Vitamin D insufficiency: implications for the immune system
Chronic kidney disease (CKD) is characterized by a loss of kidney function and dysregulation of vitamin D metabolism. Well known are the defects in final activation of vitamin D to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], resulting in renal osteodystrophy. However, in recent years, 1,25(OH)2D3 has been identified as having effects far beyond calcium and bone metabolism. In this review, specific attention is given to the effects of 1,25(OH)2D3 on the immune system and the implications of vitamin D deficiency, a feature of many patients with CKD, on immune function.
KeywordsVitamin D Immune modulation Chronic kidney disease
Vitamin D insufficiency is a term used to describe the biochemical evidence of deficiency without obvious clinical signs or symptoms, such as rickets or osteomalacia. Although there is continuing discussion about the precise levels of 25-hydroxyvitamin D3 [25(OH)D3], which define the different categories of vitamin D status, there is general agreement that vitamin D insufficiency or hypovitaminosis D is prevalent in many populations across the globe. Vitamin D insufficiency is most commonly diagnosed by a serum concentration of 25(OH)D3 between 15 and 30 ng/ml; levels <15 ng/ml are defined as vitamin D deficiency, with concentrations <5 ng/ml being severely deficient and associated with presentation of osteomalacia and rickets . Currently, serum concentrations of 30–50 ng/ml 25(OH)D3 are accepted to meet the requirements for vitamin D sufficiency; however, recent evidence suggests that the optimal serum 25(OH)D3 levels may be much higher [2, 3]. Vitamin D insufficiency has been reported to cause muscle weakness and may contribute to falls in the elderly, which—when associated with osteoporosis—increases the likelihood of fractures . However, more recent evidence indicates that vitamin D insufficiency and certainly deficiency might also be associated with diseases such as colorectal cancer, prostate cancer, multiple sclerosis, type 1 diabetes, cardiovascular diseases, and tuberculosis . In this review, we focus on the effects of vitamin D insufficiency/deficiency on the immune system.
Sources and metabolism of vitamin D
Vitamin D is an essential nutrient for mineral homeostasis and bone metabolism, being available in two distinct forms, ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). The first is a fungus/yeast-derived product and can be produced through ultraviolet (UV) exposure of foods, whereas the latter is mainly provided by UV-mediated synthesis in the epidermal layer of the skin but also by nutritional intake (e.g. fatty fish, cod liver oil, and fortified dairy products). Upon sunlight exposure, photolytic cleavage of 7-dihydrocholesterol in the skin results in the formation of previtamin D3, which is subsequently converted by a spontaneous thermal isomerization into vitamin D3 . For systemic transport, vitamin D3 and its metabolites, either produced by the skin or absorbed in the gastrointestinal tract, are bound by a carrier molecule, vitamin-D-binding protein (DBP). To become bioactive, inactive vitamin D3 is subjected to two subsequent hydroxylation steps . The first step, hydroxylation of vitamin D3 at the carbon-25 position resulting in formation of 25(OH)D3, occurs primarily in the liver and is catalyzed by 25-hydroxylase. Among the family of P450-containing enzymes (CYPs), there are several candidates to perform the first hydroxylation, including CYP27A1, CYP2C11, CYP2R1, and CYP3A4; but CYP2R1 has been demonstrated to be the high affinity 25-hydroxylase. The second hydroxylation step occurs predominantly in the proximal tubule cells of the kidney and is carried out by 1-α-hydroxylase (CYP27B1), resulting in production of the biologically active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3).
As 1,25(OH)2D3 is critically involved in the maintenance of adequate serum calcium and phosphate levels, its production is tightly controlled by these minerals. As 25-hydroxylase is poorly regulated, leading to a conversion of almost all vitamin D present into 25(OH)D3, 25(OH)D3 is the major circulating form of vitamin D and its concentration is commonly used as an indicator of vitamin D status. On the other hand, 1-α-hydroxylase activity is precisely regulated by a variety of factors, including serum calcium, phosphate, parathyroid hormone (PTH), and 1,25(OH)2D3 itself . Low serum calcium levels trigger the release of PTH from the parathyroid glands, which in turn increases renal 1-α-hydroxylase activity and subsequent 1,25(OH)2D3 production. In order to correct serum calcium levels, 1,25(OH)2D3 decreases renal calcium excretion and stimulates intestinal calcium absorption and osteoclast maturation to release calcium from the bones. When normocalcemia is obtained, PTH release is shut down and 1-α-hydroxylase activity is not longer stimulated. Other important negative regulators of 1-α-hydroxylase activity include fibroblast growth factor (FGF)-23 and Klotho proteins . 1,25(OH)2D3 itself induces expression of 24-hydroxylase (CYP24A1), the enzyme that accomplishes the first step of 1,25(OH)2D3 catabolism, eventually triggering its own degradation . This negative feedback mechanism probably serves as an internal rescue to avoid excessive vitamin D signaling.
Mechanism of action of 1,25(OH)2D3
Most biological effects of 1,25(OH)2D3 are mediated by binding of the ligand to its receptor, the vitamin D receptor (VDR), a member of the nuclear receptor superfamily . Upon ligand binding, this nuclear receptor heterodimerizes with the retinoid X receptor (RXR). Next, this complex binds to vitamin-D-responsive elements (VDREs) located in the promoter region of target genes in order to regulate their transcription. Interaction between the ligand-VDR-RXR complex and a VDRE results in the release of corepressors and recruitment of nuclear receptor coactivator proteins, thereby inducing chromatin remodeling and attracting key components of the transcription initiation complex. Alternatively, when ligand-VDR-RXR is recruited to an inhibitory VDRE, corepressors are recruited and transcription of the gene is inhibited. Besides its genomic actions, rapid nongenomic signaling events occurring within seconds or minutes upon 1,25(OH)2D3 exposure have been reported in different cell types [7, 8]. Importantly, VDR expression is not restricted to cells involved in mineral homeostasis and bone metabolism. The presence of this nuclear receptor has also been demonstrated in many other cell types, including normal cells (e.g. keratinocytes, pancreatic β-cells) and malignant cells (e.g. leukemia cells; breast, prostate, and colon cancer cells), allowing 1,25(OH)2D3 to expand its effects far beyond calcium and bone homeostasis (reviewed in ). Indeed, 1,25(OH)2D3 affects growth, differentiation status, and function of many other cell types. The discovery that various target cells of 1,25(OH)2D3 produce the enzymes necessary for vitamin D activation caused a major breakthrough in understanding the nonclassic actions of 1,25(OH)2D3. Extrarenal expression of 1-α-hydroxylase and in some cells also 25-hydroxylase allows the local auto- or paracrine production and regulation of 1,25(OH)2D3 without affecting serum concentrations of the hormone . These findings emphasize the importance of adequate vitamin D levels, not only to maintain bone health, but for global health.
Vitamin D and the immune system
Expression of the VDR and vitamin D metabolizing enzymes is not restricted to cells involved in calcium and bone metabolism. Within the immune system, VDR is expressed by almost all immune cells, including activated CD4+ and CD8+ T cells, B cells, neutrophils, and antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs) [11, 12, 13]. In addition, the key enzymes needed for vitamin D activation and degradation are expressed by certain immune cells. 1-α-hydroxylase has been detected in macrophages, DCs, and even B- and T cells [14, 15, 16, 17, 18]. Although being identical to the renal enzyme, expression of 1-α-hydroxylase in macrophages is upregulated by immune signals such as interferon (IFN)-γ and lipopolysaccharide (LPS) or viral infections (reviewed in ). In DCs, upregulation of 1-α-hydroxylase is associated with the maturation process of these cells, suggesting that local production of 1,25(OH)2D3 might serve as a negative feedback loop to prevent excessive inflammation . Simultaneously, maturation of DCs is accompanied by a decrease in VDR-expression, making them less responsive to 1,25(OH)2D3 as they mature. In contrast with 1-α-hydroxylase regulation in kidney, neither in macrophages nor in DCs is 1-α-hydroxylase activity subjected to negative feedback signals from 1,25(OH)2D3 itself, offering an explanation for the massive local production of 1,25(OH)2D3 by disease-associated macrophages seen in patients with granulomatous diseases (sarcoidosis and tuberculosis) [15, 16]. Next to 1-α-hydroxylase, expression of 25-hydroxylase and 24-hydroxylase by immune cells has been reported [16, 17, 18]. However, the ability of 1,25(OH)2D3 to induce 24-hydroxylase in monocytes and macrophages depends on the differentiation/maturation stage of the cells. Undifferentiated monocytes are highly susceptible to 1,25(OH)2D3-mediated 24-hydroxylase induction, whereas differentiated/activated macrophages are resistant .
Together, the presence of VDR and the regulated expression of vitamin-D-metabolizing enzymes in immune cells allow local conversion of vitamin D into its biologically active form. Indeed, a growing amount of data demonstrate functional responses of immune cells to inactive vitamin D metabolites, including vitamin D3 and 25(OH)D3 [16, 18, 21]. Local vitamin D metabolism from its inactive precursors offers an attractive mechanism for immune cells to reach levels of 1,25(OH)2D3, far exceeding serum concentrations in order to modulate immune responses, without affecting systemic levels of this hormone. Together, these data provide strong evidence for an auto- and/or paracrine role for 1,25(OH)2D3 in normal immune physiology.
Vitamin D: enhancer of innate immune responses
Recently, a central mechanism underlying the antimicrobial effects of 1,25(OH)2D3 was identified: 1,25(OH)2D3 induces expression of cathelicidin antimicrobial peptide (CAMP) in various cell types, including myeloid cells, keratinocytes, neutrophils, and bronchial epithelial cells, directly leading to enhanced antimicrobial activity [28, 29, 30]. Interestingly, 1,25(OH)2D3-mediated induction of CAMP was found to be an integral component of human TLR-mediated immune responses: TLR2/1-triggering of human monocytes selectively induces expression of VDR and 1-α-hydroxylase, making the cells able to convert 25(OH)D3 into active 1,25(OH)2D3 and allowing the 1,25(OH)2D3-mediated induction of CAMP . Therefore, adequate functioning of this antimicrobial mechanism critically depends on the presence of 25(OH)D3 in the medium: TLR2/1-stimulated production of CAMP was severely impaired when monocytes were cultured with serum from dark-skinned African Americans, as this serum contains low levels of 25(OH)D3 due to lower vitamin D production in dark skin. Addition of 25(OH)D3 to the medium could indeed restore impaired CAMP production. Recently, Krutzik et al. discovered IL-15 as the factor responsible for TLR2/1-mediated induction of VDR and 1-α-hydroxylase in monocytes . Remarkably, induction of CAMP is not the only mechanism linking vitamin D signaling to TLR-mediated antimicrobial responses: TLR-induced expression of another antimicrobial peptide, defensin beta 4, by monocytes requires convergence of both VDR- and IL1β-activation pathways .
Together, vitamin-D-mediated induction of antimicrobial peptides (at least partially) explains the success of UV therapy as a way to improve or even cure disease in tuberculosis patients. Nevertheless, additional mechanisms are likely to further complement the antimicrobial effects of 1,25(OH)2D3, as 1,25(OH)2D3 mediates generation of reactive oxygen species in human monocytes and macrophages . Induction of inducible nitric oxide synthase (iNOS) has been proposed as a potential mechanism by which 1,25(OH)2D3 mediates bacterial killing. However, controversial data have been obtained concerning regulation of iNOS expression by 1,25(OH)2D3. In a human macrophage-like cell line, induction of iNOS expression by the hormone was observed, whereas others reported inhibitory actions of 1,25(OH)2D3 on this enzyme [34, 35]. Remarkably, while participating in TLR-induced antimicrobial responses, 1,25(OH)2D3 induces hyporesponsiveness to PAMPS by downregulating the expression of TLR2 and TLR4 on monocytes, probably providing a negative feedback mechanism to prevent excessive TLR activation and to shut down the inflammatory response at a later stage of infection .
Vitamin D: modulator of adaptive immune responses
APCs as primary targets for 1,25(OH)2D3
In the light of adaptive immune responses, DCs have been shown to be central targets for VDR agonists [38, 39]. DC differentiation and maturation is potently inhibited by 1,25(OH)2D3, as evidenced by sustained expression of monocytic markers and decreased levels of DC markers, MHC class II, costimulatory molecules (CD40, CD80, CD86), and other maturation-induced surface markers [40, 41, 42, 43]. In addition, 1,25(OH)2D3 dramatically alters DC-derived cytokine and chemokine production: 1,25(OH)2D3-treated DCs express lower levels of IL-12 and IL-23 (known as major cytokines driving Th1- and Th17-differentiation respectively), whereas these cells secrete elevated levels of IL-10 (a cytokine exerting broad spectrum anti-inflammatory activities) and the chemokine macrophage inflammatory protein 3 alfa (MIP3α) [also known as CCL22, a chemokine involved in the recruitment of CCR4-expressing regulatory T cells (Tregs)] [40, 41, 42, 43, 44, 45, 46, 47]. Besides this, 1,25(OH)2D3 treatment of differentiating DCs also disturbs their migratory capacity in response to inflammatory and lymph-node-homing chemokines, although expression of cognate chemokine receptors remained unaffected . A large-scale analysis by our group of the protein profile of DCs using a 2D differential gel-electrophoresis (2D-DIGE) system revealed three protein groups predominantly affected by a VDR agonist, being proteins involved in cytoskeleton structure, protein biosynthesis/proteolysis, and metabolism . Alterations in cytoskeleton proteins may diminish the establishment of or deteriorate existing DC T-cell contacts and decrease secretion of cytokines by T cells.
Direct actions of 1,25(OH)2D3 on T and B lymphocytes
Besides the profound effects of 1,25(OH)2D3 on T-cell responses via modulation of DC phenotype and function, induction of VDR expression in activated T cells indicates additional direct mechanisms to shape T-cell responses (Fig. 1). In agreement with this, several groups have demonstrated the ability of 1,25(OH)2D3 to directly inhibit antigen- and lectin-stimulated proliferation of human and murine T cells and to alter their cytokine profiles: 1,25(OH)2D3 inhibits production of Th1 cytokines such as IL-2, IFN-γ, and TNF-α and favors the emergence of Th2 cells by upregulating the expression of Th2-specific transcription factors GATA-3 and c-maf and concomitant cytokines, including IL-4 [60, 61, 62, 63]. In addition, production of IL-17, the hallmark cytokine of the newly identified inflammatory Th17 cells, has been shown to be directly suppressed by VDR agonists in T cells [53, 63]. Interestingly, induction of Tregs by VDR agonists does not always require induction of tolerogenic DCs, as a combination of 1,25(OH)2D3 and dexamethasone could give rise to Tregs in vitro in the absence of APCs . These cells produced high levels of IL-10 but no IFN-γ, IL-4, or IL-5 and could potently suppress autoimmune demyelination in vivo in an antigen-specific way. In addition, 1,25(OH)2D3-induced IL-10-producing Tregs have been demonstrated to express high levels of TLR9, and triggering of this receptor abrogates their suppressive capacity, possibly reflecting an integrated mechanism for the temporal shutdown of 1,25(OH)2D3-induced Treg function when infectious agents need to be cleared .
Next to T cells, B-cell responses also are controlled by 1,25(OH)2D3. The actions of 1,25(OH)2D3 on B cells were originally believed to result from its ability to modify Th responses. However, more recently, B cells were found to be direct targets for VDR agonists. The 1,25(OH)2D3-mediated effects on B cells include inhibition of proliferation, plasma-cell differentiation, and immunoglobulin secretion, as well as memory B-cell generation and induction of apoptosis . Recently, 1,25(OH)2D3 has been identified as an important regulator of lymphocyte trafficking. DCs of the skin, which can convert locally produced vitamin D3 or 25(OH)D3 into active 1,25(OH)2D3, imprint activated T cells and terminally differentiating B cells with skin-homing properties via 1,25(OH)2D3-mediated induction of the skin-homing receptor CCR10 [18, 66].
Immune consequences of vitamin D insufficiency: clinical implications in chronic kidney disease
Suboptimal levels of 1,25(OH)2D3 are a common finding in chronic kidney disease (CKD) [67, 68]. Therefore most of these patients are at high risk of developing secondary hyperparathyroidism and other complications, including cardiovascular diseases . Importantly, CKD patients not only display impaired levels of 1,25(OH)2D3: also low circulating 25(OH)D3 levels, irrespective of disease progression, are frequently observed in these patients, which further contribute to low 1,25(OH)2D3 levels [70, 71, 72, 73]. Recent studies addressed this issue in pediatric CKD patients and found vitamin D insufficiency and deficiency to be highly prevalent among them [74, 75, 76, 77]. The lack of 25(OH)D3 can rationally be explained in cases of proteinuria, where DBP-bound 25(OH)D3 is lost, but decreased sunlight exposure and malnutrition due to chronic illness, dietary restrictions, and decreased UVB-mediated synthesis of vitamin D3 in the skin of CKD patients compared with individuals with normal kidney function have been proposed to underlie this deficit as well. Replacement therapy with 1,25(OH)2D3 or other VDR agonists in CKD is a commonly used strategy to counteract the detrimental consequences of vitamin D deficiency on mineral and bone health. However, recognition of the widespread nonclassic actions of vitamin D metabolites, together with the increasing amount of epidemiological and experimental data linking inadequate 25(OH)D3 levels to conditions closely reflecting multiple comorbidities of CKD—such as impaired immune function and increased susceptibility to malignancies, cardiovascular disease, and infections—strongly indicate a more extended role for vitamin D metabolites in the pathogenesis and treatment of CKD. Indeed, a number of studies demonstrate that vitamin D deficiency increases the risk for early mortality in hemodialysis patients [78, 79], whereas administration of VDR agonists in these patients has been associated with increased survival, independent of bone- and mineral-related parameters [80, 81, 82]. Despite these data, controversy remains, as a meta-analysis by Palmer et al. did not reveal a clear benefit of vitamin D therapy in CKD patients .
Vitamin D insufficiency and CKD-associated immune defects
Based on the recent discovery of an essential role of vitamin D in TLR-mediated host defense, it is tempting to speculate on the involvement of this mechanism in the high infection rate observed in CKD patients, with their tendency to display vitamin D deficiency. Vitamin-D-dependent antimicrobial responses might in part explain the survival benefit from increased vitamin D levels seen in hemodialysis patients. Gombart et al. found lower cathelicidin levels in patients who died from infections compared with those who survived, and they observed a modest correlation with circulating levels of 1,25(OH)2D3 but not 25(OH)D3 .
Besides its well-established role in the maintenance of mineral homeostasis and bone health, the active vitamin D metabolite, 1,25(OH)2D3, has been identified as a central regulator of normal immune functioning. In addition, the discovery of vitamin-D-activating enzymes expressed in certain immune cells, allowing a local extrarenal activation of vitamin D, has led to the emerging viewpoint of an auto- or paracrine role for vitamin D metabolites in the immune system. This concept is supported by various studies linking vitamin D deficiency or insufficiency to conditions related to dysregulated immune responses, including various autoimmune diseases and infections. Deficient levels of both 1,25(OH)2D3 and 25(OH)D3 are inherent to CKD. Given the immune abnormalities observed in CKD patients, such as systemic low-grade chronic inflammation (contributing to cardiovascular and other complications) and increased susceptibility to infections, modulation of immune responses presumably represents an important mechanism underlying improved disease outcome and mortality rates observed in patients treated with any type of vitamin D metabolite. Moreover, recognition of an auto- and/or paracrine role for vitamin D metabolites in the immune system and other sites in the body emphasizes the need for adequate levels of 25(OH)D3, not only for bone and mineral homeostasis but for global health. In addition, these findings have important implications for the current guidelines concerning vitamin D treatment in CKD patients. Current Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines recommend screening of 25(OH)D3 levels in stage 3–4 CKD patients with elevated PTH concentrations, and similar guidelines exist for stage 2–4 pediatric CKD patients [1, 89]. If serum levels of 25(OH)D3 in these patients drop <30 ng/ml, vitamin D treatment should be initiated, whereas therapy with an active vitamin D sterol (calcitriol, alfacalcidol, paricalcitol, or doxercalciferol) should be preferred at stage 5 disease. However, taking into account the high prevalence of vitamin D insufficiency during all stages of CKD and the growing appreciation of its detrimental consequences on immune and cardiovascular function, the current guidelines should be re-evaluated, considering a careful monitoring of 25(OH)D3 levels during the complete disease course and treatment of vitamin D insufficiency or deficiency from early disease stages on.
This work was supported by grants of the Flemish Research Foundation [Fonds Voor Wetenschappelijk Onderzoek (FWO) Vlaanderen G.0552.06 and G.0649.08, a postdoctoral fellowship for Conny Gysemans and Hannelie Korf and a clinical fellowship for Chantal Mathieu], of the Belgium Program on Interuniversity Poles of Attraction initiated by the Belgian State (IAP P5/17 and P6/40), of the Katholieke Universiteit Leuven (GOA 2004/10 and GOA 2009/10), of the 6th Framework Program of the European Union with acronym SAVEBETA as well as of the 7th Framework Program of the European Union with acronym NAIMIT and of the JDRF Center for Prevention of Beta Cell Therapy in Diabetes [grant number 4-2005-1327].
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