Altered glial expression of the cannabinoid 1 receptor in the subiculum of a mouse model of Alzheimer's disease.

The alteration of the endocannabinoid tone usually associates with changes in the expression and/or function of the cannabinoid CB1 receptor. In Alzheimer's disease (AD), amyloid beta (Aß)-containing aggregates induce a chronic inflammatory response leading to reactivity of both microglia and astrocytes. However, how this glial response impacts on the glial CB1 receptor expression in the subiculum of a mouse model of AD, a brain region particularly affected by large accumulation of plaques and concomitant subcellular changes in microglia and astrocytes, is unknown. The CB1 receptor localization in both glial cells was investigated in the subiculum of male 5xFAD/CB2 EGFP/f/f (AD model) and CB2 EGFP/f/f mice by immuno-electron microscopy. The findings revealed that glial CB1 receptors suffer remarkable changes in the AD mouse. Thus, CB1 receptor expression increases in reactive microglia in 5xFAD/CB2 EGFP/f/f , but remains constant in astrocytes with CB1 receptor labeling rising proportionally to the perimeter of the reactive astrocytes. Not least, the CB1 receptor localization in microglial processes in the subiculum of controls and closely surrounding amyloid plaques and dystrophic neurites of the AD model, supports previous suggestions of the presence of the CB1 receptor in microglia. These findings on the correlation between glial reactivity and the CB1 receptor expression in microglial cells and astrocytes, contribute to the understanding of the role of the endocannabinoid system in the pathophysiology of Alzheimer's disease.

Anti-inflammatory agents for smoking cessation? Focus on cognitive deficits associated with nicotine withdrawal in male mice.

Nicotine withdrawal is associated with cognitive deficits including attention, working memory, and episodic memory impairments. These cognitive deficits are a hallmark of nicotine abstinence which could be targeted in order to prevent smoking relapse. The underlying mechanisms, however, are poorly understood. In this study, memory impairment was observed in mice 4 days after the precipitation of nicotine withdrawal by the nicotinic antagonist mecamylamine. The presence of cognitive deficits correlated with microglial activation in the hippocampus and the prefrontal cortex. Moreover, an increased expression of neuroinflammatory markers including IL1ß, TNFα and IFNγ was found in both memory-related brain regions. Notably, flow cytometric analysis also revealed an enhancement of TNFα and IFNγ plasmatic levels at the same time point during nicotine withdrawal. Impaired neurogenesis, as shown by reduction in the expression of the endogenous cell proliferation marker Ki67 and the early neuron marker doublecortin, was also associated with nicotine abstinence. Treatment with the non-psychoactive cannabinoid cannabidiol abolished memory impairment of nicotine withdrawal and microglia reactivity, reduced the expression of IL1ß and IFNγ in the hippocampus and the prefrontal cortex, respectively, and normalized Ki67 levels. The nonsteroidal anti-inflammatory drug indomethacin also prevented cognitive deficits and microglial reactivity during withdrawal. These data underline the usefulness of anti-inflammatory agents to improve cognitive performance during early nicotine abstinence.

Tyrosine hydroxylase haploinsufficiency prevents age-associated arterial pressure elevation and increases half-life in mice.

Catecholamines are essential for the maintenance of physiological homeostasis under basal and stress conditions. We aim to determine the impact of deletion of a single allele of the tyrosine hydroxylase (Th) gene might have on aging arterial pressure and life-span. We found that Th haploinsufficiency prevents age-associated increase of arterial pressure (AP) in mature adult mice, and it results in the extension of the half-life of Th-heterozygous (TH-HET) mice respect to their wild-type (WT) littermates. Heart performance was similar in both genotypes. To further investigate the lack of increase in AP with age in TH-HET mice, we measured the AP response to intra-peritoneal administration of substances involved in AP regulation. The response to acetylcholine and the basal sympathetic tone were similar in both genotypes, while norepinephrine had a greater pressor effect in TH-HET mice, which correlated with altered adrenoreceptor expression in blood vessels and the heart. Furthermore, sympatho-adrenomedular response to stress was attenuated in TH-HET mice. Plasma catecholamine levels and urine glucose increased markedly in WT but not in TH-HET mice after stress. Our results showed that TH-HET mice are resistant to age-associated hypertension, present a reduction in the sympathetic response to stress and display an extended half-life.

S-endoglin expression is induced in senescent endothelial cells and contributes to vascular pathology.

Senescence of endothelial cells (ECs) may contribute to age-associated cardiovascular diseases, including atherosclerosis and hypertension. The functional and gene expression changes associated with cellular senescence are poorly understood. Here, we have analyzed the expression, during EC senescence, of 2 different isoforms (L, long; S, short) of endoglin, an auxiliary transforming growth factor (TGF)-beta receptor involved in vascular remodeling and angiogenesis. As evidenced by RT-PCR, the S/L ratio of endoglin isoforms was increased during senescence of human ECs in vitro, as well as during aging of mice in vascularized tissues. Next, the effect of S-endoglin protein on the TGF-beta receptor complex was studied. As revealed by coimmunoprecipitation assays, S-endoglin was able to interact with both TGF-beta type I receptors, ALK5 and ALK1, although the interaction with ALK5 was stronger than with ALK1. S-endoglin conferred a lower proliferation rate to ECs and behaved differently from L-endoglin in relation to TGF-beta-responsive reporters with ALK1 or ALK5 specificities, mimicking the behavior of the endothelial senescence markers Id1 and plasminogen activator inhibitor-1. In situ hybridization studies demonstrated the expression of S-endoglin in the endothelium from human arteries. Transgenic mice overexpressing S-endoglin in ECs showed hypertension, decreased hypertensive response to NO inhibition, decreased vasodilatory response to TGF-beta(1) administration, and decreased endothelial nitric oxide synthase expression in lungs and kidneys, supporting the involvement of S-endoglin in the NO-dependent vascular homeostasis. Taken together, these results suggest that S-endoglin is induced during endothelial senescence and may contribute to age-dependent vascular pathology.

Effect of different antihypertensive treatments on Ras, MAPK and Akt activation in hypertension and diabetes.

Ras GTPases function as transducers of extracellular signals regulating many cell functions, and they appear to be involved in the development of hypertension. In the present study, we have investigated whether antihypertensive treatment with ARBs (angiotensin II receptor blockers), ACEi (angiotensin-converting enzyme inhibitors) and diuretics induce changes in Ras activation and in some of its effectors [ERK (extracellular-signal-regulated kinase) and Akt] in lymphocytes from patients with hypertension without or with diabetes. ACEi treatment transiently reduced Ras activation in the first month of treatment, but diuretics induced a sustained increase in Ras activation throughout the 3 months of the study. In patients with hypertension and diabetes, ARB, ACEi and diuretic treatment increased Ras activation only during the first week. ACEi treatment increased phospho-ERK expression during the first week and also in the last 2 months of the study; however, diuretic treatment reduced phospho-ERK expression during the last 2 months of the study. In patients with hypertension and diabetes, antihypertensive treatments did not induce changes in phospho-ERK expression in lymphocytes. ACEi treatment reduced phospho-Akt expression in patients with hypertension and diabetes only in the first month of treatment. In conclusion, these findings show that antihypertensive treatments with ACEi, and diuretics to a lesser extent, modify Ras activation and some of its signalling pathways, although in different directions, whereas ARBs do not appear to have any influence on Ras signalling pathways.

Targeted genomic disruption of H-ras and N-ras has no effect on early renal changes after unilateral ureteral ligation.

PURPOSE: To assess the contribution of two different Ras monomeric GTPases isoforms H- and N-Ras in the early changes associated to obstructive nephropathy induced by unilateral ureteral obstruction (UUO). METHODS: UUO was performed in N-ras (N-ras−/−) and H-ras (H-ras−/−) knock-out mice and control (H-ras+/+/N-ras+/+) mice of C57Bl/6 background. Fibronectin, α-smooth muscle actin, cleaved caspase-3, ki-67, Ras-GTP, pERK, and pAkt expression was analyzed by western blot and/or immunohistochemistry. Ras isoforms activation and caspase activity were determined by both western blot and ELISA. RESULTS: Three days after UUO, obstructed (O) kidneys of H-ras−/−, N-ras−/−and H-ras+/+/N-ras+/+mice showed no significant differences in activated total ras, pERK1/2, pAkt, total Akt levels, fibronectin, α-SMA expression, cell proliferation, and activated caspase-3. The morphological alterations in the O kidneys, revealed by histological and immunohistochemical studies, were also similar in H-ras−/−, N-ras−/−, and H-ras+/+/N-ras+/+mice. CONCLUSIONS: These data suggest that the activation of H-ras and N-ras isoforms does not play a major role in the early renal damage induced by UUO.

Activation of Erk1/2 and Akt following unilateral ureteral obstruction.

Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.

Effect of angiotensin II and small GTPase Ras signaling pathway inhibition on early renal changes in a murine model of obstructive nephropathy.

Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis.

Delayed mTOR inhibition with low dose of everolimus reduces TGFß expression, attenuates proteinuria and renal damage in the renal mass reduction model.

BACKGROUND: The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors are widely used in solid organ transplantation, but their effect on kidney disease progression is controversial. mTOR has emerged as one of the main pathways regulating cell growth, proliferation, differentiation, migration, and survival. The aim of this study was to analyze the effects of delayed inhibition of mTOR pathway with low dose of everolimus on progression of renal disease and TGFß expression in the 5/6 nephrectomy model in Wistar rats. METHODS: This study evaluated the effects of everolimus (0.3 mg/k/day) introduced 15 days after surgical procedure on renal function, proteinuria, renal histology and mechanisms of fibrosis and proliferation. RESULTS: Everolimus treated group (EveG) showed significantly less proteinuria and albuminuria, less glomerular and tubulointerstitial damage and fibrosis, fibroblast activation cell proliferation, when compared with control group (CG), even though the EveG remained with high blood pressure. Treatment with everolimus also diminished glomerular hypertrophy. Everolimus effectively inhibited the increase of mTOR developed in 5/6 nephrectomy animals, without changes in AKT mRNA or protein abundance, but with an increase in the pAKT/AKT ratio. Associated with this inhibition, everolimus blunted the increased expression of TGFß observed in the remnant kidney model. CONCLUSION: Delayed mTOR inhibition with low dose of everolimus significantly prevented progressive renal damage and protected the remnant kidney. mTOR and TGFß mRNA reduction can partially explain this anti fibrotic effect. mTOR can be a new target to attenuate the progression of chronic kidney disease even in those nephropathies of non-immunologic origin.

Increased oxidative stress, the renin-angiotensin system, and sympathetic overactivation induce hypertension in kidney androgen-regulated protein transgenic mice.

Gender differences in the incidence and severity of hypertension have suggested the involvement of a sex-dependent mechanism. Transgenic (Tg) mice overexpressing kidney androgen-regulated protein (KAP) specifically in kidney showed hypertension associated with oxidative stress. Reactive oxygen species (ROS) are strongly implicated in the pathological signaling leading to hypertension in a framework that includes renin-angiotensin system (RAS) activation, increased sympathetic activity, and cardiac remodeling. In this report, we observed that plasma levels of angiotensin II and catecholamines were increased in KAP Tg mice, compared with wild-type animals. Systemic administration of Tempol, a membrane-permeative superoxide dismutase mimetic, reduced arterial pressure as well as urinary excretion of oxidative stress markers and reduced both angiotensin II and norepinephrine plasma levels in KAP Tg mice. Intracerebroventricular administration of Tempol also reduced arterial pressure in Tg mice. Moreover, administration of apocynin and DPI, inhibitors of NADPH oxidase, a major source of ROS, also reduced arterial pressure and both angiotensin II and norepinephrine plasma levels in Tg mice. Thus, we analyzed the involvement of the RAS and sympathetic nervous system in KAP Tg mouse hypertension. Both captopril and losartan reduced arterial blood pressure in Tg mice, as also occurred after ß-adrenergic blockade with atenolol. Also, intracerebroventricular losartan administration reduced arterial pressure in KAP Tg mice. Our data demonstrate that hypertension in male KAP Tg mice is based on increased oxidative stress, increased sympathetic activity, and RAS activation. Moreover, our results suggest a role for increased oxidative stress in the CNS as a major cause of hypertension in these animals.

Role of inflammation in túbulo-interstitial damage associated to obstructive nephropathy.

Obstructive nephropathy is characterized by an inflammatory state in the kidney, that is promoted by cytokines and growth factors produced by damaged tubular cells, infiltrated macrophages and accumulated myofibroblasts. This inflammatory state contributes to tubular atrophy and interstitial fibrosis characteristic of obstructive nephropathy. Accumulation of leukocytes, especially macrophages and T lymphocytes, in the renal interstitium is strongly associated to the progression of renal injury. Proinflammatory cytokines, NF-kappaB activation, adhesion molecules, chemokines, growth factors, NO and oxidative stress contribute in different ways to progressive renal damage induced by obstructive nephropathy, as they induce leukocytes recruitment, tubular cell apoptosis and interstitial fibrosis. Increased angiotensin II production, increased oxidative stress and high levels of proinflammatory cytokines contribute to NF-kappaB activation which in turn induce the expression of adhesion molecules and chemokines responsible for leukocyte recruitment and iNOS and cytokines overexpression, which aggravates the inflammatory response in the damaged kidney. In this manuscript we revise the different events and regulatory mechanisms involved in inflammation associated to obstructive nephropathy.

Fibroblast activation and myofibroblast generation in obstructive nephropathy.

Obstructive nephropathy is a major cause of renal failure, particularly in newborn babies and children. After urinary tract obstruction, and under the influence of mechanical forces and cytokines produced by tubular cells and cells that have infiltrated the interstitium, resident fibroblasts undergo activation and myofibroblasts are generated from bone-marrow-derived cells, pericytes and endothelial cells. In addition, selected tubular epithelial cells can become fibroblast-like cells via epithelial-mesenchymal transition. This transition is characterized by downregulation of epithelial marker proteins such as E-cadherin, zonula occludens 1 and cytokeratin; loss of cell-to-cell adhesion; upregulation of mesenchymal markers including vimentin, alpha-smooth muscle actin and fibroblast-specific protein 1; basement membrane degradation; and migration to the interstitial compartment. All the events of epithelial-mesenchymal transition are strictly regulated by complex signaling pathways. Myofibroblasts and activated fibroblasts proliferate and produce large amounts of extracellular matrix, which accumulates in the tubular interstitium; together with tubular atrophy, this accumulation leads to interstitial fibrosis. This Review examines the molecular mechanisms of fibroblast activation and epithelial-mesenchymal transition, processes that seem to be promising targets for the prevention, or even reversal, of interstitial fibrosis and renal dysfunction associated with obstructive nephropathy.

Protective effect of new nitrosothiols on the early inflammatory response to kidney ischemia/reperfusion and transplantation in rats.

Renal ischemia/reperfusion (I/R) is characterized by severe inflammatory damage. We assessed the effect of administrating recently developed nitrosothiol compounds acting as nitric oxide (NO) donors on the production of cytokines and other markers of acute inflammatory reaction in an experimental model of warm (I/R), and in a model of cold ischemia and transplant in rats. Warm ischemia was achieved by ligation of left renal pedicle for 60 min, followed by contralateral nephrectomy. NO-donors LA-803, LA-807, LA-810 were administered i.v. (1.8 micromol/kg) during 30 min before reperfusion. Cold ischemia was achieved by preservating the kidney for 24 h in Euro Collins and grafting it in consanguineous Fisher 344/Ico rats. LA-803 was administered in the preservation fluid and in the recipient rat. Reperfusion time was 4 h in warm ischemia and 3 h in cold ischemia + transplantation. Administration of LA-803, LA-807 and, in a lower proportion, LA-810 prevented from the enhanced production of tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), and interleukin-1beta (IL-1beta), the decrease in interleukin-6 (IL-6) and interleukin-10 (IL-10), the increase in tissue level of superoxide anion (SOA) and superoxide dismutase (SOD), and the increase in neutrophil infiltration induced by warm I/R. Treatment with LA-803 in animals with renal transplantation after cold ischemia was also associated with reduced plasma levels of TNF, IFN-gamma, and IL-1beta, increased plasma levels of IL-6 and IL-10, reduced renal levels of SOA and SOD, and reduced neutrophil infiltration. These data demonstrate that systemic administration of new NO-donors with nitrosothiol structure diminished inflammatory responses in a kidney subjected to warm I/R or cold ischemia and transplantation.