Effects of Paracrine and Intracellular Angiotensin II on Protein Kinase C, MAP kinase ERK1/2, and Nuclear Factor‐κB Signaling Responses in Mouse Glomerular Mesangial cells

Abstract

Angiotensin II (ANG II) acts not only as a vasoconstrictor, but also as a growth‐promoting cytokine and chemokine, proinflammatory and profibrotic factor in the kidney. Glomerular mesangial cells (MC) are a key target for endocrine, paracrine, autocrine, and intracrine ANG II. Augmentation of local paracrine and intracrine ANG II generation in MCs may induce glomerular injury by promoting MC cell growth, hypertrophy and fibrosis. In the present study, we performed phosphoproteomic characterization of ANG II‐induced protein kinase C (p‐PKCα), MAP kinase ERK1/2 (p‐ERK1/2), p38 MAPK, and nuclear factor‐κB (p65 and p50 subunits) signaling responses in murine glomerular MCs. Murine mesangial cells (MES‐13, ATCC) were grown to ~80% confluence in 6‐well plates in DMEM and Ham's F‐12 medium, containing 5% fetal bovine serum, and 1% penicillin‐streptomycin, maintained at 37°C and 95% O2/5% CO2. After starved at a serum‐free medium for 24 h, MCs were then treated with low to high levels of ANG II (0.1 to 100 nM) for 5 to 20 min, or transfected with a mitochondria‐targeting intracellular ANG II for 48 h (mito‐ECFP/ANG II, 4 μg/well), with or without pretreatment with the AT1 receptor blocker losartan (1 μM), AT2 receptor blocker PD123319 (1 μM) or both blockers. Proteins were extracted for Western blot analysis of p‐PKCα, p‐ERK1/2, p38 MAPK, and p65 and p50 subunits of NF‐κB, respectively. ANG II significantly increased p‐ERK1/2 and p‐PKCα in both dose‐ and time‐dependent manners, with peak responses to ANG II occurred at 0.1 and 1.0 nM, 5–10 min (p<0.05), respectively. ANG II also increased p38 MAPK response, but it peaked at 1 nM, 20 min (p<0.05). By contrast, ANG II appeared to decrease the NF‐κB responses at 1 nM, 10 min, which were reversed by losartan and PD123319. The expression of mito‐ECFP/ANG II selectively in the MC mitochondria significantly increased mitochondrial complex proteins (CI‐CV), which were blocked by losartan (p<0.01). Our results suggest that both paracrine and intracellular ANG II may activate AT1 receptors to induce important intracellular PKC, MAP kinase ERK1/2, p38 MAPK, and NF‐κB signaling responses in MCs, which may contribute to ANG II‐induced glomerular injury in hypertension and kidney diseases.Support or Funding InformationSupported by National Institute of Health grants, 2R01DK067299‐10A1 and 2R01DK102924‐03A1 to Zhuo, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ CAPES to Leite.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.