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3 Vitamin D and the Endothelium in General Population and Experimental Studies
M. Apetrii and A. Covic
Table 20.1 CKD-associated conditions that trigger endothelial dysfunction (ED) and vitamin D
beneficial effects upon the endothelium
Conditions associated with ED in
Increased RAAS activity and
Klotho- FGF23 axis
Vitamin D actions upon endothelial functions
1. Improves/recovers eNOS activity
2. Mitigates AGE-induced downregulation of eNOS
Downregulates NADPH oxydase
4. Anti-inflamamtory: downregulates vascular
expression of IL-6 and NF-kB
5. Inhibits endothelial cell apoptosis and promotes
6. Promotes endothelial cell migration and proliferation
7. Angiogenesis through the induction of VEGF
ED endothelial dysfunction, CKD chronic kidney disease, RAAS renin angiotensin aldosterone
system, FGF23 fibroblast growth factor 23, ADMA asymmetric dimethylarginine, eNOS endothelial nitric oxide synthase, AGE advanced glycation end-products, NADPH nicotinamide adenine
dinucleotide phosphate oxidase, SOD superoxide dismutase, IL-6 interleukin-6, NF-kB nuclear
factor-kappa B, VEGF vascular endothelium growth factor
collagen and decreased elastin fibers in the ascending aorta . Vitamin D also has
antioxidant effects on the endothelium: in human endothelial cells, reduction of cell
viability and reactive oxygen species (ROS) production due to oxidative stress is
prevented by pretreatment of endothelial cells with 1,25(OH)2D3 . Vitamin D
increases expression of the antioxidant enzyme CuZn superoxide dismutase in
endothelial cells also . The prevention of cell death is mediated by vitamin D
through reduction of apoptotic proteins and promotion of autophagy, resulting in
cell “recycling” .
In general population, data from a case-control study performed by Tarcin et al.
, pointed out that vitamin D-deficient (below 25 nmol/l) subjects had a significantly lower flow mediated dilatation (FMD) as a marker of impaired endothelial
function compared to the vitamin D-sufficient control group (mean level of 25(OH)D
= 75 nmol/l). Also, FMD significantly increased after vitamin D supplementation in
the vitamin D deficiency group [17, 18]. Vitamin D deficit also contributes to the
development of arterial stiffness: suboptimal serum 25(OH)D levels are independently associated with measures of endothelial function (increased carotid-femoral
pulse wave velocity (PWV) and augmentation index obtained through applanation
tonometry) in asymptomatic subjects, both with and without traditional CV risk
factors. ED induced by vitamin D deficiency is also mediated by vascular inflammation:
20 Vitamin D and Endothelial Function in Chronic Kidney Disease
vitamin D levels are inversely correlated with proinflammatory cytokine interleukin-6
(IL-6) expression in endothelial cells and vitamin D deficiency subjects have increased
expression of the proinflammatory NFkB transcription factor .
However, contrary to expectations, vitamin D supplementation in deficient
patients with coronary artery disease failed to show any improvement in endothelial
function. It seems that in high-risk patients that are already on medication that targets potential triggers of endothelial dysfunction (angiotensin converting enzyme,
angiotensin receptor blockers, statins), vitamin D correction/supplementation does
not bring any incremental benefit [19, 20].
Other negative effects associated with vitamin D excess include hyperphosphataemia, hypercalcaemia, medial calcification, arterial stiffness and left ventricular
hypertrophy. High levels of vitamin D increases matrix metallo-proteinase-2 expression, which degrades the extracellular matrix, opening the way for endothelial cell
migration . Angiogenesis is also promoted by vitamin D through induction of
VEGF expression in endothelial cells .
Vitamin D and the Endothelium in CKD
Impaired endothelium function is seen across the whole span of CKD, from predialysis patients to ESRD patients on dialysis to renal transplant recipients .
Endothelial cell dysfunction is a well-known culprit of cardiovascular morbidity
and it develops in CKD with remarkable frequency. In fact, CV risk rises with the
decline in the eGFR and the key mediator of this inverse relationship seems to be
represented by ED . Altered endothelial function was initially described in endstage renal disease (ESRD) patients on dialysis. Subsequently, studies have shown
that ED is actually present from the early stages of CKD. However, assessing endothelial function is challenging in renal patients since two of the main conditions that
cause renal impairment – hypertension and diabetes – are also associated with
ED. Therefore, it is difficult to establish whether abnormal endothelial function in
CKD is due to renal impairment or it is predominantly a reflection of pre-existing
vascular disease aggravated by CKD [3, 23].
In CKD patients, endothelial function is altered through various mechanisms
(Table 20.1); Traditional risk factors for cardiovascular disease like hypertension or
diabetes are joined by nontraditional risk factors, such as asymmetric dimethylarginine (ADMA), advanced glycation end-products (AGEs), pro-oxidants, all accumulating in CKD, to induce ED via eNOS uncoupling, which results not only in the
down-regulation of NO production, but also generates oxygen free radicals .
Mineral bone disorders may play an important role in endothelial function, since
correction of vitamin D, phosphate and FGF23 after renal transplantation correlates
with improved endothelium-dependent vasodilatation . Cross-sectional studies
correlated serum 25(OH)D, 1,25(OH)2D levels with endothelium-dependent
vasodilation in patients with stage 3–4 CKD and in patients with end-stage kidney
disease on dialysis, suggesting that vitamin D may have an active effect on the
vascular system .
M. Apetrii and A. Covic
The effect of vitamin D analogues on the improvement of endothelial function is
mediated by the recovery of NO activity . In a CKD-like environment in vitro,
calcitriol directly normalizes eNOS activity and also decreases the number of receptors for AGEs on the endothelial cell. Lack of coupling of AGE to their receptors
blunts AGE-mediated down-regulation of eNOS . Also, calcitriol reverses the
function of endothelial cells exposed to a CKD-like environment: calcitriol downregulates the expression of IL-6 and the activity of NFkB, thus having vascular antiinflammatory properties . These in vitro results are further confirmed by animal
models of CKD studies (sub-total nephrectomized rats) with impaired ED defined as
impaired endothelium-dependent vasorelaxation response to acethylcholine [23, 26].
The uremic environment modifies the vascular expression of a wide-range of
genes that regulate oxidative stress, hormone and immune functions, inflammation
and lipid and glucose metabolism (genes that encode apolipoprotein A-IV, fatty acid
binding protein-2, hydroxysteroid dehydrogenase, heat shock protein 1b, etc.)
finally leading to ED. VDR activators ameliorate endothelial functions also by
reversing these changes at the vascular level .
In non-dialysis patients, endothelial function deteriorates with eGFR decrease
[22, 28]. However, there are differences regarding the extent of ED between different CKD stages: ESRD patients have the worse endothelial function assessed by
brachial artery FMD compared to pre-dialysis or kidney transplantation patients. At
the same time, it seems that after renal transplantation, endothelial function is
reversed to the pre-dialysis grade of impairment, as FMD does not differ significantly between pre-dialysis CKD and post-transplantation CKD. This is probably
due to remnant kidney function impairment, preceding CV damage and immunosuppression undesirable secondary effects .
In observational studies, vitamin D is a significant positive independent predictor
of endothelial function (FMD) in non-diabetic non-dialysis CKD patients as well in
ESRD patients [28–30]. The lowest FMD is seen in vitamin D deficient patients
compared to vitamin D insufficient and vitamin D sufficient patients .
Furthermore, vitamin D deficiency is independently associated with markers of
endothelial activation such as vascular cell adhesion molecule-1 (VCAM-1) and
E-selectin in non-dialysis patients .
These results coming from cross-sectional studies were further confirmed by
clinical trials. Thus, in non-diabetic CKD patients stage 3–4 with vitamin D deficiency, 6 months supplementation with ergocalciferol significantly improved
endothelium-dependent microcirculatory function assessed at the forearm level,
proving the causal relationship between vitamin D deficiency and ED . Moreover,
ergocalciferol supplementation of CKD patients reduces oxidative stress and tissue
AGE formation, the latter being predictors of CVD in CKD . Also, cholecalciferol administration to non-diabetic, non-dialysis, vitamin D deficient CKD patients
significantly improves FMD and decreases markers of endothelial activation including inter-cellular adhesion molecule-1 (ICAM-1), VCAM-1 and E-selectin .
Paricalcitol administration (2 μg/day for 12 weeks) in patients with CKD stages 3–4
resulted in a significant change in FMD compared to the placebo group .
However, reported results regarding the endothelial benefits of vitamin D or vitamin
D analogues administration differ between trials. This may be due to differences in
20 Vitamin D and Endothelial Function in Chronic Kidney Disease
time of exposure, as paricalcitol administration for only 1 month does not have any
effect over endothelial function, irrespective of dosage (1 μg/day or 2 μg/day) .
Also, endothelial function in patients with diabetes mellitus type 2 and CKD did not
improve after paricalcitol administration (1 μg/day for 3 months). This may be due
to low dosage or it can be explained by the confounding effects of medication that
targets endothelial function (anti-hypertensive or anti-diabetic medication) .
Vitamin D and CKD-Associated Conditions That
Predispose to Endothelial Dysfunction
Vitamin D deficiency also predisposes to a wide range of conditions that cause ED
in CKD, thus having indirect effects upon the endothelium (Fig. 20.1).
Ca influx in EC
Vitamin D deficiency
Renin gene expression
Ag-II action on EC
Fig. 20.1 Various pathological conditions that link vitamin D deficiency and endothelial dysfunction
in chronic kidney disease. Abbreviations: Ag-II angiotensin II, AGE advanced glycation end products,
AT1R Angiotensin II type 1 receptor, Ca calcium, COX cyclooxygenase, EC endothelial cells, EDCF
endothelial-dependent contracting factors, eNOS endothelial nitric oxide synthase, FGF-23 fibroblast
growth factor 23, HDL-C High density lipoprotein cholesterol, IL-6 interleukin-6, LDL-C low-density
lipoprotein cholesterol, NF-kB nuclear factor k-B, PTH parathormone, RANTES regulated on activation, normal T cell expressed and secreted chemokine, RAAS renin-angiotensin system, ROS reactive
oxygen species, TACE tumor necrosis factor-α converting enzyme, TNF-α tumor necrosis factor
M. Apetrii and A. Covic
In type 2 diabetes, low circulating vitamin D levels favor poor glycemic control, and
the reduction of immature endothelial progenitor cells that are involved in endothelial repair and angiogenesis, thus promoting endothelial function impairment .
Vitamin D protects against the negative effects of AGE on eNOS activity  and
also has antiatherogenic effects by inhibiting foam cell formation . Despite this,
randomized controlled trials (RCT) of diabetic patients with suboptimal serum vitamin D levels (<30 ng/ml) did not show any significant changes induced by vitamin
D repletion regarding circulating endothelial progenitor cells, FMD or blood pressure. However, FMD was significantly ameliorated by vitamin D repletion in a posthoc analysis of diabetic patients that specifically had baseline endothelial dysfunction
. Clinical studies assessed only the circulating 25(OH)D form, while the active
intracellular form is represented by 1,25(OH)2D3. Therefore, vitamin D activation
by 1α-hydroxylase may alter the relationship between 25(OH)D and markers of
arterial function . As mentioned before, it is also possible that in diabetic
patients that are on vasoactive medication, vitamin D supplementation may not further induce any significant change.
One of the main mechanisms that is excessively activated in both hypertension and
CKD having deleterious effects on vasculature is the RAAS . Vitamin D regulates vascular tone through both genomic and non-genomic mechanisms [42, 43].
Through genomic mechanisms, calcitriol negatively regulates renin gene transcription: VDR-bound calcitriol inhibits the interaction between cyclic adenosine monophosphate (cAMP) response element binding and cAMP response element in the
renin gene promoter, thus inhibiting the transcription of renin gene . Further on,
calcitriol down-regulates angiotensin II receptor 1 (AT1R) expression in renal arteries, thus blunting the deleterious effects induced by angiotensin II binding to its
receptor: in both renal and systemic arteries, vitamin D is responsible for a reduction in angiotensin II-induced ROS excessive generation . ROS promote endothelium dysfunction and finally hypertension:  ROS alter eNOS activity, being
responsible for eNOS uncoupling and thus for impaired NO-dependent endothelial
function, ROS promote the expression of cyclooxygenases-derived contracting factors in the endothelium [3, 45, 46]. Also by genomic regulation, calcitriol downregulates the expression of oxidative-stress associated enzyme, NADPH oxidase in
renal arteries. The vascular anti-oxidative action of calcitriol also comprises upregulation of SOD-1 and SOD-2 enzymes in renal arteries. The effects of calcitriol
on NADPH oxidase and SOD expression may partially be mediated by the downregulation of AT1R, as AT1R activation is responsible for ROS production. Thus, by
regulating RAAS and ROS production, vitamin D protects both kidney and systemic vessels from ED in hypertension .