Baseline urinary metabolites predict albuminuria response to spironolactone in type 2 diabetes.

The mineralocorticoid receptor antagonist spironolactone significantly reduces albuminuria in subjects with diabetic kidney disease, albeit with a large variability between individuals. Identifying novel biomarkers that predict response to therapy may help to tailor spironolactone therapy. We aimed to identify a set of metabolites for prediction of albuminuria response to spironolactone in subjects with type 2 diabetes. Systems biology molecular process analysis was performed a priori to identify metabolites linked to molecular disease processes and drug mechanism of action. Individual subject data and urine samples were used from 2 randomized placebo controlled double blind clinical trials (NCT01062763, NCT00381134). A urinary metabolite score was developed to predict albuminuria response to spironolactone therapy using penalized ridge regression with leave-one-out cross validation. Bioinformatic analysis identified a set of 18 metabolites linked to a diabetic kidney disease molecular model and potentially affected by spironolactone mechanism of action. Spironolactone reduced UACR relative to placebo by median -42% (25th to 75% percentile -65 to 6) and -29% (25th to 75% percentile -37 to -1) in the test and replication cohorts, respectively. In the test cohort, UACR reduction was higher in the lowest tertile of the baseline urinary metabolite score compared with middle and upper tertiles -58% (25th to 75% percentile -78 to 33), -28% (25th to 75% percentile -46 to 8), -40% (25th to 75% percentile -52% to 31), respectively, P = 0.001 for trend). In the replication cohort, UACR reduction was -54% (25th to 75% percentile -65 to -50), -41 (25th to 75% percentile -46% to 30), and -17% (25th to 75% percentile -36 to 5), respectively, P = 0.010 for trend). We identified a set of 18 urinary metabolites through systems biology to predict albuminuria response to spironolactone in type 2 diabetes. These data suggest that urinary metabolites may be used as a tool to tailor optimal therapy and move in the direction of personalized medicine.

The acute blood pressure-lowering effect of amiloride is independent of endothelial ENaC and eNOS in humans and mice.

AIMS: The epithelial sodium channel (ENaC) is expressed in cultured endothelial cells and inhibitory coupling to eNOS activity has been proposed. The present study tested the hypothesis that ENaC blockers increase systemic NO-products and lower blood pressure in patients and mice, depending on eNOS. METHODS: NO-products and cGMP were measured in diabetes patient urine and plasma samples before and after amiloride treatment (20-40 mg for two days, plasma n = 22, urine n = 12 and 5-10 mg for eight weeks, plasma n = 52, urine n = 55). Indwelling catheters were implanted in the femoral artery and vein in mice for continuous arterial blood pressure and heart rate recordings and infusion. RESULTS: Treatment with amiloride for two days increased plasma and urine NO-products, while plasma cGMP decreased and urinary cGMP was unchanged in patient samples. Eight weeks of treatment with amiloride did not alter NO-products and cGMP. In mice, amiloride boli of 5, 50, and 500 µg/kg lowered heart rate and arterial blood pressure significantly and acutely. Benzamil had no effect on pressure and raised heart rate. In hypertensive eNOS-/- and L-NAME-treated mice, amiloride lowered blood pressure significantly. L-NAME infusion significantly decreased NO-products in plasma; amiloride and eNOS-deletion had no effect. An acetylcholine bolus resulted in acute blood pressure drop that was attenuated in eNOS-/- and L-NAME mice. ENaC subunit expressions were not detected consistently in human and mouse arteries and endothelial cells. CONCLUSION: Amiloride has an acute hypotensive action not dependent on ENaC and eNOS and likely related to the heart.

Predicting albuminuria response to spironolactone treatment with urinary proteomics in patients with type 2 diabetes and hypertension.

Background: The mineralocorticoid receptor antagonist spironolactone significantly reduces albuminuria in patients with diabetes. Prior studies have shown large between-patient variability in albuminuria treatment response. We previously developed and validated a urinary proteomic classifier that predicts onset and progression of chronic kidney disease. Here, we tested whether the proteomic classifier based on 273 urinary peptides (CKD273) predicts albuminuria response to spironolactone treatment. Methods: We performed a post hoc analysis in a double-blind randomized clinical trial with allocation to either spironolactone 12.5-50 mg/day (n = 57) or placebo (n = 54) for 16 weeks. Patients were diagnosed with type 2 diabetes and resistant hypertension. Treatment was an adjunct to renin-angiotensin system inhibition. Primary endpoint was the percentage change in urine albumin to creatinine ratio (UACR). Capillary electrophoresis mass spectrometry was used to quantify urinary peptides at baseline. The previously validated combination of 273 known urinary peptides was used as proteomic classifier. Results: Spironolactone reduced UACR relative to placebo by 50%, although with a large between-patient variability in UACR response (5th to 95th percentile, 7 to 312%). An interaction was detected between CKD273 and treatment assignment (ß = -1.09, P = 0.026). Higher values of CKD273 at baseline were associated with a larger reduction in UACR in the spironolactone group (ß = -0.70, P = 0.049), but not in the placebo group (ß = 0.39, P = 0.25). Stratified in tertiles of baseline CKD273, reduction in UACR was greater in the highest tertile, 63% (95% confidence interval: 35-79%), as compared with the two other tertiles combined, 16% (-17 to 40%) (P = 0.011). Conclusions: A urinary proteomics classifier can be used to identify individuals with type 2 diabetes who are more likely to show an albuminuria-lowering response to spironolactone treatment. These results suggest that urinary proteomics may be a valuable tool to tailor therapy, but confirmation in a larger clinical trial is required.

Albuminuria is associated with an increased prostasin in urine while aldosterone has no direct effect on urine and kidney tissue abundance of prostasin.

The proteinase prostasin is a candidate mediator for aldosterone-driven proteolytic activation of the epithelial sodium channel (ENaC). It was hypothesized that the aldosterone-mineralocorticoid receptor (MR) pathway stimulates prostasin abundance in kidney and urine. Prostasin was measured in plasma and urine from type 2 diabetic patients with resistant hypertension (n = 112) randomized to spironolactone/placebo in a clinical trial. Prostasin protein level was assessed by immunoblotting in (1) human and rat urines with/without nephrotic syndrome, (2) human nephrectomy tissue, (3) urine and kidney from aldosterone synthase-deficient (AS-/-) mice and ANGII- and aldosterone-infused mice, and in (4) kidney from adrenalectomized rats. Serum aldosterone concentration related to prostasin concentration in urine but not in plasma. Plasma prostasin concentration increased significantly after spironolactone compared to control. Urinary prostasin and albumin related directly and were reduced by spironolactone. In patients with nephrotic syndrome, urinary prostasin protein was elevated compared to controls. In rat nephrosis, proteinuria coincided with increased urinary prostasin, unchanged kidney tissue prostasin, and decreased plasma prostasin while plasma aldosterone was suppressed. Prostasin protein abundance in human nephrectomy tissue was similar across gender and ANGII inhibition regimens. Prostasin urine abundance was not different in AS-/- and aldosterone-infused mice. Prostasin kidney level was not different from control in adrenalectomized rats and AS-/- mice. We found no evidence for a direct relationship between mineralocorticoid receptor signaling and kidney and urine prostasin abundance. The reduction of urinary prostasin in spironolactone-treated patients is most likely the result of an improved glomerular filtration barrier function and generally reduced proteinuria.

Amiloride lowers blood pressure and attenuates urine plasminogen activation in patients with treatment-resistant hypertension.

In conditions with albuminuria, plasminogen is aberrantly filtered across the glomerular barrier and activated along the tubular system to plasmin. In the collecting duct, plasmin activates epithelial sodium channels (ENaC) proteolytically. Hyperactivity of ENaC could link microalbuminuria/proteinuria to resistant hypertension. Amiloride, an ENaC inhibitor, inhibits urokinase-type plasminogen activator. We hypothesized that amiloride (1) reduces blood pressure (BP); (2) attenuates plasminogen-to-plasmin activation; and (3) inhibits urine urokinase-type plasminogen activator in patients with resistant hypertension and type 2 diabetes mellitus (T2DM).In an open-label, non-randomized, 8-week intervention study, a cohort (n = 80) of patients with resistant hypertension and T2DM were included. Amiloride (5 mg/d) was added to previous triple antihypertensive treatment (including a diuretic and an inhibitor of the renin-angiotensin-aldosterone system) and increased to 10 mg if BP control was not achieved at 4 weeks. Complete dataset for urine analysis was available in 60 patients. Systolic and diastolic BP measured by ambulatory BP monitoring and office monitoring were significantly reduced. Average daytime BP was reduced by 6.3/3.0 mm Hg. Seven of 80 cases (9%) discontinued amiloride due to hyperkalemia >5.5 mol/L, the most frequent adverse event. Urinary plasmin(ogen) and albumin excretions were significantly reduced after amiloride treatment (P < .0001). Urokinase activity was detectable in macroalbuminuric urine, with a tendency toward reduction in activity after amiloride treatment. Amiloride lowers BP, urine plasminogen excretion and activation, and albumin/creatinine ratio, and is a relevant add-on medication for the treatment of resistant hypertension in patients with T2DM and microalbuminuria.

Plasmin in urine from patients with type 2 diabetes and treatment-resistant hypertension activates ENaC in vitro.

BACKGROUND: Aberrant filtration of plasminogen from plasma and subsequent activation to plasmin in the urinary space may activate proteolytically the epithelial sodium channel, ENaC. In conditions with chronic albuminuria, this may cause hypertension. It was hypothesized that patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension excrete plasmin(ogen) in urine in proportion to albumin and that plasmin confers to urine the ability to activate ENaC. METHOD: Patients (n = 113) with T2DM and resistant hypertension, defined as systolic blood pressure (SBP) more than 130 mmHg and/or diastolic blood pressure (DBP) more than 80 mmHg despite use of at least three drugs with one diuretic and one renin-angiotensin system inhibitor, were included. Urine was analyzed for albumin, creatinine, plasmin(ogen), protease activity, and ability to activate inward current in single collecting duct cells. RESULTS: Mean ambulatory SBP/DBP was 143 ±â€Š1/77 ±â€Š0.7 mmHg; HbA1c 7.35%; and eGFR 81.0 ml/min per 1.73 m (geometric means). Patients with microalbuminuria (39%) and macroalbuminuria (13%) displayed significantly elevated levels of urinary plasmin(ogen) normalized to urine creatinine compared with patients with normal excretion of albumin (48%). Urinary plasminogen correlated significantly to urine albumin. Western immunoblotting and gelatine zymography confirmed active plasmin in urine samples from patients with microalbuminuria and macroalbuminuria. Single collecting duct cells displayed significantly increased, amiloride-sensitive, inward current when superfused with urine from albuminuric patients compared with patients with normal albumin excretion. Urinary plasminogen/creatinine ratio correlated significantly with 24-h ambulatory blood pressure. CONCLUSION: Aberrant presence of plasmin in preurine may inappropriately activate ENaC in patients with type 2 diabetes and microalbuminuria. This may contribute to treatment-resistant hypertension.

Low dose spironolactone reduces blood pressure in patients with resistant hypertension and type 2 diabetes mellitus: a double blind randomized clinical trial.

BACKGROUND: The increased risk of cardiovascular morbidity and mortality associated with arterial hypertension is particularly pronounced in patients with type 2 diabetes mellitus. Blood pressure control is, therefore, decisively important but often not sufficiently achieved. OBJECTIVE: The primary objective of this study was to evaluate the antihypertensive effect of low dose spironolactone added to triple therapy for resistant hypertension in patients with type 2 diabetes measured by ambulatory monitoring. Secondary objectives were to evaluate the effects on glycaemic control and urinary albumin excretion as well as adverse effects. METHODS: In a multicentre, double-blind, randomized, placebo-controlled study 119 patients with blood pressure at or above 130/80 mmHg despite triple antihypertensive therapy were included. One tablet of 25 mg spironolactone or placebo was added to previous treatment and increased to two if blood pressure below 130/80 mmHg was not achieved after 4 weeks. Blood pressure was measured by ambulatory monitoring at baseline and after 16 weeks. RESULTS: The study was completed by 112 patients, 57 randomized to spironolactone and 55 to placebo. Average daytime placebo-corrected blood pressure was reduced by 8.9 (4.7-13.2)/3.7 (1.5-5.8) mmHg. Also office blood pressure, night-time, 24-h and pulse pressures were reduced significantly. Urinary albumin/creatinine ratio was significantly reduced in the spironolactone group. Glycaemic control remained unchanged. Hyperkalemia was the most frequent adverse event leading to dose reduction in three cases and discontinuation in one, whereas gynaecomastia was not reported. CONCLUSION: Low dose spironolactone exerts significant BP and urinary albumin creatinine ratio lowering effects in high-risk patients with resistant hypertension and type 2 diabetes mellitus.

Auscultatory versus oscillometric measurement of blood pressure in octogenarians.

BACKGROUND: Auscultatory measurement using a sphygmomanometer has been the predominant method for clinical estimation of blood pressure, but it is now rapidly being replaced by oscillometric measurement. OBJECTIVE: To compare blood pressure by auscultatory and oscillometric measurements in patients ≥ 80 years. METHOD: 100 patients had blood pressure measured by auscultation with a sphygmomanometer and by an electronic device using the oscillometric method. For each patient the mean of two blood pressures with each method measured within 15 min were compared. RESULTS: The mean age of participants was 85.8 years; 55.8% were women. The correlation coefficient for systolic blood pressure was 0.88 and for diastolic 0.79. Differences between auscultatory and oscillometric values were less than 10 mmHg in 70.6% of systolic blood pressures and in 83.2% for diastolic. Arrhythmia and hypertension did not influence the results, and there was no correlation between the magnitude of the differences and the level of blood pressure. CONCLUSION: Agreement between oscillometric and auscultatory measurements of blood pressure in octogenarians was found to be less than required by validation protocols. However, semi-automatic equipment, which is observer-independent, may be used even in the very elderly, particularly if multiple readings are performed.

Multimorbidity and blood pressure control in 37 651 hypertensive patients from Danish general practice.

BACKGROUND: Patients with hypertension are primarily treated in general practice. However, major studies of patients with hypertension are rarely based on populations from primary care. Knowledge of blood pressure (BP) control rates in patients with diabetes and/or cardiovascular diseases (CVDs), who have additional comorbidities, is lacking. We aimed to investigate the association of comorbidities with BP control using a large cohort of hypertensive patients from primary care practices. METHODS AND RESULTS: Using the Danish General Practice Database, we included 37 651 patients with hypertension from 231 general practices in Denmark. Recommended BP control was defined as BP <140/90 mm Hg in general and <130/80 mm Hg in patients with diabetes. The overall control rate was 33.2% (95% CI: 32.7 to 33.7). Only 16.5% (95% CI: 15.8 to 17.3) of patients with diabetes achieved BP control, whereas control rates ranged from 42.9% to 51.4% for patients with ischemic heart diseases or cerebrovascular or peripheral vascular diseases. A diagnosis of cardiac heart failure in addition to diabetes and/or CVD was associated with higher BP control rates, compared with men and women having only diabetes and/or CVD. A diagnosis of asthma in addition to diabetes and CVD was associated with higher BP control rates in men. CONCLUSION: In Danish general practice, only 1 of 3 patients diagnosed with hypertension had a BP below target. BP control rates differ substantially within comorbidities. Other serious comorbidities in addition to diabetes and/or CVD were not associated with lower BP control rates; on the contrary, in some cases the BP control rates were higher when the patient was diagnosed with other serious comorbidities in addition to diabetes and/or CVD.

The effect of low-dose spironolactone on resistant hypertension.

Our objective was to estimate the effect of addition of low-dose spironolactone to previous antihypertensive therapy in patients with resistant hypertension. Patients had 25 to 50 mg of spironolactone once daily added to the treatment of hypertension that was uncontrolled despite previous treatment with three classes of antihypertensive drugs. The effect on blood pressure was estimated by office measurements together with serum potassium and adverse effects. The data were analyzed retrospectively. A total of 544 patients were identified; 200 were excluded because of secondary hypertension, other indications for spironolactone than hypertension, previous antihypertensive therapy with less than three drugs unless demonstrated intolerance to a third drug, insufficient compliance, and lack of follow-up data. Thus, 344 cases were included in the analysis. The population was 62.1 ± 12.8 years old, 45.1% were males, and 43% had cardiovascular comorbidity. Mean blood pressure before the addition of spironolactone was 169/88 mm Hg. At 1, 3, and 6 months after the addition, blood pressure was decreased by an average of 16.6/7.0, 23.9/9.7, and 26.0/10.7 mm Hg (all P < .001). Serum potassium increased from an average of 3.7 mmol/L to 4.1 mmol/L (P < .001). Spironolactone was discontinued because of hyperkalemia in 4.1% of the cases. A total of 18% of all patients had adverse effects, which in 9.9% led to discontinuation of the drug. A total of 5.2% of the males developed gynecomastia. In conclusion, low-dose spironolactone is highly effective when added to previous treatment of patients with resistant hypertension.

Prevalence, awareness, and control of arterial hypertension in Denmark.

Hypertension is an important modifiable risk factor for cardiovascular disease. Risk is reduced by reduction of blood pressure (BP). The present survey estimated the prevalence of hypertension, awareness, treatment, and BP control in Denmark. BP was measured three times on one occasion in a representative sample (n = 7,767) of the Danish population ages 20 to 89 years. Persons with screening BP >/=140/90 mm Hg also measured BP at home. Participants with home BP >/=135/85 mm Hg in general and >/=125/75 mm Hg for patients with diabetes or renal disease were categorized as hypertensive together with those already on antihypertensive treatment. Awareness was registered by questionnaire. Treated patients with BP below relevant limits were categorized as controlled. Age-adjusted prevalence of hypertension was on the basis of screening BP 25.7% and by home BP 22.3%. Seventy-two percent of patients found hypertensive by home BP were aware of it, 64% were treated, and 57% of those treated were controlled by office BP and 68% by home BP. One-fifth of the adult Danish population was found to be hypertensive, Awareness and control of hypertension was better than in most previous reports. Control rates similar to those of clinical trials are achievable in clinical practice.

Treatment of renovascular hypertension by transluminal angioplasty--13 years experience in a single centre.

OBJECTIVE: The study is a follow-up on treatment of renovascular hypertension (RVH) with percutaneous transluminal renal angioplasty (PTRA). METHODS: Patients were screened on the basis of clinical criteria of increased probability of RVH with renography and in selected cases with renal vein renin measurements. A positive work-up suggesting a functionally important renal artery stenosis led to renal angiography and PTRA if stenosis was confirmed; in 59%, an intravascular stent was inserted. RESULTS: Outcome of treatment was classified as follows - group I: normotensive without medication; group II: with improved control of blood pressure; group III: unchanged blood pressure control. Grouping was performed immediately after treatment, at 1 month, 6 months and at the latest follow-up. One hundred-and-twenty-two patients (124 atherosclerotic and 12 fibromuscular lesions) were treated during 13 years. Immediately after PTRA the patients were grouped as follows - I: 31%, II: 59%, III: 10%. At 1 month, I: 13%, II: 72%, III: 15%; at 6 months, I: 11%, II: 74%, III: 15%, and at the latest follow-up, I: 11%, II: 78%, III: 11%. There were few significant complications, and renal function remained on average stable. CONCLUSION: PTRA is an effective treatment of RVH in patients selected by signs of a flow-restricting stenosis. Twelve percent were normotensive after angioplasty and a further 77% had better controlled hypertension. Few complications were seen and renal function was on average unchanged as measured by serum creatinine.