Meredith M. Skelton

Increased Renal Medullary H2O2 Leads to Hypertension

Abstract—We have recently reported that exaggerated oxidative stress in the renal medulla due to superoxide dismutase inhibition resulted in a reduction of renal medullary blood flow and sustained hypertension. The present study tested the hypothesis that selective scavenging of O2·− in the renal medulla would prevent hypertension associated with this exaggerated oxidative stress. An indwelling, aortic catheter was implanted in nonnephrectomized Sprague-Dawley rats for daily measurement of arterial blood pressure, and a renal medullary interstitial catheter was implanted for continuous delivery of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETC, 7.5 mg · kg−1 · d−1) and a chemical superoxide dismutase mimetic, 4-hydroxytetramethyl piperidine-1-oxyl (TEMPOL, 10 mg · kg−1 · d−1). Renal medullary interstitial infusion of TEMPOL completely blocked DETC-induced accumulation of O2·− in the renal medulla, as measured by the conversion rate of dihydroethidium to ethidium in the dialysate and by urinary excretion of 8-isoprostanes. However, TEMPOL infusion failed to prevent DETC-induced hypertension, unless catalase (5 mg · kg−1 · d−1) was coinfused. Direct infusion of H2O2 into the renal medulla resulted in increases of mean arterial pressure from 115±2.5 to 131±2.1 mm Hg, which was similar to that observed in rats receiving the medullary infusion of both TEMPOL and DETC. The results indicate that sufficient catalase activity in the renal medulla is a prerequisite for the antihypertensive action of TEMPOL and that accumulated H2O2 in the renal medulla associated with exaggerated oxidative stress might have a hypertensive consequence.

Tubulointerstitial injury and loss of nitric oxide synthases parallel the development of hypertension in the Dahl-SS rat

Objective Alterations in renal nitric oxide (NO) are involved in the hypertension of the Dahl salt-sensitive (Dahl-SS) rat. We sought to identify the kinetics and sites of expression of the major NO synthase (NOS) isoforms. Design The renal expression of the major NOS were examined in Dahl-SS and salt-resistant rats (Dahl-SR) while on a low salt (0.1% NaCl) diet at 3 and 9 weeks of age. Methods Renal biopsies from Dahl-SS and Dahl-SR rats were compared for evidence of renal injury and for alterations in expression of the NOS enzymes by quantitative immunohistochemistry. Results At 3 weeks of age Dahl-SS and Dahl-SR rats have normal renal histology and similar immunohistochemical expression of NOS1, −2, and −3. At 9 weeks Dahl-SS rats had significantly higher blood pressure than Dahl-SR rats (P < 0.005), and lower macula densa NOS1 (P < 0.05) and cortical and medullary NOS3 (P < 0.05). NOS2 was reduced in cortical tubules in biopsies showing severe tubulointerstitial damage, but was not significantly different between Dahl-SS and Dahl-SR groups as a whole. Dahl-SS rats also manifested glomerular and tubulointerstitial injury. Tubular expression of osteopontin (OPN), which is an inhibitor of NOS2, correlated with the systolic BP in individual Dahl-SS rats (r2 = 0.80, P < 0.0001). Conclusion 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.

Vasopressin reduces cardiac function and augments cardiopulmonary baroreflex resistance increases in man.

We examined the effects of physiologic infusions of arginine vasopressin (AVP) on cardiovascular hemodynamics and on reflex responses initiated by decreasing cardiopulmonary baroreceptor stimulation (with lower body negative pressure) in 10 healthy, captopril-pretreated young men (19-27 yr). Their responses were compared with those of four volunteers given isosmotic infusion. Heart rate, stroke volume, blood pressure, and forearm blood flow were measured by electrocardiography, impedance cardiography, radial artery cannulation, and strain gauge plethysmography. Two 55-min infusions of AVP at rates of 0.15 and 0.40 ng/kg per min increased average plasma concentrations from control levels of 5 pg/ml to 18 and 36 pg/ml, respectively. These infusions resulted in progressive reductions of heart rate and cardiac output and increases of forearm and total peripheral resistance. Blood pressure increases were significant only during the larger AVP infusion rate. Lower body negative pressure provoked reflex increases of total peripheral resistance. These increases were enhanced 60% during AVP infusion compared with increases during control (pre-AVP). Baseline measurements and reflex responses were unchanged by isosmotic infusions. These results demonstrate that AVP has profound effects on cardiovascular function and augments cardiopulmonary baroreflex-mediated increases of peripheral resistance in man.

Effects of Renal Perfusion Pressure on Renal Medullary Hydrogen Peroxide and Nitric Oxide Production

Studies were designed to determine the effects of increases of renal perfusion pressure on the production of hydrogen peroxide (H2O2) and NO2−+NO3− within the renal outer medulla. Sprague-Dawley rats were studied with either the renal capsule intact or removed to ascertain the contribution of changes of medullary blood flow and renal interstitial hydrostatic pressure on H2O2 and NO2−+NO3− production. Responses to three 30-minute step changes of renal perfusion pressure (from ≈85 to ≈115 to ≈145 mm Hg) were studied using adjustable aortic occluders proximal and distal to the left renal artery. Medullary interstitial H2O2 determined by microdialysis increased at each level of renal perfusion pressure from 640 to 874 to 1593 nmol/L, as did H2O2 urinary excretion rates, and these responses were significantly attenuated by decapsulation. Medullary interstitial NO2−+NO3− increased from 9.2 to 13.8 to 16.1 &mgr;mol/L, with parallel changes in urine NO2−+NO3−, but decapsulation did not significantly blunt these responses. Over the range of renal perfusion pressure, medullary blood flow (laser-Doppler flowmetry) rose ≈30% and renal interstitial hydrostatic pressure rose from 7.8 to 19.7 cm H2O. Renal interstitial hydrostatic pressure and the natriuretic and diuretic responses were significantly attenuated with decapsulation, but medullary blood flow was not affected. The data indicate that pressure-induced increases of H2O2 emanated largely from increased tubular flow rates to the medullary thick-ascending limbs of Henle and NO largely from increased medullary blood flow to the vasa recta. The parallel pressure–induced increases of H2O2 and NO indicate a participation in shaping the “normal” pressure-natriuresis relationship and explain why an imbalance in either would affect the blood pressure salt sensitivity.

Effects of daily sodium intake and ANG II on cortical and medullary renal blood flow in conscious rats

Implanted optical fibers and laser-Doppler flow measurement techniques were used for the sequential measurement of regional renal blood flow in conscious rats to determine the effects of an increase of daily NaCl intake on the renal cortical blood flow and blood flow to the outer and inner medulla. Cortical blood flow was increased significantly (32%) by the second day when NaCl intake was increased from 1 to 7 meq/day and was increased further (50%) on the second day after a further elevation of NaCl intake to 13 meq/day. Blood flow to the outer and inner medulla was not changed as NaCl intake was elevated. The increase in renal cortical flow was closely associated with significant reductions in circulating concentrations of ANG II from 31 to 16 pg/ml. Rats given a continuous infusion of nonpressor doses of ANG II (5.0 ng ⋅ kg-1 ⋅ min-1) to maintain constant plasma concentrations of ANG II as sodium intake was increased exhibited no increase of cortical flow. We conclude that reductions of plasma ANG II associated with incremental increases of daily sodium intake result in a rise of renal cortical flow. The elevated blood flow to the renal cortex may enhance sodium excretion and contribute to long-term sodium homeostasis.Implanted optical fibers and laser-Doppler flow measurement techniques were used for the sequential measurement of regional renal blood flow in conscious rats to determine the effects of an increase of daily NaCl intake on the renal cortical blood flow and blood flow to the outer and inner medulla. Cortical blood flow was increased significantly (32%) by the second day when NaCl intake was increased from 1 to 7 meq/day and was increased further (50%) on the second day after a further elevation of NaCl intake to 13 meq/day. Blood flow to the outer and inner medulla was not changed as NaCl intake was elevated. The increase in renal cortical flow was closely associated with significant reductions in circulating concentrations of ANG II from 31 to 16 pg/ml. Rats given a continuous infusion of nonpressor does of ANG II (5.0 ng.kg(-1).min-1) to maintain constant plasma concentrations of ANG II as sodium intake was increased exhibited no increase of cortical flow. We conclude that reductions of plasma ANG II associated with incremental increases of daily sodium intake result in a rise of renal cortical flow. The elevated blood flow to the renal cortex may enhance sodium excretion and contribute to long-term sodium homeostasis.

Effects of long-term vasopressin receptor stimulation on medullary blood flow and arterial pressure

Studies were carried out using instrumented unanesthetized rats to determine the long-term effects of arginine vasopressin (AVP) and a specific vasopressin V1 receptor agonist (V1AG; [Phe2, Ile3, Orn8]- vasopressin) on the renal medullary blood flow and arterial blood pressure. It was hypothesized that the hypertension observed with chronic medullary infusion of a V1 receptor agonist may be associated with a sustained reduction of blood flow, whereas infusion of AVP may fail to produce a sustained reduction of blood flow and thereby be unable to produce hypertension. Uninephrectomized Sprague-Dawley rats were prepared with implanted renal cortical and medullary optical fibers for daily measurements of cortical and medullary blood flow using laser-Doppler flowmetry techniques. An implanted renal medullary interstitial infusion catheter delivered either AVP or a specific V1AG at a dose of 2 ng ⋅ kg-1 ⋅ min-1over a period of 5 days. The V1AG produced no change of cortical blood flow but a chronic 35% reduction of medullary blood flow ( P < 0.05) and mild hypertension (11 ± 4 mmHg, P < 0.05). AVP produced only an initial, nonsignificant 1- to 2-day reduction of medullary blood flow (-13%) and failed to raise arterial pressure significantly. We conclude that a sustained V1AG response is necessary to achieve a chronic reduction of medullary blood flow and hypertension. The present data are consistent with the idea that chronic stimulation of V2receptors by AVP offsets the vasoconstrictor and hypertension actions of AVP-induced stimulation of medullary V1 receptors.Studies were carried out using instrumented unanesthetized rats to determine the long-term effects of arginine vasopressin (AVP) and a specific vasopressin V1 receptor agonist (V1AG; [Phe2, Ile3, Orn8]- vasopressin) on the renal medullary blood flow and arterial blood pressure. It was hypothesized that the hypertension observed with chronic medullary infusion of a V1 receptor agonist may be associated with a sustained reduction of blood flow, whereas infusion of AVP may fail to produce a sustained reduction of blood flow and thereby be unable to produce hypertension. Uninephrectomized Sprague-Dawley rats were prepared with implanted renal cortical and medullary optical fibers for daily measurements of cortical and medullary blood flow using laser-Doppler flowmetry techniques. An implanted renal medullary interstitial infusion catheter delivered either AVP or a specific V1AG at a dose of 2 ng . kg-1 . min-1 over a period of 5 days. The V1AG produced no change of cortical blood flow but a chronic 35% reduction of medullary blood flow (P < 0.05) and mild hypertension (11 +/- 4 mmHg, P < 0.05). AVP produced only an initial, nonsignificant 1- to 2-day reduction of medullary blood flow (-13%) and failed to raise arterial pressure significantly. We conclude that a sustained V1AG response is necessary to achieve a chronic reduction of medullary blood flow and hypertension. The present data are consistent with the idea that chronic stimulation of V2 receptors by AVP offsets the vasoconstrictor and hypertension actions of AVP-induced stimulation of medullary V1 receptors.

Dynamic convergence and divergence of renal genomic and biological pathways in protection from Dahl salt-sensitive hypertension

Chromosome 13 consomic and congenic rat strains were analyzed to investigate the pattern of genomic pathway utilization involved in protection against salt-sensitive hypertension and renal injury. Introgression of the entire Brown-Norway chromosome 13 (consomic SS-13(BN)) or nonoverlapping segments of this chromosome (congenic strains, 16 Mbp in D13Rat151-D13Rat197 or 14 Mbp in D13Rat111-D13Got22) into the genome of the Dahl salt-sensitive rat attenuated salt-induced hypertension and proteinuria. mRNA abundance profiles in the renal cortex and the renal medulla from rats receiving 0.4% or 8% NaCl diets revealed two important features of pathway recruitment in these rat strains. First, the two congenic strains shared alterations in several pathways compared with Dahl salt-sensitive rats, despite the fact that the genomic segments introgressed in the two congenic strains did not overlap. Second, even though the genomic segment introgressed in each congenic strain was a part of the chromosome introgressed in the consomic strain, pathways altered in each congenic strain were not simply a subset of those altered in the consomic. Supporting the relevance of the mRNA data, differential expression of oxidative stress-related genes among the four strains of rats was associated with differences in urinary excretion of lipid peroxidation products. The findings suggest that different genetic alterations might converge to influence shared pathways in protection from hypertension, and that, depending on the genomic context, the same genetic alteration might diverge to affect different pathways.

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

This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4) on 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 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SSNox4−/−) rats. SSNox4−/− rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) 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 with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67phox, and p22phox were found to be significantly lower (P<0.05) in SSNox4−/− compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several 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.

Dynamic cardiovascular responses to infusions of atrial natriuretic factor in humans.

We sought to demonstrate a hypotensive effect from infusions of atrial natriuretic factor (ANF) into humans and to describe the mechanism(s) of this effect. Cardiovascular and hormonal responses to human ANF-(99-126) (125 ng/kg bolus followed by a 30-minute infusion at 25 ng/kg/min) were determined in eight conscious volunteers and compared with responses of eight time-control subjects who received isotonic saline. Baseline levels of ANF (52.8 +/- 5.5 pg/ml) increased 8.8-fold after 30 minutes of ANF infusion but were unchanged in the time controls. Plasma levels of renin, aldosterone, vasopressin, sodium, potassium, and osmolality did not change during infusions. A transient 5% reduction in mean arterial pressure related to a 12% reduction in peripheral resistance was observed 10 minutes after the priming bolus of ANF. This response was not sustained during the remainder of the ANF infusion period, nor did it occur in two additional subjects who received ANF infusions without the priming bolus. Steady state responses consisted of significant reductions in central venous pressure (15%), stroke volume (13%), and cardiac output (10%), but no reduction in blood pressure. Plasma norepinephrine levels and peripheral resistance increased (34% and 9%, respectively) during ANF administration. These data indicate that steady state responses to ANF in humans consist of decreases in cardiac filling pressures, which reduce cardiac output, unload cardiopulmonary baroreceptors, and activate the sympathetic nervous system. Blood pressure is well maintained despite striking increases in plasma ANF.

Progression of Glomerular Filtration Rate Reduction Determined in Conscious Dahl Salt-Sensitive Hypertensive Rats

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.Sequential changes in glomerular filtration rate during development of hypertension in the conscious Dahl salt-sensitive rats were determined using a new method for measurement. Using a miniaturized device, disappearance curves of fluorescein isothiocyanate–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 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% (high-salt [HS]) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. Mean arterial pressure rose progressively from the last day of 0.4% (130±2 mm Hg) reaching significance by day 5 of HS and averaged 162±7 mm Hg by day 21. Urine albumin excretion was significantly elevated (×3) by day 7 of HS in Dahl salt-sensitive rats. Glomerular filtration rate reduced on day 14 of HS falling from 1.53±0.06 mL/min per 100 g body weight to 1.27±0.04. By day 21, glomerular filtration rate had fallen 28% to 1.1±0.04 mL/min per 100 g (t1/2 28.4±1.1 minute.) No significant reductions of creatinine clearance were observed throughout the study in response to HS demonstrating the insensitivity of creatinine clearance measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of glomerular filtration rate was a consequence and not a cause of the hypertension and that this noninvasive approach could be used in these conscious Dahl salt-sensitive rats for a longitudinal assessment of renal function.