RICORS2040 : The need for collaborative research in chronic kidney disease

Chronic kidney disease (CKD) is a silent and poorly known killer. The current concept of CKD is relatively young and uptake by the public, physicians and health authorities is not widespread. Physicians still confuse CKD with chronic kidney insufficiency or failure. For the wider public and health authorities, CKD evokes kidney replacement therapy (KRT). In Spain, the prevalence of KRT is 0.13%. Thus health authorities may consider CKD a non-issue: very few persons eventually need KRT and, for those in whom kidneys fail, the problem is 'solved' by dialysis or kidney transplantation. However, KRT is the tip of the iceberg in the burden of CKD. The main burden of CKD is accelerated ageing and premature death. The cut-off points for kidney function and kidney damage indexes that define CKD also mark an increased risk for all-cause premature death. CKD is the most prevalent risk factor for lethal coronavirus disease 2019 (COVID-19) and the factor that most increases the risk of death in COVID-19, after old age. Men and women undergoing KRT still have an annual mortality that is 10- to 100-fold higher than similar-age peers, and life expectancy is shortened by ~40 years for young persons on dialysis and by 15 years for young persons with a functioning kidney graft. CKD is expected to become the fifth greatest global cause of death by 2040 and the second greatest cause of death in Spain before the end of the century, a time when one in four Spaniards will have CKD. However, by 2022, CKD will become the only top-15 global predicted cause of death that is not supported by a dedicated well-funded Centres for Biomedical Research (CIBER) network structure in Spain. Realizing the underestimation of the CKD burden of disease by health authorities, the Decade of the Kidney initiative for 2020-2030 was launched by the American Association of Kidney Patients and the European Kidney Health Alliance. Leading Spanish kidney researchers grouped in the kidney collaborative research network Red de Investigación Renal have now applied for the Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS) call for collaborative research in Spain with the support of the Spanish Society of Nephrology, Federación Nacional de Asociaciones para la Lucha Contra las Enfermedades del Riñón and ONT: RICORS2040 aims to prevent the dire predictions for the global 2040 burden of CKD from becoming true

The H+-Translocating Inorganic Pyrophosphatase From Arabidopsis thaliana Is More Sensitive to Sodium Than Its Na+- Translocating Counterpart From Methanosarcina mazei

Overexpression of membrane-bound K+-dependent H+-translocating inorganic pyrophosphatases (H+-PPases) from higher plants has been widely used to alleviate the sensitivity toward NaCl in these organisms, a strategy that had been previously tested in Saccharomyces cerevisiae. On the other hand, H+-PPases have been reported to functionally complement the yeast cytosolic soluble pyrophosphatase (IPP1). Here, the efficiency of the K+-dependent Na+-PPase from the archaeon Methanosarcina mazei (MVP) to functionally complement IPP1 has been compared to that of its H+-pumping counterpart from Arabidopsis thaliana (AVP1). Both membrane-bound integral PPases (mPPases) supported yeast growth equally well under normal conditions, however, cells expressing MVP grew significantly better than those expressing AVP1 under salt stress. The subcellular distribution of the heterologously-expressed mPPases was crucial in order to observe the phenotypes associated with the complementation. In vitro studies showed that the PPase activity of MVP was less sensitive to Na+ than that of AVP1. Consistently, when yeast cells expressing MVP were grown in the presence of NaCl only a marginal increase in their internal PPi levels was observed with respect to control cells. By contrast, yeast cells that expressed AVP1 had significantly higher levels of this metabolite under the same conditions. The H+-pumping activity of AVP1 was also markedly inhibited by Na+. Our results suggest that mPPases primarily act by hydrolysing the PPi generated in the cytosol when expressed in yeast, and that AVP1 is more susceptible to Na+ inhibition than MVP both in vivo and in vitro. Based on this experimental evidence, we propose Na+- PPases as biotechnological tools to generate salt-tolerant plants.Peer reviewe

Resistencia a fármacos citotóxicos y fungicidas en levadura mediante complementación funcional de la ATPasa vacuolar por pirofosfatasas de membrana translocadoras de protones de origen vegetal

1 página.Las ATPasas vacuolares (V-ATPasas) son complejas bombas primarias de protones que juegan un papel esencial en la generación de gradientes electroquímicos en sistemas de endomembranas.Proyectos P07-CVI-03082 y BFU2010-15622 (JA, MICINN y FEDER).Peer reviewe

Uso de secuencias nucleotídicas que codifican pirofosfatasas translocadoras de protones para producir levaduras, hongos y células animales resistentes a fármacos citotóxicos y fungicidas y método para producirlas

La presente invención se refiere a una secuencia de nucleótidos de origen vegetal o microbiano que codifica para una secuencia de aminoácidos correspondiente a una pirofosfatasa translocadora de protones (H+-PPasa, miembro de una familia de proteínas con código identificativo PF03030 de la base de datos Pfam), la cual es usada para transformar o transfectar una célula de levadura (p.e. Saccharomyces cerevisiae), fúngica o animal que naturalmente no contienen esta clase de proteínas, con la finalidad de producir resistencia a fármacos o antibióticos citotóxicos y a fungicidas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de SevillaA1 Solicitud de patente con informe sobre el estado de la técnic

A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

11 pagesV-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organellesWe thank Professor Ramón Serrano for providing the anti-Pma1p antibody and yeast mutant strain RS-1144, and Professor Andrés Aguilera for providing yeast strain BJ5457. We also thank Dr Alicia Orea for excellent technical assistance with fluorescence microscopy and Ms Isabel Jiménez for yeast culturing. This work was supported by the Regional Andalusian Goverment and the Spanish Ministerio de Ciencia e Innovación financial support to PAIDI group BIO-261 [grant numbers P07-CVI-03082, BFU2007-61887 and BFU2010-15622], partially funded by the EU FEDER (Fondo Europeo de Desarrollo Regional) programme.Peer reviewe

8-Dehydrosterols induce membrane traffic and autophagy defects through V-ATPase dysfunction in Saccharomyces cerevisae

8-Dehydrosterols are present in a wide range of biologically relevant situations, from human rare diseases to amine fungicide-treated fungi and crops. However, the molecular bases of their toxicity are still obscure. We show here that 8-dehydrosterols, but not other sterols, affect yeast vacuole acidification through V-ATPases. Moreover, erg2Δ cells display reductions in proton pumping rates consistent with ion-transport uncoupling in vitro. Concomitantly, subunit Vph1p shows conformational changes in the presence of 8-dehydrosterols. Expression of a plant vacuolar H+-pumping pyrophosphatase as an alternative H+-pump relieves Vma−-like phenotypes in erg2Δ-derived mutant cells. As a consequence of these acidification defects, endo- and exo-cytic traffic deficiencies that can be alleviated with a H+-pumping pyrophosphatase are also observed. Despite their effect on membrane traffic, 8-dehydrosterols do not induce endoplasmic reticulum stress or assembly defects on the V-ATPase. Autophagy is a V-ATPase dependent process and erg2Δ mutants accumulate autophagic bodies under nitrogen starvation similar to Vma− mutants. In contrast to classical Atg− mutants, this defect is not accompanied by impairment of traffic through the CVT pathway, processing of Pho8Δ60p, GFP-Atg8p localisation or difficulties to survive under nitrogen starvation conditions, but it is concomitant to reduced vacuolar protease activity. All in all, erg2Δ cells are autophagy mutants albeit some of their phenotypic features differ from classical Atg− defective cells. These results may pave the way to understand the aetiology of sterol-related diseases, the cytotoxic effect of amine fungicides, and may explain the tolerance to these compounds observed in plants.Peer reviewe

N-terminal chimaeras with signal sequences enhance the functional expression and alter the subcellular localization of heterologous membrane-bound inorganic pyrophosphatases in yeast

Expression of heterologous multispanning membrane proteins in Saccharomyces cerevisiae is a difficult task. Quite often, the use of multicopy plasmids where the foreign gene is under the control of a strong promoter does not guarantee efficient production of the corresponding protein. Here, we show that the expression level and/or subcellular localization in S. cerevisiae of an heterologous type of multispanning membrane protein, the H+-translocating inorganic pyrophosphatase (H+-PPase), can be changed by fusing it with various suitable Nterminal signal sequences. Chimeric proteins were constructed by adding the putative Nterminal extra domain of Trypanosoma cruzi H+-PPase or the bona fide signal sequence of S. cerevisiae invertase Suc2p to H+-PPase polypeptides of different organisms (from bacteria to plants) and expressed in a yeast conditional mutant deficient in its cytosolic PPi hydrolysis activity when grown on glucose. Chimeric constructs not only substantially enhanced H+- PPase expression levels in transformed mutant cells but also allowed functional complementation in those cases in which native H+-PPase failed to accomplish it. Activity assays and Western blot analyses further demonstrated the occurrence of most H+-PPase in internal membrane fractions of these cells. The addition of N-terminal signal sequences to the vacuolar H+-PPase AVP1 from the plant Arabidopsis thaliana -a protein efficiently expressed in yeast in its natural form- alters the subcellular distribution of the chimeras suggesting further progression along the secretory sorting pathways, as shown by density gradient ultracentrifugation and in vivo fluorescence microscopy of the corresponding GFP-H+-PPase fusion proteins.Peer Reviewe

Cytotoxic drug resistance in yeast and animal cells by functional complementation of v-atpase with plant proton-translocating membrane pyrophosphatases

1 página. XIV Congresos de la Sociedad Española de Biología Celular (Málaga, 12-15 de diciembre, 2011).The vacuolar ATPase (V-ATPase) is a complex primary proton pump essential for the generation of electrochemical gradients in endomembrane systems.Project P07-CVI-15 622 03 082 and BFU2010-(JA, MICINN) co-financed by ERDF.Peer reviewe

Editorial: Pyrophosphates and Polyphosphates in Plants and Microorganisms

Phosphorus is the fifth most abundant chemical element in living cells. Microorganisms and plants take up phosphorus as dissolved (ortho)phosphate (Pi), that is often limited due to the formation of sparingly soluble complexes in soil; on the other hand, overapplication of phosphate fertilizer generally leads to the problems of eutrophication (diCenzo et al., 2017). Phosphorus usually occurs in vivo as free Pi or forming esters or diesters in metabolites and macromolecules. Protein phosphorylation also controls major metabolic pathways and cell division cycle (Li et al., 2016). Phosphate anion can react with another, releasing a molecule of water and producing a dimer, pyrophosphate (PPi, P2O 4− 7 ). More Pi residues may be added to PPi by means of this linkage, known as a “phosphoanhydride bond,” thus producing polyphosphate (polyP). Hydrolysis of phosphoanhydride bonds is thermodynamically favorable and kinetically slow, consequently, PPi and polyP are used for energy transfer and storage in many organisms. PPi and polyP also participate in metabolites like nucleoside triphosphate, inositol pyrophosphate, or activated isoprene

Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer

Cancer cells show a metabolic shift that makes them overproduce protons; this has the potential to disturb the cellular acid-base homeostasis. However, these cells show cytoplasmic alkalinisation, increased acid extrusion and endosome-dependent drug resistance. Vacuolar type ATPases (V-ATPases), together with other transporters, are responsible to a great extent for these symptoms. These multi-subunit proton pumps are involved in the control of cytosolic pH and the generation of proton gradients (positive inside) across en-docellular membrane systems like Golgi, endosomes or lysosomes. In addition, in tumours, they have been shown to play an important role in the acidification of the intercellular medium. This importance makes them an attractive target to control tumour cell proliferation. In the present review we present the major characteristics of this kind of proton pumps and we provide some recent insights on their in vivo regulation. Also, we review some of the consequences that V-ATPase inhibition carries for the tumour cell, such as cell cycle arrest or cell death, and provide a brief summary of the studies related to cancer made recently with commercially available inhibitors. In the light of recent knowledge on the regulation of this proton pump, some new approaches to impair V-ATPase function are also suggested. © 2012 Bentham Science Publishers.Peer Reviewe