Neutrophil Depletion Influences Renal Outcomes in Male Mice with Chronic Angiotensin II Infusion
Medicine and Health Sciences
Hypertension is the leading cause of morbidity and mortality worldwide; yet the exact cause for many cases remain unknown. Recent research has highlighted the significance of the immune system in the development and progression of HTN in animal models. Much of this research has focused on classical immune pathways involving antigen presenting cells and subsequent activation of the adaptive immune response (primarily CD8+ T-cells). These activated lymphocytes then support the development of HTN by mediating end-organ damage in organs tasked with regulating blood pressure and extracellular volume (such as the kidneys). In the current study, we set out to determine the role of neutrophils in a murine model of HTN – the chronic angiotensin II (AngII) infusion model. All animal experiments were performed following animal protocols approved by the Liberty University IACUC and conform to the FASEB Statement of Principles for the use of animals in research and education. Baseline blood pressure was recorded in male C57Bl/6 mice (N=10) for 3 weeks using the CODA-8 tail cuff volume pressure recording system. Neutrophil depletion was accomplished via intraperitoneal injections of a monoclonal anti-Ly6G antibody (1A8, n=5), or IgG control (2A3, n=5) every 48 hours (250μg/mouse). 18 hours following the first antibody injection, a mini-osmotic pump was implanted into the rear flank delivering 500ng/kg*min AngII. 3 days after pump implantation, blood pressure readings resumed and continued for 14 days. After 14 days, mice were individually housed in metabolic cages and urine was collected for quantification of albuminuria. Renal blood flow was estimated in anesthetized mice using contrast-enhanced ultrasonography. Following renal blood flow estimation, mice were euthanized and blood, kidney, and spleens collected for determination of leukocyte content by flow cytometry. All data were analyzed using general linear models procedures in SPSS. Significant differences in blood pressure were observed periodically during the 14 days of AngII infusion (P≤0.05 for days 8, 12, and 14); however, the overall effect of treatment (1A8 vs. 2A3) did not reach statistical significance (P<0.2). Mice receiving the 1A8 neutrophil depleting antibody also tended (P≤0.07) to have a higher urinary output and albuminuria as compared to mice receiving the isotype control (respectively). No difference in estimated renal blood flow was detected between the treatment groups. Tissue analysis by flow cytometry confirmed the depletion and/or significant reduction of circulating and splenic neutrophils in mice receiving 1A8, but not 2A3, injections after 2 weeks. In the kidneys, mice receiving the 1A8 neutrophil depleting antibody had reduced (P=0.03) CD45+ leukocyte content. Further analysis of renal single cell suspensions revealed a significant reduction (P=0.04) in B-cells and higher (P=0.03) CD8+ T-cells in 1A8 treated animals. Taken together, our data suggest that neutrophil depletion alters several renal outcomes in the AngII murine model of HTN, with neutropenic mice tending to have higher blood pressure, greater urinary output and albuminuria as compared to control mice. These effects could be mediated by the significant differences in renal leukocyte content between the treatment groups. Further studies are needed to confirm our results and delineate the molecular mechanisms responsible for altered renal function and inflammation due neutrophil depletion in this animal model.
Kelsie Smith, Hannah Ter Harr, Brittney Browning, and Joseph Christopher Gigliotti, Neutrophil Depletion Influences Renal Outcomes in Male Mice with Chronic Angiotensin II Infusion, The FASEB Journal, Volume 32, Supplemental Issue 1, April 2018, https://www.fasebj.org/doi/abs/10.1096/fasebj.2018.32.1_supplement.718.12