Browsing by Author "Marzolo, Maria-Paz"

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    Albumin Expands Albumin Reabsorption Capacity in Proximal Tubule Epithelial Cells through a Positive Feedback Loop between AKT and Megalin
    (2022) Silva-Aguiar, Rodrigo P.; Peruchetti, Diogo B.; Florentino, Lucas S.; Takiya, Christina M.; Marzolo, Maria-Paz; Dias, Wagner B.; Pinheiro, Ana Acacia S.; Caruso-Neves, Celso
    Renal proximal tubule cells (PTECs) act as urine gatekeepers, constantly and efficiently avoiding urinary protein waste through receptor-mediated endocytosis. Despite its importance, little is known about how this process is modulated in physiologic conditions. Data suggest that the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) pathway regulates PTEC protein reabsorption. Here, we worked on the hypothesis that the physiologic albumin concentration and PI3K/AKT pathway form a positive feedback loop to expand endocytic capacity. Using LLC-PK1 cells, a model of PTECs, we showed that the PI3K/AKT pathway is required for megalin recycling and surface expression, affecting albumin uptake. Inhibition of this pathway stalls megalin at EEA1(+) endosomes. Physiologic albumin concentration (0.01 mg/mL) activated AKT; this depends on megalin-mediated albumin endocytosis and requires previous activation of PI3K/mTORC2. This effect is correlated to the increase in albumin endocytosis, a phenomenon that we refer to as "albumin-induced albumin endocytosis". Mice treated with L-lysine present decreased albumin endocytosis leading to proteinuria and albuminuria associated with inhibition of AKT activity. Renal cortex explants obtained from control mice treated with MK-2206 decreased albumin uptake and promoted megalin internalization. Our data highlight the mechanism behind the capacity of PTECs to adapt albumin reabsorption to physiologic fluctuations in its filtration, avoiding urinary excretion.
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    Lipophorin receptors regulate mushroom bodies development and participate in learning, memory, and sleep in flies
    (2021) Rojo-Cortes, Francisca ; Tapia-Valladares, Victoria ; Fuenzalida-Uribe, Nicolas ; Hidalgo, Sergio ; Roa, Candy B. ; Gonzalez-Ramirez, Maria-Constanza ; Oliva, Carlos ; Campusano, Jorge M. ; Marzolo, Maria-Paz
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    Participation of OCRL1, and APPL1, in the expression, proteolysis, phosphorylation and endosomal trafficking of megalin: Implications for Lowe Syndrome
    (2022) Sandoval, Lisette; Fuentealba, Luz M.; Marzolo, Maria-Paz
    Megalin/LRP2 is the primary multiligand receptor for the re-absorption of low molecular weight proteins in the proximal renal tubule. Its function is significantly dependent on its endosomal trafficking. Megalin recycling from endosomal compartments is altered in an X-linked disease called Lowe Syndrome (LS), caused by mutations in the gene encoding for the phosphatidylinositol 5-phosphatase OCRL1. LS patients show increased low-molecular-weight proteins with reduced levels of megalin ectodomain in the urine and accumulation of the receptor in endosomal compartments of the proximal tubule cells. To gain insight into the deregulation of megalin in the LS condition, we silenced OCRL1 in different cell lines to evaluate megalin expression finding that it is post-transcriptionally regulated. As an indication of megalin proteolysis, we detect the ectodomain of the receptor in the culture media. Remarkably, in OCRL1 silenced cells, megalin ectodomain secretion appeared significantly reduced, according to the observation in the urine of LS patients. Besides, the silencing of APPL1, a Rab5 effector associated with OCRL1 in endocytic vesicles, also reduced the presence of megalin's ectodomain in the culture media. In both silencing conditions, megalin cell surface levels were significantly decreased. Considering that GSK3ss-mediated megalin phosphorylation reduces receptor recycling, we determined that the endosomal distribution of megalin depends on its phosphorylation status and OCRL1 function. As a physiologic regulator of GSK3ss, we focused on insulin signaling that reduces kinase activity. Accordingly, megalin phosphorylation was significantly reduced by insulin in wild-type cells. Moreover, even though in cells with low activity of OCRL1 the insulin response was reduced, the phosphorylation of megalin was significantly decreased and the receptor at the cell surface increased, suggesting a protective role of insulin in a LS cellular model.
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    Surface megalin expression is a target to the inhibitory effect of bradykinin on the renal albumin endocytosis
    (2021) Alves, Sarah A. S.; Florentino, Lucas S.; Teixeira, Douglas E.; Silva-Aguiar, Rodrigo P.; Peruchetti, Diogo B.; Oliveira, Ana Carolina; Scharfstein, Julio; Marzolo, Maria-Paz; Pinheiro, Ana Acacia S.; Caruso-Neves, Celso
    Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg(9)-BK, an antagonist of B1. BK induced the stall of megalin in EEA1(+) endosomes, but not in LAMP1(+) lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.
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    The p75 neurotrophin receptor evades the endolysosomal route in neuronal cells, favouring multivesicular bodies specialised for exosomal release
    (2014) Escudero, Claudia A.; Lazo, Oscal M.; Galleguillos, Carolina; Parraguez, Jose I.; Lopez-Verrilli, Maria A.; Cabeza, Carolina; Leon, Luisa; Saeed, Uzma; Retamal, Claudio; Gonzalez, Alfonso; Marzolo, Maria-Paz; Carter, Bruce D.; Court, Felipe A.; Bronfman, Francisca C.
    The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.