Redox-sensitive signaling pathways in renal cell carcinoma.

Renal cell carcinoma (RCC) is one of the most lethal urological cancers, highly resistant to chemo and radiotherapy. Obesity and smoking are the best-known risk factors of RCC, both related to oxidative stress presence, suggesting a significant role in RCC development and maintenance. Surgical resection is the treatment of choice for localized RCC; however, this neoplasia is hardly diagnosable at its initial stages, occurring commonly in late phases and even when metastasis is already present. Systemic therapies are the option against RCC in these more advanced stages, such as cytokine therapy or a combination of tyrosine kinase inhibitors with immunotherapies; nevertheless, these strategies are still insufficient. A field poorly analyzed in this neoplasia is the status of cell signaling pathways sensible to the redox state, which have been associated with the development and maintenance of RCC. This review focuses on alterations reported in the following redox-sensitive molecules and signaling pathways in RCC: mitogen-activated protein kinases, protein kinase B (AKT)/tuberous sclerosis complex 2/mammalian target of rapamycin C1, AKT/glycogen synthase kinase 3/ß-catenin, nuclear factor κB/inhibitor of κB/epidermal growth factor receptor, and protein kinase Cζ/cut-like homeodomain protein/factor inhibiting hypoxia-inducible factor (HIF)/HIF as potential targets for redox therapy.

Isoliquiritigenin Pretreatment Induces Endoplasmic Reticulum Stress-Mediated Hormesis and Attenuates Cisplatin-Induced Oxidative Stress and Damage in LLC-PK1 Cells.

Isoliquiritigenin (IsoLQ) is a flavonoid with antioxidant properties and inducer of endoplasmic reticulum (ER) stress. In vitro and in vivo studies show that ER stress-mediated hormesis is cytoprotective; therefore, natural antioxidants and ER stress inducers have been used to prevent renal injury. Oxidative stress and ER stress are some of the mechanisms of damage involved in cisplatin (CP)-induced nephrotoxicity. This study aims to explore whether IsoLQ pretreatment induces ER stress and produces hormesis to protect against CP-induced nephrotoxicity in Lilly Laboratories Cell-Porcine Kidney 1 (LLC-PK1) cells. During the first stage of this study, both IsoLQ protective concentration and pretreatment time against CP-induced toxicity were determined by cell viability. At the second stage, the effect of IsoLQ pretreatment on cell viability, ER stress, and oxidative stress were evaluated. IsoLQ pretreatment in CP-treated cells induces expression of glucose-related proteins 78 and 94 kDa (GRP78 and GRP94, respectively), attenuates CP-induced cell death, decreases reactive oxygen species (ROS) production, and prevents the decrease in glutathione/glutathione disulfide (GSH/GSSG) ratio, free thiols levels, and glutathione reductase (GR) activity. These data suggest that IsoLQ pretreatment has a moderately protective effect on CP-induced toxicity in LLC-PK1 cells, through ER stress-mediated hormesis, as well as by the antioxidant properties of IsoLQ.

Differential behavior of NF-κB, IκBα and EGFR during the renal carcinogenic process in an experimental model in vivo.

Renal cell carcinoma (RCC) is the most common type of cancer of the adult kidney. It is generally asymptomatic even at advanced stages, so opportune diagnosis is rare, making it almost impossible to study this cancer at its early stages. RCC tumors induced by ferric nitrilotriacetate (FeNTA) in rats histologically correspond to the human clear cell RCC subtype (ccRCC) and the exposure to this carcinogen during either one or two months leads to different early stages of neoplastic development. High levels of nuclear factor kappa B (NF-κB) and epidermal growth factor receptor (EGFR) as well as low levels of NF-κB inhibitor alpha (IκBα) are frequent in human RCC, but their status in FeNTA-induced tumors and their evolution along renal carcinogenesis is unclear. On this basis, in the present study NF-κB, IκBα and EGFR behavior was analyzed at different stages of the experimental renal carcinogenesis model. Similar to patients with RCC, neoplastic tissue showed high levels of p65, one of the predominant subunits of NF-κB in ccRCC and of EGFR (protein and mRNA), as well as a decrease in the levels of NF-κB's main inhibitor, IκBα, resulting in a classic oncogenic combination. Conversely, different responses were observed at early stages of carcinogenesis. After one month of FeNTA-exposure, NF-κB activity and EGFR levels augmented; but unexpectedly, IκBα also did. While after two months, NF-κB activity diminished, but EGFR and IκBα levels remained elevated. In conclusion, FeNTA-induced tumors and RCC human neoplasms are analogues regarding to the classic NF-κB, IκBα and EGFR behavior, and distinctive non-conventional combination of changes is developed at each early stage studied. The results obtained suggest that the dysregulation of the analyzed molecules could be related to different signaling pathways and therefore, to particular effects depending on the phase of the carcinogenic process.

Acute expression of the transcription factor Nrf2 after treatment with quinolinic acid is not induced by oxidative stress in the rat striatum.

Quinolinic acid (QUIN) is an excitotoxic and pro-oxidant molecule used in the study of neurodegenerative disorders because it reproduces certain biochemical characteristics present in these diseases. The use of antioxidant molecules in the QUIN model reduces cellular damage through the nuclear factor erythroid 2-related to factor 2 (Nrf2) pathway. The Nrf2 transcription factor is considered the master regulator of antioxidant genes expression, and its activation occurs by an increase in the reactive oxygen species (ROS) levels or in the presence of electrophilic compounds. However, Nrf2 activation also occurs in an oxidative stress-independent process caused by the disruption of the Keap1-Nrf2 complex by the direct interaction of Keap1 with certain proteins, such as DPP3 and p62. The aim of this study was to evaluate the effect of QUIN on Nrf2 activation over short periods of time. QUIN administration increased Nrf2 activation at 30 min in the striatum without increasing ROS production or modifying the redox cellular state. Moreover, QUIN increased Keap1 and Nrf2 nuclear levels and increased the protein-protein interaction between Keap1 and DPP3 and Keap1 and p62 30 min after QUIN administration. Finally, we found that Nrf2 activation primarily occurs in striatal neurons. Our results show that QUIN administration in vivo stimulates Nrf2 expression and activation in the absence of oxidative stress primarily in neurons and increases the interaction of p62 and DPP3 with Keap1, which could participate in Nrf2 activation.

MAPKs' status at early stages of renal carcinogenesis and tumors induced by ferric nitrilotriacetate.

Renal cell carcinoma (RCC) is asymptomatic at early stages, and thus, initial diagnosis frequently occurs at advanced or even metastatic stages, leading to a high rate of mortality. Ferric nitrilotriacetate (FeNTA)-induced RCC model is a useful tool to analyze molecular events at different stages of the carcinogenesis process in vivo. MAPKs' alterations seem to play an important role in the development and maintenance of human RCC tumors. Based on the above, p38α/ß/γ, JNK1/2, and ERK1/2 statuses were studied at early stages of FeNTA-induced renal carcinogenesis (1 and 2 months of carcinogen treatment) as well as in tumor tissue. MAPKs showed distinct response along carcinogenesis process, either as total proteins and/or as their phosphorylated forms. While the increase in total and phospho-p38α/ß levels became lower as carcinogenesis progressed, p38γ overexpression grew. Instead, total JNK2 diminished, but JNK1 was elevated at all studied times, and p-JNK1 levels increased at early stages, but not in tumors. In contrast, p-JNK2 rose at 2 months of treatment and in tumor tissue. Increased levels of p-ERK1/2 were observed at all stages analyzed. Very interestingly, at 1 and 2 months of FeNTA treatment, no alterations in MAPKs were found in liver or lung, where no primary tumors are induced with the scheme of FeNTA administration followed here. In conclusion, MAPKs' behavior evolved differentially as renal carcinogenesis advanced, even among isoforms of the same family, but it did not change in other tissues. All this strongly suggests a role of these kinases in FeNTA-induced RCC tumor development and maintenance.

Characterization of N-diethylnitrosamine-initiated and ferric nitrilotriacetate-promoted renal cell carcinoma experimental model and effect of a tamarind seed extract against acute nephrotoxicity and carcinogenesis.

Renal cell carcinoma (RCC), the commonest malignancy in adult kidney, lacks of early signs, resulting often in metastasis at first diagnosis. N-Diethylnitrosamine (DEN)-initiated and ferric nitrilotriacetate (FeNTA)-promoted RCC may be a useful experimental model, but it is not well characterized. In this study, histological alterations and oxidative stress markers were analyzed at different times throughout RCC development, histological subtype was re-evaluated in the light of current classification, and a tamarind seed extract (TSE) effect was examined. Male Wistar rats experimental groups were control, TSE, DEN, DEN+FeNTA, and TSE+DEN+FeNTA. TSE was given 2 weeks before DEN administration (200 mg/kg) and throughout the experiment. Fourteen days after DEN treatment, two FeNTA doses (9 mg Fe/kg) for acute nephrotoxicity study, and increasing FeNTA doses (3-9 mg Fe/kg) twice a week for 16 weeks for carcinogenesis protocol, were administered. In acute study, necrosis and renal failure were observed and TSE ameliorated them. Throughout carcinogenesis protocol, preneoplastic lesions were observed since 1 month of FeNTA treatment, which were more evident at 2 months, when also renal cysts and RCC were already detected. RCC tumors were obtained without changes in renal function, and clear cell histological subtype was identified in all cases. 4-Hydroxy-2-nonenal and 3-nitro-L: -tyrosine levels increased progressively throughout protocol. TSE decreased both oxidative stress markers and, although there was no statistical difference, it delayed RCC progress and decreased its incidence (21 %). This study brings an insight of the time course events in this carcinogenesis model, identifies clear cell subtype and establishes TSE renoprotective effects.

El concepto actual del sistema renina-angiotensina / Current concept of the renin-angiotensin system

El concepto tradicional del sistema renina-angiotensina (SRA) es el de un sistema andocrino que juega un papel central en el balance de agua y electrólitos y en la homeostasis de la presión arterial. El angiotensinógeno y la renina, secretados por el hígado y el riñón, respectivamente, interactúan para formar angiotensina l, la cual, a su vez, es hidrolizada por la enzima convertidora de angiotensina l para formar el péptido biológicamente activo del sistema, angiotensina II. Debido a que esta última enzima se localizó primero en la vasculatura pulmonar, se pensó que la angiotensina II se producía sólo en ese sitio y que posteriormente llegaba a sus órganos blanco por medio de la circulación. Sin embargo, gracia al desarrollo de nuevas técnicas bioquímicas y de biología molecular se ha podido documentar la presencia de los componentes del SRA, incluyendo sus ácidos ribonucleicos mensajeros (ARNm), en múltiples tejidos como riñón, vasos sanguíneos, corazón, cerebro y glándulas adrenales, entre otros. La presencia de los ARNm sugiere fuertemente que las proteínas del SRA se sintetizan localmente. Esto ha llevado a proponer que el SRA puede tener funciones paracrinas, autocrinas, e incluso intracrinas, además de las endocrinas ya bien conocidad, es decir, que el SRA puede estar involucrado en alguna función específica de cada tejido. Estos datos amplían el concepto tradicional del SRA ya que, además de su amplia distribución, se ha comprobado que solo SRA locales se regulan independientemente del sistema circulante. Por otra parte, estudiando el comportamiento de los SRA circulante y tisular ante diferentes situaciones patológicas como la hipertensión, se ha propuesto que la principal función del SRA circulante es mantener la homeostasis cardiorrenal a corto plazo, y que el control tónico de la resistencia vascular y de la función tisular local(por ejemplo, en adrenal y riñón) está reculada por los SRA locales. Finalmente, estas observaciones demuestran que el SRA tisular tiene un papel muy importante al igual que el SRA circulante.

Obtención de anticuerpos contra angiotensina II / Obtention of antibodies against angiotensin II

Se obtuvieron anticuerpos contra angiotensina II (ANG II) para medir la concentración de este octapétido por radioinmunoanálisis (RIA). La NG II se acopló a proteínas de alto peso molecular, se emulsionó con adyuvante de Freund y se inyectó intradérmicamente. Las cuatro primeras inmunozaciones se dieron a intervalos semanales. Las proteínas a las cuales se acopló el octapéptido para la primera, segunda, tercera y cuarta inmunización fueron: tiroglobulina, albúmina sérica bovina, ovoalbúmina y tiroglobulina, respectivamente. La quinta inmunización se dio doce semanas después con ANG II acoplada a hemocianina. El título del anticuerpo se determinó por RIA. Uno de los conejos inmunizados produjo un anticuerpo altamente específico, con un título de 1: 10,000 y una sensibilidad de 1.9 picogramos de ANG II. La especificidad del anticuerpo se evaluó midiendo el porcentaje en que este anticuerpo cruza péptidos relacionados estructuralmente. La reacción cruzada con [Sar, Tres] ANG II, [Sar, Gli] ANG II, [Sar, Ile] ANG II, [Val] ANG I y angiotensinógeno fue indetectable. La reacción cruzada con [Sar, Ala] ANG II, angiotensina III, [Des-Asp] ANG I, [Val] ANG II y angiotensina I fue de 3.3, 8.3, 0.2, 3.1 y 1.0% respectivamente. El anticuerpo obtenido es adecuado para medir directamente la ANG II en el plasma de ratas, así como de otras especies tales como el perro, el conejo, el ratón y el humano, cuya secuencia de aminoácidos es idéntica

Niveles de angiotensina II en ratas sometidas a estímulos del sistema renina-angiotensina / Angiotensin II concentration in rats with different stimulus that modify the renin-angiotensin system

En este trabajo se midieron los niveles de angiotensina II (ANG II) en ratas sometidas a estímulos del sistema renina angiotensina (SRA). Los estímulos usados fueron: isoproterenol, clonidina, propranolol, captopril, anestesia, deshidratación, binefrectomía, ligación ureteral y cloro de sodio al 1 y 2% en el agua de beber. En el plasma de estas ratas se midió además de la ANG II, la angiotensina I (ANG I), la actividad de renina (APR) y la concentración de renina (CPR), de angiotensinógeno (CPA) y de aldosterna (CPALDO). La anestesia, la deshidratación, el isoproterenol y el captopril aumentaron la renina y la ANG II, pero no la CPALDO. Por el contrario, la binefrectomía, el cloruro de sodio al 2%, la clonidina y el propranolol disminuyeron la renina. La clonidina disminuyó la ANG II y la binefrectomía al aumentó. El cloruro de sodio al 2% disminuyó la CPALDO y la binefrectomía la aumentó. La ligación ureteral y el cloruro de sodio al 1% no cambinaron la renina ni la ANG II, sin embargo la ligación ureteral aumentó la CPALDO. La binefrectomía, la ligación ureteral y el cloruro de sodio al 1 y 2% aumentaron la CPALDO y el captopril la disminuyó. No con todos los estímulos hubo un cambio paralelo de la ANG II y la actividad del SRA circulante. Esto puede ser explicado por cambios en la actividad del SRA tisular, por cambios en el catabolismo de la ANG II y/o por la actividad de otras enzimas, no relacionadas con el SRA, que producen ANG II