Abstract-The present study examined whether chronic increased oxidative stress within the medulla of the kidney lowers medullary blood flow and leads to hypertension. Optical fibers were implanted into the renal cortex and medulla of uninephrectomized Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, Wis) for the daily measurement of blood flow to these regions using laser-Doppler flowmetry techniques, while arterial pressure was measured from an indwelling aortic catheter. A renal medullary interstitial catheter was implanted for the continuous delivery of the superoxide dismutase (SOD) inhibitor, diethyldithiocarbamic acid (DETC), at a dose of 7.5 mg/kg/d. Renal interstitial superoxide (O 2 Ϫ ) levels were determined by perfusing an O 2 Ϫ sensitive fluorescent dye, dihydroethidium, through a microdialysis probe implanted into the medulla. Urine samples (24 hours) were collected for measurements of isoprostane excretion. The results indicate that medullary DETC infusions increased tissue O 2 Ϫ concentrations in the renal medulla (93.4Ϯ22.3,nϭ8, saline and 867.3Ϯ260.2, nϭ8, DETC; fluorescence units) and increased urinary 8-isoprostane excretion (4.1Ϯ0.4 ng/d, nϭ9, saline and 8.8Ϯ1.6 ng/d, nϭ10, DETC). Mean arterial pressure increased 24 hours after the start of intrarenal DETC infusion and remained nearly 20 mm Hg above control pressure throughout the 5 days of medullary SOD inhibition. During chronic medullary DETC infusion, medullary blood flow was significantly reduced (42.7%), whereas cortical blood flow was unchanged. Intravenous infusion of the same dose of DETC produced no changes in renal medullary or cortical blood flow or arterial blood pressure. The present experiments indicate that an increase in superoxide concentration within the renal medulla selectively reduces medullary blood flow resulting in chronic hypertension.

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.

Sequential changes in glomerular filtration rate (GFR) during development of hypertension in the conscious Dahl salt-sensitive (SS) rat were determined using a new method for measurement. Utilizing a miniaturized device, disappearance curves of fluorescein isothiocyanate (FITC)-sinistrin were measured by transcutaneous excitation and real time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure (MAP) by telemetry) were studied while maintained on a 0.4% NaCl diet and on days 2,5,7,14 and 21 after switching to 4.0% (HS) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. MAP rose progressively from the last day of 0.4% (130±2 mmHg) reaching significance by day 5 of HS and averaged 162±7 mmHg by day 21. Urine albumin excretion was significantly elevated (3×) by day 7 of HS in SS rats. GFR became reduced on day 14 of HS falling from 1.53±0.06 ml/min/100g bwgt to 1.27±0.04. By day 21, GFR had fallen 28% to 1.1±0.04 ml/min/100g bwgt (t1/2 28.4±1.1 min.) No significant reductions of creatinine clearance (Ccre) were observed throughout the study in response to HS demonstrating the insensitivity of Ccre measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of GFR was a consequence and not a cause of the hypertension and that this non-invasive approach could be used in these conscious SS rats for a longitudinal assessment of renal function.

Tubulointerstitial injury and the loss of NOS occur after birth and parallel the development of hypertension. We suggest that the structural and functional changes that occur with renal injury in the Dahl-SS rat may contribute to the development of hypertension.