month treatments with placebo or aliskiren 150 mg, 300 mg or 600 mg once daily, in random order. Primary endpoint was change in UAER; secondary endpoints included changes in 24-h BP, GFR, biomarkers and components of the renin-angiotensin-aldosterone system., Results: Placebo geometric mean UAER was 350 mg/day, mean 24-h BP was 137/81 (SD 12/9) mmHg, GFR was 85 (SD 26) ml min(-1) 1.73 m(-2). Aliskiren 150, 300 and 600 mg daily reduced UAER significantly by 36% (95% CI 17-51), 48% (33-60) and 52% (38-63) respectively (p < 0.001) compared with placebo. UAER reduction during the 600 mg dose was not significantly different from the 300 mg dose. Twenty-four-hour systolic BP was reduced by 4.5, 8.0 and 9.2 mmHg versus placebo, significant for 300 and 600 mg (p < or = 0.001). Twenty-four-hour diastolic BP was reduced by 3.0, 4.1 and 4.4 mmHg, significant versus placebo (p = 0.019, p = 0.001 and p < 0.001). GFR was reduced by 3.0, 5.1 and 6.5 ml min(-1) 1.73 m(-2). hsPRA was reduced by 63%, 70%, and 82% (p < 0.001 for all). Adverse events, most frequently dizziness and fatigue, occurred during all doses., Conclusions: In patients with type 2 diabetes mellitus, hypertension and albuminuria there is no improved antiproteinuric effect when using 600 mg aliskiren daily compared with the maximal recommended antihypertensive dose of 300 mg., Trial Registration: Clinicaltrials.gov NCT00464776, Funding: Novartis Pharma AG.

Aims/hypothesis: Type 1 diabetic patients with diabetic nephropathy have increased mortality and morbidity compared with normoalbuminuric patients. Telomere length in proliferative cells is inversely related to the total number of cell divisions, and therefore to biological age. We aimed to evaluate differences in telomere length in patients with type 1 diabetes with or without diabetic nephropathy; we also evaluated the prognostic value of telomere length., Methods: In a prospective follow-up study, 157 type 1 diabetic patients with diabetic nephropathy and a control group of 116 patients with type 1 diabetes and normoalbuminuria were followed for 11.1 years (range 0.2-12.9). Telomere length was measured from DNA samples extracted from white blood cells at baseline., Results: The mean telomere length did not differ between patients with or without diabetic nephropathy, and was similar in men and women, but was inversely correlated with age and systolic blood pressure, p < 0.05. When dividing patients into tertiles after telomere length, 36 (37%) patients died in the tertile with the shortest telomere length, 24 (28%) died in the middle tertile, and 15 (17%) of patients in the tertile with the longest telomere length died, log rank test p = 0.017. After adjustment for traditional risk factors, telomere length was still predictive of all-cause mortality., Conclusions/interpretation: In patients with type 1 diabetes we found no differences in telomere length between patients with or without diabetic nephropathy. We also found that telomere length was associated with all-cause mortality; however, confirmative studies are needed to verify our findings.

Aims/hypothesis: The purpose of this study was to evaluate the optimal renoprotective effect of ultrahigh doses of lisinopril, as reflected by short-term changes in urinary albumin excretion rate (UAER), in type 1 diabetic patients with diabetic nephropathy., Methods: At the Steno Diabetes Center, 49 type 1 diabetic patients with diabetic nephropathy completed this double-masked randomised crossover trial consisting of an initial washout period followed by three treatment periods each lasting 2 months, where all patients received lisinopril 20, 40 and 60 mg once daily in randomised order in addition to slow-release furosemide. Allocation was concealed by sequentially numbered opaque sealed envelopes. UAER, 24 h ambulatory blood pressure (ABP) and estimated GFR were determined at baseline and after each treatment period., Results: All 49 patients completed all three treatment periods. Baseline values were: UAER (geometric mean [95% CI]) 362 (240-545) mg/24 h, 24 h ABP (mean [SD]) 142 (14)/74 (8) mmHg and estimated GFR 75 (29) ml min(-1) 1.73 m(-2). Reductions in UAER from baseline were 63%, 71% and 70%, respectively, with the increasing doses of lisinopril (p < 0.001). Compared with lisinopril 20 mg there was a further reduction in UAER of 23% with lisinopril 40 mg and 19% with 60 mg, p < 0.05. ABP was reduced from baseline by 10/5, 13/7 and 12/7 mmHg (p < 0.001 vs baseline, p < 0.05 for diastolic ABP 20 vs 40 mg, otherwise NS between doses). The difference in UAER between 20 and 40 mg lisinopril was significant after adjustment for changes in ABP (p < 0.01). Two patients were excluded from the study because of an increase in plasma creatinine and one because of high BP; otherwise the study medication was well tolerated with few, mild, dose-independent adverse effects., Conclusions/interpretation: Lisinopril 40 mg once daily is generally safe and offers additional reductions in BP and UAER in comparison with the currently recommended dose of 20 mg. Lisinopril 60 mg offers no further beneficial effect., Trial Registration: ClinicalTrials.gov NCT00118976.

Aims/hypothesis: The bone-related peptide osteoprotegerin is produced by vascular cells and is involved in the process of vascular calcification. The aim of this study was to investigate the predictive value of plasma levels of osteoprotegerin in relation to mortality, cardiovascular events and deterioration in kidney function in patients with type 1 diabetes., Methods: This prospective observational follow-up study included 397 type 1 diabetic patients with overt diabetic nephropathy (243 men; age [mean+/-SD] 42.1 +/- 10.6 years, duration of diabetes 28.3 +/- 9.9 years, GFR 67 +/- 28 ml min(-1) 1.73 m(2)) and a group of 176 patients with longstanding type 1 diabetes and persistent normoalbuminuria (105 men; age 42.6 +/- 9.7 years, duration of diabetes 27.6 +/- 8.3 years)., Results: The median (range) follow-up period was 11.3 (0.0-12.9) years. Among patients with diabetic nephropathy, individuals with high osteoprotegerin levels (fourth quartile) had significantly higher all-cause mortality than patients with low levels (first quartile) (covariate-adjusted hazard ratio [HR] 3.00 [1.24-7.27]). High osteoprotegerin levels also predicted cardiovascular mortality (covariate-adjusted HR 4.88 [1.57-15.14]). Furthermore, patients with high osteoprotegerin levels had significantly higher risk of progression to end-stage renal disease than patients with low levels (covariate-adjusted HR 4.32 [1.45-12.87]). In addition, patients with high levels of plasma osteoprotegerin had an elevated rate of decline in GFR., Conclusions/interpretation: High levels of osteoprotegerin predict all-cause and cardiovascular mortality in patients with diabetic nephropathy. Furthermore, high levels of osteoprotegerin predict deterioration of kidney function towards end-stage renal disease.

Aims/hypothesis: In type 1 diabetic patients with microalbuminuria not receiving antihypertensive treatment, an increase in urinary AER (UAER) of 6-14%/year and a risk of developing diabetic nephropathy (DN) of 3-30%/year have been reported. We audited the long-term effect of blocking the renin-angiotensin-aldosterone system (RAAS) with an ACE inhibitor (ACEI) or angiotensin II receptor blocker (ARB) in microalbuminuric type 1 diabetic patients on progression of microalbuminuria and development of DN., Methods: All patients with type 1 diabetes and persistent microalbuminuria (30-300 mg/24 h) were identified (n=227) in 1995 at Steno Diabetes Center and followed for 11 years. Development of DN was defined as a UAER of >300 mg/24 h in two of three consecutive urine samples., Results: Age and duration of diabetes at baseline (mean+/-SD) were 46+/-15 and 28+/-13 years, respectively. During follow-up 14 patients emigrated and 58 (26%) died. Over the same period 79% were treated with an ACEI or ARB. There was a mean decline in UAER of 4%/year. Sixty-five patients (29%) progressed to overt DN, corresponding to 3.1%/year. However, 29 of them regressed to normo- or microalbuminuria on intensified antihypertensive treatment. Glycaemic control and blood pressure remained nearly unchanged., Conclusions/interpretation: In our outpatient clinic, the implementation of RAAS-blocking treatment in type 1 diabetic patients with microalbuminuria successfully reduced long-term progression to overt DN to a rate similar to those previously reported in randomised, double-blind intervention trials of shorter duration using RAAS blockade.

Aims/hypothesis: Activation of protein kinase C (PKC) isoforms has been implicated as a central mediator in the pathogenesis of diabetic nephropathy. Although high glucose levels stimulate catalytic activity of PKC, the effects of high glucose levels on the expression of genes encoding PKC isoforms are unknown. We sought to determine whether in addition to activation, diabetes may lead to increased transcription of two PKC isoforms that have been implicated in the pathogenesis of diabetic nephropathy, PKC-alpha and PKC-beta., Methods: Recent advances in molecular biological techniques now permit quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed., Results: Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies of diabetic patients relative to control (p<0.001). No increase in PRKC-alpha expression was seen. In addition, a correlation between renal PRKC-beta mRNA and HbA(1c) was observed in diabetic patients (r=0.63, p<0.05). There was co-localisation of PKC-beta and phospho-PKC-beta predominantly to proximal tubules. A 60% increase in PRKC-beta mRNA and peptide in cultured human proximal tubular epithelial cells exposed to high glucose (p<0.05) was seen in vitro., Conclusions/interpretation: PKC-beta is upregulated at the gene expression level in human diabetic nephropathy. PRKC-beta mRNA correlates closely with serum HbA(1c), possibly partially explaining the relationship between glycaemic control and progression of diabetic nephropathy. Archival human tissue provides a valuable resource for molecular analyses.

Aims/hypothesis: Raised N-terminal pro-B-type natriuretic peptide (NT-proBNP) is associated with a poor cardiac outcome in non-diabetic populations. Elevated NT-proBNP predicts excess morbidity and mortality in diabetic patients with an elevated urinary albumin excretion rate. This study investigated the prognostic value of NT-proBNP in a cohort of type 2 diabetic patients., Subjects, Materials and Methods: In a prospective observational follow-up study, 315 type 2 diabetic patients with normoalbuminuria (n=188), microalbuminuria (n=80) and macroalbuminuria (n=47) at baseline were followed for a median (range) of 15.5 (0.2-17.0) years. Plasma NT-proBNP concentrations were determined by immunoassay at baseline. Endpoints were overall and cardiovascular mortality., Results: Of the patients, 162 died (51%), 119 of them (74%) due to cardiovascular causes. All-cause mortality was increased in patients with NT-proBNP in the second and third tertiles (hazard ratios [95% CI] compared with the first tertile, 1.70 [1.08-2.67] and 5.19 [3.43-7.88], p<0.001). These associations persisted after adjustment for urinary albumin excretion rate, glomerular filtration rate and conventional cardiovascular risk factors (covariate adjusted hazard ratios 1.46 [0.91-2.33] and 2.54 [1.56-4.14], p<0.001). This increased mortality was attributable to more cardiovascular deaths in the second and third NT-proBNP tertile (unadjusted hazard ratios 1.63 [0.96-2.77] and 4.88 [3.01-7.91], p<0.001; covariate adjusted 1.37 [0.79-2.37] and 2.26 [1.27-4.02], p=0.01). When patients with normo-, micro- and macroalbuminuria were analysed separately, NT-proBNP levels above the median (62 ng/l) were consistently associated with increased overall and cardiovascular mortality in all three groups (p<0.001)., Conclusions/interpretation: In patients with type 2 diabetes, elevated circulating NT-proBNP is a strong predictor of the excess overall and cardiovascular mortality, this predictor status being independent of urinary albumin excretion rate and conventional cardiovascular risk factors.

Background: Anaemia is a common finding in patients with diabetes, particularly in those with overt nephropathy or renal impairment. In tertiary clinics, at least one outpatient in five with diabetes has anaemia, for whom it constitutes a significant additional burden., Discussion: Anaemia is associated with an increased risk of diabetic complications including nephropathy, retinopathy and macrovascular disease. Anaemia may also be significant in determining the outcome of heart failure and hypoxia-induced organ damage in diabetes. While several factors contribute to the increased prevalence of anaemia in diabetes, the failure of the kidney to increase erythropoietin in response to falling haemoglobin appears to be the dominant factor. Although there is a clear rationale for correcting anaemia in people with diabetes, it remains to be established whether this will lead to improved outcomes. Moreover, the balance of risks, costs, and benefits remains to be established in patients with diabetes. The Trial to Reduce Cardiovascular Events with Aranesp (darbepoetin alpha) Therapy (TREAT) is a randomised controlled trial designed to determine the impact of anaemia correction on mortality and non-fatal cardiovascular events in patients with type 2 diabetes and stage 3-4 nephropathy., Conclusion: It is anticipated that TREAT will help to define the optimal approach to the management of anaemia in diabetes.

Aims/hypothesis: We evaluated the impact of remission of nephrotic-range albuminuria (>2500 mg/24 h) (NRA) on end-stage renal disease (ESRD) and mortality in type 2 diabetic patients with nephropathy., Methods: This was a follow-up observational study involving all 79 patients (35%; 62 men, 17 women) with NRA from a cohort of type 2 diabetic patients with nephropathy that was followed for at least 3 years at the Steno Diabetes Center (n=227). Patients were followed from the onset of NRA until death or January 2005. The mean age (+/-SD) was 60+/-8 years and known diabetes duration was 14+/-7 years. Remission of NRA was defined as sustained albuminuria <600 mg/24 h for at least 1 year., Results: The duration of follow-up after onset of NRA was 6.5 years (range 2-20 years). Remission was induced in 20 (25%) of the patients, all treated with ACE inhibitors or angiotensin-II receptor blockers. Remission lasted 4.1 years (range 1-10 years) and only three patients relapsed. At the end of follow-up, only 30% (two ESRD and four deaths) of the 20 patients with remission had reached the composite endpoint of ESRD or death, in contrast to 66% (16 ESRD and 23 deaths) of the 59 patients without remission (p<0.01). Cox regression analysis revealed that remission was associated with a risk reduction of 67% (95% CI 10-87) for reaching the composite endpoint of ESRD or death and of 69% (95% CI 21-88%) for death alone. Male sex, greater age and systolic blood pressure at onset of NRA were also independently associated with an increased risk of ESRD and death., Conclusions/interpretation: Aggressive antihypertensive treatment can lead to long-term remission of NRA in a sizeable proportion of patients with type 2 diabetes. Such remission is associated with a slower progression of nephropathy and substantially improved survival.

Aims/hypothesis: We explored the impact of baseline left ventricular hypertrophy (LVH) and losartan treatment on renal and cardiovascular (CV) events in 1,513 patients from the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) trial, which studied the effects of losartan on the progression of renal disease and/or death in patients with type 2 diabetes and nephropathy., Materials and Methods: LVH was assessed using ECG criteria (Cornell product and/or Sokolow-Lyon voltage). The risk of renal or CV events was determined by a proportional hazards model fit with treatment allocation and presence of LVH. Covariates at baseline included age, sex, systolic BP, mean arterial pressure, pulse, proteinuria, serum creatinine, albumin and haemoglobin., Results: A total of 187 subjects (12%) had LVH at baseline. Treatment with losartan resulted in a significant decrease in the Cornell product (-6.2%) and Sokolow-Lyon voltage (-6.3%). LVH was shown to be significantly associated with the primary endpoint, which was a composite of doubling of serum creatinine (DSCR), endstage renal disease (ESRD) or death (hazard ratio [HR]=1.44, p=0.011), as well as with the composite renal endpoint of DSCR/ESRD (HR=1.42, p=0.031) and CV events (HR=1.68, p=0.001). Losartan treatment of patients with LVH decreased the CV as well as renal risk to a level similar to that of patients without LVH., Conclusions/interpretation: In patients with type 2 diabetes and nephropathy, LVH is associated with significantly increased risk of CV events and the progression of kidney disease. Importantly, in patients with LVH, losartan reduced the CV as well as the renal risk to a level similar to that seen in subjects without LVH.

Aims/hypothesis: Low serum adiponectin (ADPN) has been associated with increased risk of cardiovascular disease (CVD) and retinopathy in patients with type 2 diabetes mellitus. In type 1 diabetic patients, the relationship between ADPN and the presence of vascular complications is largely unknown., Methods: We investigated the relationship between serum ADPN and the presence of retinopathy, nephropathy and CVD in patients with type 1 diabetes, divided into matched groups with normoalbuminuria and no retinopathy (n=67), simplex retinopathy (n=106) or proliferative retinopathy (n=19), and nephropathy with simplex (n=62) or proliferative retinopathy (n=137). Healthy control subjects (n=25) were included., Results: Serum ADPN was increased in subjects with type 1 diabetes compared with control subjects (p<0.0001). Further, serum ADPN was higher in patients with than in those without nephropathy (p<0.0001). It was also higher in normoalbuminuric patients with than in those without proliferative retinopathy (p<0.0001). These differences remained significant after adjustment for known risk factors (p<0.03). CVD was also associated with elevated ADPN levels (p<0.05), but this difference became insignificant after risk factor adjustment. The most important predictor of serum ADPN was sex (r2=19%) in normoalbuminuric patients and GFR in patients with nephropathy (r2=18%)., Conclusion/interpretation: Patients with type 1 diabetes and microvascular complications have higher serum levels of ADPN than patients without complications. It remains to be clarified whether elevated levels of ADPN are pathogenically related to the development of microvascular complications or represent a beneficial counter-regulatory response.

Aims/hypothesis: Raised N-terminal pro-brain natriuretic peptide (NT-proBNP) is independently associated with an increased risk of death in chronic heart failure and acute coronary syndromes in nondiabetic populations. Diabetic nephropathy is characterised by an increased risk of cardiovascular morbidity and mortality. This study investigated the prognostic value of NT-proBNP in a large cohort of type 1 diabetic patients with and without diabetic nephropathy., Methods: In a prospective observational follow-up study, 198 type 1 diabetic patients with overt diabetic nephropathy (122 men, age [mean+/-SD] 41+/-10 years, duration of diabetes 28+/-8 years, GFR 74+/-33 ml min(-1)) and a matched control group of 188 patients with longstanding type 1 diabetes and persistent normoalbuminuria (114 men, age 43+/-10 years, duration of diabetes 27+/-9 years) were followed for 9.3 (0.0-9.5) years. Plasma NT-proBNP concentration was determined by immunoassay at baseline., Results: In patients with diabetic nephropathy, plasma NT-proBNP concentration was elevated to (median [range]) 110 (5-79640) ng l(-1) vs. 27 (5-455) ng l(-1) in normoalbuminuric patients (p<0.0001). Among patients with nephropathy, 39 (39%) patients with plasma NT-proBNP concentrations above the median and 12 (12%) with values below the median died from any cause (unadjusted hazard ratio 3.86 [95% CI 2.02-7.37], p<0.0001; covariate-adjusted hazard ratio 2.28 [1.04-4.99], p=0.04). This lower mortality rate was attributable to fewer cardiovascular deaths: 31 (31%) and 7 (7%) above and below the median NT-proBNP level respectively (unadjusted hazard ratio 5.25 [2.31-11.92], p<0.0001; covariate-adjusted hazard ratio 3.81 [1.46-9.94], p=0.006)., Conclusions/interpretation: Elevated circulating NT-proBNP is a new independent predictor of the excess overall and cardiovascular mortality in diabetic nephropathy patients without symptoms of heart failure.

Aims/hypothesis: We examined whether plasma N-terminal probrain natriuretic peptide (NT-proBNP) predicts cardiovascular outcome in patients with type 2 diabetes., Methods: A total of 160 microalbuminuric type 2 diabetic patients (mean age 55.1 years [SD 7.2], 119 men) were enrolled in the Steno-2 Study examining the effect of multifactorial treatment, and were divided into two groups according to baseline levels of plasma NT-proBNP below or above the median for the cohort, which was followed for an average of 7.8 years. Cardiovascular outcome was a composite of cardiovascular mortality, myocardial infarction, stroke, revascularisation procedures in the heart or legs, and amputations., Results: In the whole group, plasma NT-proBNP being above the median was associated with an increased risk of cardiovascular disease during follow-up, with an unadjusted hazard ratio of 4.4 (95% CI 2.3-8.4; p<0.0001). A decrease in plasma NT-proBNP of 10 pg/ml during the first 2 years of intervention was associated with a 1% relative reduction in the primary endpoint (p<0.001). Despite polypharmacological treatment targeting cardiovascular disease, the mean plasma NT-proBNP level increased during follow-up., Conclusions/interpretation: We conclude that high plasma NT-proBNP is a major risk marker for cardiovascular disease in patients with type 2 diabetes and microalbuminuria.

Aims/hypothesis: It has been suggested that aldosterone plays a role in the initiation and progression of renal disease independently of arterial blood pressure and plasma angiotensin II levels. We evaluated the influence of plasma aldosterone levels on progression of diabetic nephropathy during long-term blockade of the renin-angiotensin-aldosterone system., Methods: A total of 63 hypertensive patients with type 1 diabetes and diabetic nephropathy were treated with losartan, 100 mg once daily, for a mean follow-up period of 35 months. Plasma aldosterone, GFR, albuminuria and 24-h blood pressure were determined at baseline and at regular intervals during the study., Results: Patients were divided according to their increasing or decreasing levels of plasma aldosterone during long-term losartan treatment in an escape group (n=26) and a non-escape group (n=37). In the escape group, aldosterone levels increased from (geometric mean [95% CI]) 57 pg/ml (43-76 pg/ml) at 2 months, to 102 pg/ml (78-134 pg/ml) at the end of the study (p<0.01). The corresponding levels in the non-escape group were 83 pg/ml (69-102 pg/ml) and 49 pg/ml (40-60 pg/ml; p<0.01). The median rate of decline in GFR was 5.0 ml.min(-1).year(-1) (range 0.4-15.9 ml.min(-1).year(-1)) in the escape group, compared with 2.4 ml.min(-1).year(-1) (-1.6 to 11.0 ml.min(-1).year(-1)) in the non-escape group (p<0.005). The increase in plasma aldosterone correlated with the rate of decline in GFR (r(2)=0.19, p<0.001), corresponding to a decline in GFR of 1.5 ml.min(-1).year(-1) for every two-fold increase in plasma aldosterone. Pre-treatment and treatment values of plasma aldosterone were not related to albuminuria or to changes in albuminuria during the study., Conclusions/interpretation: Our data suggest that aldosterone escape during long-term blockade of the renin-angiotensin-aldosterone system is associated with an enhanced decline in GFR in patients with type 1 diabetes and diabetic nephropathy.

Aims/hypothesis: Although hyperhomocysteinaemia and methylenetetrahydrofolate reductase gene polymorphism are accepted risk factors for cardiovascular disease, their association with micro angiopathy or blood pressure in diabetic patients is still being debated. This study explores the relation between plasma homocysteine concentrations, methylenetetrahydrofolate reductase gene polymorphism, hypertension, diabetic microvascular and macrovascular complications associated with kidney function., Methods: Vascular complications, hypertension, methylenetetrahydrofolate reductase genotype (RFLP with Hinf I digestion), and total plasma homocysteine (HPLC) were investigated in 389 well-characterized Type I (insulin-dependent) diabetic patients with normal (GFR> or=75 ml x min(-1) x (1.73 m(2))(-1); n=273), or impaired renal function (GFR <75 ml x min(-1) x (1.73 m(2))(-1); n=116)., Results: Patients with microvascular and macrovascular complications showed higher total plasma homocysteine concentrations than those without complications. However, after the data for GFR (main determinant for plasma homocysteine) was adjusted we observed that plasma homocysteine concentrations greater than 8.6 micro mol/l in patients with normal GFR are not related to vascular complications, but to hypertension (8.6-11.3 micro mol/l: OR 1.9; >11.3 micro mol/l: OR 3.7). The risk for coronary heart disease (CHD) was also enhanced by a plasma homocysteine concentration greater than 11.3 micro mol/l (OR 5.9). Although the T allele was an independent determinant of plasma homocysteine, the methylenetetrahydrofolate reductase gene polymorphism was neither associated with diabetic vascular complications nor with hypertension., Conclusion/interpretation: Increased plasma homocysteine concentrations but not the T allele per se, enhance the risk of hypertension and of CHD in Danish Type I diabetic patients with normal renal function.

Patients with type 2 diabetes are especially vulnerable to both large and small vessel injury from elevated arterial blood pressure. The frequent combination of hypertension and diabetes is, therefore, associated with a high risk of cardiovascular events and end-stage renal disease. The beneficial therapeutic effects of angiotensin-converting enzyme (ACE) inhibitors, calcium channel blockers (CCBs), and other antihypertensive drugs in patients with type 2 diabetes have been well established in placebo-controlled trials, and there appears to be no evidence of differences between treatment regimens based on different drug classes. In addition, a number of major clinical trials have supported a policy of aggressive blood pressure lowering in patients with type 2 diabetes. In the UK Prospective Diabetes Study, for example, tight control of blood pressure was demonstrated to produce clinically important reductions in the risk of complications related to type 2 diabetes compared with less tight control. In most hypertensive patients, however, a reduction to a recommended level <130/85 mm Hg is unlikely to be achieved by monotherapy. Consequently, the combination of ACE inhibitors with other first-line drugs, such as CCBs, diuretics and beta-blockers, is recommended for the therapeutic management of hypertensive diabetic patients.

Aims/hypothesis: Pregnancy could damage kidney function in diabetic nephropathy. We investigated the long-term impact of pregnancy on the progression of diabetic nephropathy., Methods: Our observational follow-up study included all women patients with Type I (insulin-dependent) diabetes mellitus who developed diabetic nephropathy between 1970 and 1989 at Steno Diabetes Center (n = 93). Follow-up lasted 16 years (range 3-28) from the onset of diabetic nephropathy until death or the year 2000. A total of 26 women became pregnant after the onset of diabetic nephropathy (group A). The remaining 67 served as control subjects (group B). All patients received aggressive antihypertensive treatment (blood pressure goal < 140/90 mmHg)., Results: The two groups were comparable at onset of diabetic nephropathy regarding blood pressure, albuminuria, s-cholesterol, smoking, retinopathy and s-creatinine (mean 79(SD 23) micromol/l). The slopes of 1/s-creatinine (1000.l.micromol(-1).year(-1)) during the whole observation period were -0.39(0.40) compared with -0.41(0.70) (group A vs B-NS). The slopes of 1/s-creatinine before and after pregnancy were similar. Decline in creatinine clearance (ml/min/yr) was 3.2 (3.4) compared with 3.2 (5.1) (group A vs B -NS). At the end of follow-up, 35 % (95 %-CI:17-53) of the pregnant women had died and 19 % (7-39) had reached end stage renal disease compared to 34 % (23-45) and 24 %(14-34) of the control subjects, respectively(NS). Group A and B had similar blood pressure levels during the whole observation period: 136(13)/83(7) vs 139 (14)/85(7) mmHg (NS)., Conclusion/interpretation: Pregnancy has no adverse long-term impact on kidney function and survival in Type I diabetic patients with well-preserved kidney function (normal serum creatinine) suffering from diabetic nephropathy.

Aims/hypothesis: Reports on a putative synergism between poor glycaemic control and carriage of the angiotensin II type 1 receptor (AGTR1) C1166-allele and risk of diabetic nephropathy have been conflicting. Therefore, we investigated the interaction between long-term glycaemic control and three polymorphisms in the genes coding for AGTR1 (A1166-->C), angiotensin converting enzyme (ACE/ID) and angiotensinogen (M235T) on risk of developing diabetic nephropathy. Furthermore, we investigated the relation between a random measurement and long-term measurements of haemoglobin A1c (HbA1c)., Methods: We studied Caucasian patients with Type I (insulin-dependent) diabetes mellitus and nephropathy (120 men 74 women, age 41.1 +/- 9.6 years, diabetes duration 28 +/- 8 years) and long-standing Type I diabetic patients with persistent normoalbuminuria (112 men 69 women, age 42.5 +/- 10.0 years, diabetes duration 27 +/- 9 years). Genotyping was PCR-based and metabolic control estimated from all measurements of HbA1c done in each patient [average (range) n = 31 (6-74)]. The median observation time (range) was 13.5 (2-14) years., Results: Type I diabetic patients with a history of poor glycaemic control (HbA1c above the median, 8.7%) had an increased risk of diabetic nephropathy compared with patients with a better metabolic control, OR (95% CI): 9.2 (5.8-14.7). The magnitude of this risk was similar in carriers and non-carriers of the mutations. The risk of nephropathy in patients with HbA1c above compared with below the median in carriers of the mutant C1166-allele, D-allele, or T235-allele were 7.6 (95% CI: 3.9-14.8), 10.4 (6.0-17.8) and 9.8 (5.4-17.9), respectively. A significant correlation (r = 0.74, p < 0.001) existed between a random and long-term measurements of HbA1c with a small mean difference (limits of agreement) [0.2 (-1.8 to 2.1)%] between the two estimates., Conclusion/interpretation: Poor metabolic control is a major risk factor for diabetic nephropathy in Caucasian Type I diabetic patients. This risk was similar in carriers and non-carriers of the mutant alleles of the AGTR1 (A1166-->C), ACE/ID and angiotensinogen-M235T polymorphisms. The HbA1c value measured at random reflects rather closely average long-term HbA1c values.

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) micromol/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) micromol/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.

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.

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 scarce 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 HbA1c, 24-h urinary sodium excretion, age and known duration of diabetes were correlated with glomerular filtration rate in microalbuminuric NIDDM patients (r2 = 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.

Recombinant DNA technology allows the production of insulin analogues with faster absorption rates from subcutaneous tissue as compared to soluble human insulin. The human insulin analogue B10Asp (mono/dimeric) is absorbed twice as fast as soluble human insulin (hexameric). A double blind, randomised crossover study with a 1-month run-in period and two 2-month treatment periods was performed in 21 male insulin-dependent diabetic (IDDM) patients aged 18-40 years in order to compare the metabolic control obtained with equimolar doses of the analogue B10Asp vs soluble human insulin (Actrapid) given as mealtime insulin and intermediate acting isophane insulin (Protaphane) at bedtime. At the end of each 2-month study period, the patients were admitted to the metabolic ward. We found significantly higher plasma insulin/analogue levels after breakfast, lunch and dinner with B10Asp as compared to Actrapid (p < 0.05). The plasma insulin/analogue levels were significantly lower before lunch and dinner with B10Asp as compared to Actrapid (p < 0.05). Also, the plasma insulin/analogue level tended to be lower at bedtime when comparing B10Asp to Actrapid. The 24-h blood glucose profiles showed identical fasting blood glucose, significantly lower blood glucose after breakfast with the analogue (p < 0.05), no differences in blood glucose after lunch and dinner but a significantly higher blood glucose at midnight using the analogue (p < 0.05). The overall 24-h mean blood glucose concentrations, the daily insulin dose, HbA1c, diet, home blood glucose monitoring and frequency of hypoglycaemia were almost identical in the two treatment periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Progression in diabetic nephropathy is usually determined by repeated measurements of glomerular filtration rate and expressed as rate of decline in glomerular filtration rate. Our aim was to evaluate the agreement between rate of decline in glomerular filtration rate estimated from the Cockroft-Gault formula: (140-age)*K*body weight*(1/S-creatinine) and measured by the plasma clearance of 51Cr-EDTA. All insulin-dependent diabetic patients with diabetic nephropathy followed-up for at least 5 years with at least 5 simultaneous measurements of glomerular filtration rate, s-creatinine, and weight were included in the study. Forty-three patients (32 male/11 female), age 31 (18-61) years were enrolled. Observation period: 6.6 (5.1-9.9) years and number of investigations per patient 6 (5-16) (median(range)). Baseline glomerular filtration rate (ml/min) was 97 (30) measured and 107 (37) estimated (mean(SD))(p < 0.001) and the 95% limits of agreement were -42.0 to 20.8 ml/min. Measured and estimated glomerular filtration rate correlated significantly (r = 0.91, p < 0.00001). Rate of decline in kidney function ml.min-1.year-1 was 4.7 (3.3) measured and 4.8 (3.5) estimated (mean(SD)) (NS), but the 95% limits of agreement showed a wide range -3.9 to 3.5 ml.min-1.year-1. A significant correlation between rate of decline in measured and estimated glomerular filtration rate was present (r = 0.84, p < 0.00001). In conclusion, glomerular filtration rate is overestimated by the Cockroft-Gault formula. The mean rates of decline in glomerular filtration rate are comparable, but the limits of agreement are wide, which make the Cockroft-Gault method unacceptable for clinical purposes, i.e. monitoring progression in kidney function in the individual patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetic nephropathy is the main cause of increased mortality and morbidity in IDDM patients. The effect of antihypertensive treatment on the progression of the nephropathy is highly variable. The aim of this study was to evaluate putative predictors of the progression in diabetic nephropathy during long-term antihypertensive treatment. Eighteen hypertensive IDDM patients with diabetic nephropathy, who had not been treated previously, were followed during 3 years of treatment with captopril and frusemide or bendrofluazide. Glomerular filtration rate, arterial blood pressure, albuminuria and adjusted albuminuria were used as putative predictors of rate of decline in glomerular filtration. Fall rate in glomerular filtration rate was 4.6 (4.0) ml.min-1.year-1 (mean (SD)) during treatment. Relative change in albuminuria (ratio of first year of treatment/baseline) and albuminuria during first year of treatment were significantly correlated to fall rate in glomerular filtration rate during 3 years of treatment (r = 0.73, p < 0.001) and (r = 0.60, p < 0.01), respectively. Arterial blood pressure and glomerular filtration rate measured at baseline, during first year of treatment or relative changes in these variables did not correlate with fall rate in glomerular filtration rate during 3 years of treatment. Haemoglobin A1c, serum-cholesterol, protein intake and sodium excretion remained unchanged during treatment, and were not correlated with loss of kidney function. Reduction in albuminuria during captopril treatment predicts an attenuated rate of decline in glomerular filtration rate in early diabetic nephropathy (glomerular filtration rate > 70 ml.min-1.1.73 m-2). The finding suggests a clinical application in monitoring the efficacy of antihypertensive treatment in early diabetic nephropathy.

In an attempt to evaluate the mechanisms of proteinuria in diabetic kidney disease, we measured the renal clearances of albumin, total IgG, and IgG4 in 20 male Type 2 (non-insulin-dependent) diabetic patients with diabetic glomerulosclerosis (biopsy proven), in 10 male Type 2 diabetic patients without nephropathy (urinary albumin excretion rate < or = 30 mg/24 h), and in 10 healthy male subjects. The fractional clearance of albumin was increased in patients with nephropathy: 659 (42-4355) x 10(-6) (median (range)), compared to 2.6 (0.2-14.2) x 10(-6) in patients without nephropathy, and 2.3 (0.4-4.2) x 10(-6) in healthy subjects. The fractional clearance of total IgG (neutral) and of IgG4 (anionic) was 40-50 times higher in patients with nephropathy compared to the two other groups. The IgG/IgG4 selectivity index was not significantly different in the three groups, being: 1.12 (0.06-5.65), 1.16 (0.45-3.72) and 1.35 (0.65-3.34) in patients with nephropathy, patients without nephropathy, and healthy subjects, respectively. The IgG/albumin selectivity index was decreased in patients with nephropathy: 0.27 (0.01-1.26) compared to 1.29 (0.07-2.67) (p < 0.05) and 1.23 (0.76-7.84) (p < 0.001) in patients without nephropathy and healthy subjects, respectively. No significant change in IgG/albumin selectivity index was observed between patients without nephropathy and healthy subjects. The systolic blood pressure was elevated in the patients with nephropathy: 164 +/- 21 mm Hg (mean +/- SD) compared to patients without nephropathy: 145 +/- 20 mm Hg (p < 0.05) and to healthy subjects: 133 +/- 19 mm Hg (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

We evaluated the impact of some putative progression promoters on kidney function in albuminuric Type 2 (non-insulin-dependent) diabetic patients with biopsy-proven 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 x 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 HbA1c. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Glomerular ultrastructure was examined in a series of 20 Type 2 (non-insulin-dependent) diabetic patients with proteinuria. Reference was made to data previously obtained in non-diabetic kidney donors and in Type 1 (insulin-dependent) diabetic patients with similar degrees of proteinuria. The Type 2 diabetic patients demonstrated the changes which characterize the diabetic glomerulopathy seen in Type 1 diabetic patients: basement membrane thickening, and increase in the mesangium and mesangial matrix expressed as fraction of the glomerular volume. Among the Type 2 diabetic patients there was more variation than among the Type 1 diabetic patients, as this group included subjects with normal parameters. The group means and coefficients of variation (= SD/mean) of the glomerulopathy parameters combined in the glomerulopathy index = basement membrane thickness/10+ Vv(matrix/glom).100 were 81 (0.30) and 92 (0.15) in the two diabetic groups, clearly different from the non-diabetic index, 42 (0.16). All Type 2 diabetic patients who also had retinopathy had a glomerulopathy index above the normal range. Similar changes in glomerular composition were seen in the two diabetic groups: with increasing glomerulopathy the volume of matrix dominated over the peripheral basement membrane, and a shift in the ratio of interfaces was seen: mesangial surface towards capillary lumen increased relative to the urinary surface, and peripheral capillary surface comprised less of the total capillary surface. Data indicated marked glomerular hypertrophy, which correlated with the mesangial volume fraction, thus encompassing preserved filtration surface per glomerulus.(ABSTRACT TRUNCATED AT 250 WORDS)

The relative mortality from cardiovascular disease is on average increased five-fold in Type 2 (non-insulin-dependent) diabetic patients with diabetic nephropathy compared to non-diabetic subjects. We assessed the possible contribution of dyslipidaemia in general and elevated serum apolipoprotein(a) (apo(a)) in particular. Type 2 diabetic patients with normo-, micro- and macroalbuminuria were compared with healthy subjects. Each group consisted of 37 subjects matched for age, sex and diabetes duration. Serum creatinine in the nephropathy group was 105 (54-740) mumol/l. The prevalence of ischaemic heart disease (resting ECG, Minnesota, Rating Scale) was 57, 35, 19 and 2% in macro-, micro- and normoalbuminuric diabetic patients and healthy subjects, respectively. The prevalence of ischaemic heart disease was higher in all diabetic groups as compared to healthy subjects (p < 0.05), and higher in macroalbuminuric as compared to normoalbuminuric diabetic patients (p < 0.01). There was no significant difference between apo(a) in the four groups: 161 (10-1370), 191 (10-2080), 147 (10-942), 102 (10-1440) U/l (median (range)) in macro-, micro- and normoalbuminuric groups and healthy subjects. Serum total-cholesterol, HDL-cholesterol and LDL-cholesterol were not significantly different when comparing healthy subjects and each diabetic group. Apolipoprotein A-I was lower (p < 0.05) in all diabetic groups as compared to healthy subjects (nephropathy vs healthy subjects): 1.50 +/- 0.25 vs 1.69 +/- 0.32 g/l (mean +/- SD). Triglyceride was higher (p < 0.05) in patients with nephropathy and microalbuminuria as compared to healthy subjects (nephropathy vs healthy subjects): 2.01 (0.66-14.7) vs 1.09 (0.41-2.75) mmol/l (median (range)).(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to assess the effect of simvastatin on plasma lipoproteins and renal function in hypercholesterolaemic Type 1 (insulin-dependent) diabetic patients with diabetic nephropathy. Twenty-six hypercholesterolaemic (total cholesterol greater than or equal to 5.5 mmol/l) Type 1 diabetic patients with nephropathy were enrolled in a double-blind randomized placebo-controlled study for 12 weeks. The active treatment group (n = 14) received simvastatin (10-20 mg/day) for 12 weeks while the remaining 12 patients received treatment with placebo. The results during simvastatin treatment (baseline vs 12 weeks): total cholesterol 6.6 vs 4.8 mmol/l (p less than 0.01), LDL-cholesterol 4.25 vs 2.57 mmol/l (p less than 0.01) and apolipoprotein B 1.37 vs 1.06 mmol/l (p less than 0.01). HDL-cholesterol, and apolipoprotein A-I remained unchanged. Total cholesterol, LDL-cholesterol, HDL-cholesterol, apolipoprotein A-I, apolipoprotein B remained unchanged during placebo treatment. Albuminuria measured during the simvastatin and the placebo treatment (baseline vs 12 weeks) (the data are logarithmically transformed before analysis because of their positively skewed transformation; geometric mean (x/divided by antilog SE) is indicated) was 458 (x/divided by 1.58) vs 393 (x/divided by 1.61) and 481 (x/divided by 1.62) vs 368 (x/divided by 1.78 micrograms/min (NS). Glomerular filtration rate during simvastatin and placebo treatment (baseline vs 12 weeks) was 64 vs 63 and 72 vs 74 ml.min-1.1.73 m-2, respectively. Two patients receiving simvastatin treatment were withdrawn, one due to gastrointestinal side effects and one due to myalgia.(ABSTRACT TRUNCATED AT 250 WORDS)

A familial predisposition has been proposed as a major determinant of the increased morbidity and mortality from cardiovascular disease demonstrated in Type 1 (insulin-dependent) diabetic patients with nephropathy. We assessed this concept by studying 91 parents of Type 1 diabetic patients with nephropathy and 94 parents of aged-matched Type 1 diabetic patients with normoalbuminuria. The two groups of parents were of a similar age (58 +/- 8 vs 58 +/- 7 years). The prevalence (%) of death and cardiovascular diseases (World Health Organisation questionnaire) was 10 (4-18)% and 12 (6-21)% in parents of nephropathic patients compared to 8 (3-16)% and 13 (6-23)% in parents of normoalbuminuric Type 1 diabetic patients. The frequency of risk factors for cardiovascular disease were about the same in both groups of parents. Microalbuminuria was found in 5% and 11%, hypercholesterolaemia (greater than 6.5 mmol/l) in 25% and 26% and smokers constituted 40% and 34% of parents of patients with and without proteinuria, respectively. A familial predisposition to cardiovascular disease cannot explain the increased morbidity and mortality from cardiovascular disease in young patients with diabetic nephropathy.

The sodium retaining effect of insulin was studied in ten Type 2 (non-insulin-dependent) diabetic patients (mean age 56 (43-73) years, mean body mass index 29.5 (24.2-33.7) kg/m2) and eight age-matched control subjects (mean age 57 (43-68) years, mean body mass index 23.4 (20.8-26.6) kg/m2). The renal clearances of 99mTc-DTPA, lithium, sodium and potassium were measured over a basal period of 90 min. Then insulin was infused at a rate of 40 mU.min-1.m-2. After an equilibration period of 90 min, the clearance measurements were repeated during a new 90 min period. Blood glucose was clamped at the basal level (diabetic patients: 9.9 +/- 3.5, control subjects: 5.3 +/- 0.5 mmol/l) by a variable glucose infusion. Basal plasma insulin concentration was elevated in the diabetic patients (0.12 +/- 0.05 vs 0.05 +/- 0.02 pmol/ml, p less than 0.01). Insulin infusion resulted in comparable absolute increments in plasma insulin concentrations in the diabetic group and in the control group (0.44 +/- 0.13 vs 0.36 +/- 0.07 pmol/ml, NS). The metabolic clearance rate of glucose during the last 30 min of insulin infusion was lower in the diabetic patients (155 +/- 62 vs 320 +/- 69 ml.min-1.m-2, p less than 0.01), reflecting peripheral insulin resistance. The decline in sodium clearance during insulin infusion was similar in diabetic subjects (1.8 +/- 1.1 vs 0.7 +/- 0.4 ml.min-1.1.73 m-2, p less than 0.01) and in control subjects (1.7 +/- 0.3 vs 0.8 +/- 0.3 ml.min-1.1.73 m-2, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic predisposition to essential hypertension, represented by maximal erythrocyte sodium/lithium countertransport activity, has been suggested as a marker for the risk of developing clinical nephropathy in Type 1 (insulin-dependent) diabetes mellitus. To evaluate this hypothesis we measured arterial blood pressure and maximal sodium/lithium countertransport activity of erythrocytes in 80 parents of 49 Type 1 diabetic patients with clinical nephropathy, 78 parents of 49 normoalbuminuric patients and 17 age-matched non-diabetic individuals. The two diabetic groups were carefully matched. In the two groups of parents blood pressure and cell sodium/lithium countertransport activity showed no significant differences (137/83 vs 133/81 mm Hg and 0.33 vs 0.32 mmol/(1 cells x h) respectively). The proportion of parents who had died or received antihypertensive drugs was similar in the two groups. The patients with Type 1 diabetes had significantly higher sodium/lithium countertransport compared to the 39 non-diabetic control subjects independently of the presence or absence of nephropathy (p less than 0.002). However, patients with nephropathy tended to have higher sodium/lithium countertransport activity than normoalbuminuric patients (0.48 vs 0.41 mmol/(1 cells x h), p = 0.06). We conclude that genetic predispositions to essential hypertension and increased maximal erythrocyte sodium/lithium countertransport activity do not appear to be risk markers for the development of clinical nephropathy in Type 1 diabetic patients.

Kidney function was studied in nine, metabolically well controlled, short-term insulin-dependent male diabetics before and during glucagon infusion of 4 to 5 and 8 to 10 ng/kg/min. Glomerular filtration rate, effective renal plasma flow (steady-state infusion technique, with urinary collections, using 125I-iothalamate and 131I-iodohippurate), and urinary albumin and beta 2-microglobulin excretion rates were measured. The mean plasma glucagon concentration increased during infusion from 254 +/- 19 pg/ml to 440 +/- 31 pg/ml (low dose) and 730 +/- 52 pg/ml (high dose). Glomerular filtration rate increased in all subjects from 133 +/- 5 before the glucagon infusion to 141 +/- 4 with the low dose, and 148 +/- 7 ml/min/1.73 m2 with the high dose (p < 0.01). The increase in glomerular filtration rate correlated with the rise in plasma glucagon concentration (r = 0.67; p < 0.01). Renal plasma flow increased from 530 +/- 21 before the glucagon infusion to 555 +/- 20 with the low dose and 572 +/- 29 ml/min/1.73 m2 with the high dose (p < 0.01). Urinary beta 2-microglobulin excretion rate rose from 5.8 +/- 1.0 before infusion to 8.7 +/- 1.7 with the low dose, and 17.9 +/- 5.7 micrograms X 10(-2)/min with the high dose (p < 0.01). Urinary albumin excretion remained unchanged during the glucagon infusion. These results suggest that glucagon may contribute to the reversible elevation of glomerular filtration rate typically found in poorly regulated insulin-dependent diabetics, but not to the moderate elevation found in well controlled diabetics.

Glomerular filtration rate (GFR), renal plasma flow (RPF) and kidney volume were measured in thirteen male subjects (mean age 30 years) with short-term insulin-dependent diabetes (mean duration of disease 2.4 years) and fourteen normal male subjects (mean age 29 years). GFR and RPF were measured by constant infusion technique using I125-iothalamate and 131I-hippuran. Kidney size was determined by means of ultrasound. GFR, RPF and kidney volume were increased in the diabetic patients compared to the normal controls, 144 versus 113 ml/min X 1.73 m2 (p less than 0.0005), 627 versus 523 ml/min X 1.73 m2 (p less than 0.0025) and 278 versus 224 ml/1.73 m2 (p less than 0.0005) respectively. Combining results from diabetic patients and controls revealed a positive correlation between kidney size and GFR (r = 0.70, p less than 0.001) and between kidney size and RPF (r = 0.61, p less than 0.001). Within the groups kidney size and RPF correlated significantly in the diabetics (p less than 0.01) and the same was found for kidney size and GFR (0.025 less than p less than 0.05), while no correlations were found in the normal group. GFR and RPF correlated in the diabetics when evaluated separately (r = 0.81, p less than 0.001) and in the controls (r = 0.73, p less than 0.001). The previous and present data suggest that the mechanisms of the elevated GFR in insulin-dependent diabetics are enhanced RPF, increased transglomerular hydrostatic pressure gradient and increased glomerular ultrafiltration coefficient. The increased kidney size is probably the main cause of the above alterations in the GFR determinants.

The relationship between arterial blood pressure and diabetic nephropathy was examined in 61 Type 1 (insulin-dependent) diabetic patients (22 females and 39 males). All patients fulfilled the following criteria: persistent proteinuria (greater than 0.5 g/day), onset of diabetes before 31 years of age, age less than 42 years, serum creatinine less than 130 mumol/l, and no antihypertensive treatment. Thirty Type 1 diabetic patients without persistent proteinuria but matched for sex, age, ideal body weight and duration of diabetes, and 30 healthy subjects matched for sex, age and ideal body weight were also studied as controls. The diabetic patients with persistent proteinuria had elevated blood pressures (146/96 +/- 17/10 mmHg, mean +/- SD) compared with 123/75 +/- 11/8 mmHg in diabetic patients without persistent proteinuria, and normal subjects (120/77 +/- 6/6 mmHg; p less than 0.001 for each). Diastolic blood pressure greater than or equal to 95 mmHg was found in 51% of the group with persistent proteinuria. Elevated arterial blood pressure is frequently present in young Type 1 diabetic patients with diabetic nephropathy and normal serum creatinine.

Renal clearance of dextran of two ranges of molecular size and glomerular filtration rate (GFR, 51Cr-EDTA) were measured in seven short-term insulin-dependent diabetics (mean age 25 years). Measurements were carried out in the same patient during good and poor metabolic regulation (plasma glucose, mean +/- SEM, 6.5 +/- 0.9 and 14.8 +/- 1.5 mmol/l, respectively). GFR was elevated in all patients during poor metabolic regulation (119 +/- 6 ml/min/1.73 m2, versus 99 +/- 2 ml/min/1.73 m2 during good control, p less than 0.01). The average renal clearance of dextran with molecular weights ranging from 25,000 to 35,000 and 35,000 to 45,000 increased during poor metabolic regulation from 14.8 +/- 0.8 to 19.8 +/- 1.8 ml/min/1.73 m2, and 5.2 +/- 0.3 to 6.8 +/- 0.6 ml/min/1.73 m2, respectively (p less than 0.05). The elevated GFR and renal dextran clearance found during poor metabolic regulation were normalized within one to three weeks of effective insulin treatment. This rapid reversibility can hardly be explained by the previously demonstrated enlargement in glomerular size and filtration surface area, since these alterations remain unchanged after more than one month of insulin treatment. The metabolic regulation did not influence the size-selective properties of the glomerular wall. Therefore, we suggest that the dominating mechanism involved in the GFR and renal dextran clearance alterations is functional, viz. increased filtration pressure.

This study was undertaken to determine whether the distensibility of a passive vascular bed is reduced in Type 1 (insulin-dependent) diabetic patients with microangiopathy. The change in blood flow induced by 45 degrees head-up tilting was studied in two systems: (a) following maximal ischaemic exercise and (b) in a vascular bed locally paralysed by the injection of papaverine. Five normal subjects, six patients with long-standing Type 1 diabetes and six non-diabetic patients with severe atherosclerosis affecting the legs were studied. Blood flow was measured in the anterior tibial muscle by the isotope washout technique. The median increase in blood flow produced by tilting was greater in normal subjects than in diabetic subjects in both the locally-relaxed bed (58% and 14% respectively) and after maximal ischaemic exercise (45% and 4% respectively). In the atherosclerotic subjects, the increase in blood flow in the locally relaxed bed was 77%. The results are consistent with the hypothesis that the reduced distensibility seen in the diabetic subjects was related to the presence of microvascular disease and that the behaviour of a vascular bed relaxed by the local injection of papaverine might be an appropriate model to study this condition.