Endothelin-A Receptor Antagonism Modifies Cardiovascular Risk

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Endothelin-A Receptor Antagonism Modiﬁes Cardiovascular Risk Factors in CKD

† † ‡ ‡ † Neeraj Dhaun,* Vanessa Melville, Scott Blackwell, Dinesh K. Talwar, Neil R. Johnston, † † Jane Goddard,* and David J. Webb

*Department of Renal Medicine, Royal Inﬁrmary of Edinburgh, Edinburgh, United Kingdom; †BHF Centre of Research Excellence, University of Edinburgh, The Queen’s Medical Research Institute, Edinburgh, United Kingdom; and ‡Department of Clinical Biochemistry & Metabolic Medicine, Royal Inﬁrmary of Glasgow, Glasgow, United Kingdom BRIEF COMMUNICATION

ABSTRACT Arterial stiffness and impaired nitric oxide (NO) bioavailability contribute to the high levels has both cardiovascular and renal risk for cardiovascular disease in CKD. Both asymmetric dimethylarginine (ADMA), benefits.11–13 an endogenous inhibitor of NO production, and endothelin-1 (ET-1) oppose the Asymmetricdimethylarginine(ADMA) actions of NO, suggesting that ET-1 receptor antagonists may have a role in is an endogenous inhibitor of NO cardiovascular protection in CKD. We conducted a randomized, double-blind, synthases. By inhibiting NO formation, three-way crossover study in 27 patients with proteinuric CKD to compare the ADMA causes endothelial dysfunction, effects of the ETA receptor antagonist sitaxentan, nifedipine, and placebo on pro- vasoconstriction, elevation of BP,and proteinuria, BP, arterial stiffness, and various cardiovascular biomarkers. After 6 weeks gression ofexperimental atherosclerosis.14 of treatment, placebo and nifedipine did not affect plasma urate, ADMA, or urine ADMA concentrations are increased in ET-1/creatinine, which reflects renal ET-1 production; in contrast, sitaxentan led to patients with CKD,14 and clinical data statistically significant reductions in all three of these biomarkers. No treatment support ADMA as an independent affected plasma ET-1. Reductions in proteinuria and BP after sitaxentan treatment marker of CKD progression, cardiovascu- was associated with increases in urine ET-1/creatinine, whereas reduction in pulse- lar morbidity, and overall mortality.15–17 wave velocity, a measure of arterial stiffness, was associated with a decrease in Studies have shown a reduction in ADMA

ADMA. Taken together, these data suggest that ETA receptor antagonism may after therapy in patients with hyperten- modify risk factors for cardiovascular disease in CKD. sion and hypercholesterolemia,18,19 but not in patients with CKD. J Am Soc Nephrol 24: 31–36, 2013. doi: 10.1681/ASN.2012040355 ET-1 is a potent endogenous vasoconstrictor produced within the vasculature. It is implicated in both the CKD is common, affecting 6%–11% of and vasoconstrictor endothelin (ET) sys- development and progression of CKD.20 the population globally.1 It is strongly tems have been identiﬁed as potential Its effects are mediated via two receptors, associated with incident cardiovascular contributors to increased cardiovascular the ETA and ETB receptors; the major 2 5 disease (CVD). This increased cardio- risk in patients with CKD. These are all pathologic effects are ETA receptor me- vascular risk is not adequately explained common in a typical CKD population.3,6 diated.20 We have recently shown that by conventional (Framingham) risk fac- Epidemiologic studies report a relation- long-term selective ETA receptor tors, such as hypertension, hypercholes- ship between serum uric acid and a wide terolemia, diabetes mellitus, and smoking, variety of cardiovascular conditions, in- all of which are common in patients with cluding hypertension, diabetes mellitus, Received April 6, 2012. Accepted October 10, 2012. CKD. Thus, emerging cardiovascular risk coronary artery disease, cerebrovascular factors have been an area of intense inves- disease, and CKD.7 Indeed, serum uric Published online ahead of print. Publication date tigation.3 Arterial stiffness4 makes an im- acid is considered by some to be an in- available at www.jasn.org. portant independent contribution to dependent risk factor for both CVD8,9 Correspondence: Dr. Neeraj Dhaun, The Queen’s CVD risk in CKD, and this is promoted and CKD.10 Others have noted that an Medical Research Institute, 3rd Floor East, Room E3.23, 47 Little France Crescent, Edinburgh EH16 by both conventional and emerging car- elevated serum uric acid level predicts 4TJ, United Kingdom. Email: [email protected]. diovascular risk factors. the development of hypertension and uk 7 Hyperuricemia and a shift in the bal- CKD. Of note, emerging clinical data Copyright © 2013 by the American Society of ance of the vasodilator nitric oxide (NO) show that decreasing serum uric acid Nephrology

J Am Soc Nephrol 24: 31–36, 2013 ISSN : 1046-6673/2401-31 31 BRIEF COMMUNICATION www.jasn.org antagonist therapy using the orally active mmol/L; sitaxentan, 506621 mmol/L; 3.5460.46 pg/ml—and were not affected drug sitaxentan reduces proteinuria, BP, nifedipine, 479619 mmol/L. Baseline se- by any of the interventions (Table 1). and arterial stiffness in patients with pro- rum uric acid was inversely related to Baseline urine ET-1/creatinine levels teinuric CKD,21 effects that are poten- baseline proteinuria (r2=0.19; P=0.02). were also similar in all three phases of tially renoprotective. We hypothesized Whereas placebo and nifedipine had no the study: placebo, 761695 ag/mmol; that in this same cohort of patients with effect on serum uric acid, sitaxentan re- sitaxentan, 783684 ag/mmol; nifedipine, CKD, sitaxentan would also reduce levels duced serum uric acid by approximately 824697 ag/mmol. Although placebo and of serum uric acid, ADMA, and urine ET- 11% by study end (Figure 1A). This ef- nifedipine had no effect on urine ET-1/ 1 (as a measure of renal ET-1 production) fect was similar at weeks 3 and 6 of the creatinine, sitaxentan reduced this vari- and so provide broader cardiovascular study. In multivariate analysis, the re- able by about 19% (Figure 1C). A similar and renal protection. The current data duction in serum uric acid was not asso- effect was seen on urine ET-1 concentra- show the effects of sitaxentan, as well as ciated with changes in proteinuria, BP, tion (without correction to urine creati- placebo and an active control agent, ni- or PWV (data not shown). The reduc- nine). Neither placebo nor nifedipine fedipine, on these novel cardiovascular tion in serum uric acid was matched by affected urine ET-1, whereas sitaxentan risk factors. an increase in the fractional urinary ex- reduced this significantly (Table 1). An in- As described elsewhere,21 after 6 weeks cretion of uric acid (baseline versus week crease in urine ET-1/creatinine was asso- of dosing no significant differences were 6: 6.0%60.6% versus 7.3%60.7%; ciated with reductions in both proteinuria seen between sitaxentan and nifedipine in P=0.05). and BP (Figure 2, B and C; r=0.41; the reductions from baseline in BP mea- Baseline ADMA concentrations were P,0.05 for both), and in multivariate sures. Despite this, sitaxentan reduced the same for all three phases of the study: analysis, the change in urine ET-1/creatinine proteinuria to a significantly greater ex- placebo, 0.5260.01 mmol/L; sitaxentan, independently predicted the changes in tent than did nifedipine. Pulse-wave 0.5260.01 mmol/L; nifedipine, 0.5260.02 proteinuria and BP. velocity (PWV)—a measure of arterial mmol/L. Whereas placebo and nifedipine In addition to the important evidence stiffness—decreased to a similar degree did not affect ADMA, 6 weeks of sitaxentan of potentially renoprotective effects on with nifedipine as with sitaxentan. Pla- reduced ADMA by approximately 8% proteinuria, BP, and arterial stiffness, the cebo did not affect proteinuria, BP, or (Figure 1B). This reduction in ADMA current data show that ETA receptor an- PWV (see Table 1 for summary data). was directly correlated with a reduction tagonism selectively decreases serum Thirteen of the 27 patients took part in PWV (Figure 2A; r=0.39; P,0.05), uric acid, ADMA, and urinary ET-1 lev- in a renal substudy, which showed that and in multivariate analysis, the change els in patients with proteinuric CKD, in- sitaxentan alone reduced both GFR and in ADMA (but not changes in proteinuria, dependent of BP. These effects were seen effective filtration fraction (Table 2). Of BP, plasma ET-1, urine ET-1/creatinine, or in patients already receiving optimal note, sitaxentan did not cause increases serum uric acid) independently predicted treatment with angiotensin-converting in patients’ weight or hematocrit that the reduction in PWV after sitaxentan enzyme inhibitors and angiotensin- would suggest extracellular fluid accu- treatment. receptor blockers. These findings sug- mulation. Plasma ET-1 concentrations were sim- gest a potential role for ETA receptor Baseline serum uric acid was in the ilar at baseline in all three phases of antagonism in conferring additional frankly hyperuricemic range in all three the study—placebo, 3.5760.50 pg/ml; longer-term cardiovascular and renal phases of the study: placebo, 476620 sitaxentan, 3.6060.49 pg/ml; nifedipine, benefits in patients with CKD.

Table 1. Main study data at baseline and week 6 of each study period Placebo Sitaxentan Nifedipine Variable Baseline Week 6 Baseline Week 6 Baseline Week 6 24-hr proteinuria (g/d) 2.0660.38 2.0060.33 2.0760.34 1.4660.26a 1.9560.30 1.9960.33 Protein-to-creatinine ratio (mg/mmol) 155631 153627 157628 114623b 155627 152629 Mean arterial pressure (mmHg) 94.662.2 94.361.7 94.461.8 90.761.8c 95.562.0 91.761.7c Systolic BP (mmHg) 125.462.7 124.261.9 124.362.2 120.761.9c 125.762.4 120.761.6c Diastolic BP (mmHg) 77.961.5 77.561.2 77.961.3 74.361.3a 78. 961.5 75.761.2a PWV (m/s) 7.760.3 8.060.4 8.060.3 7.660.3c 7.960.3 7.660.3c Central augmentation index (%) 2062206220621562a 19621762 Plasma ET-1 (pg/ml) 3.660.5 3.760.6 3.660.5 3.760.5 3.560.5 3.560.5 Urine ET-1 (pg/ml) 4.560.4 4.760.4 5.160.4 4.262.1c 5.160.4 4.760.4 Values are given as predosing baseline 6 SEM. aP,0.01 for week 6 versus baseline. bP=0.01 for week 6 versus baseline. cP,0.05 for week 6 versus baseline.

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Table 2. Renal substudy data from clearance studies performed at baseline and week 6 of each study period Placebo Sitaxentan Nifedipine Variable Baseline Week 6 Baseline Week 6 Baseline Week 6 GFR (ml/min) 5667546857684868a 59685869 Effective renal blood ﬂow (ml/min) 533666 552665 511663 543673 562682 530672 Effective renal vascular resistance (mmHg/min per L) 230652 206639 236644 232648 248658 254656 Effective ﬁltration fraction (%) 19.161.1 17.961.3 20.861.0 16.660.7b 20.361.1 20.561.4 Values are given as predosing baseline 6 SEM. aP,0.05 for sitaxentan at week 6 versus sitaxentan at baseline. bP,0.01 for sitaxentan at week 6 versus sitaxentan at baseline.

Decreasing serum uric acid may re- antagonist bosentan decreased serum cross-sectional study that ADMA con- duce cardiovascular risk and CKD pro- uric acid from 353 to 305 mmol/L.25 centrations directly correlate with arterial gression.7 Treatment of asymptomatic The current data build on these studies stiffness—as measured by PVW—in a hyperuricemia improves renal func- by showing that in a randomized con- similar cohort of patients with CKD.26 tion22 and delays disease progression11 trolled trial of patients with proteinuric The current study takes this observation in patients with early CKD (stage 3). CKD, in which baseline serum uric acid further by showing for the first time In a different approach, withdrawal of levels were much higher, selective ETA that a decrease in ADMA correlates the xanthine oxidase inhibitor allopuri- receptor antagonism reduces serum with an improvement in arterial stiffness, nol from a group of patients with stable uric acid by approximately 11% (more although we recognize this correlation to CKD led to both worsening of hyperten- impressive reductions than seen in the be weak. Additionally, it is not possible to sion and acceleration of renal dysfunc- previous two studies), independent of separate independent effects of the ETA tion, although this occurred only in BP. Furthermore, as a mechanism for antagonist on arterial stiffness and patients not taking an angiotensin- thiswehaveshownanincreaseinthe ADMA in this limited number of pa- converting enzyme inhibitor.12 How- renal excretion of uric acid. tients. Because both increased ADMA ever, these studies suggesting benefits There is increasing interest in the NO and arterial stiffness independently con- of reducing serum uric acid used allopu- system and, in particular, ADMA in re- tribute to CKD progression and its asso- rinol as the therapeutic agent. More relation to both the development and pro- ciated morbidity and mortality,14,20 ET cently, the angiotensin-receptor blocker gression of CKD. Many studies in patients receptor antagonism offers a potentially losartan has been shown to decrease se- with varying degrees of CKD have con- attractive novel therapy in CKD with rum uric acid in a group of patients with firmedthat ADMAiselevatedinCKD.14,26 benefits beyond those of lowering BP type 2 diabetes and nephropathy, and Of note, data suggest that ADMA is ele- and proteinuria. this reduction was associated with a re- vated independently of renal function in Urinary ET-1 is a recognized measure 23 27 28 duction in CKD progression. ETrecep- CKD, suggesting that mechanisms other of renal ET-1 production. Selective ETA tor antagonism offers a potentially novel than impaired clearance may contribute receptor antagonism reduced renal ET-1 approach to decreasing serum uric acid to the accumulation of ADMA in this set- production at 6 weeks. Of note, the de- in patients with proteinuric CKD. ting. The ET system is upregulated in crease in BP seen with sitaxentan at 6 Only two studies have shown that ET CKD.20 There is often reciprocal upregu- weeks correlated inversely (albeit weakly) receptor antagonism reduces serum uric lation of the ETsystem20 in circumstances with urinary ET-1; that is, a greater de- acid, neither of which included patients with downregulation of NO system activ- crease in BP was seen in patients who had with CKD. Six months of treatment with ity.20 In the current study, baseline plasma less of a reduction, or even an increase, in the selective ETA receptor antagonist ADMA and ET-1 did indeed correlate renal ET-1 production. Renal ET-1 is in- atrasentan reduced serum uric acid lev- highly with each other (r2=0.56; volved in salt and water excretion29 els from 293 to 286 mmol/L in patients P,0.01), confirming the reciprocal rela- and so part of the mechanism for the with early atherosclerosis.24 Change in tionship between the NO and ETsystems. BP-lowering effect of ET receptor antag- serum uric acid was not a primary end- Few interventional studies have onism (in addition to their direct effects point in this study, and although this shownareductioninADMA.These on the vasculature) may relate to an in- was a statistically significant reduction have not included patients with CKD crease in renal ET-1 production to in- it is not clinically meaningful. In another and have suffered from being small or crease both natriuresis and diuresis. In small open-label study (n=15) in pa- lacking in rigorous methods.18,19 To our this study there was no relationship be- tients with pulmonary arterial hyperten- knowledge, the current study is the first tween changes in salt excretion and uri- sion and no control group, Ulrich and to show that ET receptor antagonism nary ET-1. A relationship similar to that colleagues showed that 6 months’ treat- may reduce circulating ADMA concen- seen with BP was also seen between the ment with the mixed ETA/B receptor trations. We have previously shown in a 6-week change in urinary ET-1 and

J Am Soc Nephrol 24: 31–36, 2013 Endothelin Antagonism and CKD 33 BRIEF COMMUNICATION www.jasn.org

proteinuria. The reduction in proteinuria with sitaxentan related to the decrease in BP (r2=0.16; P=0.04) and so this may explain part of this. The current data show for the first time that selective ETA receptor antagonism reduces novel cardiovascular risk factors in patients with proteinuric CKD established on optimal therapy. These data build on our earlier cross- sectional study, which showed that ADMA concentrations directly correlate with arterial stiffness, a powerful predic- tor of cardiovascular disease in patients with CKD. The mechanisms for these effects need to be further explored as a focus of future research. Certainly, reduction in BP is not sufficient because the active control agent nifedipine matched the decrease in BP seen with sitaxentan but did not reduce serum uric acid, ADMA, or urinary ET-1. Larger studies are needed to confirm these important findings in a group of patients at very high cardiovascular risk.

CONCISE METHODS

The rationale and design for this study have been reported in detail elsewhere.21 In brief, in a randomized, double-blind, three-way crossover study, 27 patients receiving recom- mended renoprotective treatment under- went 6 weeks of placebo; sitaxentan, 100 mg once daily; and nifedipine long-acting, 30 mg once daily. Twenty-four-hour proteinuria, urine protein-to-creatinine ratio, 24-hour ambulatory BP, and PWV (as an index of arterial stiffness) were measured at baseline, week 3, and week 6 of each treatment period. ADMA, serum urate, plasma ET-1, and urine ET-1/creatinine were also assessed at these same time points. See Supplemental Table for baseline patient characteristics.

Figure 1. Selective endothelin-A receptor antagonism reduces serum urat, ADMA and Sample Collection and Analysis urine ET-1/creatinCKDpatients. Changefrombaselinein(A) serumuric acid, (B)ADMA,and ADMA and ET-1 venous blood samples were ’ (C) urine ET-1/creatinine after 3 and 6 weeks treatment with placebo (open bar), sitaxentan collected in EDTA tubes, and urate was 6 , (speckled bar), and nifedipine (hashed bar). Values are expressed as mean SEM. *P 0.01 collected in serum tubes. These were imme- for sitaxentan versus placebo at 3 or 6 weeks; †P,0.05 for sitaxentan versus placebo at diately centrifuged at 2500 g for 20 minutes 3 weeks. at 4°C. For urine ET-1, a 20-ml aliquot of urine was collected into plain tubes with 2.5 ml of 50% acetic acid. Samples were stored at 280°C until analysis.

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Plasma ADMA concentrations were measured using an optimized and fully validated HPLC method, as previously described30 (assay variations, 1.9% and 2.3%). After extraction,31 ET-1 was determined by radioimmunoassay.32 The mean recovery of ET-1, from extraction to assay, was .90% for both plasma and urine. The intra- and interassay variations were 6.3% and 7.2%, respectively. The cross-reactivity of the anti- body was 100% with ET-1, 7% for both ET-2 and ET-3, and 10% with big ET-1. The urate assay was based on the methods of Trivedi et al.33 Uric acid is oxidized to al- lantoin by uricase with the production of hydrogen peroxide. The hydrogen peroxide reacts with 4-aminoantipyrine and 2,4,6- tribromo-3-hydroxybenzoic acid in the pres- ence of peroxidase to yield a quinoneimine dye. The resulting change in absorbance at 548 nm is proportional to the uric acid con- centration in the sample. The limits of de- tection and quantiﬁcation for the urate assay are 0.01 mmol/L and 0.015 mmol/L, respectively.

Statistical Analyses Data were stored and analyzed in Microsoft Excel, version 11.3.7 (Microsoft Corp., Red- mond, WA). Statistical analyses were performed on untransformed data. Responses were examined by repeated-measures ANOVA, and Bonferroni correction was used to assess significance at specific time points. Statistical significance was taken at the 5% level.

ADDENDUM

Sitaxentan has been voluntarily with- drawn by Pﬁzer Ltd. because of unac- ceptable adverse effects. However, the ﬁndings in this manuscript are likely to be representative of the effects of the class of selective ETA receptor antagonists.

ACKNOWLEDGMENTS

We would like to thank the study participants andNeilDavie and SimonTealofEncysive,for Figure 2. Changes in arterial stiffness, blood pressure and proteinuria following endothelin- A antagonism relate to changes in ADMA and urine ET-1/creat. Relationships between (A) their continued support through the study. percentage change in PWV against percentage change in ADMA and (B) percentage change Registration number at www.clinicaltrials. in proteinuria and (C) percentage change in mean arterial pressure against percentage gov: NCT00810732. change in urine ET-1/creatinine. MAP, mean arterial pressure; uET-1/creat, urine ET-1/ This study was funded by Encysive Phar- creatinine. maceuticals and Pﬁzer. N.D. was supported

J Am Soc Nephrol 24: 31–36, 2013 Endothelin Antagonism and CKD 35 BRIEF COMMUNICATION www.jasn.org by the British Heart Foundation (project 12. Talaat KM, el-Sheikh AR: The effect of mild hoc analysis of the reduction of endpoints in grant PG/05/91) and NHS Endowments. hyperuricemia on urinary transforming growth non-insulin-dependent diabetes mellitus with factor beta and the progression of chronic kid- the Angiotensin II Antagonist Losartan Trial. ney disease. Am J Nephrol 27: 435–440, 2007 Hypertension 58: 2–7, 2011 13. Feig DI, Soletsky B, Johnson RJ: Effect of 24. Raichlin E, Prasad A, Mathew V, Kent B, DISCLOSURES allopurinol on blood pressure of adolescents Holmes DR Jr, Pumper GM, Nelson RE, N.D., J.G., and D.J.W. have received research with newly diagnosed essential hyperten- Lerman LO, Lerman A: Efficacy and safety of – grants from Pfizer. N.D. and J.G. have held aca- sion: A randomized trial. 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36 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 31–36, 2013 Supplementary Table: Baseline patient characteristics. Values are given as mean of 3 baseline pre- treatment periods ± standard deviation. GFR: glomerular filtration rate; PCR: protein:creatinine ratio;

PWV: pulse wave velocity; cAIx: central augmentation index; ACE: angiotensin converting enzyme;

ARB: angiotensin receptor blocker. aTo convert to µmol/l, multiply by 88.4. bTo convert to mmol/l, multiply by 0.0259.

Parameter n = 27

Demographic

Age, y 48 ± 12

Male sex (%) 23 (85)

Caucasian 27 (100)

Clinical

Body mass index, kg/m2 29.3 ± 4.6

24h BP, mmHg

Systolic 125 ± 12

Diastolic 78 ± 7

Mean 94 ± 78

Creatininea, mg/dl 1.73 ± 0.85

Estimated GFR, ml/min/1.73m2 54 ± 26

Hemoglobin, g/l 136 ± 18

Serum potassium, mmol/l 4.6 ± 0.4

Cholesterolb, mg/dl 178 ± 32

1 Urinary protein excretion g/24h 2.03 ± 1.7

PCR, mg/mmol 156 ± 143

Arterial stiffness

PWV, m/s 8.3 ± 2.4

AIx, % 28 ± 12

Medications, n (%)

ACE inhibitor 18 (67)

ARB 11 (41)

ACE inhibitor + ARB 5 (19)

No ACE inhibitor or ARB 3 (11)

α blocker 6 (22)

β blocker 8 (30)

Calcium channel blocker 3 (11)

Diuretic 2 (7)

Statin 18 (67)