Machine learning and 4D-LFQ quantitative proteomic analysis explore the molecular mechanism of kidney stone formation

Nephrolithiasis, a common and chronic urological condition, exerts significant pressure on both the general public and society as a whole. The precise mechanisms of nephrolith formation remain inadequately comprehended. Nevertheless, the utilization of proteomics methods has not been employed to examine the development of renal calculi in order to efficiently hinder and manage the creation and reappearance of nephrolith. Nowadays, with the rapid development of proteomics techniques, more efficient and more accurate proteomics technique is utilized to uncover the mechanisms underlying diseases. The objective of this study was to investigate the possible alterations of HK-2 cells when exposed to varying amounts of calcium oxalate (CaOx). The aim was to understand the precise development of stone formation and recurrence, in order to find effective preventive and treatment methods.

To provide a complete view of the proteins involved in the development of nephrolithiasis, we utilized an innovative proteomics method called 4D-LFQ proteomic quantitative techniques. HK-2 cells were selected as our experimental subjects. Three groups (n = 3) of HK-2 cells were treated with intervention solutions containing 0 (negative control, NC), 1 mM, and 2 mM CaOx, respectively. For the proteins that showed differential expression, various analyses were conducted including examination of Gene Ontology (GO), Clusters of Orthologous Groups of proteins (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, enrichment analysis of protein domains, and hierarchical clustering analysis. The STRING database was used to identify the interaction network of the chosen proteins. Candidate proteins were validated using parallel reaction monitoring (PRM) in the end.

All three groups verified the repeatability of samples. According to the results of 4D-LFQ proteomic quantitative analysis, there were 120, 262, and 81 differentially expressed proteins (DEPs) in the 1 mM-VS-NC, 2 mM-VS-NC, and 2 mM-VS-1mM conditions, respectively. According to GO annotation, the functional enrichment analysis indicates that the differentially expressed proteins (DEPs) were notably enriched in promoting cell migration and the extracellular matrix, among other functions. Analysis of enrichment, based on the KEGG pathway, revealed significant enrichment of DEPs in complement and coagulation cascades, as well as in ECM-receptor (extracellular matrix-receptor) interaction and other related pathways. 14 DEPs of great interest were selected as candidate proteins, including FN1, TFRC, ITGA3, FBN1, HYOU1, SPP1, HSPA5, COL6A1, MANF, HIP1R, JUP, AXL, CTNNB1 and DSG2.The data from PRM demonstrated the variation trend of 14 DEPs was identical as 4D-LFQ proteomic quantitative analysis.

Proteomics studies of CaOx-induced HK-2 cells using 4D-LFQ proteomic quantitative analysis and PRM may help to provide crucial potential target proteins and signaling pathways for elucidating the mechanism of nephrolithiasis and better treating nephrolithiasis.