Original Report: Patient-Oriented, Translational Research American

Journal of

Nephrology

Am J Nephrol 2015;42:265–273 DOI: 10.1159/000441364

Received: June 12, 2015 Accepted: September 24, 2015 Published online: October 24, 2015

Paricalcitol, Microvascular and Endothelial Function in Non-Diabetic Chronic Kidney Disease: A Randomized Trial Kristina Lundwall Gun Jörneskog Stefan H. Jacobson Jonas Spaak Department of Clinical Sciences, Danderyd University Hospital, Karolinska Institutet, Stockholm, Sweden

Abstract Background: Vitamin D deficiency, sympathetic activation and endothelial dysfunction are associated with increased cardiovascular risk in patients with chronic kidney disease (CKD). Studies have so far failed to establish the role of vitamin D and vitamin D receptor activator (VDRA) treatment in moderate CKD. This trial was designed to assess whether VDRA treatment can ameliorate sympathetic activation and macro- and microvascular dysfunction in non-diabetic patients with moderate CKD. Methods: We conducted a randomized controlled double-blind trial using placebo, 1 or 2 μg of paricalcitol, a VDRA, for 3 months. We assessed muscle sympathetic nerve activity (MSNA) by microneurography, pulse wave velocity (PWV) by tonometry, flow mediated vasodilatation (FMD) by brachial ultrasound, skin microcirculation assessed by iontophoresis and capillary blood velocity (CBV) by videophotometric capillaroscopy. Results: Thirty-six patients with a mean age of 65 years and mean estimated glomerular filtration rate of 40 ml/min/1.73 m2 were included. We found a significant decline in endothelial

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function after 3 months, except in the group receiving 2 μg of paricalcitol. The higher dose (2 μg) seemed to attenuate the decline in microvascular endothelial function, assessed by iontophoresis of acetylcholine (p = 0.06 for all groups, p = 0.65 for the 2 μg group) and for FMD (p = 0.006 for all groups, p = 0.54 for the 2 μg group). We found a borderline significance (p = 0.05) for improved CBV in the treated groups. We found no significant changes between treatments in MSNA, PWV or albuminuria. Conclusions: Endothelial function declined significantly over 3 months in patients with moderate CKD, and this decline could be ameliorated by VDRA treatment (NCT01204528). © 2015 S. Karger AG, Basel

Introduction

Cardiovascular disease (CVD) remains the main cause of death worldwide [1]. Hyperlipidemia, smoking, hypertension and diabetes are well established as the most potent risk factors, although in recent years, chronic kidney disease (CKD) has emerged as equally important [1, 2]. At the same time, CKD including microalbuminuria is 4 times as common as diabetes, affecting 10–13% of the general population [1, 3]. Not only do CKD patients carKristina Lundwall Department of Cardiology Danderyd University Hospital SE–18288 Stockholm (Sweden) E-Mail kristina.lundwall @ gmail.com

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Key Words Vascular function · Endothelial function · Vitamin D · Chronic kidney disease · Cardiovascular risk

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Am J Nephrol 2015;42:265–273 DOI: 10.1159/000441364

Dreyer et al. [25] using ergocalciferol, in a doubleblind randomized trial, recently showed that vitamin D supplementation improves microvascular endothelial function in the skin assessed by laser Doppler flowmetry after iontophoresis of acetylcholine in patients with CKD stages 3–4. Chitalia et al. [5] using cholecalciferol and Zoccali et al. [26] using paricalcitol showed similar results on macrovascular endothelial function assessed by determining flow mediated vasodilatation (FMD) in the brachial artery. In the present double-blind placebo-controlled randomized trial, we aimed to investigate whether low- or high-dose treatment with a VDRA (paricalcitol) can ameliorate sympathetic activation and macro- and microvascular functions assessed by several state-of-the art methods in non-diabetic patients with moderate CKD.

Methods Participants We recruited patients from the Department of Nephrology at Danderyd University Hospital, Stockholm, Sweden, between June 2010 and February 2013. Patients were considered eligible if they were >20 years of age, had an estimated glomerular filtration rate (eGFR) of 15–59 ml/min/1.73 m2 calculated from plasma creatinine using the MDRD formula, with a plasma PTH level of 35–500 pg/ml, with a Ca level 30 g/l and were on stable BP medication with no change in angiotensin converting enzyme inhibitor or angiotensin receptor blocker medication during 2 months before enrolling in the trial. Exclusion criteria included nephrotic syndrome, diabetes mellitus or any treatment with vitamin D or its analogues. Patients were not to have suffered acute renal failure during the last 3 months, and they should not be expected to need dialysis within 6 months. They were also excluded if they had known renal artery stenosis, severe kidney stones, uncontrolled hypertension (repeated measures of a brachial BP >150/100 mm Hg) or any other severe disease such as active cancer, AIDS/HIV or severe congestive heart failure. The study protocol was approved by the regional Ethics Committee of Stockholm, Sweden, and all patients provided written informed consent. The trial was registered on ClinicalTrials.gov (NCT01204528; online suppl. SOLID study; for all online suppl. material, see www.karger.com/doi/10.1159/000441364). Study Design Patients were included consecutively and randomized to 3 groups: placebo, 1 μg paricalcitol daily or 2 μg paricalcitol daily. The drugs in all 3 groups were visually identical, with 2 capsules per day, taken in the morning. Participants, personnel performing the examinations and researchers were all blinded to the intervention. The study started with 2 weeks of placebo run in, followed by 12 weeks of intervention. Measurements were made at baseline and after 12 weeks. Study size was determined to 72 patients with a power of 80%; but based on variability and

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ry a high cardiovascular risk, but also have worse prognosis once they suffer a cardiovascular event [3]. This connection is usually called the cardiorenal syndrome [3]. Micro- and macrovascular endothelial dysfunction is evident from the early stages of atherosclerosis and is a potent independent predictor of cardiovascular risk [4–7]. Several studies have described endothelial dysfunction in patients with varying degree of CKD [6, 8, 9]. Both the renin angiotensin-aldosterone system (RAAS) and the sympathetic nervous system are highly activated in patients with CKD, which contribute to hypertension and cardiac disease [3, 10]. Apart from optimizing co-morbidities such as blood pressure (BP) control in hypertension, lipids in hyperlipidemia and glucose control in diabetes mellitus, no therapeutic options exist to treat endothelial dysfunction and sympathetic activation directly [1]. CKD results in low levels of activated vitamin D and disturbed mineral metabolism, and vitamin D deficiency has in several studies been correlated to an increased risk of cardiovascular events [11–13]. Vitamin D has since long been used to treat secondary hyperparathyroidism in CKD. In recent years, it has been shown that supplementation of vitamin D and treatment with vitamin D receptor activators (VDRAs) in patients with end-stage renal failure (ESRD) is associated with improved survival and reduced progression of kidney disease [14–16]. Treatment appears to lower albuminuria independent of parathyroid hormone (PTH), phosphate (Pi) and calcium (Ca) levels [15, 16]. Vitamin D supplementation or treatment in ESRD might also reduce cardiovascular events and hospitalization [13, 17]. As patients with less severe renal dysfunction also commonly have very low levels of activated vitamin D, it has been proposed that they also could benefit from vitamin D supplementation and/or treatment [18]. On the other hand, too much vitamin D may increase Ca levels, hastening vascular calcification [1]. The underlying mechanisms why vitamin D treatment/supplementation may lower cardiovascular risk in the absence of secondary hyperparathyroidism are currently being investigated. For instance, it has been shown in animal studies that vitamin D inhibits renin synthesis [19]. In humans, low plasma 25(OH)D-levels activate the RAAS [20]. Supplementation with vitamin D to lower the BP has however given inconclusive results [21]. Inflammation appears to be one of the main promoters of severe atherosclerosis observed in CKD [1]. Vitamin D has been shown to lower C-reactive protein and mediate anti-inflammatory effects by inhibiting antigen presenting cells and cytokines [21–24].

Study Procedures To avoid the influence of sun exposure and vitamin D activation, no patients were randomized during the summer (in Sweden, June–August). All examinations were performed in research laboratories at the Danderyd University Hospital, Stockholm, Sweden. Baseline examinations were performed at 2 different days, within 1 week, and the patients received the study drug after the second day of the examinations. Venous blood samples were drawn in the morning, after 12 h fasting and 20 min rest, for routine chemistry in the hospital laboratory and saved for later specialized assays. Blood samples were also checked after 1 month for plasma Ca levels. After 12 weeks of intervention, patients returned and performed identical examinations as at baseline. One month after the study was completed, Ca levels were measured again. Arterial Stiffness We used applanation tonometry to assess arterial stiffness by  pulse wave velocity (PWV) and augmentation index (AIx) (SphygmoCor, AtCor Pty, NSW, Australia). The measurements were made according to current recommendations [27]. The technique is described in detail elsewhere [28]. Large Vessel Endothelial Function Macrovascular endothelial function was assessed by FMD. The method is described in detail elsewhere [29], and the measurements were made according to current recommendations [30]. In short, ischemic reactive hyperemia was induced by a pneumatic tourniquet inflated to 250 mm Hg for 5 min, and when released, vasodilatation was measured as a change in brachial artery diameter from rest. The brachial artery diameter was measured by a vascular ultrasound device with a 9-MHz linear transducer (Vivid 7 Dimension, GE Medical system, Horten, Norway). We measured mean values at rest and 30, 60 and 90 s after cuff release. The maximal increase in mean value in brachial artery diameter after cuff release was used to calculate the percentage of increase from rest and used as a measure of FMD, ΔFMD.

cular flux expressed in arbitrary unit, AU, before, during and after drug administration. Skin microvascular flux was recorded for 10 min (ACh) and 14 min (SNP), respectively, and peak flux was determined. Capillary Blood Flow Blood cell velocity in nailfold capillaries of the great toe was measured during videophotometric capillaroscopy using crosscorrelation technique. This method has been described elsewhere [32]. In short, capillary blood velocity (CBV) was determined by 2 videophotometric windows positioned along the arterial side of the capillary axis. These windows are sensitive to variations in light intensity, thus detecting variations in optical density when blood cells and plasma gaps are passing through the capillary. The variations in light intensity are converted into an electronic signal. Given the distance between the windows and the time delay between similar events in the upstream and downstream windows, CBV can be continuously recorded using cross-correlation technique [33]. CBV was measured during resting condition and during post-occlusive reactive hyperemia. To assess resting CBV (mm/s), CBV was continuously computed during 3 min, and the mean value was calculated. Post-occlusive reactive hyperemia was performed by a small pressure cuff at the proximal phalanx of the great toe. The cuff was inflated to a pressure of 200 mm Hg for 1 min, and peak CBV (mm/s) and time to peak CBV (s) were measured following release of cuff pressure. The percentage increase of CBV during post-occlusive reactive hyperemia (CBV%) was calculated as ((peak CBV – rest CBV/rest CBV) × 100). Skin Temperature Skin temperature was continuously recorded during the measurements of CBV with a thermistor placed close to the investigated nailfold. Sympathetic Activation Muscle sympathetic nerve activity (MSNA) was recorded by microneurography as previously described [34, 35]. In short, a tungsten microelectrode was inserted percutaneously into a muscle fascicle of the peroneal nerve and the raw signal was amplified and digitized (Powerlab Neuroamp, ADInstruments, Bella Vista, Australia) and subsequently analyzed by a single blinded investigator. Heart rate was recorded by a standard electrocardiogram, and BP was measured with an automatic cuff. MSNA was quantified as bursts per minute (SNA/min) and as bursts per 100 RR intervals (SNA/RRI). MSNA was registered as primary end point and all other end points as secondary.

Skin Microvascular Function – Perfusion Imaging We used laser Doppler perfusion imaging during iontophoresis of vasoactive drugs to examine endothelium dependent and independent skin microvascular function. The technique is described elsewhere [31] and is a non-invasive technique using a small electric current for drug administration across the skin. In  short, acetylcholine (ACh; Sigma-Aldrich AB, Stockholm, Sweden) and sodium nitroprusside (SNP; Hospira, Inc., Lake Forest, Ill., USA), diluted in deionized water, were used to assess independent (SNP) and dependent (ACh) endothelial function of the skin microcirculation. Electrode chambers (LI611 Drug Delivery Electrode Imaging, Perimed, Järfälla, Sweden) were attached to the forearm and filled with either ACh (2%) or SNP (2%). A battery-powered iontophoresis controller (Perilont 382b, Perimed, Järfälla, Sweden) provided a direct current (0.1 mA for 60 s) for drug iontophoresis. ACh was delivered using an anodal charge and SNP with a cathodal charge. We then used laser Doppler imaging (Periscan PIM II, Perimed, Järfälla, Sweden) to measure the dilatation capacity of the vessels, that is, skin microvas-

Statistics To compare baseline characteristics between the 3 groups, we used 1-way analysis of variance (ANOVA) for continuous variables. Categorical variables were analyzed by the χ2 test, and with the Fisher’s exact test when counts per cell were