Macrophage polarization as a mechanism of altered kidney growth and dysfunction in perinatal rats exposed to iron deficiency
Introduction: Stressors during early development can increase the risk of chronic diseases later in life. Iron deficiency (ID) is the most common nutritional deficiency worldwide, affecting approximately 38% of pregnant women. Our lab has shown that maternal ID impacts fetal kidney development, characterized by reduced nephron endowment and impaired growth and maturation. However, the cellular mechanisms that drive altered renal development in ID are not known. Macrophages are important immune cells within the kidney. During kidney development, alternatively activated M2 macrophages promote growth by mediating cell turnover and tissue remodelling. Conversely, classically activated M1 macrophages mediate inflammatory responses, and in turn, can damage organs when inappropriately activated. Further, macrophages can switch phenotypes between M1 and M2 in response to microenvironmental cues and energy substrate availability. Polarization towards the inflammatory M1 phenotype is implicated in many disease pathologies. We hypothesized that perinatal ID impairs nephrogenesis by causing a phenotypic switch from M2 to M1 macrophages in neonatal kidneys. Methods: Female Sprague Dawley rats were fed an iron-restricted diet prior to and throughout gestation to induce maternal ID. Dams at gestational day (GD) 21 were euthanized, and tissues and hemoglobin (Hb) values from fetal offspring were collected for analysis. Kidneys were homogenized and subjected to flow cytometry to evaluate M1 and M2 macrophages. M1 populations were defined using CD68+CD86+ staining, while M2 populations were defined by CD68+CD163+ markers. Further, fluorescence activated cell sorting will be utilized to separate M1 and M2 phenotypes, and single cell RNA sequencing will be performed to examine M1/M2 genes that are altered due to ID. Immunofluorescence (IF) will also be performed to assess M1 and M2 proportions based on intracellular markers; CCR7 for M1 and arginase-1 for M2 cells. Results: Hb levels were reduced in ID pups at GD21 for both males and females (P<0.001 for both). ID resulted in decreased birth weight compared to control pups in both male and female pups (P<0.001 for both). Preliminary flow cytometry data demonstrate a 29% decrease of M2 macrophages in males (P=0.004) and a 24% decrease in females (P=0.02), compared to their respective controls. Males ID fetuses had a 12% decrease of M1 cells (P=0.04) while female ID fetuses exhibited a 17% decrease of M1 cells (P=0.01). Currently, IF is being performed to validate these results with cytoplasmic markers. Conclusion: This study will provide new insights into the mechanisms by which ID affects kidney development. Understanding macrophage biology during kidney development may lead to novel therapeutic targets to prevent long-term kidney diseases associated with ID. Drugs such as atorvastatin can be explored to correct for M1 polarization in ID kidneys, preventing renal damage. By preventing organ damage during early development, we hope to prevent the development of diseases later in life.