Increased production of superoxide anion contributes to dysfunction of the arteriovenous fistula.

Vascular access dysfunction causes morbidity in hemodialysis patients. This study examined the generation and pathobiological significance of superoxide anion in a rat femoral arteriovenous fistula (AVF). One week after AVF creation, there was increased production of superoxide anion accompanied by decreased total superoxide dismutase (SOD) and Cu/Zn SOD activities and induction of the redox-sensitive gene heme oxygenase-1. Immunohistochemical studies of nitrotyrosine formation demonstrated that peroxynitrite, a product of superoxide anion and nitric oxide, was present in increased amounts in endothelial and smooth muscle cells in the AVF. Because uncoupled NOS isoforms generate superoxide anion, and NOS coupling requires tetrahydrobiopterin (BH(4)) as a cofactor, we assessed NOS uncoupling by determining the ratio of BH(4) to dihydrobiopterin (BH(2)); the BH(4)-to-BH(2) ratio was markedly attenuated in the AVF. Because Src is a vasculopathic signaling species upstream and downstream of superoxide anion, such expression was evaluated; expression of Src and phosphorylated Src was both markedly increased in the AVF. Expression of NADPH oxidase (NOX) 1, NOX2, NOX4, cyclooxygenase (COX) 1, COX2, p47(phox), and p67(phox) was all unchanged, as assessed by Western analyses, thereby suggesting that these proteins may not be involved in increased production of superoxide anion. Finally, administration of tempol, a superoxide anion scavenger, decreased neointima formation in the juxta-anastomotic venous segment and improved AVF blood flow. We conclude that the AVF exhibits increased superoxide anion generation that may reflect the combined effects of decreased scavenging by SOD and increased generation by uncoupled NOS, and that enhanced superoxide anion production promotes juxta-anastomotic stenosis and impairs AVF function.

Regional and systemic hemodynamic responses following the creation of a murine arteriovenous fistula.

The study of hemodynamic alterations following the creation of an arteriovenous fistula (AVF) is relevant to vascular adaptive responses and hemodialysis access dysfunction. This study examined such alterations in a murine AVF created by anastomosing the carotid artery to the jugular vein. AVF blood flow was markedly increased due to reduced AVF vascular resistance. Despite such markedly increased basal blood flow, AVF blood flow further increased in response to acetylcholine. This AVF model exhibited increased cardiac output and decreased systemic vascular resistance; the kidney, in contrast, exhibited decreased blood flow and increased vascular resistance. Augmentation in AVF blood flow was attended by increased arterial heme oxygenase-1 (HO-1) mRNA and protein expression, the latter localized to smooth muscle cells of the AVF artery; AVF blood flow was substantially reduced in HO-1(-/-) mice compared with HO-1(+/+) mice. Finally, in a murine model of a representative disease known to exhibit impaired hemodynamic responses (sickle cell disease), the creation of an AVF was attended by decreased AVF flow and impaired AVF function. We conclude that this AVF model exhibits markedly increased AVF blood flow, a vasodilatory reserve capacity, increased cardiac output, decreased renal blood flow, and a dependency on intact hemodynamic responses, in general, and HO-1 expression, in particular, in achieving and maintaining AVF blood flow. We suggest that these findings support the utility of this model in investigating the basis for and the consequences of hemodynamic stress, including shear stress, and the pathobiology of hemodialysis AVF dysfunction.

Characterization of a model of an arteriovenous fistula in the rat: the effect of L-NAME.

Vascular access dysfunction contributes to the mortality of patients undergoing chronic hemodialysis. The present study analyzed the changes that evolve in a femoral arteriovenous fistula in the rat. The venous segment of this model exhibited, at 1 week, activation of pro-inflammatory transcription factors and up-regulation of pro-inflammatory, proliferative, procoagulant, and profibrotic genes; and at 4 weeks, the venous segment displayed neointimal hyperplasia, smooth muscle proliferation, and thrombus formation. These changes were accompanied by endothelial (e) nitric oxide synthase (NOS) and inducible (i) NOS up-regulation. The administration of NG-nitro-L-arginine methyl ester, an inhibitor of NOS activity, increased venous neointimal hyperplasia and pro-inflammatory gene expression (monocyte chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant-1), increased systolic blood pressure, and decreased blood flow through the fistula. In another hypertensive model, the rat subtotal nephrectomy model, venous neointimal hyperplasia in the arteriovenous fistula was also exacerbated. We conclude that this arteriovenous fistula model recapitulates the salient features observed in dysfunctional, hemodialysis arteriovenous fistulas, and that venous neointimal hyperplasia is exacerbated when this model is superimposed in two different models of systemic hypertension. Since the uremic milieu contains increased amounts of asymmetric dimethylarginine, we speculate that such accumulation of this endogenous inhibitor of NOS, by virtue of its pressor or nitric oxide-depleting effects, or a combination thereof, may contribute to the limited longevity of arteriovenous fistulas used for hemodialysis.

Quantitative mRNA analysis of serotonin 5-HT4 and adrenergic ß2 receptors in the mouse embryonic telencephalon.

The adrenergic ß2 receptor (ß2-AR) gene is embedded (nested) within the serotonin 5-HT4 receptor (5-HT4-R) gene and these two receptors can interact at the transcriptional and post-transcriptional levels. The mouse 5-HT4-R gene contains a number of exons and codes at least four mRNA splice variants (5-HT(4(a))-R, 5-HT(4(b))-R, 5-HT(4(e))-R, 5-HT(4(f))-R), whereas the ß2-AR gene is intronless. Since 5-HT4-Rs and ß2-ARs can form homodimers and heterodimers and they increase intracellular cAMP levels, these receptors may be important for integrating serotonergic and noradrenergic signals at the single-neuron level. Both 5-HT4-R and ß2-AR have been implicated in autism spectrum disorders, depression, and Alzheimer's disease. In the fetal brain, these receptors may mediate the effects of stress on neurodevelopmental processes. We used quantitative reverse-transcription PCR (qRT-PCR) to investigate the developmental expression of 5-HT4-R and ß2-AR in the mouse telencephalon at embryonic days (E) 13-18. At E13-E14, the mRNA levels of all 5-HT4-R splice variants were very low, but by E17-E18 they increased 7-fold (5- HT(4(a))-R), 5-fold (5-HT(4(b))-R), 9-fold (5-HT(4(e))-R), and 11-fold (5-HT(4(f))-R). The expression of 5-HT(4(a))-R and 5-HT(4(b))-R was rapidly upregulated between E14 and E15, at the time when the thalamocortical projections arrive in the telencephalon. This pattern was not observed in the expression of 5-HT(4(e))-R and 5-HT(4(f))-R, the mRNA levels of which showed a steady, gradual increase from E13 to E18. The ß2-AR mRNA levels were relatively high throughout the studied period of development and increased only by 70% from E13-E14 to E17-E18. These findings suggest that 5-HT4-R splice variants and ß2-ARs are differentially regulated in the embryonic telencephalon and that their relative amounts may carry developmentally important information.

Heme oxygenase activity as a determinant of the renal hemodynamic response to low-dose ANG II.

ANG II causes renal injury through hemodynamic and other effects, and pressor doses of ANG II induce heme oxygenase-1 (HO-1) as a protective response. The present studies examined the hemodynamic effects of more clinically relevant, lower doses of ANG II and the role of HO activity in influencing these effects. Under euvolemic conditions, ANG II increased arterial pressure and renal vascular resistance. ANG II did not induce oxidative stress, inflammation/injury-related gene expression, or proteinuria and did not alter extrarenal vascular reactivity. At these doses, ANG II failed to increase HO-1 or HO-2 mRNA expression or HO activity. Inhibiting HO activity in ANG II-treated rats by tin mesoporphyrin further increased renal vascular resistances, decreased renal blood flow, and blunted the rise in arterial pressure without inducing oxidative stress or altering expression of selected vasoactive/injury/inflammation-related genes; tin mesoporphyrin did not alter vasorelaxation of mesenteric resistor vessels. We conclude that in this model renal vasoconstriction occurs without the recognized adverse effects of ANG II on glomerular filtration rate, renal blood flow, oxidative stress, vascular reactivity, proteinuria, and injury-related gene expression; renal HO activity is essential in preserving perfusion of the ANG II-exposed kidney. These findings represent an uncommon example wherein function of a stressed organ (by ANG II), but not that of the unstressed organ, requires intact renal HO activity, even when the imposed stress neither induces HO-1 nor HO activity. These findings may be germane to conditions attended by heightened ANG II levels, ineffective renal perfusion, and susceptibility to acute kidney injury.

Transient expression of serotonin 5-HT4 receptors in the mouse developing thalamocortical projections.

The serotonin 5-HT(4) receptor (5-HT(4)-R) is an unusually complex G-protein coupled receptor that is likely to play important roles in brain development and that may underlie the comorbidity of central and peripheral abnormalities in some developmental disorders. We studied the expression of 5-HT(4)-Rs in the developing mouse forebrain at embryonic days 13, 15, 17, and at postnatal days 3 and 14 by using immunohistochemistry, tract tracing, and quantitative RT-PCR. The developing thalamocortical projections transiently expressed 5-HT(4)-Rs in the embryonic brain and the 5-HT(4)-R expression in the forebrain changed from axonal to somatic around birth. From embryonic days 13-17, the forebrain mRNA levels of the 5-HT(4(a))-R and 5-HT(4(b))-R splice variants increased nine- and fivefold, respectively, whereas the levels of the 5-HT(4(e))-R and 5-HT(4(f))-R variants remained relatively low throughout the studied period of embryonic development. These results suggest that during development 5-HT(4)-R expression undergoes a dynamic regulation and that this regulation may be important for the normal development of sensory and limbic processing.