Evidence of the Importance of Nox4 in Production of Hypertension in Dahl Salt-Sensitive Rats

Abstract

This study reports the consequences of knocking out NADPH oxidase 4 (Nox4) upon the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4 resulting in a loss of the ~68 kD band in Western blot analysis of renal cortical tissue of the SSNox4−/− rats. SSNox4−/− rats exhibited a significant reduction of salt-induced hypertension compared to SS rats after 21 days of 4.0% NaCl diet (134±5 vs 151±3 mmHg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3D cryoimaging revealed significantly higher redox ratios (NADH/FAD) in the kidneys of SSNox4−/− rats even when fed the 0.4% NaCl diet indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared to SS rats. Prior to the development of hypertension, RNA expression levels of NADPH oxidase subunits Nox2, p67phox, and p22phox were found to be significantly lower (p<0.05) in SSNox4−/− compared to SS rats in the renal cortex. Thus the mutation of Nox4 appears to modify transcription of a number of genes in ways that contribute to the protective effects observed in the SSNox4−/− rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SSNox4−/− rat could be the result of multiple pathways including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4.

Essential role of Kir5.1 channels in renal salt handling and blood pressure control

“…The quality of each sample was assessed using an Agilent 2100 BioAnalzyer and quantity determined by spectrophotometry (Nanodrop). Two micrograms of total RNA was reverse transcribed by random hexamer primers into cDNA (Thermo Fisher Scientific RevertAid First-Strand cDNA synthesis kit) and real-time PCR analysis was performed using 8 to 10 ng total RNA with SYBR Green chemistry on an ABI Prism 7900HT (Applied Biosystems) as previously described (59). Primer sequences are given in the supplemental data.…”

Oxidative Stress, Inflammation, and Vascular Aging in Hypertension

“…Interestingly Nox4 −/− rats showed lower Nox2, p67phox, and p22phox in the renal cortex indicating potential differential regulation or interaction between NADPH oxidase homologues. 44 This demonstrates complexity of the role of Nox4 in the regulation of vascular function and disease. Possible mechanisms linked to cardiovascular protection related to Nox4 may be linked to microRNA (miRNAs) expression.…”

Role of Nox4 and p67phox subunit of Nox2 in ROS production in response to increased tubular flow in the mTAL of Dahl salt-sensitive rats

“…We previously characterized blood pressure salt sensitivity and renal function in SS Nox4Ϫ/Ϫ and SS p67phoxϪ/Ϫ rats compared with SS rats (9,14). SS Nox4Ϫ/Ϫ and SS p67phoxϪ/Ϫ rats exhibited significant reductions (ϳ50%) in salt-induced hypertension, albumin excretion, glomerular injury, and tubular necrosis.…”