Macrophage-Specific Lactate Dehydrogenase Expression Modulates Inflammatory Function In Vitro

Background: In acute kidney injury, macrophages play a major role in regulating inflammation. Classically activated macrophages (M1) undergo drastic metabolic reprogramming during their differentiation and upregulate the aerobic glycolysis pathway to fulfill their pro-inflammatory functions. NAD+ regeneration is crucial for the maintenance of glycolysis and the most direct pathway by which this occurs is via the fermentation of pyruvate to lactate, catalyzed by lactate dehydrogenase A (LDHA). Our previous study determined that LDHA is predominantly expressed in the proximal segments of the nephron in the mouse kidney and increases with hypoxia. This study investigates the potential of LDHA as a therapeutic target for inflammation by exploring its role in macrophage function in vitro. Methods: Bone-marrow-derived macrophages (BMDMs) were isolated from myeloid-specific LDHA knockout mice derived from crossbreeding LysM-Cre transgenic mice and LDHA floxed mice. RNA sequencing and LC-MS/MS metabolomics analyses were used in this study to determine the effect of LDHA deletion on BMDM following stimulation with IFN-?. Results: LDHA deletion in IFN-? BMDMs resulted in a significant alteration of the macrophage activation and functional pathways, and change in glycolytic, cytokine, and chemokine gene expression. Metabolite concentrations associated with pro-inflammatory macrophage profiles were diminished while anti-inflammatory-associated ones were increased in LDHA KO BMDMs. Glutamate and amino sugars metabolic pathways were significantly affected by the LDHA deletion. A combined muti-omics analysis highlighted changes in Rap1 signaling, cytokine-cytokine receptor interaction, focal adhesion, and MAPK signaling metabolism pathways. Conclusions: Deletion of LDHA in macrophages results in a notable reduction in the pro-inflammatory profile and concurrent upregulation of anti-inflammatory pathways. These findings suggest that LDHA could serve as a promising therapeutic target for inflammation, a key contributor to the pathogenesis of acute kidney injury. Overall design: WT and KO mice for the LDHA gene were utilized to isolate Bone Marrow Derived Macrophages (BMDMs) which then were cultured in DMEM medium (15% FBS), with 30 ng/mL MCSF. Polarization of cells was performed using recombinant mouse interferon-gamma (IFN-?) towards M1 state and interleukin 4 (IL-4) towards M2 for 24 hours. RNA was extracted with Trizol reagent. RNA quality was checked using Eukaryote Total RNA Nano assay. Samples were sequenced by Illumina NextSeq500 Next Generation Sequencing (NGS). The resulting .fastq files were analyzed and trimmed through FastQC. Data was processed through the STAR (v2.5) pipeline for alignment using the GRCm38 mouse reference genome. Individual gene read number counting was conducted by HTseq (v. 0.6) software, generating FPKM (Fragments Per Kilobase of transcript per Million mapped reads). DESeq2/edgeR was utilized for differential expression calculation and statistical analysis.