肾性骨病方通过调节microRNA表达延缓肾性骨病进展的机制研究

Renal osteodystrophy is one of the most important complications of chronic kidney diseases (CKD) and was paid increasing attention because of its high incidence and harmfulnessas well as the limitations in late stage treatment. Our group found that low-turnover bone loss was induced in patients with early stage CKD, while irreversible high-turnover bone loss was induced in the late stage. So if effective intervention can be adopted in the early stage of CKD, the development of renal osteoporosis might probably be prohibited. Using microRNA array, we previously screened several differentially expressed microRNA in the plasma of CKD1 patients and CKD5 patients. In this study, 1) mouse model of early stage CKD and late stage CKD will be estabolished to verify the expression differences of microRNAs screened with clinical samples. 2) microRNAs overexpression will be up-regulated with microRNA agomirs, or down-regulated with microRNA antagomirs in human proximal tubule epithelial cell line (HK-2 cell), and the expressions of the target genes of microRNAs and the corresponding proteins and biological functions will be detected. 3) Adeno virus mediated microRNA mimics and inhibitors will be given to the mouse model of early stage CKD by tail intravenous injection. The development of renal osteodystrophy will be observed. 4) Kidney tonifying formula will be screened in HK-2 cells, and the effective components which can regulate microRNA expressions, target gene and protein expression and related biological function will be used for in vivo study. 5) The screened effective components and their combinations will be used in the mouse model of early stage CKD. The effect of these effective components on renal osteodystrophy will be observed.

肾性骨病是慢性肾脏疾病的重要并发症之一，因发病率高，危害性大，晚期治疗局限性而受到日益关注。本团队通过前期研究发现肾功能不全患者早期即出现低转运型骨丢失，短暂恢复后迅速发展为不可逆高转运型骨丢失。如果能在早期之前进行有效干预，则可能延迟肾性骨病的进展。团队前期利用microRNA芯片筛选出肾功能不全早期和晚期差异表达的microRNA。本研究拟在此基础上，建立肾功能不全早期和晚期小鼠模型，验证microRNA变化趋势；在肾小管上皮细胞中过表达或抑表达microRNA，验证靶基因信号通路和细胞功能变化；通过给早期肾功能不全小鼠注射病毒介导的microRNA mimics或inhibitor，观察对肾性骨病进展有无延迟。通过细胞实验，对肾性骨病临床有效的补肾中药及有效组分进行筛选，发现可以调节microRNA的有效组分并阐明调节机制，最终在肾功能不全小鼠模型中验证有效组分和优化组合的药效。

microRNA 3183可以参与调节钙离子平衡，进而调节肾小管上皮细胞凋亡，从而在肾功能不全的发展中发挥一定作用。采用HBAAV2/9-has-mir-3183局部注射，能够一定程度减轻肾功能丢失，提示microRNA3183作为肾功能不全早期干预的靶点的潜在机制。肾性骨病方早期干预能够改善CKD小鼠肾脏细胞凋亡，延缓肾性骨病的进展，缓解骨丢失。其有效组分特女真甘可以通过调节microRNA3183的表达水平，改善肾小管上皮细胞钙离子平衡状态和细胞凋亡。. HBAAV2/9-has-mir-3183单次注射对5/6肾切除小鼠的肾功能有一定的保护作用，但对骨丢失无显著作用。可能的原因是 mir-3183注射早期转染效率不够，不能早期发挥作用；或因为5/6肾切除损伤较为严重，即使mir-3183注射在一定程度上可以改善对5/6肾切除小鼠的肾功能，但仍远未达到正常水平，对骨组织仍然会造成较严重的损伤；或进入体内是否能发汇生物学作用仍未可知。因此，后期拟优化mir-3183的干预方式，以期获得更加的干预效果。. 肾性骨病方早期干预可以改善CKD小鼠骨丢失，其机制之一为减轻肾脏细胞凋亡和肾功能降低，但尚存在其他多靶点作用，从而共同达到改善CKD小鼠肾功能和骨保护作用。而特女真苷一方面可以通过调节microRNA3183的表达水平，改善肾小管上皮细胞钙离子平衡状态和细胞凋亡，但因HBAAV2/9-has-mir-3183单次注射对5/6肾切除小鼠的肾功能有一定的保护作用，但对骨丢失无显著作用，因此，特女真苷也可能还存在其他调节机制。