Environmental hyperosmolality regulates phospholipid biosynthesis in the renal epithelial cell line MDCK

Hyperosmolality is a key signal for renal physiology. On the one hand, it contributes to the differentiation of renal medullary structures and to the development of the urinary concentrating mechanism. On the other, it is a stress factor. In both cases, hyperosmolality activates processes that require an adequate extension of cellular membranes. In the present work, we examined whether hyperosmolality regulates phospholipid biosynthesis, which is needed for the membrane biogenesis in the renal epithelial cell line Madin-Darby canine kidney (MDCK). Because phospholipids are the structural determinants of all cell membranes, we evaluated their content, synthesis, and regulation in MDCK cultures subjected to different hyperosmotic concentrations of NaCl, urea, or both. Hyperosmolality increased phospholipid content in a concentration-dependent manner. Such an effect was exclusively due to changes in NaCl concentration and occurred at the initial stage of hyperosmolar treatment concomitantly with the expression of the osmoprotective protein COX-2. The hypertonic upregulation of phosphatidylcholine (PC) synthesis, the main constituent of all cell membranes, involved the transcriptional activation of two main regulatory enzymes, choline kinase (CK) and cytidylyltransferase α (CCTα) and required ERK1/2 activation. Considering that physiologically, renal medullary cells are constantly exposed to high and variable NaCl, these findings could contribute to explaining how renal cells could maintain cellular integrity even in a nonfavorable environment.Fil: Casali, Cecilia Irene. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Weber, Karen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Favale, Nicolas Octavio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Fernandez, Maria del Carmen. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentin

The restitution of an oxalate-damaged epithelium

The renal inner medulla is responsible for the hydro-saline equilibrium maintenance through water and electrolyte excretionin urine. The collecting ducts, which are involved in the urine concentration, are immersed in an extracellular matrix with thehighest body osmolarity. The hyperosmolarity is a key signal for cell differentiation and for the establishment of the urineconcentration mechanism. Moreover, renal ducts are exposed to wastes coming from blood filtration. There are severalnephrotoxic agents such as antibiotics, diuretics, antineoplastic and cytostatic agents, and renal stones. Calcium oxalate stonesare the most common type of kidney stone. The crystal aggregates are harmful for epithelial renal cells and tubular structures,and that damage could lead to the development of chronic kidney disease. Our previous results showed that differentiated renalcells treated with oxalate (Ox) for 24 h lost the typical epithelial cobblestone morphology and showed a spindle-shapedmorphology characteristic of an epithelial mesenchymal transition. After 48 h of Ox, cells started to recover their morphologyand after 72 h of Ox the epithelium was almost reestablished. The aims of the present work were to evaluate whether epithelialintegrity is disrupted after 24 h of Ox and whether epithelial differentiated characteristics are restituted after 72 h of Ox. Todo that, the renal epithelial cells MDCK were grown in a hyperosmolar environment (512 mOsm/Kg H2O) for 72 h to get adifferentiated epithelium, and then subjected to 1.5 mM Ox for 24, 48 and 72 h. After treatments, cell morphology and theexpression of differentiated epithelia markers were evaluated by fluorescence microscopy. E-cadherin, a member of adherensjunctions, was localized to the cell periphery at 24, 48 and 72 h in control conditions. After 24 h of Ox, the protein wasinternalized and its label on the periphery decreased. After 48 h of Ox, E-cadherin was localized both to the cell membranesand to the cytoplasm, while after 72 h of Ox the label was mainly at the cell periphery. In control cells the apical marker gp135was localized at apical cell surface, while in cells treated with 24 h of Ox gp135 apical staining was reduced. After 48 h of Ox,the percentage of cells expressing apical gp135 started to increase reaching values like control conditions at 72 h. Finally,primary cilium was evidenced by acetylated-tubulin immunofluorescence. Control cells showed a high percentage of ciliatedcells, while it decreased upon treatment with 24 h of Ox. After 48 h of Ox, the cells started to recover the primary cilium, andafter 72 h of Ox, the percentage of ciliated cells reached control values. The results showed that the treatment with 24 h of Oxinduces dedifferentiation and after 72 h of the cell damage there is a restitution of the differentiated epithelia. The next goal isto elucidate the molecular mechanisms involved in the restitution of the oxalate-damaged epithelium.Fil: Sendyk, Dylan. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Pescio, Lucila Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Fernández Tomé, M. C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Casali, Cecilia Irene. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaLVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research and XVI Annual Meeting of the Argentinean Society for General MicrobiologyVirtualArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularAsociación Civil de Microbiología Genera

X-box binding protein 1 (XBP1): A key protein for renal osmotic adaptation: Its role in lipogenic program regulation

In renal cells, hyperosmolarity can induce cellular stress or differentiation. Both processes require active endoplasmic reticulum (ER)-associated protein synthesis. Lipid biosynthesis also occurs at ER surface. We showed that hyperosmolarity upregulates glycerophospholipid (GP) and triacylglycerol (GL-TG) de novo synthesis. Considering that massive synthesis of proteins and/or lipids may drive to ER stress, herein we evaluated whether hyperosmolar environment induces ER stress and the participation of inositol-requiring enzyme 1α (IRE1α)-XBP1 in hyperosmotic-induced lipid synthesis. Treatment of Madin-Darby canine kidney (MDCK) cells with hyperosmolar medium triggered ER stress-associated unfolded protein response (UPR). Hyperosmolarity significantly increased xbp1 mRNA and protein as function of time; 24 h of treatment raised the spliced form of XBP1 protein (XBP1s) and induced its translocation to nuclear compartment where it can act as a transcription factor. XBP1 silencing or IRE1α ribonuclease (RNAse) inhibition impeded the expression of lipin1, lipin2 and diacylglycerol acyl transferase-1 (DGAT1) enzymes which yielded decreased GL-TG synthesis. The lack of XBP1s also decreased sterol regulatory element binding protein (SREBP) 1 and 2. Together our data demonstrate that hyperosmolarity induces IRE1α → XBP1s activation; XBP1s drives the expression of SREBP1 and SREBP2 which in turn regulates the expression of the lipogenic enzymes lipin1 (LPIN1) and 2 (LPIN2) and DGAT1. We also demonstrated for the first time that tonicity-responsive enhancer binding protein (TonEBP), the master regulator of osmoprotective response, regulates XBP1 expression. Thus, XBP1 acts as an osmoprotective protein since it is activated by high osmolarity and upregulates lipid metabolism, membranes generation and the restoration of ER homeostasis.Fil: Casali, Cecilia Irene. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Malvicini, Ricardo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Erjavec, Luciana Cecilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Parra, Leandro Gastón. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Artuch, Ayelen. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Fernández Tome, María C.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Molecular mechanisms underlying resveratrol effect on renal osmoprotection: modulation of COX-2 expression

Resveratrol (RSV) is a polyphenol naturally present in several plants. Nowadays it is sold as an over-the-counter dietarysupplement due to its antioxidant, anti-inflammatory and antitumoral effects. Paradoxically, it has been documented that RSVmay also present pro-oxidizing and pro-proliferative effects. In fact, some studies suggest that RSV treatment can result inopposite effects depending on the cell type, its concentration, or the treatment time. Particularly in renal tissue, animal injurymodels described RSV beneficial effects, while studies with chronic intake of RSV observed nephrotoxicity. Hence, RSVeffects on renal tissue are still controversial. Due to the urinary concentrating mechanism, renal medullary interstitium presentsan elevated osmolality that can abruptly change depending on the hydric state of the body, reaching values up to 800-1200mOsm/kg H2O. To survive in this environment, renal cells activate protective pathways. We have demonstrated that renalepithelial cell line MDCK undergoes an adaptive process during the first 24h of hyperosmolarity, in which the transcription ofthe osmoprotective gene cyclooxygenase 2 (COX-2) is activated, among others. After 48h these cells are already adapted andbegin to differentiate, acquiring a polarized epithelium morphology. In this work we evaluate RSV effect on adaption anddifferentiation mechanisms, focusing particularly on COX-2 role. To do this, MDCK cells were pretreated with differentconcentrations of RSV (1, 5, 10, 25 µM) and cultured in hyperosmolar medium (~512 mOsm/kg H2O) for 24 and 48h. Cellswere harvested to obtain cell number and viability. Cell cycle, immunofluorescence (IF), western blot and RT-PCR analysiswere performed. We found that RSV significantly decreased cell number in a concentration-dependent manner at 24 and 48h.Cell cycle analysis revealed that RSV increased S-phase and Sub-G0 cell population. In addition, treated cells did not reachtypical epithelium morphology. COX-2 mRNA and protein levels were surprisingly upregulated by RSV at 24 and 48h, andIF revealed an accumulation of the protein in cytoplasmic granules. To investigate the pathways leading to this upregulation,we indirectly evaluated TonEBP, NF-κB and ERK1/2 pathways, which are activated by hyperosmolarity; and SIRT1implication, a target of RSV. TonEBP target genes mRNA did not show any significant change under RSV treatment, whileNF-κB target gene mRNA presented an increase similar to that of COX-2 mRNA. Moreover, NF-κB IF revealed an increasein its nuclear localization. Regarding ERK1/2, treatment with ERK1/2 selective inhibitor (U0126) completely blocked COX2 protein expression. These results suggest that in renal cells RSV pretreatment decreased cell number and impeded typicalcell morphology acquisition; but it increased COX-2 expression, possibly through NF-κB and ERK1/2 activation.Fil: Erjavec, Luciana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Parra, Leandro Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Morel Gómez, Emanuel Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Lampropulos, T.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Casali, Cecilia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Fernandez, Maria del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaLVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research and XVI Annual Meeting of the Argentinean Society for General MicrobiologyVirtualArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularAsociación Civil de Microbiología Genera

XBP-1 regulation of arachidonic acid and glicerolipids metabolism in renal epithelial cells under osmotic stress

Hyperosmolarity is a key controversial signal for renal cells. Under physiological conditions, it induces renal celldifferentiation and maturation of urine concentrating system. However, abrupt changes in environmental osmolarity may also induce cell stress that can lead to death. To adapt and survive in such adverse conditions, renal cells implement differentosmoprotective mechanisms that includes both the upregulation of cyclooxygenase-2 (COX-2) expression and prostaglandins(PGs) synthesis from arachidonic acid (AA), and a coordinated increase in phospholipids (PL) and triacylglycerides (TAG)biosynthesis. We previously shown that hyperosmolarity induces ER stress and activates the unfolded protein response (UPR)in Madyn Darby Canine Kidney Cells (MDCK) through IRE1α-XBP1s pathway, and that XBP1s modulates lipid synthesisregulating lipogenic enzymes expression. In the present work we evaluated how XBP1s modulates phospholipase A2(PLA2)/COX-2/PGs pathway and its relationship with lipid synthesis induction under osmotic stress. MDCK cells weresubjected to hyperosmolarity (298-512 mOsm/kg H2O) for different periods of time (0, 12, 24 and 48 h) and treated withdifferent PLA2 (cPLA2, iPLA2 and sPLA2) and IRE1α inhibitors. RT-PCR studies showed that hyperosmolarity increasedcPLA2 expression at 24 and 48 h but did not upregulate iPLA2 expression. Inhibition of cPLA2 but not iPLA2 nor sPLA2prevented hyperosmolarity-induced lipid synthesis and lipid droplets accumulation. Furthermore, IRE1α RNase activityinhibition was accompanied by a decrease in cPLA2 and COX-2 but not in iPLA2 expression evaluated by RT-PCR. Instead,western blot analysis showed a significant increase in COX-2 protein levels when xbp1 (u) splicing was blocked by IRE1αinhibitor. Our findings suggest that the UPR modulates glycerolipids metabolism under osmotic stress by regulatingcPLA2/COX-2/PGs axis.Fil: Parra, Leandro Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Casali, Cecilia Irene. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Erjavec, Luciana Cecilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Morel Gómez, Emanuel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Setton, Clara Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Fernandez, Maria del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaLVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research and XVI Annual Meeting of the Argentinean Society for General MicrobiologyVirtualArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularAsociación Civil de Microbiología Genera

SPARC inhibition accelerates NAFLD-associated hepatocellular carcinoma development by dysregulating hepatic lipid metabolism

Background and aims: Non-alcoholic fatty liver (NAFLD) and its more serious form non-alcoholic steatohepatitis increase risk of hepatocellular carcinoma (HCC). Lipid metabolic alterations and its role in HCC development remain unclear. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is involved in lipid metabolism, NAFLD and diabetes, but the effects on hepatic lipid metabolism and HCC development is unknown. The aim of this study was to evaluate the role of SPARC in HCC development in the context of NAFLD. Methods: Primary hepatocyte cultures from knockout (SPARC−/−) or wild-type (SPARC+/+) mice, and HepG2 cells were used to assess the effects of free fatty acids on lipid accumulation, expression of lipogenic genes and de novo triglyceride (TG) synthesis. A NAFLD-HCC model was stabilized on SPARC−/− or SPARC+/+ mice. Correlations among SPARC, lipid metabolism-related gene expression patterns and clinical prognosis were studied using HCC gene expression dataset. Results: SPARC−/− mice increases hepatic lipid deposits over time. Hepatocytes from SPARC−/− mice or inhibition of SPARC by an antisense adenovirus in HepG2 cells resulted in increased TG deposit, expression of lipid-related genes and nuclear translocation of SREBP1c. Human HCC database analysis revealed that SPARC negatively correlated with genes involved in lipid metabolism, and with poor survival. In NAFLD-HCC murine model, the absence of SPARC accelerates HCC development. RNA-seq study revealed that pathways related to lipid metabolism, cellular detoxification and proliferation were upregulated in SPARC−/− tumour-bearing mice. Conclusions: The absence of SPARC is associated with an altered hepatic lipid metabolism, and an accelerated NAFLD-related HCC development.Fil: Onorato, Agostina Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Fiore, Esteban Juan. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Bayo Fina, Juan Miguel. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Casali, Cecilia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Fernandez, Maria del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rodriguez, Marcelo Maximiliano. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Domínguez, Luciana María. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Argemi, Josepmaría. Universidad de Navarra; EspañaFil: Hidalgo, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Favre, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: García, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Atorrasagasti, María Catalina. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; ArgentinaFil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Ciencias Biomédicas. Instituto de Investigaciones en Medicina Traslacional. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Medicina Traslacional; Argentin

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Coordinate regulation between the nuclear receptor peroxisome proliferator-activated receptor-γ and cyclooxygenase-2 in renal epithelial cells

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors involved in lipid metabolism and glucose utilization, in cell growth, differentiation and apoptosis, and in the regulation of pro-inflammatory genes expression such as cyclooxygenase-2 (COX-2). PPARγ is the main isoform in the renal inner medulla where it is believed to possess nephroprotective actions. In this kidney zone, COX-2 acts as an osmoprotective gene and its expression is modulated by changes in interstitial osmolarity. In the present work we evaluated whether hyperosmolar-induced COX-2 expression is modulated by PPARγ in renal epithelial cells MDCK subjected to high NaCl medium. The results presented herein show that ligand-activated PPARγ repressed COX-2 expression. But more important, the present findings show that hyperosmolar medium decreased PPARγ protein and increases the PPARγ phosphorylated form, which is inactive. ERK1/2 and p38 activation precedes PPARγ disappearance and induced-COX-2 expression. Therefore, the decrease in PPARγ expression is required for hyperosmotic induction of COX-2. We also found that PGE2, the main product of COX-2 in MDCK cells, induced these changes in PPARγ protein. Our results may alert on the long term use of thiazolidinediones (TZD) since they could affect renal medullary function that depends on COX-2 for cellular protection against osmotic stress.Fil: Casali, Cecilia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Weber, Karen. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Faggionato, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Morel Gómez, Emanuel Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Fernandez, Maria del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentin

Are prostaglandins involved in the restitution of an oxalatedamaged epithelium?

Renal collecting ducts, which are involved in the urine concentration mechanism, are immersed in an extracellular matrix withthe highest body osmolarity. This hyperosmolarity is a key signal for renal cell differentiation and for the establishment of theurine concentration mechanism. However, hyperosmolarity can induce cell death when there is a great osmolarity change.Renal cells activate adaptive and protective mechanisms to survive in the hyperosmolar environment. One important cellmechanism is the expression of osmoprotective genes such as cyclooxygenase 2 (COX2). Moreover, renal ducts are exposedto wastes coming from blood filtration that include nephrotoxic drugs and kidney stones. Calcium oxalate stones are the mostcommon type of kidney stone. Crystal aggregates are harmful for epithelial renal cells and tubular structures, and the damagecould lead to renal kidney disease. Our prior results showed that oxalate modulates COX2 mRNA and protein expression inrenal differentiated epithelial cells, but the role of this protein is still unknown. The aim of the present work is to evaluatewhether prostaglandins, the COX2 products, are involved in the regeneration mechanism of differentiated renal epithelial cellsdamaged with oxalate. To do that, renal epithelial cells MDCK were grown in a hyperosmolar environment (512 mOsm/KgH2O) for 72 h to get a differentiated epithelium and then subjected to 1.5 mM oxalate (Ox) for 24, 48 and 72 h. To inhibitCOX2, 10 µM NS398 was added 30 min before Ox treatment; and to restore the inhibition, PGE2 (10-5, 10-6and 10-7 M) wasadded 30 min after Ox addition. After treatment, cells were harvested, counted and cell viability was determined. Cellmorphology and COX2 expression was also evaluated. Cells treated with 24 h of Ox showed a spindle-shaped morphologycharacteristic of an epithelial mesenchymal transition (EMT) and NS398 addition before Ox treatment did not allow theseEMT. After 48 h of Ox cells started to recover their typical epithelial morphology. Cell treated with NS398 before Ox showeda cobblestone morphology, but gaps in the monolayer were observed. Control conditions showed the typical epithelialcobblestone morphology after 24 and 48 h. PGE2 addition to cells treated with NS398 and Ox did not allow the EMT at 24and 48h. Moreover, PGE2 treated cells showed a morphology characteristic of an epithelial cells (cobblestone). Ox decreasedthe number of cells at 24 h and 48 h compared to controls. The treatment with NS398 before Ox addition caused a slightdecrease of cell numbers at 24 h but not at 48 h. PGE2 addition did not affect cell number at 24 and 48 h. Cell viability did notchange after all treatments. NS398 induced COX2 expression and the addition of PGE2 slightly decreased it. The resultsshowed that PGE2 may be implicated in the restitution of the differentiated epithelia damaged with oxalate, but furtherexperiments are needed to elucidate the molecular mechanisms involved.Fil: Casali, Cecilia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Morel Gómez, Emanuel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Erjavec, Luciana Cecilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Parra, Leandro Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Marino, José Ignacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Fernández Tomé, M. C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaLVII SAIB Meeting; XVI SAMIGE MeetingArgentinaSociedad Argentina de Investigación en Bioquímica y Biología Molecula

TAG synthesis and storage under osmotic stress. A requirement for preserving membrane homeostasis in renal cells

Hyperosmolarity is a controversial signal for renal cells. It can induce cell stress or differentiation and both require an active lipid metabolism. We showed that hyperosmolarity upregulates phospholipid (PL) de novo synthesis in renal cells. PL synthesis requires fatty acids (FA), usually stored as triglycerides (TAG). PL and TAG de novo synthesis utilize the same initial biosynthetic route: sn-glycerol 3P (G3P) → phosphatidic acid (PA) → diacylglycerol (DAG). In the present work, we evaluate how such pathway contributes to PL and TAG synthesis in renal cells subjected to hyperosmolarity. Our results show an increase in PA and DAG formation under hyperosmotic conditions; augmented DAG production, due to lipin enzyme activity, lead to the increase of both TAG and PL. However, at early stages (24 and 48 h), most of the de novo synthesized DAG was directed to PL synthesis; longer treatments downregulated PL synthesis and the DAG formed was mainly driven to TAG synthesis. Hyperosmolarity induced ACC and FASN transcription which mediated FA de novo synthesis. New FA molecules were stored in TAG. Silencing experiments revealed that hyperosmotic-induction of lipin-1 and -2 was mediated by SREBP1. Interestingly, SREBP1 knockdown also dropped SREBP2, indicating a modulatory action between both isoforms. Impairing SREBP activity leads to a decline in TAG levels but not PL. Membrane homeostasis is maintained through the adequate PL synthesis and renewal and constitute a protective mechanism against hyperosmolarity. The present data reveal the relevance of TAG synthesis and storage for PL synthesis in renal cells.Fil: Weber, Karen. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Casali, Cecilia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gaveglio, Virginia Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Pasquaré, Susana Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Morel Gómez, Emanuel Dario. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Parra, Leandro Gastón. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Erjavec, Luciana Cecilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perazzo, Cecilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Fernandez, Maria del Carmen. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin