Urinary cytokine profiles according to the site of blockade of the renin-angiotensin system in nephrectomized rats / Perfil de citocinas urinárias de acordo com o local de bloqueio do sistema renina angiotensina em ratos nefrectomizados

Abstract Introduction: It is still unknown how the pharmacological inhibition of the Renin Angiotensin System (RAS) impacts the levels of inflammation and fibrosis biomarkers. Objective: This study sought to evaluate the effect of enalapril, candesartan and aliskiren on urinary levels of cytokines in a model of chronic kidney disease (CKD). Methods: Male Wistar rats were submitted to surgical removal of ¾ of renal parenchyma to induce CKD (¾ nephrectomy), or subjected to sham surgery (control). Animals were then randomized into five groups: Sham surgery receiving vehicle; ¾ Nephrectomy receiving vehicle; ¾ Nephrectomy receiving enalapril (10 mg/kg); ¾ Nephrectomy receiving candesartan (10 mg/kg) and ¾ Nephrectomy receiving aliskiren (10 mg/kg). Urine output, water intake, mean arterial pressure (MAP) and urinary concentrations of creatinine, urea, albuminuria, Na+, K+, interleukin (IL) -1β, IL-6, IL-10 and transforming growth factor beta (TGF-β) were measured. Results: Nephrectomy significantly impaired renal function, increased MAP and altered the levels of all evaluated cytokines in urine. Enalapril, candesartan and aliskiren improved renal function and decreased MAP and IL-6 when compared to vehicle-treated nephrectomized group. Candesartan and aliskiren decreased IL-1β, while only candesartan reduced TGF-β and only aliskiren increased IL-10. Conclusion: Enalapril, candesartan and aliskiren presented similar effects on improving renal function and reducing MAP and urinary levels of IL-6 in rats with CKD. On the other hand, cytokine profile differed according to the treatment, suggesting that differential mechanisms were triggered in response to the site of RAS blockade.

Resumo Introdução: Ainda não se sabe como a inibição farmacológica do Sistema Renina Angiotensina (SRA) afeta os níveis de biomarcadores de inflamação e fibrose. Objetivo: Este estudo pretendeu avaliar o efeito de enalapril, candesartan e alisquireno sobre os níveis urinários de citocinas em um modelo de doença renal crônica (DRC). Métodos: Ratos Wistar machos foram submetidos à remoção cirúrgica de ¾ do parênquima renal para induzir DRC (nefrectomia), ou submetidos à cirurgia fictícia (controle). Animais foram então randomizados em cinco grupos: Cirurgia fictícia recebendo veículo; Nefrectomia recebendo veículo; Nefrectomia recebendo enalapril (10 mg/kg); Nefrectomia recebendo candesartan (10 mg/kg) e Nefrectomia recebendo alisquireno (10 mg/kg). Débito urinário, ingesta hídrica, pressão arterial media (PAM) e concentrações urinárias de creatinina, ureia, albumina, Na+, K+, interleucina (IL) -1β, IL-6, IL-10 e fator de transformação e crescimento beta (TGF-β) foram medidas. Resultados: A nefrectomia comprometeu significativamente a função renal, aumentou a PAM e alterou os níveis de todas as citocinas avaliadas na urina. Enalapril, candesartan e alisquireno melhoraram a função renal e diminuíram a PAM e a IL-6 quando comparado aos grupo de animais nefrectomizados tratados com veículo. Candesartan e alisquireno reduziram IL-1β, enquanto somente candesartan diminuiu o TGF-β e somente alisquireno aumentou a IL-10. Conclusão: Enalapril, candesartan e alisquireno apresentaram efeitos similares em relação à melhora da função renal e redução da PAM e dos níveis urinários de IL-6 em ratos com DRC. Por outro lado, o perfil de citocinas diferiu de acordo com o tratamento, sugerindo que diferentes mecanismos sejam desencadeados em resposta ao local de bloqueio do SRA.

A memória hiperglicêmica no rim diabético: marcas metabólicas, moleculares e epigenéticas / The hyperglycemic memory in diabetic kidney: metabolic, molecular, and epigenetic marks

A nefropatia diabética (ND) é uma das complicações microvasculares do diabetes e consiste no dano ao parênquima renal por consequência de uma série de fatores hemodinâmicos e moleculares. A ocorrência de ND e de outras complicações mesmo em indivíduos sob adequado controle glicêmico tem sido associada a um fenômeno conhecido como memória metabólica. Neste trabalho foram investigadas vias bioquímicas e moleculares persistentemente alteradas no rim de animais diabéticos tratados após um período inicial de hiperglicemia, com o propósito de entender os mecanismos envolvidos na memória metabólica. Para tanto, ratos com diabetes induzida por estreptozotocina foram mantidos hiperglicêmicos durante 4 semanas (período curto) ou 12 semanas (período longo) e posteriormente tratados com insulina isoladamente ou combinada com metformina (100mg/kg/dia) durante as 4 (período curto) ou 12 (período longo) semanas seguintes. Todos os animais tratados tiveram os seus níveis glicêmicos e função renal normalizados. Os tratamentos também foram capazes de normalizar os níveis elevados de malonaldeído no rim, bem como a excreção aumentada dos adutos de DNA 8-oxo-2'-desoxiguanosina (8-oxodG) e N2-carboxietil-2'- desoxiguanosina (CEdG) na urina observados nos animais diabéticos. Níveis aumentados de 8-oxodG foram detectados em DNA mitocondrial (mtDNA), mas não em DNA nuclear, de animais diabéticos apenas no período curto de estudo e também foram normalizados após o controle glicêmico. Nós identificamos uma via gradualmente alterada durante o curso do diabetes que permanece persistentemente alterada após o controle glicêmico tardio. Essa via compreende um declínio precoce do clearance de ácido úrico e expressão da pAMPK, seguida pelo acúmulo de fumarato, expressão aumentada de TGF-ß, expressão reduzida de PGC-1α e redução da metilação e hidroximetilação do mtDNA. A redução persistente do clearance de ácido úrico em animais diabéticos tratados pode sustentar as alterações bioquímicas renais prolongadas observadas após o controle glicêmico, e essa regulação é provavelmente mediada pela redução sustentada da expressão de pAMPK e pela indução de inflamação. Este trabalho propõe a primeira consideração do possível papel da hiperuricemia e das alterações bioquímicas subjacentes como parte da memória metabólica na nefropatia diabética

Diabetic nephropathy is one of the diabetes microvascular complications, and it consists on the damage to the renal parenchyma due to several hemodynamic and molecular factors. The occurrence of diabetic nephropathy and other complications even in those individuals under tight glycemic control has been associated to a phenomenon known as metabolic memory. Here we investigated biochemical and molecular pathways persistently altered in the kidney of diabetic animals treated after a previous period of hyperglycemia, aiming to understand underlying mechanisms in metabolic memory. Streptozotocin-induced diabetic rats were maintained hyperglycemic during 4 (short period) or 12 weeks (long period), and then they were treated with insulin alone or combined with metformin (100 mg/kg/day) for the following 4 or 12 weeks, respectively. All the treated animals had them glycemic levels and renal function normalized. The treatments were also able to control enhanced kidney malondialdehyde levels, as well as the increased urine excretion of the DNA adducts 8-oxo-2'- deoxyguanosine (8-oxodG) and N2-carboxyethyl-2'-deoxyguanosine seen in diabetic animals. Increased levels of 8-oxodG were detected in mitochondrial DNA, but not in nuclear DNA of diabetic animals in the short period, and were also recovered after glycemic control. We have identified a kidney pathway that is gradually altered during the course of diabetes and remains persistently changed after late glycemic control. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-ß expression, reduced PGC-1α expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This work proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy

Efecto del aliskireno sobre la angiogénesis en modelo de membrana alantocoriónica (MAC) de pollo / Effect of aliskiren on angiogenesis in chick embryo chorioallantoic membrane (CAM) assay

La acción antiangiogénica de los inhibidores del receptor de angiotensina II (ARA II), ha sido documentada previamente, sin embargo, no ha sido descrita la relación entre angiogénesis e inhibidores directos de la renina (DRIs), los cuales participan regulando el sistema renina-angiotensina-aldosterona (SRAA). El objetivo fue demostrar el efecto antiangiogénico de aliskireno, un DRI, en membranas alantocoriónicas (MAC) de pollo, para lo cual fueron instilados aliskireno y enalapril sobre MAC en distintas concentraciones para realizar su comparación posterior. En secciones histológicas seriadas se registró el número de vasos sanguíneos presentes en 9000 µm2 bajo microscopio de luz a máximo aumento, y se realizó análisis estadístico utilizando ANOVA y el test de Tukey para demostrar posibles diferencias. Los receptores tratados con aliskireno, en ambas concentraciones utilizadas, presentaron menor densidad vascular, en comparación con los controles, siendo ésta estadísticamente significativa a mayor concentración. Aliskireno en concentraciones altas tiene un efecto antiangiogénico en un modelo experimental de MAC. Este hallazgo plantea la necesidad de estudios posteriores, dada la proyección que podría tener el uso inhibidores directos de la renina. A partir de estos resultados, se podría pensar en la factibilidad del uso de aliskireno para la modulación de la angiogénesis en diversas enfermedades crónicas no transmisibles.

Angiogenesis is the formation of new blood vessels from pre-existing ones. Antiangiogenic effect of angiotensin receptor blockers has been reported, however, the relationship between direct renin inhibitors and angiogenesis has not been well described. To assess the antiangiogenic effect of aliskiren, a direct renin inhibitor, on chick embryo chorioallantoic membrane (CAM) assay. Aliskiren and enalapril were instilled in different concentrations and compared. In serial histological sections, the number of blood vessels per 9000 µm2 area under observation through optical microscope using maximum zoom, was registered. Statistical analysis using Anova and Tukey test in order to show possible differences, was performed. Receptors treated with aliskiren presented lower vascular density, which was statistically significant when a higher concentration was administered. High concentrations of aliskiren have an antiangiogenic effect on CAM assay. This finding means further studies are needed, because of the usefulness direct renin inhibitors could have. These results, also, might enhance the possibility of using aliskiren for regulating angiogenesis in the context of non-transmissible chronic diseases.

Renin-angiotensin system blockers regulate the metabolism of isolated fat cells in vitro

Due to the presence of the renin-angiotensin system (RAS) in tissues and its specific influence on white adipose tissue, fat cells are possible targets of pharmacological RAS blockers commonly used as anti-hypertensive drugs. In the present study, we investigated the effects of different RAS blockers on fat cell metabolism, more specifically on lipolysis, lipogenesis and oxidation of energy substrates. Isolated primary adipocytes were incubated with different RAS blockers (aliskiren, captopril and losartan) in vitro for 24 h and lipolysis, lipogenesis and glucose oxidation capacities were determined in dose-response assays to a β-adrenergic agonist and to insulin. Although no change was found in lipolytic capacity, the RAS blockers modulated lipogenesis and glucose oxidation in a different way. While captopril decreased insulin-stimulated lipogenesis (−19% of maximal response and −60% of insulin responsiveness) due to reduced glucose derived glycerol synthesis (−19% of maximal response and 64% of insulin responsiveness), aliskiren increased insulin-stimulated glucose oxidation (+49% of maximal response and +292% of insulin responsiveness) in fat cells. Our experiments demonstrate that RAS blockers can differentially induce metabolic alterations in adipocyte metabolism, characterized by a reduction in lipogenic responsiveness or an increase in glucose oxidation. The impact of RAS blockers on adipocyte metabolism may have beneficial implications on metabolic disorders during their therapeutic use in hypertensive patients.

Concomitant inhibition of renin angiotensin system and Toll-like receptor 2 attenuates renal injury in unilateral ureteral obstructed mice

BACKGROUND/AIMS: There has been controversy about the role of Toll-like receptor 2 (TLR2) in renal injury following ureteric obstruction. Although inhibition of the renin angiotensin system (RAS) reduces TLR2 expression in mice, the exact relationship between TLR2 and RAS is not known. The aim of this study was to determine whether the RAS modulates TLR2. METHODS: We used 8-week-old male wild type (WT) and TLR2-knockout (KO) mice on a C57Bl/6 background. Unilateral ureteral obstruction (UUO) was induced by complete ligation of the left ureter. Angiotensin (Ang) II (1,000 ng/kg/min) and the direct renin inhibitor aliskiren (25 mg/kg/day) were administrated to mice using an osmotic minipump. Molecular and histologic evaluations were performed. RESULTS: Ang II infusion increased mRNA expression of TLR2 in WT mouse kidneys (p < 0.05). The expression of renin mRNA in TLR2-KO UUO kidneys was significantly higher than that in WT UUO kidneys (p < 0.05). There were no differences in tissue injury score or mRNA expression of monocyte chemotactic protein 1 (MCP-1), osteopontin (OPN), or transforming growth factor beta (TGF-beta) between TLR2-KO UUO and WT UUO kidneys. However, aliskiren decreased the tissue injury score and mRNA expression of TLR2, MCP-1, OPN, and TGF-beta in WT UUO kidneys (p < 0.05). Aliskiren-treated TLR2-KO UUO kidneys showed less kidney injury than aliskiren-treated WT UUO kidneys. CONCLUSIONS: TLR2 deletion induced activation of the RAS in UUO kidneys. Moreover, inhibition of both RAS and TLR2 had an additive ameliorative effect on UUO injury of the kidney.