J. Carlos Romero

Increased Oxidative Stress in Experimental Renovascular Hypertension

The pathophysiological mechanisms responsible for maintenance of chronic renovascular hypertension remain undefined. Excess angiotensin II generation may lead to release of reactive oxygen species and increased vasoconstrictor activity. To examine the potential involvement of oxidation-sensitive mechanisms in the pathophysiology of renovascular hypertension, blood samples were collected and renal blood flow measured with electron-beam computed tomography in pigs 5 and 10 weeks after induction of unilateral renal artery stenosis (n=7) or sham operation (n=7). Five weeks after procedure, plasma renin activity and mean arterial pressure were elevated in hypertensive pigs. Levels of prostaglandin F2&agr; (PGF2&agr;)–isoprostanes, vasoconstrictors and markers of oxidative stress, also were significantly increased (157±21 versus 99±16 pg/mL;P <0.05) and correlated with both plasma renin activity (r =0.83) and arterial pressure (r =0.82). By 10 weeks, plasma renin activity returned to baseline but arterial pressure remained elevated (144±10 versus 115±5 mm Hg;P <0.05). Isoprostane levels remained high and still correlated directly with the increase in arterial pressure (r =0.7) but not with plasma renin activity. Stenotic kidney blood flow was decreased at both studies. In shock-frozen cortical tissue, ex vivo endogenous intracellular radical scavengers were significantly decreased in both kidneys. The present study demonstrates, for the first time, that in early renovascular hypertension, an increase in plasma renin activity and arterial pressure is associated with increased systemic oxidative stress. When plasma renin activity later declines, PGF2&agr;-isoprostanes remain elevated, possibly due to local activation or slow responses to angiotensin II, and may participate in sustenance of arterial pressure. Moreover, oxidation-sensitive mechanisms may influence ischemic and hypertensive parenchymal renal injury.

Distinct Renal Injury in Early Atherosclerosis and Renovascular Disease

Background—Atherosclerotic renovascular disease may augment deterioration of renal function and ischemic nephropathy compared with other causes of renal artery stenosis (RAS), but the underlying mechanisms remain unclear. This study was designed to test the hypothesis that concurrent early atherosclerosis and hypoperfusion might have greater early deleterious effects on the function and structure of the stenotic kidney. Methods and Results—Regional renal hemodynamics and function at baseline and during vasoactive challenge (acetylcholine or sodium nitroprusside) were quantified in vivo in pigs by electron-beam computed tomography after a 12-week normal (n=7) or hypercholesterolemic (HC, n=7) diet, RAS (n=6), or concurrent HC and a similar degree of RAS (HC+RAS, n=7). Flash-frozen renal tissue was studied ex vivo. Basal cortical perfusion and single-kidney glomerular filtration rate (GFR) were decreased similarly in the stenotic RAS and HC+RAS kidneys, but tubular fluid reabsorption was markedly impaired only in HC+RAS. Perfusion responses to challenge were similarly blunted in the experimental groups. Stimulated GFR increased in normal, HC, and RAS (38.3±3.6%, 36.4±7.6%, and 60.4±9.3%, respectively, P <0.05), but not in HC+RAS (6.5±15.1%). These functional abnormalities in HC+RAS were accompanied by augmented perivascular, tubulointerstitial, and glomerular fibrosclerosis, inflammation, systemic and tissue oxidative stress, and tubular expression of nuclear factor-&kgr;B and inducible nitric oxide synthase. Conclusions—Early chronic HC+RAS imposes distinct detrimental effects on renal function and structure in vivo and in vitro, evident primarily in the tubular and glomerular compartments. Increased oxidative stress may be involved in the proinflammatory and progrowth changes observed in the stenotic HC+RAS kidney, which might potentially facilitate the clinically observed progression to end-stage renal disease.

Cyclosporine-induced hypertension after transplantation

OBJECTIVE To describe the features and mechanisms of posttransplantation hypertension and suggest appropriate management of the disorder. DESIGN We review our own experience and reports from the literature on hypertension in cyclosporine A (CSA)-treated transplant recipients. RESULTS Soon after immunosuppression with CSA and corticosteroids, hypertension develops in most patients who undergo transplantation. The blood pressure increases, which are usually moderate, occur universally because of increased peripheral vascular resistance. Disturbances in circadian patterns of blood pressure lead to loss of the normal nocturnal decline, a feature that magnifies hypertensive target effects. Changes in blood pressure sometimes are severe and associated with rapidly developing target injury, including intracranial hemorrhage, left ventricular hypertrophy, and microangiopathic hemolysis. The complex mechanisms that underlie this disorder include alterations in vascular reactivity that cause widespread vasoconstriction. Vascular effects in the kidney lead to reduced glomerular filtration and impaired sodium excretion. Many of these changes affect local regulation of vascular tone, including stimulation of endothelin and suppression of vasodilating prostaglandins. Effective therapy includes use of vasodilating agents, often calcium channel blocking drugs. Caution must be exercised to avoid interfering with the disposition of CSA or aggravating adverse effects relative to kidney and electrolyte homeostasis. CONCLUSION Recognition and treatment of CSA-induced hypertension and vascular injury are important elements in managing the transplant recipient.

Low-Dose Angiotensin II Increases Free Isoprostane Levels in Plasma

Chronic intravenous infusion of subpressor doses of angiotensin II causes blood pressure to increase progressively over the course of several days. The mechanisms underlying this response, however, are poorly understood. Because high-dose angiotensin II increases oxidative stress, and some compounds that result from the increased oxidative stress (eg, isoprostanes) produce vasoconstriction and antinatriuresis, we tested the hypothesis that a subpressor dose of angiotensin II also increases oxidative stress, as measured by 8-epi-prostaglandin F(2alpha) (isoprostanes), which may contribute to the slow pressor response to angiotensin II. To test this hypothesis, we infused angiotensin II (10 ng/kg per minute for 28 days via an osmotic pump) into 6 conscious normotensive female pigs (30 to 35 kg). We recorded mean arterial pressure continuously with a telemetry system and measured plasma isoprostanes before starting the angiotensin II infusion (baseline) and again after 28 days with an enzyme immunoassay. Angiotensin II infusion significantly increased mean arterial pressure from 121+/-4 to 153+/-7 mm Hg (P<0. 05) without altering total plasma isoprostane levels (180.0+/-24.3 versus 147.0+/-29.2 pg/mL; P=NS). However, the plasma concentrations of free isoprostanes increased significantly, from 38.3+/-5.8 to 54.7+/-10.4 pg/mL (P<0.05). These results suggest that subpressor doses of angiotensin II increase oxidative stress, as implied by the increased concentration of free isoprostanes, which accompany the elevation in mean arterial pressure elevation. Thus, isoprostane-induced vasoconstriction and antinatriuresis may contribute to the hypertension induced by the slow pressor responses of angiotensin II.

Temporal analysis of signaling pathways activated in a murine model of two-kidney, one-clip hypertension

Unilateral renal artery stenosis (RAS) leads to atrophy of the stenotic kidney and compensatory enlargement of the contralateral kidney. Although the two-kidney, one-clip (2K1C) model has been extensively used to model human RAS, the cellular responses in the stenotic and contralateral kidneys, particularly in the murine model, have received relatively little attention. We studied mice 2, 5, and 11 wk after unilateral RAS. These mice became hypertensive within 1 wk. The contralateral kidney increased in size within 2 wk after surgery. This enlargement was associated with a transient increase in expression of phospho-extracellular signal-regulated kinase (p-ERK), the proliferation markers proliferating cell nuclear antigen and Ki-67, the cell cycle inhibitors p21 and p27, and transforming growth factor-beta, with return to baseline levels by 11 wk. The size of the stenotic kidney was unchanged at 2 wk but progressively decreased between 5 and 11 wk. Unlike the contralateral kidney, which showed minimal histopathological alterations, the stenotic kidney developed progressive interstitial fibrosis, tubular atrophy, and interstitial inflammation. Surprisingly, the stenotic kidney showed a proliferative response, which involved largely tubular epithelial cells. The atrophic kidney had little evidence of apoptosis, despite persistent upregulation of p53; expression of cell cycle regulatory proteins in the stenotic kidney was persistently increased through 11 wk. These studies indicate that in the 2K1C model, the stenotic kidney and contralateral, enlarged kidney exhibit a distinct temporal expression of proteins involved in cell growth, cell survival, apoptosis, inflammation, and fibrosis. Notably, an unexpected proliferative response occurs in the stenotic kidney that undergoes atrophy.

Combination of Hypercholesterolemia and Hypertension Augments Renal Function Abnormalities

Hypercholesterolemia and hypertension are both risk factors for end-stage renal disease. This study was designed to examine whether their coexistence augmented impairment in renal function and redox status. Regional renal hemodynamics and function in response to vasoactive challenges with acetylcholine or sodium nitroprusside were quantified by using electron-beam computed tomography in pigs after 12 weeks of either a normal (n=10) or hypercholesterolemic (n=10) diet, renovascular hypertension (n=7), or combined hypercholesterolemia+hypertension (n=6). The hypercholesterolemic and hypercholesterolemic+hypertensive groups had significantly increased serum cholesterol levels, whereas in the hypertensive and hypercholesterolemic+hypertensive groups, mean arterial pressure was significantly elevated compared with the group fed a normal diet. Basal regional renal perfusion and glomerular filtration rates were similar among the groups. In response to acetylcholine, cortical perfusion increased in normal animals (15.6±4.7%, P =0.002) but not in hypercholesterolemic or hypertensive animals (8.0±7.4% and 8.2±5.9%, respectively;P >0.05). Moreover, in the hypercholesterolemic+hypertensive group, cortical perfusion response was further attenuated (2.5±4.8%, P =0.02) and significantly different from the group fed a normal diet (P <0.05). The response to sodium nitroprusside followed a similar pattern, and the impairment was augmented in the hypercholesterolemic+hypertensive group. The functional abnormalities in hypercholesterolemia or hypertension were associated with a decrease in systemic and/or renal tissue levels of oxygen radical scavengers that was again accentuated in hypercholesterolemia+hypertension. These results demonstrate that concurrent hypercholesterolemia and hypertension have a greater detrimental effect on renal perfusion responses compared with hypercholesterolemia or hypertension alone, associated with a marked pro-oxidant shift in redox status. These effects may potentially augment renal functional impairment and play a role in the initiation and progression of renal injury in hypertension and atherosclerosis.

Deficient production of nitric oxide induces volume-dependent hypertension.

AIM To study the influence of nitric oxide on renal function. DESIGN Nitric oxide synthesis was inhibited and the effects on renal parameters were determined. METHODS Nitric oxide synthesis was progressively blocked by the intravenous administration of increasing doses of NG-nitro-arginine methylester (L-NAME) and then (c)GMP was administered. RESULTS The blockade of nitric oxide synthesis first induced a marked fall in urinary sodium excretion, and later, a sustained increase in mean arterial pressure. These effects were reversed by 8-bromide cGMP. Nitric oxide-dependent cGMP formation was higher in the inner medulla than in any other part of the renal parenchyma, and the inhibition of nitric oxide synthesis significantly decreased both pressure- and volume expansion-induced natriuresis. CONCLUSIONS Both the natriuretic and vasodilator tone maintained by nitric oxide are ultimately due to the production of cGMP. Nitric oxide-induced formation of cGMP appears to be the major factor that links changes in renal medullary circulation to those of sodium excretion. Sufficient inhibition of nitric oxide synthesis to decrease sodium excretion without altering blood pressure induces volume-dependent hypertension because blood pressure is elevated by an increased sodium intake.

Impaired renal vascular endothelial function in vitro in experimental hypercholesterolemia

Hypercholesterolemia (HC) induces alterations in systemic vascular reactivity, which can manifest as an attenuated endothelium-dependent relaxation, partly consequent to an impairment in nitric oxide (NO) activity. To determine whether experimental HC has a similar effect on renal vascular function, renal artery segments obtained from pigs fed a HC (n=5) or normal (n=5) diet were studied in vitro. Endothelium-dependent relaxation was examined using increasing concentrations of acetylcholine (Ach), calcium ionophore A23187, and Ach following pre-incubation with N(G)-monomethyl-L-arginine or L-arginine (L-ARG). The NO-donor diethylamine (DEA) was used to examine smooth muscle relaxation response and cyclic GMP generation in endothelium-denuded vessels. The expression of endothelial NO synthase (eNOS) in the renal arteries was examined using Western blotting. Endothelium-dependent relaxation to Ach was significantly attenuated in the HC group compared to normal (53.3+/-9.1 vs. 98.8+/-3.7%, P<0.005), but normalized after pre-incubation with L-ARG (82.3+/-13.8%, P=0.21). Receptor-independent endothelium-dependent relaxation to A23187 was also significantly blunted in HC (75.2+/-10.5 vs. 115.5+/-4.2%, P<0. 017). Smooth muscle relaxation and cyclic GMP generation in response to DEA were greater in denuded HC vessels, while relaxation of intact vessels to nitroprusside was unaltered. In the HC vessels eNOS was almost undetectable. In conclusion, experimental HC attenuates in vitro endothelium-dependent relaxation of the porcine renal artery, possibly due to low bioavailability of NO. These vascular alterations in HC could play a role in the pathogenesis of renal disease or hypertension, supporting a role for HC as a risk factor for renovascular disease.

Visualization of Three-Dimensional Nephron Structure With Microcomputed Tomography

The three‐dimensional architecture of nephrons in situ and their interrelationship with other nephrons are difficult to visualize by microscopic methods. The present study uses microcomputed X‐ray tomography (micro‐CT) to visualize intact nephrons in situ. Rat kidneys were perfusion‐fixed with buffered formalin and their vasculature was subsequently perfused with radiopaque silicone. Cortical tissue was stained en bloc with osmium tetroxide, embedded in plastic, scanned, and reconstructed at voxel resolutions of 6, 2, and 1 μm. At 6 μm resolution, large blood vessels and glomeruli could be visualized but nephrons and their lumens were small and difficult to visualize. Optimal images were obtained using a synchrotron radiation source at 2 μm resolution where nephron components could be identified, correlated with histological sections, and traced. Proximal tubules had large diameters and opaque walls, whereas distal tubules, connecting tubules, and collecting ducts had smaller diameters and less opaque walls. Blood vessels could be distinguished from nephrons by the luminal presence of radiopaque silicone. Proximal tubules were three times longer than distal tubules. Proximal and distal tubules were tightly coiled in the outer cortex but were loosely coiled in the middle and inner cortex. The connecting tubules had the narrowest diameters of the tubules and converged to form arcades that paralleled the radial vessels as they extended to the outer cortex. These results illustrate a potential use of micro‐CT to obtain three‐dimensional information about nephron architecture and nephron interrelationships, which could be useful in evaluating experimental tubular hypertrophy, atrophy, and necrosis. Anat Rec, 2007.

Role of Endothelin in the Pathogenesis of Hypertension

In 1985, investigators characterized a potent vasoconstrictor of endothelial origin called endothelin (ET). Subsequently, 3 peptides were recognized that had a comparable molecular structure but different receptors that mediate potent vasoconstrictive and mild vasodilative effects. The renal effects are characterized by natriuresis despite renal vasoconstriction. This effect, along with the stimulation of ET by high sodium intake, suggests that ET may be responsible for maintaining sodium balance when the renin-angiotensin system is depressed. Endothelin is activated in desoxycorticosterone acetate salt hypertension models and salt-sensitive hypertension. However, ET involvement with spontaneous hypertension models and renovascular hypertension in rats appears minimal. In humans, the role of ET appears similar to that in experimental animals; in both, ET regulates salt metabolism. Salt-sensitive patients exhibit a blunted renal ET-1 response during sodium load. The role of ET in humans has been investigated using nonspecific ET receptor blockers that inhibit the vasoconstrictive and vasodilative components of ET. However, the effects of ET blockade should be investigated with ET subtype A receptor blockers that mediate vasoconstriction alone. Effects of ET blockade also should be evaluated with respect to stimulation of oxidative stress and tissue damage, important mechanisms responsible for tissue fibrosis. This review offers the clinician a balanced view on the hypertensive mechanisms involved with activation of ET and associated clinical implications.