维生素D3通过VDR-miR-125b-Slc26a6通路促进肾脏草酸钙结石形成的机制研究

Urolithiasis is one of the most common diseases of the urinary system, and the prevalence of urolithiasis is increasing in the worldwide. In our long-term clinical work and basic research, we found that high intakes of vitamin D3 is the risk factor of renal calculi. Elevated serum 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations correlated with recurrent calcium kidney stone-formers and implicated in the pathogenesis of hypercalciuria. Vitamin D metabolism, oxalate excretion, and the potential regulated mechanisms have not been defined. Here, we describe a VDR-miRNA-Slc26a6 signaling pathway in the kidney that underlies intestine oxalate absorption inhibition. After treatment with Vitamin D3 reagents, we show that the expression of miR-125b is upregulated through a transcriptional mechanism mediated by VDR-microRNA and associated promoter activation. Luciferase assay was conducted to verify that Slc26a6 was the directly target of miR-125b, then transfected with miR-125b mimics could suppress Slc26a6 expression. Also, treatment of Caco-2 cell with Vitamin D3 stimulated the transcription of PRKCD and ATP activity, which have been shown to suppress the expression of Slc26a6. With Ion chromatograph techniques, we showed that the concentration of oxalate transiently increase when the transport gene Slc26a6 was inhibited in the Caco-2 cell. Furthermore, vitamin D3 could promote intestine Ca2+ absorption by activating Ca2+ transporter, including TRPV5, CALB1 and Slc8a1, the positive regulator of the transcellular pathway. In addition, our research also set up the first zebrafish animal model of renal calculi, by exogenous intake of vitamin D3, we can observe the ethylene glycol-induced calcium oxalate stone formation in the zebrafish model. Moreover, calcium oxalate calculi would be obversed in zebrafish which SLC26a6 gene was knocked out with Cas-9 technique. Together, our results define an important signaling cascade that, vitamin D3 mediate intestine oxalate imbalance through regulating VDR-miRNA-Slc26a6 pathway which promoting kidney stone formation. This study intends to explore the mechanism of vitamin D3 promoting stone formation, so as to enrich the molecular biological mechanism of the pathogenesis of renal calculi, provide new strategies and accurate targets for the treatment of renal calculi.

泌尿系结石是泌尿系统最常见的疾病之一，全球发病率日益增高。在长期的临床工作和基础研究中我们发现，大量摄取维生素D3是肾结石发病危险因素。本项目将围绕维生素D3促进草酸钙结石形成的相关机制进行深入研究，通过其对VDR-miR-125b-SLC26a6通路，VDR-ATP-PRKCD-SLC26A6和VDR-miR-125b -ESRRA两条旁路的作用以及维生素D3对Ca2+转运蛋白的作用，进而观察其对小肠细胞草酸和Ca2+水平变化的影响。此外，本项目还国际首次建立了斑马鱼肾结石动物模型，利用该模型通过外源性摄入维生素D3来观察其对乙二醇所诱导的斑马鱼草酸钙结石形成的影响，以及应用Cas-9技术行斑马鱼SLC26a6基因敲除观察草酸钙结石形成。本研究拟探索维生素D3促进结石生成的相关机制，从而丰富肾结石发病相关分子生物学机制，为肾结石精准化治疗提供新的策略和靶点。

泌尿系结石是一类常见且容易复发的代谢紊乱疾病。草酸钙结石占据了肾结石的绝大部分，经常伴随着复杂的高草酸尿和高钙尿，而且血液或尿液中草酸浓度的细微变化也会影响草酸钙结石形成的风险。尽管最近的研究表明血清1,25-二羟基维生素D3水平升高会诱导高钙尿的发生，进而增加罹患肾脏草酸钙结石的风险，但尚不清楚其是否对血浆/尿草酸盐改变也有潜在影响。人类临床研究和多种体内小鼠/体外细胞系模型的结果表明，维生素D3可能通过改变VDR/miR-125b/Slc26a6轴增加肾脏草酸钙晶体的形成和沉积进而诱导肾钙质沉着症。机制研究表明，维生素D3/VDR可能通过转录调节其宿主基因MIR99AHG来增加miR-125b的表达，而miR-125b可通过直接结合草酸转运蛋白Slc26a6的3'-UTR区降低Slc26a6的表达。D3/VDR/miR-125b/Slc26a6轴改变的结果可能会增加尿草酸水平，从而增加肾脏CaOx晶体的形成和沉积。重要的是，维生素D3/VDR增加的miR-125b表达可以通过增加CYP24A1的表达抑制D3的降解，或者通过靶向其3'-UTR来抑制VDR的表达，形成正反馈机制。我们还发现这一正反馈循环可能会被miR-125b与VDR 3'-UTR结合处的SNP位点rs55774542破坏，改变罹患肾结石的敏感性。另外，本项目在小鼠模型上给予小分子miR-125b拮抗剂进行的临床前研究结果也表明抑制miR-125b可通过减少尿草酸水平来抑制肾脏CaOx晶体的形成和沉积，这可能有助于我们开发新的方法来更好地抑制肾钙质沉着症，进而防治草酸钙肾结石病。