新型钙离子通道TRPV5在遗传性高钙尿结石大鼠肾组织中的表达

目的 探讨新型钙离子通道(TRPV5)在遗传性高钙尿结石(GHS)大鼠肾组织的表达水平及其在尿石形成中的作用. 方法 取3月龄清洁级雄性GHS大鼠模型4只,体质量250～300 g,另取月龄和体质量相仿的正常雄性SD大鼠4只作为对照组,处死后取新鲜肾脏组织,Western blot检测TRPV5蛋白表达水平,实时荧光定量PCR检测其基因表达水平. 结果 GHS大鼠TRPV5蛋白表达水平0.30±0.05,低于对照组大鼠0.59±0.08;对照组大鼠TRPV5 mRNA基因表达水平为GHS组大鼠的12.5倍(分别为1.00和0.08),组间差异均有统计学意义(P＜0.05). 结论 TRPV5可能是尿钙重吸收重要的调控分子,其表达水平下降可能是特发性高钙尿症及尿石形成的重要发病机制.     

遗传高钙尿结石大鼠肾组织钙敏感受体mRNA异常表达及其意义

目的 观察肾钙敏感受体（CaSR）在特发性高钙尿症（IH）发病中的作用。方法 雄性遗传性高钙尿结石（GHS）大鼠和正常野生型SD大鼠各6只，相同的饮食和饲养条件下，检测各组大鼠血Ca^2＋、P5^＋、1，25（OH）2D3和连续2个24h尿Ca^2＋的水平，使用半定量逆转录-聚合酶链反应（RT-PCR）检测肾组织钙敏感受体mRNA表达，PCR产物纯化后进行DNA直接测序。结果两组大鼠血Ca^2＋、P5^＋、1，25．（OH）2D3水平相似，均在正常范围内，而GHS组平均24h尿Ca^2＋排泄量为正常对照（NC）组的3．36倍[分别为（2．66±0．64）、（0．79±0．06）mg／d]，两组比较差异有统计学意义（t=5．82，P〈0．01）；GHS组CaSR mRNA平均相对吸光度（A）值为1．354±0．108，NC组为0．602±0．218，差异有统计学意义（t=6．19，P〈0．01）；CaSR基因第7外显子区域cDNA测序结果与Genebank（Accession：NM-016996）公布的碱基序列比较，GHS组有4只大鼠存在第3072位碱基G／A替换，使第1024位谷氨酸（Glu）变为赖氨酸（Lys），其余2只和NC组均与Genebank公布的碱基序列一致。结论 肾组织CaSRmRNA高表达及其第7外显子Glu1024Lys替换可能是特发性高钙尿症发病的分子机制之一。     

BMP2及Msx2在特发性高钙尿肾结石患者肾乳头组织表达的研究

目的：研究骨形态发生蛋白2（BMP2）及成骨样细胞转录因子Msx2在特发性高钙尿（IH）肾结石患者肾乳头组织中表达以及探讨其在IH患者结石形成中作用机制。方法：筛选特发性高钙尿肾结石患者8例（IH组）,排除各种已知可能影响血清钙或者尿钙的继发疾病;选择同期因肾肿瘤或非结石所致的无功能肾需行肾切除术的患者8例（NC组）。分别取16例患者肾乳头组织若干,各标本应用实时荧光定量PCR检测BMP2和Msx2mRNA的表达,并应用Westernblot测定两组蛋白质表达水平。结果：IH组BMP2的mRNA表达量为（1．491±0．121）,而NC组BMP2的tuRNA为（1．032±0．034）,两组间表达量差异有统计学意义（P〈0．05）;而1H组与NC组Msx2的mRNA表达量分别为（1．432±0．091）和（1．015±0．017）,两组数据差异有统计学意义（P〈0．05）。Westernblotting检测BMP2蛋白提示NC组和IH组蛋白质表达量分别为（1．475±0．042）和（1．681±0．153）,两组数据差异有统计学意义（P〈0．05）;测定Msx2蛋白水平表达显示NC组为（1．531±0．134）,而IH组（1．603±0．156）,两者差异无统计学意义（P〉0．05）。结论：特发性高钙尿（IH）肾结石患者肾乳头BMP2和Msx2mRNA表达增强为间质异位钙化特征,BMP2信号通路在特发性高钙尿结石患者Randall钙斑形成中具有一定作用。     

BMP2和MSX2在特发性高钙尿肾结石患者肾乳头组织表达的研究

目的:研究骨形态发生蛋白2(BMP2)及成骨样细胞转录因子MSX2在特发性高钙尿(IH)肾结石患者肾乳头组织中的表达,探讨其在IH患者结石形成中的作用机制。方法:筛选特发性高钙尿肾结石患者8例(IH组),排除各种已知可能影响血清钙或尿钙的继发疾病;选择同期因肾肿瘤或非结石所致的无功能肾需行肾切除术的患者8例(NC组)。分别取16例患者肾乳头组织若干,各标本应用实时荧光定量PCR检测BMP2和MSX2mRNA的表达,并应用Western blot测定两组蛋白质表达水平。结果:IH组BMP2的mRNA表达量为1.491±0.121,而NC组BMP2的mRNA为1.032±0.034,两组间表达量差异有统计学意义(P<0.05);而IH组与NC组MSX2的mRNA表达量分别为1.432±0.091和1.015±0.017,两组比较差异有统计学意义(P<0.05)。Western blotting检测BMP2蛋白提示NC组和IH组蛋白质表达量分别为1.475±0.042和1.681±0.153,两组比较差异有统计学意义(P<0.05);测定MSX2蛋白水平表达显示NC组为1.531±0.134,而IH组为1.603±0.156,两者比较差异无明显统计学意义(P>0.05)。结论:IH肾结石患者肾乳头BMP2和MSX2mRNA表达增强为间质异位钙化特征,BMP2信号通路在IH结石患者Randall钙斑形成中具有一定作用。     

大鼠交感节后神经元间钙调蛋白的差异性表达及其意义

目的 观察大鼠不同节段交感节后神经元的钙调蛋白(CaM)的表达,探讨其与交感神经元电生理特征的关系.方法 SD大鼠,采集交感神经链中的颈上神经节,颈胸神经节及腹腔神经节,采用逆转录-聚合酶链反应(RT-PCR)和免疫组织化学在mRNA和蛋白质水平分别观察并比较神经元钙调蛋白的表达.结果 钙调蛋白在所选取的3个节段神经元中均有表达,颈上神经节组、颈胸神经节组及腹腔神经节组在mRNA水平的相对表达强度比值分别为1.191 50±0.05009、1.18270±0.053 93、1.03090±0.06624,在蛋白质水平的相对表达强度分别为63.388 60±3.309 38、61.435 70±2.87909、48.77490±3.08877.其表达水平在颈上和颈胸神经节之间差异无统计学意义(q=0.6631、3.5366,P＞0.05),颈上和颈胸神经节的表达均明显高于腹腔神经节(q=11.554、36.0912、10.9209、31.1996,P＜0.05).结论 交感神经不同节段之间钙调蛋白表达存在的差异可能是交感神经元间电生理差异的基础之一.     

遗传特发性高钙尿大鼠模型的建立

目的建立能够稳定遗传的特发性高钙尿大鼠模型,初步探讨特发性高钙尿的发生机理。方法筛选血钙、磷及1,25(OH)2D3水平正常而24h尿钙显著升高的雌、雄性SD大鼠,反复繁殖传代,直至子代鼠稳定出现高钙尿。采用免疫组织化学方法(SP法)检测模型鼠小肠黏膜维生素D受体表达情况。结果93%(14/15)的第七代模型雄鼠和92%(12/13)的雌鼠2个24h尿钙排泄值均高于对照组两个标准差以上,分别为(2.56±0.86)mg/dvs.(1.12±0.18)mg/d,(2.86±1.16)mg/dvs.(1.15±0.12)mg/d(F=27.10,P<0.01;F=21.16,P<0.01),而血清中钙、磷及1,25-(OH)2D3无明显异常(F<2.22,P>0.05)。模型鼠小肠黏膜VDR表达显著增强(F=14.23,P<0.01)。结论本模型鼠可作为研究特发性高钙尿症的理想动物模型,小肠黏膜VDR过度表达可能是特发性高钙尿的发病机理。     

钙敏感受体基因第7外显子单核苷酸多态性与特发性高钙尿症的关系

目的了解钙敏感受体（CaSR）基因单核苷酸多态性与特发性高钙尿（IH）症的关系，探讨特发性高钙尿发病的分子机制。方法提取76例湖北地区汉族特发性高钙尿患者及126例健康对照者外周血标本中基因组DNA，应用聚合酶链反应（PER）结合DNA测序方法检测并分析了CaSR基因的第7外显子3个簇集的多态性位点单核苷酸多态性分布。结果特发性高钙尿病例和对照组CaSR基因的第7外显子第986、990多态性位点等位基因频率分布均符合Hardy-Wein-berg定律，其基因型分布频率在特发性高钙尿患者和正常对照者中差异无统计学意义（P〉0．05），第1011位未见有多态改变。但特发性高钙尿组组内CaSR 990位GG纯合子和RG杂合子个体的24h尿钙排泄量较RR纯合子明显增高，差异有统计学意义（P〈0．05）。结论CaSR基因不是特发性高钙尿的易感主基因，但第7外显子990位A／G单核苷酸多态性能影响尿钙排泄，可能是特发性高钙尿中调节钙排泄的遗传成分之一。     

心肌钙敏感受体在高位脊髓损伤大鼠心肌损伤中的作用

目的 探讨心肌钙敏感受体(CasR)在高位脊髓损伤大鼠心肌损伤中的作用.方法 健康雄性SD大鼠18只,体重250 ～ 300 g,采用随机数字表法,将大鼠随机分为2组:假手术组(S组,n＝6)和高位脊髓损伤组(SCI组,n＝12).采用砝码(10 g)从5 cm高处沿中空玻璃管垂直自由落下撞击C7脊髓的方法制备高位脊髓损伤模型.SCI组于高位脊髓损伤后12和24h(T1,2)时,S组于T1时取血样,测定血清肌酸激酶(CK)和CK-MB活性,取心肌组织,在透射电镜下观察心肌超微结构,分别采用荧光定量PCR法和Western blot法检测心肌CaSR mRNA及其蛋白的表达水平.结果 与S组比较,SCI组血清CK和CK-MB活性升高,心肌CaSR mRNA及其蛋白表达上调(P<0.05);与T1时比较,SCI组T2时血清CK活性升高,CK-MB活性降低,心肌CaSR mRNA表达上调(P<0.05),CaSR蛋白表达差异则无统计学意义(P＞0.05).SCI组T1,2时心肌超微结构呈现不同程度的损伤改变.结论 大鼠高位脊髓损伤后心肌CaSR表达上调,该变化可能是高位脊髓损伤后心肌损伤的机制之一.     

青少年特发性脊柱侧凸患者椎旁肌中钙调蛋白的表达及临床意义

目的:探讨青少年特发性脊柱侧凸(AIS)患者椎旁肌钙调蛋白的表达及其对肌浆网钙离子通道的影响。方法:AIS患者共30例,Cobb角40°～100°,其中Cobb角≥50°者15例(A组),Cobb角<50°者15例(B组);对照组(C组)5例患者均为非脊柱侧凸病例。于手术中取A、B组患者顶椎区两侧椎旁肌、取C组患者非病变部位两侧椎旁肌作为标本进行分子生物学分析,采用RT-PCR方法检测所有标本钙调蛋白和肌浆网钙离子通道的mRNA表达量。结果:A、B组患者椎旁肌钙调蛋白mRNA的表达比C组高(P<0.05),并且A组患者凸侧椎旁肌钙调蛋白mRNA显著高于凹侧(P<0.05),而B组患者其凹凸侧钙调蛋白mRNA没有显著性差异(P>0.05);C组椎旁肌肌浆网钙离子通道mRNA的表达比A组患者高(P<0.05),A组患者凸侧椎旁肌肌浆网钙离子通道mRNA显著低于凹侧(P<0.05),而B组患者其凹凸侧肌浆网钙离子通道mRNA没有显著性差异。结论:钙调蛋白mRNA在AIS患者椎旁肌中的表达较无脊柱侧凸者高,而且其表达随着侧凸程度的加重而增高;AIS患者椎旁肌肌浆网钙离子通道表达有异常,钙调蛋白的过度表达可能导致了钙离子通道的损伤。     

EphrinA1、缺氧诱导因子-1α和血管内皮生长因子基因及其蛋白产物在大鼠肺缺血再灌注损伤模型中的表达

目的 探讨肺缺血再灌注损伤(LIRI)中EphrinA1、缺氧诱导因子(HIF)-1α和血管内皮生长因子(VEGF)mRNA和蛋白的表达及其意义.方法 大鼠左侧开胸,阻断左肺门根部60min,再灌注6 h.分为单纯缺血组和缺血再灌注组及相应的手术对照组和空白对照组.评价EphrinA1、HIF-1α和VEGF的mRNA和蛋白表达水平.结果 各因子mRNA水平在单纯缺血组、缺血再灌注组及相应的手术对照组之间差异无统计学意义(P＞0.05),VEGF mRNA表达水平甚至有所减低(P＜0.05);各因子蛋白表达在缺血再灌注组明显高于其他各组(P＜0.05),单纯缺血组与各手术对照组之间差异无统计学意义(P＞0.05);缺血再灌注组的EphrinA1、HIF-1α和VEGF蛋白表达水平相关性检验显示各因子表达水平两两之间均显著相关.结论 EphrinA1和HIF-1α、VEGF参与LIRI发生在蛋白表达水平,mRNA表达水平和蛋白表达水平不一致;上述因子蛋白表达主要发生在再灌注期间,各个因子表达之间明显相关.     

Adenovirus‐delivered microRNA targeting the vitamin D receptor reduces intracellular Ca2+ concentrations by regulating the expression of Ca2+‐transport proteins in renal epithelial cells

What’s known on the subject? and What does the study add? Experimental data have shown that VDR overexpression in the duodenum and kidney cortex is a biological characteristic of genetic hypercalciuric stone‐forming rats (GHS rat), and a link between idiopathic calcium stone formation and the microstatellite marker D12S339 (near the VDR locus) has been proven in humans. Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin‐D28k and PMCA1b in NRK cell lines. VDR knockdown results in a decrease in intracellular Ca²⁺ concentration in NRK cell lines. The effect of the elevated VDR level in the kidney on hypercalciuria and the underlying mechanisms need to be further addressed.

OBJECTIVE

? To determine the effects of vitamin D receptor (VDR) on hypercalciuria and the mechanisms underlying such effects.

MATERIALS AND METHODS

? The adenovirus vector‐delivered microRNA targeting rat VDR was constructed. We infected the normal rat kidney epithelial cell line NRK (Cellbank, China) with the adenovirus and then collected the cells at 0, 48, 72, 96, 120 h after infection. The mRNA and protein levels of VDR and VDR‐dependent epithelial Ca²⁺ transport proteins were detected using real‐time polymerase chain reaction and Western blot assays, respectively. ? Fluorescent Ca²⁺ indicator Fluo‐4 NW (Fluo‐4 NW calcium assay kit, Molecular Probes, Invitrogen, USA) and laser scanning confocal microscope (Olympus, FV500‐IX71, Japan) were used to detect the cytosolic free Ca²⁺ concentration at different time points after infection.

RESULTS

? The mRNA and protein level of VDR, transient receptor potential vanilloid receptor subtype 5 (TRPV5), calbindin‐D_28k and plasma membrane Ca²⁺‐ATPase (PMCA1b) in infected NRK cells was significantly lower at 72 and 96 h after infection than that in control cells. ? There was no significant difference between the two groups in the mRNA and protein level of TRPV6 and the Na⁺/Ca²⁺‐exchanger (NCX1). ? Furthermore, VDR knockdown results in a decrease in intracellular Ca²⁺ concentration ([Ca²⁺]i) in NRK cell lines.

CONCLUSIONS

? Our study shows that VDR can positively regulate the mRNA and protein expression of TRPV5, calbindin‐D28k and PMCA1b, but not of TRPV6 or NCX1, in NRK cell lines. VDR knockdown results in a decrease in [Ca²⁺]i in NRK cell lines. ? The effect of the elevated VDR level in the kidney on hypercalciuria and the mechanisms underlying need to be further addressed.     

Aromatase deficiency causes altered expression of molecules critical for calcium reabsorption in the kidneys of female mice *.

Kidney stones increase after menopause, suggesting a role for estrogen deficiency. ArKO mice have hypercalciuria and lower levels of calcium transport proteins, whereas levels of the klotho protein are elevated. Thus, estrogen deficiency is sufficient to cause altered renal calcium handling. INTRODUCTION: The incidence of renal stones increases in women after menopause, implicating a possible role for estrogen deficiency. We used the aromatase deficient (ArKO) mouse, a model of estrogen deficiency, to test the hypothesis that estrogen deficiency would increase urinary calcium excretion and alter the expression of molecular regulators of renal calcium reabsorption. MATERIALS AND METHODS: Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n = 5-12/group) were used to measure urinary calcium in the fed and fasting states, relative expression level of some genes involved in calcium reabsorption in the distal convoluted tubule by real-time PCR, and protein expression by Western blotting or immunohistochemistry. Plasma membrane calcium ATPase (PMCA) activity was measured in kidney membrane preparations. ANOVA was used to test for differences between groups followed by posthoc analysis with Dunnett's test. RESULTS: Compared with WT, urinary Ca:Cr ratios were elevated in ArKO mice, renal mRNA levels of transient receptor potential cation channel vallinoid subfamily member 5 (TRPV5), TRPV6, calbindin-D28k, the Na+/Ca+ exchanger (NCX1), and the PMCA1b were significantly decreased, and klotho mRNA and protein levels were elevated. Estradiol treatment of ArKO mice normalized urinary calcium excretion, renal mRNA levels of TRPV5, calbindin-D(28k), PMCA1b, and klotho, as well as protein levels of calbindin-D28k and Klotho. ArKO mice treated with estradiol had significantly greater PMCA activity than either untreated ArKO mice or WT mice. CONCLUSIONS: Estrogen deficiency caused by aromatase inactivation is sufficient for renal calcium loss. Changes in estradiol levels are associated with coordinated changes in expression of many proteins involved in distal tubule calcium reabsorption. Estradiol seems to act at the genomic level by increasing or decreasing (klotho) protein expression and nongenomically by increasing PMCA activity. PMCA, not NCX1, is likely responsible for extruding calcium in response to in vivo estradiol hormonal challenge. These data provide potential mechanisms for regulation of renal calcium handling in response to changes in serum estrogen levels.     

Abundance and activity of Ca2+-ATPase in hypercalciuric children

The plasma membrane Ca²⁺-ATPase (PMCA) is one of the main regulators of cell Ca²⁺ homeostasis. The aim of our study was to determine whether the abundance and activity of PMCA are altered in erythrocytes of children with idiopathic hypercalciuria. Twenty-four children with idiopathic hypercalciuria (13 girls and 11 boys, mean age 10.6±4.8 years; mean urinary calcium concentration 0.85±0.20 mmol/mmol creatinine) and 30 healthy age-matched children were enrolled. PMCA protein abundance was determined by Western blot analysis. Enzyme activity was determined spectrophotometrically. The abundance of PMCA did not differ in hypercalciuric patients from that of control subjects (98±22% vs 100±18%). Moreover, the activity was not different between the studied groups (3141±1494 vs 2953±780 nmol ATP/mg protein/h). The extent of hypercalciuria did not correlate with enzyme abundance or activity. Assuming that erythrocytes may reflect the renal tubular transporting processes, our data suggest that other Ca²⁺-transport mechanisms than PMCA might be involved in the development of idiopathic hypercalciuria in children. Received: 5 December 2000 / Revised: 2 May 2001 / Accepted: 3 May 2001     

The localization of PMCA1b in epithelial cells and aposomes of the rat coagulating gland is influenced by androgens

BACKGROUND: Rat coagulating gland epithelial cells export proteins by an apocrine secretion mode within membrane blebs arising from the apical plasma membrane. Using a pan-PMCA antibody, we have recently shown the plasma membrane Ca(2+)-ATPase (PMCA) being part of the apical plasma membrane of epithelial cells and incorporated into the aposomal membrane. The mRNA of PMCA isoforms 1 and 4 respectively, have been detected by RT-PCR in rat coagulating gland. METHODS: In order to identify which PMCA isoform is integrated into aposomes during apocrine secretion and whether or not PMCA export is influenced by androgens RT-PCR, in situ hybridization, Western blotting, and immunofluorescence experiments were performed. RESULTS: PMCA1b is the isoform which is expressed and located in the apical plasma membrane of coagulating gland epithelial cells and is integrated into the aposomal membrane. In contrast, PMCA4 mRNA and protein are restricted to the stroma. Androgen deprivation by castration within 14 days leads to an accumulation of PMCA1b in coagulating gland epithelium, while aposomes are not detected anymore. CONCLUSIONS: We showed for the first time that PMCA isoform 1b is released via aposomes of the epithelial cells of the rat coagulating gland and that the localization of PMCA1b in the epithelial cells is influenced by androgens.     

1,25-dihydroxyvitamin D(3)-independent stimulatory effect of estrogen on the expression of ECaC1 in the kidney

Estrogen deficiency results in a negative Ca(2+) balance and bone loss in postmenopausal women. In addition to bone, the intestine and kidney are potential sites for estrogen action and are involved in Ca(2+) handling and regulation. The epithelial Ca(2+) channel ECaC1 (or TRPV5) is the entry channel involved in active Ca(2+) transport. Ca(2+) entry is followed by cytosolic diffusion, facilitated by calbindin-D(28K) and/or calbindin-D(9k), and active extrusion across the basolateral membrane by the Na(+)/Ca(2+)-exchanger (NCX1) and plasma membrane Ca(2+)-ATPase (PMCA1b). In this transcellular Ca(2+) transport, ECaC1 probably represents the final regulatory target for hormonal control. The aim of this study was to determine whether 17beta-estradiol (17beta-E(2)) is involved in Ca(2+) reabsorption via regulation of the expression of ECaC1. The ovariectomized rat model was used to investigate the regulation of ECaC1, at the mRNA and protein levels, by 17beta-E(2) replacement therapy. Using real-time quantitative PCR and immunohistochemical analyses, this study demonstrated that 17beta-E(2) treatment at pharmacologic doses increased renal mRNA levels of ECaC1, calbindin-D(28K), NCX1, and PMCA1b and increased the protein abundance of ECaC1. Furthermore, the involvement of 1,25-dihydroxyvitamin D(3) in the effects of 17beta-E(2) was examined in 25-hydroxyvitamin D(3)-1alpha-hydroxylase-knockout mice. Renal mRNA expression of calbindin-D(9K), calbindin-D(28K), NCX1, and PMCA1b was not significantly altered after 17beta-E(2) treatment. In contrast, ECaC1 mRNA and protein levels were both significantly upregulated. Moreover, 17beta-E(2) treatment partially restored serum Ca(2+) levels, from 1.63 +/- 0.06 to 2.03 +/- 0.12 mM. In conclusion, this study suggests that 17beta-E(2) is positively involved in renal Ca(2+) reabsorption via the upregulation of ECaC1, an effect independent of 1,25-dihydroxyvitamin D(3).     

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.     

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.