Hypertension is the leading cause of morbidity and mortality from stroke, myocardial infarction, heart failure, and chronic kidney disease. Despite the importance of blood pressure control, the pathogenesis of essential hypertension remains poorly understood. In the past several years it has become clear that sodium can accumulate in the interstitium, particularly in the skin and skeletal muscle and that these modestly elevated concentrations of sodium can drive immune cell activation. Our laboratory has recently described a new pathway by which extracellular sodium activates NADPH oxidase in dendritic cells and showed that this promotes isolevuglandin-adducts that are recognized as non-self and evoke an immune response. I propose that salt stabilizes NADPH oxidase subunits, specifically p22phox, via serum and glucocorticoid-regulated kinase 1 (SGK1) in dendritic cells (DCs), which leads to the promotion of hypertension.
In Aim 1, I will test the hypothesis that stabilization of p22phox protein in response to salt is dependent on SGK1 and to determine if this promotes DC activation and hypertension. In this aim I will use mice in which we have deleted SGK1 specifically in DCs. In the first part of this aim, I will demonstrate if this increased sodium indeed enhances stability of the NADPH oxidase protein subunits and if this is dependent on SGK1. In a second series of experiments, I will examine the effect of SGK1 on the phenotype of DCs. DCs will be analyzed by flow cytometry and for superoxide production by electron spin resonance. In additional experiments, I will examine the in vivo role of SGK1 in DCs. I will perform adoptive transfer of dendritic cells co-treated with mannitol or high salt into nave mice and measure blood pressure by radiotelemetry. I predict that deletion of SGK1 prevents NADPH oxidase subunit stabilization, production of superoxide, and increase is blood pressure with low dose angiotensin II.
In aim 2, I will determine if NADPH oxidase subunits p22phox, p47phox, and/or gp91phox are ubiquitinated in response to salt via SGK1, and to determine if this promotes DC activation and hypertension. In these studies, we will perform immunoprecipitation of the NADPH oxidase subunits p22phox, p47phox and gp91phox. We will use mass spectrometry to identify ubiquitinated lysines of the NADPH oxidase subunits. In additional experiments, we will assess p22phox, p47phox, and gp91phox ubiquitination in vivo utilizing a rodent model of salt-sensitive hypertension. I predict that NADPH oxidase subunits will be stabilized during high salt treatment, and that genetic deletion of SGK1 will prevent this in DCs. This will advance our understanding of hypertension and will provide new therapeutic directions for this disease.
Hypertension and cardiovascular disease is a major cause of morbidity and mortality in western societies. Our project seeks to understand how inflammation causes blood vessel and kidney damage in hypertension.
