Human Physiology. Workbook

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Alteraciones vasculares en la insuficiencia renal crónica. Papel del endotelio.

RESUMEN El síndrome urémico resulta de la disminución marcada de la función renal. Este estado mantenido conduce a la insuficiencia renal crónica, que se define como la destrucción progresiva e irreversible de las nefronas. En la insuficiencia renal crónica se dan cita diferentes factores de riesgo cardiovascular, la propia uremia supone un factor de riesgo. La causa principal de morbi-mortalidad en estos pacientes deriva de alteraciones en el sistema cardiovascular. El tratamiento mediante hemodiálisis ha supuesto la prolongación de la vida para estos enfermos, pero a la vez, ha acarreado problemas relacionados con alteraciones cardiovasculares. En la presente tesis se estudian las vías de síntesis de factores endoteliales que pueden estar alteradas por la enfermedad y que pueden ser las responsables, al menos en parte, de las alteraciones vasculares que padecen estos enfermos. Nos centramos en la participación de los factores endoteliales relajantes: prostaciclina, NO y EDHF, observando que la uremia produce una disminución de su síntesis o liberación en respuesta a estímulos vasodilatadores y vasoconstrictores. Las alteraciones vasculares que puede ocasionar el tratamiento con hemodiálisis, ha puesto de manifiesto que aunque disminuye la participación de NO en la respuesta vascular, aumenta la de EDHF. Estos resultados son importantes, pues suponen la aparición de un mecanismo compensatorio, capaz de mejorar la función endotelial. El estudio de la expresión de genes implicados de manera directa o indirecta en la reactividad vascular sugiere que la enfermedad podría tener su origen en un fallo metabólico, puesto que nuestros resultados muestran una menor expresión génica de DDAH-1. Esta enzima es la encargada de metabolizar las toxinas que se acumulan en la uremia y que son inhibidores enzimáticos, causando gran parte de la disfunción vascular y alteraciones neurológicas que aparecen en esta enfermedad. Las modificaciones enzimáticas que hemos observado en la insuficiencia renal crónica podrían ser dianas terapéuticas para mejorar la calidad de vida en estos enfermos. __________________________________________________________________________________________________Uremia is an established risk factor for cardiovascular and cerebrovascular accidents. Hemodialysis improves the lifes quality of these patients, but this treatment also produces vascular alterations. We examined the relaxant and contractile response in forearm veins from patients with chronic renal failure before hemodialysis, on chronic hemodialysis and in veins obtained from donors (controls). We study the role of vasodilator prostaglandins (PGI2), nitric oxide (NO) and endothelium derived hyperpolarizing factor (EDHF). The contribution of these factores is diminished in chronic renal failure. This effect might contribute to, or even cause, some of the vascular features of this condition. Hemodialysis improves the endothelial function because of an augmented participation of EDHF. The study of gene expression showed a metabolic failure in the enzyme that degrades the uremic toxins. The low expression of this enzyme, dimethylarginine dimethylamino hydrolase (DDAH), could explain some vascular features and could be a therapeuthic strategy for improving lifes quality of these patients

Contractile responses of human thyroid arteries to vasopressin

Aims: In the present study we investigated the intervention of nitric oxide and prostacyclin in the responses to vasopressin of isolated thyroid arteries obtained from multi-organ donors. Main methods: Paired artery rings from glandular branches of the superior thyroid artery, one normal and the other deendothelised, were mounted in organ baths for isometric recording of tension. Concentration-response curves to vasopressin were determined in the absence and in the presence of either the vasopressin V1 receptor antagonist d(CH2)5Tyr(Me)AVP (10−8 M), the nitric oxide synthase inhibitor NG-monomethyl-Larginine (L-NMMA, 10−4 M), or the inhibitor of prostaglandins indomethacin (10−6 M). Key findings: In artery rings under resting tension, vasopressin produced concentration-dependent, endotheliumindependent contractions. The vasopressin V1 receptor antagonist d(CH2)5Tyr(Me)AVP (10−8 M) displaced the control curve to vasopressin 19-fold to the right in a parallelmanner. The contractile response to vasopressinwas unaffected by L-NMMA or by indomethacin. Significance: Vasopressin causes constriction of human thyroid arteries by stimulation of V1 vasopressin receptors located on smooth muscle cells. These effects are not linked to the presence of an intact endothelium or to the release of nitric oxide or prostaglandins. The constriction of thyroid arteries may be particularly relevant in certain pathophysiological circumstances in which vasopressin is released in amounts that could interfere with the blood supply to the thyroid gland

Accumulation of symetric dimethylarginine in hepatorenal syndrome.

In patients with cirrhosis, nitric oxide (NO), asymmetric dimethylarginine (ADMA), and possibly symmetric dimethylarginine (SDMA) have been linked to the severity of the disease. We investigated whether plasma levels of dimethylarginines and NO are elevated in patients with hepatorenal syndrome (HRS), compared with patients with cirrhosis without renal failure (no- HRS). Plasma levels of NO, ADMA, SDMA, and L-arginine were measured in 11 patients with HRS, seven patients with no-HRS, and six healthy volunteers. SDMA concentration in HRS was higher than in no-HRS and healthy subjects (1.47 6 0.25 vs. 0.38 6 0.06 and 0.29 6 0.04 lM, respectively; P , 0.05). ADMA and NOx concentrations were higher in HRS and no-HRS patients than in healthy subjects (ADMA, 1.20 6 0.26, 1.11 6 0.1, and 0.53 6 0.06 lM, respectively; P , 0.05; NOx, 94 6 9.1, 95.5 6 9.54, and 37.67 6 4.62 lM, respectively; P , 0.05). In patients with HRS there was a positive correlation between serum creatinine and plasma SDMA (r2¼0.765, P , 0.001) but not between serum creatinine and ADMA or NOx. The results suggest that renal dysfunction is a main determinant of elevated SDMA concentration in HRS. Accumulation of ADMA as a result of impaired hepatic removal may be the causative factor initiating renal vasoconstriction and SDMA retention in the kidney. Exp Biol Med 231:7075, 200

Tyrosine Phosphorylation Modulates the Vascular Responses of Mesenteric Arteries from Human Colorectal Tumors

The aim of this study was to analyze whether tyrosine phosphorylation in tumoral arteries may modulate their vascular response. To do this, mesenteric arteries supplying blood flow to colorectal tumors or to normal intestine were obtained during surgery and prepared for isometric tension recording in an organ bath. Increasing tyrosine phosphorylation with the phosphatase inhibitor, sodium orthovanadate produced arterial contraction which was lower in tumoral than in control arteries, whereas it reduced the contraction to noradrenaline in tumoral but not in control arteries and reduced the relaxation to bradykinin in control but not in tumoral arteries. Protein expression of VEGF-A and of the VEGF receptor FLT1 was similar in control and tumoral arteries, but expression of the VEGF receptor KDR was increased in tumoral compared with control arteries. This suggests that tyrosine phosphorylation may produce inhibition of the contraction in tumoral mesenteric arteries, which may increase blood flow to the tumor when tyrosine phosphorylation is increased by stimulation of VEGF receptors

Neuronal effects of Sugammadex in combination with Rocuronium or Vecuronium.

Rocuronium (ROC) and Vecuronium (VEC) are the most currently used steroidal non-depolarizing neuromuscular blocking (MNB) agents. Sugammadex (SUG) rapidly reverses steroidal NMB agents after anaesthesia. The present study was conducted in order to evaluate neuronal effects of SUG alone and in combination with both ROC and VEC. Using MTT, CASP-3 activity and Western-blot we determined the toxicity of SUG, ROC or VEC in neurons in primary culture. SUG induces apoptosis/necrosis in neurons in primary culture and increases cytochrome C (CytC), apoptosis-inducing factor (AIF), Smac/Diablo and Caspase 3 (CASP-3) protein expression. Our results also demonstrated that both ROC and VEC prevent these SUG effects. The protective role of both ROC and VEC could be explained by the fact that SUG encapsulates NMB drugs. In BBB impaired conditions it would be desirable to control SUG doses to prevent the excess of free SUG in plasma that may induce neuronal damage. A balance between SUG, ROC or VEC would be necessary to prevent the risk of cell damag

Effects of asymmetric dimethylarginine on renal arteries in portal hypertension and cirrhosis

AIM. To evaluate the effects of asymmetric dimethylarginine (ADMA) in renal arteries from portal hypertensive and cirrhotic rats. METHODS. Rat renal arteries from Sham (n = 15), pre-hepatic portal hypertension (PPVL; n = 15) and bile duct ligation and excision-induced cirrhosis (BDL; n = 15) were precontracted with norepinephrine, and additional contractions were induced with ADMA (10-6-10-3 mol/L), an endogenous inhibitor of nitric oxide (NO) synthase. Concentration-response curves to acetylcholine (1 × 10-9-3 × 10-6 mol/L) were determined in precontracted renal artery segments with norepinephrine in the absence and in the presence of ADMA. Kidneys were collected to determine the protein expression and activity of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes ADMA. RESULTS. In renal arteries precontracted with norepinephrine, ADMA caused endothelium-dependent contractions. The pD2 values to ADMA were similar in the Sham and PPVL groups (4.20 ± 0.08 and 4.11 ± 0.09, P > 0.05, respectively), but were lower than those of the BDL group (4.79 ± 0.16, P < 0.05). Acetylcholine-induced endothelium-dependent relaxation that did not differ, in terms of pD2 and maximal relaxation, among the 3 groups studied. Treatment with ADMA (3 × 10-4 mol/L) inhibited acetylcholine-induced relaxation in the 3 groups, but the inhibition was higher (P < 0.05) in the BDL group compared with that for the Sham and PPVL groups. The mRNA and protein expression of DDAH-1 were similar in kidneys from the three groups. Conversely, DDAH-2 expression was increased (P < 0.05) in PPVL and further enhanced (P < 0.05) in the BDL group. However, renal DDAH activity was significantly decreased in the BDL group. CONCLUSION. Cirrhosis increased the inhibitory effect of ADMA on basal- and induced-release of NO in renal arteries, and decreased DDAH activity in the kidney

Sugammadex, a neuromuscular blockade reversal agent, causes neuronal apoptosis in primary cultures.

Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (< 3% in rats), and thus no relevant central nervous toxicity is expected. However the blood brain barrier permeability can be altered under different conditions (i.e. neurodegenerative diseases, trauma, ischemia, infections, or immature nervous system). Using MTT, confocal microscopy, caspase-3 activity, cholesterol quantification and Western-blot we determine toxicity of Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria- dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types

Nitric oxide mediates abnormal responsiveness of thyroid arteries in methimazole-treated patients

Objective: We studied the intervention of nitric oxide (NO), prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor (EDHF) in mediating responses to acetylcholine in thyroid arteries from euthyroid (E) and methimazole-treated (MT) patients. Design and methods: Branches of the superior thyroid artery were obtained from 19 E patients and 17 MT patients (euthyroid at the time of surgery) undergoing total thyroidectomy or hemithyroidectomy. Artery rings were suspended in organ baths for isometric recording of tension. Results and conclusions: Acetylcholine caused endothelium-dependent relaxation of greater magnitude in arteries from MT patients (pD2 7.68±0.19 in E and 8.17±0.26 in MT, P<0.05). The relaxation was unaffected by indomethacin and partially reduced by the NO synthase inhibitor L-NMMA. This reduction was higher in arteries from MT patients (50±6 %) as compared to E patients (36±6 %) (P<0.05). Inhibition of K+ channels using apamin combined with charybdotoxin or high K+ solution abolished the relaxation resistant to L-NMMA and indomethacin. The maximal contractions to noradrenaline (in percentage of the response to 100 mM KCl) were lower in MT than in E patients (57±10 and 96±8, respectively, P<0.05). The hyporesponsiveness to noradrenaline in arteries from MT patients was corrected by L-NMMA. The results indicate: (1) thyroid arteries from MT patients show increased relaxation to acethylcholine and decreased contraction to noradrenaline due to overproduction of NO; (2) EDHF plays a prominent role in acetylcholine-induced relaxation through activation of Ca2+-activated K+ channels; (3) the abnormal endothelium-dependent responses in arteries from MT patients are not corrected by medical treatment

PPARγ as an indicator of vascular function in an experimental model of metabolic syndrome in rabbits

Background and aims: Underlying mechanisms associated with vascular dysfunction in metabolic syndrome (MetS) remain unclear and can even vary from one vascular bed to another. Methods: In this study, MetS was induced by a high-fat, high-sucrose diet, and after 28 weeks, aorta and renal arteries were removed and used for isometric recording of tension in organ baths, protein expression by Western blot, and histological analysis to assess the presence of atherosclerosis. Results: MetS induced a mild hypertension, pre-diabetes, central obesity and dyslipidaemia. Our results indicated that MetS did not change the contractile response in either the aorta or renal artery. Conversely, vasodilation was affected in both arteries in a different way. The aorta from MetS showed vascular dysfunction, including lower response to acetylcholine and sodium nitroprusside, while the renal artery from MetS presented a preserved relaxation to acetylcholine and an increased sensitivity to sodium nitroprusside. We did not find vascular oxidative stress in the aorta from MetS, but we found a significant decrease in PPARγ, phospho-Akt (p-Akt) and phospho-eNOS (p-eNOS) protein expression. On the other hand, we found oxidative stress in the renal artery from MetS, and PPARγ, Akt and p-Akt were overexpressed. No evidence of atherosclerosis was found in arteries from MetS. Conclusions: MetS affects vascular function differently depending on the vessel. In the aorta, it decreases both the vasodilation and the expression of the PPARγ/Akt/eNOS pathway, while in the renal artery, it increases the expression of PPARγ/Akt signalling pathway without decreasing the vasodilation