Renal and systemic effects of endothelin-1 in diabetic-hypertensive rats.

The Cohen-Rosenthal Diabetic Hypertensive rat (CRDH) is a unique animal model in which genetic hypertension and diabetes developed after crossbreeding of Cohen diabetic rats sensitive substrain (CDR) and spontaneously hypertensive rats (SHR). The present study examined: 1) The acute effects of ET-1 on the systemic and renal hemodynamics in CRDH rats, CDR, and SHR; 2) The expression of ET-1 and its receptors in the renal tissue of CRDH rats. Intravenous injection of ET-1 (1.0 nmol/kg) into anesthetized SHR rats resulted in a significant immediate depressor response (mean arterial pressure (MAP) decreased from 165 ± 3 to 124 ± 12 mmHg, p < 0.0001) followed by a minor hypertensive phase (MAP increased to 170 ± 2 mmHg). Simultaneously, the administration of ET-1 caused a significant decrease in renal blood flow (RBF) from 5.8 ± 0.9 ml/min to 3.2 ± 0.5 ml/min (p = 0.026). These responses were blunted in CRDH rats and CDR. Analysis of intra-renal blood flow by laser-Doppler in CRDH rats revealed that ET-1 injection caused a decrease in cortical blood flow (Δ = -12 ± 2.9%). However, in contrast to its well-known renal medullary vasodilatory effect, ET-1 produced a significant decline in the medulla blood flow (Δ = -17.5 ± 3.4%) (p = 0.0125). These findings suggest that CDR and CRDH rats have reduced sensitivity to vascular and renal action of ET-1. Furthermore, in the CRDH rats, the expected ET-1-induced medullary vasodilatation was abolished and even reversed into prolonged vasoconstriction.

Hyperbaric oxygen treatment improves GFR in rats with ischaemia/reperfusion renal injury: a possible role for the antioxidant/oxidant balance in the ischaemic kidney.

BACKGROUND: Ischaemic kidney injury continues to play a dominant role in the pathogenesis of acute renal failure (ARF) in many surgical and medical settings. A major event in the induction of renal injury is related to the generation of oxygen-free radicals. Hyperbaric oxygen therapy (HBO) is indicated for treatment of many ischaemic events but not for ARF. Therefore, the present study examined the effects of HBO on kidney function and renal haemodynamics in rats with ischaemic ARF. METHODS: Renal ischaemia was induced by unilateral renal artery clamping (45 min) in rats. Within 24 h following ischaemia, rats were treated twice with HBO of 100% O(2) at 2.5 absolute atmospheres for 90 min each (+HBO). Untreated rats (-HBO) served as a control. Forty-eight hours later, GFR, RBF and endothelial-dependent vasorelaxation were measured. In addition, the immunoreactive staining of 4-hydroxy-2-noneal (4-HNE), a major product of endogenous lipid peroxidation, and superoxide dismutase (SOD) were assessed. RESULTS: In the -HBO group, GFR was reduced by 94% compared with the untouched normal kidney (ischaemic: 0.06 +/- 0.03 ml/min, normal: 1.02 +/- 0.13 ml). In contrast, in the +HBO group, GFR of the ischaemic kidney (0.36 +/- 0.07 ml/min) was reduced only by 68% compared with the contralateral normal kidney (1.12 +/- 0.12 ml/min). In line with these findings, HBO improved the vasodilatory response to ACh as expressed in enhancement of both total and regional renal blood flow. In addition, HBO reduced the formation of 4-HNE by 33% and 76% and increased SOD by 30% and 70% in the cortex and outer stripe region of the medulla of the ischaemic kidney, respectively. CONCLUSION: HBO attenuates the decline in GFR following renal ischaemia, and improves endothelial-dependent vasorelaxation, suggesting that treatment with HBO may be beneficial in the setting of ischaemic ARF.

Effects of novel vasopressin receptor antagonists on renal function and cardiac hypertrophy in rats with experimental congestive heart failure.

Arginine vasopressin (AVP) plays an important role in renal hemodynamic alterations, water retention, and cardiac remodeling in congestive heart failure (CHF). The present study evaluated the acute and chronic effects of vasopressin V(1a) receptor subtype (V(1a)) and vasopressin V(2) receptor subtype (V(2)) antagonists on renal function and cardiac hypertrophy in rats with CHF. The effects of acute administration of SR 49059 [(2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide)] (0.1 mg/kg) and SR 121463B (1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzenesulfonyl]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy)cyclohexane]indol-2-one, fumarate; equatorial isomer) (0.3 mg/kg), V(1a) and V(2) antagonists, respectively, on renal function, and of chronic treatment (3.0 mg/kg/day for 7 or 28 days, via osmotic minipumps or p.o.), on water excretion and cardiac hypertrophy were studied in rats with aortocaval fistula and control rats. CHF induction increased plasma AVP (12.8 +/- 2.5 versus 32.2 +/- 8.3 pg/ml, p < 0.05). Intravenous bolus injection of SR 121463B to controls produced dramatic diuretic response (from 5.5 +/- 0.8 to 86.3 +/- 21.9 microl/min; p < 0.01). In contrast, administration of SR 49059 did not affect urine flow. Likewise, administration of SR 121463B, but not SR 49059, to rats with CHF significantly increased urinary flow rate from 20.8 +/- 6.4 to 91.6 +/- 26.5 microl/min (p < 0.01). The diuretic effects of SR 121463B were associated with a significant decline in urinary osmolality and insignificant change of Na+ excretion. In line with its acute effects, chronic administration of SR 121463B to CHF rats increased daily urinary volume 2 to 5-fold throughout the treatment period. Both SR 121463B and SR 49059 significantly reduced heart weight in CHF rats when administered for 4 weeks, but not 1 week. These results suggest that V(2) and V(1a) antagonists improve water balance and cardiac hypertrophy in CHF and might be beneficial for the treatment of water retention and cardiac remodeling in CHF.

Desferrioxamine and zinc-desferrioxamine reduce lens oxidative damage.

Our purpose was to investigate the quality and morphology of cultured bovine lenses after exposure to hyperbaric oxygen (HBO) in the presence or absence of desferrioxamine (DFO) or zinc-desferrioxamine (Zn-DFO). Intact bovine lenses were cultured and exposed to HBO of 100% oxygen at 2.5 ATA for 120 min. One hundred and fifty lenses were included in the present study. Lenses were divided into study groups of 25 lenses each: (1a) HBO-exposed lenses; (1b) control lenses extracted from the contralateral eyes of group 1a and exposed to normal room air. (2a) HBO-exposed lenses treated with DFO; (2b) control lenses extracted from the contralateral eyes of group 2a exposed to normal room air in the presence of DFO (3a) HBO-exposed lenses treated with Zn-DFO; (3b) control lenses extracted from the contralateral eyes of group 3a, exposed to normal room air in the presence of Zn-DFO. Lens optical quality and structural changes were assessed. Oxygen toxicity to lenses was demonstrated by decreased light transmission, increase in focal length variability and a decrease in morphological integrity. Light intensity measurements showed a distinct pattern in control lenses. A different pattern was noticed for hyperbaric oxygen-exposed lenses. Focal length variability values were stable in control lenses and increased significantly in oxygen-exposed lenses. Structural damage to lenses was demonstrated by the appearance of bubbles between lens' fibers possibly demonstrating failure of lens tissue to cope with oxygen load. All measured parameters showed that both Zn-DFO and DFO attenuated the oxidative damage. The effect of DFO was small whereas Zn-DFO demonstrated a significantly stronger effect. Treatment of hyperbaric oxygen-exposed lenses with DFO only marginally reduced the oxidative damage. Treatment with Zn-DFO was superior in reducing the oxidative damage to lenses. These results indicate a possible role for Zn-DFO in the prevention of cataracts.

Involvement of Rho kinase pathway in the mechanism of renal vasoconstriction and cardiac hypertrophy in rats with experimental heart failure.

Rho-dependent kinases serve as downstream effectors of several vasoconstrictor systems, the activities of which are upregulated in congestive heart failure (CHF). We evaluated renal and cardiac effects of Y-27632, a highly selective Rho kinase inhibitor, in an experimental model of volume-overload CHF. Effects of acute administration of Y-27632 (0.3 mg/kg) on renal hemodynamic and clearance parameters and effects of chronic treatment (10.0 mg.kg(-1).day(-1) for 7 days via osmotic minipumps) on cardiac hypertrophy and cumulative Na+ excretion were studied in male Wistar rats with aortocaval fistula and control rats. The Y-27632-induced decrease in renal vascular resistance (from 40.4 +/- 4.6 to 26.0 +/- 3.1 resistance units, P < 0.01) in CHF rats was associated with a significant increase in total renal blood flow (+34%) and cortical and medullary blood flow (approx +37 and +27%, respectively). These values were significantly higher than those in control rats and occurred despite a decrease in mean arterial pressure (-15 mmHg). Despite the marked renal vasodilatory effect, Y-27632 did not alter glomerular filtration rate and renal Na+ excretion. Chronic administration of Y-27632 did not alter daily or cumulative renal Na+ excretion in CHF rats but was associated with a significant decrease in heart-to-body weight ratio, an index of cardiac hypertrophy: 0.32 +/- 0.007, 0.46 +/- 0.017, and 0.37 +/- 0.006% in control, CHF, and CHF + Y-27632 rats, respectively. The findings suggest that Rho kinase-dependent pathways are involved in the mechanisms of renal vasoconstriction and cardiac hypertrophy in rats with volume-overload heart failure. Selective blockade of these signaling pathways may be considered an additional tool to improve renal perfusion and attenuate cardiac hypertrophy in heart failure.

[Oxygen effect on ocular lens].

BACKGROUND: Cataract is the leading cause of preventable blindness worldwide. Clinical observations and laboratory results have shown that oxygen has a possible toxic role in cataract formation. AIM: The aim of the present study was to demonstrate, measure and characterize the damage caused to bovine lenses in organ culture as a result of their exposure to hyperbaric oxygen pressure. MATERIALS AND METHODS: Twenty bovine lenses exposed to hyperbaric pressure were compared to 20 control lenses. Lenses were kept in an organ culture for 14 days. Each day the focusing ability of the exposed lenses was compared to controls. The comparison was based on the amount to which the focus point of each measured ray diverged from the focus point of the lens. Lenses were also examined under the microscope and morphologic changes in study lenses were compared to controls. RESULTS: A statistically significant difference in focusing ability between the study and control lenses was observed. The difference became larger during the incubation period indicating an accumulation of damage. The damage resulted from the peripheral but not the central part of the lenses. The morphologic changes observed under the microscope matched the damage profile of the focusing ability. CONCLUSIONS: Oxygen has a possible role in cataract formation. The effect of oxygen is cumulative. The route of damage formation follows the diffusion of oxygen into the lens.

Effects of endothelin-1 on systemic and renal hemodynamics in hypertensive-diabetic rats (CRDH), diabetic rats (CDR), and hypertensive rats (SHR).

The Cohen-Rosenthal diabetic hypertensive rat is a unique animal model in which genetic hypertension and diabetes developed after crossbreeding of a sensitive substrain of Cohen diabetic rats and spontaneously hypertensive rats (SHR) and feeding them a copper-poor sucrose diet. This study examined the acute effects of endothelin-1 on the systemic and renal hemodynamics in Cohen-Rosenthal diabetic hypertensive rats, Cohen diabetic rats and spontaneously hypertensive rats. Intravenous injection of endothelin- 1 (1.0 nmol/kg) into anesthetized SHR resulted in a significant immediate depressor response in mean arterial pressure [from 165 +/- 3 mmHg to 124 +/- 12 mmHg (P < 0.0001)] followed by a minor hypertensive phase (mean arterial pressure increased to 170 +/- 2 mmHg). Simultaneously, the administration of endothelin-1 caused a significant decrease in renal blood flow from 5.8 +/- 0.9 mL/minute to 3.2 +/- 0.5 mL/minute (P = 0.026). These responses were blunted in Cohen-Rosenthal diabetic hypertensive rats and Cohen diabetic rats. Analysis of intrarenal blood flow by laser-Doppler in Cohen-Rosenthal diabetic hypertensive rats revealed that endothelin-1 injection caused a decrease in cortical blood flow (Delta = -12 +/- 2.9%). However, in contrast to its well known renal medullary vasodilatory effect, endothelin-1 produced a significant decline in the medulla blood flow (Delta = -17.5 +/- 3.4%) (P = 0.0125). These findings suggest that Cohen diabetic rats and Cohen-Rosenthal diabetic hypertensive rats have reduced sensitivity to the vascular and renal action of endothelin-1. Furthermore, in the Cohen-Rosenthal diabetic hypertensive rats the expected endothelin- 1-induced medullary vasodilation was abolished and even reversed into prolonged vasoconstrictor response.

Vascular endothelial growth factor (VEGF) fails to improve blood flow and to promote collateralization in a diabetic mouse ischemic hindlimb model.

BACKGROUND: Angiogenic therapy with vascular endothelial growth factor (VEGF) has been proposed as a treatment paradigm for patients suffering from an insufficiency of collateral vessels. Diabetes is associated with increase in the production of VEGF and therefore additional VEGF may not be beneficial. Accordingly, we sought to determine the efficacy of VEGF therapy to augment collateral formation and tissue perfusion in a diabetic mouse ischemic hindlimb model. METHODS: Diabetic and non-diabetic mice were studied in parallel for the efficacy of VEGF administration. Diabetes was induced with streptozotocin. Hindlimb ischemia was produced by severing the left iliac artery. An outlet tube from an osmotic infusion pump with placebo/500 micrograms of plasmid-DNA encoding VEGF was fenestrated and tunneled into the left quadriceps muscle. RESULTS: VEGF induced more rapid and complete restoration of blood flow in normal mice. However, in the setting of diabetes there was no difference between VEGF vs. placebo in the rate or adequacy of flow restoration. There was a significant increase in smooth muscle actin and Factor-VIII antigen densities in diabetic animals and in animals which received VEGF. CONCLUSIONS: Angiogenic therapy with VEGF in the setting of diabetes does not appear to have the beneficial effects seen in the absence of diabetes.

Lenticular oxygen toxicity.

PURPOSE: To investigate the possible toxic effect of oxygen on lenses in an organ culture. METHODS: Bovine lenses were exposed to four different combinations of ambient pressure and oxygen concentration in an organ culture throughout a 7-day period. Lens transparency, histology, enzymatic activities, and photomicrographs were compared in study and control groups. RESULTS: No differences were observed between study and control lenses in all measured parameters in a group subjected to a single exposure of 100% oxygen under increased (i.e., hyperbaric) ambient conditions and a group exposed repeatedly to high ambient pressure and normal oxygen partial pressure. Decreased lenticular transparency and enzymatic activities along with structural changes were observed in lenses exposed repeatedly to 100% oxygen concentration under both normal and increased ambient pressures. The observed changes were oxygen-load-dependent: the higher the oxygen partial pressure and the longer the time of exposure, the more severe the changes observed. Optical and structural changes in the lens occurred in a centripetal orientation: the greater the oxygen load, the more central the damage. CONCLUSIONS: High oxygen load has a toxic effect on bovine lenses in organ culture. These effects appear to be cumulative: the higher the oxygen partial pressure and the greater the number of exposures, the more severe the changes observed in the lenses. Changes marking toxicity follow the route of oxygen diffusion into the lens, from the periphery to the center. Cautious interpretation of the results may indicate a role of oxygen (and/or its derivatives) in human cataract formation.

Restoration of blood flow by using continuous perimuscular infiltration of plasmid DNA encoding subterranean mole rat Spalax ehrenbergi VEGF.

The optimal vector, regulatory sequences, and method of delivery of angiogenic gene therapy are of considerable interest. The Spalax ehrenbergi superspecies live in subterranean burrows at low oxygen tensions and its tissues are highly vascularized. We tested whether continuous perimuscular administration of Spalax vascular endothelial growth factor (VEGF) DNA could increase tissue perfusion in a murine hindlimb ischemia model. Placebo or VEGF +/- internal ribosome entry site (IRES) was continuously administrated perimuscularly in the ischemic zone by using an infusion pump. None of the mice in the VEGF-treated group (>50 microg) developed visible necrosis vs. 33% of the placebo group. Microscopic necrosis was observed only in the placebo group. Spalax VEGF muscular infiltration resulted in a faster and more complete restoration of blood flow. The restoration of blood flow by VEGF was dose-dependent and more robust and rapid when using the VEGF-IRES elements. The flow restoration using continuous perimuscular infiltration was faster than single i.m. injections. Vessel density was higher in the VEGF and VEGF-IRES (-) groups compared with the placebo. Continuous perimuscular administration of angiogenic gene therapy offers a new approach to restore blood flow to an ischemic limb. Incorporation of an IRES element may assist in the expression of transgenes delivered to ischemic tissues. Further studies are needed to determine whether VEGF from the subterranean mole rat Spalax VEGF is superior to VEGF from other species. If so, 40 million years of Spalax evolution underground, including adaptive hypoxia tolerance, may prove important to human angiogenic gene therapy.

Role of myocardial inducible nitric oxide synthase in contractile dysfunction and beta-adrenergic hyporesponsiveness in rats with experimental volume-overload heart failure.

BACKGROUND: Whereas nitric oxide (NO) has been implicated in the pathophysiology of heart failure (HF), the significance and functional role of different NO synthase (NOS) isoforms in this pathology are controversial. Our aim was to study in the myocardium of rats with volume-overload-induced HF the expression, activity, and localization of endothelial (eNOS) and inducible (iNOS) isoforms and the involvement of iNOS in depressed cardiac contractile properties, intracellular Ca(2+) ([Ca(2+)](i)) transients, and beta-adrenergic hyporesponsiveness. METHODS AND RESULTS: HF was induced by aortocaval fistula (ACF). Compensated and decompensated subgroups of HF were selected on the basis of daily sodium excretion. ACF induced cardiac hypertrophy in rats with compensated (36%) and decompensated (76%) HF. Whereas in HF rats, cardiac eNOS expression and activity were unchanged, iNOS expression and activity increased approximately 2-fold. iNOS immunostaining was observed in ventricular myocytes of compensated and decompensated HF rats but not in controls. Isoproterenol-positive inotropic and lusitropic effects were markedly attenuated in papillary muscle of HF rats, more pronouncedly in decompensated than in compensated rats. Isoproterenol-induced increases in the rates of [Ca(2+)](i) activation and relaxation were also depressed in ACF rats. Selective iNOS blockade by N-(3-(aminomethyl)benzylacetamidine improved the attenuated beta-adrenergic responsiveness in HF rats. CONCLUSIONS: Our findings indicate that myocardial iNOS is activated in rats with volume-overload HF and suggest that increased iNOS activity contributes to depressed myocardial contractility and beta-adrenergic hyporesponsiveness.