University of Groningen Towards personalized cardiovascular risk management in renal transplant recipients de Vries, Laura Victorine

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(1)University of Groningen. Towards personalized cardiovascular risk management in renal transplant recipients de Vries, Laura Victorine. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2018 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): de Vries, L. V. (2018). Towards personalized cardiovascular risk management in renal transplant recipients. Rijksuniversiteit Groningen.. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 18-07-2021.

(2) Towards Personalized Cardiovascular Risk Management in Renal Transplant Recipients. Laura V. de Vries.

(3) Laura V. de Vries Towards Personalized Cardiovascular Risk Management in Renal Transplant Recipients PhD dissertation, University of Groningen, the Netherlands Financial support by the University of Groningen, University Medical Center Groningen, Groningen Graduate School of Medical Sciences, Dutch Kidney Foundation, Nederlandse Transplantatie Vereniging, and Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged. Further financial support for the printing of this thesis was kindly provided by Astellas Pharma B.V., Chiesi Pharmaceuticals B.V., ChipSoft, Eurocept Homecare, Fresenius Medical Care Nederland B.V., Noord Negentig Accountants & Belastingadviseurs, Sanofi, Spark Holland B.V., and Van der Meer Accountants & Adviseurs. Cover design & lay-out: Evelien Jagtman (evelienjagtman.com) Printed by: Gildeprint Drukkerijen, Enschede ISBN: 978-94-6233-890-6 (printed version) ISBN: 978-94-034-0503-2 (digital version) © Copyright 2018, L.V. de Vries, Groningen, the Netherlands All rights reserved. No part of this publication may be reproduced, copied, modified, stored in a retrieval system, or transmitted in any form without prior written permission of the author..

(4) Towards Personalized Cardiovascular Risk Management in Renal Transplant Recipients Proefschrift. ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 11 april 2018 om 14.30 uur. door. Laura Victorine de Vries geboren op 3 augustus 1986 te Zeist.

(5) Promotores Prof. dr. S.J.L. Bakker Prof. dr. G.J. Navis Prof. dr. I.P. Kema Beoordelingscommissie Prof. dr. J.J. Homan van der Heide Prof. dr. R.J. Porte Prof. dr. P. de Vos.

(6) Paranimfen Drs. M.A. van den Boomgaard Dr. J.C. Tanis.

(7) r. te. ap. Ch 4. ter. ap. Ch. 5. Chapter 6. Cha pt. er 3. Chapter 2.

(8) CONTENTS. 1. Chapter 1 Introduction and Aims of Thesis. 11. Chapter 2 Effects of Dietary Sodium Restriction in Kidney Transplant Recipients Treated With Renin-Angiotensin-Aldosterone System Blockade: A Randomized Clinical Trial Am J Kidney Dis. 2016 Jun;67(6):936-44. 33. Chapter 3 Aldosterone, From (Patho)Physiology to Treatment in Cardiovascular and Renal Damage Curr Vasc Pharmacol. 2011 Sep;9(5):594-605. 53. Chapter 4 Twenty-four Hour Urinary Cortisol Excretion and the Metabolic Syndrome in Prednisolone-Treated Renal Transplant Recipients Steroids. 2017 Sep 8;127:31-39. 83. Chapter 5 Endogenous Glucocorticoid Metabolites and Mortality in Prednisolone-Treated Renal Transplant Recipients Submitted. 113. Chapter 6 The Tryptophan-Kynurenine Pathway, Systemic Inflammation, and Long-Term Outcome after Kidney Transplantation Am J Physiol Renal Physiol. 2017 Aug 1;313(2):F475-F486. 137. Chapter 7 Summary and General Discussion. Chapter 8 Nederlandse Samenvatting (voor niet-ingewijden) Appendix Dankwoord / Acknowledgements About the Author Author Affiliations . 169. 193 205 215 219.

(9) ITHACA When you set out for distant Ithaca, fervently wish your journey may be long, — full of adventures and with much to learn. Of the Laestrygones and the Cyclopes, of the angry god Poseidon, have no fear: these you shall not encounter, if your thought remains at all times lofty, — if select emotion touches you in body and spirit. Not the Laestrygones, not the Cyclopes, nor yet the fierce Poseidon, shall you meet, unless you carry them within your soul, — unless your soul should raise them to confront you. Fervently wish your journey may be long. May they be numerous — the summer mornings when, pleased and joyous, you will be anchoring in harbours you have never seen before. Stay at the populous Phoenician marts, and make provision of good merchandise; coral and mother of pearl; and ebony and amber; and voluptuous perfumes of every kind, in lavish quantity. Sojourn in many a city of the Nile, and from the learned learn and learn amain..

(10) At every stage bear Ithaca in mind. The arrival there is your appointed lot. But hurry not the voyage in the least: ’twere better if you travelled many years and reached your island home in your old age, being rich in riches gathered on the way, and not expecting more from Ithaca. Ithaca gave you the delightful voyage: without her you would never have set out: and she has nothing else to give you now. And though you should find her wanting, Ithaca will not surprise you; for you will arrive wise and experienced, having long since perceived the unapparent sense in Ithacas.. Κωνσταντίνος Π. Καβάφης, 1911 (origineel in Grieks) Vertaling: John Cavafy, 2003.

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(12) Chapter 1 INTRODUCTION AND AIMS OF THESIS.

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(14) Introduction and Aims of Thesis. INTRODUCTION. 1. Kidney transplantation Since the first successful operation in man in 1954, kidney transplantation has evolved from an experimental therapy to the treatment of choice for most patients with endstage renal disease (ESRD). Kidney transplantation offers a significant survival benefit to patients with ESRD and improves their quality of life as compared with patients who remain dependent on dialysis.1,2 As an example, the expected unadjusted remaining lifetime for a 40 year old patient on dialysis is 12.0 years, whereas this is 26.2 years for a renal transplant recipient (RTR) of the same age (Figure 1).3 The number of kidney transplantations performed each year in the Netherlands has continued to grow over the past decades and increased from 587 in 2002 to 984 in 2015.4 In line with this, the total number of patients in the Netherlands who now live with a functioning kidney transplant is around 16,000 and increasing.4,5. Figure 1. Expected remaining lifetime for the general population (green dots), renal transplant recipients (blue squares), and dialysis patients (pink triangles). Data from the ERA-EDTA Annual Report 2014.3. 13.

(15) Chapter 1. Long-term survival after kidney transplantation One year graft survival after kidney transplantation has steadily improved from approximately 40% in the 1970s to more than 90% to date.6 This is mainly the result of improved surgical techniques and advancements in immunosuppressive therapy. However, despite these improvements in short-term outcomes, there has been surprisingly little improvement in long-term outcomes over the past decades.7,8 Moreover, observed improvements in long-term survival are mostly attributable to improvements in survival in the first months after transplantation as is depicted in Figure 2, where lines for graft survival for the different year cohorts run almost parallel beyond 6 months after transplantation. Currently, in the Netherlands, the 5-year allograft survival rate for living donor transplants is 84%, and for deceased donor transplants this is only 70%.5 In the US, for example, these survival rates are even slightly worse.2 The causes of long-term kidney allograft loss are multifactorial. In about half of successfully operated patients, the kidney transplant fails due to diverse causes, including recurrent primary kidney disease, and calcineurin inhibitor toxicity. The other half of allograft losses occurs because the recipient dies with a functioning kidney transplant.9,10. Figure 2. Allograft survival of first kidney transplants according to different years of transplantation. Data from the Collaborative Transplant Study; figure is available online at: http://www.ctstransplant.org (Figure: K-14001-0217).. 14.

(16) Introduction and Aims of Thesis. Cardiovascular disease after kidney transplantation Cardiovascular disease (CVD) is the primary cause of death of RTR, preceding infection and malignancy. Beyond one year after transplantation, age- and sex-adjusted death rates in RTR are several times higher than in the general population, primarily due to an excess in CVD.11,12 The most common causes of cardiovascular death are myocardial infarction, left ventricular hypertrophy, sudden cardiac arrest, and stroke.12-14 Interestingly, the cardiovascular risk profile of RTR differs from that of the general population or patients with chronic kidney disease (CKD). Many RTR already have several traditional cardiovascular risk factors before transplantation, which likely contributed to the development and progression of their underlying kidney disease in the first place (Table 1). Unfortunately, these risk factors are only partially remitted following successful transplantation. In fact, the prevalence of these traditional risk factors, such as for instance hypertension, is generally higher than in the general population or other high risk populations such as diabetes or CKD,11,15,16 and the underlying mechanisms, as well as response to treatment may be different. Moreover, after transplantation new transplantation-related risk factors emerge, such as remaining subnormal kidney function, viral infections, and the use of immunosuppressive drugs (i.e. corticosteroids and calcineurin inhibitors) (Table 1 and Figure 3).12-14 The risk of adverse effects of treatment with these drugs is considerable, because of the narrow therapeutic window between efficacy and toxicity. For this reason, calcineurin inhibitors are titrated by close monitoring of drug levels, but for corticosteroids, currently, no such monitoring is available, and accordingly treatment with these drugs is more or less ‘one-size-fits-all’.17 The consequences of the differences in risk profile between RTR and other populations have not been systematically investigated, and hence, cardiovascular risk management in this population is still largely based on studies in other populations, for example patients with hypertension, diabetes or CKD.16 This might well be an underlying factor in the high CV morbidity and mortality in this population. Therefore, in order to improve long-term outcome after kidney transplantation, we are in need of comprehensive strategies to reduce increased cardiovascular risk, ideally addressing both traditional and transplantation-related risk factors, and to provide adequate transplantation-specific guidelines for cardiovascular risk management in RTR. In addition, better personalization of risk management could greatly benefit from tools (such as biomarkers) that can guide better personalization of treatment in RTR.. 15. 1.

(17) Chapter 1. Table 1. Cardiovascular (CV) risk factors in renal transplant recipients. Non-modifiable risk factors age sex (male > female) ethnicity family history prior CV history Potentially modifiable risk factors Traditional. Traditional risk factors. risk factors. in the transplantation setting. obesity insulin resistance / diabetes. post-transplant weight gain → obesity ↑ insulin resistance / diabetes ↑. Transplantation-related risk factors time on dialysis delayed graft function. hypertension. hypertension ↑. acute rejection episodes. dyslipidemia. sodium sensitivity ↑. reduced kidney function. anemia. physical inactivity ↑. smoking. chronic inflammation / immune activation ↑. high alcohol intake high sodium intake physical inactivity. proteinuria new-onset diabetes viral infections (e.g. CMV) corticosteroid use calcineurin inhibitor use. chronic inflammation. Figure 3. Accumulation of traditional and transplantation(tx)-related cardiovascular risk factors in renal transplant recipients, adding to increased cardiovascular risk.. 16.

(18) Introduction and Aims of Thesis. Hypertension after kidney transplantation – The pressure is on Of all traditional and transplantation-related cardiovascular risk factors, hypertension is the most prevalent. Up to 90% of RTR have high blood pressure or are treated with antihypertensive drugs.12,18 There are many factors contributing to hypertension after kidney transplantation, among which are general risk factors such as an unfavorable metabolic profile (i.e. weight excess, dyslipidemia, and insulin resistance), male sex, and age, but also transplantation-related risk factors such as increased sympathetic nerve activity or vascular calcification, reduced kidney function, and treatment with calcineurin inhibitors and/or corticosteroids.19,20 Treatment of hypertension after kidney transplantation mostly involves pharmacological treatment with a combination of different antihypertensive drugs, which are chosen based on co-morbidity, efficacy, and interactions with other drugs.20,21 Mostly calcium channel blockers, beta- and alpha-blockers, and diuretics are prescribed.21 Treatment with renin-angiotensin-aldosterone system (RAAS) blockade, such as angiotensin converting enzyme inhibitors or angiotensin receptor blockers, has largely been avoided in RTR, because two meta-analyses of otherwise inconclusive data pointed toward an advantage of calcium channel blockers over RAAS blockers for the management of hypertension in this population.21,22 Moreover, intrinsic effects of RAAS blockade on glomerular filtration rate may mimic rejection. Therefore, many transplant physicians are still reluctant to prescribe them. However, RAAS blockers have been shown to significantly reduce proteinuria in RTR and evidence suggests an advantage of prolonged treatment with this type of drugs in RTR.23-25 Alternatives to pharmacological treatment of hypertension Despite extensive pharmacological treatment, blood pressure management in RTR often remains inadequate. This is illustrated by a recent study using data of the Netherlands Organ Transplant Registry, which showed that in the Netherlands only 23% of RTR meet blood pressure recommendations (Figure 4).18 Similarly, in a large international cohort (29,751 patients) of the Collaborative Transplant Study, up to 55% of RTR did not reach the goal for blood pressure control.26 Each 10-mmHg incremental rise in systolic blood pressure independently increases the risk for death and death-censored allograft failure in RTR by 18% and 17%, respectively.27 Therefore, next to intensifying pharmacological treatment, it is important to identify other modifiable risk factors which allow for intervention. Corticosteroids and calcineurin inhibitors form the cornerstone of post-transplant immunosuppression, but they are widely known to cause hypertension. Therefore, it is important to optimize their dosing regimens to accomplish optimal immunosuppressive effects on the one hand, with as little as possible adverse effects on the other. To be able to do so, gaining knowledge on the dose-response curves and acquiring tools for dose titration are of great importance. Treatment 17. 1.

(19) Chapter 1. with calcineurin inhibitors is already closely monitored and doses are continuously adapted to frequently measured blood levels of these drugs. However, treatment with corticosteroids is entirely different. In the Netherlands, the current treatment regimen for corticosteroids encompasses a ‘one-size-fits-all’ approach with an empirical dose of usually 7.5 mg prednisolone per day, irrespective of body size and/or steroid sensitivity. The main reason for this approach is that there is currently no way to guide intensity of treatment.17 Thus, personalization of corticosteroid treatment could be an interesting strategy to reduce blood pressure (as well as other adverse effects of corticosteroids, such as glucose intolerance, osteoporosis and sarcopenia), as will be outlined in the paragraphs below. Nevertheless, since treatment with either calcineurin inhibitors and/or corticosteroids will likely remain necessary in the majority of RTR in the near future, alternative strategies to reduce blood pressure also have to be considered. Lifestyle interventions, such as weight reduction, increasing physical activity, cessation of smoking, and reduction of sodium intake, have shown to be effective in reducing blood pressure in other populations and have great potential to reduce blood pressure in RTR. However, they have only been sparsely studied in this population.. Figure 4. Blood pressure (BP) targets and the use of antihypertensive drugs in renal transplant recipients in the Netherlands. Reprinted with permission from Dobrowolski et al.16. 18.

(20) Introduction and Aims of Thesis. Sodium restriction as therapeutic strategy Dietary sodium restriction effectively reduces blood pressure and proteinuria in patients with chronic kidney disease (CKD).28-31 Moreover, several studies have shown that low sodium intake is associated with much better kidney disease and cardiovascular outcomes in patients with CKD.32 Therefore, the KDOQI and DASH guidelines advocate a maximum sodium intake of 100 mmol per day for all patients with kidney disease. Despite these recommendations, average sodium intake in RTR largely exceeds this recommendation, with intakes of 150 to 200 mmol per day.18,33-36 Moreover, treatment with calcineurin inhibitors and corticosteroids, in addition to decreased kidney function and prevalent obesity, may render blood pressure even more sodium sensitive in RTR compared with patients with CKD. This is illustrated by a recent study in 660 Dutch RTR, showing an independent association of sodium intake with blood pressure.33 In addition, recent studies showed that treatment with the calcineurin inhibitor tacrolimus increases renal tubular sodium absorption.37 Thus, although evidence points towards potential benefits of dietary sodium restriction in RTR, no randomized clinical trials studying dietary sodium restriction in RTR are available to date. Sodium status and aldosterone High sodium intake is even more deleterious when it is accompanied by high serum aldosterone. Aldosterone is one of the main effector hormones of the RAAS, and its main function is to restore volume status in times of sodium and/or volume depletion. It does so by activating the mineralocorticoid receptor, leading to increased renal tubular sodium and water reabsorption and potassium excretion. Therefore, increased aldosterone production leads to hypertension and volume overload. In addition, aldosterone is known to exert pro-fibrotic and pro-inflammatory effects on the vasculature. Interestingly, detrimental effects of aldosterone are only observed in states of primary increase in aldosterone concentrations, rather than states in which increased aldosterone concentrations are secondary to volume depletion. For example, in patients with resistant hypertension the effects of high sodium intake on proteinuria are most pronounced in patients with the highest aldosterone.38 In contrast, in case of hyperaldosteronism secondary to volume depletion, such as routine low-sodium intake in Yanomami Indians or Gitelman or Bartter syndrome with renal sodium loss, hypertension and cardiovascular damage are absent.39,40 Taken together, these data suggest that aldosterone mostly exerts adverse effects when its serum concentration is inappropriately high for the prevailing sodium status. Corticosteroids – Two sides of the medal Another important contributor to hypertension and cardiovascular disease in RTR is treatment with corticosteroids. Corticosteroids were among the first drugs used to 19. 1.

(21) Chapter 1. prevent and treat rejection after kidney transplantation, and are still used to date.41,42 The most often used corticosteroids after transplantation are prednisone and its bioactive metabolite prednisolone, and in the University Medical Center Groningen (UMCG) prednisolone is exclusively used. Prednisolone exerts its immunosuppressive effects by binding to the glucocorticoid receptor (GR), of which cortisol is the natural ligand. It also binds to the mineralocorticoid receptor (MR), of which aldosterone is the natural ligand. Through its ability to bind both GR and MR, prednisolone causes a wide range of side effects, including weight gain, lipid derangement, glucose intolerance, and hypertension41,42, thereby adding to the increased cardiovascular risk after kidney transplantation. Therefore, there has been a great effort to get rid of corticosteroids as part of maintenance immunosuppressive regimens after kidney transplantation.8,42 Nevertheless, it has recently been concluded that corticosteroids have to remain part of the immunosuppressive regimen in order to maintain low acute rejection rates and optimal long-term allograft survival.43,44 As mentioned earlier, corticosteroid dosing regimens unfortunately remain empiric to date, usually with fixed doses independent of either body size and/or steroid sensitivity.17 Therefore, tools are needed to monitor and personalize corticosteroid therapy in order to reduce corticosteroid-related adverse effects. Cortisol synthesis and metabolism Corticosteroids are synthetic derivatives of endogenous cortisol. Because of their strong structural similarity to endogenous cortisol, they are able to bind the GR and interfere in cortisol synthesis and metabolism (Figure 5 and 6). Under physiological conditions, cortisol synthesis is regulated by the hypothalamus-pituitary-adrenal (HPA) axis. When this axis is activated, the hypothalamus secretes corticotropin releasing hormone (CRH), which stimulates the release of adrenocorticotropic hormone (ACTH) by the pituitary, which then stimulates cortisol synthesis by the adrenal glands. Cortisol, in turn, exerts inhibitory effects on the hypothalamus and pituitary via a negative feedback mechanism, thereby regulating its own production (Figure 6). Cortisol is metabolized to biologically inactive cortisone by the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2), whereas its counterpart 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) regenerates cortisol back from cortisone (Figure 5 and 6).45,46 Both cortisol and cortisone are acted upon by 5α- and 5β-reductases and 3α-hydroxysteroid dehydrogenase, ultimately leading to generation of tetrahydrocortisol (THF), allo-tetrahydrocortisol (allo-THF), and tetrahydrocortisone (THE) (Figure 6). Especially 11β-HSD enzymes play a pivotal role in systemic cortisol availability, with 11β-HSD1 in the liver generating about 30-40% of daily cortisol production, and 11βHSD2 in the kidney inactivating a similar portion.45. 20.

(22) Introduction and Aims of Thesis. 1. Figure 5. Structural similarities and differences between endogenous cortisol and cortisone, and exogenous prednisolone and prednisone. Green and pink circles indicate differences between activated (green) and inactivated (pink) corticosteroids; dashed blue circles indicate differences between endogenous cortisol and cortisone, and exogenous prednisolone and prednisone. Abbreviations: 11β-HSD1, 11beta-hydroxysteroid dehydrogenase type 1; 11β-HSD2, 11beta-hydroxysteroid dehydrogenase type 2; MW, molecular weight.. Corticosteroids and cortisol metabolism Chronic prednisolone treatment after kidney transplantation is known to suppress the HPA axis, leading to reduced endogenous cortisol synthesis by the adrenal gland (Figure 6).47-49 Moreover, recent studies in other populations suggest that exogenous corticosteroids could also interfere in cortisol metabolism by altering 11β-HSD enzyme activity (Figure 6).50-52 To date, the majority of RTR in the UMCG are still treated with prednisolone. Therefore, it would be interesting to investigate whether HPA axis activity and 11β-HSD enzyme activities are altered in these prednisolone-treated RTR compared to subjects of the general population. In addition, it would be even more interesting to investigate whether the degree to which prednisolone alters cortisol production and metabolism is related to the degree of prednisolone exposure in these patients, and thus with metabolic side effects and risk of (cardiovascular) mortality long-term after kidney transplantation. 21.

(23) Chapter 1. Figure 6. Cortisol synthesis and metabolism, and supposed effects of prednisolone. Abbreviations: CRH, corticotropin releasing hormone; ACTH, adrenocorticotropic hormone; 11β-HSD1, 11beta-hydroxysteroid dehydrogenase type 1; 11β-HSD2, 11beta-hydroxysteroid dehydrogenase type 2; THF, tetrahydrocortisol; alloTHF, allo-tetrahydrocortisol; THE, tetrahydrocortisone; PRED, prednisolone.. Systemic inflammation – A new player in the field A major effect of corticosteroids is to suppress inflammation, not only locally in transplanted organs, but also systemically, in the organism hosting the transplanted organ. Interestingly, systemic inflammation is increasingly acknowledged as a risk factor for cardiovascular morbidity and mortality in the general population.53-55 In RTR, systemic inflammation is also known to influence outcome. For example, high sensitivity C-reactive protein (CRP) has been found to be independently associated with major cardiovascular events and all-cause mortality in RTR.56,57 In addition, it was found to be associated with long-term allograft failure after kidney transplantation.58,59 22.

(24) Introduction and Aims of Thesis. The tryptophan-kynurenine pathway An interesting pathway that is tightly linked to systemic inflammation and corticosteroid exposure is the kynurenine pathway. In contrast to CRP, which is a more constitutional marker of inflammation, the kynurenine pathway may be more open to modification, because it is the major metabolic pathway of the essential amino-acid tryptophan. Under physiological conditions, tryptophan is metabolized to kynurenine by tryptophan 2,3-dioxygenase (TDO) in the liver. However, under inflammatory conditions, extra-hepatic indoleamine 2,3-dioxygenase (IDO) is activated and additionally metabolizes tryptophan to kynurenine.60,61 In the next step of the pathway, kynurenine is metabolized to cytotoxic 3-hydroxykynurenine by kynurenine 3-monooxygenase (KMO) (Figure 7).61 Both IDO and KMO enzymes are activated by pro-inflammatory stimuli and are expressed in a variety of tissues and immune cells.62 Interestingly, in experimental animal studies, IDO activation by pro-inflammatory stimuli is enhanced by GR and MR activation by exogenous dexamethasone, corticosterone and aldosterone, suggesting that corticosteroids interact with inflammatory stimuli to enhance kynurenine synthesis.63. Figure 7. The tryptophan-kynurenine pathway. High-lighted in dark pink is the toxic kynurenine metabolite 3-hydroxykynurenine.. 23. 1.

(25) Chapter 1. Modification of the kynurenine pathway as therapeutic strategy Kynurenine and particularly down-stream cytotoxic 3-hydroxykynurenine are thought to play an important role in systemic inflammation. As such, accumulation of kynurenine metabolites has been linked to the development of atherosclerosis and cardiovascular disease,64-69 particularly in patients with kidney disease.70-72 Because the kynurenine pathway is thought to play a role in the pathophysiology of many inflammation-related diseases, there is currently great interest in ways to modify this pathway. Initially, inhibition of IDO gained most interest, because this enzyme catalyzes the first and rate-limiting step of the pathway.73,74 However, recently inhibition of KMO gained more interest, because this would more directly block production of cytotoxic 3-hydroxykynurenine.73-76 In RTR, activation of the kynurenine pathway has been associated with increased risk of acute rejection.77,78 Thus, modification of the kynurenine pathway seems a promising strategy to reduce systemic inflammation and subsequent risk of cardiovascular disease in other populations, and might too in RTR. However, not much is known of the role of the kynurenine pathway in systemic inflammation in stable RTR and how this affects long-term survival of both patient and allograft.. 24.

(26) Introduction and Aims of Thesis. OUTLINE AND AIMS OF THIS THESIS Cardiovascular risk is greatly increased in RTR, which is due to an interaction of traditional and transplantation-related cardiovascular risk factors, and impairs long-term patient and allograft survival after transplantation. In addition, in the setting of kidney transplantation, several traditional cardiovascular risk factors are more prevalent, more severe, or less responsive to treatment than in non-transplanted patients. However, few adequate specific guidelines for cardiovascular risk management in RTR exist, and current guidelines are mainly based on strategies for other (high risk) populations. Therefore, the overall aim of this thesis is to make the first steps towards personalized cardiovascular risk management in RTR. More specifically, this thesis aims to identify modifiable risk factors that allow for intervention and development of RTR-specific treatment strategies, which ideally address both traditional and transplantation-related cardiovascular risk factors. In addition, it aims to identify biomarkers that allow for personalization of treatment of the individual transplant recipient. Hypertension is the most prevalent of all cardiovascular risk factors in RTR, and sodium intake is known to be an important contributing factor. In Chapter 2 we investigate the effects of dietary sodium restriction on blood pressure and albuminuria in stable RTR. Using a randomized cross-over design, we compare a sodium restricted diet with a normal sodium diet. High sodium intake is especially deleterious when serum aldosterone concentrations are also high. In Chapter 3 we review the effects of aldosterone on the kidney and vasculature, and the interaction of sodium status with aldosterone. In addition, we review potential therapeutic strategies to reduce the combined effects of these evil twins. Prednisolone treatment after kidney transplantation is associated with numerous metabolic side effects, including hypertension, which contribute to increased cardiovascular risk in RTR. It is also known to suppress endogenous cortisol production, by suppressing the hypothalamus-pituitary-adrenal (HPA) axis. In addition, prednisolone treatment has been suggested to alter systemic cortisol exposure by interfering in the enzymes that (in)activate cortisol, the 11-beta hydroxysteroid dehydrogenases (11β-HSDs). In Chapter 4 we investigate whether HPA axis activity, as measured by 24h urinary cortisol excretion, is altered in prednisolone-treated RTR, and whether the degree of HPA axis suppression is related to metabolic side effects of prednisolone. In Chapter 5 we go one step further, and zoom in not only on the effects of prednisolone on the HPA axis, but also on 11β-HSD activity, and compare RTR to healthy controls. By using 25. 1.

(27) Chapter 1. the 24h urinary cortisol metabolite profile to assess these parameters, we investigate whether altered HPA axis and 11β-HSD activity is associated with long-term (cardiovascular) mortality in prednisolone-treated RTR. Finally, we shed our light on the effects of systemic inflammation on long-term outcome after kidney transplantation. To this end, we study activation of the pro-inflammatory tryptophan-kynurenine pathway, and its association with long-term outcome after kidney transplantation in Chapter 6.. 26.

(28) Introduction and Aims of Thesis. REFERENCES 1. . Ponton P, Rupolo GP, Marchini F, et al: Quality-of-life change after kidney transplantation. Transplant Proc 33(1-2):1887-1889, 2001. 2. . Wolfe RA, Ashby VB, Milford EL, et al: Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med 341(23):1725-1730, 1999. 3. . ERA-EDTA Registry: Annual report 2014.. 4. . Stichting REgistratie NIerfunctievervanging NEderland (RENINE); available from http://www.nefrovisie.nl/nefrodata.. 5. . Nederlandse Orgaantransplantatie Registratie (NOTR); available from www.transplantatiestichting.nl.. 6. . Hariharan S, Johnson CP, Bresnahan BA, Taranto SE, McIntosh MJ, Stablein D: Improved graft survival after renal transplantation in the united states, 1988 to 1996. N Engl J Med 342(9):605-612, 2000. 7. . Meier-Kriesche HU, Schold JD, Srinivas TR, Kaplan B: Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era. Am J Transplant 4(3):378-383, 2004. 8. . Sayegh MH, Carpenter CB: Transplantation 50 years later--progress, challenges, and promises. N Engl J Med 351(26):2761-2766, 2004. 9. . Pascual M, Theruvath T, Kawai T, Tolkoff-Rubin N, Cosimi AB: Strategies to improve long-term outcomes after renal transplantation. N Engl J Med 346(8):580-590, 2002. 10. . Nankivell BJ, Kuypers DR: Diagnosis and prevention of chronic kidney allograft loss. Lancet 378(9800):1428-1437, 2011. 11. . Oterdoom LH, de Vries AP, van Ree RM, et al: N-terminal pro-B-type natriuretic peptide and mortality in renal transplant recipients versus the general population. Transplantation 87(10):1562-1570, 2009. 12. . Ojo AO: Cardiovascular complications after renal transplantation and their prevention. Transplantation 82(5):603-611, 2006. 13. . Neale J, Smith AC: Cardiovascular risk factors following renal transplant. World J Transplant 5(4):183195, 2015. 14. . Marcen R: Cardiovascular risk factors in renal transplantation--current controversies. Nephrol Dial Transplant 21 Suppl 3:iii3-8, 2006. 15. . Vanrenterghem YFC, Claes K, Montagnino G, et al: Risk factors for cardiovascular events after successful renal transplantation. Transplantation 85(2):209-216, 2008. 16. . Shirali AC, Bia MJ: Management of cardiovascular disease in renal transplant recipients. Clin J Am Soc Nephrol 3(2):491-504, 2008. 17. . Bergmann TK, Barraclough KA, Lee KJ, Staatz CE: Clinical pharmacokinetics and pharmacodynamics of prednisolone and prednisone in solid organ transplantation. Clin Pharmacokinet 51(11):711-741, 2012. 18. . Dobrowolski LC, Bemelman FJ, van Donselaar-van der Pant KA, Hoitsma AJ, ten Berge IJ, Krediet CT: Treatment efficacy of hypertension in kidney transplant recipients in the netherlands. Neth J Med 72(5):258-263, 2014. 19. . Hesselink DA, Hoorn EJ: Improving long-term outcomes of kidney transplantation: The pressure is on. Neth J Med 72(5):248-250, 2014. 20. . Thomas B, Taber DJ, Srinivas TR: Hypertension after kidney transplantation: A pathophysiologic. 27. 1.

(29) Chapter 1. approach. Curr Hypertens Rep 15(5):458-469, 2013 21. . Cross NB, Webster AC, Masson P, O’Connell PJ, Craig JC: Antihypertensive treatment for kidney transplant recipients. Cochrane Database Syst Rev (3):CD003598. doi(3):CD003598, 2009. 22. . Hiremath S, Fergusson D, Doucette S, Mulay AV, Knoll GA: Renin angiotensin system blockade in kidney transplantation: A systematic review of the evidence. Am J Transplant 7(10):2350-2360, 2007. 23. . Paoletti E, Bellino D, Marsano L, Cassottana P, Rolla D, Ratto E: Effects of ACE inhibitors on long-term outcome of renal transplant recipients: A randomized controlled trial. Transplantation 95(6):889895, 2013. 24. . Ibrahim HN, Jackson S, Connaire J, et al: Angiotensin II blockade in kidney transplant recipients. J Am Soc Nephrol 24(2):320-327, 2013. 25. . Heinze G, Mitterbauer C, Regele H, et al: Angiotensin-converting enzyme inhibitor or angiotensin II type 1 receptor antagonist therapy is associated with prolonged patient and graft survival after renal transplantation. J Am Soc Nephrol 17(3):889-899, 2006. 26. . Opelz G, Zeier M, Laux G, Morath C, Dohler B: No improvement of patient or graft survival in transplant recipients treated with angiotensin-converting enzyme inhibitors or angiotensin II type 1 receptor blockers: A collaborative transplant study report. J Am Soc Nephrol 17(11):3257-3262, 2006. 27. . Kasiske BL, Anjum S, Shah R, et al: Hypertension after kidney transplantation. Am J Kidney Dis. 28. . Vogt L, Waanders F, Boomsma F, de Zeeuw D, Navis G: Effects of dietary sodium and hydrochloro-. 43(6):1071-1081, 2004 thiazide on the antiproteinuric efficacy of losartan. J Am Soc Nephrol 19(5):999-1007, 2008 29. . Slagman MC, Waanders F, Hemmelder MH, et al: Moderate dietary sodium restriction added to angiotensin converting enzyme inhibition compared with dual blockade in lowering proteinuria and blood pressure: Randomised controlled trial. BMJ 343:d4366, 2011. 30. . Kwakernaak AJ, Krikken JA, Binnenmars SH, et al: Effects of sodium restriction and hydrochlorothiazide on RAAS blockade efficacy in diabetic nephropathy: A randomised clinical trial. Lancet Diabetes Endocrinol 2(5):385-395, 2014. 31. . McMahon EJ, Bauer JD, Hawley CM, et al: A randomized trial of dietary sodium restriction in CKD. J Am Soc Nephrol 24(12):2096-2103, 2013. 32. . Vegter S, Perna A, Postma MJ, Navis G, Remuzzi G, Ruggenenti P: Sodium intake, ACE inhibition, and progression to ESRD. J Am Soc Nephrol 23(1):165-173, 2012. 33. . van den Berg E, Geleijnse JM, Brink EJ, et al: Sodium intake and blood pressure in renal transplant recipients. Nephrol Dial Transplant 27(8):3352-3359, 2012. 34.. Prasad GV, Huang M, Nash MM, Zaltzman JS: Role of dietary salt intake in posttransplant hypertension with tacrolimus-based immunosuppression. Transplant Proc 37(4):1896-1897, 2005. 35. . Ramesh Prasad GV, Huang M, Nash MM, Zaltzman JS: The role of dietary cations in the blood. 36. . Moeller T, Buhl M, Schorr U, Distler A, Sharma AM: Salt intake and hypertension in renal transplant. pressure of renal transplant recipients. Clin Transplant 20(1):37-42, 2006 patients. Clin Nephrol 53(3):159-163, 2000 37. . Hoorn EJ, Walsh SB, McCormick JA, et al: The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension. Nat Med 17(10):1304-1309, 2011. 38. . Pimenta E, Gaddam KK, Pratt-Ubunama MN, et al: Relation of dietary salt and aldosterone to urinary. 39. . Calo LA, Puato M, Schiavo S, et al: Absence of vascular remodelling in a high angiotensin-II state. protein excretion in subjects with resistant hypertension. Hypertension 51(2):339-344, 2008 (bartter’s and gitelman’s syndromes): Implications for angiotensin II signalling pathways. Nephrol Dial Transplant 23(9):2804-2809, 2008 40. . 28. Nowaczynski W, Oliver WJ, Neel JV: Serum aldosterone and protein-binding variables in yanomama.

(30) Introduction and Aims of Thesis. indians: A no-salt culture as compared to partially acculturated guaymi indians. Clin Physiol Biochem 3(6):289-306, 1985 41. . Hurley HA, Haririan A: Corticosteroid withdrawal in kidney transplantation: The present status. Expert Opin Biol Ther 7(8):1137-1151, 2007. 42. . Srinivas TR, Meier-Kriesche HU: Minimizing immunosuppression, an alternative approach to reduc-. 43. . Haller MC, Royuela A, Nagler EV, Pascual J, Webster AC: Steroid avoidance or withdrawal for kidney. ing side effects: Objectives and interim result. Clin J Am Soc Nephrol 3 Suppl 2:S101-S116, 2008 transplant recipients. Cochrane Database Syst Rev 22(8):1-165, 2016 44. . Knight SR, Morris PJ: Steroid avoidance or withdrawal after renal transplantation increases the risk of acute rejection but decreases cardiovascular risk. A meta-analysis. Transplantation 89(1):1-14, 2010. 45. . Chapman K, Holmes M, Seckl J: 11beta-hydroxysteroid dehydrogenases: Intracellular gate-keepers of tissue glucocorticoid action. Physiol Rev 93(3):1139-1206, 2013. 46. . Tomlinson JW, Walker EA, Bujalska IJ, et al: 11beta-hydroxysteroid dehydrogenase type 1: A tis-. 47. . Boots JMM, van den Ham ECH, Christiaans MHL, van Hooff JP: Risk of adrenal insufficiency with. sue-specific regulator of glucocorticoid response. Endocr Rev 25(5):831-866, 2004 steroid maintenance therapy in renal transplantation. Transplant Proc 34(5):1696-7, 2002 48. . Bromberg JS, Alfrey EJ, Barker CF, et al: Adrenal suppression and steroid supplementation in renal transplant recipients. Transplantation 51(2):385-390, 1991. 49. . Broersen LHA, Pereira AM, Jorgensen JOL, Dekkers OM: Adrenal insufficiency in corticosteroids use: Systematic review and meta-analysis. J Clin Endocrinol Metab 100(6):2171-80, 2015. 50. . Werumeus Buning J, van Faassen M, Brummelman P, et al: Effects of hydrocortisone on the regulation of blood pressure: Results from a randomized controlled trial. The Journal of Clinical Endocrinology & Metabolism 101(10):3691-3699, 2016. 51. . Sherlock M, Behan LA, Hannon MJ, et al: The modulation of corticosteroid metabolism by hydrocortisone therapy in patients with hypopituitarism increases tissue glucocorticoid exposure. European Journal of Endocrinology 173(5):583-593, 2015. 52. . Tomlinson JW, Stewart PM: Cortisol metabolism and the role of 11beta-hydroxysteroid dehydrogenase. Best Practice & Research.Clinical Endocrinology & Metabolism 15(1):61-78, 2001. 53. . Kaplan RC, Frishman WH: Systemic inflammation as a cardiovascular disease risk factor and as a potential target for drug therapy. Heart Dis 3(5):326-332, 2001. 54. . Eapen DJ, Manocha P, Patel RS, et al: Aggregate risk score based on markers of inflammation, cell stress, and coagulation is an independent predictor of adverse cardiovascular outcomes. J Am Coll Cardiol 62(4):329-337, 2013. 55. . Wilson PW: Evidence of systemic inflammation and estimation of coronary artery disease risk: A population perspective. Am J Med 121(10 Suppl 1):S15-20, 2008. 56. . Bakri RS, Afzali B, Covic A, et al: Cardiovascular disease in renal allograft recipients is associated with elevated sialic acid or markers of inflammation. Clin Transplant 18(2):201-204, 2004. 57. . Dahle DO, Mjoen G, Oqvist B, et al: Inflammation-associated graft loss in renal transplant recipients. Nephrol Dial Transplant 26(11):3756-3761, 2011. 58. . van Ree RM, Gross S, Zelle DM, et al: Influence of C-reactive protein and urinary protein excretion on prediction of graft failure and mortality by serum albumin in renal transplant recipients. Transplantation 89(10):1247-1254, 2010. 59. . van Ree RM, Oterdoom LH, de Vries AP, et al: Elevated levels of C-reactive protein independently predict accelerated deterioration of graft function in renal transplant recipients. Nephrol Dial Transplant 22(1):246-253, 2007. 29. 1.

(31) Chapter 1. 60. . Ozaki Y, Edelstein MP, Duch DS: Induction of indoleamine 2,3-dioxygenase: A mechanism of the antitumor activity of interferon gamma. Proc Natl Acad Sci U S A 85(4):1242-1246, 1988. 61. . Takikawa O, Yoshida R, Kido R, Hayaishi O: Tryptophan degradation in mice initiated by indoleamine 2,3-dioxygenase. J Biol Chem 261(8):3648-3653, 1986. 62. . Yamazaki F, Kuroiwa T, Takikawa O, Kido R: Human indolylamine 2,3-dioxygenase. its tissue distribution, and characterization of the placental enzyme. Biochem J 230(3):635-638, 1985. 63. . Brooks AK, Lawson MA, Smith RA, Janda TM, Kelley KW, McCusker RH: Interactions between inflammatory mediators and corticosteroids regulate transcription of genes within the kynurenine pathway in the mouse hippocampus. J Neuroinflammation 13(1):98-016-0563-1, 2016. 64. . Eussen SJ, Ueland PM, Vollset SE, et al: Kynurenines as predictors of acute coronary events in the hordaland health study. Int J Cardiol 189:18-24, 2015. 65. . Pedersen ER, Midttun O, Ueland PM, et al: Systemic markers of interferon-gamma-mediated immune activation and long-term prognosis in patients with stable coronary artery disease. Arterioscler Thromb Vasc Biol 31(3):698-704, 2011. 66. . Pedersen ER, Svingen GF, Schartum-Hansen H, et al: Urinary excretion of kynurenine and tryptophan, cardiovascular events, and mortality after elective coronary angiography. Eur Heart J 34(34):2689-2696, 2013. 67. . Pedersen ER, Tuseth N, Eussen SJ, et al: Associations of plasma kynurenines with risk of acute myocardial infarction in patients with stable angina pectoris. Arterioscler Thromb Vasc Biol 35(2):455-462, 2015. 68. . Sulo G, Vollset SE, Nygard O, et al: Neopterin and kynurenine-tryptophan ratio as predictors of coronary events in older adults, the hordaland health study. Int J Cardiol 168(2):1435-1440, 2013. 69. . Zuo H, Ueland PM, Ulvik A, et al: Plasma biomarkers of inflammation, the kynurenine pathway, and risks of all-cause, cancer, and cardiovascular disease mortality: The hordaland health study. Am J Epidemiol 183(4):249-258, 2016. 70. . Sallee M, Dou L, Cerini C, Poitevin S, Brunet P, Burtey S: The aryl hydrocarbon receptor-activating effect of uremic toxins from tryptophan metabolism: A new concept to understand cardiovascular complications of chronic kidney disease. Toxins (Basel) 6(3):934-949, 2014. 71. . Pawlak K, Domaniewski T, Mysliwiec M, Pawlak D: Kynurenines and oxidative status are independently associated with thrombomodulin and von willebrand factor levels in patients with end-stage renal disease. Thromb Res 124(4):452-457, 2009. 72. . Pawlak K, Domaniewski T, Mysliwiec M, Pawlak D: The kynurenines are associated with oxidative stress, inflammation and the prevalence of cardiovascular disease in patients with end-stage renal disease. Atherosclerosis 204(1):309-314, 2009. 73. . Dounay AB, Tuttle JB, Verhoest PR: Challenges and opportunities in the discovery of new therapeutics targeting the kynurenine pathway. J Med Chem 58(22):8762-8782, 2015. 74. . Zadori D, Veres G, Szalardy L, et al: Inhibitors of the kynurenine pathway as neurotherapeutics: A patent review (2012-2015). Expert Opin Ther Pat 26(7):815-832, 2016. 75. . Mole DJ, Webster SP, Uings I, et al: Kynurenine-3-monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis. Nat Med 22(2):202-209, 2016. 76. . Smith JR, Jamie JF, Guillemin GJ: Kynurenine-3-monooxygenase: A review of structure, mechanism, and inhibitors. Drug Discov Today 21(2):315-324, 2016. 77. . Brandacher G, Cakar F, Winkler C, et al: Non-invasive monitoring of kidney allograft rejection through IDO metabolism evaluation. Kidney Int 71(1):60-67, 2007. 78. . Lahdou I, Sadeghi M, Daniel V, et al: Increased pretransplantation plasma kynurenine levels do not protect from but predict acute kidney allograft rejection. Hum Immunol 71(11):1067-1072, 2010. 30.

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(34) Chapter 2 EFFECTS OF DIETARY SODIUM RESTRICTION IN KIDNEY TRANSPLANT RECIPIENTS TREATED WITH RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM BLOCKADE: A RANDOMIZED CLINICAL TRIAL. Laura V. de Vries Linn C. Dobrowolski Jacqueline J.O.N. van den Bosch Ineke J. Riphagen C.T. Paul Krediet Frederike J. Bemelman Stephan J.L. Bakker Gerjan Navis Am J Kidney Dis. 2016; 67: 936-944..

(35) Chapter 2. ABSTRACT BACKGROUND: In patients with chronic kidney disease receiving renin-angiotensin-aldosterone system (RAAS) blockade, dietary sodium restriction is an often-used treatment strategy to reduce blood pressure (BP) and albuminuria. Whether these effects extend to kidney transplant recipients is unknown. We therefore studied the effects of dietary sodium restriction on BP and urinary albumin excretion (UAE) in kidney transplant recipients receiving RAAS blockade. STUDY DESIGN: Two-center randomized cross-over trial. SETTING & PARTICIPANTS: Stable outpatient kidney transplant recipients with creatinine clearance >30 mL/min, BP ≥ 120/80 mmHg, receiving stable RAAS blockade therapy. INTERVENTION: 6-week regular-sodium diet (target, 150 mmol/24h) and a 6-week low-sodium diet (target, 50 mmol/24h). OUTCOMES & MEASUREMENTS: Main outcome parameters were systolic and diastolic BP, UAE, and estimated glomerular filtration rate (eGFR) at the end of each diet period. Dietary adherence was assessed by 24h urinary sodium excretion. RESULTS: We randomly assigned 23 kidney transplant recipients, of whom 22 (mean age, 58 ± 8 [SD] years; 50% men; mean eGFR, 51 ± 21 mL/min/1.73 m2) completed the study. One patient withdrew from the study because of concerns regarding orthostatic hypotension on the low-sodium diet. Sodium excretion decreased from 164 ± 50 mmol/24h during the regular-sodium diet to 87 ± 55 mmol/24h during the low-sodium diet (mean difference, -77 [95% CI, -110 to -44] mmol/24h; P<0.001). Sodium restriction significantly reduced systolic BP from 140 ± 14 mmHg to 129 ± 12 mmHg (mean difference, -11 [95% CI, -14 to -7] mmHg; P<0.001), diastolic BP from 86 ± 8 mmHg to 79 ± 8 mmHg (mean difference, -7 [95% CI, -10 to -5] mmHg; P<0.001). We found no significant effect on natural log (ln)-transformed UAE (mean difference, -0.03 [95% CI, -0.6 to 0.6] ln(mg/24h); P=0.9) or eGFR. LIMITATIONS: No hard end points; small study; small proportion of patients willing to test the intervention; adherence to sodium diet was achieved in 86% of patients. CONCLUSIONS: In stable kidney transplant recipients receiving RAAS blockade, dietary sodium restriction effectively reduces BP without affecting eGFR. Dietary sodium restriction is relevant to BP management in kidney transplant recipients receiving RAAS blockade. 34.

(36) Sodium Restriction in Renal Transplant Recipients. INTRODUCTION Hypertension and albuminuria are common after kidney transplantation and are major risk factors for cardiovascular disease and transplant failure in this population.1-3 Up to 90% of kidney transplant recipients have high blood pressure (BP) or use antihypertensive drugs,4-6 and up to 40% have albuminuria.7,8 In patients with native chronic kidney disease (CKD), renin-angiotensin-aldosterone system (RAAS) blockade is the standard of care for the treatment of hypertension and albuminuria.9-12 Meanwhile, this class of drugs has largely been avoided in kidney transplant recipients because two meta-analyses of otherwise inconclusive data pointed towards an advantage of calcium channel blockers instead of RAAS blockers for BP control in this population.13,14 However, this view changed recently when data became available from two well-conducted clinical trials in kidney transplant recipients suggesting an advantage of prolonged treatment with RAAS blockade in kidney transplant recipients.15,16 High sodium intake has been shown to blunt the antihypertensive and antiproteinuric effects of RAAS blockade.17 Interestingly, moderate dietary sodium restriction potentiates RAAS blockade efficacy and effectively reduces BP and proteinuria in patients with diabetic18 and nondiabetic CKD.19,20 Moreover, several studies show that low sodium intake is associated with much better kidney disease and cardiovascular outcomes in patients with CKD.21,22 However, there are indications of a U-shaped association of sodium intake with outcomes, with increased risks at both very low and excessive sodium intakes.23,24 Therefore, the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (NKF-KDOQI) and Dietary Approaches to Stop Hypertension (DASH) guidelines advocate a maximum sodium intake of 100 mmol/day.10,25 Despite these recommendations, average sodium intake in kidney transplant recipients is about 150 to 200 mmol/day.26-29 Furthermore, treatment with calcineurin inhibitors and corticosteroids, in addition to decreased kidney function and prevalent obesity, may render BP even more sodium sensitive in kidney transplant recipients compared with patients with CKD in general.30-32 In line with this, a recent cross-sectional study of 660 kidney transplant recipients showed a strong association of sodium intake with BP.26 Therefore, although dietary sodium restriction might have beneficial effects in kidney transplant recipients, evidence from clinical trials is lacking. The aim of this randomized cross-over clinical trial is therefore to assess the effects of dietary sodium restriction on BP and urinary albumin excretion (UAE) in stable outpatient kidney transplant recipients receiving RAAS blockade. 35. 2.

(37) Chapter 2. MATERIALS AND METHODS Study Design This is a two-center cross-over randomized clinical trial performed at the University Medical Center Groningen (UMCG) and the Academic Medical Center Amsterdam, the Netherlands, January 2012 to May 2014. The study protocol was in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the UMCG (METc 2011/131). The study was conducted according to the guidelines of Good Clinical Practice. Written informed consent was obtained from each patient before inclusion. The CONSORT (Consolidated Standards of Reporting Trials) checklist was used as a reporting reference.33,34 Participants We screened all kidney transplant recipients who visited the outpatient nephrology transplantation clinic of the UMCG and Academic Medical Center Amsterdam and who had undergone kidney transplantation at least 1 year before. Kidney transplant recipients were invited to participate in the study if they were 18 years or older and had stable kidney transplant function with creatinine clearance of at least 30 mL/min and BP ≥ 120/80 mmHg. An angiotensin-converting enzyme inhibiter or angiotensin receptor blocker had to be part of their antihypertensive regimen. For safety reasons, we excluded kidney transplant recipients with systolic BP (SBP) ≥ 180 mmHg or diastolic BP (DBP) ≥ 100 mmHg. Other exclusion criteria were use of a calcineurin inhibitor withdrawal regimen or corticosteroid withdrawal regimen or (suspected) rejection of the transplant; accordingly, the immunosuppressive regimen was kept stable throughout the study. Furthermore, pregnancy or lactation, active malignancy, insufficient mastery of the Dutch language to participate in the study, or participation in another intervention study during or within a month prior to this study were also exclusion criteria. Immunosuppressive Regimen After Transplantation Standard immunosuppression consisted of the following: cyclosporine standard formulation (Sandimmune; Novartis Pharma bv; 10 mg/kg; trough levels of 175-200 µg/L for the first 3 months, 150-175 µg/L 3-12 months post-transplantation, and 100-150 µg/L thereafter) combined with prednisolone (starting with 20 mg/day, rapidly tapered to 10 mg/day) in kidney transplant recipients who underwent transplantation January 1988 to February 1993; cyclosporine microemulsion (Neoral; Novartis Pharma bv; 10 mg/kg; trough levels the same as for Sandimmune) and prednisolone in kidney transplant recipients who underwent transplantation March 1993 to May 1997; mycophenolate mofetil (Cellcept; Roche bv; 2 g/day) was added in May 1997 and used to date; tacrolimus (Prograft; Astellas Pharma bv; 0.25 mg/kg; trough levels of 8-12 µg/L 36.

(38) Sodium Restriction in Renal Transplant Recipients. for the first 3 months, 6-10 µg/L 3-12 months post-transplantation, and 4-8 µg/L thereafter) replaced cyclosporine as standard therapy in March 2011. Dietary Intervention A schematic overview of the study design is shown in Figure 1. At inclusion, participants were assigned to a 6-week period of a regular-sodium diet or low-sodium diet. A cross-over design was used, such that each patient received 6 weeks of both diets. To prevent systematic errors resulting from the cross-over design, diet order was assigned randomly. For allocation, a computer-generated list of random numbers was used. The study protocol did not include a wash-out period between diet periods because one of the study arms was regular sodium intake and the study arms lasted 6 weeks, making it unlikely that carry-over effects would occur. Sodium intake during the low-sodium diet was targeted at 50 mmol/day (~1,200 mg of sodium or 3 g of sodium chloride [NaCl] per day). Sodium intake during the regular-sodium diet was targeted at 150 mmol/day (~3,600 mg of sodium or 9 g of NaCl per day) because average sodium intake of kidney transplant recipients in the Netherlands is about 150 mmol/day.6,26 To increase feasibility and adherence, participants received individualized dietary counseling by the research physicians, which focused on remaining as close as possible to the participant’s nutritional preferences and habits. During the regular-sodium diet, participants were advised to maintain normal habits regarding sodium intake. During the low-sodium diet, participants were advised not to add salt to their food and to replace sodium-rich products with low-sodium or sodium-free alternatives. In addition, they were instructed to maintain an isocaloric diet and stable protein intake. Adherence to the sodium diet was monitored by measuring urinary sodium excretion in 24h urine samples half-way through and at the end of each 6-week period. To ensure adequate 24h urine collection, participants were carefully instructed to start 24h urine collection with emptying of the bladder, record the time of voiding, and collect all subsequent urine through the next 24 hours and include the next morning’s specimen on the day of their visit to the outpatient clinic. After each measurement, participants received oral feedback on their sodium intake and dietary advice. Adequate adherence to the study diet was defined as having at least a 35-mmol reduction in 24h sodium excretion (equaling an intake reduction of 2 g of NaCl) from the regular-sodium diet to the low sodium diet, based on the difference between 24h sodium excretion assessed at 6 and 12 weeks. By study design, antihypertensive medication was not changed during the study periods unless patients experienced severe orthostatic complaints. If patients contacted us with such complaints, they were asked to visit the outpatient clinic, where it was decided whether antihypertensive medication had to be reduced based on combined information for BP and severity of the complaints.. 37. 2.

(39) Chapter 2. Figure 1. Study design. Abbreviations: BP, blood pressure measurement; lab, laboratory measurements; U24, 24h urine collection.. Measurements Measurements were performed at baseline and after 6 and 12 weeks (Figure 1). At each visit, fasting blood and 24h urine samples of the preceding day were taken, and anthropometry (including height, weight, and waist and hip circumference) and BP measurement were performed. Waist and hip circumferences were measured as previously described.35 BP was measured at 1-minute intervals for 15 minutes with a semiautomatic device (Dinamap; GE Medical Systems) while the patient was left alone in a room in a semi-supine position.20,26,36 After 15 minutes of measurements, we discarded the last BP measurement to avoid confounding and used the mean of the second-to-last 4 readings for analysis of study BP. Blood electrolytes, lipids, and proteins and urinary electrolytes were measured by using an automated multianalyzer (Modular; Roche Diagnostics). Urinary albumin was measured with a turbidimetric assay using benzethonium chloride (Modular). N-Terminal pro-brain natriuretic peptide (NT-proBNP) was measured with a chemiluminescence sandwich immunoassay (Elecsys; Roche Diagnostics), aldosterone was measured with a competitive fixed-time solid-phase radioimmunoassay (Coat-a-Count; Siemens Medical Solutions Diagnostics), and renin was measured with a chemiluminescence sandwich immunoassay (Liaison; DiaSorin). Second-center (Academic Medical Center Amsterdam) samples were stored at -80°C and analyzed in the UMCG using analytic methods as described. Estimated glomerular filtration rate (eGFR) was calculated using the 4-variable MDRD (Modification of Diet in Renal Disease) Study equation. Relevant donor and transplantation-related characteristics were obtained from patients’ medical records.. 38.

(40) Sodium Restriction in Renal Transplant Recipients. Outcomes Primary outcome parameters were SBP and DBP. The secondary outcome parameter was UAE. Tertiary outcome parameters were creatinine clearance, eGFR, serum creatinine, body weight, serum sodium, potassium, urea, total cholesterol, NT-proBNP, renin, and aldosterone and urinary excretion of urea, calcium, potassium, and creatinine. Statistical Analysis Our study was primarily powered to detect an effect on SBP. We calculated a sample size of 22 patients (by cross-over design) to detect a difference of 8 mmHg,37 with standard deviation of 7 mmHg, in SBP with a power of 90%. A drop-out rate of 10% (=2 kidney transplant recipients) was taken into account. Therefore, 24 kidney transplant recipients were intended to be included in the study. Statistical analyses were performed with SPSS, version 22.0, for Windows (SPSS Inc) and GraphPad Prism, version 5.0 (GraphPad Software Inc). Normally distributed variables are given as mean ± standard deviation; non-normally distributed variables, as median and interquartile range; and categorical variables as absolute number and percentage. A 2-tailed P<0.05 was considered statistically significant. We tested for differences between participants and non-participants using t tests for normally distributed data, Mann-Whitney U tests for non-normally distributed data, and χ2 tests for nominal data. We found no significant differences between participants and non-participants in terms of sex, transplant vintage, body weight, body mass index, creatinine clearance, eGFR, serum creatinine, proteinuria, and sodium intake values. Participants were slightly older compared with non-participants (mean age, 58 ± 8 years vs 52 ± 13 years; P=0.004). To estimate the effects of sodium restriction on clinical parameters, we used linear mixed-effect models for repeated measurements, including a Bonferroni correction, using the unstructured covariance structure with ‘diet’ and ‘sequence’ as fixed effects and ‘participant’ as random effect. Skewed data were logarithmically transformed before statistical analysis. We checked for potential carry-over or sequence effects by means of linear mixed models with ‘diet’ and ‘sequence’ and their interaction ‘diet x sequence’ as fixed effects and ‘participant’ as random effect.. 39. 2.

(41) Chapter 2. RESULTS Patient Characteristics Of 181 eligible kidney transplant recipients, 25 gave written informed consent. Of these 25 kidney transplant recipients, two withdrew consent before randomization. Of the 23 who were randomly assigned, one patient withdrew halfway through the low-sodium period because of orthostatic hypotension (Figure 2). For the primary analysis, we analyzed data for all 22 participants who completed the study according to the intention-to-treat principle. We performed additional per-protocol analyses for the primary and secondary outcome parameters, in which we first excluded participants who were non-adherent to the study diet (n=19 in analysis), subsequently excluded participants who required cessation of 1 or more classes of their antihypertensive medication (n=20 in analysis), and finally excluded both groups, leaving 17 kidney transplant recipients for the final per-protocol analysis. At baseline, mean age was 58 ± 8 (standard deviation) years, 50% were men, mean creatinine clearance was 70 ± 32 mL/min, and median UAE was 40 (interquartile range, 16-141) mg/24h. All participants used RAAS blockade as antihypertensive treatment, and 18 of 22 (82%) used one or more antihypertensive drug beyond RAAS blockade (Table 1). All participants used prednisolone as maintenance immunosuppressive therapy, with the addition of cyclosporine (36%) or tacrolimus (18%) and/or mycophenolate mofetil (68%) or azathioprine (9%).. Figure 2. Study inclusion flow chart.. 40.

(42) Sodium Restriction in Renal Transplant Recipients. Dietary Adherence Sodium excretion was significantly reduced from 164 ± 50 mmol/24h during the regular-sodium diet to 87 ± 56 mmol/24h during the low-sodium diet (P<0.001; Table 2). This 77-mmol reduction in sodium excretion equaled a reduction of ~2 g of sodium or 4.5 g of salt (NaCl) per day. We found no significant change in natural log (ln)-transformed urinary creatinine excretion between the regular- and low-sodium diets (0.0; 95% CI, -0.1 to 0.1 ln(g/24h); P=0.9; Table 2), indicating accurate 24h urine sample collection. Adherence to the study diet, defined as having at least a 35-mmol reduction in 24h sodium excretion from the regular-sodium to the low-sodium diet, based on 24h sodium excretion at 6 and 12 weeks, was achieved in 19 of 22 (86%) participants. Primary Outcome Parameter: Blood Pressure During the regular-sodium diet, mean SBP was 140 ± 14 mmHg and mean DBP was 86 ± 8 mmHg. Sodium restriction significantly reduced SBP (mean difference, -11 [95% CI, -14 to -7] mmHg; P<0.001; Table 2) and DBP (mean difference, -7 [95% CI, -10 to -5] mmHg; P<0.001; Table 2). We found no significant carry-over or sequence effects for SBP and DBP. Both SBP and DBP decreased in 20 of 22 (91%), remained stable in 1 of 22 (4.5%), and increased in 1 of 22 (4.5%) participants (Figure 3A-B). Orthostatic hypotension was present in none of the participants during the regular-sodium diet, whereas it was present in 5 participants during the low-sodium diet. Of these 5 participants, 2 needed tapering of their antihypertensive regimen to resolve the orthostatic hypotension. In the first of these 2 participants, hydrochlorothiazide dosage was halved to 12.5 mg once daily, metoprolol dosage was halved to 25 mg twice daily, and treatment with doxazosin, 4 mg, was discontinued. In the second, metoprolol dosage was halved to 50 mg twice daily, and treatment with hydrochlorothiazide, 12.5 mg, was discontinued. Both these participants remained on RAAS blockade during the entire study period. Finally, one participant withdrew from the study because of orthostatic hypotension. Secondary Outcome Parameter: Urinary Albumin Excretion We found no significant reduction in ln(UAE) (mean difference, -0.03 [95% CI, -0.6 to 0.6] ln(mg/24h); P=0.9; Table 2). We also found no significant carry-over or sequence effects for UAE. Individual data show that UAE decreased in 13 of 22 (59%), remained stable in 1 of 22 (5%), and increased in 8 of 22 (36%) participants (Figure 3C). Tertiary Outcome Parameters We found no significant effect of sodium restriction on creatinine clearance (mean difference, -1 [95% CI, -9 to 6] mL/min; P=0.7), eGFR (mean difference, -0.5 [95% CI, -0.3 to 2] mL/min/1.73 m2; P=0.7), or natural log-transformed serum creatinine (mean difference, 0.02 [95% CI, -0.03 to 0.07] ln(mg/dL); P=0.4; Table 2). Body weight signifi41. 2.

(43) Chapter 2. cantly decreased from 83 ± 15 to 81 ± 14 kg (mean difference, -2 [95% CI, -3 to -1] kg; P<0.001; Table 2). We found a non-significant increase in plasma renin (P=0.1) and a significant increase in plasma aldosterone concentrations (P<0.001; Table 2). Serum sodium concentration decreased during dietary sodium restriction, but remained well within the reference range (Table 2).. Figure 3. Change in [A] systolic blood pressure (SBP), [B] diastolic blood pressure (DBP), and [C] urinary albumin excretion (UAE) for individual kidney transplant recipients in response to sodium restriction. Each dot represents an individual patient (N=22). Abbreviations: LS, low-sodium diet; RS, regular-sodium diet.. Per-Protocol Analyses We performed per-protocol analyses for the primary and secondary outcome parameters and creatinine clearance, in which we excluded participants with diet non-adherence or protocol deviations from the analyses. For SBP and DBP, results of the per-protocol analyses were not materially different from those of the primary analysis. The same held true for creatinine clearance (Table 3). Interestingly, we found a trend between sodium restriction and ln(UAE) after exclusion of participants who were non-adherent to the study diet and those who needed cessation of antihypertensive medication (mean difference, -0.4 [95% CI, -0.9 to 0.0] ln(mg/24h); P=0.07; Table 3).. 42.

(44) Sodium Restriction in Renal Transplant Recipients. Table 1. Baseline characteristics of intention-to-treat population. Intention-to-treat population (n=22) Patient demographics Age, yrs. 58 ± 8. Male sex, n (%). 11 (50). Body composition Height, m. 1.74 ± 0.10. Weight, kg. 83 ± 14. Hip, cm. 103 ± 10. Waist, cm. 101 ± 12. BMI, kg/m2. 27.1 ± 3.6. 2. Blood pressure SBP, mmHg. 138 ± 15. DBP, mmHg. 85 ± 9. Number of antihypertensive drugs 1, n (%). 4 (18). 2, n (%). 6 (27). 3, n (%). 9 (41). 4, n (%). 3 (14). Type of antihypertensive drugs RAAS blockade, n (%). 22 (100). Calcium channel blockade, n (%). 6 (27). β-blockade, n (%). 11 (50). α-blockade, n (%). 3 (14). Diuretic, n (%). 12 (55). Kidney function Serum creatinine, mg/dL Creatinine clearance, mL/min eGFR, mL/min/1.73m2 Urinary albumin excretion, mg/24h. 1.4 [1.2-1.6] 70 ± 32 51 ± 21 40 [15-142]. Transplant characteristics Transplant vintage, yrs. 7.3 [3.1-11.5]. Living donor, n (%). 11 (50). Prior dialysis, n (%). 15 (68). Immunosuppression Tacrolimus, n (%). 4 (18). Cyclosporine, n (%). 8 (36). Mycophenolate mofetil, n (%). 15 (68). Azathioprine, n (%) Prednisolone dose, mg. 2 (9) 7.5 [7.5-10]. Values for categorical variables are given as number (percentage); values for continuous variables are given as mean ± standard deviation or median [interquartile range]. Conversion factor for units: creatinine in mg/ dL to μmol/L, ×88.4. Abbreviations: α, alpha-receptor; β, beta-receptor; BMI, body mass index; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; RAAS, renin-angiotensin-aldosterone system; SBP, systolic blood pressure.. 43.

(45) Chapter 2. Table 2. Effect of sodium restriction on clinical and biochemical parameters, intention-to-treat analysis. Values after interventiona. Treatment effectb. RS (n=22). LS (n=22). LS vs. RS. P-value. 164 ± 50. 87 ± 56. -77 (-110, -44). <0.001. SBP, mmHg. 140 ± 14. 129 ± 12. -11 (-14, -7). <0.001. DBP, mmHg. 86 ± 8. 79 ± 8. -7 (-10, -5). <0.001. Sodium excretion, mmol/24h Blood pressure. Body composition Weight, kg. 83 ± 15. 81 ± 14. -2 (-3, -1). <0.001. Hip, cm. 104 ± 10. 103 ± 9. -0.8 (-1.7, -0.1). 0.06. Waist, cm. 101 ± 13. 99 ± 11. -2 (-4, 1). 0.1. 1.4 [1.2-1.7]. 1.4 [1.2-1.8]. 0.34 ± 0.37. 0.36 ± 0.39. 0.02 (-0.03, 0.07). 0.4. 50 ± 18. 49 ± 20. -0.5 (-0.3, 2). 0.7. -1 (-9, 6). 0.7. -0.03 (-0.6, 0.6). 0.9 0.003. Kidney function Serum creatinine, mg/dL ln(serum creatinine), ln(mg/dL) eGFR, mL/min/1.73m2 Creatinine clearance, mL/min UAE, mg/24h ln(UAE), ln(mg/24h). 67 ± 25. 66 ± 27. 29 [11-99]. 22 [13-94]. 3.5 ± 1.8. 3.4 ± 1.5. Blood Sodium, mmol/L. 141 ± 4. 139 ±4. -2 (-3, -1). Potassium, mmol/L. 4.1 ± 0.5. 4.1 ± 0.6. 0.0 (-0.2, 0.2). 0.8. Urea, mmol/L. 10.8 ± 4.6. 11.5 ± 6.3. 0.6 (-0.7, 2.0). 0.3. Albumin, g/L. 43 ± 3. 44 ± 2. 0.4 (-0.8, 1.5). 0.5. Total protein, g/L. 68 ± 4. 69 ± 4. 0.4 (-1.4, 2.2). 0.7. 196 ± 29. 193 ± 32. -4 (-11, 4). 0.3. 133 [71-321]. 128 [55-273] -0.1 (-0.4, 0.1). 0.3. 0.2 (-0.1, 0.5). 0.1. 0.5 (0.3, 0.8). <0.001. 0.0 (-0.1, 0.1). 0.9. -0.1 (-0.2, 0.0). 0.2. -0.1 (-0.2, 0.0). 0.1. -0.2 (-0.4, 0.1). 0.3. Total cholesterol, mg/dL NT-proBNP, ng/L ln(NT-proBNP), ln(ng/L) Renin, IU/mL ln(Renin), ln(IU/mL) Aldosterone, pmol/L ln(Aldosterone), ln(pmol/L). 5.0 ± 0.9. 4.9 ± 1.0. 105 [47-241]. 153 [72-337]. 4.6 ± 1.2. 4.8 ± 1.3. 276 [149-514]. 476 [264-759]. 5.6 ± 0.9. 6.1 ± 0.9. 1.2 [1.1-1.5]. 1.3 [1.1-1.5]. Urine Creatinine, g/24h ln(Creatinine), ln(g/24h) Urea, mmol/24h ln(Urea), ln(mmol/24h) Potassium, mmol/24h ln(Potassium), ln(mmol/24h) Calcium, mmol/24h ln(Calcium), ln(mmol/24h). 0.2 ± 0.3. 0.2 ± 0.2. 426 [326-480]. 376 [303-432]. 6.0 ± 0.3. 5.9 ± 0.2. 82 [66-104]. 71 [57-90]. 4.4 ± 0.3. 4.3 ± 0.4. 2.5 [1.3-3.8]. 1.4 [0.9-3.8]. 0.7 ±1.1. 0.6 ±0.9. Variables with a skewed distribution were ln-transformed before analyses. Conversion factors for units: creatinine in mg/dL to μmol/L, ×88.4; total cholesterol in mg/dL to mmol/L, ×0.02586. Abbreviations: DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; LS, low sodium diet; NT-proBNP, n-terminal pro-brain natriuretic peptide; RS, regular sodium diet; SBP, systolic blood pressure; UAE, urinary albumin excretion. a Data are presented as unadjusted mean ± standard deviation or median [interquartile range]. b Data are mean differences (95% CI) obtained from linear mixed-effect models for repeated measurements.. 44.

(46) Sodium Restriction in Renal Transplant Recipients. Table 3. Effect of sodium restriction on primary and secondary outcome parameters, per protocol analysis. Values after interventiona. Treatment effectb. n. RS. LS. LS vs RS. P-value. KTR with diet non-adherence excluded. 19. 139 ± 14. 128 ± 12. -11 (-14, -8). <0.001. KTR with AHT cessation excluded. 20. 140 ± 15. 129 ± 11. -10 (-14, -6). <0.001. Both subgroups above excluded. 17. 139 ± 14. 129 ± 12. -11 (-14, -7). <0.001. KTR with diet non-adherence excluded. 19. 86 ± 8. 78 ± 8. -8 (-10, -6). <0.001. KTR with AHT cessation excluded. 20. 87 ± 7. 79 ± 7. -8 (-10, -5). <0.001. Both subgroups above excluded. 17. 86 ± 8. 78 ± 7. -8 (-11, -6). <0.001. Systolic blood pressure, mmHg. 2. Diastolic blood pressure, mmHg. ln(Urinary albumin excretion), ln(mg/24h) KTR with diet non-adherence excluded. 19. 3.4 ± 1.9. 3.3 ± 1.5. -0.01 (-0.7, 0.7). 0.9. KTR with AHT cessation excluded. 20. 3.7 ± 1.7. 3.4 ± 1.5. -0.4 (-0.7, 0.0). 0.06. Both subgroups above excluded. 17. 3.7 ± 1.7. 3.2 ± 1.5. -0.4 (-0.9, 0.0). 0.07. KTR with diet non-adherence excluded. 19. 70 ± 25. 68 ± 27. -2 (-11, 7). 0.6. KTR with AHT cessation excluded. 20. 66 ± 25. 66 ± 28. 0 (-8, 8). 0.9. Both subgroups above excluded. 17. 69 ± 24. 68 ± 28. -1 (-11, 9). 0.9. Creatinine clearance, mL/min. Data are presented as unadjusted mean ± standard deviation or median [interquartile range]. b Data are mean differences (95% CI) obtained from linear mixed-effect models for repeated measurements. Variables with a skewed distribution were ln-transformed before analyses. Abbreviations: AHT, antihypertensive treatment; LS, low sodium diet; RS, regular sodium diet. a. 45.

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