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.