Vitamin D Receptor Activators and Clinical Outcomes in Chronic Kidney Disease

Vitamin D deficiency appears to be an underestimated risk factor for cardiovascular disease in patients with chronic kidney disease. Evidence from both basic science and clinical studies supports the possible protective role of vitamin D beyond its effect on mineral metabolism. Toxicity of pharmacologic doses of active vitamin D metabolites, in particular calcitriol, is mainly due to the possibility of positive calcium and phosphorus balance. Therefore, vitamin D analogs have been developed, which suppress PTH secretion and synthesis with reduced calcemic and phosphatemic effects. Observational studies suggest that in hemodialysis patients the use of a vitamin D receptor (VDR) activator, such as calcitriol, doxercalciferol, paricalcitol, or alfacalcidol, is associated with a reduced mortality when compared with nonusers of any VDR activator. In this article the existing literature on the topic is reviewed, although a more robust answer to the question of whether or not VDR activators have beneficial effects in hemodialysis patients will hopefully come from a randomized controlled trial.


Introduction
Chronic kidney disease (CKD) is associated with increased cardiovascular events and mortality when the glomerular filtration rate declines below 60 mL/min [1][2][3]. One significant event in CKD patients is the development of calcitriol deficiency secondary to the reduction/absence of kidney α1-hydroxylase which mediates the final hydroxylation step of 25(OH)vitamin-D to 1,25-(OH) 2 vitamin-D, or calcitriol [4,5]. 1,25-(OH) 2 vitamin-D deficiency causes parathyroid hyperplasia and increased parathyroid hormone [6]; the consequent hyperparathyroidism and hyperphosphatemia are important risk factors for mortality in CKD patients [4,7]. Accordingly, vitamin D treatment is associated with a reduced rate of cardiovascular diseases and mortality [5,8].
Several studies also underline the side effects of calcitriol treatment, such as hypercalcemia and hyperphosphatemia, which carry an increased risk of cardiovascular calcifications. Compared to calcitriol, vitamin D analogs, such as paricalcitol, cause less hypercalcemia and hyperphosphatemia because of less bone resorption and less intestinal absorption [9,10]. Calcitriol and vitamin D analogs are better identified as Vitamin D receptor activators.
In addition to suppression of PTH, use of vitamin D receptor activators has been associated with other effects: reduced hospitalization and mortality, prevention of cardiovascular diseases, vascular calcification and atherosclerosis, inhibition of the rennin-angiotensin system, preservation form cellular senescence, improved endothelial function, reduced tubular interstitial fibrosis, and reduced inflammatory status.
Studies about vitamin D receptor activators and clinical outcomes (Table 1) in chronic kidney disease are reviewed in this article. (Table 2). Coyne et al. [11] found that 91% of patients treated with paricalcitol reached two consecutive PTH level reductions of 30% or greater versus 13% of placebo patients (P < .001), while incidences of hypercalcemia, hyperphosphatemia were not significantly different between two groups. Martin et al. [12] demonstrated that 68% of patients treated with paricalcitol had a 30% decrease in serum PTH for 4 consecutive weeks-without evidence of hypercalcemia and hyperphosphatemia-versus 8% of patients treated with placebo (P < .001) (12). Lindberg et al. [13] showed, in an open-label study, that PTH levels fell into target range by month 5 without episodes of hypercalcemia and hyperphosphatemia. (Table 2). A multicenter, double-blind RCT conducted by Sprague et al. [14] demonstrated that paricalcitol patients have ≥50% and faster reduction in baseline PTH versus calcitriol patients; they also showed that hypercalcemic episodes were 18% for paricalcitol versus 33% for calcitriol (P < .01). In a retrospective study, Mittman et al. [15] found that PTH levels were significantly lower for paricalcitol versus calcitriol (247 versus 190 pg/mL) while episodes of hypercalcemia and hyperphosphatemia were significantly fewer for paricalcitol versus calcitriol. A crossover study conducted by Coyne et al. [16] demonstrated that suppression of PTH at 36 hours was significantly greater after administration of 160 μg of paricalcitol (63.6% ± 2.3%) versus calcitriol (but similar after administration of 160 μg of paricalcitol), while the increase of serum calcium is greater in calcitriol group. Lund et al. [17] in a single-center, double-blind, activecontrolled, randomized, crossover trial observed that fractional intestinal calcium absorption was significantly lower after paricalcitol versus calcitriol. Finally, Mittman et al. [18] in a 2-year, single-center crossover study demonstrated that conversion from calcitriol to paricalcitol resulted in lower serum calcium (P < .001), lower serum phosphorus (P < .05), reduced PTH (P = .001) and serum alkaline phosphatase (P < .001). (Tables 3 and 4). In a crossover study comparing paricalcitol and doxercalciferol, Joist et al. [19] observed a similar suppression of PTH, while serum phosphorus was significantly higher using doxercalciferol. In a doubleblind randomized study, Frazão et al. [20], in an openlabel doxercalciferol treatment (16 weeks), and randomized, double-blinded treatment with doxercalciferol or placebo (8 weeks), found that 80% of doxercalciferol patients showed a 70% reduction in PTH levels from baseline, although serum calcium and phosphate levels increased respectively from 9.2 to 9.7 mg/dL and from 5.4 to 5.9 mg/dL. Coburn et al. [21] in a randomized, double-blinded, placebo-controlled, multicenter trial in 55 patients with stage 3 or 4 CKD showed that iPTH levels decreased more in doxercalciferol treatment versus placebo (P < .001); no significant differences in mean serum calcium or phosphorus were observed between the two groups. In a randomized study, Zisman et al. [22] demonstrated that in patients on a maintenance dose of paricalcitol, dosing doxercalciferol at 55-60% of the paricalcitol dose results in comparable inhibition of PTH, with similar incidences of hypercalcemia and hyperphosphatemia. Comparing maxacalcitol and calcitriol, Hayashi et al. [23] found no significant differences between the two groups in serum iPTH and phosphorus concentration, while serum calcium was significantly higher in the maxacalcitol versus calcitriol group during early treatment, but not at the end of treatment. Shiizaki et al. [24], in a study conducted in 5/6 nephrectomized rats treated by a direct injection of maxacalcitol into the parathyroid gland, found a significant decrease of PTH versus rats treated by vehicle, along with upregulation of both VDR and CaSR in the parathyroid tissue; no differences in calcium and phosphorus levels were observed between two groups. Kazama et al. [25] found that both maxacalcitol and calcitriol significantly decreased plasma intact PTH levels and increased serum Ca levels, but PTH levels were significantly lower in the maxacalcitol group after 24 weeks of treatment. In addition, serum phosphate levels were significantly higher in the calcitriol group. Thus, these authors proposed maxacalcitol as a possible less phosphatemic active vitamin D agent which might reduce the risk of extraskeletal calcification [25]. Oyama et al. [26] treated patients with maxacalcitol intravenously and found that lower pretreatment plasma iPTH and calcium levels, but not phosphorus levels, were predictor of the response to treatment with maxacalcitol. On the other hand, serum levels of phosphorus did not significantly increase during treatment. (Table 5). Several studies addressed the issue of parathyroid hyperplasia (Table 5).

Parathyroid Hyperplasia
Okuno et al. [27] demonstrated that the responsiveness to maxacalcitol therapy of secondary hyperparathyroidism is dependent on parathyroid gland size and that the simple measurement of maximum parathyroid gland diameter by ultrasonography may be useful for predicting responsiveness to maxacalcitol treatment. They suggest that glands larger than 11 mm do not adequately respond to treatment. Shiizaki et al. [28] also studied 20 patients with SHPT and enlarged parathyroid glands treated by percutaneous maxacalcitol 4 International Journal of Nephrology  Serum phosphorus levels did not significantly increase. Phosphorus levels were not predictive of the response to treatment with maxacalcitol. PTH levels fell promptly and significantly and were well controlled for one year. Serum calcium levels rose significantly, but within a physiological range. Hypercalcemia (33.1%) was resolved or ameliorated after the withdrawal or dose reduction of maxacalcitol.

Saito et al. [30] 2010
An outpatient treatment regimen using percutaneous maxacalcitol injection therapy (PMIT) on a weekly basis for 4-6 weeks following dialysis.
Intact PTH decreased from 797 to 253 pg/mL. Ultrasonographic examination detected a gradual reduction in parathyroid gland volume from 1.27 to 0.24 cm 3 . injection therapy, which significantly decreased the serum intact-PTH level and parathyroid gland volume for at least 12 weeks. Akizawa and Kurokawa [29], in a trial on the longterm administration of maxacalcitol, found that PTH levels fell promptly and were well controlled for one year, with doses ranging from 2.5 to 20 mg per dialysis. Serum calcium levels rose significantly, but within a physiological range; episodes of hypercalcemia were present in 33% of patients. Saito et al. [30] proposed an outpatient treatment regimen using percutaneous maxacalcitol injection therapy on a weekly basis for 4-6 weeks following dialysis. They found no major complications and intact parathyroid hormone decreased from 797 ± 178 pg/mL to 253 ± 25 pg/mL, while the parathyroid gland volume gradually decreased from 1.27 ± 1.06 cm 3 to 0.24 ± 0.15 cm 3 .

Vitamin D Receptor Activators, Mineral Metabolism, and
Bone Disorders ( Table 6). Activation of the vitamin D receptor plays a role in bone metabolism and treatment with VDR activators may favorably affect bone disease. Slatopolsky et al. [31] studied uremic rats to assess the efficacy of paricalcitol in prevention and treatment of renal osteodystrophy. Paricalcitol resulted effective in preventing and suppressing hyperparathyroidism induced by uremia and enhanced by   [34] analyzed the observational studies on the association between use of VDR activators and mortality. They underscored the absence of randomized controlled trials but considered the available observational studies "quite robust and consistent". The hypothesis of a positive, clinically significant effect of treatment with VDR activators is supported by the presence of plausible mechanisms that might explain their observed benefit in patients on dialysis, beyond their classic role in bone and mineral metabolism. Specifically, these include inhibition of renin biosynthesis, modulation of arterial function, positive effects on left ventricular hypertrophy, attenuation of insulin resistance, potential positive impact on immune function, reduced incidence of cancer, and other potential mechanisms [34]. A seminal study on the possible relationship between paricalcitol treatment and reduced mortality was published by Teng et al. [35]. They designed a historical cohort study to compare the 36-month survival rate among hemodialysis patients receiving treatment with paricalcitol versus calcitriol: mortality rate among patients receiving paricalcitol was significantly lower versus patients receiving calcitriol (P < .001). Tentori et al. [36], conduced on 7731 patients to compare calcitriol, paracaclcitol and doxercalciferol. They demonstrated that mortality rates were similar in patients treated with doxercalciferol and paricalcitol, while higher in patients treated with calcitriol (P < .001). Thus, the survival benefit in chronic kidney disease patients, independent of the effects on parathyroid hormone and calcium levels, appears to be better with the use of vitamin D analogs (paricalcitol and doxercalciferol), followed by the use of calcitriol, and the worst survival is associated with no VDR activation therapy. The mechanisms underlying the cardiovascular and survival benefit of VDR activators are still under active investigation. Several different potential factors could play a role, as VDR has been identified in more than 30 different tissues in the human body, including the vasculature [8]. Ablation of the 1alpha-hydroxylase gene in mice led to hypertension, cardiac hypertrophy, and systolic dysfunction, and this cardiac phenotype was rescued with exogenous 1,25vitD administration. 1,25vitD plays a protective role in the cardiovascular system by repressing the renin-angiotensin system independent of extracellular calcium or phosphorus. PRIMO study (Paricalcitol Benefits in Renal Disease Induced Cardiac Morbidity Study) [40] Ongoing RCT, oral paricalcitol compared to placebo in 220 predialysis patients (GFR 15-45 ml/min) affected by mild-to-moderate LVH and an LV ejection fraction >50%

Study ongoing
Mizobuchi et al. [41] 2007 Uremic rats (5/6 NX rats) were given calcitriol, paricalcitol, or doxercalciferol 3/week for 1 month Calcitriol and doxercalciferol, but not paricalcitol, increased vascular calcification in uremic rats. The different effects of VDRA on vascular calcification are independent of an effect on Ca and P. Doxercalciferol significantly increased the Ca × P product and the aortic calcium content. A lower doxercalciferol did not increase the calcium-phosphate product but increased the aortic calcium content. (Table 8). One hypothesis derived from the available observational studies suggests that systemic activation of VDRs may have direct effects on the cardiovascular system to decrease mortality in patients with chronic kidney disease [8]. Vitamin D and its analogs may play a role in preserving the cardiovascular system and reducing vascular calcification. In accordance with this concept, Levin and Li [54] suggested that Vitamin D deficiency might be an underestimated risk factor for cardiovascular disease in chronic kidney disease. They also underscore that evidence from both basic science and clinical studies supports the possible protective role of vitamin D beyond its effect on mineral metabolism. Bodyak et al. [37] studied Dahl salt-sensitive rats fed a high-salt diet (6% NaCl for 6 weeks) and receiving paricalcitol, showing lower heart and lung weights, reduced left ventricular mass, posterior wall thickness and end diastolic pressures, and increased fractional shortening. Xiang et al. [38] studied VDR knockout mice and they showed that cardiac renin mRNA levels were significantly increased, suggesting that the cardiac hypertrophy in VDR knock-out mice is a consequence of the activation of both the systemic and cardiac rennin angiotensin system and that 1,25-dihydroxyvitamin D 3 regulates cardiac functions. In another gene knock-out study by Zhou et al. [39], ablation of the 1alpha-hydroxylase gene in mice led to hypertension, cardiac hypertrophy, and systolic dysfunction, and this cardiac phenotype was rescued with exogenous 1,25-dihydroxyvitamin D administration. The authors concluded that calcitriol plays a protective role in the cardiovascular system by repressing the reninangiotensin system independent of extracellular calcium or phosphorus. In humans, the PRIMO study (Paricalcitol Capsules Benefits in Renal Failure Induced Cardiac MOrbidity Study), a multinational, randomized, double-blinded trial with oral paricalcitol compared to placebo, is ongoing [40]: its primary outcome measure is to investigate the effects of paricalcitol on progression or regression of left ventricular hypertrophy in Stage 3B/4 chronic kidney disease subjects, 8 International Journal of Nephrology A study in atherosclerotic mice to investigate the protective effect of paricalcitol combined with angiotensin-converting enzyme inhibitor (enalapril) on aortic oxidative injury ApoE-deficient mice developed hypertension which was prevented by enalapril or enalapril + paricalcitol treatment (not by paricalcitol alone). Atherosclerotic plaque in the aorta of ApoE-deficient mice was prevented by paricalcitol, enalapril, and paricalcitol + enalapril treatments through the evaluation of changes in left ventricular mass index, in a time frame of 48 weeks. Mizobuchi et al. [41] studied uremic rats treated with calcitriol, paricalcitol, or doxercalciferol and found that calcitriol and doxercalciferol, but not paricalcitol, increase vascular calcification in uremic rats; in particular the different effects of VDR activators on vascular calcification appear to be independent of serum calcium and phosphate levels. (Table 9). Husain et al. [42] conducted a study in atherosclerotic mice to investigate the protective effect of paricalcitol combined with angiotensinconverting enzyme inhibition (by enalapril) on aortic oxidative injury. They found that ApoE-deficient mice developed hypertension, which was prevented by enalapril or by the combined enalapril and paricalcitol treatment, but not by paricalcitol alone. On the other hand, atherosclerotic plaque in the aorta of ApoE-deficient mice was prevented by paricalcitol, enalapril, and paricalcitol + enalapril treatments. Combination therapy afforded greater protection against aortic inflammatory and oxidative injury in atherosclerosis than monotherapy. This observation underscores the role of VCR activators not only as PTH suppressors but also as an essential treatment in patients with chronic kidney diseases, which are notoriously more exposed to cardiovascular diseases and atherosclerosis. (Table 10). Agarwal et al. [43] in three double-blind, randomized, placebo controlled studies in patients with chronic kidney disease stage 3 and 4, found a reduction in proteinuria in 51% of paricalcitol patients versus 25% of placebo patients (P < .01). The demonstration of a reduction in proteinuria associated with paricalcitol treatment, independent of concomitant use of agents that block the renin angiotensin system RAA, suggests that paricalcitol is a potential pharmacologic means of reducing proteinuria in chronic kidney disease. As a consequence, de Zeeuw et al. [44] designed a multinational, placebo-controlled, doubleblind trial (VITAL study): patients affected by type 2 diabetes and albuminuria receiving angiotensin-converting enzyme inhibitors or angiotensin receptor blockers were randomized to receive placebo, 1 μg/day paricalcitol, or 2 μg/day paricalcitol. They demonstrated that patients treated with 2 μg of paricalcitol showed an early and sustained reduction in urinary albumin-to-creatinine ratio versus placebo (P < .05). In another, smaller double-blind randomized study, Fishbane et al. [45] randomized 61 patients with estimated glomerular filtration rate from 15 to 90 mL/min/1.73 m 2 and protein excretion greater than 400 mg/24 h to receive paricalcitol, 1 mcg/day, or placebo: changes in protein excretion from baseline to last evaluation were +2.9% for controls and −17.6% for the paricalcitol group (P < .05). Zhang et al. [46] studied streptozotocin-(STZ-) induced diabetic mice and discovered that treatment with losartan and paricalcitol completely prevented albuminuria, restored glomerular filtration barrier structure, and markedly reduced glomerulosclerosis, preventing renal injury in diabetic nephropathy. Thus, evidence is available suggesting that inhibition of the rennin angiotensin system with combination of vitamin D analogs and rennin angiotensin system inhibitors effectively prevents renal injury in diabetic nephropathy and it may be associated with improved renal protection. (Table 11). Paricalcitol inhibits the rennin-angiotensin system. Freundlich et al. [47], in a study in remnant kidney model of chronic renal failure (5/6 nephrectomy) mice, administrated paricalcitol and found that paricalcitol decreases angiotensinogen, renin, renin receptor, and vascular endothelial growth factor mRNA levels in the remnant kidney by 30-50 percent versus untreated animals. Bodyak et al. [37] also found that paricalcitol significantly reduced cardiac renin expression in Dahl salt-sensitive rats. (Table 12). Tan et al. [48], using a mouse model of obstructive nephropathy, found that paricalcitol attenuates renal tubulo-interstitial fibrosis. Paricalcitol reduced infiltration of T cells and macrophages in the obstructed kidney and this inhibition of inflammatory cell infiltration was accompanied by a decreased expression of RANTES and TNF-alpha. Their results suggest that paricalcitol inhibits renal inflammatory infiltration and RANTES expression by promoting VDRmediated sequestration of NF-kappaB signaling. Wang et al. [49] treated with the VDR agonist doxercalciferol dietinduced obese mice, presenting proteinuria, renal mesangial expansion, accumulation of extracellular matrix proteins, and activation of oxidative stress. Treatment with doxercalciferol decreased proteinuria, podocyte injury, mesangial expansion, and extracellular matrix protein accumulation. The VDR agonist also decreased macrophage infiltration, oxidative stress, proinflammatory cytokines, and profibrotic growth factor. In addition, it prevented the activation of the renin-angiotensin-aldosterone system including the angiotensin II type 1 receptor and the mineralocorticoid Zhang et al. [46] 2008 Study in streptozotocin-(STZ-) induced diabetes model mice Treatment with losartan and paricalcitol completely prevented albuminuria, restored glomerular filtration barrier structure, and markedly reduced glomerulosclerosis, effectively preventing renal injury in diabetic nephropathy receptor. An additional novel finding of this study is that the VDR activator decreased the accumulation of neutral lipids (triglycerides and cholesterol) and the expression of enzymes that mediate fatty acid and cholesterol synthesis. (Table 13). In a pilot trial, Alborzi et al. [50] randomized patients in 3 groups receiving oral paricalcitol 0, 1, or 2 mcg/day. They observed a reduction of high sensitivity C-reactive protein and of albuminuria in patients treated with paricalcitol, with a mechanism independent of its effects on hemodynamics or PTH suppression, as no differences were observed in iothalamate clearance, 24-hour ambulatory blood pressure, or PTH levels. Eleftheriadis et al. [51] also found that basal TNF-alpha concentration and basal IL-8 concentration were reduced by paricalcitol. These studies therefore suggest that paricalcitol also has immunomodulatory properties, another reason for administration of paricalcitol in patients with chronic renal failure. Indeed, patients with chronic kidney disease have chronic inflammation in the cardiovascular system and a reduced immunity to infections. (Table 14). VDR activators have been shown to modulate inflammation, thrombosis, and vasolidation, which are associated with endothelial dysfunction [55], and they have a potential for treatment of cardiovascular disease, including the improvement of endothelial dysfunction, which increases cardiovascular disease risk in chronic kidney disease [56]. Wu-Wong et al. [52] suggest that VDR activation improves endothelial function. They studied uremic rats (5/6 nephrectomized rat) and demonstrated that the uremia-impaired aortic relaxation was improved by paricalcitol (with a short duration of treatment, 2 weeks), in a dose-dependent manner, independent of serum PTH levels or blood pressure. PTH suppression alone did not improve endothelial function since in a separate experiment cinacalcet suppressed PTH without affecting endothelialdependent vasorelaxation. The role of phosphate in uremic Wang et al. [49] 2011 Study in mice with diet-induced obesity, treated with doxercalciferol.

Endothelial Function
Doxercalciferol decreased proteinuria, podocyte injury, mesangial expansion, extracellular matrix protein accumulation, macrophage infiltration, oxidative stress, proinflammatory cytokines, and profibrotic growth factor. In addition, it prevented the activation of the renin-angiotensin-aldosterone system. VDR activation also decreased the accumulation of neutral lipids (triglycerides and cholesterol) and the expression of enzymes that mediate fatty acid and cholesterol synthesis. A pilot trial in 24 patients randomized to 3 groups to receive 0, 1, or 2 mcg of paricalcitol orally for 1 month.

Eleftheriadis et al. [51] 2010
A study in 10 healthy volunteers; peripheral blood mononuclear cells (PBMC) were cultured for 48 hours in presence or not of lipopolysaccharide (LPS) and in the presence or not of paricalcitol. TNF-alpha and IL-8 produced by PBMC were measured.
Basal TNF-alpha concentration and IL-8 concentrations were reduced by paricalcitol. Paricalcitol also blunted the TNF-alpha concentration increase induced by LPS. Paricalcitol reduced to its basal level the IL-8 concentration increase by LPS. The in vitro inhibition of TGF-alpha and IL-8 by paricalcitol confirms the immunomodulatory properties of this vitamin D analogue. Wu-Wong et al. [52] 2010 Study in uremic rats (5/6 NX rats), treated for two weeks with paricalcitol.
Uremia impaired aortic relaxation was improved by paricalcitol in a dose-dependent manner, independent of serum PTH levels or blood pressure. PTH suppression alone did not improve endothelial function since in a separate experiment cinacalcet suppressed PTH without affecting endothelial-dependent vasorelaxation.
subjects is important to consider. In the study by Wu-Wong et al. [52] uremic rats had normal serum phosphate levels. In a previous study, Karavalakis et al. [53] reported that, in the 5/6 nephrectomized rats fed a special diet that induced severe hyperphosphatemia, paricalcitol at 0.2 μg/kg reduced vasoconstriction but increased vascular calcification. The improvement of endothelial function by VDR activators may be one of the mechanisms responsible for the cardiovascular benefit associated with these agents in chronic kidney disease.

Acknowledgment
L. Gravellone and M. A. Rizzo contributed equally to the preparation of this paper.