In liver cirrhosis, elevated levels of NO and ROS (reactive oxygen species) might greatly favour the generation of peroxynitrite. Peroxynitrite is a highly reactive oxidant and it can potentially alter the vascular reactivity and the function of different organs. In the present study, we evaluated whether peroxynitrite levels are related to the progression of renal vascular and excretory dysfunction during experimental cirrhosis induced by chronic BDL (bile-duct ligation) in rats. Experiments were performed at 7, 15 and 21 days after BDL in rats and in rats 21 days post-BDL chronically treated with L -NAME ( N G -nitro- L -arginine methyl ester). Sodium balance, BP (blood pressure), basal RPP (renal perfusion pressure) and the renal vascular response to PHE (phenylephrine) and ACh (acetylcholine) in isolated perfused kidneys were measured. NO levels were calculated as 24-h urinary excretion of nitrites, ROS as TBARS (thiobarbituric acid-reacting substances), and peroxynitrite formation as the renal expression of nitrotyrosine. BDL rats had progressive sodium retention, and decreased BP, RPP and renal vascular responses to PHE and ACh in the time following BDL. They also had increasing levels of NO and ROS, and renal nitrotyrosine accumulation, especially in the medulla. All of these changes were either prevented or significantly decreased by chronic L -NAME administration. In conclusion, these results suggest that the increasing levels of peroxynitrite might contribute to the altered renal vascular response and sodium retention in the development of the experimental biliary cirrhosis. Moreover, the beneficial effects of decreasing NO synthesis are, at least in part, mediated by anti-peroxinitrite-related effects.

In the present study, we have analysed the mechanisms of Ca 2+ entry and release in platelets obtained from BDL (bile-duct-ligated) rats, 11–13 days and 4 weeks after surgery. Platelets were washed and loaded with fura-2, and [Ca 2+ ] i (cytosolic Ca 2+ concentration) was determined in cell suspensions by means of fluorescence spectroscopy. Basal [Ca 2+ ] i was similar in platelets from BDL rats compared with those from their respective controls, both in the absence and presence of extracellular Ca 2+ . Platelet stimulation with thrombin in the absence and presence of extracellular Ca 2+ induced a rapid rise in [Ca 2+ ] i that was of greater magnitude in platelets from BDL rats than in controls. Ca 2+ storage was significantly elevated in platelets from BDL rats, as well as the activity of SERCA (sarcoplasmic/endoplasmic-reticulum Ca 2+ -ATPase). Capacitative Ca 2+ entry, as evaluated by inhibition of SERCA with thapsigargin, was also altered in platelets from BDL rats, having lower rates of Ca 2+ entry. In conclusion, chronic BDL alters intracellular Ca 2+ homoeostasis in platelets, such that an enhanced Ca 2+ release is evoked by thrombin, which may be due to an increased amount of Ca 2+ stored in the intracellular organelles and secondary to an enhanced activity of SERCA. These alterations are already evident before cirrhosis has completely developed and occurs during the cholestasis phase.

The present study investigates the effects of chronic administration of ACEIs (angiotensin-converting-enzyme inhibitors; either zofenopril or enalapril) in combination with a diruetic (hydrochlorothiazide) on BP (blood pressure) increase and renal injury induced by L -NAME ( N G -nitro- L -arginine methyl ester), an inhibitor of NO (nitric oxide) synthesis. Rats were untreated or received L -NAME alone, L -NAME+zofenopril+hydrochlorothiazide or L -NAME+enalapril+hydrochlorothiazide for 8 weeks. L -NAME treatment resulted in marked elevation in BP and mortality. Treatment with either ACEI and diuretic prevented the increase in BP induced by L -NAME, reduced the death rate and improved excretory parameters. Renal injury in the L -NAME group was severe, but, in the groups treated with either ACEI and diuretic, glomerular and tubulointerstitial lesions were not observed and the intensity, number and size of vessels affected was reduced. However, the efficacy of zofenopril+diuretic was superior to that of enalapril+diuretic in reducing vascular alterations. Oxidative stress indices and the expression of NO synthase and nitrotyrosine were normalized by the treatments. In conclusion, the combined treatment of zofenopril or enalapril with hydrochlorothiazide completely prevented the development of arterial hypertension induced by L -NAME. Renal morphological and functional alterations in the hypertensive animals were also almost completely normalized, but the treatment with zofenopril+diuretic produced a more complete organ protection. The protective effect is related to an activation of endothelial NO synthase expression and to a normalization of the oxidative stress parameters due to the inhibition of angiotensin II.

Nitric oxide (NO) has diverse physiological and pathophysiological effects. The roles of NO in the renal and cardiac dysfunction found in cirrhosis are reviewed. In the kidneys of experimental animals with cirrhosis, several lines of evidence speak in favour of an enhanced production of NO, through the activation of both endothelial constitutive and inducible isoforms of NO synthase. In contrast with the situation in normal animals, inhibition of NO synthesis in rats with cirrhosis improves sodium and water excretion via blood pressure-dependent and -independent mechanisms, which indicates that the renal sodium and water retention of cirrhosis is related to an excess of NO production. The deleterious effect of excessive NO on the kidney may be mediated by peroxynitrite, a potent oxidant that is readily formed whenever superoxide anions and the ·NO radical are produced together. The peroxidation of arachidonic acid by peroxynitrite leads to the formation of F 2a -isoprostanes, which are powerful renal vasoconstrictors. F 2a -isoprostane levels are correlated with the severity of liver injury during cirrhosis. However, whether peroxynitrite or F 2a -isoprostanes are the elusive mediator of the NO-induced renal alterations in cirrhosis remains to be firmly established. NO is also involved in cardiac contractility, probably in the normal heart as well as in disease conditions such as non-cirrhotic and cirrhotic cardiomyopathy. In the latter state, evidence suggests that inducible NO synthase attenuates ventricular contractility, mediated by cGMP. Another gas that transduces its signal through cGMP, carbon monoxide, is also likely to play a role in cirrhotic cardiomyopathy, but the nature of the interaction between NO and carbon monoxide in this syndrome remains unclear.

The mechanisms that mediate hyporesponsiveness to vasoconstrictors in liver cirrhosis are not completely established. In the present study we have explored the role of NO and potassium channels by studying the pressor response to methoxamine in rats with carbon tetrachloride-induced cirrhosis with ascites. Experiments were performed in the isolated and perfused mesenteric arterial bed of control rats and of cirrhotic rats with ascites. Pressor responses to methoxamine, an α-adrenergic agonist, were analysed under basal conditions, after inhibition of guanylate cyclase with Methylene Blue (MB; 10 µ M), after inhibition of NO synthesis with N G -nitro- L -arginine ( L -NNA; 100 µ M) and after blockade of potassium channels with tetraethylammonium (TEA; 3 mM). Compared with those from controls, preparations from cirrhotic rats showed a lower pressor response to methoxamine (maximum: controls, 114.4±6.8 mmHg; cirrhotic rats, 74.7±7.3 mmHg). Pretreatment with MB or L -NNA increased the responses in both groups, but without correcting the lower than normal response of the cirrhotic rats. Pretreatment with TEA alone did not modify the responses as compared with the untreated groups. Pretreatment with TEA plus MB or TEA plus L -NNA also potentiated the responses, and the responses of the cirrhotic animals were greater than those of the groups treated with MB or L -NNA alone. However, no treatment completely normalized the lower response of the mesenteries from cirrhotic animals, suggesting that factors other than NO and potassium channels also participate, although to a lesser degree, in the lower pressor response of the mesenteric arterial bed of animals with cirrhosis. These results confirm that NO and potassium channels are important mediators of the lower vascular pressor response of the mesenteric bed of cirrhotic rats with ascites. This effect seems to be mediated by the NO-dependent formation of cGMP and by the NO-dependent and -independent activation of potassium channels.

In the present study we have characterized the evolution of changes in systemic haemodynamics (thermodilution in conscious animals) and sodium balance (metabolic cages) in a model of liver cirrhosis induced by chronic bile duct ligation (BDL). Mean arterial pressure (BDL, 111.5±4.7 mmHg; sham-operated, 122.9±3.0 mmHg) and peripheral vascular resistance (BDL, 2.63±0.08 units; sham-operated, 2.93±0.09 units) were lower in BDL rats from day 12 after surgery and decreased progressively throughout the following days. Portal hypertension was evident earlier in BDL rats and was maintained throughout the study period. Cardiac index (BDL, 58.8±3.9 ml·min -1 ·100 g -1 ; sham-operated, 43.9±1.5 ml·min -1 ·100 g -1 ) and stroke volume (BDL, 147.2±12.7 ml·beat -1 ·100 g -1 ; sham-operated, 109.0±4.2 ml·beat -1 ·100 g -1 ) were significantly elevated in the BDL rats only from day 18 after surgery. There were no significant differences in sodium balance between the groups until day 16 after surgery, at which time BDL animals started to retain significantly more sodium than the controls. Sodium retention increased progressively, and at day 20 BDL rats had retained 0.7 mmol/100 g more than the control animals (accumulated retention: BDL, 2.2±0.2 mmol/100 g; sham-operated, 1.5±0.2 mmol/100 g). Plasma renin activity and aldosterone concentration were not elevated in the BDL animals at days 12, 16 or 20 after surgery. These data indicate that the BDL rat model shows early portal hypertension, peripheral vasodilation and arterial hypotension, several days before sodium retention is detectable, and in the absence of changes in plasma levels of renin and aldosterone. Overall, these data suggest that, in the BDL rat model, sodium retention is secondary to portal hypertension and peripheral vasodilation.

1. The role of nitric oxide as mediator of the vascular alterations present in different models of experimental liver cirrhosis is controversial. In the present study, we evaluated the role of nitric oxide and that of the endothelium in the response to phenylephrine and acetylcholine of isolated aortic rings from chronic bile duct-ligated (29 days) rats and their corresponding controls. Experiments were performed in rings with or without endothelium, in rings pretreated with N -ω-nitro-l-arginine methyl ester (10 −4 mol/l) to inhibit nitric oxide synthesis and in rings pretreated with aminoguanidine (10 −4 mol/l) to inhibit inducible nitric oxide synthesis. 2. Under basal conditions, the maximum absolute tension developed in response to cumulative addition of phenylephrine was significantly decreased in rings from bile duct-ligated animals (1.62 ± 0.06 g) compared with the control rings (2.15 ± 0.099). This hyporesponsiveness to phenylephrine of rings from bile duct-ligated animals was corrected after treatment with N -ω-nitro-l-arginine methyl ester and reduced, but not completely eliminated, in rings without endothelium. In contrast, aminoguanidine did not modify the lower response to phenylephrine rings from bile duct-ligated animals. ED 50 values were not different between groups under any experimental conditions. 3. The endothelium-dependent vasodilatation to acetylcholine in phenylephrine-constricted rings was similar in both groups of animals, control and bile duct ligated, under all experimental conditions. N -ω-nitro-l-arginine methyl ester pretreatment and removal of the endothelium completely abolished the response to acetylcholine in cirrhotic and control rings. 4. These results demonstrate that in aortic rings from cirrhotic, bile duct-ligated rats, increased production of nitric oxide, mainly of endothelial origin, is responsible for the lower contractile response to phenylephrine. Our data, however, do not support the involvement of the inducible nitric oxide synthase isoform in this alteration. In contrast, endothelial vasodilatory response to acetylcholine is not altered in this model of cirrhosis, which indicates that not all mechanisms of nitric oxide release are abnormal.

1. Renal responses to changes in renal perfusion pressure were studied in anaesthetized hyperthyroid (thyroxine, 300 μg day −1 kg −1 ) and hypothyroid (methimazole, 0.03% via drinking water) rats to determine whether an abnormality in the pressure-diuresis-natriuresis phenomenon is involved in the resetting of kidney function in these disorders. 2. There were no significant differences between control and hypothyroid rats with respect to the relationships between renal perfusion pressure and absolute or fractional water and sodium excretion. However, in hyperthyroid rats the pressure-diuresis-natriuresis mechanism was impaired. 3. Renal blood flow and glomerular filtration rate were well autoregulated and there were no differences between control and hypothyroid rats at every level of renal perfusion pressure. A significantly lower glomerular filtration rate was observed in hyperthyroid rats when data were expressed per gram kidney weight, but glomerular filtration rate was similar to that of control rats when normalized by body weight. 4. The shift in the pressure-diuresis-natriuresis response of hyperthyroid rats is mainly due to an increase in tubular reabsorption. Blunting of the renal pressure-diuresis-natriuresis mechanism in hyperthyroid rats may represent the functional resetting of the kidney necessary for sustained hypertension. However, a normal pressure-natriuresis response was observed in hypothyroid rats, in which blood pressure was markedly reduced.

1. The purpose of the present investigation was to determine whether an abnormality of the renal papillary circulation is present in a well-established model of cirrhosis without ascites (carbon tetrachloride/phenobarbital). 2. Compared with the control animals, cirrhotic rats showed a reduced diuretic (61.0 ±5.1 versus 18.0 ±2.5%) and natriuretic (67.8 ±8.3 versus 29.6 ±3.6%) response to a volume expansion (3% body weight infusion of 0.9% NaCl). The volume expansion-induced increase in renal interstitial hydrostatic pressure was also blunted in the cirrhotic rats (control 9.3 ±0.9 versus cirrhotic 6.1±1.0 mmHg) and there were no differences in mean arterial blood pressure, renal blood flow or glomerular filtration rate between control and cirrhotic animals. 3. Papillary plasma flow was determined by the 125 I-albumin accumulation technique and expressed as mlmin −1 100 g −1 . In the basal state, papillary plasma flow was significantly lower in cirrhotic rats (59.1 ±4.4, n = 9) than in the control animals (81.8 ±6.9, n = 9). An isotonic saline expansion similar to the one described above significantly increased papillary plasma flow in control rats (108.4±9.1, n = 7) but did not change it in cirrhotic rats (60.2 ±4.9, n = 6). 4. Our results indicate the existence of a selective alteration in the renal papillary circulation in cirrhotic rats, both in the basal state and after a well-established vasodilatory stimulus. The reduced papillary plasma flow of the cirrhotic animals, probably mediated through changes in renal interstitial hydrostatic pressure, may participate in the sodium and water retention that precedes the development of ascites and may be an important mechanism mediating the blunted renal response to extracellular volume expansion.