Am J Physiol Regul Integr Comp Physiol 308: R208–R218, 2015. First published December 4, 2014; doi:10.1152/ajpregu.00409.2014.

Tetrahydrobiopterin lowers muscle sympathetic nerve activity and improves augmentation index in patients with chronic kidney disease Jeanie Park,1,2 Peizhou Liao,3 Salman Sher,4 Robert H. Lyles,3 Don D. Deveaux,1,2 and Arshed A. Quyyumi4 1

Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; 2Research Service Line, Department of Veterans Affairs Medical Center, Decatur, Georgia; 3Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, Georgia; and 4Cardiology Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia Submitted 26 September 2014; accepted in final form 1 December 2014

Park J, Liao P, Sher S, Lyles RH, Deveaux DD, Quyyumi AA. Tetrahydrobiopterin lowers muscle sympathetic nerve activity and improves augmentation index in patients with chronic kidney disease. Am J Physiol Regul Integr Comp Physiol 308: R208–R218, 2015. First published December 4, 2014; doi:10.1152/ajpregu.00409.2014.—Chronic kidney disease (CKD) is characterized by overactivation of the sympathetic nervous system (SNS) that contributes to cardiovascular risk. Decreased nitric oxide (NO) bioavailability is a major factor contributing to SNS overactivity in CKD, since reduced neuronal NO leads to increased central SNS activity. Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase that increases NO bioavailability in experimental models of CKD. We conducted a randomized, double-blinded, placebo-controlled trial testing the benefits of oral sapropterin dihydrochloride (6R-BH4, a synthetic form of BH4) in CKD. 36 patients with CKD and hypertension were randomized to 12 wk of 1) 200 mg 6R-BH4 twice daily ⫹ 1 mg folic acid once daily; vs. 2) placebo ⫹ folic acid. The primary endpoint was a change in resting muscle sympathetic nerve activity (MSNA). Secondary endpoints included arterial stiffness using pulse wave velocity (PWV) and augmentation index (AIx), endothelial function using brachial artery flow-mediated dilation and endothelial progenitor cells, endotheliumindependent vasodilatation (EID), microalbuminuria, and blood pressure. We observed a significant reduction in MSNA after 12 wk of 6R-BH4 (⫺7.5 ⫾ 2.1 bursts/min vs. ⫹3.2 ⫾ 1.3 bursts/min; P ⫽ 0.003). We also observed a significant improvement in AIx (by ⫺5.8 ⫾ 2.0% vs. ⫹1.8 ⫾ 1.7 in the placebo group, P ⫽ 0.007). EID increased significantly (by ⫹2.0 ⫾ 0.59%; P ⫽ 0.004) in the 6R-BH4 group, but there was no change in endothelial function. There was a trend toward a reduction in diastolic blood pressure by ⫺4 ⫾ 3 mmHg at 12 wk with 6R-BH4 (P ⫽ 0.055). 6R-BH4 treatment may have beneficial effects on SNS activity and central pulse wave reflections in hypertensive patients with CKD. sympathetic activity; autonomic function; blood pressure; nitric oxide; vascular stiffness

is characterized by chronic sympathetic nervous system (SNS) overactivation (14, 21, 45), which is a major contributing factor to increased cardiovascular (CV) risk in this population. SNS overactivity increases CV risk not only by increasing blood pressure (BP), but also by factors independent of increased BP, including vascular inflammation and hypertrophy (9, 55), arterial stiffness (19), arrhythmogenesis (60), insulin resistance (35), and progression of myocardial and renal sclerosis (4, 46).

CHRONIC KIDNEY DISEASE (CKD)

Address for reprint requests and other correspondence: J. Park, Renal Division, Emory Univ. School of Medicine, 1639 Pierce Dr., WMB 338, Atlanta, GA 30322 (e-mail: [email protected]). R208

Current therapeutic options to combat SNS overactivation include central (clonidine) and peripheral (␤-blockers and ␣-blockers) sympatholytic medications. However, treatment with these agents in CKD patients is often complicated by adverse side effects, including hypotension, orthostasis, fatigue, and erectile dysfunction. In addition, peripheral sympatholytics cause a reflex increase in central sympathetic nerve activation (12, 22, 57), as evidenced by increased muscle sympathetic nerve activity (MSNA), which reflects central SNS activity. Therefore, these medications may have adverse or equivocal effects on long-term CV risk. Indeed, there is no evidence that ␤-blocker therapy improves mortality risk in CKD patients without heart disease (5, 7), and long-term ␤-blocker use is associated with development of insulin resistance and hyperlipidemia (6, 12). Therefore, there is a need to investigate adjunctive or alternative therapies that safely ameliorate SNS overactivation in CKD. One potential alternative strategy for reducing SNS activity is by increasing nitric oxide (NO) bioavailability. Neuronal NO has a tonic inhibitory effect on central sympathetic activation (8, 51). Therefore, a reduction in NO bioavailability leads to overactivation of the SNS when the sympathoinhibitory effect of NO is reduced; conversely, increasing NO bioavailability results in greater restraint of central SNS activity. CKD patients have significantly decreased NO bioavailability (68), and NO deficiency is a major contributing factor in the pathogenesis of chronic SNS overactivity in CKD (65). In addition, decreased NO bioavailability contributes to other CV risk factors in CKD, including endothelial dysfunction, arterial stiffness, and hypertension (11, 51). Thus, strategies to improve NO bioavailability in CKD have the potential to impact CV risk by reducing central SNS activation, as well as improving arterial compliance, endothelial function, and BP. A novel therapeutic approach for improving NO bioavailability is via treatment with sapropterin dihydrochloride (6RBH4, BioMarin). 6R-BH4 is the synthetic form of the naturally occurring enzyme tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS) in the catalytic reaction that forms NO (44, 52, 54). BH4 supplementation has been shown to increase NO bioavailability and improve BP and endothelial function in animal models of chronic renal failure (48, 49, 64); however, 6R-BH4 has never previously been tested in humans with kidney disease. In addition, the effects of 6R-BH4 supplementation on SNS activity are unknown. Therefore, we conducted a 12-wk randomized, placebo-controlled double-blinded clinical trial (NCT 1356966), testing the hypothesis that 6R-BH4 supplementation lowers SNS activity, http://www.ajpregu.org

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

as well as improves arterial stiffness, endothelial function, and BP in patients with CKD. METHODS

Study Population Forty-nine male veteran participants with hypertension and CKD Stage 2 or 3 were recruited and enrolled from outpatient clinics at the Atlanta Veterans Affairs (VA) Medical Center. Stage 3 CKD was defined as an estimated glomerular filtration rate (eGFR) (34) between 30 and 59 ml·min⫺1·1.73 m⫺2, and Stage 2 CKD was defined as an eGFR between 60 and 89 ml·min⫺1·1.73 m⫺2 with concomitant urinary microalbumin:creatinine ratio greater than 30 mg/g. Inclusion criteria included stable renal function defined as no greater than a 10% fluctuation in eGFR within the prior 3 mo and stable antihypertensive medication regimen for 3 mo prior to enrollment. Exclusion criteria included severe CKD (eGFR ⬍30 cm·l⫺1·min⫺1), diabetes; HIV infection; clinical evidence of congestive heart failure or ejection fraction below 35%; any history of past coronary artery; cerebrovascular, aortic, or peripheral vascular disease; symptomatic heart disease determined by electrocardiogram, stress test, and/or history; hepatic enzyme concentrations greater than 2 times the upper limit of normal; severe anemia with hemoglobin level ⬍10 g/dl; history of nephrolithiasis; any serious systemic disease that might influence survival; current treatment with clonidine, levodopa, or methotrexate; BP greater than 160/90 mmHg; BP less than 110/60; change in medications or surgery within the past 3 mo; drug or alcohol abuse; previous treatment or hypersensitivity to 6R-BH4. Study Design This was a prospective, randomized, placebo-controlled, doubleblind clinical trial (clinicaltrials.gov identifier NCT1356966). After obtaining written informed consent, baseline MSNA, office and ambulatory BP, basic metabolic panel, and urinary microalbumin and creatinine were obtained. On a separate day, baseline brachial artery flow-mediated dilatation (FMD), endothelium-independent vasodilation (EID), pulse wave analysis, pulse wave velocity, and blood samples for progenitor cell (PC) were obtained. All measurements were obtained in a quiet, temperate (⬃21°C) environment, after abstaining from food, caffeine, smoking, and alcohol for at least 12 h, and exercise for at least 24 h. Participants in whom an adequate baseline MSNA neurogram was unable to be obtained, did not proceed to the clinical trial. After baseline assessments, participants were randomly assigned to receive 6R-BH4 200 mg twice daily with folic acid 1 mg daily, or an identical placebo twice daily with folic acid 1 mg daily. All participants were given folic acid because folic acid enhances the binding affinity of BH4 to NOS and also enhances the regeneration of BH4 from inactive BH2 (41). Participants were instructed to dissolve study pills in water or apple juice and to take with food. Interim visits were performed at weeks 1, 3, 6, 9, and 12, to assess compliance via pill count, assess for adverse effects, and measure BP and laboratory measures of serum electrolytes and kidney function. At the end of the trial, between weeks 9 and 12, baseline measurements were repeated under identical conditions. Participants were instructed not to change medication regimen, dietary, or exercise habits for the duration of the study. This study was approved by the Emory University Institutional Review Board, and the Atlanta VA Medical Center Research and Development Committee. Measurements and Procedures Blood pressure. Office BP was measured at baseline and at each follow-up visit after 5 min of rest in a seated position with the arm supported at heart level using an appropriately sized cuff. BP was measured with an automated device (Dinamap) using the oscillometric method. Each data point of BP was obtained as an average of three consecutive BP measurements separated by 5 min.

R209

Muscle sympathetic nerve activity. Multiunit postganglionic MSNA was recorded directly from the peroneal nerve by microneurography, as previously described (58, 61). Participants were placed in a supine position, and the leg was positioned for microneurography. A tungsten microelectrode (tip diameter 5–15 ␮m) (Bioengineering, University of Iowa) was inserted into the nerve, and a reference microelectrode was inserted subcutaneously 1–2 cm from the recording electrode. The signals were amplified (total gain: 50,000 – 100,000), filtered (700-2,000 Hz), rectified, and integrated (time constant 0.1 s) to obtain a mean voltage display of sympathetic nerve activity (Nerve Traffic Analyzer, model 662C-4, University of Iowa, Bioengineering) that was recorded by the LabChart 7 Program (PowerLab 16sp, ADInstruments). Continuous ECG was recorded simultaneously with the neurogram using a bioamp system. Beat-to-beat arterial BP was measured concomitantly using a noninvasive monitoring device that detects digital blood flow via finger cuffs and translates blood flow oscillations into continuous pulse pressure waveforms and beat-to-beat values of BP (CNAP, CNSystems) (29, 30). Absolute values of BP were internally calibrated using a concomitant upper arm BP reading and were calibrated at the start and every 15 min throughout the study. The tungsten microelectrode was manipulated to obtain a satisfactory nerve recording that met previously established criteria (16, 17, 38). After 10 min of rest, baseline BP, heart rate, respiratory rate, and MSNA were recorded continuously for 10 min. Arterial stiffness. Pulse wave analysis and PWV were assessed using ultrasound measurements of the radial, carotid, and femoral arteries, using the SphygmoCor Pulse Wave Velocity system (PWV Medical). In brief, peripheral pressure waveforms were recorded from the radial artery at the wrist, using applanation tonometry with a high-fidelity micromanometer. After 20 sequential waveforms were acquired, a validated generalized transfer function was used to generate the corresponding central aortic pressure waveform, and the degree of pressure augmentation due to the reflected wave form from the periphery was measured. Central AIx was calculated as the augmented pressure divided by the central pulse pressure from the central pressure waveform. Central AIx was also corrected for heart rate using a standardized value of 75 beats per min (bpm). Carotidfemoral artery PWV was determined by measuring transcutaneous Doppler flow velocity recordings carried out over the common carotid artery and the femoral artery. The distance between the recording sites was measured manually and divided by the time interval between the EKG R-waves from the recorded waveforms at each site, to arrive at a measurement of velocity (m/s). Brachial artery flow-mediated dilatation. Participants were placed in a supine position, and a forearm occlusion cuff was placed around the forearm. For each measurement, baseline images of the brachial artery were obtained using an 13-MHz high-resolution ultrasound transducer (Acuson) longitudinally 2–10 cm above the antecubital fossa. Electrocardiogram gating was used to capture end-diastolic arterial diameters, and the average diameter and blood velocity for three cardiac cycles was recorded and analyzed for baseline values. The cuff was then rapidly inflated to suprasystolic levels (50 mmHg above systolic pressure) for 5 min (E-20 rapid cuff inflator; D. E. Hokanson) to create a flow stimulus in the brachial artery. Doppler ultrasound was used to measure peak hyperemic blood velocity during the first 10 s after cuff release, and then diameter measurements were captured at 60 and 90 s after cuff release. Calculations of FMD were based on the 60- and 90-s measurements, and at peak hyperemic diameters. Images were digitized, and arterial diameters were measured with customized software (Medical Imaging Applications). All measurements were obtained and analyzed by a single investigator (SS) blinded to the clinical status of the participant. FMD was expressed as the % change in artery diameter from baseline: (peak hyperemic diameter ⫺ baseline diameter)/baseline diameter. Shear rate was calculated as 4⫻ peak blood velocity/arterial diameter, both at baseline and at peak hyperemia. FMD values were also normalized

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R210

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

for peak hyperemic shear rate. After 20 min of rest to reestablish baseline conditions, EID was assessed by obtaining ultrasound images of the brachial artery just before, and 3–5 min after the administration of 0.4 mg of sublingual nitroglycerin. EID is expressed as the %change in artery diameter from baseline. Progenitor cells. Circulating progenitor-enriched populations were estimated by measuring the expression of surface antigens using direct flow cytometry analysis (BD FACSCanto II flow cytometer), as previously described (3). Approximately 300 ml of venous blood (anticoagulant: EDTA) was incubated with fluorochrome-labeled monoclonal mouse antihuman antibodies, namely, FITC-CD34 (BD Biosciences), PerCP-CD45 (BD Biosciences), PE-VEGF2R (R&D system-also known as “Kinase insert domain receptor-KDR”), and APC-CD133 (Miltenyi) for 15 min. Red blood cells were removed by lysis in 1.5 ml of ammonium chloride lysing buffer, which was added to the sample and incubated for an additional 10 min. The lysis process was stopped by adding 1.5 ml of staining medium (PBS with 3% heat-inactivated serum and 0.1% sodium azide). Five million events were acquired from the cytometer with FlowJo software (Treestar) used for subsequent analysis of accumulated data. Absolute cell counts for target cell subsets were determined using an indirect cell estimation method. The total absolute white cell count and absolute counts for lymphocytes, monocytes, and granulocytes were determined using a Coulter ACT/Diff cell counter (Beckman Coulter). The absolute mononuclear cell count was then estimated as the sum of lymphocytes and monocytes. The frequency of our target cell populations was reported as a subset of the mononuclear cell compartments. This frequency (% of mononuclear cell compartments) was then multiplied by the absolute count of mononuclear cells to calculate the absolute count of our target cells, which are a component of the mononuclear cell compartment and reported as cell counts per milliliter. Circulating cell populations commonly considered to be enriched for hematopoietic and endothelial progenitors include those expressing CD34⫹, CD133⫹, and VEGF2R⫹ surface markers among a CD45medium population, either singly or in combination. The CD34⫹ population represents hematopoietic progenitors from which subsequent lineages, including endothelial progenitors, are derived. The CD133⫹ subset of CD34⫹ cells are believed to include immature or early progenitors as the marker subsequently disappears as they mature, whereas CD34 positivity with VEGF2R markers is widely considered to be a population enriched for endothelial progenitors. Thus, we examined the frequency in the peripheral circulation of CD34⫹, dual-positive CD34⫹/CD133⫹, dual-positive CD34⫹/ VEGF2R⫹, and triple-positive CD34⫹/CD133⫹/VEGF2R⫹ cell populations among the CD45medium population. Samples were repeatedly analyzed on two occasions by two technicians. Study Endpoints The primary endpoint was the change in MSNA from baseline to end-of-study. Secondary outcomes included central AIx, PWV, brachial artery FMD, EID, endothelial PC counts, BP measurements at interval visits, and microalbuminuria. Data Analysis Muscle sympathetic nerve activity. MSNA and ECG data were exported from the LabChart data acquisition system to WinCPRS (Absolute Aliens, Turku, Finland) for analysis. R-waves were detected and marked from the continuous ECG recording. MSNA bursts were automatically detected by the program using the following criteria: 3:1 burst-to-noise ratio within a 0.5-s search window, with an average latency in burst occurrence of 1.2–1.4 s from the previous R-wave. After automatic detection, the ECG and MSNA neurograms were visually inspected for accuracy of detection by a single investigator (J. Park). MSNA was expressed as burst frequency (bursts/min), and burst incidence (bursts/100 heartbeats).

Statistical Analysis Statistical analysis was performed using the R Language 3.0.1. Unless otherwise noted, within-group changes in variables of interest are reported in terms of means ⫾ SE. Within-group changes were assessed via nonparametric Wilcoxon signed rank tests, while twogroup Wilcoxon rank sum tests were conducted to assess whether median within-group changes differed between the 6R-BH4 and placebo groups. For the primary outcome variable (change in MSNA from baseline to 12 wk), multiple linear regression was used to determine whether a significant unadjusted two-group comparison was maintained after controlling for age and body mass index (BMI) as covariates. For outcome variables that displayed no difference in within-group changes from baseline to 12 wk, mixed linear models were also applied to estimate trends over time within groups and to compare these trends across groups. RESULTS

Study Enrollment and Baseline Characteristics Among the 49 patients who met the eligibility criteria and were enrolled, 32 were ultimately randomized to receive 6RBH4 (n ⫽ 18) vs. placebo (n ⫽ 14) (Fig. 1). All participants also received folic acid 1 mg daily. Table 1 depicts baseline and clinical characteristics of the study population. All participants were male and African-American, except for one Caucasian in the 6R-BH4 group, and one Caucasian in the placebo group. Mean serum creatinine and estimated glomerular filtration rate (eGFR) were similar between groups, and the cause of CKD was hypertension in the majority of both groups. The mean number of antihypertensive medications was 2.2 among the 6R-BH4 group, and 2.4 among the placebo group, and the majority of both groups were treated with calcium channel blockers and angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (ACE/ARB). Baseline systolic BP was significantly lower in the 6R-BH4 group by ⬃10 mmHg compared with the placebo group, and the baseline serum potassium was slightly lower in the placebo group. All other baseline hemodynamics, MSNA, measures of endothelial function, arterial stiffness, and oxidative stress, were similar between groups. Primary End Point The primary endpoint was the change in resting levels of MSNA assessed via direct peroneal nerve recordings of efferent sympathetic nerve discharge directed to the muscle using the microneurography technique. After 12 wk of treatment, there was a significant difference in change in resting MSNA between groups (P ⫽ 0.003; Fig. 2). Whereas MSNA decreased significantly (by ⫺7.5 ⫾ 2.1 bursts/min; P ⫽ 0.007) in the 6R-BH4 group, there was no significant change in MSNA in the placebo group (⫹3.2 ⫾ 1.3 bursts/min P ⫽ 0.175). Results were similar when analyzed as burst incidence (bursts/ 100 heartbeats). After adjustment for age and BMI, the difference remained significant (P ⫽ 0.032). Results were also similar in subgroup analyses among patients treated and not treated with ACE/ARB. 6R-BH4 treatment was associated with a significant reduction in MSNA in both patients treated with ACE/ARB and those not treated with ACE/ARB (data not shown).

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R211

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE Screened for Eligibility (n=1055)

Consented (n=49) Unable to Obtain Baseline MSNA (n=6) Did not follow-up to start pills (n=5) Blood Pressure too high (n=2) Blood Pressure too low (n=3) Withdrew Consent (n=1) Randomized (n=32)

Allocated to 6R-BH4 (n=18)

Allocated to Placebo (n=14)

Unable to Obtain End of Study MSNA (n=2) Withdrew due to nausea (n=1) Withdrawn by PI for Acute Kidney Injury (n=1) FMD not done (n=4) PWA/PWV not done (n=4)

Unable to Obtain End of Study MSNA (n=1) Withdrew due to dysgeusia (n=1) Withdrew due to unrelated trauma (n=1) FMD not done (n=5) PWA/PWV not done (n=4)

Included in Primary Analysis (n=14) Included in Secondary Analyses (n=18)

Included in Primary Analysis (n=11) Included in Secondary Analyses (n=14)

Secondary End Points Secondary end points included arterial stiffness measured by central AIx and PWV; endothelial function assessed by brachial artery flow-mediated dilatation FMD and endothelial PC; EID assessed via brachial artery vasodilatory response to nitroglycerin (NTG); microalbuminuria; and BP throughout the study period. There was a significant difference in change in arterial stiffness measured as central AIx, and central AIx corrected for heart rate (AIx at 75) between the groups (Fig. 3). Central AIx decreased in the 6R-BH4 group (by ⫺5.8 ⫾ 2.0% vs. ⫹1.8 ⫾ 1.7% in placebo group; P ⫽ 0.007), and similarly, central AIx at 75 decreased in the 6R-BH4 group (by ⫺3.2 ⫾ 2.1% vs. ⫹1.1 ⫾ 1.2%; P ⫽ 0.046). There was no significant difference in the change in PWV between the groups. (P ⫽ 0.53; Fig. 3A). Brachial artery diameters, peak blood velocities, and shear rates at baseline and peak hyperemia are given in Table 2. There was no significant difference in change in brachial artery FMD (P ⫽ 0.878) and FMD normalized for hyperemic shear rate (P ⫽ 0.439) between groups. In contrast, there was a significant increase in EID (by ⫹2.0 ⫾ 0.59%; P ⫽ 0.004) in the 6R-BH4 group, whereas no significant change was observed within the control group (Fig. 3B). Table 2 also gives central aortic pressures before and after treatment. There were no significant differences in change in central systolic, diastolic (CDP), and central mean pressure between the groups. There was a trend toward a reduction in CDP among the 6R-BH4 treatment group by ⫺4 ⫾ 3 mmHg (P ⫽ 0.059). There were also no significant differences in change in peripheral systolic BP (SBP) or mean arterial pressure (MAP) throughout the study period between groups (Fig. 4, A and C). We observed a trend toward a reduction in diastolic BP (DBP, by ⫺4 ⫾ 3 mmHg at 12 wk) among the 6R-BH4 group

Fig. 1. Flow diagram depicting patient enrollments, withdrawals, and completion. MSNA, muscle sympathetic nerve activity; 6R-BH4, oral sapropterin (a synthetic form of BH4); FMD, flow-mediated dilation; PWA, pulse wave analysis; PWV, pulse wave velocity.

compared with the placebo group, but this did not reach statistical significance (P ⫽ 0.055; Fig. 4B). There were no significant differences in change in kidney function, microalbuminuria, or PC counts between groups (Table 3), although there was a small but significant decrease in serum potassium within the 6R-BH4 group. For variables that showed no significant within-group change, analyses based on mixed linear models utilizing outcome variable assessments at intervening time points yielded qualitatively similar conclusions to the simpler baseline vs. 12 wk comparisons (results not shown). Adverse Events Overall, 6R-BH4 was well tolerated with few adverse events. Among patients assigned to 6R-BH4, one patient withdrew from the study due to nausea and gastroesophageal reflux, which resolved after cessation of the study drug. Another patient was withdrawn by the PI for acute kidney injury (AKI) after week 3 of the study. The patient was noted to have an increase in serum creatinine from a baseline of 2.23 mg/dl to 2.75 mg/dl at the week 6 interim visit, which was subsequently repeated and confirmed. Concomitantly, he had a reduction in SBP from 141 mmHg at baseline to 113 mmHg, and DBP from 86 mmHg to 64 mmHg. The cause of AKI was deemed secondary to decreased renal perfusion from low BP. After cessation of the study drug, patient’s BP and renal function returned to baseline. Among patients assigned to placebo, one patient withdrew due to dysgeusia, and a second patient withdrew due to trauma unrelated to the study. An additional two patients from the treatment group and one patient from the placebo group were not included in the primary analysis because an adequate MSNA neurogram was unable to be obtained at the end of study.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R212

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

Table 1. Baseline characteristics of study population, stratified by treatment Characteristic

6R-BH4 (n ⫽ 18)

Placebo (n ⫽ 14)

P Value

Age, yr Sex (men/women) Race, n (%) Black White Weight, kg Body mass index, kg/m2 Biochemical parameters Sodium, mEq/l Potassium, mEq/l Bicarbonate, mEq/l Urea, mg/dl Creatinine, mg/dl Estimated GFR, ml/min per 1.73 m2 Urinary microalbumin:creatinine, mg/g Cause of chronic kidney disease, n (%) Hypertension Nephrotoxic drugs ADPKD Glomerulonephritis Unknown Antihypertensive medications, n (%) Calcium channel blockers ACEI/ARB Diuretics ␤-blockers Aldosterone receptor blockers ␣-blockers Hydralazine Number of antihypertensive medications Lipid-lowering drugs, n (%) Baseline hemodynamics Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Mean arterial pressure, mmHg Heart rate, beats/min Baseline muscle sympathetic nerve activity Burst frequency, bursts/min Burst incidence, bursts/100 heartbeats Baseline measures of arterial stiffness Pulse wave velocity, m/s Central AIx, % Central AIx at 75 bpm⫺1, % Baseline brachial artery flow-mediated dilation, %

57.5 ⫾ 1.3 18/0

55.2 ⫾ 2.2 14/0

0.357

17 (94%) 1 (6%) 108.3 ⫾ 4.6 32.7 ⫾ 1.2

13 (93%) 1 (7%) 102.5 ⫾ 4.2 32.3 ⫾ 1.3

0.854 0.854 0.373 0.824

139 ⫾ 1 4.0 ⫾ 0.07 25 ⫾ 1 21 ⫾ 2 1.81 ⫾ 0.087 49.8 ⫾ 3.5 148 ⫾ 54 10 (56) 2 (11) 1 (6) 1 (6) 4 (22)

140 ⫾ 1 3.6 ⫾ 0.2 27 ⫾ 1 17 ⫾ 2 1.62 ⫾ 0.07 54.3 ⫾ 2.7 163 ⫾ 58 8 (57) 1 (7) 1 (7) 0 (0) 4 (29)

0.497 0.030 0.130 0.228 0.107 0.350 0.854 0.928 0.702 0.854 0.370 0.681

11 (61) 11 (61) 6 (33) 6 (33) 2 (11) 3 (17) 1 (6) 2.2 ⫾ 0.2 4 (22)

8 (57) 11 (79) 8 (57) 5 (36) 1 (7) 0 (0) 1 (7) 2.4 ⫾ 0.2 4 (29)

0.821 0.290 0.178 0.888 0.702 0.109 0.854 0.489 0.681

131 ⫾ 3 84 ⫾ 2.6 99 ⫾ 3 63 ⫾ 2

140 ⫾ 3 83 ⫾ 2 102 ⫾ 2 66 ⫾ 4

0.044 0.884 0.455 0.452

47.0 ⫾ 2.4 78.6 ⫾ 3.5

45.1 ⫾ 2.6 75.1 ⫾ 4.3

0.593 0.522

8.9 ⫾ 0.4 26.9 ⫾ 2.9 17.9 ⫾ 2.8 2.28 ⫾ 0.55

9.6 ⫾ 0.8 22.8 ⫾ 1.4 16.3 ⫾ 1.0 3.96 ⫾ 1.21

0.451 0.265 0.647 0.187

Values with plus/minus sign are expressed as means ⫾ SE. 6R-BH4, oral sapropterin (a synthetic form of BH4); GFR, glomerular filtration rate; ADPKD, autosomal dominant polycystic kidney disease; ACEI, angiotensin-converting enzyme inhibitor; ARB, ANG receptor blocker. AIx refers to augmentation index. AIx at 75 bpm⫺1 is corrected for heart rate. Significant P values are shown in bold.

DISCUSSION

This is the first investigation testing the effects of 6R-BH4 therapy on SNS activity in humans. In this randomized, double-blinded, placebo-controlled trial, we demonstrate that 12 wk of 6R-BH4 treatment significantly lowers resting MSNA in CKD patients. We also demonstrate that 6R-BH4 concomitantly and significantly improves arterial wave reflection and vasodilatory response to NTG. There were no significant changes in SBP and MAP, and a strong trend toward a reduction in DBP with 6R-BH4 treatment. We observed no significant changes in endothelial function assessed via brachial artery FMD and PC counts, renal function, or microalbuminuria with 6R-BH4 treatment. Our findings demonstrate that 6R-BH4 may be one novel strategy for lowering SNS activity in humans with CKD. CKD is characterized by chronic SNS overactivation that contributes to and correlates with mortality risk in this population (69).

Since SNS overactivity increases CV risk via multiple mechanisms both dependent and independent of BP (35, 55, 60), novel strategies for lowering sympathetic nerve activity could have clinical implications on long-term risk in these patients. The mechanisms by which 6R-BH4 lowers SNS activity in CKD patients are unclear, but increased neuronal NO likely plays a role. Neuronal NO has a tonic inhibitory effect on central SNS activation (8, 51), and therefore, decreased NO bioavailability, as characteristic of chronic renal failure, can lead to overactivation of sympathetic nerve activity. BH4 is a cofactor for NOS function, essential for the pathway leading to hydroxylation of L-arginine, resulting in formation of L-citrulline and NO (44). When there is a relative deficiency of BH4, a state of “NOS uncoupling” from L-arginine occurs, in which superoxide is generated from the oxygenase domain of NOS rather than NO. Thus, in BH4-deficient states, NOS uncoupling leads to a shift in balance, resulting in depletion of NO and

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R213

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

*P=0.003

30 20

40

**

30 20

12 WK

generation of oxidative stress. BH4 deficiency contributes to reduced NO bioavailability in a variety of diseases, including hypertension, diabetes, smoking, hyperlipidemia, atherosclerosis, aging, and chronic renal failure (44, 52, 54). Endogenous BH4 levels were significantly lower in humans with kidney disease (66), and BH4 treatment increased NO bioavailability in animal models of chronic renal failure. Together, these findings suggest that CKD patients have a relative deficiency in BH4 that leads to NOS uncoupling, decreased NO bioavailability, and subsequent overactivation of central SNS activity, which can be ameliorated with long-term supplementation of 6R-BH4. Further, our finding that 6R-BH4 reduces SNS activity but has no effect on endothelial function suggests that BH4 may have a relatively greater effect on neuronal (nNOS) than endothelial NOS (eNOS). Concomitant with a reduction in SNS activity, we observed that 6R-BH4 treatment significantly improves central AIx, a measure of the augmentation of central aortic pressure by the reflected pulse wave. Although PWV, the gold-standard measurement of arterial stiffness, was not significantly altered by 12 wk of 6R-BH4 treatment, we observed a significant improvement in AIx, which is another validated measure of arterial stiffness (42). Increased arterial stiffness leads to an increase in the amplitude of the reflected pressure wave to the left ventricle (i.e., central AIx), leading to increased wall stress, elevated left ventricular afterload, and potentiation of atherosclerosis, thereby increasing CV risk (42). Indeed, AIx is an independent risk factor for early coronary atherosclerotic disease, CV events, and all-cause mortality (59, 63). In CKD, increased AIx independently predicts CV and all-cause mortality (36) and is associated with a more rapid progression of renal failure (62). Thus, our finding that 6R-BH4 treatment improves central AIx in CKD patients may have clinical implications that could impact long-term CV and renal outcomes. Although PWV and AIx are both measures of arterial stiffness, we observed a reduction in central AIx that was independent of a change in PWV after treatment with 6R-BH4. PWV is largely reflective of stiffness of large arteries, whereas AIx may also be influenced by vascular tone of the small muscular arteries and arterioles (32). Prior studies have shown that NTG and ANG II (vasoactive substances that act predominantly to alter the tone of the small muscular arteries and arterioles, with little effect on the aorta) are capable of producing large changes in AIx without any effect on PWV. Indeed, sympathetic nerve activation may have greater effect on vascular tone

BL

12 WK

of small arteries and arterioles, which may explain why 6RBH4 treatment was associated with a significant reduction in sympathetic nerve activity with a concomitant reduction in AIx, but without a significant change in PWV. One mechanism by which 6R-BH4 treatment may improve AIx is via reduction in sympathetic nerve activity, as demonstrated in this study. Prior studies have demonstrated a significant positive relationship between MSNA and AIx in normo-

A

6

6

4

4

2

2

0

0

-2

-2

-4

-4

-6

-6

*

-8

-8

*

-10 AIx

Change in PWV, m/s

40

50

Change in Central AIx, %

50

Fig. 2. Change in MSNA from baseline (BL) to 12 wk (WK) of treatment with placebo vs. 6R-BH4. Open circles depict individual values at baseline and 12 wk for each study participant, while closed circles depict the mean values at baseline and 12 wk within each group. *P ⬍ 0.05 denoting statistically significant difference in the change in MSNA between groups. **P ⬍ 0.05 denoting statistically significant difference within the 6RBH4 group from baseline to 12 wk.

60

-10 AIx at 75

PWV

Placebo 6R-BH4

B 5

*

0

EID, %

60

BL

6R-BH4

70

MSNA, bursts/minute

MSNA, bursts/minute

70

Placebo

-5

-10

-15 Placebo

6R-BH4

Fig. 3. Mean change in measures of arterial stiffness (A) and endothelium independent vasodilatation (EID) (B) after 12 wk of treatment with placebo vs. 6R-BH4. There was a significantly greater reduction in central augmentation index (AIx), AIx corrected for heart rate (AIx at 75), and a significant improvement in EID in the 6R-BH4 group compared with placebo. There was no difference in change in pulse wave velocity (PWV) between the groups. *P ⬍ 0.05.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R214

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

Table 2. Values for brachial artery flow-mediated dilatation and central aortic pressures 6R-BH4

Brachial Artery FMD Baseline arterial diameter, mm Baseline blood velocity, m/s Baseline shear rate, s⫺1 ⫻ 103 Hyperemic arterial diameter, mm Hyperemic blood velocity, m/s Hyperemic shear rate, s⫺1 ⫻ 103 FMD, % Normalized FMD, %/s⫺1 ⫻ 103 Central aortic pressures, mmHg Central systolic pressure Central diastolic pressure Central mean pressure

Placebo

Before Drug

After Drug

Before Drug

After Drug

4.47 ⫾ 0.12 0.75 ⫾ 0.03 0.68 ⫾ 0.04 4.57 ⫾ 0.12 1.40 ⫾ 0.06 1.24 ⫾ 0.07 2.26 ⫾ 0.54 1.85 ⫾ 0.45

4.46 ⫾ 0.11 0.75 ⫾ 0.04 0.67 ⫾ 0.04 4.50 ⫾ 0.12 1.62 ⫾ 0.20 1.46 ⫾ 0.20 1.97 ⫾ 0.65 1.50 ⫾ 0.58

4.61 ⫾ 0.18 0.75 ⫾ 0.07 0.67 ⫾ 0.08 4.79 ⫾ 0.17 1.35 ⫾ 0.11 1.15 ⫾ 0.13 3.96 ⫾ 1.21 3.27 ⫾ 0.99

4.47 ⫾ 0.20 0.67 ⫾ 0.05 0.61 ⫾ 0.05 4.61 ⫾ 0.35 1.29 ⫾ 0.07 1.15 ⫾ 0.11 3.38 ⫾ 1.26 2.83 ⫾ 1.05

123 ⫾ 4 85 ⫾ 3 100 ⫾ 3

120 ⫾ 4 81 ⫾ 2 97 ⫾ 3

129 ⫾ 3 86 ⫾ 3 103 ⫾ 3

126 ⫾ 5 85 ⫾ 3 103 ⫾ 3

Values are expressed as means ⫾ SE. FMD %, flow-mediated dilatation as % change from baseline diameter. Normalized FMD, FMD normalized to peak hyperemic shear rate.

tensive men (13) and postmenopausal women (23), suggesting that increased SNS activity may be causally related to higher amplitudes of the reflected aortic wave forms in men and older women. In vitro studies have shown that adrenergic stimulation induces extracellular matrix and adventitial fibroblast proliferation in human vascular smooth muscle cells, leading to structural changes that affect vascular compliance (43, 67). In humans, SNS overactivity is associated with vascular wall hypertrophy, suggesting that tonic elevations in sympathetic innervation may lead to arterial remodeling and structural changes associated with arterial stiffness. In addition to inducing long-term structural changes, sympathetic innervation may modulate functional compliance of blood vessels acutely, as prior studies have shown that compliance and viscoelastic properties of the vasculature in the human forearm are acutely altered by alpha adrenergic receptor stimulation (20). Acute elevations in SNS activity induced by the cold pressor test lead to marked reductions in arterial compliance in humans (10), and SNS activation under basal conditions exerts a marked tonic restraint on arterial distensibility in healthy humans and those with atherosclerotic disease (19). Moreover, maneuvers that reduce chronic sympathetic overactivity, such as removal of pheochromocytoma and renal nerve ablation, have been shown to significantly improve measures of arterial stiffness, independent of effects on BP (27, 47). Finally, our finding that a reduction in MSNA occurred concomitantly with a reduction in peripheral and central AIx without a significant change in BP, lends further evidence that SNS overactivity may have a contributory and independent role in the pathogenesis of arterial stiffness among patients with CKD that can be improved with amelioration of SNS overactivation by 6R-BH4 treatment. The role of NO in modulation of arterial stiffness is unknown, and data are conflicting. Prior reports show that both exogenous NO and local NO production improve human iliac artery distensibility (53) independent of BP changes, whereas others report that inhibition of basal NO increased BP and AIx to the same degree as norepinephrine, suggesting that the effects of NO on arterial stiffness are due to changes in BP alone and not due to an independent effect of NO on the vasculature (56). Our surprising finding that brachial artery FMD, another measure that is dependent on endothelial NO bioavailability, is not improved by 6R-BH4 treatment supports

the notion that endothelial NO may have a less important role in improving pulse wave reflections via 6R-BH4 in CKD. We also found that PC counts, which are also dependent on endothelial NOS function (2) and correlated with improved CV outcomes (39), were not improved with 6R-BH4 treatment. In contrast to our current findings, prior studies in humans with other chronic diseases, as well as animal models of CKD have shown improvement in endothelial function with BH4 treatment. In rats with chronic renal failure induced by 5/6 nephrectomy, BH4 supplementation prevented development of hypertension and increased vascular eNOS expression (48, 49). BH4 restored endothelium-dependent vasodilation in rats with ischemic acute renal failure (31) and with chronic renal failure when given in conjunction with L-arginine (64). In humans with poorly controlled hypertension, daily oral 6R-BH4 supplementation significantly improved BP and endothelial function (50). Similarly, oral 6R-BH4 treatment ameliorated endothelial dysfunction and oxidative stress in humans with hypercholesterolemia (15). Acute intravenous infusions of 6R-BH4 improved endothelial function via increased NO bioavailability in patients with type 2 diabetes (26), coronary artery disease (37), and smokers (25). The reasons underlying the discrepancy in our findings, which demonstrate a lack of improvement in endothelial function with 6R-BH4 treatment compared with prior reports in other patient populations, are unclear, but we speculate on several possibilities. First, our study is the first to examine the effects of 6R-BH4 on endothelial function in CKD patients, who have vascular abnormalities that are distinct from other patient groups. In addition to intimal lesions, patients with reduced renal function are particularly prone to medial vascular calcification due to abnormalities in calcium and phosphorus metabolism, treatment with activated vitamin D, and deficiency of calcification inhibitors (18). In such a setting, 6R-BH4 may have less capacity to induce changes in vasodilatory response to endothelial NO. Second, we conducted a randomized double-blind placebo-controlled trial testing chronic effects after long-term (12 wk) oral supplementation, whereas most previous reports did not include a placebo group, were not blinded, or tested only acute effects after a single intravenous infusion of BH4. One prior study in hyperlipidemic patients (15) did utilize a randomized controlled design in a smaller number of

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R215

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

Change in SBP, mm Hg

A

10

Placebo BH4

0

-10

-20

0

2

4

6

8

10

12

14

Weeks of Treatment

Change in DBP, mm Hg

B

10

0

-10

-20

0

2

4

6

8

10

12

14

Weeks of Treatment

C

8

Table 3. Laboratory data

6

Change in MAP, mm Hg

Whether 6R-BH4 improves arterial baroreflex sensitivity remains unknown. Although we observed no changes in NO-mediated vasodilation, we observed a small but significant improvement in EID with 6R-BH4. EID, or the vasodilatory response to NTG, is dependent on structural and functional properties of vascular smooth muscle cells, rather than on endothelium-derived NO (1). Increased SNS activation increases vascular smooth muscle cell contraction and restrains vasodilatory responses (24, 28, 40). As such, the decrease in sympathetic nerve activity concomitant with an improvement in EID observed with 6RBH4 suggests that decreased sympathetic innervation may lead to a greater capacity for vasodilation in response to NTG when neurogenic constraint is reduced. Despite a significant reduction in sympathetic activity, there were no significant differences in change in SBP or MAP with treatment between the groups; however, we did observe a trend toward a reduction in DBP (P ⫽ 0.055). A larger sample size may have detected significant reductions in DBP among the treatment group. In addition, the baseline BP was well controlled in the study population, making it less likely to observe significant changes in interval BP measurements, in contrast to prior studies that were conducted in patients with poorly controlled BP at baseline (50). Alternatively, the degree of inhibition of sympathetic nerve activity with this dose of 6R-BH4 may not have been sufficient to significantly affect the variability of BP under normal conditions. Adaptations such as altered vascular alpha adrenergic sensitivity or activation of the renin-angiotensin-aldosterone system may have also prevented a significant change in resting BP. Interestingly, one CKD

4

Measurement

2 0 -2 -4 -6 -8 -10 -12

Baseline

12 Weeks

P Value

1.81 ⫾ 0.09 1.62 ⫾ 0.07

1.75 ⫾ 0.11 1.62 ⫾ 0.05

0.346 1.000

21 ⫾ 2 17 ⫾ 2

19 ⫾ 2 17 ⫾ 2

0.276 0.969

4.0 ⫾ 0.1 3.6 ⫾ 0.2

3.8 ⫾ 0.1 3.7 ⫾ 0.1

0.025 0.682

148 ⫾ 54 163 ⫾ 58

157 ⫾ 40 137 ⫾ 61

0.348 0.278

4.17 ⫾ 0.94 3.07 ⫾ 0.71

3.94 ⫾ 0.71 3.09 ⫾ 0.63

0.898 0.898

2.00 ⫾ 0.40 1.74 ⫾ 0.44

1.97 ⫾ 0.39 1.64 ⫾ 0.38

0.966 0.831

0.05 ⫾ 0.01 0.04 ⫾ 0.009

0.01 ⫾ 0.006 0.03 ⫾ 0.006

0.813 0.759

0.01 ⫾ 0.006 0.01 ⫾ 0.004

0.01 ⫾ 0.004 0.02 ⫾ 0.004

0.389 0.344

Renal Function and Electrolytes

0

2

4

6

8

10

12

14

Weeks of Treatment Fig. 4. Mean change in systolic blood pressure (SBP; A), diastolic blood pressure (DBP; B), and mean arterial pressure (MAP; C) during study period in patients treated with placebo vs. 6R-BH4.

patients for a shorter duration, but a larger dosage (400 mg twice daily) of BH4. Whether larger doses of 6R-BH4 might modulate endothelial function in CKD is unclear. Our finding that 6R-BH4 significantly lowers sympathetic nerve activity, but has no effect on endothelial function or microalbuminuria, suggests that 6R-BH4 treatment may have a relatively greater effect on neuronal NOS, compared with eNOS or renal NOS. Alternatively, rather than exerting direct central effects, 6RBH4 may act peripherally to improve arterial baroreflex function, thereby reducing resting sympathetic nerve activity.

Creatinine, mg/dl 6R-BH4 group Placebo group Blood urea nitrogen, mg/dl 6R-BH4 group Placebo group Potassium, mg/dl 6R-BH4 group Placebo group Urinary microalbumin:creatinine, mg/g 6R-BH4 group Placebo group

Progenitor Cells ⫹

CD34 , cells/␮l 6R-BH4 group Placebo group CD34⫹/CD133⫹, cells/␮l 6R-BH4 group Placebo group CD34⫹/VEGF2R⫹, cells/␮l 6R-BH4 group Placebo group CD34⫹/CD133⫹/VEGF2R⫹, cells/␮l 6R-BH4 group Placebo group

Values are expressed as the means ⫾ SE. VEGF2R, vascular endothelial growth factor 2 receptor. A significant P value is shown in bold.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R216

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

patient randomized to 6R-BH4 was withdrawn from the study after experiencing hemodynamically significant reduction in BP, which resolved after cessation of the study drug, suggesting that 6R-BH4 may have a BP-lowering effect in some CKD patients. We acknowledge several limitations. First, the study population was composed of only men, without diabetes, and primarily African-American, which may limit generalizability. Whether 6R-BH4 improves SNS overactivity in a more typical population with CKD, including women, diabetics, and other racial groups, is unknown. Secondly, the study population had wellcontrolled BP and low levels of microalbuminuria at baseline. Whether 6R-BH4 treatment might have greater effects on BP and proteinuria in CKD patients with higher levels of these parameters at baseline is unknown, but likely. Similarly, patients were on multiple antihypertensive medications that were not discontinued for participation in the study. There were no significant differences in antihypertensive regimen between the treatment and control groups, and participants were carefully followed and monitored to ensure that no adjustments of BP medications occurred during the study period. However, the majority of patients in both groups were treated with inhibitors of the renin-angiotensin system, which have been shown to improve endothelial function and reduce SNS activity (33). Therefore, 6R-BH4 treatment may have less of an effect on these parameters in patients already treated with these agents. In addition, only one dose (200 mg twice per day) of 6R-BH4 was tested. This dosage was chosen on the basis of a prior study in hypertensive patients that reported that a dosage of 200 mg twice daily of 6R-BH4 led to significant improvements in BP and endothelial function (50). In the same study, a smaller total daily dosage of 200 mg did not result in improved BP or endothelial function. However, even higher doses (400 mg twice per day) were used in a different study in hyperlipidemic patients that demonstrated improvements in endothelial function and inflammatory biomarkers (15) and whether doubling the dose might have greater effects on endothelial function, BP, and oxidative stress in CKD patients remains unknown. Lastly, FMD was determined by measuring brachial artery diameter intermittently, rather than continuously, after cuff release; therefore, peak hyperemia may have occurred at a time point that was not captured in this technique. Although FMD measurements were performed uniformly in all subjects across time by a single investigator, continuous measurements of arterial diameter may have improved the detection of peak hyperemia. In conclusion, this study demonstrates that 6R-BH4 significantly lowers sympathetic activity and improves AIx and EID in patients with CKD Stage II and III. We also observed a trend toward a reduction in DBP with 6R-BH4 treatment. The treatment was safe and well tolerated, with few adverse effects. These findings warrant larger studies testing the potential benefits of 6R-BH4 treatment on long-term CV and renal outcomes in patients with CKD. ACKNOWLEDGMENTS We gratefully acknowledge BioMarin for supplying the 6R-BH4 study drug and matching placebo pills. An abstract presenting a preliminary version of these findings was presented at the Experimental Biology Meeting (2014).

GRANTS This work was supported by National Institutes of Health Grant K23 HL-098744 (to J. Park); Satellite health care, a not-for-profit renal care provider; the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Clinical Studies Center, Decatur, Georgia; the Atlanta Research and Education Foundation; and Public Health Service Grant UL1 RR025008 from the Clinical and Translational Science Award program, National Institutes of Health, National Center for Research Resources. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: J.P. and A.A.Q. conception and design of research; J.P., S.S., and D.D.D. performed experiments; J.P., P.L., and R.H.L. analyzed data; J.P., D.D.D., and A.A.Q. interpreted results of experiments; J.P. prepared figures; J.P. drafted manuscript; J.P., P.L., S.S., R.H.L., and A.A.Q. edited and revised manuscript; J.P., P.L., S.S., R.H.L., D.D.D., and A.A.Q. approved final version of manuscript. REFERENCES 1. Adams MR, Robinson J, McCredie R, Seale JP, Sorensen KE, Deanfield JE, Celermajer DS. Smooth muscle dysfunction occurs independently of impaired endothelium-dependent dilation in adults at risk of atherosclerosis. J Am Coll Cardiol 32: 123–127, 1998. 2. Aicher A, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, Zeiher AM, Dimmeler S. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 9: 1370 –1376, 2003. 3. Al Mheid I, Corrigan F, Shirazi F, Veledar E, Li Q, Alexander WR, Taylor WR, Waller EK, Quyyumi AA. Circadian variation in vascular function and regenerative capacity in healthy humans. J Am Heart Assoc 3: e000845, 2014. 4. Amann K, Rump LC, Simonaviciene A, Oberhauser V, Wessels S, Orth SR, Gross ML, Koch A, Bielenberg GW, Van Kats JP, Ehmke H, Mall G, Ritz E. Effects of low dose sympathetic inhibition on glomerulosclerosis and albuminuria in subtotally nephrectomized rats. J Am Soc Nephrol 11: 1469 –1478, 2000. 5. Badve SV, Roberts MA, Hawley CM, Cass A, Garg AX, Krum H, Tonkin A, Perkovic V. Effects of ␤-adrenergic antagonists in patients with chronic kidney disease: a systematic review and meta-analysis. J Am Coll Cardiol 58: 1152–1161, 2011. 6. Bakris GL, Fonseca V, Katholi RE, McGill JB, Messerli FH, Phillips RA, Raskin P, Wright JT Jr, Oakes R, Lukas MA, Anderson KM, Bell DS. Metabolic effects of carvedilol vs metoprolol in patients with type 2 diabetes mellitus and hypertension: a randomized controlled trial. JAMA 292: 2227–2236, 2004. 7. Bakris GL, Hart P, Ritz E. Beta blockers in the management of chronic kidney disease. Kidney Int 70: 1905–1913, 2006. 8. Bergamaschi CT, Campos RR, Lopes OU. Rostral ventrolateral medulla: A source of sympathetic activation in rats subjected to long-term treatment with L-NAME. Hypertension 34: 744 –747, 1999. 9. Bevan RD. Trophic effects of peripheral adrenergic nerves on vascular structure. Hypertension 6: III19 –III26, 1984. 10. Boutouyrie P, Lacolley P, Girerd X, Beck L, Safar M, Laurent S. Sympathetic activation decreases medium-sized arterial compliance in humans. Am J Physiol Heart Circ Physiol 267: H1368 –H1376, 1994. 11. Cardillo C, Kilcoyne CM, Quyyumi AA, Cannon RO, 3rd, Panza JA. Selective defect in nitric oxide synthesis may explain the impaired endothelium-dependent vasodilation in patients with essential hypertension. Circulation 97: 851–856, 1998. 12. Carella AM, Antonucci G, Conte M, Di Pumpo M, Giancola A, Antonucci E. Antihypertensive treatment with ␤-blockers in the metabolic syndrome: a review. Curr Diabetes Rev 6: 215–221, 2010. 13. Casey DP, Curry TB, Joyner MJ, Charkoudian N, Hart EC. Relationship between muscle sympathetic nerve activity and aortic wave reflection characteristics in young men and women. Hypertension 57: 421–427, 2011. 14. Converse RL Jr, Jacobsen TN, Toto RD, Jost CM, Cosentino F, Fouad-Tarazi F, Victor RG. Sympathetic overactivity in patients with chronic renal failure. N Engl J Med 327: 1912–1918, 1992.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE 15. Cosentino F, Hurlimann D, Delli Gatti C, Chenevard R, Blau N, Alp NJ, Channon KM, Eto M, Lerch P, Enseleit F, Ruschitzka F, Volpe M, Luscher TF, Noll G. Chronic treatment with tetrahydrobiopterin reverses endothelial dysfunction and oxidative stress in hypercholesterolaemia. Heart 94: 487–492, 2008. 16. Delius W, Hagbarth KE, Hongell A, Wallin BG. General characteristics of sympathetic activity in human muscle nerves. Acta Physiol Scand 84: 65–81, 1972. 17. Delius W, Hongell A, Hongell A, Wallin BG. Manoeuvres affecting sympathetic outflow in human muscle nerves. Acta Physiol Scand 84: 82–94, 1972. 18. Disthabanchong S. Vascular calcification in chronic kidney disease: Pathogenesis and clinical implication. World J Nephrol 1: 43–53, 2012. 19. Failla M, Grappiolo A, Emanuelli G, Vitale G, Fraschini N, Bigoni M, Grieco N, Denti M, Giannattasio C, Mancia G. Sympathetic tone restrains arterial distensibility of healthy and atherosclerotic subjects. J Hypertens 17: 1117–1123, 1999. 20. Frances MF, Goswami R, Rachinsky M, Craen R, Kiviniemi AM, Fleischhauer A, Steinback CD, Zamir M, Shoemaker JK. Adrenergic and myogenic regulation of viscoelasticity in the vascular bed of the human forearm. Exp Physiol 96: 1129 –1137, 2011. 21. Grassi G, Quarti-Trevano F, Seravalle G, Arenare F, Volpe M, Furiani S, Dell’Oro R, Mancia G. Early sympathetic activation in the initial clinical stages of chronic renal failure. Hypertension 57: 846 –851, 2011. 22. Grassi G, Seravalle G, Stella ML, Turri C, Zanchetti A, Mancia G. Sympathoexcitatory responses to the acute blood pressure fall induced by central or peripheral antihypertensive drugs. Am J Hypertens 13: 29 –34, 2000. 23. Hart EC, Charkoudian N, Joyner MJ, Barnes JN, Curry TB, Casey DP. Relationship between sympathetic nerve activity and aortic wave reflection characteristics in postmenopausal women. Menopause 20: 967– 972, 2013. 24. Hart EC, Wallin BG, Barnes JN, Joyner MJ, Charkoudian N. Sympathetic nerve activity and peripheral vasodilator capacity in young and older men. Am J Physiol Heart Circ Physiol 306: H904 –H909, 2014. 25. Heitzer T, Brockhoff C, Mayer B, Warnholtz A, Mollnau H, Henne S, Meinertz T, Munzel T. Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: evidence for a dysfunctional nitric oxide synthase. Circ Res 86: E36 –E41, 2000. 26. Heitzer T, Krohn K, Albers S, Meinertz T. Tetrahydrobiopterin improves endothelium-dependent vasodilation by increasing nitric oxide activity in patients with Type II diabetes mellitus. Diabetologia 43: 1435–1438, 2000. 27. Hering D, Lambert EA, Marusic P, Ika-Sari C, Walton AS, Krum H, Sobotka PA, Mahfoud F, Bohm M, Lambert GW, Esler MD, Schlaich MP. Renal nerve ablation reduces augmentation index in patients with resistant hypertension. J Hypertens 31: 1893–1900, 2013. 28. Higashi Y, Sasaki S, Nakagawa K, Kimura M, Noma K, Matsuura H, Hara K, Goto C, Oshima T, Chayama K. Excess norepinephrine impairs both endothelium-dependent and -independent vasodilation in patients with pheochromocytoma. Hypertension 39: 513–518, 2002. 29. Ilies C, Bauer M, Berg P, Rosenberg J, Hedderich J, Bein B, Hinz J, Hanss R. Investigation of the agreement of a continuous non-invasive arterial pressure device in comparison with invasive radial artery measurement. Br J Anaesth 108: 202–210, 2012. 30. Jeleazcov C, Krajinovic L, Munster T, Birkholz T, Fried R, Schuttler J, Fechner J. Precision and accuracy of a new device (CNAPTM) for continuous non-invasive arterial pressure monitoring: assessment during general anaesthesia. Br J Anaesth 105: 264 –272, 2010. 31. Kakoki M, Hirata Y, Hayakawa H, Suzuki E, Nagata D, Tojo A, Nishimatsu H, Nakanishi N, Hattori Y, Kikuchi K, Nagano T, Omata M. Effects of tetrahydrobiopterin on endothelial dysfunction in rats with ischemic acute renal failure. J Am Soc Nephrol 11: 301–309, 2000. 32. Kelly RP, Millasseau SC, Ritter JM, Chowienczyk PJ. Vasoactive drugs influence aortic augmentation index independently of pulse-wave velocity in healthy men. Hypertension 37: 1429 –1433, 2001. 33. Klein IH, Ligtenberg G, Oey PL, Koomans HA, Blankestijn PJ. Enalapril and losartan reduce sympathetic hyperactivity in patients with chronic renal failure. J Am Soc Nephrol 14: 425–430, 2003. 34. Levey AS, Coresh J, Greene T, Stevens LA, Zhang YL, Hendriksen S, Kusek JW, Van Lente F. Using standardized serum creatinine values in the modification of diet in renal disease study equation for estimating glomerular filtration rate. Ann Intern Med 145: 247–254, 2006.

R217

35. Levick SP, Murray DB, Janicki JS, Brower GL. Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart. Hypertension 55: 270 –276, 2010. 36. London GM, Blacher J, Pannier B, Guerin AP, Marchais SJ, Safar ME. Arterial wave reflections and survival in end-stage renal failure. Hypertension 38: 434 –438, 2001. 37. Maier W, Cosentino F, Lutolf RB, Fleisch M, Seiler C, Hess OM, Meier B, Luscher TF. Tetrahydrobiopterin improves endothelial function in patients with coronary artery disease. J Cardiovasc Pharmacol 35: 173–178, 2000. 38. Mano T, Iwase S, Toma S. Microneurography as a tool in clinical neurophysiology to investigate peripheral neural traffic in humans. Clin Neurophysiol 117: 2357–2384, 2006. 39. Mavromatis K, Aznaouridis K, Al Mheid I, Veledar E, Dhawan S, Murrow JR, Forghani Z, Sutcliffe DJ, Ghasemzadeh N, Alexander RW, Taylor WR, Quyyumi AA. Circulating proangiogenic cell activity is associated with cardiovascular disease risk. J Biomol Screen 17: 1163– 1170, 2012. 40. McGowan CL, Murai H, Millar PJ, Notarius CF, Morris BL, Floras JS. Simvastatin reduces sympathetic outflow and augments endotheliumindependent dilation in non-hyperlipidaemic primary hypertension. Heart 99: 240 –246, 2013. 41. Moens AL, Kass DA. Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb Vasc Biol 26: 2439 –2444, 2006. 42. Nichols WW. Clinical measurement of arterial stiffness obtained from noninvasive pressure waveforms. Am J Hypertens 18: 3S–10S, 2005. 43. O’Callaghan CJ, Williams B. The regulation of human vascular smooth muscle extracellular matrix protein production by ␣- and ␤-adrenoceptor stimulation. J Hypertens 20: 287–294, 2002. 44. Ozkor MA, Quyyumi AA. Tetrahydrobiopterin. Curr Hypertens Rep 10: 58 –64, 2008. 45. Park J, Campese VM, Nobakht N, Middlekauff HR. Differential distribution of muscle and skin sympathetic nerve activity in patients with end-stage renal disease. J Appl Physiol 105: 1873–1876, 2008. 46. Perlini S, Palladini G, Ferrero I, Tozzi R, Fallarini S, Facoetti A, Nano R, Clari F, Busca G, Fogari R, Ferrari AU. Sympathectomy or doxazosin, but not propranolol, blunt myocardial interstitial fibrosis in pressure-overload hypertrophy. Hypertension 46: 1213–1218, 2005. 47. Petrak O, Strauch B, Zelinka T, Rosa J, Holaj R, Vrankova A, Kasalicky M, Kvasnicka J, Pacak K, Widimsky J Jr. Factors influencing arterial stiffness in pheochromocytoma and effect of adrenalectomy. Hypertens Res 33: 454 –459, 2010. 48. Podjarny E, Benchetrit S, Rathaus M, Pomeranz A, Rashid G, Shapira J, Bernheim J. Effect of tetrahydrobiopterin on blood pressure in rats after subtotal nephrectomy. Nephron Physiol 94: 6 –9, 2003. 49. Podjarny E, Hasdan G, Bernheim J, Rashid G, Green J, Korzets Z. Effect of chronic tetrahydrobiopterin supplementation on blood pressure and proteinuria in 5/6 nephrectomized rats. Nephrol Dial Transplant 19: 2223–2227, 2004. 50. Porkert M, Sher S, Reddy U, Cheema F, Niessner C, Kolm P, Jones DP, Hooper C, Taylor WR, Harrison D, Quyyumi AA. Tetrahydrobiopterin: a novel antihypertensive therapy. J Hum Hypertens 22: 401–407, 2008. 51. Sander M, Chavoshan B, Victor RG. A large blood pressure-raising effect of nitric oxide synthase inhibition in humans. Hypertension 33: 937–942, 1999. 52. Schmidt TS, Alp NJ. Mechanisms for the role of tetrahydrobiopterin in endothelial function and vascular disease. Clin Sci (Lond) 113: 47–63, 2007. 53. Schmitt M, Avolio A, Qasem A, McEniery CM, Butlin M, Wilkinson IB, Cockcroft JR. Basal NO locally modulates human iliac artery function in vivo. Hypertension 46: 227–231, 2005. 54. Schulz E, Jansen T, Wenzel P, Daiber A, Munzel T. Nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension. Antioxid Redox Signal 10: 1115–1126, 2008. 55. Seals DR, Dinenno FA. Collateral damage: cardiovascular consequences of chronic sympathetic activation with human aging. Am J Physiol Heart Circ Physiol 287: H1895–H1905, 2004. 56. Stewart AD, Millasseau SC, Kearney MT, Ritter JM, Chowienczyk PJ. Effects of inhibition of basal nitric oxide synthesis on carotid-femoral pulse wave velocity and augmentation index in humans. Hypertension 42: 915–918, 2003.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org

R218

TETRAHYDROBIOPTERIN IN CHRONIC KIDNEY DISEASE

57. Tank J, Diedrich A, Schroeder C, Stoffels M, Franke G, Sharma AM, Luft FC, Jordan J. Limited effect of systemic beta-blockade on sympathetic outflow. Hypertension 38: 1377–1381, 2001. 58. Vallbo AB, Hagbarth KE, Torebjork HE, Wallin BG. Somatosensory, proprioceptive, and sympathetic activity in human peripheral nerves. Physiol Rev 59: 919 –957, 1979. 59. van Sloten TT, Schram MT, van den Hurk K, Dekker JM, Nijpels G, Henry RM, Stehouwer CD. Local stiffness of the carotid and femoral artery is associated with incident cardiovascular events and all-cause mortality: the hoorn study. J Am Coll Cardiol 63: 1739 –1747, 2014. 60. Volders PG. Novel insights into the role of the sympathetic nervous system in cardiac arrhythmogenesis. Heart Rhythm 7: 1900 –1906, 2010. 61. Wallin BG, Fagius J. Peripheral sympathetic neural activity in conscious humans. Annu Rev Physiol 50: 565–576, 1988. 62. Weber T, Ammer M, Gunduz D, Bruckenberger P, Eber B, Wallner M. Association of increased arterial wave reflections with decline in renal function in chronic kidney disease stages 3 and 4. Am J Hypertens 24: 762–769, 2011. 63. Weber T, Auer J, O’Rourke MF, Kvas E, Lassnig E, Berent R, Eber B. Arterial stiffness, wave reflections, and the risk of coronary artery disease. Circulation 109: 184 –189, 2004.

64. Yamamizu K, Shinozaki K, Ayajiki K, Gemba M, Okamura T. Oral administration of both tetrahydrobiopterin and L-arginine prevents endothelial dysfunction in rats with chronic renal failure. J Cardiovasc Pharmacol 49: 131–139, 2007. 65. Ye S, Nosrati S, Campese VM. Nitric oxide (NO) modulates the neurogenic control of blood pressure in rats with chronic renal failure (CRF). J Clin Invest 99: 540 –548, 1997. 66. Yokoyama K, Tajima M, Yoshida H, Nakayama M, Tokutome G, Sakagami H, Hosoya T. Plasma pteridine concentrations in patients with chronic renal failure. Nephrol Dial Transplant 17: 1032–1036, 2002. 67. Zhang H, Faber JE. Trophic effect of norepinephrine on arterial intimamedia and adventitia is augmented by injury and mediated by different ␣1-adrenoceptor subtypes. Circ Res 89: 815–822, 2001. 68. Zoccali C. Traditional and emerging cardiovascular and renal risk factors: an epidemiologic perspective. Kidney Int 70: 26 –33, 2006. 69. Zoccali C, Mallamaci F, Parlongo S, Cutrupi S, Benedetto FA, Tripepi G, Bonanno G, Rapisarda F, Fatuzzo P, Seminara G, Cataliotti A, Stancanelli B, Malatino LS. Plasma norepinephrine predicts survival and incident cardiovascular events in patients with end-stage renal disease. Circulation 105: 1354 –1359, 2002.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00409.2014 • www.ajpregu.org