Soy licenciado en Biología por la Universidad de Murcia, con un máster en Ensayos Clínicos. Terminé mi doctorado en el programa de Ciencias Biomédicas de la Universidad de Murcia en el año 2008. 20 años de experiencia en investigación en ámbito académico e industria privada en los que he desarrollado conocimientos en todas las áreas de investigación básica y clínica en el marco de las enfermedades cardiovasculares y renales, síndrome metabólico e hipertensión. Obtuve un puesto de investigación postdoctoral competitivo en el Children's National Medical Center en Washington, DC, en 2009. Continué con mi segundo puesto de becario postdoctoral en el División de Nefrología de la Universidad de Maryland (Baltimore) y conseguí un puesto estable como científico de investigación en la Universidad George Washington 3 años después. He trabajado como científico senior en el Children's National Health system, departamento de Medicina Genética, dirigiendo un laboratorio en el estudio de los mecanismos y vías involucradas en la patogénesis de la enfermedad renal poliquística y las nefropatías de la diabetes. He realizado un curso de formación y trabajado en la empresa GeneDX y en el diagnostico genetico y el análisis de la patogenicidad de variantes encontradas en pacientes. He obtenido el Contrato Saavedra Fajardo en el año 2020 de la Fundación Séneca (Murcia, España) para continuar mi línea de investigación en enfermedades renales durante los próximos 3 años, como investigador principal en el BioMedical Research Institute of Murcia (IMIB-Arrixaca) (Murcia, España). Tengo experiencia en el diseño y desarrollo de ensayos clínicos, diagnostico genético y larga experiencia en el laboratorio de proteómica y análisis molecular, técnicas de biología, y el manejo de animales de laboratorio y cultivos celulares. En este momento, tengo 23 publicaciones científicas en Pudmed publicadas en los últimos años, de las cuales 9 soy autor de correspondencia, y 4 en cuatro de ellas soy último autor, demostrando mi capacidad de desarrollar proyectos como investigador principal. Soy miembro del equipo de investigación de cinco proyectos de investigación financiados por el NIH y un Proyecto de transferencia financiado por una empresa privada sobre un tratamiento experimental en hipertensión. En dos ocasiones, obtuve financiación como investigador principal de la Fundación Nacional del Riñón de Maryland en los años 2013-2014, y 2014-2015. Tengo cuarenta y ocho contribuciones a reuniones, incluyendo pósters y presentaciones orales, en las reuniones más prestigiosas del campo. He sido galardonado con seis premios a la excelencia investigadora y un premio competitivo por la asistencia al congreso, y mi trabajo ha sido seleccionado diez veces como primer o último autor en presentaciones orales de selección competitiva en las mejores reuniones del mundo de la enfermedad cardiovascular, renal y de la hipertensión, lo que demuestra la calidad investigadora de mi trabajo. Dirijo un proyecto que muestra los efectos protectores sobre las enfermedades renales de un péptido llamado ND-13. La Universidad George Washington y la Universidad de Tel Aviv proporcionaron la financiación para una patente sobre los efectos protectores del ND-13 como posible tratamiento para prevenir la fibrosis e inflamación renal. Tengo una red establecida de colaboraciones con varios grupos de investigación en el campo. He sido co-mentor de numerosos estudiantes durante mi período como científico de investigación. Soy revisor de importantes revistas en el campo como la hipertensión y miembro de varias asociaciones de científicos como y miembro de la junta directiva de la Asociación de Científicos Españoles en los Estados Unidos (ECUSA) y he sido por 5 años director de la sección de Política Científica.
Biología molecular, celular y genética, Fisiología y Farmacología, Medicina
BioMedical ResearchInstitute of Murcia (IMIB-Arrixaca)
Title: Role of DJ-1/Nrf2 pathway in the prevention of diabetic nephropathy.
Diabetic nephropathy (DN) is associated with cardiovascular disease and is the most common cause of renal failure worldwide. Currently, the development of the renal disease in diabetes patients cannot be prevented with the current pharmacological therapies. Therefore, new approaches of therapy and targets are urgently required (Higgins 2014).
Renal oxidative stress and inflammation are two of the most important factors involved in the pathogenesis of DN and other cardiovascular complications in diabetes (Giacco 2010). Inflammation, and their consequence oxidative stress, is considering mayor factor to trigger fibrosis and key in the development and progression of DN (Navarro-González 2008). Members of the inflammasome family such as the nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) are involved to the inflammatory immune response, stimulates the inflammatory cascade reaction, which is crucial for the pathogenesis of DN (Qiu YY 2016).
DJ-1 (also known as Park 7), which was initially identified as an autosomal recessive gene associated with Parkinson disease, is expressed in brain, heart, kidney, liver, pancreas, and skeletal muscle in rodents and humans (Nagakubo 1997).DJ-1 is a multifunctional oxidative stress response protein that functions as a redox-sensitive chaperone with intrinsic antioxidant properties, especially in the mitochondria, and regulates the expression of several antioxidant genes (Liu 2008, Zhou 2005). DJ-1 is present mainly in the cytoplasm and to a lesser extent in the mitochondria; however, upon an oxidant challenge more of the DJ-1 translocate into the mitochondria and protect mitochondrial function (Junn 2009).
Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates the expression of several antioxidant genes. Nrf2 has also been reported to inhibit the development and progression of several diseases affecting the kidney (Shelton 2013). Nrf2 attenuates NFkappaB-inflammatory response, and suppresses macrophage inflammatory response (Li 2008, Kobayashi 2016).We have reported that renal-selective silencing of DJ-1 in mice impairs D2R-mediated antioxidant response and increases blood pressure (BP) that is associated with decreased renal Nrf2 expression and activity (Cuevas 2012, Cuevas 2015). In addition, mice with DJ-1 selectively silenced in the kidney and mice with germline deletion of DJ-1 (DJ-1-/-) have high BP, renal damage and decreased expression and activity of Nrf2 (Cuevas 2015), suggesting that DJ-1 can inhibit renal reactive oxygen species (ROS) production, at least in part, via the activation of Nrf2-antioxidant genes. It has reported recently that activation of DJ-1/Nrf2 pathway could be involved in the pathogenesis of diabetic nephropathy in rats (Sun 2016).
Therefore, the goal of this study is determine if DJ-1/Nrf2 pathway may be a new approach of treatment on renal diseases.
We have reached an agreement to collaborate with Dr. Daniel Offen, Professor of Neuroscience, Tel-Aviv University, Israel. Dr. Offen's laboratory has developed a peptide from the amino acid sequence of DJ-1. The active areas of DJ-1 were studied and the most effective protected area was selected; a 13 amino acid chain from DJ-1. To achieve cell permeability, the 13 amino acid chain was fused to a 7-amino acid cell-penetrating peptide. The resulting 20 amino acid compound was named: ND-13. It has been repeatedly demonstrated that neuronal cultures treated with ND-13 are protected from the effects of exposure to Parkinson's disease (PD)-relevant neurotoxins and other diseases (Glat 2015, Lev 2015a, Lev 2015b). ND-13 reduces apoptosis and inactivates the pro-apoptotic caspase-3 in neuronal cell lines exposed to neurotoxic insults. The cells treated with ND-13 activated the Nrf2 pathway, resulting in the increased expression of Nrf2-induced antioxidant genes (Lev 2015b), similar to our findings in the kidney (Cuevas 2015). This project will study the effect of ND-13 on the prevention of DN.
Antioxidant treatment in human diseases: The redox state is the balance between the production of free radicals, and highly reactive species and antioxidant defenses. Disturbances in the normal redox state lead to overproduction of ROS which may damage all components of the cell (Craige 2011). Oxidative stress is involved in the development of numerous cardiovascular diseases, such diabetic nephropathy (DN). Inflammation and oxidative stress are known to cause renal damage, and there are numerous reports supporting the benefits of antioxidant treatment in kidney diseases (Jun 2013, Pergola 2011). However, despite the numerous studies in basic science and animal models, it has been difficult to demonstrate the role of the oxidative stress and ROS production in the pathogenesis of human diseases. Several antioxidant drugs have been studied in clinical trials for the last 20 years to determine if they can prevent the deleterious effects of oxidative stress in different cardiovascular and renal diseases. However, adverse effects have been described in several studies. For example, α-tocopherol and β-carotene treatment (5.3 years) in men with prior myocardial infarction did not show protective effects on major coronary events but was associated with more deaths from coronary heart disease (Rapola 1997). A meta-analysis of studies in more than 135,000 subjects showed that high doses of vitamin E increased mortality (Miller 2005). Antioxidant treatment in hypertensive humans has shown benefit in a limited number of patients (Duffy 1999, Mullan 2002). However, larger studies have not found a clear beneficial effect of antioxidant vitamins on the development or treatment of high blood pressure (Czernichow 2005, Kim 2002). These results have questioned the oxidative stress theory in human diseases. What is evident from these data is that the role of the ROS in the cardiovascular system is more complex than expected and new approaches have to be formulated to resolve these controversies.
Side effects of antioxidant treatment in human: Several studies have shown evidence that oxidative stress could also have beneficial effects in cell function and the excessive antioxidants could have deleterious consequences. The undesirable effects of chronic antioxidant treatment may be due to the fact that ROS is important in cellular signaling (Cuevas 2019). One interesting study has shown that in physical exercise, the small but continuous stimulation of ROS production enhances defenses and induces the expression of antioxidant enzymes, and vitamin C administration prevents this beneficial effect of physical exercise in rats (Gomez-Cabrera 2008). In this context, it has been described that impaired local pro-inflammatory response in the adipocyte tissue could have deleterious consequences by increasing lipid accumulation (Gancz 2012). Moreover, a master transcription factor of pro-inflammatory proteins such as NFĸB could have beneficial effects dependent on the timing, duration of activation, and cellular context (Gordon 2011).
Enzymes that produce ROS can regulate the activity of the several proteins that oxidize the SH of cysteine groups (Huot 1997, Chung 2013, Evans 2003), indicating that ROS could act as a second messenger in the regulation of different cellular functions. In addition to this, some reports have shown that H2O2 is an important ROS product in the kidney, as it promotes endothelial angiogenesis (Craige 11) which protects against chronic load-induced cardiac stress (Zhang 2010). ROS is the main regulator of the vasoconstriction of the afferent arteriole, ROS constricting the afferent arteriole when the SBP is increased, thus, maintaining a relatively constant glomerular capillary pressure which protects against glomerular damage even if blood pressure continues to be elevated (Bidani 2009). Long-term antioxidant expression could increase the dilatation of renal afferent arterioles, increasing the glomerular capillarity pressure and producing glomeruli damage which impairing renal function. Albuminuria is marker of glomeruli function and it is increased is some patients treated long term antioxidant medication such as bardoxolone (Rossing 2013, Himmelfarb 2013). Therefore, moderate but not excessive ROS production could be protective against renal injury and long-term inhibition of ROS production could have deleterious consequences (Cuevas 2019).
Nrf2 activation in renal diseases:Nrf2 is known for its ability to regulate the expression of several enzymes, the effects of several drugs that increased Nrf2 expression have been proved in cardiovascular and renal diseases. Nrf2 inducers such as resveratrol (Tomé-Carneiro 2013) and bardoxolone (Pergola 2011) were initially shown to have protectives effect in cardiovascular diseases. Bardoxolene improved renal function in humans with advanced chronic kidney disease and type 2 diabetes. However, bardoxolone was withdrawn from clinical trials because of increased rates of heart failure and cardiovascular events, hypertension, and albuminuria, among others (Rossing 2013, Himmelfarb 2013).
Our published data have shown that SBP and renal MDA expressions, which are increased in DJ1-/- mice, were normalized by bardoxolone but had no effect in their wild-type littermates (Cuevas 2015). Bardoxolone treatment increased renal Nrf2 expression in both wild-type (118±17%) and DJ1-/- mice (112±49), suggesting that the effect of bardoxolone is due to an increase in Nrf2 expression that normalized oxidative stress-mediated hypertension caused by DJ-1 silencing (Cuevas 2015). However, bardoxolone caused widespread renal tubular and glomerulus damage that was much greater in in DJ1-/- mice than wild-type littermates (data do not show). This side effect of bardoxolone has been described previously in rat models (Zoja 2013, Vaziri 2015). Our previous reports showed that DJ-1 does not increase Nrf2 expression activity in normal conditions, because DJ-1 avoids the Nrf2 degradation increasing his activity only in pathological conditions where Nrf2 pathway is activated (Cuevas 2015) which could avoid the undesirable consequences of chronic Nrf2 activation. Therefore, in this study we will also study the possible pathways involved in these side effects of chronic activation of Nrf2. We believe that upregulation of DJ-1 via ND-13 could be an appropriate approach to increased Nrf2 and minimize the side effects produced by other Nrf2 inducers such as bardoxolone.
Preliminary data:Unilateral ureter obstruction (UUO) is a useful model to examine the mechanisms involved in renal tubule interstitial fibrosis in vivo (Chevalier 2009). UUO was used as a model of renal disease to determine if ND-13 has a protective effect. Our preliminary data show that ND-13 treatment prevented the fibrosis in the UUO model in C57Bl/6 control mice but not in DJ-1-/- mice, suggesting that ND-13 could have a role to prevent connective tissue deposition in the kidney parenchyma, but not to reverse this process (Figure 1A). The amount of urinary NGAL (aka lipocalin and oncogene 24p3), a marker of renal injury, was also increased by UUO which was prevented by ND-13 (Figure 1B). UUO was associated with an increase in the renal mRNA expression of the inflammatory markers TNF alpha and IL-6, the fibrosis marker Collagen 1a1, and TGF beta, which is an important factor associated with both renal inflammation and fibrosis (Sutariya 2016). The increases in the renal expressions of TNF alpha and IL-6, and TGF beta associated with UUO were decreased by ND-13 in both C57Bl/6 control mice and DJ-1-/- mice; ND-13 also prevent the increased of Col1al caused by UUO in C57Bl/6 but not DJ-1-/- mice (Figure 2). In addition, pre-treatment of ND-13 prevent the papillary necrosis induced by UUO in C57Bl/6, evidencing the capacity of ND-13 to reduces apoptosis as have been demonstrate in others tissues (Lev 2015b) Therefore, our preliminary data suggest that ND-13 has protective effects on renal injury, fibrosis and inflammation which are considered as crucial mechanisms in the pathogenesis of several renal diseases.
Methodology section.
Specific aim 1will study the protective role of ND-13 on renal inflammation, fibrosis and renal injury in DN.
Hypothesis.ND-13 has protective properties on oxidative stress and inflammation associated with the pathogenesis of DN.
Specific Aim 1.1will test the hypothesis that ND-13 inhibits fibrosis, renal injury and inflammation and attenuates the pathology in DN.
Methods. Streptozotocin (STZ) induces beta-cell damage and diabetes in mice. Diabetes mice model STZ induced has been successfully evaluated by insulin treatment and is considered as good model to study the human renal damage in diabetes (Islam 2013). DN will be induced in C57Bl/6 mice via injection of STZ (50 mg/kg, pH 4.5, dissolved in sodium citrate) as has been reported previously (Yang 2015). The experiments will be perform with 3 groups, one group control, one group with DN induced STZ and treated with scrambled peptide and other group with DN mice induced by STZ and treated by ND-13.
The mRNA and protein expression of caspase-1 as well as interleukin (IL)-1β, IL-18 and the activation of NLRP3 inflammasome will be determine in all groups to study the role of this pathways in DN and the effects of ND-13 on the inactivation of the NLRP3 inflammasome. Infiltration of CD4, CD8 cells and macrophages in the kidney sections, Nrf2 expression and activity, mRNA and protein expressions of pro-inflammatory factors, markers of the vital mitochondrial processes, such as mitophagy, fusion and fission and mitochondrial biosynthesis will be quantified. MtDNA will be quantifying in plasma and urine of the all animals. Renal morphology, fibrosis, urinary oxidative stress markers, and micro-albumin in urine, creatinine concentration in serum and urine, KIM-1, microalbuminuria and NGAL, ATP levels in renal cortical tissue will be also determined. Insulin and creatinine clearances will be used to estimate glomerular filtration rate. The levels of Glucose in blood also will be determined.
Specific Aim 1.2will determine the mechanism involved in the ND-13 protective effects on renal diseases. Methods. Human renal proximal tubular treated with ND-13 in the presence or not of H2O2, or containing 20 mM mannitol will be study as described previously (Czajka 2015) to determine the consequences of the oxidative stress induced by H2O2 and high glucose concentration on cells functions and Nrf2 activity. These experiments will help to determine the mechanisms involved on the protective effect of ND-13. The expression of inflammatory factor, mitochondrial function markers, Nrf2 expression activity, oxidative stress markers and mtDNA levels will be measured in cell homogenates.
Specific aim 2in order to determine the possible translational application of these finds in human, the renal and cardiovascular side effects of ND-13 compare with bardoxolone will study in long term period of treatment in C57Bl/6 mice. Hypothesis. ND-13, on the contrary that Bardoxolone, has not side effects on renal and cardiovascular function in mice. Methods. C57Bl/6 mice will be treated with vehicle, ND-13 and bardoxolone for 20 weeks as has been described in previous reports (Cuevas 2015). The markers described in specific aim 1 will be determined in all these animals.
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1. New method to silencing target genes in the kidney.
Publicación: Cuevas S, Zhang Y, Yang Y, Escano C, Asico L, Jones JE, Armando I, Jose PA. Role of renal DJ-1 in the pathogenesis of hypertension associated with increased reactive oxygen species production. Hypertension. 2012 Feb;59(2):446-52.
2. Dopamine 2 Receptors regulate the expression of DJ-1 in the Kidney, which is necessary to keep the renal redox balance and normal blood pressure.
Publicación: Cuevas S, Zhang Y, Yang Y, Escano C, Asico L, Jones JE, Armando I, Jose PA. Role of renal DJ-1 in the pathogenesis of hypertension associated with increased reactive oxygen species production. Hypertension. 2012 Feb;59(2):446-52
3. DJ-1 up regulates Nrf2 in the kidney only in pathological conditions.
Publication:Cuevas S, Yang Y, Konkalmatt P, Asico L, Feranil J, Jones J, Armando I, Jose PA .Nrf2 mediates the Oxidative-Dependent Hypertension associated to the Depletion of Renal DJ-1. Hypertension. Hypertension. 2015 Jun;65(6):1251-7.
4. Novel mechanic of blood pressure regulation that demonstrate that Antioxidants Can Induce Salt-Sensitive Hypertension.
Publication: Cuevas S, Asico LD, Jose PA, Konkalmatt P. Renal Hydrogen Peroxide Production Prevents Salt-Sensitive Hypertension J Am Heart Assoc. 2020 Jan 7;9(1):e013818.
5. Role of ND-13 in the prevention of renal diseases.
Publication: De Miguel C, Kraus AC, Saludes MA, Konkalmatt P, Ruiz Domínguez A, Asico LD, Latham PS, Offen D, Jose PA, Cuevas S. Int J Mol Sci. 2020 Sep 24;21(19):7048. doi: 10.3390/ijms21197048.
mechanisms in the pathogenesis of kidney injury. Thus, ND-13 may be a new therapeutic approach to prevent renal diseases.
