A stop-flow comprehensive two-dimensional HK-2 and HK-2/CIKI cell membrane chromatography comparative analysis system for screening the active ingredients from Pyrrosia calvata (Bak.) Ching against crystal-induced kidney injury

Abstract Crystal-induced kidney injury (CIKI) is the fundamental pathological change during nephrolithiasis, although the molecular mechanism is still unclear. Pyrrosia calvata (Bak.) Ching has been used in folk medicine to treat urolithiasis for years. To clarify the pharmacodynamic substances and the mechanism of its antiurolithiasis effects, in this study, a novel, stop-flow, comprehensive, two-dimensional (2D) HK-2 and HK-2/CIKI cell membrane chromatography (CMC) comparative analysis system was developed to screen for the potential active ingredients from Pyrrosia calvata (Bak.) Ching against CIKI. The comprehensive 2D CMC comparative analysis system showed satisfactory selectivity, and eight ingredients were screened and identified by this system. Among them, mangiferin exhibited higher affinity for the HK-2/CIKI CMC column than the HK-2 CMC column and was selected for further efficacy verification. Cell proliferation assays showed that mangiferin could protect HK-2 cell viability after stimulation with sodium oxalate (NaOX). Additionally, in a rodent model of CIKI, mangiferin decreased the deposition of calcium oxalate (CaOX) crystals in mouse kidneys, alleviated the pathological damage to kidney tissue, and inhibited the upregulation of OPN, MCP1, and CD44 expression caused by CaOX crystals. The established comprehensive 2D CMC comparative analysis system can be applied to screen active ingredients with disease specificity from traditional Chinese medicine (TCM) and is suitable for other cell models.