Changes in microparticle profiles by vitamin D receptor activation in chronic kidney disease – a randomized trial

The importance of the close association between chronic kidney disease (CKD) and cardiovascular disease (CVD), the cardiorenal syndrome, is becoming increasingly recognised among nephrologists and cardiologists [1‐3]. The mechanisms underlying this association have been explored during the last decade, identifying an accelerated vascular disease with early endothelial dysfunction [4], and subsequent vascular calcification progressing to arterial stiffening and remodelling [3, 5‐8].

Vitamin D is currently regarded as a hormone with multiple pleiotropic effects, and the deficiency of the activated form in CKD has attracted substantial attention [9‐12]. Vitamin D seems to have endothelial protective properties, downregulating both oxidative stress and the inflammatory response, and upregulating the key enzyme for endothelial function, eNOS [13‐17]. Treatment or supplementation with vitamin D compounds to CKD patients seems to lower albuminuria [18‐20]. Regarding measures of vascular function, several studies and a recent meta-analysis by our group have shown positive effects on endothelial function [10, 21‐24], although this might be debated in lower doses [25, 26]. Regarding measures of arterial stiffness, the next step in the vascular disease process, most [27, 28] but not all [10, 11] studies have been negative.

Microparticles are small vesicles shedded from the parent cell membrane as a response to activation of the cell, but also during stress and apoptosis [29‐31]. Evidence is emerging showing that MPs are biologically active, affecting cells locally and mediating intercellular communication [29, 30, 32‐34]. The mechanism for their release, apoptosis, cell damage or activation, seems to induce different patterns of markers such as CD31, CD62E, CD62P, or CD144 on their surface [29, 31, 35]. Depending on their origin and surface molecules, they exhibit opposite effects on apoptosis, inflammation and oxidative stress [29, 31‐33, 36, 37].

Levels of CD31-positive-CD41-negative (CD31⁺ CD41⁻) and CD144-positive (CD144⁺) endothelial MPs (EMPs) correlate with physiological measurements of vascular function, such as flow mediated vasodilatation (FMD) and pulse wave velocity (PWV) [38‐42]. The marked endothelial dysfunction in CKD patients is consequently reflected in higher levels of CD144⁺ and CD31⁺CD41⁻EMPs compared to healthy controls [38, 43‐45]. For CD144⁺, and CD31⁺CD41⁻ EMPs, elevated levels are associated with cardiovascular morbidity and mortality in atherosclerosis, kidney failure, pulmonary hypertension and heart failure [39, 46‐49].

Intercellular adhesion molecule 1 (ICAM-1; CD54) and vascular cell adhesion molecule 1 (VCAM-1; CD106) are expressed on endothelial cells and on EMPs from activated and proinflammatory cells [31, 35]. They control the recruitment and migration of white blood cells to the site of inflammation and play an important role in the atherosclerotic process [50]. Some studies indicate that levels of soluble ICAM-1 and VCAM-1 correlate with cardiovascular events and death in CKD patients [51] [52, 53], to intima media thickness in hemodialysis (HD) patients [54], and to measures of dyslipidaemia [52, 53]. ICAM-1 and VCAM-1 are therefore considered to reflect the level of endothelial proinflammatory and proatherosclerotic activation in the endothelium [50].

This is a sub-study of the SOLID trial in which we show that active vitamin D attenuates a decline in endothelial function measured by FMD, and reduces levels of proinflammatory cytokines [15, 22]. The effects of vitamin D treatment on MPs and their expression of surface proteins in CKD patients have until now not been studied. We therefore hypothesized that these protective vascular effects of vitamin D treatment would be reflected in the concentrations of ICAM-1 and VCAM-1 positive MPs, and potentially also in the concentrations of CD62E⁺ EMPs, as well as other cellspecific MPs.

Paricalcitol treatment for 12 weeks to CKD patients in stage 3–4 induces a decline relative to placebo in concentrations of ICAM-1 positive MPs, where soluble ICAM-1 is a marker known to be implicated in atherosclerosis and to correlate with risk of future cardiovascular events [53]. This is in line with our previous findings in the SOLID study, with a maintained endothelial function and reduced inflammation in patients treated with 2 μg of paricalcitol [15, 22]. These findings were also accompanied by sustained concentrations of cell activation induced EMPs and PMPs, as well as levels of LMPs in the 2 μg treatment group, whereas the MP concentrations in the placebo group decreased for all measured cell specific MPs.

It is of importance to know the subtype of MPs investigated to correctly interpret the results, however this is not always clearly stated. For EMPs, Jimenez et al. [35] showed that when apoptosis was induced in endothelial cells in-vitro, activation markers on the endothelial cells and cell activation induced CD62E+ EMPs remained stable, while apoptotic markers and the production of apoptotic EMPs increased rapidly. In patients with acute coronary syndrome, three studies have documented a pattern with high concentrations of apoptotic EMPs [35, 55, 56], probably reflecting acute endothelial cell injury due to ischemia, whereas other MP subtypes were not described.

There are few studies performed that investigate changes in EMPs and their expression by an intervention. Two in-vitro studies [14, 17] on endothelial cell lines show lower levels of CD31+ EMPs after vitamin D treatment likely due to a protective effect against apoptosis. Two clinical studies on atorvastatin treatment in non-CKD patients have shown a rise in CD144⁺ EMPs by treatment [57, 58]. Interestingly, statins are believed to have similar endothelial cell protective effects as vitamin D, reducing oxidative stress and upregulating eNOS [59]. Mobarrez et al. interpreted the statin induced rise in EMPs as a protection against apoptosis, and a more healthy and reactive endothelium. On the other hand, Tehrani et al. [58] have shown an impaired endothelial dependent microcirculatory function in patients with type 1 diabetes during high dose statin treatment, although in that case it might have been due to an impaired glucose control. Augustine et al. [60] reported in a study of apoptotic EMP- and PMP-response to dobutamine stress echocardiography, that patients with signs of coronary disease (wall motion abnormalities) did not react with elevated CD31⁺ EMPs or PMPs, whereas patients with a normal stress-test did. These results seem to indicate, at least in some settings, that a dysfunctional endothelium might be less reactive, not producing more MPs in response to stress or other events, in the same way as in healthy controls or treated patients.

Concentrations of MPs expressing ICAM-1 in our patients were declining in the treatment groups with a significant interaction with treatment. ICAM-1 is an activation marker, implicated in the atherosclerotic process [50] and is believed to correlate to atherosclerotic events [51‐53]. Our results are in line with previous findings [21, 61] of decreasing levels of soluble ICAM-1 with vitamin D treatment in patients with CKD, believed to be a sign of a less pro-atherosclerotic endothelium.

We found an overall decline in the levels of cell-specific MP subtypes, however with sustained levels for the 2 μg treatment group. For CD62E⁺ EMPs there were sustained levels in both treatment groups with decreasing levels in placebo treated patients. This finding is especially interesting since we have previously shown, in these patients, that treatment maintained endothelial function measured by FMD [22]. In line with former interventional studies, decreasing levels of activation-induced EMPs may indicate less reactive potential in a chronically dysfunctional endothelium. Vitamin D treatment might then be protective against this ongoing process. However, these findings demand further research.

High levels of CD31⁺ 41⁻ and CD144⁺ EMPs seem to correlate to endothelial dysfunction and to future risk of cardiovascular morbidity and mortality [38‐42, 46‐49]. It is however unknown how changes over time relate to risk and treatment. Previous results are difficult to directly relate to our findings since only one of these studies investigated CD62⁺ EMPs [46], and since we, in the present study, did not use healthy controls. However, we do have a well characterized population, with sustained endothelial function and decreasing levels of proinflammatory cytokines in the 2 μg treatment group. Our findings with reduced concentrations of ICAM-1 positive MPs and sustained EMP, PMP and LMP concentrations might, in the light of our previous findings, be interpreted as a protective effect of paricalcitol on the vasculature.

The main limitations of the present randomized double blind study is that it is of short duration and with a small number of participants, limiting the generalizability. Despite the randomized design, there were differences in age, and differences in absolute numbers of CV disease in the three groups. Since all outcome measures, as well as measures of kidney function and laboratory parameters, were well matched at baseline, we do not believe that these differences have affected the results. However, we do acknowledge this as a limitation since age and previous CV disease is of great importance in the development of vascular disease. Samples were frozen and thawed for the analyses, however the techniques used should be robust for this issue. MPs are still new as biomarkers and there are very few interventional studies performed, making the results more difficult to interpret. This is to our knowledge the first study reporting on changes in MP concentrations in a randomized interventional trial in CKD patients, and our findings needs further investigations.

In this study we show that active vitamin D treatment with paricalcitol in CKD patients reduce concentrations of ICAM-1 positive MPs, indicating a less pro-atherosclerotic endothelium. The previously reported maintained endothelial function measured by FMD in the 2 μg treatment group, was associated with maintained concentrations of cell-activation induced EMPs, PMPs and LMPs, which might be interpreted as markers of an endothelium with preserved reactive potential.