Renoprotective role of the vitamin D receptor in diabetic nephropathy

1,25-Dihydroxyvitamin D3 negatively regulates the renin-angiotensin system (RAS), which plays a critical role in the development of diabetic nephropathy. We tested if mice lacking the vitamin D receptor (VDR) are more susceptible to hyperglycemia-induced renal injury. Diabetic VDR knockout mice developed more severe albuminuria and glomerulosclerosis due to increased glomerular basement membrane thickening and podocyte effacement. More fibronectin (FN) and less nephrin were expressed in the VDR knockout mice compared to diabetic wild-type mice. In receptor knockout mice, increased renin, angiotensinogen, transforming growth factor-beta (TGF-beta), and connective tissue growth factor accompanied the more severe renal injury. 1,25-Dihydroxyvitmain D3 inhibited high glucose (HG)-induced FN production in cultured mesangial cells and increased nephrin expression in cultured podocytes. 1,25-Dihydroxyvitmain D3 also suppressed HG-induced activation of the RAS and TGF-beta in mesangial and juxtaglomerular cells. Our study suggests that receptor-mediated vitamin D actions are renoprotective in diabetic nephropathy.     

Improved renal allograft survival with vitamin D receptor polymorphism

BACKGROUND: Polymorphisms in genes, coding for proteins involved in immune response, or the pathogenesis of atherosclerosis may influence immunological and non-immunological mechanisms that lead to allograft loss. Vitamin D receptor (VDR) agonists reduce allograft rejection in animal models, and there are a number of functional polymorphisms in VDR. METHODS: In all, 379 renal transplant recipients were genotyped for VDR (FokI & ApaI) polymorphisms, and the association of each genotype with renal allograft survival and acute rejection was determined. RESULTS: There was significantly improved allograft survival for patients who were homozygous or heterozygous for the VDR FokI T allele (Hazard Ratio [HR] = 0.488, p < 0.001). CONCLUSION: The association of VDR FokI T allele with improved renal allograft survival is a unique observation. The finding is in keeping with data showing the prevention of chronic allograft rejection with the use of Vitamin D receptor agonists.     

Mechanism and function of high vitamin D receptor levels in genetic hypercalciuric stone-forming rats.

The functional status and mechanism of increased VDR in GHS rats were investigated. Basal VDR and calbindins were increased in GHS rats. 1,25(OH)(2)D(3) increased VDR and calbindins in controls but not GHS rats. VDR half-life was prolonged in GHS rats. This study supports the mechanism and functional status of elevated VDR in GHS rats. INTRODUCTION: Genetic hypercalciuric stone-forming (GHS) rats form calcium kidney stones from hypercalciuria arising from increased intestinal calcium absorption and bone resorption and decreased renal calcium reabsorption. Normal serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels and increased vitamin D receptor (VDR) protein suggest that high rates of expression of vitamin D-responsive genes may mediate the hypercalciuria. The mechanism of elevated VDR and state of receptor function are not known. MATERIALS AND METHODS: GHS and non-stone-forming control (NC) male rats (mean, 249 g), fed a normal calcium diet, were injected intraperitoneally with 1,25(OH)2D3 (30 ng/100 g BW) or vehicle 24 h before cycloheximide (6 mg/100 g, IP) and were killed 0-8 h afterward. Duodenal VDR was measured by ELISA and Western blot, and duodenal and kidney calbindins (9 and 28 kDa) were measured by Western blots. RESULTS AND CONCLUSIONS: Duodenal VDR protein by Western blot was increased 2-fold in GHS versus NC rats (633 +/- 62 versus 388 +/- 48 fmol/mg protein, n = 4, p < 0.02), and 1,25(OH)2D3 increased VDR and calbindins (9 and 28 kDa) further in NC but not GHS rats. Duodenal VDR half-life was prolonged in GHS rats (2.59 +/- 0.2 versus 1.81 +/- 0.2 h, p < 0.001). 1,25(OH)2D3 prolonged duodenal VDR half-life in NC rats to that of untreated GHS rats (2.59 +/- 0.2 versus 2.83 +/- 0.3 h, not significant). This study supports the hypothesis that prolongation of VDR half-life increases VDR tissue levels and mediates increased VDR-regulated genes that result in hypercalciuria through actions on vitamin D-regulated calcium transport in intestine, bone, and kidney.     

维生素D受体在糖尿病肾病足细胞损伤及蛋白尿缓解中的作用

目的探讨维生素D受体(VDR)在糖尿病肾病(DKD)足细胞中的表达水平及在足细胞损伤及蛋白尿缓解中的作用。方法(1)本研究纳入了65例诊断患有2型糖尿病(伴或不伴蛋白尿)的患者,并纳入了25例年龄和性别相匹配的健康体检者为对照组。根据白蛋白/肌酐(ACR)的尿排泄比例对2型糖尿病患者进行分组,分别为无蛋白尿(ACR<30 mg/g,n=24)、微量白蛋白尿(ACR 30~300 mg/g,n=18)和临床蛋白尿(ACR>300 mg/g,n=23)。另选择25例经肾活检确诊的DKD患者作为DKD组。正常肾脏组织标本均取自泌尿外科同一时期肾脏肿瘤切除患者10例。将各组检测指标进行对比,同时采用实时定量PCR、ELISA法和免疫组化法检测VDR在各组患者的血液、尿液样本和肾脏组织中的表达情况,以及使用Pearson相关分析分析VDR与尿蛋白的相关性。(2)在2型糖尿病肾病小鼠模型中对上述结果进行验证,将遗传背景均为C57BLKs/J的雄性db/db小鼠及同窝出生的db/m小鼠,随机分为正常对照组(A组)、DKD对照组(B组)、DKD二甲基亚砜处理组(C组)、DKD帕立骨化醇(VDR激动剂)处理组(D组),C、D组连续腹腔注射处理8周,对照组不做任何处理。小鼠10周龄时开始连续干预8周,在小鼠22周龄(开始干预后12周)检测各组小鼠体重、血、尿生化指标对比;Western印迹法检测β⁃catenin、VDR的变化;免疫荧光观察足细胞标志蛋白podocin及足细胞损伤蛋白α⁃SMA的表达变化。结果(1)与正常健康对照组相比,无蛋白尿组、微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿组的糖尿病患者相比,微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05)。(2)与正常健康对照组相比,无蛋白尿糖尿病组和DKD组患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿糖尿病组患者相比,DKD组患者血浆中VDR的mRNA和蛋白水平亦较低(均P<0.05)。(3)免疫组化结果显示,DKD组肾组织中VDR的表达明显少于正常对照组。(4)DKD患者血浆中VDR mRNA相对水平与ACR呈负相关(r=-0.342,P<0.05)。(5)各组尿液上清液中VDR的水平与血浆中的水平呈相反趋势。(6)Western印迹结果显示,B组、C组肾小球足细胞β⁃catenin蛋白表达高于D组(均P<0.05),VDR蛋白的表达低于D组(均P<0.05);免疫荧光结果显示,B组、C组肾小球足细胞podocin的表达低于D组(均P<0.05),α⁃SMA的表达高于D组(均P<0.05)。结论VDR高表达缓解DKD足细胞损伤及蛋白尿。     

Regulation of parathyroid vitamin D receptor expression by extracellular calcium

Low extracellular calcium (Ca) stimulates parathyroid hormone (PTH) secretion and also increases the renal synthesis of calcitriol (CTR), which is known to decrease PTH production. This study began with the hypothesis that the parathyroid cell response to CTR may be modulated by extracellular Ca concentration through an effect on parathyroid cell vitamin D receptor (VDR). In the present study, rat parathyroid glands were incubated in low (0.6 mM) and high (1.5 mM) Ca concentration. The parathyroid VDRmRNA was higher in 1.5 than 0.6 mM Ca. Furthermore, this effect was not observed in incubated slices of kidney cortex and medulla, tissues which also possess both Ca and vitamin D receptors. Experiments were also performed to evaluate the effect of Ca on VDR expression in vivo. Male Wistar rats received intraperitoneal injections of CaCl(2) or a single intramuscular injection of EDTA to obtain 6 h of hypercalcemic (ionized Ca, 1.4 to 1.6 mM) or hypocalcemic (ionized Ca, 0.85 to 0.95 mM) clamp; a third group of rats was used as control. A small dose of CTR was administered to hypercalcemic rats to match the serum CTR levels of hypocalcemic rats. Parathyroid gland VDRmRNA and VDR protein were increased in hypercalcemic rats as compared with hypocalcemic rats. Increasing doses of CTR upregulated VDRmRNA and VDR only in hypercalcemic rats. Additional experiments showed that the decrease in VDR in hypocalcemic rats prevented the inhibitory effect of CTR on PTHmRNA. In conclusion, our study shows that extracellular Ca regulates VDR expression by parathyroid cells independently of CTR and that by this mechanism hypocalcemia may prevent the feedback of CTR on the parathyroids.     

New insights into mineral and skeletal regulation by active forms of vitamin D

Recent studies in mice using genetic approaches have shed new light on the physiological effects of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) and the vitamin D receptor (VDR) in skeletal and mineral homeostasis, and on their interaction with calcium. These studies in mice with targeted deletion of the 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha(OH)ase), and of the VDR or of double mutants, have shown the discrete effects of calcium in inhibiting parathyroid hormone secretion and in enhancing bone mineralization, but overlapping effects of calcium and 1,25(OH)(2)D on inhibiting parathyroid growth and on normal development of the cartilaginous growth plate. The 1,25(OH)(2)D/VDR system is essential, however, in enhancing intestinal calcium absorption and in optimally increasing osteoclastic activation. In addition, the 1,25(OH)(2)D/VDR system has important anabolic effects on bone, thus defining a dual role for this system in bone turnover. These studies are revealing functions of the vitamin D/VDR system which have relevance for new concepts of the pathophysiology of renal bone disease and, in particular, of the adynamic bone disorder, and for the development of new analogs of the active form of vitamin D, which have less calcemic activity and greater skeletal anabolic effects.     

Elevated vitamin D receptor levels in genetic hypercalciuric stone‐forming rats are associated with downregulation of Snail

Patients with idiopathic hypercalciuria (IH) and genetic hypercalciuric stone‐forming (GHS) rats, an animal model of IH, are both characterized by normal serum Ca, hypercalciuria, Ca nephrolithiasis, reduced renal Ca reabsorption, and increased bone resorption. Serum 1,25‐dihydroxyvitamin D [1,25(OH)₂D] levels are elevated or normal in IH and are normal in GHS rats. In GHS rats, vitamin D receptor (VDR) protein levels are elevated in intestinal, kidney, and bone cells, and in IH, peripheral blood monocyte VDR levels are high. The high VDR is thought to amplify the target‐tissue actions of normal circulating 1,25(OH)₂D levels to increase Ca transport. The aim of this study was to elucidate the molecular mechanisms whereby Snail may contribute to the high VDR levels in GHS rats. In the study, Snail gene expression and protein levels were lower in GHS rat tissues and inversely correlated with VDR gene expression and protein levels in intestine and kidney cells. In human kidney and colon cell lines, ChIP assays revealed endogenous Snail binding close to specific E‐box sequences within the human VDR promoter region, whereas only one E‐box specifically bound Snail in the rat promoter. Snail binding to rat VDR promoter E‐box regions was reduced in GHS compared with normal control intestine and was accompanied by hyperacetylation of histone H₃. These results provide evidence that elevated VDR in GHS rats likely occurs because of derepression resulting from reduced Snail binding to the VDR promoter and hyperacetylation of histone H₃. © 2010 American Society for Bone and Mineral Research.     

A new enzyme-linked immunosorbant assay for the measurement of human vitamin D receptor

The hormonal actions of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are mediated by its cognate receptor protein, the vitamin D receptor (VDR). Despite the growing importance of the VDR system as a modulator of cell growth and differentiation, convenient assays for quantitative measurement of VDR are not readily available, and [(3)H]1,25(OH)(2)D(3) ligand binding assays remain the standard method. In this paper, we present data to validate and characterize the usefulness of a new VDR enzyme-linked immunosorbant assay (ELISA) kit developed for the measurement of VDR in biological samples. In this assay, samples are added to microtitration wells coated with anti-VDR antibody and incubated with a second anti-VDR antibody that is biotinylated. The antibody receptor complex is then detected with streptavidin-labeled horseradish peroxidase followed by incubation with a chromogenic substrate, tetramethylbenzidine. The assay was found to be sensitive and accurate for measurements of VDR and compared favorably with the conventional radioligand binding assay (RBA). The interassay variation ranged from 5% to 25% and the intraassay variation was less than 5%. The ELISA presents several advantages over existing methodology, including the use of nonradioactive detection systems, lower protein and sample volume requirements, as well as convenience and speed. The assay can be completed in as short a time as 3 h, avoiding overnight incubations. Data are also presented to demonstrate the ability of the ELISA to detect both occupied and unoccupied VDR, making it a valuable research tool in settings where 1,25(OH)(2)D(3) is present. However, the ELISA, as currently formulated, is only useful for the detection of human VDR.     

Identification of the vitamin D receptor in various cells of the mouse kidney

The kidney is the major, if not sole, site for the production of 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the biologically active form of vitamin D that can stimulate calcium reabsorption in the kidney and may provide renoprotective benefits. The biological effects of 1,25(OH)(2)D(3) are mediated through a nuclear hormone receptor, known as the vitamin D receptor (VDR). It is well accepted that the VDR is present in the distal renal convoluted tubule cells; however, whether VDR is present in other kidney cell types is uncertain. Using a highly specific and sensitive anti-VDR antibody, we determined its distribution in the mouse kidney by immunohistochemistry. Our results show that the VDR is not only present in the distal but is also found in the proximal tubules, but at 24-fold lower levels. The VDR was also found in the macula densa of the juxtaglomerular apparatus, glomerular parietal epithelial cells, and podocytes. In contrast, the VDR is either very low or absent in interstitial fibroblasts, glomerular mesangial cells, and juxtaglomerular cells. Thus, identification of VDR in the proximal tubule, macula densa, and podocytes suggests that 1,25(OH)(2)D(3) plays a direct role in these cells under normal conditions.     

Vitamin D in chronic kidney disease: a systemic role for selective vitamin D receptor activation

Hyperparathyroidism occurs in most patients during the progression of chronic kidney disease (CKD) and one of its initiating events, reduced serum levels of 1,25-dihydroxyvitamin D, results from a decrease in renal 1alpha hydroxylase activity, which converts 25-hydroxyvitamin D to its activated form. The combination of persistently high parathyroid hormone (PTH) and low 1,25-dihydroxyvitamin D is associated with bone loss, cardiovascular disease, immune suppression and increased mortality in patients with end-stage kidney failure. Recent studies in dialysis patients suggest that paricalcitol, a selective activator of the vitamin D receptor (VDR), is associated with a more favorable efficacy to side effect profile than calcitriol, with less morbidity and better survival. One hypothesis derived from such studies suggests that systemic activation of VDRs may have direct effects on the cardiovascular system to decrease mortality in CKD. Although current guidelines for regulating serum calcium, phosphate and PTH recommend specific interventions at the various stages of CKD to prevent or postpone irreversible parathyroid disease and decrease cardiovascular morbidity and mortality, emerging data suggest that vitamin D therapy may prolong survival in this patient population by mechanisms that are independent of calcium, phosphate and PTH. It is suggested that a re-evaluation of current treatment recommendations is needed and that future research should focus on mechanisms that distinguish potential tissue specific benefits of selective VDR activators in patients with CKD.     

Correlation of enhanced cell proliferation with decreased density of vitamin D receptor in parathyroid hyperplasia in chronic dialysis patients

Summary: The decreased density of the vitamin D receptor (VDR) plays an important role in the pathogenesis and progression of parathyroid hyperplasia in renal failure. In chronic dialysis patients, VDR density is less in nodular hyperplasia than in diffuse hyperplasia and the difference of cell proliferation has been also suggested by DNA analysis. to prove a more direct correlation between VDR density and cell proliferation, VDR density and proliferating cell nuclear antigen (PCNA) were detected in situ by immunohistochemistry in serial sections of surgically excised parathyroid glands from 10 chronic dialysis patients. Among 28 excised glands, 20 glands were nodular hyperplasia and eight glands were diffuse hyperplasia. Vitamin D receptor positive cells were much fewer in nodular hyperplasia (13.1 ± 4.8%) than in diffuse hyperplasia (383 ± 5.6%). In contrast, mean PCNA positive cell numbers per one 400 x field were much higher in nodular hyperplasia (2.0± 1.2) than in diffuse hyperplasia (0.1±0.2). These two parameters, simultaneously detected in the same area of the serial sections, showed strong negative correlation (r= -0.719, P <0.0001). Remarkable differences in VDR and PCNA were evident between nodules and the surrounding diffuse hyperplasia in the same section. These data suggest more direct relationship between the decrease of VDR density and parathyroid cell proliferation in chronic renal failure as a pathophysiological mechanism.     

The effects of vitamin D receptor polymorphism on secondary hyperparathyroidism and bone density after renal transplantation.

Immunosuppresive treatment and secondary hyperparathyroidism (SHPT) are considered among the most important pathogenetic factors for postrenal transplant bone disease. The aim of this study was to investigate the relationships among vitamin D receptor (VDR) gene polymorphism, parathyroid hormone (PTH) levels, and bone density in renal transplant recipients. We enrolled 69 patients (47 men and 22 women; mean age, 47 +/- 11 years) who had undergone kidney transplantation 51 +/- 5 months before. All patients underwent an evaluation of the main biochemical parameters of bone metabolism as well as bone densitometry. VDR alleles were typed by a polymerase chain reaction (PCR) assay based on a polymorphic BsmI restriction site. When the patients were categorized according to the VDR genotype (BB, Bb, and bb), serum creatinine, and the cumulative doses of immunosuppressive drugs were similar across the groups. PTH levels higher than 80 pg/ml were found in 53.6% of the patients, with the highest values being detected in the bb VDR genotype (p < 0.05). PTH was significantly correlated to urinary type I collagen cross-linked N-telopeptide (NTx) values. Bone density was low in the whole population; however, spinal bone density was lower in the bb subgroup (p < 0.02). In the whole population, only PTH (p < 0.05) and body mass index (BMI; p < 0.01) were independent predictors of spinal bone density. When grouping the patients by the VDR gene polymorphism, only PTH continued to be an independent predictor of spinal bone density in the bb allele subgroup (R2 adj. = 0.17). We can conclude that the VDR genotype polymorphism affects bone density of renal transplant recipients via its effects on the severity of SHPT.     

Differential effects of 20-epi vitamin D analogs on the vitamin D receptor homodimer.

Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C-terminal anti-VDR antisera were screened for their ability to interact with analog-bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor alpha (rhRXR alpha). Like calcitriol, analog-bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20-epi conformations, produced homodimer complexes that were 3- to 6-fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C-terminal anti-VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060- and CB1093-bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXR alpha extracts. Although new heterodimer complexes appeared in a time-dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C-terminal protein conformation that may partially explain the varied biological responses of these compounds.