Storage temperature and differing methods of sample preparation in the measurement of urinary albumin

Summary Microalbuminuria is a predictor of persistent proteinuria, renal failure and cardiovascular disease and therefore accurate determination of urinary albumin concentration is important. We examined the stability of albumin in urine under different conditions of storage, temperature and sample preparation. There was no significant difference in urinary albumin concentration between fresh urine and urine stored at either 4°C or 20°C for up to 7 days. Similarly in urine samples from diabetic patients there was no significant difference in albumin concentration at levels ranging from 1.3 to 1999.3 mg/l between fresh urine at 4°C and urine stored frozen for 1 week, 1 month or 6 months. Neither storage temperature (−20°C or −40°C) nor centrifugation of sample prior to assay made a significant difference to the albumin concentration. Multiple freezing and thawing of urine samples during 6 weeks of storage at −20°C made no difference to albumin concentrations. Storage of urine samples in either polypropylene, polystyrene or borosilicate glass tubes did not result in a significant change in urinary albumin concentration after either 1 week or 1 month at −20°C although, after 1 month of storage, urinary albumin concentrations tended to be lower by an average of approximately 7%. In tubes to which gelatine had been added this was reduced to 4%. We conclude that fresh urine can be kept at 4°C or 20°C for up to 7 days. Frozen urine samples can be stored for up to 6 months before assay without any loss of albumin concentration. Polypropylene, polystyrene or borosilicate glass tubes are acceptable containers for short-term storage and samples can simply be thoroughly thawed and vortex mixed immediately prior to assay.     

Blood rheology and cardiovascular risk factors in type 1 diabetes: relationship with microalbuminuria.

Whole blood and plasma viscosity, erythrocyte aggregation and deformability, plasma fibrinogen, lipids, lipoproteins, apolipoproteins, and measures of blood glucose control were compared between 21 Type 1 diabetic patients with microalbuminuria (overnight albumin excretion rate 30-200 micrograms min-1) and 21 patients with albumin excretion below this range matched for age, sex, and duration of diabetes. Patients with microalbuminuria had significantly higher glycosylated haemoglobin (9.4 +/- 1.6 (+/- SD) vs 7.9 +/- 1.8% (normal range 5.0 to 7.6%)), total-cholesterol (5.6 +/- 1.1 vs 4.6 +/- 1.3 mmol l-1), apolipoprotein B (0.82 +/- 0.21 vs 0.66 +/- 0.14 g l-1), and apolipoprotein B:A1 ratio (0.58 +/- 0.18 vs 0.50 +/- 0.15) than those without microalbuminuria (all p less than 0.05). HDL-cholesterol was also raised (1.71 +/- 0.46 vs 1.43 +/- 0.37 mmol l-1, p less than 0.05). Lipoprotein(a) concentration was possibly higher in the microalbuminuric group (median (95% Cl) 105 (82-140) vs 72 (52-114) mg l-1, p = 0.06). No differences were seen in any of the rheological measurements. These results confirm the presence of potentially atherogenic lipoprotein changes in Type 1 diabetic patients with microalbuminuria, but suggest that altered blood rheology does not predate the development of nephropathy.     

Inhibitory effect of clofibrate on arginine-induced insulin secretion in chemical diabetes

Summary Clofibrate, 3g/die for 10 consecutive days, significantly reduced arginine-induced IRI release in chemical diabetics and in normal controls, whether of normal body weight or obese. Blood glucose levels were not affected by clofibrate. In agreement with previous findings, it would appear that clofibrate, administered for short periods, does not lead to a decrease in glucose tolerance. However, studies relating to the effect of chronic clofibrate administration in chemical diabetes are needed in order to be sure that prolonged inhibition of IRI secretion does not lead to overt diabetes.     

Continuous veno-venous hemofiltration for the treatment of contrast-induced acute renal failure after percutaneous coronary interventions.

Acute renal failure (ARF) requiring hemodialysis after percutaneous coronary interventions (PCI) is a serious complication with poor prognosis. Hemodialysis-induced hypotension may have deleterious cardiovascular effects, especially in high-risk patients. Ultrafiltrate removal and simultaneous fluid replacement with a solution similar to plasma for high-volume controlled hydration can be obtained with hemodynamic stability by continuous veno-venous hemofiltration (CVVH). We prospectively assessed the safety and effectiveness of percutaneous CVVH (Y-shaped double-lumen catheter, circuit originating from and terminating in the femoral vein) in 33 consecutive patients (23 men and 10 women; mean age, 69 +/- 9 years) who, after PCI, developed oligo-anuric ARF, associated in 20 of them with congestive heart failure. All patients received a concomitant infusion of furosemide (500-1000 mg/day) and dopamine (2 microg/kg/min). During CVVH, the average fluid volume replacement and body fluid net reduction were 1000 +/- 247 and 75 +/- 48 ml/hr, respectively. Treatment with CVVH continued for 4.7 +/- 2.7 days and corrected fluid overload in all cases. No patient experienced systemic hypotension or hypovolemia. Diuresis recovered in 32 (97%) patients, who showed a parallel improvement of renal function parameters. One patient required chronic dialysis. In-hospital and 1-year mortality was 9.1% and 27.3%, respectively. In conclusion, our data indicate that CVVH is a safe and effective therapy of radiocontrast-induced ARF following PCI. It temporarily replaces renal function without deleterious cardiovascular effects, allowing the kidney to recover from the nephrotoxic injury. However, despite promising early results, large randomized trials are required to define the role of CVVH in ARF after PCI.     

Elevated serum levels of macrophage-derived cytokines precede and accompany the onset of IDDM

Summary To determine whether cytokines could have a role in the development of insulin-dependent diabetes mellitus (IDDM), we measured serum levels of cytokines derived from T helper 1 (interleukin-2 and interferon-), T helper 2 (interleukin-4 and inter-leukin-10) lymphocytes and macrophages (tumour necrosis factor-, interleukin-1 and interleukin-1 ) in patients before and after the onset of IDDM. Recently diagnosed IDDM patients had significantly higher levels of interleukin-2, interferon-, tumour necrosis factor- and interleukin-1 than patients with either long-standing IDDM, non-insulin-dependent diabetes (NIDDM), Graves' disease, or control subjects (p<0.05 for all). Compared with control subjects, patients with long-standing IDDM and those with NIDDM had higher interleukin-2 and tumour necrosis factor- levels (p<0.01 for all). Interleukin-4 and interleukin-10 were detectable in sera of patients with Graves' disease only, while interleukin-1 was not detectable in the serum of any control or test subject. To investigate whether high cytokine levels precede the onset of IDDM, we studied 28 non-diabetic identical co-twins of patients with IDDM, followed-up prospectively for up to 6 years after the diagnosis of the index. Levels of tumour necrosis factor- and interleukin-1 were elevated above the normal range more frequently in the eight twins who developed diabetes than in those 20 who did not (p<0.005). Analysis of T helper 1 and T helper 2 profiles of the twin groups did not reveal a clear difference between prediabetic twins and twins remaining non-diabetic. These results support the notion that T helper 1 lymphocytes may play a role in the development of IDDM. This is associated with release of macrophage-derived cytokines, which is also a feature of the prediabetic period. The lack of evidence of a dominant T helper 1 profile of cytokine release before diabetes onset suggests that additional events, activating this arm of the cellular immune response, are required in the immediate prediabetic period.Abbreviations IL Interleukin - IFN- interferon-gamma - T_H T helper - ICA islet-cell antibody - GAD glutamic acid decar-boxylase - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus - TNF- tumour necrosis factor-alpha - CTLL-16 murine cytotoxic cell line     

Predictors of Congestive Heart Failure after Treatment with an Endothelin Receptor Antagonist

Background and objectives

The Avosentan on Time to Doubling of Serum Creatinine, End Stage Renal Disease or Death (ASCEND) trial tested the renoprotective effect of the endothelin receptor antagonist avosentan in patients with diabetes and nephropathy, but the study was terminated due to an excess of congestive heart failure (CHF) events in the avosentan arms, likely due to fluid retention. The aim of this study was to identify risk markers of CHF after treatment with avosentan.

Design, setting, participants, & measurements

In a post hoc analysis of the ASCEND trial (N=1392 participants), we assessed which baseline characteristics predicted CHF risk during avosentan treatment. Furthermore, postrandomization changes between baseline and the first available measurement of body weight and hemoglobin were examined as potential clinical indicators of fluid retention for their relationship with CHF development.

Results

Relative to placebo, avosentan increased CHF risk (hazard ratio, 2.76; 95% confidence interval, 1.68 to 4.54). The avosentan-related CHF risk was higher with lower baseline cholesterol levels (P interaction=0.003) and concomitant statin use (P interaction=0.06), whereas it was lower with a lower estimated GFR (P interaction=0.04). Patients allocated to avosentan had a median body weight increase of 0.6 kg (interquartile range, 0.0 to 2.0 kg) and a median hemoglobin decrease of 1.4 g/dl (interquartile range, −2.1 to −0.7 g/dl) at the first postrandomization measurement. The body weight increase induced by avosentan was associated with CHF development (P interaction=0.04), whereas hemoglobin decrease was not (P interaction=0.64). The increase in body weight was particularly pronounced in patients with a cardiovascular disease history and in patients using statins.

Conclusions

In avosentan-treated patients, body weight increase, but not hemoglobin decrease, was associated with CHF development, indicating that close body weight monitoring could provide an early signal of CHF development in future trials with endothelin receptor antagonists.     

Effect of metergoline,a powerful and long-acting antiserotoninergic agent,on insulin secretion in normal subjects and in patients with chemical diabetes

Summary The effect of metergoline on insulin secretion has been evaluated in normal subjects and in patients with chemical diabetes. The repeated administration of metergoline, 2 mg at four-hour intervals to give a total of 24 mg, has enhanced insulin secretion in response to i. v. glucose in normal subjects but not in chemical diabetics. No changes in blood glucose pattern were observed. Under similar conditions, metergoline administration caused a slight but significant decrease in arginine-induced insulin release, both in normal subjects and in chemical diabetics. These results support the concept of a serotoninergic control of insulin secretion and suggest that serotonin exerts different effects on insulin release according to the different stimuli.     

Experience of malignancies with oral glucose-lowering drugs in the randomised controlled ADOPT (A Diabetes Outcome Progression Trial) and RECORD (Rosiglitazone Evaluated for Cardiovascular Outcomes and Regulation of Glycaemia in Diabetes) clinical trials

Aims/hypothesis  

Observational and mechanistic studies have suggested a possible relationship between treatment with metformin and decreased incidence of cancer in participants with type 2 diabetes. We extracted data for malignancies from the ADOPT (A Diabetes Outcome Progression Trial) and RECORD (Rosiglitazone Evaluated for Cardiovascular Outcomes and Regulation of Glycaemia in Diabetes) randomised controlled clinical trials, in which the efficacy and/or safety of metformin was assessed in comparison with sulfonylureas and rosiglitazone.     

Podocyte number in normotensive type 1 diabetic patients with albuminuria

We estimated glomerular cell number in 50 normotensive type 1 diabetic patients with raised albumin excretion rate (AER) and investigated any change after 3 years in a subgroup of 16 placebo-treated patients. Biopsies from 10 normal kidney donors were used as controls. Mesangial and endothelial cell number was increased in the 50 diabetic patients at the start of the study compared with control subjects. There was no difference in podocyte number. Glomerular volume was increased in diabetic patients, but surface area of glomerular basement membrane (GBM) underlying the podocytes did not differ between groups. AER correlated positively with mesangial cell number in microalbuminuric patients (r = 0.44, P = 0.012) and negatively with podocyte number in proteinuric patients (r = -0.48, P = 0.040). In the 16 placebo-treated patients, glomerular volume increased after 3 years owing to matrix accumulation and increased GBM surface area. Although overall cell number did not differ significantly from baseline, the decrease in podocyte number during follow-up correlated with AER at follow-up (r = -0.72, P = 0.002). In conclusion, cross-sectional analysis of podocyte number in type 1 diabetic patients with raised AER but normal blood pressure shows no significant reduction compared with nondiabetic control subjects. Longitudinal data provide evidence for an association between podocyte loss and AER, but whether cellular changes are a response to, a cause of, or concomitant with the progression of nephropathy remains uncertain.