Supplementary Material for: Role of mitophagy in ischemia-reperfusion renal injury: new insights from bioinformatics analysis

Introduction: Mitophagy is central to acute kidney injury (AKI) pathogenesis. Elucidating its molecular interplay with AKI is crucial for novel therapeutics. Methods: This study is based on transcriptome sequencing combined with single-cell sequencing, and applies bioinformatics analysis. Finally, it is verified by in vitro, in vivo and clinical specimen experiments. Results: In transcriptome analysis, combining protein-protein interaction mapping with machine intelligence algorithms, we screened out two mitophagy-related differentially expressed genes (MitoDEGs), Solute Carrier Family 3 Member 2 (SLC3A2) and Thioredoxin (TXN). The immunological analysis revealed a notable rise in monocyte infiltration in the immune microenvironment of ischemia-reperfusion renal injury (IRI)-AKI. Spearman analysis indicated hub MitoDEGs were positively correlated with pro-inflammatory immune cell infiltration, and negatively correlated with anti-inflammatory or regulatory immune cell infiltration. Based on the highest binding score, 506-26-3 CTD (Gamma-linolenic acid, GLA) was determined to be the top promising therapeutic candidate. At the single-cell level, hub MitoDEGs were mainly expressed in proximal tubular. In cell experiments, mitophagy was inhibited after hypoxia-reoxygenation, SLC3A2 matched earlier results, while TXN was contrary to the previous analysis results. In the IRI-AKI rat experiments, the findings regarding hub MitoDEGs aligned with our prior analysis, revealing a decrease in the expression of genes associated with mitophagy. Consequently, we directed our attention to the expression levels of SLC3A2 in clinical cases of AKI, where we observed a notable increase. Conclusion: Our research indicates that SLC3A2 could be a crucial target for enhancing IRI-AKI through the modulation of the mitophagy pathway.