Mineralocorticoid receptor antagonism in acutely decompensated heart failure

1 João Pedro Melo Marques Pinho Ferreira Mineralocorticoid Receptor Antagonism in Acutely Decompensated Heart Failure Tese de Candidatura ao grau de Doutor em Ciências Médicas, submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador: – Doutor Henrique Cyrne Carvalho; Professor Auxiliar Convidado com Agregação no Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Coorientadores: - Doutor Paulo Bettencourt; Professor Catedrático Convidado da Faculdade de Medicina da Universidade do Porto - Dra. Irene Marques; Assistente Convidada no Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. 2 3 De acordo com o disposto no n.º 1 do artigo 34.º do Decreto-Lei n.º 74/2006, publicado em Diário da República, 1.ª série, n.º 60 de 24 de Março de 2006, e republicado pelo Decreto-Lei n.º 115/2013, publicado em Diário da República, 1.ª série, n.º 151 de 7 de Agosto de 2013, que procede à terceira alteração ao Decreto-Lei n.º 74/2006, de 24 de março de 2006, constam nesta tese os artigos já publicados/aceites para publicação e os submetidos, que a seguir se discriminam: I. Ferreira JP, Santos M, Almeida S, Marques I, Bettencourt PM, Carvalho H. Mineralocorticoid Receptor Antagonism in Acutely Decompensated Chronic Heart Failure. Eur J Intern Med 2014; 25: 67–72. II. Ferreira JP, Santos M, Almeida S, Marques I, Bettencourt PM, Carvalho H. Tailoring diuretic therapy in acute heart failure: insight on early diuretic response predictors. Clin Res Cardiol 2013; 102: 745-753. III. Ferreira JP, Santos M, Almeida S, Marques I, Bettencourt PM, Carvalho H. The role of albuminuria as a non-invasive marker for congestive acutely decompensated chronic heart failure and the spironolactone effect in elderly Portuguese: a non-randomized trial. Nephrology 2014; 19: 149–156. IV. Ferreira JP, Santos M, Almeida S, Marques I, Bettencourt PM, Carvalho H. HighDose Spironolactone Changes Renin and Aldosterone Levels in Acutely Decompensated Heart Failure. Cor et Vasa 2014; in press. V. Ferreira JP, Santos M, Almeida S, Marques I, Bettencourt PM, Carvalho H. High Sensitivity Troponin T: A Biomarker for Diuretic Response in Decompensated Heart Failure Patients? Cardiol Res Pract vol. 2014, Article ID 269604. doi:10.1155/2014/269604. VI. Ferreira JP, Santos M, Almeida S, Marques I, Bettencourt PM, Carvalho H. Urinary Sodium to Potassium Ratio: Biomarker of Mineralocorticoid Receptor Antagonism in Decompensated Heart Failure. Submitted. VII. Ferreira JP, Santos M, Oliveira JC, Marques I, Bettencourt PM, Carvalho H. The Influence of Spironolactone on Matrix Metalloproteinases in Acute Decompensated Heart Failure. Submitted. 4 5 Agradecimentos Ao Professor Doutor Henrique Cyrne Carvalho, que se mostrou disponível sempre que necessário, com ajudas preciosas, quer em pequenas dicas, quer em fazer acontecer. Uma relação baseada na confiança, com a liberdade necessária para a criatividade florescer. Ao Professor Doutor Paulo Bettencourt, pela crítica concisa e assertiva, pela disponibilidade permanente e acima de tudo por me ajudar a quebrar barreiras interinstituições. À Dra. Irene Marques por me ajudar a implementar o processo dentro do Hospital de Santo António e por me ajudar a vencer os obstáculos que surgiram no percurso. Ao amigo e colega Mário Santos, pela crítica ponderada, pelas questões pertinentes, pelas correções minuciosas dos textos, pela ajuda estatística e pela procura permanente de novos caminhos. À Professora Doutora Sofia Almeida pela ajuda na análise estatística. Ao Dr. José Carlos Oliveira por ter aberto as portas do laboratório a este estudo, com armazenamento e análises de amostras que não são efetuadas por rotina e na gestão imaculada da seroteca. Ao Sr. Fernando Santos por ter separado os soros criteriosamente, de forma a que não fossem trocados ou perdidos. A todos os enfermeiros que colheram as amostras fora de horas! A todos os colegas que colaboraram no estudo, sem eles teria sido impossível. To Professor Faiez Zannad, Professor Patrick Rossignol and Dr. Nicolas Girerd for the discussion, support, and most of all for the interest in the work of a young unknown clinical scientist. Thank you! Muito obrigado! 6 Contents Introduction .................................................................................................................................. 7 Setting and Methods ................................................................................................................ 17 Aims ................................................................................................ Erro! Marcador não definido. Papers ........................................................................................................................................ 23 I. Mineralocorticoid Receptor Antagonism in Acutely Decompensated Chronic Heart Failure .......................................................................................................................... 24 II. Tailoring diuretic therapy in acute heart failure: insight on early diuretic response predictors ............................................................................................................................... 44 III. The Role of Albuminuria as a Non-Invasive Marker for Congestive Acutely Decompensated Chronic Heart Failure and the Spironolactone Effect in Elderly Portuguese: a Non-Randomized Trial ............................................................................... 64 IV. High-Dose Spironolactone Changes Renin and Aldosterone Levels in Acutely Decompensated Heart Failure............................................................................................ 85 V. High Sensitivity Troponin T: A Biomarker for Diuretic Response in Decompensated Heart Failure Patients? ........................................................................ 104 VI. Urinary Sodium to Potassium Ratio: Biomarker of Mineralocorticoid Receptor Antagonism in Decompensated Heart Failure ............................................................... 125 VII. The Influence of Spironolactone on Matrix Metalloproteinases in Acute Decompensated Heart Failure.......................................................................................... 145 General Discussion and Limitations .................................................................................... 161 Conclusions ............................................................................................................................. 165 Abstract .................................................................................................................................... 167 Resumo .................................................................................................................................... 169 7 Introduction 8 Heart failure (HF) is the leading cause of hospitalization in patients older than 65 years of age and a major public health problem1. Hospital admissions for acutely decompensated heart failure (ADHF) are frequent and accompanied by high percentages of hospital complications and mortality2. There is a paucity of data derived from controlled clinical trials to define optimal treatment for patients with acute HF which is largely based on loop diuretics and vasodilators for congestion relief3. The pathophysiology of ADHF is characterized by abnormal neurohormonal activation, leading to an increase of sodium and water retention, arterial vasoconstriction, and activation of inflammatory cascades4,5. Excessive neurohormonal activation may lead to several harmful effects6, including renal dysfunction and myocardial injury7. The recognition of neurohormonal activation in HF has led to the development of therapies able to inhibit inappropriate neurohormonal enhancement, particularly the renin-angiotensin-aldosterone system (RAAS). The advent of these therapies had high impact on morbidity and mortality of these patients4. Despite the use of angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and beta-blockers (BBs), neurohormonal activation remains inappropriately elevated in patients hospitalized with ADHF8-10. Furthermore, there is growing evidence suggesting that aldosterone may only transiently be suppressed with ACEIs/ARBs. This phenomenon termed “aldosterone breakthrough” can have important clinical consequences given aldosterone sodiumretaining, profibrotic, and inflammatory properties11-13. The addition of mineralocorticoid receptor antagonists (MRAs) on top of ACEIs/ARBs and BBs has been shown to improve clinical outcomes14. The use of MRAs reduced morbidity and mortality in patients with chronic, severe systolic heart failure (HF)14, in chronic HF with mild symptoms15, and after myocardial infarction16. The improved morbidity and mortality observed in these trials is thought to be in part due to suppression of neurohormonal activation by MRAs, suggesting a potential interest in early initiation of MRAs in the ADHF setting. Considering the anti-hypertensive and diuretic properties of MRAs, the ideal candidate for this therapy should be hyperor normotensive and fluid overloaded, without evidence of cardiogenic shock. In addition, patients with systemic congestion, and/or with predominantly right-sided HF may have hepatic congestion and consequently have impaired clearance of neurohormonal factors8, providing an attractive subset of patients likely to take the most benefit out of MRA therapy. Mineralocorticoid Receptor Antagonists and Diuretic Resistance Resistance to loop diuretics has been associated to a much higher plasma aldosterone concentration17-19. Chronic administration of loop diuretics has been shown 9 to cause hypertrophy of the distal nephron and increase expression of the sodium chloride cotransporter, which is an aldosterone-induced protein. These structural changes can lead to loop diuretic resistance and these effects are potentially reversed by a mineralocorticoid antagonist20. Congestion refractory to oral diuretics may be responsible for up to one-third of hospital admissions due to ADHF1, and unresolved congestion may contribute to the high readmission rates observed in these patients1. Furthermore, approximately 30% of patients admitted for ADHF develop diuretic resistance, defined as reduced diuresis and natriuresis in response to a constant high dose of loop diuretics21. Strategies to overcome this clinical problem still have limited success. The use of diuretics to treat persistent congestion may lead to kidney injury and worsening renal function. Venovenous ultrafiltration is an option in these patients, however it is an expensive and invasive procedure that requires hospitalization. Most importantly, this technique did not reduce mortality or hospitalizations for HF compared to strategy of stepped diureticbased therapy22. Therefore, the use of this technique does not seem justified for patients hospitalized for ADHF, worsened renal function, and persistent congestion. The use of natriuretic doses of MRAs in this setting may be an attractive alternative to reverse diuretic resistance and inappropriate RAAS activation23. Mineralocorticoid Receptor Antagonists May Prevent Myocardial Injury, Remodelling and Fibrosis Myocardial injury, indicated by troponin release, is common in ADHF7,24-27. The ADHF episodes are associated with increased mechanical strain on the heart, activation of neurohormonal systems, and increased and oxidative stress28. These stimuli are known to mediate myocardial injury and accelerating myocyte loss28. Improvements in analytical sensitivity have transformed circulating troponin from a biomarker that was only detectable in a minority of patients to one that is detectable in the vast majority of patients with HF25. The high sensitivity troponin-T (hsTnT) test can detect very small changes in the circulating troponin levels25,29. Elevations in baseline troponin levels were demonstrated to be independent predictors of events during the acute hospitalization (worsening or persistent HF, death, and increased length of stay) and also independent predictors of post-discharge outcomes24,27,30-33. Accordingly, changes in troponin status during initial treatment for ADHF have been proposed as potentially important targets for drug development34. Mineralocorticoid receptor antagonists decrease myocardial fibrosis, inflammation, oxidative stress, apoptosis, neurohormonal activation, and remodelling in animal models and patients with HF35,36. The anti-fibrotic influence of MRAs in patients with ischaemic myocardial necrosis provides an additional therapeutic benefit as 16 hospitalized for acute heart failure: analysis from the EVEREST trial. Eur J Heart Fail. 2013. 51. Eudy RJ, Sahasrabudhe V, Sweeney K, et al. The use of plasma aldosterone and urinary sodium to potassium ratio as translatable quantitative biomarkers of mineralocorticoid receptor antagonism. J Transl Med. Vol 9. England2011:180. 52. Canessa CM, Schild L, Buell G, et al. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature. 1994;367(6462):463-467. 53. Bailey MA, Mullins JJ, Kenyon CJ. Mineralocorticoid and glucocorticoid receptors stimulate epithelial sodium channel activity in a mouse model of Cushing syndrome. Hypertension. 2009;54(4):890-896. 54. Brandish PE, Chen H, Szczerba P, Hershey JC. Development of a simplified assay for determination of the antimineralocorticoid activity of compounds dosed in rats. J Pharmacol Toxicol Methods. 2008;57(2):155-160. 55. Kagawa CM, Jacobs RS, Jr. Mineralocorticoid effects of 9 alphafluorodeoxycorticosterone in adrenalectomized rats. Proc Soc Exp Biol Med. 1960;104:60-62. 17 Setting and Methods 18 This project was designed to answer the questions elaborated in the Introduction. a) Is high-dose spironolactone safe in ADHF? b) Can spironolactone overcome diuretic resistance? c) Can spironolactone mitigate myocardial and renal injury during the ADHF episode? d) Can spironolactone reduce turnover and fibrosis biomarkers? e) Does high-dose spironolactone affect the RAAS? f) Is there an easily available surrogate marker for spironolactone use? In order to answer these questions we designed a prospective, nonrandomized, experimental, single-centre, and single-blinded trial conducted in Centro Hospitalar do Porto enrolling participants between February 2012 and February 2013. Patients were eligible for enrollment if they presented with decompensation of chronic HF with symptoms leading to hospitalization. HF was diagnosed on the basis of the presence of history of chronic heart failure and at least one symptom (dyspnea, orthopnea, or edema) and one sign (rales, peripheral edema, ascites, or pulmonary vascular congestion on chest radiography). Exclusion criteria were: chronic use of MRAs, cardiac surgery within 60 days of enrollment, cardiac mechanical support, cardiac resynchronization-therapy within the last 60 days, comorbid conditions with an expected survival of less than 6 months, acute myocardial infarction at time of hospitalization, hemodynamically significant uncorrected primary cardiac valvular disease, patients requiring intravenous vasodilators or inotropic agents, supine systolic arterial blood pressure <90 mmHg, serum creatinine level >1.5 mg/dL, serum potassium level >5.0 mmol/L, hemoglobin level <9 g/dL, and sepsis. Patients were non-randomly assigned in a sequential 1:1 ratio to the intervention or standard treatment. Chief investigator was responsible to assess the eligibility criteria and to allocate the intervention after being contacted by the patient assistant physician. Patients were blinded to the intervention allocation. Assistant physicians were not blinded to intervention allocation. Assistant physicians were Attending Physicians or Fellows of Internal Medicine or Cardiology depending on the ward where each patient was admitted. The assistant physicians evaluated the clinical signs and symptoms and registered their evaluation in the clinical diaries and then transcribed to our database by the authors. Patients were assigned to either oral spironolactone (minimum and maximum initial dose of 50 - 100 mg/d, according to assistant physician) plus standard AHF 19 therapy or standard AHF therapy alone. Standard AHF therapy included intra-venous (i.v.) furosemide (bolus or continuous infusion), digoxin, ACEi, ARB, nitrates, and/or non-invasive ventilation (NIV), according to attending physician decision. At day 2, the attending physician had the option of adjusting spironolactone dose on the basis of the clinical judgment and laboratory results. At this time, the physician could decrease the dose by 50%, to a minimum of 50 mg/d, or maintain the same strategy. Patient`s clinical status was prospectively recorded, by the assistant physicians, according to previous defined parameters. An assessment of biomarkers, including plasma creatinine (pCr), ions, Nterminal pro-brain natriuretic peptide (NTproBNP), high sensitivity troponin T (hsTnT) and microalbuminuria was performed at a central core laboratory at admission day (day 1) and day 3. Clinical assessment and routine analyses were performed daily during hospital stay. All patients performed a transthoracic echocardiography within 72 hours upon admission. Left-ventricular ejection fraction was estimated by transthoracic echocardiography using the biplane Simpson method. Patients characteristics are describe in Table 1. 20 Table 1. Baseline Characteristics of the Study Population Control Group Spironolactone Group p Value Age (yrs) 78,8 ± 9,3 73,2 ± 11,7 0,01 Male sex – no. (%) 17 (34) 22 (44) 0,31** Ejection Fraction (%) 45,5 ± 10,7 41,4 ± 12,4 0,08** HFrEF – no. (%) 13 (26) 18 (36) 0,28** HgB (g/dL) 12,2 ± 1,8 12,7 ± 2,3 0,22 Etiology of Heart Failure – no. (%) Ischemic 24 (48) 26 (52) 0,69** Non-Ischemic 26 (52) 24 (48) 0,84** Basal NYHA class – no. (%) II 41 (82) 44 (88) 0,4** III 9 (18) 6 (12) 0,4** History of Atrial Fibrillation or Flutter - no. (%) 34 (68) 25 (50) 0,07** Outpatients Medications – no. (%) Furosemide 37 (74) 35 (70) 0,65** ACE Inhibitors 24 (48) 18 (36) 0,22** Beta-Blockers 26 (52) 10 (20) <0,01** Outpatients Oral Dose (mg) Furosemide 69,2 ± 37,8 68 ± 30 0,89 ACE Inhibitors 5 ± 3 4,3 ± 2,5 0,43 Beta-Blockers 4,4 ± 2,7 4,5 ± 1,1 0,90 BMI ≥ 30 (Kg/m2) – no. (%) 14 (28) 19 (38) 0,29** Diabetes Mellitus – no. (%) 25 (50) 20 (40) 0,31** Glycated Hemoglobin (%) 6,9 ± 0,7 7,1 ± 1,2 0,43** Obstructive Sleep Apnea Syndrome – no. (%) 5 (10) 13 (26) 0,32** AHF precipitant (n) Undertreatment 34 (68) 29 (58) 0,30** Dysrythmia 10 (20) 9 (18) 0,80** Non-compliance 6 (12) 8 (16) 0,56** NSAIDs 0 4 (8) 0,04** Continuous variables are presented as mean value ± standard deviation [SD], p value. Categorical variables are presented as absolute number (%), p value. **Chi-square test. HgB = hemoglobin; ACE = angiotensin-converting enzyme; BMI = Body Mass Index; NYHA = New York Heart Association; AHF = Acute Heart Failure; NSAID = NonSteroidal Anti-Inflammatory Drug; HFrEF, heart failure with reduced ejection fraction. 21 Outcomes 22 The outcomes of our study were: a) To test the influence of high-dose spironolactone in congestion assessment, evaluating the proportion of patients who were free of congestion at day 3 (defined as jugular venous pressure of < 8 cm, no orthopnea and no peripheral edema), weight change and also in congestion surrogate markers variation, such as N-terminal pro-brain natriuretic peptide (NT-pro BNP); b) To test the safety of spironolactone use in ADHF, assessing creatinine and potassium change between day 1 and day 3; c) To test the influence of spironolactone in surrogate markers such as albuminuria, hsTnT, uNa/K, MMP2, renin and aldosterone. 23 Papers 24 I. Mineralocorticoid Receptor Antagonism in Acutely Decompensated Chronic Heart Failure 25 João Pedro Ferreira, MD1, Mário Santos, MD1, Sofia Almeida, PhD2, Irene Marques, MD1, Paulo Bettencourt, MD, PhD3, Henrique Carvalho, MD, PhD1 1 Centro Hospitalar do Porto, 2 Climate Change Impacts, Adaptation and Mitigation Research Group (CC-IAM), Faculdade de Ciências, Universidade de Lisboa, 3 Centro Hospitalar de São João Correspondence: João Pedro Ferreira, Centro Hospitalar do Porto, Internal Medicine Department, Largo Prof. Abel Salazar 4099-001 Porto, Portugal. Contacts: Telephone – 00351222077500; Fax – 00351222053218; E-mail – [email protected] Abstract Background/Objectives: Mineralocorticoid receptor antagonists (MRAs) use in acutely decompensated chronic heart failure (ADCHF) may improve congestion through diuretic effect and prevent neurohormonal activation. We aimed to evaluate the clinical effect and safety of spironolactone in ADCHF. Methods: Prospective, experimental, single-centre, and single-blinded trial. Patients were treated with: standard ADCHF therapy or oral spironolactone 50 - 100 mg/d plus standard ADCHF therapy. Results: During 1 year period, 100 patients were enrolled, 50 included in the treatment group. Mean (SD) spironolactone dose (mg) at day 1 was 94,5 ± 23,3 and at day 3 was 62,7 ± 24,3. Worsening renal function (increase in pCr ≥ 0,3 mg/dL from day 1 to day 3) was more likely to occur in control group (20% vs. 4%; p = 0,038), serum potassium did not differ between groups, and plasma NTproBNP had a significant decrease in spironolactone group at day 3 (median [IQR], 2488 [4579] vs. 1555 [1832]; p = 0,05). Furthermore, a greater proportion of patients in the treatment group were free of congestion at day 3: less edema, rales, jugular venous pressure (JVP) and orthopnea (all, p < 0,05). In addition, a significantly higher proportion of patients were on oral furosemide at day 3 (44% vs. 82%; p < 0,001). Conclusions: Our study supports the safety of high dose spironolactone in ADCHF and suggests a positive impact in the resolution of congestion. The important findings of our pilot study need to be confirmed in larger trials. 32 Conclusion Our study shows that treating ADHF patients with spironolactone was safe. It was also associated with an earlier resolution of the congestive signs and with a more pronounced NTproBNP reduction. Despite its exploratory nature, our study highlights the need to improve the treatment of ADHF and points out the direction of future investigation towards MRAs. Acknowledgements The authors acknowledge the lab technicians, specially Mr. Fernando Santos for technical assistance and to all physicians collaborating in the study. Disclosures The authors have no conflicts to disclose. 33 Tables Table 1. Baseline Characteristics of the Study Population Control Group Spironolactone Group p value Age (yrs) 78,8 ± 9,3 73,2 ± 11,7 0,01 Male sex – no. (%) 17 (34) 22 (44) 0,31** Ejection Fraction (%) 45,5 ± 10,7 41,4 ± 12,4 0,08 Left Atrial Size (mm) 47,4 ± 5,3 46,3 ± 7,1 0,40 Charlson Index (pts) 6, 1 ± 1,1 5,9 ± 0,9 0,38 HgB (g/dL) 12,2 ± 1,8 12,7 ± 2,3 0,22 Albumin (mg/dL) 3,7 ± 0,4 3,6 ± 0,4 0,63 TSH (mUI/L) 2,8 ± 3,0 2,6 ± 2,8 0,79 Etiology of Heart Failure – no. (%) Ischemic 24 (48) 26 (52) 0,69** Non-Ischemic 26 (52) 24 (48) 0,84** Basal NYHA class – no. (%) II 41 (82) 44 (88) 0,40** III 9 (18) 6 (12) 0,40** History of Atrial Fibrillation or Flutter - no. (%) 34 (68) 25 (50) 0,07** Outpatients Medications – no. (%) Furosemide 37 (74) 35 (70) 0,65** ACE Inhibitors 24 (48) 18 (36) 0,22** Beta-Blockers 26 (52) 10 (20) 0,001** Outpatients Oral Dose (mg) Furosemide 69,2 ± 37,8 68 ± 30 0,89 ACE Inhibitors 5 ± 3 4,3 ± 2,5 0,43 Beta-Blockers 4,4 ± 2,7 4,5 ± 1,1 0,90 ADHERE: inhospital mortality risk – no. (%) Low 44 (88) 40 (80) 0,27** Intermediate 2 2 (4) 3 (6) 0,65** Intermediate 3 4 (8) 7 (14) 0,34** EFFECT: Heart Failure Mortality Risk Prediction – no. (%) Low 4 (8) 8 (16) 0,22** Intermediate 27 (54) 25 (50) 0,69** High 18 (36) 17 (34) 0,83** BMI ≥ 30 (Kg/m2) – no. (%) 14 (28) 19 (38) 0,29** 34 Diabetes Mellitus – no. (%) 25 (50) 20 (40) 0,31** Glycated Hemoglobin (%) 6,9 ± 0,7 7,1 ± 1,2 0,43 Obstructive Sleep Apnea Syndrome – no. (%) 5 (10) 13 (26) 0,32** Non-Invasive Ventilation – no. (%) 7 (14) 10 (20) 0,42** AHF precipitant (n) Undertreatment 34 (68) 29 (58) 0,30** Dysrythmia 10 (20) 9 (18) 0,80** Non-compliance 6 (12) 8 (16) 0,56** NSAIDs 0 4 (8) 0,04** Continuous variables are presented as mean value ± standard deviation [SD], p value. Categorical variables are presented as absolute number (%), p value. **Chi-square test. HgB = hemoglobin; ACE = angiotensin-converting enzyme; BMI = Body Mass Index; NYHA = New York Heart Association; AHF = Acute Heart Failure; NSAID = NonSteroidal Anti-Inflammatory Drug. 35 Table 2. Study End-Points. Control Group Spironolactone Group p Value Heart Rate (beats/min) Day 1 91,2 ± 24,7 96,1 ± 23,9 0,30 Day 3 74,9 ± 12,4 77,9 ± 11,4 0,20 SBP (mmHg) Day 1 140,5 ± 23,9 139 ± 27,9 0,80 Day 3 122 ± 15,6 121,9 ± 16,8 0,97 RR (cycles/min) Day 1 35 [5] 33,5 [6] 0,90* Day 3 20 [2] 18 [3] < 0,001* BMI (Kg/m2) Day 1 29,3 ± 5,7 29,5 ± 6,6 0,90 Day 3 28,1 ± 5,4 27,7 ± 6,6 0,76 Peripheral Edema – no. (%) Day 1 50 (100) 50 (100) Day 3 34 (68) 17 (34) 0,001** Rales – no. (%) Day 1 50 (100) 50 (100) Day 3 38 (76) 17 (34) <0,001** JVP ≥ 8 cm – no. (%) Day 1 32 (64) 28 (56) 0,41** Day 3 5 (10) 0 0,02** Orthopnea – no. (%) Day 1 50 (100) 50 (100) - Day 3 12 (24) 2 (4) 0,004** pCr (mg/dL) Day 1 Day 3 1,15 ± 0,27 1,23 ± 0,43 1,03 ± 0,29 1,06 ± 0,33 0,026 0,035 pCr Change(mg/dL) Day 3 – Day 1 0,075 ± 0,3 0,038 ± 0,17 0,47 Increase in pCr ≥ 0,3 mg/dL from Day 1 to Day 3 – no. (%) 10 (20) 2 (4) 0,038** pUrea Day 1 Day 3 59,32 ± 22,27 67,08 ± 27,09 51,10 ± 18,63 57,54 ± 23,03 0,048 0,061 pUrea Change (mg/dL) Day 3 – Day 1 7,7 ± 22,9 6,4 ± 17,9 0,75 Albuminuria (mg/g) Day 1 Day 3 73,50 [196,5] 60,16 [203] 54,05 [203,7] 27,9 [80,2] 0,521* 0,118* Albuminuria Change (mg/g) Day 3 – Day 1 - 7,3 [45,8] - 10,1 [71,2] 0,32* Albuminuria ratio Day 3 / Day 1 0,9 [0,8] 0,7 [0,7] 0,19* FENa (%) Day 1 1,4 [2] 2,4 [3,5] 0,24* Day 3 1,2 [2] 1,6 [2,5] 0,27* FEUr (%) Day 1 38,9 ± 12,7 38,2 ± 11,1 0,47 36 Day 3 39,5 ± 13,2 37,1 ± 10,5 0,31 UNa/K Ratio Day 1 2,7 [3,7] 3,6 [4,7] 0,18* Day 3 2,1 [3,1] 4,0 [3,9] 0,007* Serum Potassium (mmol/L) Day 1 4,1 ± 0,4 4,0 ± 0,6 0,33 Day 3 3,9 ± 0,5 4,1 ± 0,5 0,15 Hypokalemia (mmol/L) at Day 3 – no. (%) 13 (26) 7 (14) 0,13** Serum Sodium (mmol/L) Day 1 140 [7] 141 [4] 0,9* Day 3 141,2 ± 4,3 140,2 ± 3,5 0,2 Serum Ionized Calcium (mmol/L) Day 1 1,2 [0,1] 1,2 [0,1] 0,6* Day 3 1,2 [0,1] 1,2 [0,1] 0,1* Serum Magnesium (mmol/L) Day 1 0,84 [0,1] 0,8 [0,1] 0,2* Day 3 0,87 [0,1] 0,85 [0,1] 0,6* ProBNP (pg/ml) Day 1 3102 [6408] 2701 [3541] 0,17* Day 3 2488 [4579] 1555 [1832] 0,05* TnT (ng/mL) Day 1 0,034 [0,035] 0,03 [0,032] 0,5* Day 3 0,032 [0,036] 0,029 [0,028] 0,3* TnT Reduction (ng/mL) Day 3 – Day 1 - 0,0005 [0,01] - 0,001 [0,01] 0,57* CkMB (UI/L) Day 1 15,5 [9] 14 [9] 0,14* Day 3 14 [8] 12 [8] 0,13* CkMB Reduction (UI/mL) Day 3 – Day 1 - 1,7 ± 6,2 - 2,3 ± 4,3 0,63 IV Furosemide Dose (mg) Day 1 80 [20] 80 [30] 0,86* Day 3 60 [20] 60 [30] 0,22* Oral Furosemide at Day 3 – no. (%) 22 (44) 41 (82) < 0,001** ACE Inhibitors Dose (mg) Day 1 2,5 [3,8] 2,5 [2,5] 0,75* Day 3 2,5 [3,8] 2,5 [3,8] 0,72* ACE Inhibitors – no. (%) Day 1 19 (38) 25 (50) 0,20** Day 3 30 (60) 31 (62) 0,80** Beta-Blockers Dose (mg) Day 1 2,5 [0] 2,5 [2,5] 0,95* Day 3 2,5 [2,5] 2,5 [1,25] 0,46* Beta-Blockers - no. (%) Day 1 21 (42) 16 (32) 0,30** Day 3 27 (54) 30 (60) 0,50** Length of Stay (days) 9 [5] 8 [5] 0,8* Continuous variables are presented as mean value ± standard deviation [SD], p value or median [inter-quartile range, IQR], p value. Categorical variables are presented as absolute number (%), p value. *Non-parametric test; **Chi-square test. 37 SBP = systolic blood pressure; RR = respiratory rate; JVP = jugular venous pressure; BMI = Body Mass Index; PaO2/FiO2 = partial pressure arterial oxygen/fraction inspired oxygen; pCr = plasma creatinine; FENa = spot urine fractional excretion of sodium; FEUr = spot urine fractional excretion of urea; UNa/K = urinary sodium to potassium ratio; proBNP = N-terminal pro brain natriuretic peptide; TnT = high-sensitivity troponin T; CkMB = creatine kinase-MB; ACE = angiotensin-converting enzyme. 38 Figures Graph 1. Changes in Congestive Signs and Patients Taking Oral Furosemide at Day 3 (%) in the Control and Spironolactone Groups. p = 0,001 p < 0,001 p = 0,02 p = 0,004 p < 0,001 -40 -20 0 20 40 60 80 100 120 Peripheral Edema Rales JVP ≥ 8 cm Orthopnea Oral Furosemide at Day 3 Control Group Spironolactone Group 39 Graph 2. Changes in Serum Potassium (K+) from Day 1 to Day 3 in the Control and Spironolactone Groups. 40 Graph 3. Changes in Mean Plasma Levels of NT Pro-Brain Natriuretic Peptide (NTproBNP) from Day 1 to Day 3 in the Control and Spironolactone Groups. 41 References 1. Mann DL, Bristow MR. Mechanisms and models in heart failure: The biomechanical model and beyond. Circulation. United States; 2005:2837-2849. 2. Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, Bittman R, Hurley S, Kleiman J, Gatlin M. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Eengl Jj Mmed. United States: 2003 Massachusetts Medical Society; 2003:1309-1321. 3. Zannad F, McMurray JJ, Krum H, van Veldhuisen DJ, Swedberg K, Shi H, Vincent J, Pocock SJ, Pitt B. Eplerenone in patients with systolic heart failure and mild symptoms. N Engl J Med. 2011;364:11-21 4. Gheorghiade M, Pang PS. Acute heart failure syndromes. J am coll cardiol. United States; 2009:557-573. 5. Albaghdadi M, Gheorghiade M, Pitt B. Mineralocorticoid receptor antagonism: Therapeutic potential in acute heart failure syndromes. Eur heart j. England; 2011:2626-2633. 6. Aronson D, Burger AJ. Neurohormonal prediction of mortality following admission for decompensated heart failure. Am j cardiol. United States; 2003:245-248. 7. Schmidt BM, Sammer U, Fleischmann I, Schlaich M, Delles C, Schmieder RE. Rapid nongenomic effects of aldosterone on the renal vasculature in humans. Hypertension. United States; 2006:650-655. 8. Farquharson CA, Struthers AD. Aldosterone induces acute endothelial dysfunction in vivo in humans: Evidence for an aldosterone-induced vasculopathy. Clin Sci (Lond). 2002;103:425-431 9. De Luca L, Fonarow GC, Adams KF, Jr., Mebazaa A, Tavazzi L, Swedberg K, Gheorghiade M. Acute heart failure syndromes: Clinical scenarios and pathophysiologic targets for therapy. Heart Fail Rev. 2007;12:97-104 10. Struthers AD. Aldosterone: Cardiovascular assault. Am heart j. United States; 2002:S2-7. 11. Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: Development and validation. J Chronic Dis. 1987;40:373-383 12. Fonarow GC, Adams KF, Jr., Abraham WT, Yancy CW, Boscardin WJ. Risk stratification for in-hospital mortality in acutely decompensated heart failure: Classification and regression tree analysis. Jama. United States; 2005:572-580. 48 and red cell distribution width (RDW) at day 1; proportion of inpatients on spironolactone, ACEi, and BB. Statistical Analysis Comparison between groups (versus [vs]) was performed using parametric, non-parametric tests, or chi-square tests, as appropriate. Continuous variables are expressed as mean (standard deviation, SD) or median (inter-quartile range, IQR). Categorical variables are expressed in absolute numbers (no.) and proportions (%). Association between different variables was tested by univariate analysis. Variables with significant association were tested by multivariate analysis in a stepwise manner. Predictors of outcome were identified by logistic regression analyses. Significant association was defined by a p value < 0,05. Statistical analysis was performed using SPSS software (version 19, Chicago, IL, USA). Results Mean ± SD age was 76,0 ± 10,9 years. Sixty-one patients were female. All patients had congestive signs at admission. After three days of inpatient treatment, 16 (16%) patients maintained or increased i.v. furosemide dose (SDR). This group of patients had more indirect signs of fluid overload (rales: 87,5% vs 48,8%, p = 0,004; peripheral edema: 81,3% vs 45,2%, p = 0,008; orthopnea: 37,5% vs 9,5%, p = 0,008; JVP ≥ 8 cm: 18,8% vs 2,4%, p = 0,006). Body mass index also increased in SDR group (mean ± SD, 31,3 ± 5,4 vs 27,8 ± 6,2, p = 0,038). The other 84 patients had FDR, greater congestion relief and a decrease in i.v. furosemide dose or oral route furosemide switch (dose reduction – no. [%] = 21 [25]; oral route – no. [%] = 63 [75]) – table 1. We did not find any significant differences between both groups (SDR vs FDR) regarding baseline characteristics, comorbidities, and BMI - table 1. All patients received i.v. bolus furosemide. Combination therapy with thiazide diuretics was not used. Intravenous bolus furosemide dose (mg) on admission day did not differ between groups (mean ± SD, 72,5 ± 20,5 vs 76,4 ± 21,8). Univariate analysis of the variables potentially linked to SDR is shown in table 2. Admission day covariates predicting SDR were: higher levels of pUr (mean ± SD, 69,6 ± 20,9 vs 52,5 ± 19,8, p = 0,002); higher levels of pUr / pCr ratio (mean ± SD, 58,3 ± 15,2 vs 49,6 ± 15,1, p = 0,036); higher levels of albuminuria (median [IQR], 131,5 [396,9] vs 47,1 [143,6], p = 0,011) – figure 1.; higher levels of RDW (median 49 [IQR], 16,0 [1,9] vs 15,1 [1,5], p = 0,039); lower levels of HgB (mean ± SD, 11,5 ± 1,8 vs 12,6 ± 2,1, p = 0,04); and higher levels of hsTnT (median [IQR], 0,05 [0,05] vs 0,03 [0,03], p = 0,026). Higher levels of pCr at day 1 are also likely to predict SDR (mean ± SD, 1,21 ± 0,28 vs 1,06 ± 0,28, p = 0,06) and a trend to higher levels of renin on admission day was found in SDR patients (8,1 [14,3] vs 4,1 [6,7], p = 0,098). During the first three days of hospitalisation the covariates associated with SDR were: higher levels of pCr at day 3 (mean ± SD, 1,40 ± 0,46 vs 1,1 ± 0,36, p = 0,004); increment in pCr ≥ 0,3 mg/dL from day 1 to day 3 (37,5% vs 7,1%, p = 0,001) – figure 2.; higher levels of pUr at day 3 (mean ± SD, 79,7 ± 24,9 vs 59,0 ± 24,3, p = 0,002); higher levels of albuminuria at day 3 (median [IQR], 123,9 [358,4] vs 26,4 [85,2], p = 0,001) – figure 1.; higher levels of NTproBNP at day 3 (median [IQR], 3013 [4116] vs 1701 [2563], p = 0,009); NTproBNP maintenance or increment (37,5% vs 13,1%, p = 0,017) – figure 3.; higher levels of hsTnT at day 3 (median [IQR], 0,06 [0,04] vs 0,03 [0,03], p = 0,004). Patients treated with spironolactone had FDR (12,5% vs 57,1%, p = 0,001) – figure 4. Such differences were not found in patients taking ACEi and BB. Multivariate analysis results are shown in table 3. The strongest independent early predictors of SDR were: pUr (OR [95%CI], 1,04 [1,01 – 1,07], p = 0,006), and redcell distribution width (RDW) (OR [95%CI], 1,47 [1,07 – 2,02], p = 0,018). NTproBNP increase or decrease by less than 30% from day 1 to day 3 was the strongest independent factor associated with SDR (OR [95%CI], 4,84 [1,14 – 20,55], p = 0,032). Noteworthy, no use of spironolactone increases the risk of SDR (OR [95%CI], 5,98 [1,17 – 30,42], p = 0,031). Time to oral furosemide (in days) was longer in patients with SDR (mean ± SD, 5,9 ± 1,7 vs 3,6 ± 2,2, p < 0,0001) and hospital length of stay (in days) was also longer in SDR group (mean ± SD, 10,9 ± 3,2 vs 8,5 ± 3,3, p = 0,008). No deaths or complete treatment failure occurred. Discussion Our study suggests that higher levels of pUr and RDW at admission can predict a subset of ADCHF patients with a slow diuretic response. Moreover, patients with little reduction or increase in NTProBNP levels during the first few days of hospitalisation needed to maintain high doses of i.v. diuretics. On the contrary, patients taking spironolactone had faster diuretic response. Urea is freely filtered through the glomerulus and undergoes substantial tubular reabsorption9. This reabsorption of pUr is flow dependent so that more urea is reabsorbed at lower urine flow rates10. The neurohormonal response in HF involves the 50 nonosmotic secretion of arginine vasopressin (AVP), stimulation of the RAAS and sympathetic nervous system10, which cause decrease of renal blood flow by renal vasoconstriction, and increased proximal tubular reabsorption of sodium and water. These changes will result in increased urea reabsorption caused by slow tubular flow in the collecting duct and higher AVP plasma concentration11, 12. This neurohormonal activation is exacerbated by loop diuretics because they block sodium chloride absorption at the macula densa and consequently increase renin secretion by the juxtaglomerular apparatus 13. In HF patients with higher pUr, neurohormonal activation with high-dose loop diuretics can be greater than the withdrawal of neurohormonal activation resulting from decongestion, leading to a poorer survival9, 14. Components of the neurohormonal axis are not routinely measured in HF patients. Therefore, the rise in pUr may serve as an indirect marker of neurohormonal activation10. Higher plasma concentrations of plasma renin activity are associated with increased risk of death in HF, as occurred with the higher admission pUr values and changes in pUr values during hospitalization10. Volume management strategies based on pUr concentration or other markers of renal neurohormonal activation is a potential field for clinical investigation. In this regard, the use of natriuretic doses of potassium-sparing diuretics may allow both the minimization of loop diuretic doses and the maintenance of euvolemia9. Furthermore, an elevated admission pUr / Cr ratio has been associated with an increased incidence of post-discharge WRF, independent of the discharge glomerular filtration rate (GFR)15. In our study, high pUr and pUr / pCr ratio at admission predicted SDR. High renin also seemed to predict SDR. These results at day 1 are independent of the use of i.v. diuretics. These findings can lead to an early identification of patients who should have tighter follow-up and individualized diuretic approach, particularly the use of high dose spironolactone which showed to increase diuretic response in this pool of patients without severe renal dysfunction. Red blood cell distribution width (RDW) is a percentual measure of the variability in the size of circulating erythrocytes, recorded during a standard complete blood count16. Disorders related to ineffective erythropoiesis or increased destruction cause greater heterogeneity in size and a higher RDW17. High RDW may reflect nutritional deficiencies, bone marrow dysfunction, or systemic inflammation, representing an integrative measure of the pathological processes occurring in HF18. Higher RDW was shown to be a strong independent predictor of greater morbidity and mortality in patients with chronic HF17, 19, but the mechanism underlying the association between RDW and death in patients with HF is unclear18. Higher RDW level at discharge as also associated with a worse long-term outcome in patients hospitalized with acute heart failure (AHF), regardless of anemia status18. In our population higher 51 RDW predicted SDR, to the best of our knowledge this was the first report of this association. Excessive activation of the RAAS causes glomerular hypertension and proteinuria20. A close correlation between increased excretion of urinary albumin and mortality in chronic HF has been reported21, 22. Our results suggests that increased albuminuria may also predict a subset of SDR in ADCHF patients. An elevation in cardiac troponin indicates the presence of myocyte injury or death23. Myocyte loss is recognized to be a prominent pathophysiological mechanism in the evolution of cardiac dysfunction24. The pathophysiological factors that that are thought to be responsible for ongoing myocyte injury include neurohormonal activation and abnormalities in inflammatory cytokines, oxidative and mechanical stress25. An association between higher mortality and positive troponin in patients with ADCHF has been demonstrated26, 27. In our study higher levels of hsTnT at admission also predict SDR, adding an additional marker for early identification of patients who will require early implementation of aggressive therapy and monitorization. Worsening renal function with persistence of congestive signs is associated with SDR. This observation is consistent with recent findings suggesting that the link between high-dose diuretics and poor outcomes may reflect the severity of the illness rather than an independent harmful effect of diuretics6. We also found an association between maintenance or increase in NTProBNP and SDR, suggesting that NTProBNP is a reliable biomarker for the evaluation of treatment efficacy and might help clinicians to reconsider treatment strategies in the first days of hospitalization. This is another potential role for this biomarker, since variations in NTproBNP levels during hospitalization are also associated with higher hospital readmission rates and 6-month mortality28. Our study has several limitations that need to be considered. First, it was a single-centre investigation of a small sample size. Second, the decision to withdraw diuretic therapy was based on subjective assessment of congestive signs and symptoms so we cannot rule out the inter-observer variability. However, in real-life patients, the decision to step down diuretic therapy is also based on subjective clinical evaluation. Finally, the external validity of our conclusions is limited to patients with mild renal failure HF patients since pCr level ≤ 1,5 mg/dl was an inclusion criteria. Conclusion High RDW and high levels of pUr at admission are strong predictors of slower diuretic response. No change or increase in NTproBNP and worsening renal function in the first three days of treatment are associated with slower diuretic response. On the 52 other hand, the use of high dose spironolactone is associated with faster diuretic response. Our findings warrant further investigation to establish the value of these predictors in guiding diuretic strategies in hospitalised ADCHF patients. Acknowledgements The authors acknowledge the lab technicians, especially Mr. Fernando Santos for technical assistance and to all physicians collaborating in the study. Disclosures The authors have no conflicts to disclose. 53 Tables Table 1. Baseline Characteristics and Differences in Congestive Signs at Day 1 and Day 3 Furosemide Maintenance or Increase (n=16) Furosemide Decrease or Oral Administration (n=84) p Value Age (yrs) 78,8 ± 6,9 75,5 ± 11,4 0,13 Male Sex – no. (%) 5 (31,3) 34 (40,5) 0,49** Diabetes Mellitus – no. (%) 10 (62,5) 35 (41,7) 0,13** COPD – no. (%) 2 (12,5) 15 (17,9) 0,46** Dementia – no. (%) 3 (18,8) 9 (10,7) 0,40** Sleep Apnea – no. (%) 3 (42,9) 15 (40,5) 0,61** BMI (%) 29,7 ± 5,5 29,4 ± 6,3 0,83 Charlson Index (pts) 6,38 ± 0,6 5,9 ± 1,1 0,29 Ischemic Etiology for Heart Failure – no. (%) 10 (62,5) 40 (47,6) 0,28** Furosemide Ambulatory Dose (mg) 68,3 ± 30,1 68,8 ± 33,6 0,98 Outpatients on ACEi – no. (%) 8 (50) 34 (40,5) 0,48** Outpatients on Betablockers – no. (%) 7 (43,8) 50 (59,5) 0,24 Left Atrial Size (mm) 48,6 ± 5,3 46,6 ± 6,4 0,42 Ejection Fraction (%) 43,0 ± 11,2 43,6 ± 11,9 0,86 Rales – no. (%) Day 1 Day 3 16 (100) 14 (87,5) 84 (100) 41 (48,8) - 0,004** Peripheral Edema – no. (%) Day 1 Day 3 16 (100) 13 (81,3) 84 (100) 38 (45,2) - 0,008** Orthopnea – no. (%) Day 1 Day 3 16 (100) 6 (37,5) 84 (100) 8 (9,5) - 0,003** JVP ≥ 8 cm – no. (%) Day 1 Day 3 12 (75) 3 (18,8) 48 (57,1) 2 (2,4) 0,18 0,006** BMI (Kg/m2) Day 1 Day 3 29,7 ± 5,5 31,3 ± 5,4 29,4 ± 6,3 27,8 ± 6,2 0,83 0,038 Continuous variables are presented as mean value ± standard deviation [SD], p value. Categorical variables are presented as absolute number (%), p value. **Chi-square test. COPD = chronic obstructive pulmonary disease; BMI = Body Mass Index; ACEi = angiotensin-converting enzyme inhibitors; JVP = jugular venous pressure. 54 Table 2. Univariate Analysis for Furosemide Response Predictors, Furosemide Response Associations, Time to Oral Furosemide and Hospital Length of Stay Furosemide Maintenance or Increase Furosemide Decrease or Oral Administration p Value IV Furosemide at Day 1 (mg) 72,5 ± 20,5 76,4 ± 21,8 0,51 pCr (mg/dL) Day 1 Day 3 1,21 ± 0,28 1,40 ± 0,46 1,06 ± 0,28 1,1 ± 0,36 0,06 0,004 Increase in pCr ≥ 0,3 mg/dL from Day 1 to Day 3 – no. (%) 6 (37,5) 6 (7,1) 0,001** pUrea (mg/dL) Day 1 Day 3 69,6 ± 20,9 79,7 ± 24,9 52,5 ± 19,8 59,0 ± 24,3 0,002 0,002 pUrea Change from Day 1 to Day 3 10,1 ± 30,0 6,5 ± 19,4 0,52 pUrea to pCr ratio Day 1 Day 3 58,3 ± 15,2 58,8 ± 12,4 49,6 ± 15,1 54,3 ± 16,7 0,036 0,32 Albuminuria (mg/g) Day 1 Day 3 131,5 [396,9] 123,9 [358,4] 47,1 [143,6] 26,4 [85,2] 0,011* 0,001* Albuminuria Change from Day 1 to Day 3 -13,3 [67,8] -7,8 [56,1] 0,84* Serum Potassium (mmol/L) Day 1 Day 3 3,9 ± 0,5 3,8 ± 0,6 4,1 ± 0,5 4,1 ± 0,5 0,28 0,08 Serum Sodium (mmol/L) Day 1 Day 3 140,6 ± 4,1 140,8 ± 4,7 140,5 ± 4,5 140,7 ± 3,8 0,98 0,89 HgB at Day 1 (g/dL) 11,5 ± 1,8 12,6 ± 2,1 0,04 RDW at Day 1 16,0 [1,9] 15,1 [1,5] 0,039* Albumin at Day 1 (mg/dL) 3,8 ± 0,4 3,6 ± 0,4 0,08 NTproBNP (pg/mL) Day 1 Day 3 3390 [4511] 3013 [4116] 2698 [4577] 1701 [2563] 0,26 0,009* NTproBNP Decrease by Less than 30% or Increase from Day 1 to Day 3 – no. (%) 6 (37,5) 11 (13,1) 0,017** hsTnT (ng/mL) Day 1 Day 3 0,05 [0,05] 0,06 [0,04] 0,03 [0,03] 0,03 [0,03] 0,026* 0,004* hsTnT Change from Day 1 to Day 3 -0,0005 [0,01] -0,001 [0,01] 0,51* Aldosterone at Day 1 (ng/dL) 34 [98,3] 35,5 [75,7] 0,84* Renin at Day 1 (pg/mL) 8,1 [14,3] 4,1 [6,7] 0,098* Inpatients on Spironolactone – no. (%) 2 (12,5) 48 (57,1) 0,001** Inpatients on ACEi – no (%) 7 (43,8) 37 (44) 0,98** 55 Inpatients on Beta-Blockers – no (%) 7 (43,8) 30 (35,7) 0,54** Time to Oral Furosemide (days) 5,9 ± 1,7 3,6 ± 2,2 <0,0001 Hospital Length of Stay (days) 10,9 ± 3,2 8,5 ± 3,3 0,008 Continuous variables are presented as mean value ± standard deviation [SD], p value or median [inter-quartile range, IQR], p value. Categorical variables are presented as absolute number (%), p value. *Non-parametric test; **Chi-square test. pCr = plasma creatinine; pUrea = plasma urea; HgB = hemoglobin; RDW = red cell distribution width; NTProBNP = N-terminal pro-brain natriuretic peptide; hsTnT = highsensitivity troponin T; ACEi = angiotensin-converting enzyme inhibitors. 56 Table 3. Multivariate Analysis: Odds Ratios (OR) and 95% Confidence Intervals (95%CI) for Predictors and Factors Associated with Slow Diuretic Response. Furosemide Maintenance or Increase, OR (95%CI) p Value RDW at Day 1 1,47 (1,07 – 2,02) 0,018 pUrea at Day 1 1,04 (1,01 – 1,07) 0,006 No Spironolactone Use 5,98 (1,17 – 30,42) 0,031 NTProBNP Increase or Decrease by Less than 30% from Day 1 to Day 3 4,84 (1,14 – 20,55) 0,032 Increase in pCr ≥ 0,3 mg/dL from Day 1 to Day 3 4,07 (0,85 – 19,45) 0,079 pUrea at Day 3 1,02 (1,00 – 1,05) 0,087 RDW = red cell distribution width; pUrea = plasma urea; NTproBNP = N-terminal probrain natriuretic peptide; pCr = plasma creatinine; pUrea = plasma urea. 57 Figures Figure 1. Comparison of Albuminuria (mg/g) in Slow versus Fast Diuretic Responders at Day 1 and Day 3 of Hospitalization. Albuminuria (mg/g) SDR Day 1 FDR p = 0,011 Day 3 p = 0,001 SDR FDR Legend: SDR, Slow Diuretic Responders; FDR, Fast Diuretic Responders 64 III. The Role of Albuminuria as a Non-Invasive Marker for Congestive Acutely Decompensated Chronic Heart Failure and the Spironolactone Effect in Elderly Portuguese: a NonRandomized Trial 65 João Pedro Ferreira, MD1, Mário Santos, MD1, Sofia Almeida, PhD2, Irene Marques, MD1, Paulo Bettencourt, MD, PhD3, Henrique Carvalho, MD, PhD1 1 Centro Hospitalar do Porto, 2 Climate Change Impacts, Adaptation and Mitigation Research Group (CC-IAM), Faculdade de Ciências, Universidade de Lisboa, 3 Centro Hospitalar de São João Correspondence: João Pedro Ferreira, Centro Hospitalar do Porto, Internal Medicine Department, Largo Prof. Abel Salazar 4099-001 Porto, Portugal. Contacts: Telephone – 00351222077500; Fax – 00351222053218; E-mail – [email protected] Abstract Background/Objectives: Albuminuria is a robust, validated cardiovascular risk factor. It is a simple and widely available test that was shown to be a powerful and independent predictor of prognosis in chronic heart failure. Mineralocorticoid receptor antagonists may reduce the acute and chronic harmful effects of mineralocorticoid receptor activation on the kidney. The objectives of the trial were to compare the effect of spironolactone versus standard acutely decompensated heart failure (ADHF) therapy on albuminuria and to investigate the role of albuminuria as a prognostic marker in patients with ADHF. Methods: Secondary analysis of a prospective, interventional study including 100 patients with ADHF. Fifty patients were non-randomly assigned to spironolactone 100 mg/day plus standard ADHF therapy (intervention group) or standard ADHF therapy alone (control group). Results: Patients in control group were older, had higher creatinine and urea levels, and had higher proportion of microalbuminuria (all, p < 0,05). Paired comparison of baseline and day 3 log albuminuria within each group, showed a more pronounced decrease in the intervention group (1,79 ± 0,75 to 1,59 ± 0,67, p = 0,003 vs. 1,89 ± 0,70 to 1,79 ± 0,74, p = 0,096). In addition, the proportion of patients with normoalbuminuria increased from baseline to day 3 in spironolactone group (20 (40%) to 27 (54%), p < 001), accordingly the number of patients in the micro and macroalbuminuria groups was reduced. Day 1 albuminuria was positively correlated with day 1 N-terminal pro-brain natriuretic peptide (0,260 [0,105 to 0,758], p = 0,009). Conclusions: High-dose spironolactone added to standard ADHF therapy is likely to induce a more pronounced albuminuria decrease and a significant reduction in the proportion of micro and macroalbuminuria. 66 Key-words: Acute heart failure. Albuminuria. Acute kidney injury. Introduction Albuminuria is a robust, validated cardiovascular (CV) risk factor. It is independently associated with major adverse events such as stroke, myocardial infarction (MI) and CV death1. Screening for increased albuminuria is recommended for risk stratification of patients with diabetes and hypertension in order to adequate the treatment aggressiveness2,3. Albuminuria determination is a simple and widely available test that was shown to be important for the risk stratification of patients with heart failure (HF), and a powerful and independent predictor of prognosis in HF4. In these patients, increased albuminuria could be a consequence of the increased neurohormonal activation, endothelial injury, systemic inflammation, and renal dysfunction5,6. These mechanisms are also involved in the pathophysiology of acute decompensated heart failure (ADHF) 4,7, 8 and have a multitude of effects in the kidney, including arteriolar vasoconstriction and increased tubular reabsorption of sodium and urea9,10. However, the prognostic value of albuminuria is not yet established in this clinical setting. Mineralocorticoid receptor antagonists (MRAs) have been shown to attenuate ventricular and vascular hypertrophy, potassium and magnesium loss, glomerulosclerosis, renal interstitial fibrosis and proteinuria in patients with chronic kidney disease (CKD)11,12. Thus, MRAs may reduce the acute and chronic harmful effects of mineralocorticoid receptor activation on the kidney, effectively augment diuresis in diuretic-resistant patients, and attenuate the development of the vasomotor nephropathy in ADHF patients13. Our aims were to compare the effect of spironolactone versus standard acutely decompensated heart failure (ADHF) therapy on albuminuria and to investigate the role of albuminuria as a prognostic marker in patients with ADHF. Methods Study Design We analysed data from a previous prospective, interventional, clinical trial that we performed. In that study we enrolled 100 consecutive patients who presented in a Portuguese tertiary hospital with ADCHF, between February 2012 and February 2013. Patients were eligible for enrollment if they presented with decompensation of chronic HF with symptoms leading to hospitalization. ADCHF was diagnosed on the basis of the presence of history of chronic HF and at least one symptom (dyspnea, orthopnea, or edema) and one sign (rales, peripheral edema, ascites, or pulmonary vascular congestion on chest radiography). Patients were non-randomly assigned in a 67 sequential 1:1 ratio to spironolactone plus standard ADCHF therapy or standard ADCHF therapy alone, 50 patients within each arm (i.e. patients were alternatively assigned to spironolactone arm or standard ADCHF therapy arm in a sequential manner - the first patient to one arm and the next to the other arm. This sequence was repeated until we reach 100 patients, 50 patients within spironolactone group and 50 patients within control group). Patients were blinded to the allocation, and the clinicians were not blinded to the allocation. The recommended spironolactone dose was 100 mg/day, however the assistant physician could decrease the spironolactone dose to 50 mg/day after 48h upon admission. After 72h the study was open label. Furosemide dose and route of administration was adjusted clinically according to the hydration status of the patients – Figure 1. Exclusion criteria were: chronic use of mineralocorticoid receptor antagonists (MRAs), cardiac surgery within 60 days of enrollment, cardiac mechanical support, cardiac resynchronization-therapy within the last 60 days, comorbid conditions with an expected survival of less than 6 months, acute MI at time of hospitalization, hemodynamically significant uncorrected primary cardiac valvular disease, patients requiring intravenous vasodilators or inotropic agents, supine systolic arterial blood pressure <90 mmHg, plasma creatinine (pCr) level >1,5 mg/dL, serum potassium level >5,0 mmol/L, hemoglobin (HgB) level <9 g/dL, and sepsis. Institutional review board or ethics committee approval was obtained. All patients provided written informed consent to participate in the study. Clinical assessment of participants Patient`s clinical status including physical examination and was prospectively recorded by the same assistant physician at day 1 and day 3. Medications and respective dosages were prospectively recorded by the investigators according to the assistant physician prescriptions. Blood and spot urine samples were collected in the first 24 hours (h) after admission (day 1) of the patient to the hospital, and the day 3 samples were collected between 72 and 96 h of hospitalization. Samples were analysed at a central core laboratory, and included pCr, plasma urea (pUr), electrolytes, N-terminal pro-brain natriuretic peptide (NTproBNP), high-sensitivity troponin T (hsTnT) and albuminuria. Clinical assessment and routine analyses were performed daily during hospital stay. Estimated glomerular filtration rate (eGFR) was determined using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation14. All patients performed a transthoracic echocardiography within 72 hours upon admission. Left ventricular ejection fraction (LVEF) was calculated according to biplane Simpson method. 68 Albuminuria and urine creatinine were measured using the COBAS INTEGRA Tina-quant Albumin Gen.2 urine application (Roche Diagnostics) and expressed as mg/g of Cr. We categorized albuminuria using standard cut-off points: normoalbuminuria (<30 mg/gCr), microalbuminuria (30-299 mg/gCr), and macroalbuminuria (≥300mg/gCr). Variable definitions We studied albuminuria regarding the following covariates: comorbidities such as diabetes mellitus (DM), chronic obstructive pulmonary disease (COPD), and sleep apnea; body mass index (BMI); heart rate (HR); systolic blood pressure (SBP); atrial fibrillation (AFib); HF etiology; echocardiographic parameters such as EF; furosemide dose, proportion of patients on angiotensin converting enzyme inhibitors (ACEi), betablockers (BB), and spironolactone; pCr, pUr, NTproBNP, hsTnT, sodium, potassium; HgB and serum albumin. Statistical Analysis Normally distributed continuous variables are expressed as mean ± standard deviation (SD), and skewed distributions are presented as median [inter-quartile range, IQR]. Because of the positively skewed distributions of BMI, HR, SBP, pCr, eGFR, NTproBNP, hsTnT, and albuminuria, these variables were log transformed for analysis. Categorical variables are expressed in absolute numbers (no.) and proportions (%). Comparison between groups was performed using parametric, non-parametric tests, or chi-square tests, as appropriate. A p value < 0,05 was considered statistically significant. The relationships between baseline characteristics, day 3 and changes (Δ) in albuminuria during the first 3 days and baseline, day 3 or Δ in other variables were tested by univariate analysis linear regression. Factors with a probability value of ≤0.10 by single variable linear regression analyses were included in a multivariable linear regression analyses together with age and sex. In other words, simple linear regression analyses were performed to examine the relationship between baseline characteristics, day 3 and Δ in other variables and albuminuria. Thereafter, a multivariable linear regression analysis was performed, which included age, sex, and the factors with a probability value of ≤0.10 in the bivariate analyses. Simple linear regression values are presented as non-adjusted coefficient (NAC) plus 95% confidence interval (95%CI). Multiple regression values are presented as adjusted coefficient (AC) plus (95%CI). 69 Statistical analysis was performed using SPSS software (version 19, Chicago, IL, USA). Results Baseline Patients Characteristics in Treatment and Control Groups Patients in control group were older (78,8 ± 9,3 versus [vs.] 73,2 ± 11,7 years, p = 0,01), had higher creatinine and urea levels (1,15 ± 0,27 vs. 1,03 ± 0,30, p = 0,026 and 59,32 ± 22,27 vs. 51,10 ± 18,63, p = 0,048), and had higher proportion of microalbuminuria (58% vs. 38%, p = 0,045). No differences between groups were found regarding sex, DM, COPD, dementia, sleep apnea, NIV, IHD, AFib, LVEF, BMI, HR, SBP, potassium, sodium, HgB, albumin, NTproBNP, hsTnT, albuminuria, furosemide dose and in the proportion of patients on ACEi and BB – Table 1. Spironolactone Influence on Albuminuria Dynamic Changes Change in albuminuria levels from baseline to day 3 was not significantly different between groups, despite the reduction was more pronounced in spironolactone group (-0,09 ± 0,40 vs. -0,20 ± 0,44, p = 0,226) – Table 2. However, paired comparison of baseline and day 3 log albuminuria within each group, showed a more pronounced decrease in the intervention group (1,79 ± 0,75 to 1,59 ± 0,67, p = 0,003 - within spironolactone group - and 1,89 ± 0,70 to 1,79 ± 0,74, p = 0,096 - within control group) – Figure 2. In addition, the proportion of patients with normoalbuminuria increased from baseline to day 3 in spironolactone group (20 (40%) to 27 (54%), p < 001), accordingly the number of patients in the micro and macroalbuminuria groups was reduced (19 (38%) to 16 (32%), p = 0,014 and 11 (22%) to 7 (14), p < 0,001, respectively) – Figure 3. Albuminuria Correlations By bivariate analysis, day 1 albuminuria was positively correlated with day 1 NTproBNP (0,260 [0,105 to 0,758], p = 0,009). Day 3 albuminuria was positively correlated with day 3 BMI (0,254 [0,025 to 0,047], p = 0,011), SBP (0,447 [0,006 to 0,027], p < 0,001), log hsTnT (0,220 [0,037 to 0,849], p = 0,028), and log pCr (0,248 [0,266 to 2,185), p = 0,013), and negatively correlated with log eGFR (-0,251 [-1,915 to -0,246], p = 0,012). Albuminuria decrease was negatively correlated with beta-blocker use during hospitalization (-0,243 [-0,355 to – 0,036], p = 0,015). By multivariate analysis, day 1 albuminuria correlated with NTproBNP (0,298 [0,167 to 0,830], p = 0,004), and day 3 albuminuria correlated with SBP (0,394 [0,009 to 0,025], p < 0,001) and log hsTnT (0,198 [0,043 to 0,759], p = 0,028) – Table 3. 70 Discussion Our study showed a more pronounced albuminuria decrease and a significant reduction in the proportion of micro and macroalbuminuria in ADHF patients submitted to spironolactone treatment. Increased albuminuria in patients with HF was found to be associated with increased risk of adverse clinical outcomes, including death4,15. Most patients in the Studies of Left Ventricular Dysfunction (SOLVD) had a urine dipstick test for protein at baseline16. Of 5487 (81% of total) tested, 177 (3%) had proteinuria. This subset of patients was more symptomatic, had higher blood pressure, higher prevalence of DM, and also greater left ventricular systolic dysfunction. Urinary albumin to creatinine ratio (UACR) was measured at baseline and during follow-up in the Candesartan in Heart failure: Assessment of Reduction in Mortality and morbidity (CHARM) Programme4. Of 2310 patients, 1349 (58%) had a normal UACR, 704 (30%) had microalbuminuria, and 257 (11%) had macroalbuminuria. Patients with an increased UACR were older, had more cardiovascular comorbidity, worse renal function, and a higher prevalence of DM than did those with normoalbuminuria. Elevated UACR was associated with increased risk of the composite outcome and death even after adjustment for other prognostic variables including renal function, DM, and HgBA1c. Interestingly, albuminuria per se has been associated with subsequent heart failure, even in individuals with few cardiovascular risk factors and UACR within the normal range17. Despite the strong body of evidence in chronic HF, few studies approach albuminuria in the acute HF setting. In a recently published study8, albuminuria was assessed at day 1 and 7 of hospitalization in 115 patients presenting with acute HF. Nearly 70% of patients had elevated UACR at admission. UACR decreased significantly after 7 days of treatment (from 83 to 22 mg/gCr, p <0,0001). The decrease was correlated with serum NTproBNP and bilirubin, but not with changes in renal function. Despite the evidence that effective HF treatment may reduce albuminuria during the acute episode, our study points towards an additional and interesting potential effect of MRAs in this setting. Neurohormonal activation causes vasoconstriction of both the afferent and efferent renal arterioles, and stimulates mesangial contraction in glomeruli, thus diminishing the glomerular filtration surface18. Excessive activation of the renin–angiotensin–aldosterone system (RAAS) causes glomerular hypertension and proteinuria, whereas its inhibition reduces proteinuria19,20. Excessive activation of the sympathetic nervous system also causes proteinuria21,22. Therefore, inhibition of neurohormonal activation is determinant to improve outcomes in patients with HF23-28. In our study, the increased albuminuria at admission might also reflect an excessive neurohormonal activation. This hypothesis is reinforced by the 71 greater decrease in albuminuria observed in patients submitted to spironolactone treatment. The MRAs have shown to reduce proteinuria in CKD patients already on ACEis and ARBs, however with an increased risk for hyperkalemia29, and a reduction of albuminuria was also observed with MRAs in the setting of chronic heart failure30. However, to the best of our knowledge our results are the first to demonstrate a greater reduction in albuminuria in ADHF patients submitted to spironolactone treatment. The rationale for this effect may rely on effective neurohormonal blockade, particularly on the activated the RAAS9, suggesting a potential renal protective effect of this treatment. Studies in animals have shown that increasing renal venous pressure leads to a reduction in glomerular filtration, which was probably mediated by a decreased renal perfusion31. Increased renal vein pressure in heart failure patients showed a marked reduction in renal blood flow as well as water and salt excretion32,33. Acute HF was correlated with higher albuminuria in patients with acute myocardial infarction34. Thus one can hypothesize that elevation of albuminuria can be a surrogate marker of severe volume overload. This hypothesis is reinforced by the positive correlation of albuminuria with NTproBNP at admission. Increased albuminuria probably has a hemodynamic basis in HF, particularly when renal venous congestion is associated with reduced renal blood flow, since urinary albumin excretion was inversely related to renal blood flow in patients with heart failure35,36. In addition, renal venous congestion caused proteinuria in dogs37. These findings support albuminuria as a surrogate marker of volume overload. High-dose spironolactone has shown to potentiate NTproBNP reduction and probably greater congestion relief in ADHF patients38, these findings provide additional clues to explain the observed reduction of albuminuria in patients submitted to spironolactone treatment – these patients may have greater congestion relief in addition to the effective neurohormonal blockade. Our study has several limitations that need to be considered. First, it was a single-centre investigation of a small sample size. Second, we used a single spot urine sample to determine albuminuria, which can be insufficient, as it might fluctuate. Finally, the external validity of our conclusions is limited to normo-hypertensive and fluid overloaded HF patients with mild renal failure, since all these factors were considered inclusion criteria. On the other hand, our conclusions can be reproducible in this set of patients widely common in clinical practice. Conclusions High-dose spironolactone added to standard ADHF therapy is likely to induce a more pronounced albuminuria decrease and a significant reduction in the proportion of 72 micro and macroalbuminuria. Further studies are required to explore the role of albuminuria as a prognostic marker in ADHF. Acknowledgements The authors acknowledge the lab technicians, especially Mr. Fernando Santos for technical assistance and to all physicians collaborating in the study. Disclosures The authors have no conflicts of interest to disclose. 73 Tables Table 1. Baseline Population Characteristics, Laboratory Results and Medications in Treatment and Control Groups Control Group (n = 50) Spironolactone Group (n = 50) p Value Age (yrs) 78,8 ± 9,3 73,2 ± 11,7 0,010 Male Sex – % 34 44 0,31** Diabetes Mellitus - % Glycated HgB (%) 50 6,9 ± 0,7 40 7,1 ± 1,2 0,31** 0,43 COPD - % 10 26 0,32** Dementia - % 16 8 0,22** Sleep Apnea - % 10 26 0,32** Non-Invasive Ventilation - % 14 20 0,42** Ischemic Heart Disease -% 48 52 0,69** Atrial Fibrillation - % 68 50 0,07** LV Ejection Fraction (%) 45,5 ± 10,7 41,4 ± 12,4 0,08 LV Ejection Fraction ≥ 40% - % 70 64 0,52** Body Mass Index (Kg/m2) 29,3 ± 5,7 29,5 ± 6,6 0,90 Heart Rate (bpm) 91,2 ± 24,7 96,1 ± 23,9 0,30 SBP (mmHg) 140,5 ± 23,9 139 ± 27,9 0,80 Plasma Creatinine (mg/dL) 1,15 ± 0,27 1,03 ± 0,30 0,026 eGFR (mL/min/1,73 m2) 54,48 ± 16,45 68,28 ± 23,55 0,001 Plasma Urea (mg/dL) 59,32 ± 22,27 51,10 ± 18,63 0,048 Serum Potassium (mmol/L) 4,1 ± 0,4 4,0 ± 0,6 0,33 Serum Sodium (mmol/L) 140,52 ± 5,04 140,56 ± 3,65 0,964 Hemoglobin (g/dL) 12,2 ± 1,8 12,7 ± 2,3 0,22 Albumin (mg/dL) 3,7 ± 0,4 3,6 ± 0,4 0,63 NTproBNP (pg/mL) 3102 [1797 – 8204] 2701 [1463 – 5004] 0,167* hsTnT (ng/mL) 0,034 [0,023 – 0,059] 0,030 [0,018 – 0,049] 0,490* Albuminuria (mg/g) 73,50 [28,83 – 225,45] 54,05 [17,43 – 221,10] 0,521* Normoalbuminuria - % 26 40 0,137** Microalbuminuria - % 58 38 0,045** Macrolbuminuria - % 16 22 0,444** IV Furosemide Dose (mg/d) 75,60 ± 20,72 76,00 ± 25,50 0,927 ACEi – no. (%) 19 (38) 25 (50) 0,201** Ramipril Eq. Dose (mg/d) 3,16 ± 2,21 3,15 ± 1,94 0,990 Beta-Blocker - % 21 (42) 16 (32) 0,302** Bisoprolol Eq. Dose (mg/d) 2,98 ± 1,01 3,05 ± 1,20 0,847 Spironolactone Dose (mg/d) - 94,5 ± 23,3 - Continuous variables are presented as mean value ± standard deviation [SD], p value or median [inter-quartile range, IQR], p value. Categorical variables are presented as absolute number (%), p value. *Non-parametric paired sample test; ** Chi-square test. Legend: COPD = chronic obstructive pulmonary disease; LV = left ventricular; eGFR = estimated glomerular filtration rate; NTproBNP = N-terminal pro brain natriuretic peptide; hsTnT = high sensitivity troponin T; IV = intra-venous; ACEi = angiotensin converting enzyme inhibitors. 80 Figure 3. Comparison of the Prevalence of Normo, Micro, and Macroalbuminuria at Baseline and Day 3 within Spironolactone Group. Legend: Normoalbuminuria: 20 (40%) to 27 (54%), p < 001; Microalbuminuria: 19 (38%) to 16 (32%), p = 0,014; Macroalbuminuria: 11 (22%) to 7 (14), p < 0,001. Data are presented in absolute number (%). Comparison between groups was performed using chi-square test. 20 (40) 19 (38) 11 (22) 27 (54) 16 (32) 7 (14) 0 5 10 15 20 25 30 Normoalbuminuria Microalbuminuria Macroalbuminuria Baseline Day 3 81 References 1. Gerstein HC, Mann JF, Yi Q, et al. Albuminuria and risk of cardiovascular events, death, and heart failure in diabetic and nondiabetic individuals. JAMA. Vol 286. United States2001:421-426. 2. Mansia G, De Backer G, Dominiczak A, et al. 2007 ESH-ESC Guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). Blood Press. Vol 16. Norway2007:135232. 3. Standards of medical care in diabetes--2013. Diabetes Care. Vol 36 Suppl 1. 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High-Dose Spironolactone Changes Renin and Aldosterone Levels in Acutely Decompensated Heart Failure 86 João Pedro Ferreira, MD1, Mário Santos, MD1, Sofia Almeida, PhD2, Irene Marques, MD1, Paulo Bettencourt, MD, PhD3, Henrique Carvalho, MD, PhD1 1 Centro Hospitalar do Porto, 2 Climate Change Impacts, Adaptation and Mitigation Research Group (CC-IAM), Faculdade de Ciências, Universidade de Lisboa, 3 Centro Hospitalar de São João Correspondence: João Pedro Ferreira, Centro Hospitalar do Porto, Internal Medicine Department, Largo Prof. Abel Salazar 4099-001 Porto, Portugal. Contacts: Telephone – 00351222077500; Fax – 00351222053218; E-mail – [email protected] Abstract Background: In acutely decompensated heart failure (ADHF) patients higher aldosterone levels correlate with worse post-discharge outcomes, suggesting that further modulation of the mineralocorticoid system during or immediately after hospitalization might favourably improve outcomes. Methods and Results: This was an observational, retrospective secondary analysis of a study including 100 patients with ADHF. In that study 50 patients were submitted to spironolactone treatment (50 – 100 mg/day). A higher proportion of patients with renin levels above 16,5 pg/mL and aldosterone levels above 100 ng/dL was observed in subjects submitted to spironolactone treatment (44,7% vs. 66,7% and 56% vs. 64,7%, respectively, both p < 0,05). In the group of patients submitted to spironolactone treatment the proportion of patients with renin and aldosterone levels above the cutoff had a significant increase from baseline to day 3 (24% to 32% and 16% to 44%, respectively, both p < 0,05). Log renin and aldosterone were higher in patients with renin and aldosterone levels above the cutoff point (both p < 0,05). Conclusions: High-dose spironolactone added to standard ADHF therapy induces an additional increase in renin and aldosterone levels. Whether higher levels of renin and aldosterone due to the reactive response to full MRA still have prognostic value requires further investigation. Key-words: mineralocorticoid receptor antagonism; renin; aldosterone; acute heart failure. 87 Introduction The use of mineralocorticoid receptor antagonists (MRAs) has demonstrated to improve outcomes and reduce mortality in chronic heart failure (HF) and postmyocardial infarction1-3. The benefit observed with MRAs is probably due to excessive neurohormonal activation blockade. Particularly, aldosterone is probably essential for the progression of HF. Higher aldosterone levels were found in patients with chronic HF when compared with controls, and were found to be associated with poor outcome4-7. A rise in aldosterone levels was also observed in the acute myocardial infarction setting8,9, and likewise associated with worse outcomes in this setting10. In acutely decompensated heart failure (ADHF) patients with ejection fraction (EF) < 40%, higher aldosterone levels correlate with worse post-discharge outcomes11, suggesting that further modulation of the mineralocorticoid system during or immediately after hospitalization might favourably improve outcomes. Regarding this matter, high dose spironolactone as add-on therapy in the acutely decompensated heart failure (ADHF) setting has demonstrated to be safe and likely to provide greater symptomatic relief translated into a more pronounced decrease in natriuretic peptides12. We used an ADHF model to study the influence of the MRA spironolactone on renin and aldosterone. The aim of this study is to demonstrate the renin and aldosterone associations and changes before and after spironolactone introduction. Methods Study Design This study is based on analysed data from a previous prospective, interventional, clinical trial that we performed12. In that study we enrolled 100 consecutive patients who presented in a Portuguese tertiary hospital with ADHF, between February 2012 and February 2013. They were non-randomly assigned in a sequential 1:1 ratio to spironolactone plus standard ADHF therapy or standard ADHF therapy alone, 50 patients within each arm (i.e. patients were alternatively assigned to spironolactone arm or standard ADHF therapy arm in a sequential manner - the first patient to one arm and the next to the other arm. This sequence was repeated until we reach 100 patients, 50 patients within spironolactone group and 50 patients within control group). Patients were blinded to the allocation, and the clinicians were not blinded to the allocation. The recommended spironolactone dose was 100 mg/day, however the assistant physician could decrease the spironolactone dose to 50 mg/day 88 after 48h upon admission. After 72h the study was open label. Furosemide dose and form of administration was performed according to the treating physician. Patients were eligible for enrollment if they presented with decompensation of chronic HF with symptoms leading to hospitalization. All patients presented at the emergency department severely symptomatic in NYHA class IV. ADHF was diagnosed on the basis of the presence of history of chronic HF, at least one symptom (dyspnea, orthopnea, or edema), one sign (rales, peripheral edema, ascites, or pulmonary vascular congestion on chest radiography) and elevated natriuretic peptides. Exclusion criteria were: chronic use of MRAs, cardiac surgery within 60 days of enrollment, cardiac mechanical support, cardiac resynchronization-therapy within the last 60 days, comorbid conditions with an expected survival of less than 6 months, acute MI at time of hospitalization, hemodynamically significant uncorrected primary cardiac valvular disease, patients requiring intravenous vasodilators or inotropic agents, supine systolic arterial blood pressure <90 mmHg, plasma creatinine (pCr) level >1,5 mg/dL, serum potassium level >5,0 mmol/L, hemoglobin (HgB) level <9 g/dL, and sepsis. Institutional review board or ethics committee approval was obtained. All patients provided written informed consent to participate in the study. Study assessments Patient`s clinical status including physical examination and was prospectively recorded by the same assistant physician at day 1 and day 3. Medications and respective dosages were prospectively recorded by the investigators according to the assistant physician prescriptions. Blood and spot urine samples were collected in the first 24 hours (h) after admission (day 1) of the patient to the hospital. The first dose of spironolactone was only administered after the first sample was collected. Fifty patients had daily oral spironolactone according to the study protocol described above. The day 3 samples were collected between 72 and 96 h of hospitalization. All samples were collected in the morning with the patient in supine position, and first-morning spot urine was used. All patients had low-salt, low-calorie hospital diet. Extra fruit and vegetables administration was not allowed. An assessment of biomarkers (including pCr, plasma urea [pUr], electrolytes, N-terminal pro-brain natriuretic peptide [NTproBNP], highsensitivity troponin T [hsTnT] and proteinuria) was performed at a central core laboratory at day 1 and day 3. Clinical assessment and routine analyses were performed daily during hospital stay. All patients performed a transthoracic echocardiography within 72 hours upon admission. Left ventricle ejection fraction (LVEF) was calculated according to biplane Simpson method. 89 Aldosterone was measured using radioimmunoassay (RIA) Coat-a-Count® (Siemens) and renin with RIA (DiaSource®). Variable definitions We defined high renin levels when values were above the 16,5 pg/mL cutoff, and high aldosterone levels when values were above the 100 ng/dL cutoff. The manufacturer suggested a cutoff of 16,5 pg/mL for renin and a cutoff of 160 ng/dL for aldosterone. We lowered aldosterone cutoff to 100 ng/dL to increase test sensitivity, although levels above 160 ng/dL are more specific, we might miss important information, since p.e. in the EVEREST trial only 33,2% of patients had aldosterone levels above 160 ng/dL11, and increased mortality was also observed in lower aldosterone quartiles. We studied aldosterone (ng/dL) and renin (pg/mL) regarding the following covariates: age; sex; diabetes mellitus (DM); ischemic HF; EF (%); atrial fibrillation (AF); systolic blood pressure (SBP); intravenous (IV) furosemide dose; proportion of patients with IV furosemide at day 3; proportion of patients on angiotensin converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARB), beta-blockers (BB), and spironolactone; pCr (mg/dL), pUr (mg/dL), NTproBNP (pg/mL), hsTnT (ng/mL), sodium (mmol/L), potassium (mmol/L), uNa/K ratio, proteinuria (g/g), red-cell distribution width (RDW), HgB (g/dL), and length of stay. Statistical Analysis Normally distributed continuous variables are expressed as mean ± standard deviation (SD), and skewed distributions are presented as median [inter-quartile range, IQR]. Because of the positively skewed distributions of aldosterone, renin, pCr, proteinuria, RDW, NTproBNP, hsTnT and uNa/K ratio, these variables were log transformed for analysis. Categorical variables are expressed in proportions (%). Comparison between groups was performed using parametric, non-parametric tests, or chi-square tests, as appropriate. Significant association was defined by a probability (p) value ≤0,05. Statistical analysis was performed using SPSS software (version 19, Chicago, IL, USA). 96 Table 2. Comparison Between Normal and High Renin and Aldosterone Values at Admission. Renin < 16,5 (n=80) Renin ≥ 16,5 (n=20) p Value Aldosterone < 100 (n=77) Aldosterone ≥ 100 (n=23) p Value Age 75,99 ± 10,33 76,05 ± 13,13 0,982 74,91 ± 10,70 76,65 ± 10,88 0,066 Male Sex – no. (%) 30 (37,5) 9 (45) 0,539 35 (45,5) 4 (17,4) 0,015 DM – no. (%) 38 (47,5) 7 (35) 0,315 34 (44,2) 11 (47,8) 0,756 EF <40% – no. (%) 26 (32,5) 5 (25) 0,517 25 (32,5) 6 (26,1) 0,562 Ischemic HF – no. (%) 37 (46,3) 13 (65) 0,134 37 (48,1) 13 (58,5) 0,476 AF – no. (%) 42 (52,5) 17 (85) 0,008 44 (57,1) 15 (65,2) 0,490 SBP 140,93 ± 26,08 135,25 ± 25,11 0,383 140,84 ± 26,72 136,26 ± 22,94 0,459 Beta Blocker – no. (%) 31 (38,8) 5 (25) 0,252 27 (35,1) 9 (39,1) 0,722 ACEi/ARB – no. (%) 34 (42,5) 8 (40) 0,839 32 (41,6) 10 (43,5) 0,870 Sodium 140,75 ± 4,41 139,70 ± 4,26 0,340 140,84 ± 4,54 139,52 ± 3,69 0,205 Potassium 4,05 ± 0,50 3,98 ± 0,55 0,586 3,97 ± 0,47 4,24 ± 0,57 0,023 Log Creatinine 0,02 ± 0,11 0,02 ± 0,14 0,888 0,02 ± 0,12 0,04 ± 0,11 0,495 Log Proteinuria -0,44 ± 0,40 -0,45 ± 0,47 0.962 -0,51 ± 0,39 -0,23 ± 0,42 0,008 Log RDW 1,18 ± 0,03 1,21 ± 0,06 0,004 1,18 ± 0,04 1,20 ± 0,04 0,047 Log NTproBNP 3,49 ± 0,45 3,41 ± 0,35 0,444 3,42 ± 0,41 3,67 ± 0,47 0,014 Log UNa/K ratio 0,50 ± 0,38 0,22 ± 0,31 0,003 0,49 ± 0,37 0,29 ± 0,37 0,026 Log Aldosterone 1,53 ± 0,43 1,70 ± 0,49 0,135 1,39 ± 0,34 2,15 ± 0,12 <0,001 Log Renin 0,52 ± 0,33 1,46 ± 0,22 <0,001 0,69 ± 0,48 0,78 ± 0,50 0,402 Log hsTnT -1,47 ± 0,39 -1,47 ± 0,33 0,995 -1,47 ± 0,39 -1,47 ± 0,34 0,988 Urea 53,78 ± 19,68 60,95 ± 24,68 0,170 52,44 ± 20,52 64,48 ± 19,60 0,014 Hemoglobin 12,50 ± 1,98 12,16 ± 2,44 0,512 12,39 ± 2,06 12,59 ± 2,17 0,689 Log Length of Stay 0,89 ± 0,17 1,01 ± 0,15 0,003 0,91 ± 0,16 0,91 ± 0,19 0,958 Legend: DM = diabetes mellitus; EF = left ventricular ejection fraction; HF = heart failure; AF = atrial fibrillation; SBP, systolic blood pressure; ACEi/ARB = angiotensin converting enzyme inhibitors/angiotensin receptor blockers; RDW = red cell distribution width; NTproBNP = N-terminal pro brain natriuretic peptide; UNa/K = urinary sodium to potassium; hsTnT = high sensitivity troponin T. 97 Table 3. Comparison Between Normal and High Renin and Aldosterone Values at Day 3. Renin < 16,5 (n=76) Renin ≥ 16,5 (n=24) p Value Aldosterone < 100 (n=66) Aldosterone ≥ 100 (n=34) p Value Age 76,58 ± 9,61 74,17 ± 14,25 0,346 78,02 ± 9,28 72,09 ± 12,70 0,009 Male Sex – no. (%) 28 (36,8) 11 (45,8) 0,431 25 (37,9) 14 (41,2) 0,749 DM – no. (%) 37 (48,7) 8 (33,3) 0,188 29 (43,9) 16 (47,1) 0,766 EF <40% – no. (%) 22 (28,9) 9 (37,5) 0,430 18 (27,3) 13 (38,2) 0,262 Ischemic HF – no. (%) 36 (47,4) 14 (58,3) 0,241 30 (45,5) 20 (68,8) 0,205 AF – no. (%) 43 (56,6) 16 (66,7) 0,264 42 (63,6) 17 (50) 0,189 SBP 123,55 ± 16,73 116,96 ± 13,52 0,082 122,55 ± 15,76 120,85 ± 17,20 0,623 Beta Blocker – no. (%) 46 (60,5) 11 (45,8) 0,205 38 (57,6) 19 (55,9) 0,871 ACEi/ARB – no. (%) 48 (63,2) 13 (54,2) 0,431 41 (62,1) 20 (58,8) 0,749 IV Furosemide – no. (%) 27 (35,5) 10 (41,7) 0,587 25 (37,9) 12 (35,4) 0,800 Spironolactone – no. (%) 34 (44,7) 16 (66,7) 0,050 28 (56) 22 (64,7) 0,035 Spironolactone Dose 63,89 ± 24,96 60,29 ± 23,48 0,620 60,00 ± 23,31 66,30 ± 25,68 0,355 Sodium 141,11 ± 3,85 139,33 ± 4,05 0,055 141,12 ± 4,33 139,82 ± 2,96 0,121 Potassium 4,01 ± 0,56 4,14 ± 0,49 0,299 4,00 ± 0,50 4,10 ± 0,61 0,402 Log Creatinine 0,03 ± 0,14 0,05 ± 0,16 0,675 0,02 ± 0,14 0,07 ± 0,14 0,058 Log Proteinuria -0,46 ± 0,38 -0,59 ± 0,31 0.132 -0,49 ± 0,35 -0,49 ± 0,41 0,955 Log RDW 1,18 ± 0,03 1,21 ± 0,06 0,007 1,19 ± 0,04 1,18 ± 0,04 0,658 Log NTproBNP 3,27 ± 0,50 3,29 ± 0,51 0,881 3,28 ± 0,49 3,24 ± 0,52 0,679 Log UNa/K ratio 0,42 ± 0,40 0,34 ± 0,37 0,378 0,44 ± 0,38 0,31 ± 0,40 0,120 Log Aldosterone 1,64 ± 0,45 1,94 ± 0,51 0,007 1,47 ± 0,38 2,21 ± 0,19 <0,001 Log Renin 0,59 ± 0,34 1,52 ± 0,25 <0,001 0,69 ± 0,45 1,07 ± 0,54 <0,001 Log hsTnT -1,51 ± 0,33 -1,52 ± 0,42 0,838 -1,51 ± 0,33 -1,51 ± 0,39 0,942 Urea 60,86 ± 25,20 66,92 ± 26,32 0,312 60,70 ± 26,83 65,44 ± 22,64 0,380 Hemoglobin 12,52 ± 2,11 12,16 ± 1,96 0,465 12,38 ± 2,11 12,53 ± 2,03 0,728 Log Length of Stay 0,89 ± 0,17 0,99 ± 0,13 0,008 0,89 ± 0,18 0,96 ± 0,13 0,043 Legend: DM = diabetes mellitus; EF = left ventricular ejection fraction; HF = heart failure; AF = atrial fibrillation; SBP, systolic blood pressure; ACEi/ARB = angiotensin converting enzyme inhibitors/angiotensin receptor blockers; RDW = red cell distribution width; NTproBNP = N-terminal pro brain natriuretic peptide; UNa/K = urinary sodium to potassium; hsTnT = high sensitivity troponin T. 98 Table 4. Comparison of Renin and Aldosterone Levels at Admission and Day 3 within Spironolactone and Control Groups Day 1 Day 3 Spironolactone Control p Value Spironolactone Control p Value Renin > 16,5 – no. (%) 12 (24) 8 (16) 0,317 16 (32) 8 (16) 0,050 Aldosterone > 100 – no. (%) 8 (16) 15 (30) 0,096 22 (44) 12 (24) 0,035 Note: Day 1 analysis were performed before spironolactone administration 99 Figures Figure 1. Comparison of Urinary Sodium to Potassium Ratio According to Renin and Aldosterone Levels at Admission. Legend: Renin = Log Renin; Aldosterone = Log Aldosterone; U Na/K = urinary sodium to potassium. Patients with higher renin and aldosterone levels at admission had lower urinary sodium to potassium ratios. <16,5 pg/mL ≥16,5 pg/mL <100 ng/dL ≥100 ng/dL 100 Figure 2. Comparison of Renin and Aldosterone Levels Before and After Spironolactone Treatment Legend: Results are presented in percentage (%) of total. Renin is expressed in pg/mL and Aldosterone in ng/dL. 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High Sensitivity Troponin T: A Biomarker for Diuretic Response in Decompensated Heart Failure Patients? 105 João Pedro Ferreira1, Mário Santos1, Sofia Almeida2, Irene Marques1, Paulo Bettencourt3, Henrique Carvalho1 1 Centro Hospitalar do Porto, Porto, Portugal 2 Climate Change Impacts, Adaptation and Mitigation Research Group (CC-IAM), Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal 3 Centro Hospitalar de São João, Porto, Portugal Correspondence: João Pedro Melo Maques Pinho Ferreira, Centro Hospitalar do Porto, Internal Medicine Department, Largo Prof. Abel Salazar 4099-001 Porto, Portugal. Contacts: Telephone – 00351222077500; Fax – 00351222053218; E-mail – [email protected] Abstract Background: Patients presenting with acutely decompensated heart failure (ADHF) and positive circulating cardiac troponins were found to be a high-risk cohort. The advent of high-sensitive troponins resulted in a detection of positive troponins in a great proportion of heart failure patients. However, the pathophysiological significance of this phenomenon is not completely clear. Objectives The aim of this study is to determine the early evolution and clinical significance of high-sensitivity troponin T (hsTnT) in ADHF. Methods: Retrospective, secondary analysis of a prospective study including 100 patients with ADHF. Results: Globally, high-sensitivity troponin T decreased from day 1 to day 3 (p = 0,039). However, in the subgroup of patients who remained decompensated no significant differences in hsTnT from day 1 to day 3 were observed (p = 0,955), whereas in successfully compensated patients a significant reduction in hsTnT levels was observed (p = 0,025). High sensitivity troponin T decrease was correlated with NTproBNP reduction (p = 0,007). Patients with hsTnT increase had longer length of stay (p = 0,033). Conclusions: Episodes of ADHF are associated with transient increases in the blood levels of hsTnT that are reduced with effective acute episode treatment. The decrease in hsTnT can translate less myocardial damage along with favourable ADHF treatment. Key-words: Acute Heart Failure. High-Sensitivity Troponin T. Biomarkers. N-Terminal Pro-Brain Natriuretic Peptide 112 based on subjective clinical evaluation. Our study protocol defined that the first blood sample would be collected in the first 24 h, so at the time of venous blood sampling patients could had been treated already with diuretics. Although we are not comparing diuretic-naïve patients at day 1 measurements, the overall effect of this bias would be an underestimated difference between day 1 and day 3, which does not significantly affect the internal validity of our study conclusions. Finally, the external validity of our conclusions is limited to normo-hypertensive and fluid overloaded HF patients with normal or mildly impaired renal function, since all these factors were considered inclusion criteria. On the other hand, our conclusions can be reproducible in this set of patients widely common in clinical practice. Conclusions Episodes of ADHF are associated with transient increases in the blood levels of hsTnT that are reduced with effective acute episode treatment. The decrease in hsTnT and NTproBNP can translate ventricular wall stress relief and less myocardial damage along with favourable ADHF treatment. Further studies are needed to examine the value of combining necrosis markers and natriuretic peptides in the clinical management of ADHF patients. Disclosures The authors have no conflicts of interest to disclose. Acknowledgements The authors acknowledge the lab technicians, especially Mr. Fernando Santos for technical assistance and to all physicians collaborating in the study. 113 Tables Table 1. Population Characteristics, and Comparison of Clinical Variables, Laboratory Results and Medications Between Admission Day (Day 1) and Day 3 Age (yrs) 76,0 ± 10,88 Male Sex – % 39 Diabetes Mellitus - % Glycated HgB (%) 45 7,02 ± 0,96 COPD - % 17 Dementia - % 12 Sleep Apnea - % 18 Non-Invasive Ventilation - % 17 Ischemic Heart Disease -% 50 Atrial Fibrillation - % 59 LV Ejection Fraction (%) 43,46 ± 11,73 LV Ejection Fraction ≥ 40% - % 68 Day 1 Day 3 p Value Body Mass Index (Kg/m2) 29,44 ± 6,17 28,35 ± 6,23 < 0,001 Heart Rate (bpm) 93,65 ± 24,35 76,41 ± 11,96 < 0,001 SBP (mmHg) 139,79 ± 25,86 121,97 ± 16,2 < 0,001 Plasma Creatinine (mg/dL) 1,04 [0,89 – 1,31] 1,06 [0,85 – 1,40] 0,082* eGFR (mL/min/1,73 m2) 58,0 [44,0 – 72,0] 58,0 [39,25 – 72,75] 0,171* Plasma Urea (mg/dL) 55,21 ± 20,84 62,3 ± 25,47 0,001 Serum Potassium (mmol/L) 4,03 ± 0,51 4,04 ± 0,54 0,95 Serum Sodium (mmol/L) 140,54 ± 4,38 140,68 ± 3,95 0,72 Hemoglobin (g/dL) 12,43 ± 2,07 - - Albumin (mg/dL) 3,68 ± 0,40 - - NTproBNP (pg/mL) 2750 [1672 – 6032] 1835 [902 – 3837] < 0,001* hsTnT (ng/mL) 0,033 [0,019 – 0,050] 0,030 [0,018 – 0,051] 0,039* IV Furosemide - % 100 37 < 0,001** IV Furosemide Dose (mg/d) 75,80 ± 21,52 67,57 ± 25,54 0,001 Oral Furosemide - % 0 63 - Oral Furosemide Dose (mg/d) 0 74,6 ± 28,1 - Furosemide Dose Reduction or Oral Route - % - 84 - ACEi - % 44 61 < 0,001** Ramipril Eq. Dose (mg/d) 3,15 ± 2,04 3,36 ± 2,14 0,474 Beta-Blocker - % 37 57 < 0,001** Bisoprolol Eq. Dose (mg/d) 3,01 ± 1,08 2,96 ± 1,89 0,474 Spironolactone - % 50 50 1** Spironolactone Dose (mg/d) 94,50 ± 23,31 62,74 ± 24,33 < 0,001 Continuous variables are presented as mean value ± standard deviation [SD], p value or median [inter-quartile range, IQR], p value. Categorical variables are presented as % of total (100 patients), p value. *Non-parametric paired sample test; ** Chi-square test. Legend: COPD = chronic obstructive pulmonary disease; LV = left ventricular; eGFR = estimated glomerular filtration rate; NTproBNP = N-terminal pro brain natriuretic peptide; hsTnT = high sensitivity troponin T; IV = intra-venous; ACEi = angiotensin converting enzyme inhibitors. 114 Table 2. Comparison of TnT Levels Between Patients Who Responded to Diuretic Therapy versus Patients Who Needed to Increase Diuretic Dose Furosemide Maintenance or Increase (n=16) Furosemide Decrease or Oral Administration (n=84) p Value Between Groups hsTnT (ng/mL) Day 1 Day 3 Δ hsTnT 0,046 [0,033 to 0,087] 0,055 [0,032 to 0,072] -0,0005 [-0,043 to 0,004] 0,032 [0,017 to 0,048] 0,028 [0,017 to 0,045] -0,0010 [-0,020 to 0,002] 0,026* 0,004* 0,51* p Value Within Group p Value Within Group 0,955* 0,025* Continuous variables are presented as median [inter-quartile range, IQR], p value. *Non-parametric test. Legend: hs TnT = high-sensitivity troponin T 115 Table 3. Comparison of hsTnT Values Below (Negative) and Above (Positive) the 99th Percentile (≥ 0,014 ng/mL) Day 1 Negative hsTnT – no. (%) Positive hsTnT – no. (%) Total p Value Day 3 Negative hsTnT – no. (%) 10 (76,9) 3 (3,4) 13 (13) < 0,001** Positive hsTnT – no. (%) 3 (23,1) 84 (96,6) 87 (87) < 0,001** Total 13 (13) 87 (87) 100 ** Chi-square test. Legend: hsTnT = high sensitivity troponin T. 116 Table 4. Associations With Log hsTnT at Day 1, Day 3, and Changes Between Day 1 and Day 3 (Δ) Nonadjust ed Coefficient for Log hsTnT 95%CI p Value Adjusted Coefficient for Log hsTnT 95%CI p Value Age 0,119 -0,001 to 0,005 0,240 Male Sex -0,066 -0,086 to 0,043 0,515 DM 0,095 -0,033 to 0,093 0,349 HgBA1c 0,058 -0,046 to 0,067 0,707 LVEF 0,089 -0,001 to 0,004 0,376 Ischemic HF -0,078 -0,087 to 0,038 0,442 Beta Blocker -0,004 -0,065 to 0,062 0,969 ACEi 0,023 -0,057 to 0,072 0,820 Spironolactone -0,116 -0,099 to 0,026 0,251 BMI Day 1 -0,205 -0,025 to 0,000 0,042 Day 3 -0,087 -0,016 to 0,006 0,391 Δ BMI -0,008 -0,019 to 0,018 0,937 HR Day 1 0,063 -0,002 to 0,004 0,533 Day 3 -0,078 -0,008 to 0,004 0,438 Δ HR 0,013 -0,001 to 0,001 0,899 SBP Day 1 0,102 -0,001 to 0,004 0,314 Day 3 0,098 -0,002 to 0,006 0,333 Δ SBP 0,207 0,000 to 0,003 0,039 Log NTproBNP Day 1 0,481 0,267 to 0,574 <0,001 0,400 0,185 to 0,513 <0,001 Day 3 0,486 0,218 to 0,464 <0,001 0,381 0,146 to 0,389 <0,001 Δ Log NTproBNP 0,267 0,044 to 0,276 0,007 - - - Log Albuminuria 117 Day 1 0,131 -0,035 to 0,172 0,193 Day 3 0,220 0,012 to 0,203 0,028 0,088 -0,041 to 0,128 0,311 Δ Log Albuminuria 0,099 -0,038 to 0,113 0,325 Log eGFR Day 1 -0,275 -1,231 to - 0,216 0,006 0,165 -0,503 to 1,372 0,360 Day 3 -0,399 -1,232 to - 0,455 <0,001 0,034 -0,812 to 0,957 0,870 Δ Log eGFR 0,068 0,203 to 0,413 0,502 Log pCr Day 1 0,345 0,500 to 1,704 <0,001 0,270 -0,224 to 1,951 0,118 Day 3 0,439 0,630 to 1,503 <0,001 0,256 -0,393 to 1,641 0,226 Δ Log pCr -0,040 -0,443 to 0,297 0,696 pUrea Day 1 0,309 0,002 to 0,009 0,002 0,116 -0,002 to 0,007 0,342 Day 3 0,382 0,003 to 0,008 <0,001 0,121 -0,002 to 0,005 0,335 Δ pUrea -0,172 -0,003 to 0,000 0,087 Albumin at Day 1 -0,049 -0,099 to 0,060 0,626 Hemoglobin at Day 1 0,076 -0,009 to 0,021 0,451 Day 1 values are compared with day 1 hsTnT; day 3 values are compared with day 3 hsTnT; Δ, age, sex, DM, HgBA1c, LVEF, ischemic HF, and medications are compared with changes (Δ) in hsTnT between day 1 and day 3 (day 3 – day 1). Legend: DM = diabetes mellitus; HgBA1c = glycated hemoglobin; LVEF = left ventricular ejection fraction; HF = heart failure; ACEi = angiotensin converting enzyme inhibitors; BMI = body mass index; HR = heart rate; SBP = systolic blood pressure; NTproBNP = N-terminal pro brain natriuretic peptide; hsTnT = high sensitivity troponin T; eGFR = estimated glomerular filtration rate; pCr = plasma creatinine; pUrea = plasma urea; Δ = changes between day 3 and day 1 (day 3 – day 1). 118 Table 5. Determinants of hsTnT Dichotomic Changes hsTnT Decrease (n = 63) Increase ( = 37) p Value Age (years) 75,94 ± 11,92 76,11 ± 8,97 0,940 Male Sex – no. (%) 22 (34,9) 17 (45,9) 0,275** DM – no. (%) 24 (38,1) 21 (56,8) 0,070** HGA1c (%) 6,93 ± 0,94 7,13 ± 1,00 0,475 Sleep Apnea – no. (%) 7 (36,8) 11 (44) 0,632** NIV – no. (%) 10 (15,9) 7 (18,9) 0,695** IHD – no. (%) 32 (50,8) 18 (48,6) 0,836** AF – no. (%) 31 (49,2) 28 (75,7) 0,009** LVEF (%) 43,37 ± 12,68 43,62 ± 10,08 0,917 LVEF ≥ 40% - no. (%) 41 (65,1) 26 (70,3) 0,594** HgB (g/dL) 12,32 ± 1,95 12,62 ± 2,28 0,478 Albumin (mg/dL) 3,68 ± 0,41 3,67 ± 0,39 0,924 Δ BMI (Kg/m2) -1,08 ± 1,70 -1,10 ± 1,76 0,964 Δ HR (bpm) -17,05 ± 20,71 -17,57 ± 29,15 0,917 Δ SBP (mmHg) -18,56 ± 23,32 -16,57 ± 27,63 0,702 Δ pCr (mg/dL) 0,03 [-0,1 to 0,18] 0,02 [-0,06 to 0,11] 0,803* Δ eGFR (ml/min/1,73m2) -2,0 [-9,0 to 7,0] -1,0 [-11,0 to 6,0] 0,937* Δ pUrea (mg/dL) 7,40 ± 20,59 6,59 ± 20,65 0,851 Δ NTproBNP (pg/mL) -1167 [-2337 to - 367] -379 [-1273 to 319,5] 0,003* Δ hsTnT (ng/mL) -0,004 [-0,014 to - 0,001] 0,004 [0,002 to 0,009] <0,001* Δ Albuminuria (mg/g) -6,10 [-38,50 to 2,40] -23,70 [-90,75 to 11,05] 0,337* IV Furosemide at Day 1 (mg) 78,83 ± 21,61 74,05 ± 21,53 0,537 IV Furosemide Dose Maintenance or Increase at Day 3 – no. (%) 9 (14,3) 7 (18,9) 0,542** ACEi – no. (%) 30 (47,6) 14 (37,8) 0,341** Beta Blocker – no (%) 22 (34,9) 15 (40,5) 0,574** Spironolactone – no. (%) 30 (47,6) 20 (54,1) 0,534** Length of Stay (days) 8,0 [6,0 to 11,0] 9,0 [7,0 to 12,0] 0,033* Continuous variables are presented as mean value ± standard deviation [SD], p value or median [inter-quartile range, IQR], p value. Categorical variables are presented as absolute number (%), p value. *Non-parametric paired sample test; ** Chi-square test. DM = diabetes mellitus; HgBA1c = glycated hemoglobin; NIV = non-invasive ventilation; IHD = ischemic heart disease; AF = atrial fibrillation; HgB = hemoglobin; BMI = body mass index; HR = heart rate; SBP = systolic blood pressure; eGFR = estimated glomerular filtration rate; pCr = plasma creatinine; pUrea = plasma urea; 119 NTproBNP = N-terminal pro brain natriuretic peptide; hsTnT = high sensitivity troponin T; IV = intra-venous; ACEi = angiotensin converting enzyme inhibitors; Δ = changes between day 3 and day 1 (day 3 – day 1). 120 Figures Figure 1. Differences in hsTnT Between Faster Diuretic Responders and Slower Diuretic Responders at Day 1 and Day 3 Legend: hsTnT = high sensitivity troponin T (ng/mL) 121 Figure 2. Correlation Between Δ Log hsTnT and Δ Log NT-pro BNP Legend: hsTnT = high sensitivity troponin T (ng/mL); NT-pro BNP = N-terminal probrain natriuretic peptide (pg/mL); Δ = changes between day 3 and day 1 (day 3 – day 1). r = 0,267; p = 0,007 Δ Log hsTnT Δ Log NTproBNP 128 assigned to spironolactone arm or standard ADCHF therapy arm in a sequential manner - the first patient to one arm and the next to the other arm. This sequence was repeated until we reach 100 patients, 50 patients within spironolactone group and 50 patients within control group). Patients were blinded to the allocation, and the clinicians were not blinded to the allocation. The recommended spironolactone dose was 100 mg/day, however the assistant physician could decrease the spironolactone dose to 50 mg/day after 48h upon admission. After 72h the study was open label. Furosemide dose and form of administration was liberal. Patients were eligible for enrollment if they presented with decompensation of chronic HF with symptoms leading to hospitalization. ADHF was diagnosed on the basis of the presence of history of chronic HF and at least one symptom (dyspnea, orthopnea, or edema) and one sign (rales, peripheral edema, ascites, or pulmonary vascular congestion on chest radiography). Exclusion criteria were: chronic use of MRAs, cardiac surgery within 60 days of enrollment, cardiac mechanical support, cardiac resynchronization-therapy within the last 60 days, comorbid conditions with an expected survival of less than 6 months, acute MI at time of hospitalization, hemodynamically significant uncorrected primary cardiac valvular disease, patients requiring intravenous vasodilators or inotropic agents, supine systolic arterial blood pressure <90 mmHg, plasma creatinine (pCr) level >1,5 mg/dL, serum potassium level >5,0 mmol/L, hemoglobin (HgB) level <9 g/dL, and sepsis. Institutional review board or ethics committee approval was obtained. All patients provided written informed consent to participate in the study. Study assessments Patient`s clinical status including physical examination and was prospectively recorded by the same assistant physician at day 1 and day 3. Medications and respective dosages were prospectively recorded by the investigators according to the assistant physician prescriptions. Blood and spot urine samples were collected in the first 24 hours (h) after admission (day 1) of the patient to the hospital. The first dose of spironolactone was only administered after samples collection. Fifty patients had daily oral spironolactone according to the study protocol described above. The day 3 samples were collected between 72 and 96 h of hospitalization. All samples were collected in the morning with the patient in supine position, and first-morning spot urine was used. All patients had low-salt, low-calorie hospital diet. Extra fruit and vegetables administration was not allowed. An assessment of biomarkers (including pCr, plasma urea [pUr], electrolytes, N-terminal pro-brain natriuretic peptide [NTproBNP], high-sensitivity troponin T [hsTnT] 129 and proteinuria) was performed at a central core laboratory at day 1 and day 3. Clinical assessment and routine analyses were performed daily during hospital stay. All patients performed a transthoracic echocardiography within 72 hours upon admission. Left ventricle ejection fraction (EF) was calculated according to biplane Simpson method. Aldosterone was measured using radioimmunoassay (RIA) Coat-a-Count® (Siemens) and renin with RIA (DiaSource). Variable definitions We studied aldosterone (ng/dL) and renin (pg/mL) and their variation (delta) regarding the following covariates: age; sex; diabetes mellitus (DM); ischemic HF; EF (%); systolic blood pressure (SBP); intravenous (IV) furosemide dose; proportion of patients with furosemide dose reduction or switch to oral route from baseline to day 3; proportion of patients on angiotensin converting enzyme inhibitors (ACEi), angiotensin receptor blockers (ARB), beta-blockers (BB), and spironolactone; pCr (mg/dL), pUr (mg/dL), NTproBNP (pg/mL), hsTnT (ng/mL), sodium (mmol/L), potassium (mmol/L), uNa/K ratio, proteinuria (g/g) and HgB (g/dL). Statistical Analysis Normally distributed continuous variables are expressed as mean ± standard deviation (SD), and skewed distributions are presented as median [inter-quartile range, IQR]. Because of the positively skewed distributions of aldosterone, renin, pCr, proteinuria, RDW, NTproBNP, hsTnT and uNa/K ratio, these variables were log transformed for analysis. Categorical variables are expressed in proportions (%). Comparison between groups was performed using parametric, non-parametric tests, or chi-square tests, as appropriate. Significant association was defined by a probability (p) value ≤0,05. Correlations of log aldosterone and log renin with the mentioned variables were first examined by single variable linear or logistic regression and presented as nonadjusted coefficient (NAC) and 95% confidence interval [95%CI]. Factors with a p value ≤0,05 by single variable regression analyses were included in a multivariable linear regression model, presented as adjusted coefficient (AC) [95%CI]. Statistical analysis was performed using SPSS software (version 19, Chicago, IL, USA). 130 Results Baseline Characteristics, Medications and Lab Results Mean ± SD age of the 100 patients admitted due to ADHF was 76,0 ± 10,9 years. Thirty-nine (39%) patients were male; 50 patients had documented ischemic heart disease (IHD); 32 had EF < 40%; spironolactone was administered to 50 patients starting on the admission day. Patient characteristics, and comparison of systolic blood pressure (SBP), lab results and medication dosage between admission day (day 1) and the third day of inpatient treatment is shown in Table 1. Plasma urea, proteinuria, NTproBNP and hsTnT reduced between day 1 and day 3 (all, p < 0,05). Furosemide dose was reduced from day 1 to day 3 (75,80 ± 21,52 vs. 67,57 ± 25,54, p < 0,001). Spironolactone dose was also reduced according to study protocol (94,50 ± 23,31 vs. 62,74 ± 24,33). The proportion of patients on ACEi/ARB, BB and oral furosemide increased from day 1 to day 3 (all, p < 0,05). Renin and aldosterone increased during the study period (these results were not compared because day 1 samples were all collected before spironolactone administration and at day 3 fifty patients were on spironolactone). No changes were found between day 1 and day 3 regarding pCr and uNa/K ratio – Table 1. Day 1 Aldosterone and Renin Correlations Day 1 log aldosterone was positively correlated with ischemic HF (3,029 [1,197 to 7,668], p = 0,019), pUr (0,231 [0,001 to 0,009], p = 0,021), and log proteinuria (0,215 [0,021 to 0,443], p = 0,031). These associations remained significant after adjustment – Table 2. Day 1 log renin was negatively correlated with log uNa/K ratio (-0,332 [-0,671 to -0,184], p=0,001) and SBP (-0,223 [-0,008 to -0,001], p = 0,026). Correlation was positive with ischemic HF (2,743 [1,150 to 6,542], p=0,023). After adjustment log renin remained significantly correlated with log uNa/K ratio and SBP – Table 2. Aldosterone, Renin, and uNa/K ratio Changes Previous to spironolactone administration (day 1) log aldosterone levels did not differ between groups, although a trend to higher log aldosterone levels was observed in control group (1,484 ± 0,436 vs. 1,651 ± 0,446, p=0,062), this tendency was inverted after 3 days of spironolactone use (1,789 ± 0,461 vs. 1,654 ± 0,500, p=0,166) with higher levels of aldosterone in spironolactone group and a significant variation in the log aldosterone levels (0,305 ± 0,484 vs. 0,004 ± 0,432, p=0,001) – Table 3. At day 1 log renin levels were not significantly different between groups, but in this case higher log renin levels were observed in spironolactone group (0,801 ± 0,491 vs. 0,625 ± 0,475, p=0,071), these differences were accentuated after spironolactone 131 administration (0,934 ± 0,547 vs. 0,704 ± 0,454, p=0,025) – Table 3. A similar scenario was found in uNa/K ratio analysis with significant differences being achieved after spironolactone administration (0,487 ± 0,394 vs. 0,312 ± 0,370, p=0,024) – Table 3. Day 3 Aldosterone and Renin Correlations Within Spironolactone and Control Groups Within control group, both day 3 log aldosterone and log renin were correlated with log uNa/K ratio (-0,450 [-0,955 to -0,257], p = 0,001 and -0,347 [-0,756 to 0,091], p = 0,014, respectively) – Table 4. and Figure 1; log aldosterone was also associated with log pUr (0,302 [0,069 to 1,588], p = 0,033) – Table 4., and log renin was correlated to log pCr (0,297 [0,062 to 1,750], p = 0,036), and negatively correlated to beta-blocker use (-0,290 [-0,512 to -0,011], p = 0,041) – Table 4. Within spironolactone group log aldosterone and log renin were correlated (0,359 [0,074 to 0,530], p = 0,011) – Table 4.; log aldosterone was also positively correlated with furosemide dose reduction (0,334 [0,142 to 1,415], p = 0,018), and log renin was negatively correlated with serum sodium (-0,337 [-0,094 to -0,010], p = 0,017) – Table 4. Associations Between Delta (Δ) Log Renin, Δ Log Aldosterone and Δ Log UNa/K ratio Changes (day 3 – day 1) in log aldosterone and log renin were correlated with the changes in log uNa/K ratio within spironolactone group (-0,288 [-0,518 to -0,009], p=0,042 and -0,512 [-0,740 to -0,256], p <0,001, respectively) – Table 5. Na/K ratio: Receiver Operating Curve Analysis Na/K ratio showed a sensitivity of 86% and a specificity of 44% (area under curve [AUC], 0,657 [0,550 to 0,765], p = 0,007) in predicting spironolactone use – Figure 2. Comparison Between Spironolactone and Control Groups Regarding Potential Confounders No differences were observed regarding SBP, serum sodium and potassium, ACEi/ARB and beta-blocker use, and i.v. furosemide dose. A greater proportion of patients were on oral furosemide at day 3 within spironolactone group (22 vs. 41, p < 0,001). Discussion The present study tested if uNa/K ratio was independently associated with plasma levels of aldosterone and renin, and if the levels of these biomarkers changed with the introduction of spironolactone in a cohort of patients with ADHF, half of them treated with spironolactone. Renin, aldosterone and uNa/K ratio levels increased in the 132 group of patients submitted to spironolactone treatment. In this group of patients, the variation in aldosterone and renin levels from baseline to day 3 were correlated with the variation in the uNa/K ratio levels. In addition, uNa/K ratio showed a fair accuracy for spironolactone use. Taken together, our results support the potential usefulness of this simple urinary measure as biomarker of MRA. Aldosterone regulates sodium balance by increasing the expression of the epithelial Na+ channel and the Na+/K+-ATPase pump found on the distal nephron epithelial cells11, promoting reabsorption of Na+ and the excretion of K+12. Previous reports had indicated a measurable effect of MRA on uNa/K ratio consistent with effects of aldosterone on electrolyte balance13,14. Mineralocorticoid receptor blockade elicited an increase in uNa/K ratio in rats in a dose-dependent manner10, supporting the use of uNa/K ratio as a translatable biomarker of MRA with the potential to enable dose selection for clinical trials10. In our study, renin was negatively associated with uNa/K ratio at day 1, and at day 3 both renin and aldosterone were independently associated with uNa/K ratio in the group of patients not submitted to spironolactone treatment. These findings are consistent with the RAAS effects on distal nephron, i.e. higher levels of renin and aldosterone lead to higher Na+ reabsorption and K+ excretion, decreasing uNa/K ratio15. In addition, we also observed an increase of uNa/K ratio in the group of spironolactone-treated patients, despite the higher increase in aldosterone and renin levels in this group of patients, suggesting a compensatory response to MRA10,16,17. These observations were supported by the significant correlation between the variation (day 3 – baseline) in aldosterone and renin levels with the variation in the uNa/K ratio levels. Together, these dynamic changes demonstrate an effective MRA by spironolactone and suggest that uNa/K ratio can serve as a surrogate biomarker for MRA. No significant differences between groups (spironolactone vs. control) were observed regarding ACEi/ARB use, IV furosemide dose, SBP, plasma potassium, serum sodium or diet, therefore it is likely that the observed dynamic changes were due to the spironolactone effect. Noteworthy, a higher proportion of patients on oral furosemide at day 3 was observed in spironolactone group, potentially translating a faster diuretic response within this group18, however the less aggressive diuretic strategy would theoretically decrease urinary sodium excretion leading to a lower uNa/K, yet this was not observed in our study, by the contrary, spironolactone treated patients had higher uNa/K ratio corroborating that the observed dynamic changes in uNa/K ratio were driven by MRA effect. Our preliminary results, are the first to show the potential usefulness of uNa/K as a surrogate marker for MRA. The implications of these observations have the 133 potential to be translated into clinical trials and also to daily practice, helping in the MRA dose titration and clinical effect monitorization. In our study uNa/K ratio has demonstrated to have a fair accuracy in determining spironolactone use, with patients on spironolactone having higher uNa/K ratio (high sensitivity), but the test has not demonstrated to be accurate in excluding patients without spironolactone treatment (low specificity). Whether the accuracy of the test can be increased with higher MRA dose or tighter potential confounder control (in a randomized and controlled trial) needs to be determined in future studies. However we find our results very encouraging towards the use of this inexpensive and readily available biomarker. We validated uNa/K ratio against aldosterone and renin plasma concentrations. Although there are some caveats in the interpretation of these tests, they are recommended and used in standard clinical practice to screen and diagnose diseases associated to increased SRAA activity. The median aldosterone and renin levels found in our study were higher than the median values found in previous reports of patients with chronic HF4,5,7,19 and acute HF9. This finding may be explained by the lower proportion (only 44%) of the patients on baseline ACEi/ARB, potentially leading to higher levels of renin and aldosterone19,20, similar to the levels found before the widespread use of the RAAS inhibitors7,8. MRA induced an increase in serum aldosterone and renin levels. These findings are concordant with previous reports in which the physiological elevation in plasma renin activity (PRA) and aldosterone were demonstrated in response to eplerenone and spironolactone treatment10,16,17,21,22. Our study has several limitations that need to be considered. First, it was a single-centre of a small sample size study. Second, diuretic therapy is could independently increase both renin and aldosterone plasma concentrations and uNa/K ratio. However, this would attenuate the inverse correlation between these variables that we found. Third, the plasma renin and aldosterone measure the circulating RAAS ativity, while uNa/K ratio is influenced by systemic and intra-renal SRAA activity. Fourth, whether the accuracy of uNa/K ratio as a biomarker for MRA use can be increased with higher MRA doses or tighter variable control is yet to be determined. Finally, our inclusion criteria by restricting the enrolment of patients with hyperkalemia, severe impaired renal function limit the external validity of our conclusions. Conclusions In an ADHF patients cohort, we demonstrated that uNa/K ratio is independently associated with renin and aldosterone at different time points and is also influenced by MRA therapy. Therefore, our findings disclose the potential role of uNa/K 134 ratio as a non-invasive and inexpensive biomarker of MRA therapy in acute HF patients. Nevertheless, how clinical management will be tailored according to the activation level of the RAAS is still unclear. Acknowledgements The authors acknowledge the lab technicians for technical assistance and to all physicians collaborating in the study. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Disclosures The authors have no conflicts of interest to disclose. 135 Tables Table 1. Population Characteristics, Laboratory Results and Spironolactone Dose at Admission Day (Day 1) and Day 3 Age (yrs) 76,0 ± 10,88 Male Sex – % 39 DM - % 45 Ischemic HF - % 50 EF < 40 - % 32 Hemoglobin (g/dL) 12,43 ± 2,07 Day 1 Day 3 p Value SBP (mmHg) 139,79 ± 25,86 121,97 ± 16,2 < 0,001 IV Furosemide - % 100 37 < 0,001** IV Furosemide Dose (mg/d) 75,80 ± 21,52 67,57 ± 25,54 0,001 Oral Furosemide - % 0 63 - Oral Furosemide Dose (mg/d) 0 74,6 ± 28,1 - ACEi - % 44 61 < 0,001** Ramipril Eq. Dose (mg/d) 3,15 ± 2,04 3,36 ± 2,14 0,474 Beta-Blocker - % 37 57 < 0,001** Bisoprolol Eq. Dose (mg/d) 3,01 ± 1,08 2,96 ± 1,89 0,474 Spironolactone - % 50 50 1 Spironolactone Dose (mg/d) 94,50 ± 23,31 62,74 ± 24,33 < 0,001 Plasma Creatinine (mg/dL) 1,04 [0,89 – 1,31] 1,06 [0,85 – 1,40] 0,082* Plasma Urea (mg/dL) 55,21 ± 20,84 62,3 ± 25,47 0,001 Plasma Potassium (mmol/L) 4,03 ± 0,51 4,04 ± 0,54 0,950 Serum Sodium (mmol/L) 140,54 ± 4,38 140,68 ± 3,95 0,72 Proteinuria (g/g) 0,289 [0,188 – 0,629] 0,299 [0,160 – 0,970] 0,045* NTproBNP (pg/mL) 2750 [1672 – 6032] 1835 [902 – 3837] < 0,001* UNa/K ratio 3,04 [1,52 – 5,76] 2,80 [1,50 – 4,78] 0,341* Renin (pg/mL) 4,35 [2,30 – 10,78] 5,34 [3,14 – 16,30] - ** Aldosterone (ng/dL) 35,0 [12,0 – 92,5] 67,0 [21,3 – 125,0] - ** hsTnT (ng/mL) 0,033 [0,019 – 0,050] 0,030 [0,018 – 0,051] 0,039* Continuous variables are presented as mean value ± standard deviation [SD], p value or median [inter-quartile range, IQR], p value. Categorical variables are presented as absolute number (%), p value. *Non-parametric paired sample test. **These results were not compared because day 1 samples were all collected before spironolactone administration and at day 3 fifty patients were on spironolactone. Note: day 1 analysis were collected before spironolactone administration. Legend: DM = diabetes mellitus; HF = heart failure; EF = ejection fraction; ACEi/ARB = angiotensin converting enzyme inhibitors/angiotensin receptor blockers; NTproBNP = N-terminal pro brain natriuretic peptide; UNa/K = urinary sodium to potassium; hsTnT = high sensitivity troponin T. 136 Table 2. Day 1 Log Aldosterone and Log Renin Associations NAC for Log Aldosterone 95%CI p Value AC for Log Aldosterone 95%CI p Value Age 0,102 -0,004 to 0,120 0,311 Male Sex 0,458 -0,180 to 1,162 0,100 DM 0,965 -0,398 to 2,342 0,938 EF <40% 0,515 -0,194 to 1,370 0,184 Ischemic HF 3,029 1,197 to 7,668 0,019 0,259 0,068 to 0,393 0,006 SBP -0,154 -0,006 to 0,001 0,126 IV Furosemide Dose 0,096 -0,002 to 0,006 0,340 Beta Blocker 1,690 -0,669 to 4,271 0,267 ACEi/ARB 1,088 -0,455 to 2,659 0,853 Spironolactone -0,187 -0,342 to 0,008 0,062 Log Creatinine 0,150 -0,170 to 1,310 0,130 Urea 0,231 0,001 to 0,009 0,021 0,200 0,000 to 0,008 0,034 Potassium 0,145 -0,047 to 0,303 0,149 Sodium -0,011 -0,032 to 0,009 0,267 Log Proteinuria 0,215 0,021 to 0,443 0,031 0,202 0,020 to 0,416 0,031 Log NTproBNP 0,189 -0,008 to 0,397 0,059 Log UNa/K ratio -0,180 -0,440 to 0,020 0,073 Log Renin 0,156 -0,039 to 0,323 0,122 Log hsTnT -0,032 -0,273 to 0,198 0,754 Hemoglobin 0,087 -0,024 to 0,062 0,388 NAC for Log Renin 95%CI p Value AC for Log Renin 95%CI p Value Age 0,001 -0,008 to 0,010 0,804 Male Sex 1,217 -0,533 to 2,777 0,642 DM 0,634 -0,277 to 1,449 0,280 EF <40% 0,691 -0,283 to 1,685 0,416 Ischemic HF 2,743 1,150 to 6,542 0,023 0,173 -0,010 to 0,347 0,064 SBP -0,223 -0,008 to -0,001 0,026 -0,232 -0,008 to - 0,001 0,013 IV Furosemide Dose -0,09 -0,002 to 0,007 0,372 Beta Blocker 0,639 -0,270 to 1,514 0,309 ACEi/ARB 1,100 -0,487 to 2,488 0,819 Spironolactone 0,181 -0,016 to 0,368 0,071 Log Creatinine 0,108 -0,377 to 1,263 0,286 Urea 0,169 -0,001 to 0,009 0,092 Potassium -0,100 -0,289 to 0,096 0,322 Sodium -0,034 -0,026 to 0,019 0,740 Log Proteinuria -0,064 -0,312 to 0,160 0,525 Log NTproBNP -0,038 -0,268 to 0,183 0,709 Log UNa/K ratio -0,332 -0,671 to -0,184 0,001 -0,322 -0,651 to - 0,178 0,001 Log 0,156 -0,039 to 0,323 0,122 137 Aldosterone Log hsTnT -0,031 -0,055 to 0,040 0,759 Hemoglobin -0,031 -0,055 to 0,040 0,759 Note: day 1 analysis were collected before spironolactone administration. Legend: NAC = non-adjusted coefficient; AC = adjusted coefficient; DM = diabetes mellitus; LVEF = left ventricular ejection fraction; HF = heart failure; ACEi/ARB = angiotensin converting enzyme inhibitors/angiotensin receptor blockers; NTproBNP = N-terminal pro brain natriuretic peptide; UNa/K = urinary sodium to potassium; hsTnT = high sensitivity troponin T. Table 3. Comparison Between Groups (Spironolactone vs. No Spironolactone) at Day 1 and Day 3 144 12. Bailey MA, Mullins JJ, Kenyon CJ. Mineralocorticoid and glucocorticoid receptors stimulate epithelial sodium channel activity in a mouse model of Cushing syndrome. Hypertension. 2009;54(4):890-896. 13. Brandish PE, Chen H, Szczerba P, Hershey JC. Development of a simplified assay for determination of the antimineralocorticoid activity of compounds dosed in rats. J Pharmacol Toxicol Methods. 2008;57(2):155-160. 14. Kagawa CM, Jacobs RS, Jr. Mineralocorticoid effects of 9 alphafluorodeoxycorticosterone in adrenalectomized rats. Proc Soc Exp Biol Med. 1960;104:60-62. 15. Ogiyama Y, Miura T, Watanabe S, et al. Circadian rhythm of urinary potassium excretion during treatment with an angiotensin receptor blocker. J Renin Angiotensin Aldosterone Syst. 2013. 16. Krum H, Nolly H, Workman D, et al. Efficacy of eplerenone added to reninangiotensin blockade in hypertensive patients. Hypertension. 2002;40(2):117123. 17. Menard J, Gonzalez MF, Guyene TT, Bissery A. Investigation of aldosteronesynthase inhibition in rats. J Hypertens. 2006;24(6):1147-1155. 18. Ferreira JP, Santos M, Almeida S, Marques I, Bettencourt P, Carvalho H. Tailoring diuretic therapy in acute heart failure: insight into early diuretic response predictors. Clin Res Cardiol. 2013. 19. Vantrimpont P, Rouleau JL, Ciampi A, et al. Two-year time course and significance of neurohumoral activation in the Survival and Ventricular Enlargement (SAVE) Study. Eur Heart J. 1998;19(10):1552-1563. 20. Cleland JG, Dargie HJ, Hodsman GP, et al. Captopril in heart failure. A double blind controlled trial. Br Heart J. 1984;52(5):530-535. 21. Ortiz RM, Graciano ML, Seth D, Awayda MS, Navar LG. Aldosterone receptor antagonism exacerbates intrarenal angiotensin II augmentation in ANG IIdependent hypertension. Am J Physiol Renal Physiol. 2007;293(1):F139-147. 22. Rousseau MF, Gurne O, Duprez D, et al. Beneficial neurohormonal profile of spironolactone in severe congestive heart failure: results from the RALES neurohormonal substudy. J Am Coll Cardiol. 2002;40(9):1596-1601. 145 VII. The Influence of Spironolactone on Matrix Metalloproteinases in Acute Decompensated Heart Failure 146 João Pedro Ferreira, MD1, Mário Santos, MD1, José Carlos Oliveira, MD1, Irene Marques, MD1, Paulo Bettencourt, MD, PhD2, Henrique Carvalho, MD, PhD1 1 Centro Hospitalar do Porto, Porto, Portugal, 2 Centro Hospitalar de São João, Porto, Portugal Correspondence: João Pedro Ferreira, Centro Hospitalar do Porto, Internal Medicine Department, Largo Prof. Abel Salazar 4099-001 Porto, Portugal. Contacts: Telephone – 00351222077500; Fax – 00351222053218; E-mail – [email protected] Abstract Background: Matrix metalloproteinases (MMPs) are a family of enzymes important for the resorption of extracellular matrices (ECM), control of vascular remodelling and repair. Increased activity of MMP2 has been demonstrated in heart failure (HF) and in acutely decompensated heart failure (ADHF) a decrease of circulating MMPs has been demonstrated along with successful treatment. Objectives: Our aims are to test the influence of spironolactone in MMP2 levels. Methods: Secondary analysis of a prospective, interventional study including 100 patients with ADHF. Fifty patients were non-randomly assigned to spironolactone 100 mg/day plus standard ADHF therapy (intervention group) or standard ADHF therapy alone (control group). Results: Patients within spironolactone group were younger, had lower creatinine and urea levels (all p < 0,05). Baseline MMP2, NT-pro BNP and weight did not differ between spironolactone and control groups. A trend to a more pronounced decrease of MMP2 from baseline to day 3 was observed in spironolactone group (-21 [-50 to 19] vs 1,5 [-26 to 38] ng/mL, p = 0,06), NT-pro BNP and weight also had a greater decrease in spironolactone group. The proportion of patients with a decrease in MMP2 levels from baseline to day 3, was also likely to be greater in spironolactone group (50% vs 66,7%), but without statistical significance. Correlations between MMP2, NT-pro BNP and weight variation were not statistically significant. Conclusions: MMP2 levels are increased in ADHF. Patients submitted to spironolactone treatment may have greater reduction in MMP2 levels. Key-words: matrix metalloproteinase-2; decompensated heart failure; spironolactone. 147 Introduction Matrix metalloproteinases (MMPs) are a family of zinc-dependent interstitial enzymes important for the resorption of extracellular matrices (ECM) in both health and disease1. ECM are a dynamic structure central to the control of vascular remodelling and repair1, mostly due to the ability of MMPs to reabsorb and digest excessive amounts of ECM responsible for structural disruption2,3. Elevated MMPs promote loss of cardiac contractility via cell proteolysis and alterations in the ECM, contributing to cardiac and extra-cardiac remodelling processes4. In fact, clinical and experimental heart failure (HF) models of dilated and ischemic cardiomyopathy demonstrated an increased activity of matrix metalloproteinase-2 (MMP2)2,5-7. In patients with HF, increased levels of MMP2 were associated with all-cause mortality8. Concordantly, in the acutely decompensated heart failure (ADHF) setting a decrease of circulating MMPs has been demonstrated along with successful ADHF treatment3,9. Previous studies have suggested a therapeutic benefit of spironolactone in ADHF setting10. But no studies had looked to the effect of spironolactone in the ECM remodelling. In the present study, we aimed to examine the influence of spironolactone on the ECM remodeling in ADHF patients. We hypothesized that MMP-2 plasma levels of ADHF patients will have steeper decrease if spironolactone is added to standard treatment. Methods Study Design We analysed data from a previous pilot, prospective, interventional, clinical trial that we performed. That study was performed between February 2012 and February 2013 and during that period we enrolled 100 consecutive patients who presented into a Portuguese tertiary hospital with ADHF. Patients were eligible for enrollment if they presented with decompensation of chronic HF with symptoms leading to hospitalization. ADHF was diagnosed on the basis of the presence of history of chronic HF and at least one acute symptom (dyspnea, orthopnea, or edema) and one sign (rales, peripheral edema, ascites, or pulmonary vascular congestion on chest radiography). Patients were non-randomly assigned in a sequential 1:1 ratio to spironolactone plus standard ADHF therapy or standard ADHF therapy alone, 50 patients within each arm. Patients were alternatively assigned to spironolactone arm or standard ADHF therapy arm in a sequential manner - the first patient to one arm and the next to the other arm. This sequence was repeated until we reach 100 patients, 50 patients within spironolactone group and 50 patients within control group. Patients were 148 blinded to the allocation, but not the clinicians. The recommended spironolactone dose was 100 mg/day, however the assistant physician could decrease the spironolactone dose to 50 mg/day after 48h upon admission. Furosemide dose and route of administration was adjusted clinically according to the hydration status of the patients. Exclusion criteria were: chronic use of mineralocorticoid receptor antagonists, cardiac surgery within 60 days of enrollment, cardiac mechanical support, cardiac resynchronization-therapy within the last 60 days, comorbid conditions with an expected survival of less than 6 months, acute myocardial infarction at time of hospitalization, hemodynamically significant uncorrected primary cardiac valvular disease, patients requiring intravenous vasodilators or inotropic agents, supine systolic arterial blood pressure <90 mmHg, plasma creatinine level >1,5 mg/dL, serum potassium level >5,0 mmol/L, hemoglobin level <9 g/dL, and sepsis. Institutional review board or ethics committee approval was obtained. All patients provided written informed consent to participate in the study. Clinical assessment of participants Patient`s clinical status including physical examination was prospectively recorded by the same assistant physician at day 1 and day 3. Medications and respective dosages were prospectively recorded by the investigators according to the assistant physician prescriptions. Blood samples were collected in the first 24 hours (h) after admission (baseline) of the patient to the hospital, and the day 3 samples were collected between 72 and 96 h of hospitalization. Samples were analysed at a central core laboratory, and included plasma creatinine and urea, electrolytes, NT-pro BNP and MMP2. Clinical assessment and routine analyses were performed daily during hospital stay. Estimated glomerular filtration rate (eGFR) was determined using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation11. All patients performed a transthoracic echocardiography within 72 hours upon admission. Left ventricular ejection fraction (LVEF) was calculated according to biplane Simpson method. MMP2 was measured by enzyme-linked immunoabsorbent assays (ELISA) - Quantikine Elisa Human MMP-2 Immunoassay, by the manufacturer R&D Systems, Inc®. The normal range of MMP2 values published by the manufacturer are median [inter-quartile range25-75], 199 [161 – 301] ng/mL. The assay sensitivity is 0,047 ng/mL. Of the 100 studied patients we analysed baseline (day 1) and day 3 blood samples from 87. Thirteen (13) samples were not analysed due to transport and/or sampling processing errors. Samples were collected in the morning with patients supine. Serum was separated and stored at -80 ºC until sample analysis. 149 Variable definitions We classified patients according to spironolactone use and their response to diuretic therapy. We studied the relationships between baseline characteristics, day 3 and changes (Δ, difference between day 3 and baseline values) in MMP2, NT-proBNP and weight regarding the spironolactone use and diuretic response. Statistical Analysis Normally distributed continuous variables are expressed as mean ± standard deviation (SD), and skewed distributions are presented as median [inter-quartile range25-75, IQR]. Categorical variables are expressed in proportions (%). Comparison between groups was performed using parametric (independent samples t-test), non-parametric (Mann-Whitney test), or Chi-square tests, as appropriate. Correlations of MMP2 were examined by single variable linear regression and presented as correlation coefficient and 95% confidence interval [95%CI]. A p value < 0,05 was considered statistically significant. Statistical analysis was performed using SPSS software (version 19, Chicago, IL, USA). Results Baseline Patients Characteristics in Control and Spironolactone Groups Patients in control group were older (78,8 ± 9,3 versus [vs.] 73,2 ± 11,7 years, p = 0,01), and had higher creatinine and urea levels (1,15 ± 0,27 vs. 1,03 ± 0,30 mg/dL, p = 0,026 and 59,32 ± 22,27 vs. 51,10 ± 18,63 mg/dL, p = 0,048). No differences between groups were found regarding sex, diabetes mellitus, chronic obstructive pulmonary disease, dementia, sleep apnea, non-invasive ventilation, ischemic heart disease, atrial fibrillation, LVEF, weight, systolic blood pressure, potassium, sodium, hemoglobin, albumin, NT-pro BNP, MMP2, furosemide dose, hospital length of stay, and in the proportion of patients on angiotensin converting enzyme inhibitors and betablockers – Table 1. Spironolactone Influence on MMP2, NT-pro BNP and Weight Dynamic Changes No differences between control and spironolactone groups were observed regarding baseline and day 3 MMP2 levels – Table 2. However, MMP2 decreased from baseline to day 3 in spironolactone group, while in control group MMP2 levels 150 increased, leading to a tendency for a reduction in MMP2 levels in spironolactone group (1,5 [-26 to 38] vs. -21 [-50 to 19] ng/mL, p = 0,06) – Table 2. and Figure 1. The proportion of patients in which a decrease in MMP2 levels, from baseline to day 3, was observed was also greater in spironolactone group, however this difference did not reach statistical significance – number (%), 21 (50) in controls versus 30 (66,7) in spironolactone group, p = 0,115. No differences were observed in NT-pro BNP levels at baseline, however at day 3 the group of patients who underwent spironolactone treatment presented lower levels of NT-pro BNP (248 [923 – 5502] vs. 1555 [722 – 2554] pg/mL, p = 0,05). No differences between groups were observed in the variation of NT-pro BNP levels probably due to the lower levels (although not significantly lower) of NT-pro BNP at baseline in the spironolactone group, leading to a smaller amplitude of variation in this group – Table 2. A greater weight decrease was also observed in the spironolactone-treated patients (-2,9 ± 2,4 vs. -4,8 ± 2,8 Kg, p < 0,001) – Table 2. Δ MMP2, Δ NT-pro BNP and Δ Weight Correlations No significant correlations were observed between Δ in MMP2, NT-pro BNP and weight - Table 3. Discussion In the present study we observed an increased baseline levels of MMP2 in patients with ADHF. Those patients treated with spironolactone showed a tendency to a greater reduction of MMP2 levels. These results are consistent with previous findings demonstrating the impact of mineralocorticoid receptor antagonists on the ECMs remodelling and highlight the potential interest of spironolactone in the treatment of ADHF where those mechanisms are strongly exacerbated. Increased serum levels of MMP2 have been demonstrated in the ADHF setting3,9. In our study the median [IQR] MMP2 levels at admission day were 260 [225 - 312] ng/mL. These values are above the normal range defined by the manufacturer, 199 [161 – 301] ng/mL, and are concordant with previous reports on patients with HF decompensation. A previous study by Shirakabe A., et al3, also showed increased serum MMP2 levels in ADHF, with a rapid decrease along with HF compensation. Furthermore, an interventional placebo-controlled trial performed by Tziakas DN., et al9, showed a significant reduction in MMP2 levels in the group of patients treated with levosimendan. In animal models, exacerbated neurohormonal activation leads to an increase in the levels of several myocardial MMPs subtypes12-14. MMPs are important for proteolysis that can affect the composition of ECM and consequently myocardial 151 remodelling. Additionally, increased ECM turnover may be associated with pathological myocardial remodelling, that may be accelerated in decompensated HF3,15. Consequently, a reduction in markers of ECM turnover may serve as a surrogate marker for deceleration of myocardial turnover and remodelling. Our study showed a greater decrease of MMP2 in the group of patients submitted to spironolactone treatment. Mineralocorticoid receptor antagonists improve survival and reduce morbidity in patients with heart failure with reduced ejection fraction, and mild-to-severe symptoms, and in patients with left ventricular systolic dysfunction and heart failure after acute myocardial infarction16-18, additionally, used in natriuretic doses, the mineralocorticoid receptor antagonists are likely to improve congestion in ADHF with good tolerability and few side effects19. Several proposed mechanisms explain how MRAs improve HF outcomes, and these pathways include a reduction of myocardial remodelling20. Our study provides important information towards a better understanding of ECM turnover processes. The steeper MMP2 reduction observed in patients submitted to spironolactone treatment provides a real demonstration of potential mitigation of harmful remodelling through spironolactone use. Interestingly, patients without MMP2 reduction or increase, after an acute HF episode, had poorer prognosis3. Therefore, changes in MMP2 levels are a potentially useful prognostic marker in patients admitted due to ADHF. NT-pro BNP is a well-validated, widely used, and very accurate biomarker for the diagnosis and risk stratification of HF21. Patients submitted to spironolactone treatment had lower levels of NT-pro BNP at day 3, and a more pronounced weight reduction when compared to controls, and patients with slower diuretic response had higher NT-pro BNP levels at day 3 of hospitalization, a tendency to lower NT-pro BNP reduction, and less weight loss. However, the changes in MMP2 values did not correlate with the variation in NT-pro BNP or weight. The small sample size, the NTproBNP and MMP2 elevated variance, and the different mechanisms influencing those biomarkers may all explain the absence of this correlation. Several limitations in our study should be noticed. First, this was a singlecentre, non-randomized trial with a small number of patients with mixed HF etiologies and treatments. Second, this post-hoc analysis has limitations inherent to observational studies. Third, the decision to withdraw diuretic therapy was based on subjective assessment of congestive signs and symptoms so we cannot rule out the interobserver variability. However, in real-life patients, the decision to step down diuretic therapy is also based on subjective clinical evaluation. Fourth, our study excluded HF patients significant renal impairment, since plasma creatinine level of less than 1,5 mg/dL was an inclusion criteria, leading to a potential selection of a subset of low risk 152 patients, which can affect the external validity of our results. Fifth, the group of patients submitted to spironolactone treatment were younger and had lower plasma creatinine and urea levels which can positively affect the response to this drug. Finally, only MMP2 was evaluated and other forms of MMPs may have different effects and responses in ADHF patients. Conclusion The present study showed that MMP2 levels can be increased in ADHF, and that patients treated with spironolactone may have greater reduction in MMP2 levels. Whether these findings have prognostic significance requires further investigation. Acknowledgements The authors acknowledge the lab technicians, especially Mr. Fernando Santos for technical assistance and to all physicians collaborating in the study. Funding Matrix Metalloproteinases kits were supported by a grant from Johnson & Johnson. Disclosures The authors have no conflicts of interest to disclose. 153 Tables Table 1. Baseline Population Characteristics, Laboratory Results, Medications, and Hospital Length of Stay in Treatment and Control Groups Control Group (n = 50) Spironolactone Group (n = 50) p Value Age (yrs) 78,8 ± 9,3 73,2 ± 11,7 0,010 Male Sex – % 34 44 0,31** Diabetes Mellitus - % 50 40 0,31** COPD - % 10 26 0,32** Dementia - % 16 8 0,22** Sleep Apnea - % 10 26 0,32** Non-Invasive Ventilation - % 14 20 0,42** Ischemic Heart Disease -% 48 52 0,69** Atrial Fibrillation - % 68 50 0,07** LV Ejection Fraction < 40% - % 56 68 0,22** Weight (Kg) 75,6 ± 16,3 76,1 ± 16,4 0,89 SBP (mmHg) 140,5 ± 23,9 139 ± 27,9 0,80 Plasma Creatinine (mg/dL) 1,15 ± 0,27 1,03 ± 0,30 0,03 eGFR (mL/min/1,73 m2) 54,5 ± 16,5 68,3 ± 23,6 0,001 Plasma Urea (mg/dL) 59,3 ± 22,3 51,1 ± 18,6 0,05 Serum Potassium (mmol/L) 4,1 ± 0,4 4,0 ± 0,6 0,33 Serum Sodium (mmol/L) 140,5 ± 5,0 140,6 ± 3,7 0,96 Hemoglobin (g/dL) 12,2 ± 1,8 12,7 ± 2,3 0,22 Albumin (mg/dL) 3,7 ± 0,4 3,6 ± 0,4 0,63 NTproBNP (pg/mL) 3102 [1797 – 8204] 2701 [1463 – 5004] 0,17* MMP2 (ng/mL) 260 [226 – 299] 268 [207 – 336] 0,52* IV Furosemide Dose (mg/d) 75,6 ± 20,7 76,0 ± 25,5 0,93 ACEi/ARB – % 38 50 0,20** Beta-Blocker - % 42 32 0,30** Spironolactone - % - 100 - Spironolactone Dose (mg/d) - 94,5 ± 23,3 - Hospital Length of Stay (days) 9,0 ± 3,7 8,7 ± 3,0 0,59 Continuous variables are presented as mean value ± standard deviation [SD], p value or median [inter-quartile range, IQR], p value. Categorical variables are presented as absolute number (%), p value. *Non-parametric paired sample test; ** Chi-square test. Legend: COPD - chronic obstructive pulmonary disease; LV - left ventricular; eGFR - estimated glomerular filtration rate; NT-pro BNP - N-terminal pro brain natriuretic peptide; hsTnT - high sensitivity troponin T; MMP2 - matrix metalloproteinase -2; IV - intra-venous; ACEi - angiotensin converting enzyme inhibitors