1型糖尿病骨缺失中维生素D代谢酶表达的改变和肾脏钙转运蛋白的变化

Alteration of Vitamin D Metabolic Enzyme Expression and Calcium Transporter Abundance in Kidney Involved in Type 1 Diabetes-Induced Bone Loss

目的 调查实验引起的糖尿病小鼠维生素D代谢酶表达的改变以及肾脏中分子间钙转运蛋白的变化.方法 雄性DBA/2J小鼠被连续5天注射链脲霉素（实验组）和空载体（对照组）,Zhang.Y等使用边缘定量CT测量骨密度,用番红O染色观察骨组织形态学变化,通过实时定量PCR和Western blotting来研究目的 基因及蛋白表达的变化情况.结果 与对照组相比,实验组1型糖尿病可导致泌尿系大量钙的排泄和胫骨干骺端近端及股骨远端骨小梁的丢失.显微结构也显示骨质丢失与骨小梁恶化有关,定量PCR显示糖尿病小鼠肾脏内mRNA水平的表达为10周后25-羟化维生素D-24-羟化酶下调,20周后25-羟化维生素D-1α-羟化酶上调.另外,实验组小鼠体内肾脏瞬时感受器电位V6、质膜Ca-ATP酶（PMCA1b）、维生素D受体（VDR）基因mRNA表达水平均下降.Western blotting分析表明实验组小鼠肾脏内PMCA1b和VDR蛋白表达显著下降.结论 该实验局限性在于缺乏血清中维生素D、甲状旁腺激素和磷水平的研究,然而现有的研究支持1型糖尿病引起骨丢失的潜在机制可能是维生素D代谢酶的改变和肾脏转运蛋白表达的下降.

Objective To introduction the purpose of this study was to investigate the changes of the expression of vitamin D metabolic enzymes and transcellular calclum-transporting proteins in kidneys from mice with experimentally induced diabetes. Methods Male DBA/2J mice were injected with either vehicle （control） or streptozotocin （STZ） daily for five consecutive days by Zhang. Y et al. Bone mineral density was measured by peripheral quantitative computerized tomography, and bone histomorphology was analysed by Safranin O staining. Real-time PCR and Western blotting were applied to determine the expression of target genes and proteins. Results Type 1 diabetes produced high urinary calcium excretion and loss of trabecular bone measured at the proximal metaphysis of the tibia and the distal femur. Bone loss was associated with deterioration of trabeeular bone microstructure. Quantified PCR results showed that mRNA expression level in the kidney of diabetic mice for 25-hydroxyvitamin D- 24-hydroxylase was downregulated at week 10,while those for 25-hy- droxyvitamin D-la-hydroxylase were upregulated at week 20. In addition,mRNA expression levels for renal transient receptor potential V6,plasmamembrane Ca-ATPase （PMCA）lb,and vitamin D receptor （VDR） genes were decreased in STZ-treated mice. Western blot analysis showed that protein expression of PMCAlb and VDR was significantly decreased in kidneys from STZ-treated mice compared to that of controls. Conclusion The limitation in this study was the lack of vi- tamin D,parathyroid hormone,and phosphorus levels in serum. However,the present study supports the conclusion that the underlying mechanism contributing to type 1 diabetes-assoclated bone loss may be alterations of vitamin D metabolic enzyme expression and associated decreases in expression of renal calcium transporters.