Supplementary Material for: High serum phosphate promotes cognitive impairment in uremia rats via mediating vascular calcification

Introduction: Cognitive impairment (CI) is common in patients with end-stage renal disease (ESRD), but the mechanism of uremia-associated CI is poorly recognized. Methods: In this study, we constructed uremia rat models, treated with high level phosphate (Pi) and vascular calcification inhibitor sodium thiosulphate (STP). Assessed the spatial learning and memory by Morris Water Maze (MWM), detected the vascular calcification (VC) by histological staining and monitored the osteogenic factors ALP and Runx2 expressions in Uremia + Pi and Uremia + Pi + STP rats through qPCR, Western blotting analysis and Immunohistochemistry analysis. Moreover, we examined the calcium content, and the ALP and Runx2 expressions in VSMCs treated with Pi and STP. Results: The results showed that both the learning and memory abilities and the spatial exploration ability of the rats in Uremia + Pi group were significantly decreased with enhanced expressions of ALP and Runx2, calcium content and ALP activity, however, STP supplementation significantly alleviated the spatial learning and memory damage, reduced the ALP and Runx2 expressions, calcium content and ALP activity. Moreover, Pi treatment significantly increased the calcium and MDA content in VSMCs, and ALP and Runx2 expressions, STP addition reversed these changes, indicating high phosphate level induced VC development. Conclusion: Taking together, these results indicated that elevated serum phosphate might promote VC in uremic rats by activating the expressions of ALP and Runx2, thereby causing damages to the kidneys and complications of uremia such as CI. Therefore, this study improved understanding to possible pathogenic mechanisms of uremia-induced CI, and will be beneficial to optimize the prevention and treatment strategy for CI in uremia patients.

引言：认知功能障碍（Cognitive impairment, CI）在终末期肾病（End-stage renal disease, ESRD）患者中较为常见，但尿毒症相关性认知功能障碍的发病机制尚未得到充分阐明。
方法：本研究构建尿毒症大鼠模型，分别给予高浓度磷酸盐（Phosphate, Pi）与血管钙化抑制剂硫代硫酸钠（Sodium thiosulphate, STP）干预。通过莫里斯水迷宫（Morris Water Maze, MWM）评估大鼠的空间学习与记忆能力；采用组织学染色检测血管钙化（Vascular calcification, VC）情况；并通过实时荧光定量PCR（quantitative real-time polymerase chain reaction, qPCR）、蛋白质印迹法（Western blotting）及免疫组化（Immunohistochemistry）分析，检测尿毒症+Pi组与尿毒症+Pi+STP组大鼠中成骨因子碱性磷酸酶（Alkaline phosphatase, ALP）与Runt相关转录因子2（Runt-related transcription factor 2, Runx2）的表达水平。此外，本研究还检测了经Pi与STP处理的血管平滑肌细胞（Vascular smooth muscle cells, VSMCs）中的钙含量，以及ALP与Runx2的表达水平。
结果：结果显示，尿毒症+Pi组大鼠的学习记忆能力与空间探索能力均显著下降，同时ALP、Runx2表达水平、钙含量及ALP活性均显著升高；而给予STP干预后，大鼠的空间学习记忆损伤得到显著缓解，ALP与Runx2表达水平、钙含量及ALP活性均显著降低。此外，Pi处理可显著升高VSMCs中的钙与丙二醛（Malondialdehyde, MDA）含量，并上调ALP与Runx2的表达；STP处理可逆转上述变化，表明高磷酸盐水平可诱导血管钙化的发生发展。
结论：综合上述结果，本研究表明血清磷酸盐水平升高可通过激活ALP与Runx2的表达，促进尿毒症大鼠的血管钙化，进而造成肾脏损伤及尿毒症相关并发症（如认知功能障碍）。本研究加深了对尿毒症诱导认知功能障碍潜在发病机制的理解，可为优化尿毒症患者认知功能障碍的防治策略提供理论支撑。