María Inés Rosón

Vascular reactivity in diabetic rats: effect of melatonin.

The aim of the present study was to evaluate the in vitro contractile response of rat aorta in mild and severe type I diabetes and the effect of melatonin on it. Aortic rings were obtained from male Wistar rats injected with streptozotocin 8–12 wks earlier. Rats were divided into three groups: non‐diabetic rats (NDR), mildly diabetic rats (MDR) and severely diabetic rats (SDR). Dose–response curves for acetylcholine‐induced, endothelium‐related relaxation of aortic rings (after previous exposure to phenylephrine) and for serotonin‐induced vasoconstriction were conducted in the presence or absence of 10–5 mol/L melatonin. This protocol was repeated with rings preincubated in a high glucose solution (44 mmol/L). The contractile response to phenylephrine decreased in SDR, an effect counteracted by preincubation with high glucose. Melatonin decreased phenylephrine‐induced vasoconstriction in MDR and counteracted the effect of high glucose in SDR. Acetylcholine‐evoked relaxation decreased significantly after exposure to a high glucose in SDR, this effect being counteracted by melatonin. Serotonin‐induced vasoconstriction decreased in SDR and augmented in MDR, but only after exposure to high glucose. Melatonin reduced the maximal tension of aortic contraction after serotonin in MDR, both under basal conditions and after preincubation in a high glucose solution. The results support the existence of differences in vasomotor responses as a function of the diabetes state and of an improvement of contractile performance in diabetic rats after exposure to melatonin at a pharmacological concentration (in terms of circulating melatonin levels but not necessarily for some other fluids or tissues).

Controversy about COOPERATE ABPM Trial Data

mm Hg, 63% between 120 and 150 mm Hg, and the remaining 19% more than 150 mm Hg. A similar incidence was observed among treatment groups. Interestingly, the high variability of the systolic BP values was more characteristic among patients who were able to effectively restrict their salt intake. As compared with the systolic values, the nighttime values were constant throughout the measurements, partly because the drugs were taken at night or bedtime. Naoyuki Nakao, MD, PhD Division of Nephrology Rokko Island Hospital Koh-Yoh Cho Naka 2-11 Higashinada, Kobe, Japan

Nitric Oxide and Superoxide Anions in Vascular Reactivity of Renovascular Hypertensive Rats

The present study intends to define the role of the endothelium derived relaxing factor nitric-oxide (EDRF-NO) and the reactive oxygen intermediates in hypersensitivity to 5-hydroxytryptamine (5-HT) observed in abdominal aorta rings of two kidney-two clip hypertensive rats. Methylene Blue (which blocks production of cGMP by EDRF-NO) and Nw-nitro-L-arginine (which inhibits EDRF-NO synthesis), both shifted 5-HT dose-response curves to the left and completely abolished the differences in sensitivity to the agonist. The aortic perfusion with Krebs-Alcohol 20% (v/v) suppressed vascular relaxation to Ach (10(-5) M) and also abolished differences in sensitivity to 5-HT. These results suggest that a lower availability of EDRF-NO accounts for a higher 5-HT sensitivity in vessels of hypertensive rats. On the contrary, ridogrel (inhibitor of tromboxane-synthase and blocker of PGH2 and TxA2 receptors) did not suppress the hypersensitivity to 5-HT. In addition, since the superoxide anion (O2-) inactivates EDRF-NO, the effects of Superoxide dismutase (SOD) and Catalase (CAT) added in the bath were analyzed. Significant changes in sensitivity (P < 0.005) were found only for vessels of hypertensive rats (SOD depressing and CAT increasing sensitivity to 5-HT). Complementary, SOD activity was evaluated in the aorta homogenates and was found to be significantly lower in the hypertensive rats [(differences between hypertensive and sham rats, mU.mg wet weight tissue-1: 7 days after clipping, -183 +/- 67 (n = 11), P < 0.02; 21 days, -160 +/- 70 (n = 9), p < 0.05]. Results would indicate: 1. Lower EDRF-NO availability in vessels of the hypertensive animals which would account for higher sensitivity to 5-HT; 2. Such a lower EDRF-NO might depend, in part, upon its greater inactivation by O2- anions; 3. A greater presence of O2- anions in the vessels of hipertensive rats that might be favored by the lower SOD activity concentration in the vascular wall.

Different protective actions of losartan and tempol on the renal inflammatory response to acute sodium overload

The aim of this work was to study the role of local intrarenal angiotensin II (Ang II) and the oxidative stress in the up‐regulation of pro‐inflammatory cytokines expression observed in rats submitted to an acute sodium overload. Sprague–Dawley rats were infused for 2 h with isotonic saline solution (Control group) and with hypertonic saline solution alone (Na group), plus the AT1 receptor antagonist losartan (10 mg kg−1 in bolus) (Na–Los group), or plus the superoxide dismutase mimetic tempol (0.5 mg min−1 kg−1) (Na–Temp group). Mean arterial pressure, glomerular filtration rate, and fractional sodium excretion (FENa) were measured. Ang II, NF‐κB, hypoxia inducible factor‐1α (HIF‐1α), transforming growth factor β1 (TGF‐β1), smooth muscle actin (α‐SMA), endothelial nitric oxide synthase (eNOS), and RANTES renal expression was evaluated by immunohistochemistry. Ang II, NF‐κB, and TGF‐β1 and RANTES early inflammatory markers were overexpressed in Na group, accompanied by enhanced HIF‐1α immunostaining, lower eNOS expression, and unmodified α‐SMA. Losartan and tempol increased FENa in sodium overload group. Although losartan reduced Ang II and NF‐κB staining and increased eNOS expression, it did not restore HIF‐1α expression and did not prevent inflammation. Conversely, tempol increased eNOS and natriuresis, restored HIF‐1α expression, and prevented inflammation. Early inflammatory markers observed in rats with acute sodium overload is associated with the imbalance between HIF‐1α and eNOS expression. While both losartan and tempol increased natriuresis and eNOS expression, only tempol was effective in restoring HIF‐1α expression and down‐regulating TGF‐β1 and RANTES expression. The protective role of tempol, but not of losartan, in the inflammatory response may be associated with its greater antioxidant effects. J. Cell. Physiol. 224:41–48, 2010

Renal Protective Role of Atrial Natriuretic Peptide in Acute Sodium Overload-Induced Inflammatory Response

Background: The present study was performed to explore the effect of exogenous infusions of atrial natriuretic peptide (ANP) on the early inflammatory response during acute sodium overload in normal rats. Methods: Sprague Dawley rats were exposed to acute sodium overload (Na 1.5 M). Nonhypotensive doses of ANP (1 and 5 µg ·kg–1 ·h–1) were infused simultaneously with sodium or after sodium infusion in order to evaluate prevention or reversion of the inflammatory response, respectively. We determined inflammation markers in renal tissue by immunohistochemistry. Results: Creatinine clearance was not reduced in any case. Sodium tubular reabsorption increased after sodium overload (334.3 ± 18.7 vs. control 209.6 ± 27.0 mEq·min–1, p < 0.05) without changes in mean arterial pressure. This increase was prevented (228.9 ± 26.4; p < 0.05) and reversed (231.5 ± 13.9; p < 0.05) by ANP-5 µg ·kg–1 ·h–1. Sodium overload increased the expression of: RANTES (38.4.3 ± 0.8 vs. 2.9 ± 0.6%, p < 0.001), transforming-growth-factor-β1 (35.3 ± 1.0 vs. 5.0 ± 0.7%, p < 0.001), α-smooth muscle actin (15.6 ± 0.7 vs. 3.1 ± 0.3%, p < 0.001), NF-ĸB (9.4 ± 1.3 to 2.2 ± 0.5 cells/mm2, p < 0.001), HIF-1α (38.2 ± 1.7 to 8.4 ± 0.8 cells/mm2, p < 0.001) and angiotensin II (35.9 ± 1.3 to 8.2 ± 0.5%, p < 0.001). ANP-5 µg ·kg–1 ·h–1 prevented and reversed inflammation: RANTES (9.2 ± 0.5 and 6.9 ± 0.7, p < 0.001); transforming growth factor-β1 (13.2 ± 0.7 and 10.2 ± 0.5, p < 0.001) and α-smooth muscle actin (4.1 ± 0.4 and 5.2 ± 0.4, p < 0.001). Both prevention and reversion by ANP were associated with downregulation of NF-ĸB (3.2 ± 0.4 and 2.8 ± 0.5, p < 0.001) and angiotensin II (8.2 ± 0.5 and 9.1 ± 0.7, p < 0.001) and diminished hypoxia evaluated through HIF-1α expression (8.4 ± 0.8 and 8.8 ± 0.7, p < 0.001). Conclusion: Our study provides evidence supporting a protective role of ANP in both prevention and reversion of renal inflammation in rats with acute sodium overload.

Renal dopaminergic system: Pathophysiological implications and clinical perspectives

Fluid homeostasis, blood pressure and redox balance in the kidney are regulated by an intricate interaction between local and systemic anti-natriuretic and natriuretic systems. Intrarenal dopamine plays a central role on this interactive network. By activating specific receptors, dopamine promotes sodium excretion and stimulates anti-oxidant and anti-inflammatory pathways. Different pathological scenarios where renal sodium excretion is dysregulated, as in nephrotic syndrome, hypertension and renal inflammation, can be associated with impaired action of renal dopamine including alteration in biosynthesis, dopamine receptor expression and signal transduction. Given its properties on the regulation of renal blood flow and sodium excretion, exogenous dopamine has been postulated as a potential therapeutic strategy to prevent renal failure in critically ill patients. The aim of this review is to update and discuss on the most recent findings about renal dopaminergic system and its role in several diseases involving the kidneys and the potential use of dopamine as a nephroprotective agent.

Atrial natriuretic peptide behaviour and myocyte hypertrophic profile in combined pressure and volume-induced cardiac hypertrophy.

Objective To investigate cardiomyocyte hypertrophy and hormonal profile in cardiac hypertrophy resulting from sequentially applied overloads. Methods We studied Sprague–Dawley rats with renovascular hypertension (RV), where pressure overload predominates, or deoxycorticosterone acetate (DOCA)-salt (DS), where volume overload predominates, at 2 and 4 weeks of treatment, and the combination of both models in inverse sequence: RV 2 weeks/DS 2 weeks (RV2/DS2) and DS 2 weeks/RV 2 weeks (DS2/RV2), and their sham controls (Sh). Results Blood pressure and cardiomyocyte diameter increased to a similar extent in RV and DS at 2 and 4 weeks and in combined models. Cardiomyocyte length increased remarkably in the DS4 group. Circulating atrial natriuretic peptide (ANP) was elevated in all hypertensive groups after 2 and 4 weeks. The RV2/DS2 group showed similar plasma ANP levels to RV4, but DS2/RV2 exhibited a three-fold increase in ANP levels (P < 0.001 versus Sh4, DS2 and DS4). Atrial ANP mRNA remained unchanged in all groups. DS treatment alone or in combination with RV increased left ventricular ANP mRNA, meanwhile only RV treatment increased left ventricular B-type natriuretic peptide (BNP) mRNA. Ventricular ANP expression levels, but not circulating ANP, correlated with both cardiomyocyte diameter (r = 0.859, P < 0.01) and length (r = 0.848, P < 0.01). Renal expression of natriuretic peptide receptor C (NPR-C) was unchanged in RV4 but decreased to a similar extent in the DS4 group and both combined treatments. Conclusion Morphometric patterns seem to be more related to the paracrine function of the heart than to the secretion of ANP and the endocrine function. Plasma ANP in the DS2/RV2 group could indicate a different evolution of the remodelling process. ANP expression seems to be a more sensitive marker for volume than for pressure overload.

Contrasting effects of norepinephrine and 5-hydroxytryptamine on contractility of abdominal aorta of two kidney-two clip hypertensive rats. Effects of inhibitors of arachidonic acid metabolic enzymes.

This study intends to: 1) define reactivity in vessels of two kidney-two clip (2K2C) hypertensive rats (6-11 days after clipping); 2) determine the possible involvement of prostaglandins in modulating contractile vascular responses. Cumulative dose-response curves to norepinephrine (NE), 5-hydroxytryptamine (5-HT) and potassium chloride (KCl) were elicited on helical strips of abdominal aorta both in the absence and in the presence of prostacyclin synthetase (transylcypromine, TCP, 0.25mM) or cyclooxygenase (indomethacin, IND, 0.014 mM and acetylsalicylic acid, ASA, 0.20 mM) inhibitors Vessels of hypertensive animals developed significantly less tension to NE (n = 21) but higher tension and lower ED50 in response to 5-HT (n = 9) than sham control rat vessels. Force development to KCl (n = 9) was not statistically different between hypertensive and sham vessels. Vascular responses were decreased with the inhibitors, but the contrasting effects of NE and 5-HT on clip vessels were maintained. Threshold doses of PGE2 significantly reversed the effect of IND but not that of TCP on NE responses. Threshold doses of PGI2 had no significant effect on NE and 5-HT responses under TCP. The results would indicate: 1) different functional alterations for contractions to NE and 5-HT appear to have developed in vessels of 2K2C hypertensive rats; 2) PGE2 effectively contributes to modulation of NE responses in rat aorta strips; 3) these experiments suggest that prostaglandins do not play a significant role in the altered contractility of vessels in hypertensive rats.

High-sodium diet promotes a profibrogenic reaction in normal rat kidneys: effects of Tempol administration

BACKGROUND Studies carried out in vitro have recently shown that salt loading induces an increasing mechanical stretch and a flow-induced superoxide production in the thick ascending limb of Henles loop. In this regard, we hypothesized that the oxidative stress induced by salt overload could stimulate inflammatory and fibrogenic signaling pathways in normal rats. METHODS Sprague Dawley rats were fed with an 8% NaCl high- (HS) or 0.4% NaCl normal-salt (NS) diet for 3 weeks, with or without Tempol (T) administration (1 mM, administered in drinking water). Mean arterial pressure (MAP), glomerular filtration rate (GFR) and urinary sodium excretion (UVNa) were measured. NAD(P)H oxidase p47phox, angiotensin II (Ang II), transforming growth factor ß1 (TGF-ß1), a-smooth muscle actin (a-SMA) and nuclear factor-kappa B (NF-kB) expression were evaluated in renal tissues by immunohistochemistry. RESULTS A high NaCl diet produced a slight but significant increase in MAP and enhanced UVNa and oxidative stress. Administration of a high NaCl diet induced the overexpression of TGF-ß1, a-SMA and NF-?B in cortex and medulla, while Ang II increased in proximal convoluted tubules, and decreased in cortical collecting ducts. Tempol administration prevented these changes and simultaneously normalized MAP accompanied by an enhancement in GFR and UVNa. CONCLUSION The results showed that a high NaCl diet is able to produce a renal profibrotic response also in normal rats, which could be associated with oxidative stress rather than intrarenal Ang II expression.

Impaired response to insulin associated with protein kinase C in chronic fructose-induced hypertension

A fructose-enriched diet induces an increase in blood pressure associated with metabolic alterations in rats. Our hypothesis was that an increase in protein kinase C (PKC) activation, reported in the acute period of fructose overload, and an impaired vessels response to vasoactive substances contribute to maintain elevated blood pressure levels in the chronic period. The aims of this study were to investigate in this animal model of hypertension: (1) if the increase in PKC activation was also found in the chronic stage; (2) the involvement of nitric oxide and insulin in the vessels response; and plasma atrial natriuretic factor and nitrites/nitrates (nitric oxide metabolites) behavior. We evaluated the effects of: PKCstimulator 12,13-phorbol dibutyrate, phenylephrine, insulin, nitric oxide synthase-inhibitor N G -nitro- L arginine methyl esther (L-NAME) and PKC-inhibitor Calphostin C on aortic rings responses of Sprague- Dawley rats: fructose-fed and control. The fructose-fed group showed higher contractility to 12,13- phorbol dibutyrate than the control group in aortic rings pre-incubated with insulin, and this difference disappeared with L-NAME. The response to phenylephrine in rings pre-incubated with Calphostin C was decreased in the fructose-fed group and increased with Calphostin C plus L-NAME. Fructose-fed rats showed higher levels of plasma atrial natriuretic factor and nitrites/nitrates than controls. In conclusion, chronic fructose feeding seems to develop an impaired response to insulin, dependent on nitric oxide, suggesting a PKC alteration. Vasorelaxant agents, such as atrial natriuretic factor and nitric oxide, would behave as compensatory mechanisms in response to high blood pressure.