Cardiovascular outcomes with antihypertensive therapy in type 2 diabetes: an analysis of intervention trials.

In studies of antihypertensive therapy, relative cardiovascular (CV) risk reduction is largely independent of attained systolic blood pressure (SBP). How this translates to absolute risk reduction (ARR) in diabetes is not clear. We have compared 5-year CV outcomes in 10 studies of intensive versus moderate or active versus placebo therapy in subjects with type 2 diabetes and attained SBP < or ⩾ 140 mm Hg. Attained SBP ⩾ 140 mm Hg occurred in five early studies (HOT n = 1001, UKPDS n = 1148, SHEP n = 583, SYSTEUR n = 422, MICRO_HOPE n = 3377) and attained SBP<140 mm Hg occurred in five recent studies (ABCD-NT n = 480, ADVANCE n = 11,140 INVEST n = 4266, ACCORD n = 4733, ROADMAP n= 4447). In each study, ARR was calculated from group mean data and expressed as % change in CV events over 5 years per 10 mm Hg decrease in attained SBP. In studies with attained SBP ⩾ 140 mm Hg, ARR was 13 ± 2.6% per 10 mm Hg, and the number needed to treat (NNT) to prevent one event in 5 years was 8. In studies with attained SBP < 140 mm Hg, ARR was 1.6 ± 1 .9% per 10 mm Hg (P = 0.0007), and NNT was 68. The present analysis indicates that CV outcomes reach a plateau after attaining SBP of 140 mm Hg in patients with type 2 diabetes.

Estimating dual-energy X-ray absorptiometry-derived total body skeletal muscle mass using single-slice abdominal magnetic resonance imaging in obese subjects with and without diabetes: a pilot study.

BACKGROUND/OBJECTIVES: Single-slice abdominal computed tomography or magnetic resonance imaging (MRI) performed to measure visceral adipose tissue in individuals with obesity and diabetes mellitus can also image skeletal muscle. The aim of this study was to validate a method developed in cancer patients using a single abdominal cross-sectional image to estimate fat-free mass (FFM) and appendicular lean tissue mass index (LTMI), a total body skeletal muscle mass surrogate, in an obese cohort of subjects with and without type 2 diabetes. SUBJECTS/METHODS: In total, 49 obese subjects (22 with diabetes) recruited into a weight loss study underwent dual-energy X-ray absorptiometry (DXA) and abdominal MRI at baseline. DXA-derived FFM and LTMI were compared with skeletal muscle area at the level of the third lumbar vertebra (L3) on MRI. RESULTS: L3 skeletal muscle area correlated with FFM (R (adj) (2)=0.825; P<0.001) and LTMI (R (adj) (2)=0.6; P<0.001). A simple formula, previously shown to predict LTMI in cancer patients, produced a good estimation of LTMI from L3 skeletal muscle area (95% confidence interval -3.70, 2.56%) in our obese cohort. Equations incorporating age, sex, height, weight and diabetic status improved the relationship between L3 skeletal muscle area and estimated FFM (r=0.976, P<0.001) and LTMI (r=0.879, P<0.001). CONCLUSION: A single-axial slice at the L3 level can be used to estimate FFM and LTMI in obese diabetic and non-diabetic subjects, allowing precise analysis of body composition using a single imaging modality in clinical research and practice.

The clinical significance of hyperfiltration in diabetes.

Glomerular filtration rate is commonly elevated in early diabetes and patients with this symptom are arbitrarily considered to have hyperfiltration. The prevalence of hyperfiltration in type 1 diabetes varies from less than 25% to more than 75%. The corresponding figures in type 2 diabetes are significantly lower, ranging between 0% and more than 40%. Several factors, methodological and biological, may contribute to the wide variation in estimates of hyperfiltration prevalence. Methodological differences in measurement and evaluation of GFR apply in particular to the handling of plasma disappearance curves of filtration markers. Biological factors that may influence GFR in the hyperfiltration range include glycaemic control, diabetes duration, BMI, sex, pubertal status in type 1 diabetes and age in type 2 diabetes. Hyperglycaemia may influence GFR and albuminuria, and may therefore confound the evaluation of hyperfiltration as an independent risk factor for diabetic nephropathy. Adequate assessment of the relationship between glycaemic control, GFR and AER therefore requires serial measurements of all three variables followed by multivariate analysis. A recent meta-analysis of ten type 1 diabetes studies concluded that the presence of hyperfiltration at baseline more than doubled the risk of developing micro- or macroalbuminuria at follow-up. However, not all studies allowed for confounding factors or regression dilution bias. Future studies will therefore need to address the independent role of hyperfiltration, not only in the evolution of albuminuria, but also in the subsequent decline of GFR.

Salt supplementation blunts the blood pressure response to telmisartan with or without hydrochlorothiazide in hypertensive patients with type 2 diabetes.

AIMS/HYPOTHESIS: We assessed the effects of sodium chloride (NaCl) supplementation on the blood pressure response to treatment with telmisartan with or without hydrochlorothiazide in hypertensive patients with type 2 diabetes and habitually high (HDS, sodium excretion >200 mmol/24 h on two out of three consecutive occasions) or low (LDS, sodium excretion <100 mmol/24 h on two out of three consecutive occasions) salt intake. METHODS: Patients received 4 weeks of telmisartan followed by 4 weeks of telmisartan plus hydrochlorothiazide. In a double-blind randomised fashion, patients received sodium chloride (NaCl, 100 mmol/24 h) or placebo capsules in addition to their habitual salt intake during the last 2 weeks of telmisartan and telmisartan plus hydrochlorothiazide therapy. The protocol was repeated with NaCl and placebo capsules administered in reverse order to allow each participant to act as his or her own control. At 0, 4, 8, 14, 18 and 22 weeks, 24 h ambulatory blood pressure (ABP) and 24 h urine collections were performed. RESULTS: No statistically significant differences were seen in the ABP response in the LDS vs HDS groups to any of the interventions (p = 0.58). NaCl supplementation reduced the effect of telmisartan with or without hydrochlorothiazide on systolic BP by approximately 50% (-5.8 mmHg during NaCl supplementation vs -11.3 mmHg during placebo, mean difference 5.6 mmHg [95% CI 1.7-9.4 mmHg], p = 0.005), irrespective of habitual salt intake. By contrast, addition of hydrochlorothiazide increased the antihypertensive effect of telmisartan on systolic BP by approximately 35% (p = 0.048) in both groups of patients. CONCLUSIONS/INTERPRETATION: NaCl supplementation blunts the effectiveness of telmisartan with or without hydrochlorothiazide in hypertensive patients with type 2 diabetes, independently of habitual low or high salt intake.

New and old markers of progression of diabetic nephropathy.

The onset of diabetic nephropathy is characterised by a rise in albumin excretion rate (AER) and/or a transient rise in glomerular filtration rate (GFR) (hyperfiltration). Without intervention AER increases exponentially and there is a linear decrease in GFR after onset of overt nephropathy. In overt nephropathy, AER is a predictor of decline in GFR and the early AER response to antihypertensive therapy correlates with long-term decline in GFR. AER can be measured by immunoassay or by other techniques including HPLC. However, HPLC assays result in higher levels of AER in normal subjects compared with immunoassayable AER. Recent data suggest that there are distinct albuminuric and non-albuminuric pathways to renal impairment in type 1 and type 2 diabetes. In type 2 diabetes, the non-albuminuric pathway may explain a decline in GFR to <60 ml/min/1.73 m(2) in approximately one in four subjects after accounting for the use of renin angiotensin system inhibitors. In established nephropathy (chronic kidney disease (CKD) stages 3 and 4), plasma cystatin C based estimates of GFR are marginally superior to creatinine based estimates. However, cystatin C clearly outperforms creatinine based estimates of GFR decline at GFR levels >60 ml/min/1.73 m(2) (CKD stages 1 and 2). Other potential markers of progression of diabetic nephropathy include transforming growth factor beta (TGFbeta) and connective tissue growth factor (CTGF). However, long-term studies are needed to define their roles as markers of progression. Diabetic nephropathy is likely to be more susceptible to intervention at an early stage and accurate estimation of GFR is already possible with cystatin C. However, improved formulas for estimating GFR based on using creatinine or other markers are still required, because this may still provide the most cost effective approach applicable to existing clinical practice.

The accuracy of cystatin C and commonly used creatinine-based methods for detecting moderate and mild chronic kidney disease in diabetes.

BACKGROUND: The accuracy of measuring serum cystatin C levels for detecting various stages of chronic kidney disease (CKD) in diabetes is still unclear. METHODS: In a cross-sectional study of 251 subjects, a reference glomerular filtration rate (GFR) was measured using (99c)Tc-DTPA plasma clearance (iGFR). Multivariate analysis was used to identify independent clinical and biochemical associations with serum cystatin C and iGFR levels. The diagnostic accuracy of cystatin C and commonly used creatinine-based methods of measuring renal function (serum creatinine, the MDRD four-variable and Cockcroft-Gault formulae) for detecting mild and moderate CKD was also compared. RESULTS: In the entire study population the same five variables, age, urinary albumin excretion rates, haemoglobin, history of macrovascular disease and triglyceride levels were independently associated with both cystatin C and iGFR levels. A serum cystatin C level cut-off > 82.1 nmol/l (1.10 mg/l) had the best test characteristics as a screening tool for detecting moderate CKD (< 60 ml/min per 1.73 m(2)) when compared with creatinine-based methods. At the upper threshold for mild CKD (< 90 ml/min per 1.73 m(2)), cystatin C also had greater diagnostic accuracy than creatinine, but had similar diagnostic accuracy when compared with creatinine-based formulae for predicting renal function. CONCLUSIONS: This study suggests that the clinical and biochemical parameters associated with serum cystatin C levels are closely linked to those associated with GFR and highlights the potential usefulness of screening for moderate or mild CKD in subjects with diabetes by simply measuring serum cystatin C levels.

Estimating glomerular filtration rate in diabetes: a comparison of cystatin-C- and creatinine-based methods.

AIMS/HYPOTHESIS: We compared the predictive performance of a GFR based on serum cystatin C levels with commonly used creatinine-based methods in subjects with diabetes. SUBJECTS, MATERIALS AND METHODS: In a cross-sectional study of 251 consecutive clinic patients, the mean reference (plasma clearance of (99m)Tc-diethylene-triamine-penta-acetic acid) GFR (iGFR) was 88+/-2 ml min(-1) 1.73 m(-2). A regression equation describing the relationship between iGFR and 1/cystatin C levels was derived from a test population (n=125) to allow for the estimation of GFR by cystatin C (eGFR-cystatin C). The predictive performance of eGFR-cystatin C, the Modification of Diet in Renal Disease 4 variable formula (MDRD-4) and Cockcroft-Gault (C-G) formulas were then compared in a validation population (n=126). RESULTS: There was no difference in renal function (ml min(-1) 1.73 m(-2)) as measured by iGFR (89.2+/-3.0), eGFR-cystatin C (86.8+/-2.5), MDRD-4 (87.0+/-2.8) or C-G (92.3+/-3.5). All three estimates of renal function had similar precision and accuracy. CONCLUSIONS/INTERPRETATION: Estimates of GFR based solely on serum cystatin C levels had the same predictive potential when compared with the MDRD-4 and C-G formulas.

Renal hyperfiltration in type 2 diabetes: effect of age-related decline in glomerular filtration rate.

AIMS/HYPOTHESIS: We sought to characterise the effect of the age-related decline of GFR on hyperfiltration in type 2 diabetes and to identify clinical characteristics associated with hyperfiltration. MATERIALS AND METHODS: GFR was measured in 662 type 2 diabetic patients by plasma disappearance of 99 m-technetium-diethylene-triamine-penta-acetic acid. The prevalence of hyperfiltration was calculated using both an age-unadjusted GFR threshold of >130 ml min(-1) 1.73 m(-2) and an age-adjusted threshold incorporating a decline of 1 ml min(-1) year(-1) after the age of 40. The hyperfiltering patients were compared with type 2 diabetic subjects who had a GFR between 90 and 130 ml min(-1) 1.73 m(-2) and were matched for age, sex and disease duration to allow for identification of modifiable factors associated with hyperfiltration. RESULTS: The prevalence of hyperfiltration was 7.4% when age-unadjusted and 16.6% when age-adjusted definitions were used. The age-unadjusted vs -adjusted prevalence rates for hyperfiltration were 50 vs 50%, 12.9 vs 23.4% and 0.3 vs 9.0% for patients aged <40 years, 40 to 65 years and >65 years, respectively. Both the age-unadjusted and -adjusted hyperfiltration groups had lower mean diastolic blood pressure and lower serum creatinine levels than the control groups. Although the age-unadjusted hyperfiltration group had larger kidneys compared to the control group, this difference was no longer significant when the age-adjusted definition was used. There were no differences in HbA(1)c, mean arterial pressure, antihypertensive use, insulin therapy, dyslipidaemia, frequency of macro- or microvascular complications, BMI, urinary sodium, urea and albumin excretion between the groups. CONCLUSIONS/INTERPRETATION: Hyperfiltration was still more common among younger patients with type 2 diabetes even after adjusting for the expected age-related decline in GFR. Hyperfiltration was associated with a lower mean diastolic blood pressure independent of age.

Super high flux hemodialysis at high dialysate flows: an ex vivo assessment.

BACKGROUND AND OBJECTIVES: The removal of cytokines by standard hemofiltration is limited. Super high flux membranes may significantly improve removal even when used in dialysis mode. We sought to measure cytokine clearance using a large surface super high-flux membrane and a standard hemodialysis setting. SETTING: ICU laboratory of a tertiary institution. SUBJECTS: Six healthy volunteers. METHODS: Blood form healthy volunteers was incubated for 4 hours with E. coli endotoxin to stimulate cytokine production. Cytokine containing blood was then circulated through a dialysis circuit at 3 different dialysate flow rates. Blood and dialysate were sampled for cytokine and albumin measurements and calculation of clearances. RESULTS: Super high-flux dialysis achieved high median cytokine clearances (IL-1 clearance of 106 ml/min, IL-6 clearance of 66.8 ml/min, IL-8 clearance of 61.7 ml/min and TNF clearance of 36.1 ml/min). Increasing dialysate flow rate from 300 to 500 ml/min did not significantly increase cytokine clearances. Albumin clearances however were between 2.7 and 5.4 ml/min. CONCLUSIONS: Cytokine dialysis is feasible at high dialysate flow rates yielding high cytokine clearances. Albumin loss, however, is appreciable and may require separate supplementation in the clinical setting.

Beta2-microglobulin clearance with super high flux hemodialysis: an ex vivo study.

BACKGROUND: Beta2m accumulation induces disease in patients with end-stage renal failure (ESRF). Thus, its removal from patients with ESRF appears desirable. Current dialysis technology, however, has limited effectiveness. AIMS: To measure beta2m clearance with a novel super high flux membrane. DESIGN: Ex vivo experimental study. SETTING: Intensive Care Laboratory of Tertiary institution. SUBJECTS: Six volunteers. MEASUREMENTS AND RESULTS: At a blood flow of 300 ml/min, the clearance of beta2-MG increased from 113.5 +/- 38.5 ml/min with a dialysate flow rate of 200 ml/min to 184.8 +/- 61.1 ml/min with a flow rate of 300 ml/min and 195.0 +/- 60.0 ml/min with a 500 ml/min flow rate. The clearance of albumin was 4.5 ml/min with a dialysate flow rate of 200 ml/min, 5.2 ml/min for a flow rate of 300 ml/min and 5.8 ml/min for a flow rate of 500 ml/min. CONCLUSIONS: High levels of beta2m clearance can be achieved with a super high flux membrane while albumin losses remain limited.

Super high flux hemofiltration: a new technique for cytokine removal.

OBJECTIVE: To test whether hemofiltration using a hemofilter with large pores (super high flux hemofiltration) achieves effective cytokine removal. DESIGN: : Ex vivo study. SETTING: Laboratory of an intensive care unit in a tertiary hospital. PATIENTS AND PARTICIPANTS: Five healthy volunteers. INTERVENTIONS: Blood was spiked with 1 mg of endotoxin and then circulated through a closed hemofiltration circuit with a large pore polyamide super high flux hemofilter (nominal cut-off point: 100 kDa). Hemofiltration was conducted at 1 l/h or 6 l/h of ultrafiltrate flow. Samples were taken from the arterial, venous and ultrafiltration sampling ports. MEASUREMENTS AND RESULTS: Sieving coefficients (SC) above 0.6 were achieved for interleukin (IL)-1beta, IL-6 and IL-10 and SCs above 0.3 were achieved for IL-8 and TNF-alpha at 1 l/h. SCs of all cytokines (except IL-1) were reduced when the ultrafiltration rate was increased from 1 l/h to 6 l/h ( p<0.01), but cytokine clearances still increased ( p<0.01). The highest SC for albumin was 0.1 at 1 l/h and fell to 0.01 at 6 l/h. No adsorption of cytokines and albumin was observed. CONCLUSION: High volume ultrafiltration using a super high flux filter achieved cytokine clearances comparable to, or greater than, those currently achieved for urea during standard continuous renal replacement therapy.

Cytokine removal with a large pore cellulose triacetate filter: an ex vivo study.

OBJECTIVE: To test the hypothesis that hemofiltration using a new large pore cellulose triacetate hemofilter can achieve effective ultrafiltration of cytokines. DESIGN: Ex-vivo study. SETTING: Laboratory of Intensive Care Unit in tertiary hospital. SUBJECTS: Six healthy volunteers. INTERVENTIONS: Blood from 6 volunteers was incubated for 4 hours with 1 mg of endotoxin and then circulated through a closed hemofiltration circuit with a large pore cellulose triacetate hemofilter (nominal cut-off point: 60 kilodaltons). Hemofiltration was conducted at 1 L/h or 6 L/h of ultrafiltrate (UF) flow at the start of extra-corporeal circulation, and after 2 and 4 hours. Samples were taken from the arterial, venous and UF sampling ports. MEASUREMENTS AND MAIN RESULTS: IL-Ibeta, IL-6, IL-8, IL-10, TNFalpha, and albumin were measured. Sieving coefficients (SC) above 0.6 were achieved for IL-Ibeta and IL-6 and SCs above 0.3 were achieved for IL-8 and TNF-alpha at 1 L/h. Sieving coefficients of all cytokines (except IL-10, p=0.22) were reduced when the ultrafiltration rate was increased from IL/h to 6 L/h (p<0.01), but the increase in ultrafiltration rate resulted in an overall increase in the clearance of all cytokines (p<0.001). The highest SC for albumin was 0.07 at 4 hours at 1 L/h, and fell to 0.01 at 6 L/h. The SCs for IL-8 fell at 4 hours (p<0.01), but the SCs for other cytokines did not change. No adsorption of cytokines and albumin was observed. CONCLUSION: High volume hemofiltration (HVHF) using a new large pore cellulose triacetate filter achieved cytokine clearances greater than those reported with currently available hemo filters.

Renoprotective effects of a novel inhibitor of advanced glycation.

AIMS/HYPOTHESIS: ALT-946, an inhibitor of advanced glycation with a minimal inhibitory effect on nitric oxide synthase, was compared with aminoguanidine in experimental diabetic nephropathy. METHODS: In vitro and in vivo assays were used to assess the ability of ALT-946 to inhibit AGE-protein cross-link formation. Diabetic animals were randomly allocated into groups receiving aminoguanidine for 32 weeks, ALT-946 or vehicle (untreated). As a delayed intervention protocol, an additional diabetic group was treated with ALT-946 from week 16 to week 32 of the study. Non-diabetic rats were studied concurrently. Systolic blood pressure, body weight, plasma glucose, glycated haemoglobin and urinary albumin excretion were measured serially. Accumulation of advanced-glycation end products in the kidney was assessed by immunohistochemistry. RESULTS: The ALT-946 inhibitor was more potent than aminoguanidine in inhibiting AGE-protein cross-linking both in vitro and in vivo. Increased albuminuria observed in diabetic rats was attenuated in all three treatment groups. We found no difference in body weight, blood pressure or glycaemic control with any of the treatments. The untreated diabetic group had a twofold increase in glomerular staining for advanced-glycation end products compared with the diabetic groups which received treatment. CONCLUSION/INTERPRETATION: ALT-946 is a potent inhibitor of advanced renal glycation end-product accumulation and reproduces the renoprotective effects of aminoguanidine. Therefore, ALT-946 should be considered as a treatment for preventing or retarding diabetic nephropathy.

Aminoguanidine and ramipril prevent diabetes-induced increases in protein kinase C activity in glomeruli, retina and mesenteric artery.

This study investigated the effects of insulin therapy, inhibition of advanced glycation end-product formation with aminoguanidine and angiotensin-converting enzyme inhibition with ramipril on diabetes-related increases in protein kinase C (PKC) activity in the streptozotocin-diabetic rat. PKC activity in the glomeruli, retina and mesenteric artery was increased by 1.5-2-fold after induction of diabetes, and this increase was maintained over 24 weeks. Treatment with insulin at 2 units or 6 units per day attenuated glomerular PKC in proportion to the level of glycohaemoglobin after 4 weeks of diabetes (r=0.68, P<0.0001). The higher dose of insulin prevented the diabetes-related increase in glomerular PKC activity, although blood glucose levels were not normalized. After 8 weeks of diabetes, ramipril completely prevented the diabetes-related increases in PKC activity in the glomeruli, retina and mesenteric artery. By contrast, aminoguanidine treatment resulted in no inhibition of glomerular PKC activity, partial inhibition of retinal PKC activity and complete inhibition of mesenteric artery PKC activity. After 24 weeks of diabetes, both aminoguanidine and ramipril prevented the diabetes-related increases in PKC activity in all three tissues, in parallel with suppression of albuminuria by both agents. Aminoguanidine also prevented diabetes-related increases in retinal permeability at 16 weeks. These results suggest that the organ-protective effects of insulin, aminoguanidine and ramipril in diabetes may be mediated, at least in part, through the differential inhibition of PKC activity in various tissues.

Angiotensin converting enzyme inhibition reduces retinal overexpression of vascular endothelial growth factor and hyperpermeability in experimental diabetes.

AIMS/HYPOTHESIS: Angiotensin converting enzyme (ACE) inhibition has been recently suggested to have retinoprotective actions in diabetic patients but the mechanism of this effect is not known. In vitro, angiotensin II stimulates expression of vascular endothelial growth factor (VEGF), a permeability-inducing and endothelial cell specific angiogenic factor which has been implicated in the pathogenesis of diabetic retinopathy in humans and in experimental animals. We sought to determine the effects of ACE inhibition on retinal VEGF expression and permeability in experimental diabetic retinopathy. METHODS: Streptozotocin-induced diabetic rats and control animals were assigned at random to receive ACE inhibitor treatment or vehicle. At 24 weeks the retinal VEGF protein gene expression was assessed by northern blot analysis and in situ hybridisation. Retinal permeability to albumin was measured using a double isotope technique. RESULTS: Experimental diabetes was associated with cell specific two to fourfold increase in retinal VEGF protein gene expression (p < 0.01) and a 2-fold increase in retinal vascular permeability to albumin (p < 0.01). The localization of VEGF expression in the retina was not altered in animals with experimental diabetes. Angiotensin converting enzyme inhibitor treatment of diabetic rats reduced diabetes-associated changes in VEGF gene expression and vascular permeability. CONCLUSION/INTERPRETATION: These findings implicate the renin-angiotensin system in the VEGF overexpression and hyperpermeability which accompany diabetic retinopathy and provide a potential mechanism for the beneficial effects of ACE inhibition in diabetic retinal disease.

Proteolysis of insulin-like growth factor-binding protein-3 is increased in urine from patients with diabetic nephropathy.

The insulin-like growth factor (IGF) system has been implicated in the development of experimental diabetic nephropathy. IGF-binding protein-3 (IGFBP-3) modulates IGF actions, and proteolysis decreases its binding affinity for IGFs. The aim of this study was to explore the possibility that proteolysis of IGFBP-3 may be altered in diabetic nephropathy and may therefore modify the intrarenal effects of IGFs. IGFBP-3 proteolysis in urine from diabetic patients with normo- [albumin excretion rate (AER), <20 microg/min], micro- (AER, 20-200 microg/min), and macroalbuminuria (AER, >200 microg/min) was studied in 34 patients with noninsulin-dependent diabetes mellitus (NIDDM), 14 patients with insulin-dependent diabetes mellitus, and 9 controls. Urine samples were analyzed by Western ligand blotting and IGFBP-3 immunoblotting. Protease activity was quantitated using [125I]IGFBP-3 as a substrate. WLB showed three main bands (40-46, 35, and 26 kDa) in control urine and a fainter 18-kDa band. All but the 35-kDa band were immunoreactive with the IGFBP-3 antiserum. The same pattern of IGFBPs was seen in urine from normoalbuminuric diabetic patients. However, the urine of diabetic patients with micro- and macroalbuminuria contained little or no intact 40- to 46-kDa IGFBP-3. In patients with noninsulin-dependent diabetes mellitus, urinary IGFBP-3 protease activity in micro- (n = 13) and macroalbuminuric patients (n = 12; mean +/- SD[SCAP], 75 +/- 25% and 84 +/- 24%) was significantly higher than that in normoalbuminuric patients (29 +/- 9%; P = 0.0001). Similar results were observed in patients with insulin-dependent diabetes mellitus. Proteolytic activity in diabetic urine was due to a serine protease. In conclusion, diabetic nephropathy was associated with IGFBP-3 proteolysis in urine. As similar changes were not observed in patients' sera, this is likely to reflect changes in the kidney or urinary tract, resulting in increased local IGF bioavailability, and therefore may contribute to the structural changes of diabetic nephropathy.

Prevention of albuminuria by aminoguanidine or ramipril in streptozotocin-induced diabetic rats is associated with the normalization of glomerular protein kinase C.

This study examined whether the prevention of diabetes-related albuminuria by aminoguanidine (AG) or ramipril (RAM) may be mediated by a common post-glomerular basement membrane renal intracellular mechanism involving protein kinase C (PKC). The renal handling of albumin was examined over 24 weeks in control and streptozotocin (STZ)-induced diabetic rats. A radioimmunoassay (RIA) that measures intact albumin, and intravenously injected tritium-labeled rat serum albumin, was used to assess the proportion of intact albumin and albumin fragments in urine. Diabetes was induced in male Sprague-Dawley rats by the intravenous administration of STZ at a dose of 50 mg/kg. Age-matched control rats received buffer alone. Diabetes was characterized by an increase in blood glucose (>15 mmol/l), an increase in GHb (means at 24 weeks 29.3+/-1.1%; control 6.1+/-0.1%, P<0.005), an increase in glomerular filtration rate (GFR) (4.13+/-0.15 ml/min; control 3.54+/-0.19 ml/min, P<0.005), an increase in intact albumin excretion rate (expressed as geometric mean 11.64 times/divided by 2.11 mg/24 h; control 0.74 times/divided by 1.57 mg/24 h, P<0.005) as measured by RIA, and an increase in glomerular PKC activity (26.83+/-2.38 pmol x mg(-1) x min(-1); control 14.6+/-2.99 pmol x mg(-1) x min(-1), P<0.005). Treatment of diabetic rats with either AG or RAM prevented the rise in intact albuminuria and glomerular PKC activity. Renal lysosomal cathepsin activity decreased in diabetic rats and this was not prevented by AG or RAM. Neither drug affected glycemic control or GFR, but RAM reduced systolic blood pressure (BP), whereas AG did not. These data indicate that urinary excretion of intact albumin and albumin-derived fragments in diabetes may be modulated independently of glycemic control (AG and RAM) and systolic BP (RAM). While both drugs are known for their different mechanisms of action, the fact that both prevent diabetes-related increases in glomerular PKC activity and albuminuria supports the hypothesis that PKC plays a central role in the development of diabetic nephropathy.

Effect of diabetes and aminoguanidine therapy on renal advanced glycation end-product binding.

BACKGROUND: Advanced glycation end-products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy, and aminoguanidine (AG) has been shown to decrease the accumulation of AGEs in the diabetic kidney. METHODS: This study investigates changes in AGE binding associated with diabetes in the rat kidney using in vitro and in vivo autoradiographic techniques. Male Sprague-Dawley rats were randomized into control and diabetic groups with and without AG treatment and were sacrificed after three weeks. Frozen kidney sections (20 microm) were incubated with [125I]-AGE-RNase or [125I]-AGE-BSA. To localize the AGE binding site, in vivo autoradiography was performed by injection of 15 microCi of [125I]-AGE-BSA into the abdominal aorta of the rat. RESULTS: Low-affinity binding sites specific for AGEs in the renal cortex (IC50 = 0.28 microm) were detected by in vitro autoradiography. There was a significant increase in [125I]-AGE binding in the diabetic kidney, which was prevented by AG treatment. Emulsion autoradiography revealed that binding was localized primarily to proximal tubules in the renal cortex. Renal AGE levels, as assessed by fluorescence or by radioimmunoassay, were increased after three weeks of diabetes. This increase was attenuated by AG therapy. CONCLUSIONS: AGE binding sites are present within the proximal tubules of the kidney and appear to be modulated by endogenous AGE levels. It remains to be determined if these binding sites represent receptors involved in clearance of AGEs or are linked to pathogenic pathways that lead to the development of diabetic nephropathy.

Alterations in renal degradation of albumin in early experimental diabetes in the rat: a new factor in the mechanism of albuminuria.

1. Albumin is normally excreted as a mixture of intact protein and fragments that are produced during renal passage. The purpose of this study was to investigate the ratio of intact versus degraded forms of excreted albumin to ascertain whether changes in this ratio could account for the apparent increase in albumin excretion seen in diabetes, as measured by standard radioimmunoassay techniques. 2. Four-week male Sprague-Dawley rats with streptozotocin-induced diabetes and age-matched control rats were intravenously injected with [3H]albumin. Urine collected over 2 h was analysed by size exclusion chromatography and radioimmunoassay. A standard radioimmunoassay found a 7-fold increase in albumin excretion rate in diabetic rats, whereas there was only a 2-fold increase in albumin excretion (intact plus fragments). Urine analysed by size exclusion chromatography showed severe degradation for control rats (% monomer=4+/-2%); in diabetic rats there was a significant amount of monomer albumin excreted, along with moderately degraded and heavily degraded albumin (% monomer=17+/-5%). 3. This study has shown that the radioimmunoassay, which specifically detects intact albumin, considerably underestimates the amount of total urinary albumin which consists of intact and degraded material. The increase in albumin excretion rate observed in diabetes as measured by radioimmunoassay is mainly due to a change in the amount of intact albumin excreted and this is specifically due to the inhibition of albumin degradation at a post-glomerular site and not due to the onset of any type of glomerular 'shunt' pathway.