María R. Trolliet

Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

Oxidative Stress and Renal Dysfunction in Salt-Sensitive Hypertension

Hypertension is a risk factor for the development of end-stage renal disease. The mechanisms underlying hypertensive nephropathy are poorly understood. There is evidence, however, that in hypertension there is an accumulation of partially reduced oxygen and its derivatives, known collectively as reactive oxygen species, which may contribute to progressive renal dysfunction. In the present study, we assess the contribution of oxidative stress in the development of salt-dependent hypertensive nephrosclerosis. Going beyond previous end point studies, which inferred renal function either indirectly or only qualitatively, we have determined oxidative stress concurrently with direct and quantitative measurements of renal function (via inulin and p-aminohippuric acid clearances). Moreover, in this time-dependent study, the measurements have been taken under low- as well as high-salt diets. As was expected from previous studies, in the Dahl salt-sensitive rat, a high-salt diet (8% NaCl) resulted in the development of hypertension, in a decreased glomerular filtration rate, and in a decreased renal plasma flow as compared with the normotensive control, the Dahl salt-resistant rat. In addition, however, we found clear evidence for the accumulation of reactive oxygen species in renal tissue homogenates of Dahl salt-sensitive rats on the high-salt diet. Our time-dependent protocol also indicated that renal oxidative stress follows, in time, the development of hypertension. We also found that after 2 weeks of increased salt loading, Dahl salt-sensitive rats excreted less cyclic guanosine monophosphate and NOx than Dahl salt-resistant rats on the same diet. It is known that urinary cyclic guanosine monophosphate and NOx represent the activity and stable derivatives of renal NO·, respectively, and that they closely correlate with renal vascular resistance. Therefore, our results suggest that, in the Dahl salt-sensitive rat, increased oxidative stress is associated with salt-dependent hypertensive nephrosclerosis and that decreased NO· bioavailability may represent a common factor responsible for the vascular and glomerular dysfunction.

Salt-induced hypertension in Dahl salt-resistant and salt-sensitive rats with NOS II inhibition.

Although recent evidence suggests that reduced nitric oxide (NO) production may be involved in salt-induced hypertension, the specific NO synthase (NOS) responsible for the conveyance of salt sensitivity remains unknown. To determine the role of inducible NOS (NOS II) in salt-induced hypertension, we treated Dahl salt-resistant (DR) rats with the selective NOS II inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) for 12 days. Tail-cuff systolic blood pressures rose 29 +/- 6 and 42 +/- 8 mmHg in DR rats given 150 and 300 nmol AMT/h, respectively (P < 0.01, 2-way ANOVA) after 7 days of 8% NaCl diet. We observed similar results with two other potent selective NOS II inhibitors, S-ethylisourea (EIT) and N-[3-(aminomethyl)benzyl]acetamidine hydrochloride (1400W). Additionally, AMT effects were independent of alterations in endothelial function as assessed by diameter change of mesenteric arterioles in response to methacholine using videomicroscopy. We, therefore, conclude from these data that NOS II is important in salt-induced hypertension.

Prevention of hypertension and renal dysfunction in Dahl rats by α-tocopherol

&NA; Although hypertension is a risk factor for the development of end‐stage renal disease, not all hypertensive patients progress to develop renal dysfunction. The mechanisms underlying hypertensive nephropathy are poorly understood. The authors have recently shown that the development of hypertension and renal dysfunction is accompanied by an accumulation of partially reduced oxygen and its derivatives, known collectively as reactive oxygen species. In the present study, the effect of a lipid‐soluble antioxidant on the development of salt‐dependent hypertensive nephropathy was evaluated in the Dahl rat. It was found that a high‐salt diet (8% NaCl) led to the development of hypertension, increased renal oxidative stress (superoxide production and 8‐epi‐prostaglandin F2&agr;), and decreased glomerular filtration rate and renal plasma flow in the Dahl salt‐sensitive (DSS) rat, and that these adverse effects of salt were prevented by supplementing the high‐salt diet with 1000 U/kg chow of &agr;‐tocopherol. It is well known that urinary cyclic guanosine monophosphate (cGMP) levels are lower in hypertensive DSS rats than in Dahl salt‐resistant (DSR) rats on a high‐salt diet. Most surprisingly, when supplemented with &agr;‐tocopherol, DSS rats on an 8% NaCl diet were able to excrete as much cGMP as DSR rats. Taken together, these findings suggest that, in the DSS rat, salt‐dependent hypertensive nephropathy and decreased nitric oxide bioavailability are associated with increased oxidative stress, and that antioxidants can preclude these adverse effects of salt feeding, and consequently, prevent salt‐dependent hypertension and nephropathy.

Nitric Oxide and Hypertension

Although hypertension has been studied extensively for decades, its etiology remains an enigma. An increase in systemic intravascular pressure or systemic hypertension can result from changes in two basic hemodynamic parameters: (1) a decrease in intravascular size as occurs during vasoconstriction, or (2) an increase in intravascular volume. The latter may occur as a result of either increased salt and water retention or cardiac output. Nitric oxide (NO·) has emerged as a critically important agent in the regulation of vascular tone, renal fluid and volume regulation, and cardiac function. Consequently, an alteration in NO· action on vessel tone, volume regulation, or heart function can lead to hypertension (Fig. 1).