NMR-Based Metabolomics of Rat Hippocampus, Serum, and Urine in Two Models of Autism.

Autism spectrum disorders (ASDs) are increasingly diagnosed as developmental disabilities of unclear etiology related to genetic, epigenetic, or environmental factors. The diagnosis of ASD in children is based on the recognition of typical behavioral symptoms, while no reliable biomarkers are available. Rats in whom ASD-like symptoms are due to maternal administration of the teratogenic drugs valproate or thalidomide on critical day 11 of pregnancy are widely used models in autism research. The present studies, aimed at detecting changes in the levels of hydrophilic and hydrophobic metabolites, were carried out on 1-month-old rats belonging to the abovementioned two ASD models and on a control group. Analysis of both hydrophilic and hydrophobic metabolite levels gives a broader view of possible mechanisms involved in the pathogenesis of autism. Hippocampal proton magnetic resonance (MRS) spectroscopy and ex vivo nuclear magnetic resonance (NMR) analysis of serum and urine samples were used. The results were analyzed using advanced statistical tests. Both the results of our present MRS studies of the hippocampus and of the NMR studies of body fluids in both ASD models, particularly from the THAL model, appeared to be consistent with previously published NMR results of hippocampal homogenates and data from the literature on autistic children. We detected symptoms of disturbances in neurotransmitter metabolism, energy deficit, and oxidative stress, as well as intestinal malfunction, which shed light on the pathogenesis of ASD and could be used for diagnostic purposes. These results confirm the usefulness of the noninvasive techniques used in ASD studies.

Arginine and tetrahydrobiopterin supplementation in rats with salt-induced blood pressure increase: minor hypotensive effect but improvement of renal haemodynamics.

High salt (HS) intake can lead to hypertension, probably the result of the predominance of vasoconstrictor reactive oxygen species over vasodilator nitric oxide (NO). We aimed to examine if the supposed NO deficiency and the resultant blood pressure increase could be corrected by supplementation of L-arginine, the substrate, and tetrahydrobiopterin (BH4), a co-factor of NO synthases. Wistar rats without known genetic background of salt sensitivity were exposed to HS diet (4%Na) for 10 or 26 days, without or with supplementation with oral L-arginine, 1.4 mg/kg b.w. daily, alone or together with intraperitoneal BH4, 10 mg/kg daily. Systolic blood pressure (SBP, tail-cuff method) was measured repeatedly and found to increase ~40 mmHg after 26 days; L-arginine and BH4 did not significantly attenuate this increase. At the end of chronic studies, in anaesthetized rats the diet- and treatment-induced changes in renal haemodynamics were assessed. HS diet selectively decreased (-30%, P < 0.03) the inner medullary blood flow (IMBF, laser-Doppler flux) without changing total or cortical renal perfusion. Arginine supplementation tended to raise all renal circulatory parameters, and distinctly increased IMBF, to 61% above the HS diet level (P < 0.05). In conclusion, unlike in confirmed genetically determined salt-dependent hypertension, L-arginine and BH4 supplementation failed to attenuate the SBP increase observed after exposure to HS diet. On the other hand, arginine increased total and regional renal perfusion, especially IMBF. This suggests that the delivery of arginine increased intrarenal NO synthesis, an action of renoprotective potential which presumably countered the harmful influence of the local tissue oxidative stress.

High salt intake increases blood pressure in normal rats: putative role of 20-HETE and no evidence on changes in renal vascular reactivity.

UNLABELLED: Background/Aims . High salt (HS) intake may elevate blood pressure (BP), also in animals without genetic salt sensitivity. The development of salt-dependent hypertension could be mediated by endogenous vasoactive agents; here we examined the role of vasodilator epoxyeicosatrienoic acids (EETs) and vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE). METHODS: In conscious Wistar rats on HS diet systolic BP (SBP) was examined after chronic elevation of EETs using 4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (c-AUCB), a blocker of soluble epoxide hydrolase, or after inhibition of 20-HETE with 1-aminobenzotriazole (ABT). Thereafter, in acute experiments the responses of renal artery blood flow (Transonic probe) and renal regional perfusion (laser-Doppler) to intrarenal acetylcholine (ACh) or norepinephrine were determined. RESULTS: HS diet increased urinary 20-HETE excretion. The SBP increase was not reduced by c-AUCB but prevented by ABT until day 5 of HS exposure. Renal vasomotor responses to ACh or norepinephrine were similar on standard and HS diet. ABT but not c-AUCB abolished the responses to ACh. Conclusions . 20-HETE seems to mediate the early-phase HS diet-induced BP increase while EETs are not engaged in the process. Since HS exposure did not alter renal vasodilator responses to Ach, endothelial dysfunction is not a critical factor in the mechanism of salt-induced blood pressure elevation.

Interaction of nitric oxide and the cytochrome P-450 system on blood pressure and renal function in the rat: dependence on sodium intake.

AIM: Interaction was examined of nitric oxide (NO) and cytochrome P-450 (CYP-450)-dependent arachidonic acid derivatives, 20-HETE and EETs, in control of arterial pressure (MABP) and renal function. Modification of this interaction by changing sodium intake was also studied. METHODS: On low, standard or high Na diet (LS, STD and HS rats respectively) effects of sequential blockade of NO synthases (NOS) and CYP-450 enzyme activity on MABP, renal blood flow (RBF, Transonic probe), renal medullary perfusion (MBF, laser-Doppler technique), medullary tissue NO (selective electrode) and renal excretion were examined in anaesthetized rats. All NOS were blocked with N(ϖ) -nitro-l-arginine methyl ester (l-NAME), the neuronal NOS with S-methyl-l-thiocitrulline (SMTC), and CYP-450 with 1-aminobenzotriazole (ABT). RESULTS: In each diet group the baseline MABP was highest in rats pre-treated with l-NAME. CYP-450 inhibition significantly decreased MABP only in LS (-9%) and HS rats (-22%) pre-treated with l-NAME. This MABP decrease correlated directly with the dietary sodium content (r = 0.644, P < 0.001). CYP-450 inhibition decreased RBF in LS and HS rats (not in HS pre-treated with l-NAME). Acute exclusion of CYP-450 significantly increased MBF only in STD, SMTC pre-treated rats; in HS group it significantly increased medullary tissue NO by about 1.0 nA. The post-ABT changes in renal excretion occurred in LS and HS rats, irrespective of the status of NO synthesis. CONCLUSIONS: Both NO- and CYP-450-dependent agents contribute to blood pressure and kidney function control, however, the role of 20-HETE and EETs becomes crucial only under conditions of high sodium intake or after NOS inhibition.