Marinus G.A. Baggen

Effect of losartan on microalbuminuria in normotensive patients with type 2 diabetes mellitus - A randomized clinical trial

Context Angiotensin-receptor antagonists reduce renal and cardiovascular complications in patients with type 2 diabetes and hypertension. The effect of these drugs on normotensive patients with type 2 diabetes is not known. Contribution This 10-week randomized, placebo-controlled trial showed that in normotensive diabetic patients receiving losartan, albumin excretion rate was significantly reduced and blood pressure slightly decreased. Cautions The study included patients with systolic blood pressure up to 150 mm Hg and was too brief to evaluate progression of clinical renal disease. Angiotensin-receptor antagonists hold promise in preserving renal function in patients with type 2 diabetes without marked hypertension, but they warrant further study. The Editors Diabetes mellitus is an important cause of nephropathy, end-stage renal disease, and cardiovascular events. Nephropathy occurs in about 40% of patients with type 2 diabetes, and 25% to 40% of diabetic patients in Europe and the United States develop end-stage renal disease. Increased urinary albumin excretion, the first marker of kidney damage, carries an increased risk for renal as well as cardiovascular disease (1-5). Because reduction in microalbuminuria is associated with marked renal protection, the delay or retardation of the disease process is important in the management of diabetes mellitus (1). Angiotensin-converting enzyme (ACE) inhibitors reduce albumin excretion in both normotensive and hypertensive patients with type 1 or type 2 diabetes. The antiproteinuric effects appear to be at least partly independent of the reduction in blood pressure caused by these agents (6-11), although some studies have not confirmed this finding (12). Angiotensin II is thought to play a prominent role in the pathogenesis of diabetic nephropathy. Therefore, both preventing the formation of angiotensin II by ACE inhibition and blockade of the angiotensin receptor might be renoprotective. The trials of angiotensin-receptor antagonists in hypertensive patients with type 1 or 2 diabetes mellitus and microalbuminuria showed a reduction in albumin excretion, regardless of pretreatment levels (13-20). Recently, the angiotensin-receptor antagonist irbesartan retarded progression from microalbuminuria to overt proteinuria in hypertensive patients with type 2 diabetes (21). In RENAAL (The Reduction of Endpoints in NIDDM [noninsulin-dependent diabetes mellitus] with the Angiotensin II Antagonist Losartan study) and IDNT (Irbesartan Diabetic Nephropathy Trial), losartan and irbesartan, respectively, reduced proteinuria and slowed the progression of diabetic nephropathy in hypertensive patients with type 2 diabetes (22-24). Therefore, therapy that interferes with the reninangiotensinaldosterone system should probably be initiated when microalbuminuria develops in order to reduce albumin excretion and the associated risk for overt nephropathy. All previous studies on this subject investigated hypertensive patients. Therefore, we conducted a multicenter randomized, double-blind, placebo-controlled, clinical trial with forced dose titration to investigate the effects of losartan on urinary albumin excretion rate in normotensive patients with type 2 diabetes and microalbuminuria. Secondary objectives were to determine the time course of the antiproteinuric effect, the optimal dose of losartan, effects on creatinine clearance and blood pressure, and the safety and tolerability of losartan in normotensive patients. Methods Study Design In 19 outpatient clinics in the Netherlands, we conducted a 20-week randomized, placebo-controlled trial. The trial consisted of a 5-week screening and washout period, a 10-week double-blind treatment period, and a 5-week placebo washout period. The study was performed according to the guidelines of good clinical practice and was approved by the institutional review board at each center. All patients gave written informed consent. Patients From March 1999 through August 2001, all outpatients with type 2 diabetes mellitus, microalbuminuria, and a sitting blood pressure of 150/90 mm Hg or less were invited to participate. Type 2 diabetes mellitus was defined as diabetes diagnosed at age older than 30 years or controlled by diet or blood glucoselowering agents for at least 6 months. Microalbuminuria was defined as a urinary albumin excretion rate of 20 to 200 g/min. The current definition of normotension is a blood pressure less than 140/90 mm Hg, with a blood pressurelowering target of less than 130/80 mm Hg in hypertensive adults with diabetes mellitus (25, 26); however, we developed the protocol for this study in 1998, when normotension was defined as a sitting blood pressure of 160/90 mm Hg or less. Because of this and the fact that the mean baseline blood pressures appeared to be 135.9/78.8 mm Hg for the losartan group and 138.3/80.3 mm Hg for the placebo group, our study sample can be considered normotensive. We excluded patients with a myocardial infarction in the preceding 6 months, cerebrovascular events within the past year, unstable angina pectoris, or symptomatic heart failure; patients with electrocardiographic abnormalities (atrioventricular conduction disturbances, sick sinus syndrome, atrial fibrillation, or other clinically significant rhythm disturbances), acute renal failure, chronic glomerulonephritis, polycystic kidney disease, or a serum creatinine level greater than 150 mol/L (1.7 mg/dL); and patients with a glycosylated hemoglobin level greater than 10% or other relevant laboratory abnormalities. Concomitant use of antihypertensive agents, ophthalmic preparations containing -blocking agents, steroids, or lithium was not allowed. Eligible patients were included in the screening and washout period (weeks 5 to 0). In patients not taking blood pressurelowering or renoprotective medication, microalbuminuria was assessed by two sequential 24-hour urine collections before the randomization visit (week 0). Patients could be randomly assigned if the average of these two measurements confirmed microalbuminuria as defined above. Eligible patients who were receiving ACE inhibitors, angiotensin-receptor antagonists, or other antihypertensive agents first entered a 5-week washout period. In these patients, microalbuminuria was assessed after this period. If therapy with such medication had been initiated to normalize known hypertension, the patient was excluded. Interventions Patients who met all study criteria were randomly assigned in a double-blind fashion to receive losartan, 50 mg once daily, or matching placebo (week 0). After 5 weeks of treatment, patients initially assigned to receive 50 mg of losartan had fixed-dose titration to 100 mg of losartan; patients initially assigned to receive placebo continued to receive placebo. Treatment was continued at this dose for another 5 weeks, after which all patients were switched to single-blind placebo for the last 5 weeks. At the end of this washout period, patients returned for their final visit (week 15). The study sponsor, Merck, Sharpe & Dohme, provided each center with the study medication and randomly assigned the patients in blocks of four. The investigators dispensed the study medication and provided the patient with the first available allocation number. In each center, the allocation numbers were assigned in consecutive order. The patients had an equal probability of assignment to either group. The randomization list was inaccessible to all investigators. No randomization code was broken, so all investigators and participants remained blinded to treatment assignment during the entire study. Throughout the study, patients received the standard of care for diabetes. They were instructed not to change their physical exercise patterns significantly because doing so may influence proteinuria. Outcomes The primary end point was the change in urinary albumin excretion rate from baseline to week 10. Secondary end points were change in albumin excretion rate at week 5, change in creatinine clearance from baseline to week 10, and change in systolic and diastolic blood pressures at weeks 5 and 10. Tertiary end points were safety and tolerability of losartan. Measurements At each visit, the same investigator measured blood pressure and pulse. Blood pressure was measured by a sphygmomanometer, with the patient seated for 5 minutes before the first measurement. Three replicate measurements, obtained 1 minute apart, were averaged. In each center, microalbuminuria was measured just before every visit in two sequential 24-hour urine collections by using an immunonephelometric assay on an automated analyzer (Beckman Coulter, Brea, California). The measurements of the two samples were averaged. Complete laboratory evaluations (biochemistry, hematology) were conducted at weeks 0 and 10; at weeks 5 and 15, only renal function and electrolytes were checked. A virtual central laboratory recalculated all laboratory results, including microalbuminuria, and corrected them for possible bias. At weeks 0, 5, 10, and 15, adverse events were recorded, and medication use and adherence were checked by counting pills. Patients were considered adherent if at least 75% of the study drug had been taken. Statistical Analysis Primary and secondary end points were analyzed in the full-analysis study sample, which consisted of all patients randomly assigned (intention-to-treat analysis). The statistical method used was the generalized linear mixed model for longitudinal data, which compares the differences in trends over time between both treatment groups (27). Baseline values were used as covariates in this model, and outcomes were adjusted by using treatment, visit, center, and the corresponding interaction terms as fixed effects. A generalized linear mixed model was also applied to evaluate whether the observed changes in albumin excretion rate were consistent across different baseline blood pressures. Furthermore, an

Effects of doxazosin on lipids, lipoprotein lipases, and cholesterol synthesis in the golden hamster.

The effects of treatment with adrenoceptor blockers on sites regulating lipid metabolism were studied in golden hamsters. In hamsters fed a standard chow, doxazosin, propranolol, and atenolol did not affect plasma cholesterol or triglycerides. After hypercholesterolemia was induced by feeding a cholesterol-enriched diet, doxazosin lowered plasma cholesterol by 12%. Lipoprotein lipase activity in adipose tissue and in the heart was not changed by any of the treatments. Hepatic lipase activity in the liver and blood was lowered by 31% in the doxazosin-treated animals. Hepatic cholesterol synthesis, measured as acetate incorporation into cholesterol and hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase activity, was also lowered in the doxazosin-treated hamsters. After norepinephrine administration to cholesterol-fed hamsters, atenolol increased (+8%) and doxazosin decreased (-35%) plasma triglycerides. Plasma cholesterol levels and hepatic cholesterol synthesis were no longer significantly affected by doxazosin. In norepinephrine-treated animals, adipose tissue lipoprotein lipase activity was enhanced (+30%) by doxazosin. Hepatic lipase activity in plasma and liver, which was lowered by norepinephrine, was increased by doxazosin. In hamsters not treated with norepinephrine, adrenoceptor blockers had no effect on plasma insulin or thyroid hormone, but with norepinephrine, levels of both insulin and thyroid hormone were increased by doxazosin. These data indicate that selective α1-inhibition with doxazosin may interfere with lipid metabolism at several regulatory sites. The effects depend to a large extent on nutritional and hormonal status. Doxazosin might exert these effects partly via influences on other hormones.

Circulating and liver-bound salt-resistant hepatic lipases in the golden hamster

The serum of male golden hamsters was found to contain a circulating triacylglycerol hydrolase activity (serum lipase). In vitro, the enzyme activity was slightly activated by 1 M NaCl (+20%) and inhibited by rat serum (-29%). The hamster liver contained an enzyme with similar characteristics (liver lipase). This enzyme was released into the circulation after intravenous administration of heparin. Both lipase activities were further characterized and compared. The serum lipase had a pH optimum of 9, which was higher than that of the liver enzyme (pH 8.0). The serum enzyme did not bind to Sepharose-heparin columns in contrast to the liver lipase, which could be eluted from the column with 0.75 M NaCl. A polyclonal antibody preparation raised against the heparin-releasable salt-resistant lipase from rat liver inhibited both the hamster serum enzyme and the liver enzyme completely. The affinity of the antibodies towards the hamster enzymes was lower than the affinity towards the rat liver enzyme, but similar with that towards the hamster enzymes in the serum and the liver. A panel of five monoclonal antibodies raised against the rat enzyme did not bind either of the hamster enzymes. If the hamsters were fed a normal lab chow, the lipase activity in the serum amounted up to 110 +/- 20 mU (mean +/- S.D., n = 16) per ml serum (about 600 mU per animal), the liver contained 200 +/- 41 mU per g tissue (total about 800 mU per animal). In animals fed a cholesterol-enriched diet, the serum activity increased by 82% and the liver activity by 27%.

The IGF‐I system and the renal and haemodynamic effects of losartan in normotensive patients with type 2 diabetes mellitus: a randomized clinical trial

Objective  Losartan has been shown to protect the diabetic kidney, at least partly independent of changes in blood pressure. Imbalances in the IGF‐I system are associated with the development of diabetic nephropathy. We investigated whether renal as well as haemodynamic effects of losartan are associated with changes in the IGF‐I system in normotensive patients with type 2 diabetes mellitus (T2DM).

Inhibition of hepatic cholesterol synthesis by the alpha1-adrenoceptor blocker doxazosin in the hypercholesterolemic golden hamster

The effect of treatment with the alpha 1-specific adrenoceptor blocker, Doxazosin, on lipid parameters was studied in male Golden hamsters fed a cholesterol-enriched diet. Within 1 week the Doxazosin-treated animals had a lower plasma (-12%) and hepatic (-30%) cholesterol content than the cholesterol-fed controls. De novo cholesterol synthesis in the liver was lowered by 39% in the Doxazosin-treated animals. These data indicate that the reported beneficial effect of alpha 1-blockade on plasma cholesterol levels may be due to lowering of the hepatic cholesterol synthesis.

The effect of Synacthen administration on lipoprotein lipase activity in the epididymal fat pad of the rat

Conflicting data have been reported on the influence of (excess) glucocorticoids on lipoprotein lipase (LPL) activity in adipose tissue. To solve this problem hypercorticism was induced in rats by treatment for varying periods with Synacthen, a synthetic corticotrophin-1-24 preparation, and LPL was measured in the epididymal fat pads using different methods. In extracts of defatted tissue preparations from overnight fasted rats treated for 3 days with Synacthen we observed an increase in LPL activity (acetone-ether powder LPL) to values similar to those found in normally fed controls. In contrast, the heparin-elutable part of LPL activity in the tissue was not influenced by the Synacthen treatment. This activity remained significantly lower in overnight fasted animals, Synacthen treated or not, than in normally fed rats. Adrenalectomy lowered the acetone-ether powder LPL activity of the epididymal adipose tissue in fasted as well as in fed rats. In fasted rats it prevented the stimulation of the LPL activity by Synacthen.

Effects of synacthen on lipid metabolism in the perfused epididymal fat pad of the rat.

Rats were treated with Synacthen, a synthetic corticotrophin analogue, to induce hypercorticism. The epididymal fat pad was selectively cannulated and perfused. In fasted rats acetone ether powder lipoprotein lipase (LPL) activity rose during treatment to levels found in fed controls. In fed animals no further rise in LPL activity was observed during Synacthen treatment. However, the heparin-elutable LPL activity did not change during this treatment in fasted nor fed animals. Pharmacologic levels of insulin in the perfusion medium caused an increase in heparin-releasable LPL activity as a percentage of total fat pad LPL activity (15% v 48%). Hydrolysis of chylomicrons was higher in fasted three days treated animals then in controls (10 +/- 4% v 2 +/- 2%). In this group a higher uptake of liberated free fatty acids was found (2.6 +/- 1.5% v 1.0 +/- 0.5% in controls). The increase in hydrolysis rate and uptake of fatty acids in the treated fasted animals could not be explained by an increase in releasable LPL activity. Fatty acid release from the fat pad was lower in treated animals than in controls (fasted and fed), basally as well as after adrenalin stimulation. The observation that the epididymal fat pad retains its weight during hypercorticism may therefore be ascribed to an increased influx of fatty acids from increased hydrolysis of TG-rich particles and to an inhibited efflux of fatty acids from the adipocyte. The discrepancy between the LPL activity extractable from an acetone ether powder and the heparin releasable LPL activity suggests impairment of the transport of LPL from the adipocyte to the heparin releasable pool at the endothelium.