THE ROLE OF FERROPTOTIC STRESS IN MODULATING THE EFFECTS OF PRO-INFLAMMATORY MACROPHAGES DURING SEPSIS ASSOCIATED ACUTE KIDNEY INJURY

Sepsis associated – AKI [SA-AKI] is one of the leading causes of mortality in patients across the world, with its underlying pathogenic mechanisms being attributed to excessive immune response by the body. Macrophages, which are the primary responders of infections, serve as primary mediators of septic shock such as nitric oxide [NO] (via the iNOS pathway), TNF-α, and IL-1. NO acts as a vasodilator throughout the body, but suppresses the eNOS pathway in the glomerulus, leading to vasoconstriction and shunting of blood to the renal cortex, resulting in medullary ischemia and aggravating renal failure. NO produced in the glomerulus due to the infiltration of circulating macrophages can also cause podocyte injury and endothelial dysfunction. Iron metabolism has shown promise for its immunomodulatory properties, with pathways such as heme oxygenase-1 having crucial roles in regulating innate immune responses. This study aims to understand the role of ferroptotic stress in modulating macrophage phenotype and its effect on the damage caused by NO, thereby potentially alleviating podocyte injury and preserving slit diaphragm integrity.

2D monoculture and coculture models were used to study the crosstalk between macrophages, iron metabolism, and the different cells of the glomerulus. RAW 264.7 cell line and macrophages differentiated from THP-1 cell line were used to represent mouse and human macrophages respectively. Conditionally immortalized mouse podocytes, human podocytes, and human mesangial cells were used in coculture studies to understand the effect of macrophages and their cytokines on the glomerulus. Ferroptosis was induced using erastin and RSL-3, with ferroptosis inhibitors like ferrostatin-1 being used to establish cause and effect. Griess assay was used to estimate NO levels in culture supernatants after different treatments, and ELISA was used to estimate pro-inflammatory and anti-inflammatory cytokines of macrophages. Immunostaining and Western blotting were used to quantify protein expressions on the glomerular cells to understand the extent of injury, while qPCR was employed to interpret changes at the genetic level.

RAW 264.7 macrophages were co-treated with different concentrations of IFNγ and LPS for 48 hours to stimulate M1 polarization and NO production. 24 hours post treatment, 10 μM erastin was added to induce ferroptosis in the presence of iNOS inducers, and NO levels were measured after 24 hours of erastin treatment. Results showed a significant decrease in the levels of NO, suggesting that ferroptosis can play a regulatory role in the production of NO by macrophages through the iNOS pathway.

Reduction in the levels of NO produced by macrophages upon treatment with ferroptosis inducers provides evidence of a novel mechanism, wherein ferroptosis plays a protective role in sepsis. The promising results with the immune cells ensure that future experiments in this study will focus on the effect of iron modulated M1 macrophage cytokine treatment on human podocytes and their health. This paves way for novel investigations into the various pathways of iron metabolism that could play a role in immunomodulation, and their resultant effects on alleviating glomerular injury in the context of SA-AKI.