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Albumin-associated free fatty acids induce macropinocytosis in podocytes.

ABSTRACT: Podocytes are specialized epithelial cells in the kidney glomerulus that play important structural and functional roles in maintaining the filtration barrier. Nephrotic syndrome results from a breakdown of the kidney filtration barrier and is associated with proteinuria, hyperlipidemia, and edema. Additionally, podocytes undergo changes in morphology and internalize plasma proteins in response to this disorder. Here, we used fluid-phase tracers in murine models and determined that podocytes actively internalize fluid from the plasma and that the rate of internalization is increased when the filtration barrier is disrupted. In cultured podocytes, the presence of free fatty acids (FFAs) associated with serum albumin stimulated macropinocytosis through a pathway that involves FFA receptors, the G?/G? complex, and RAC1. Moreover, mice with elevated levels of plasma FFAs as the result of a high-fat diet were more susceptible to Adriamycin-induced proteinuria than were animals on standard chow. Together, these results support a model in which podocytes sense the disruption of the filtration barrier via FFAs bound to albumin and respond by enhancing fluid-phase uptake. The response to FFAs may function in the development of nephrotic syndrome by amplifying the effects of proteinuria.

SUBMITTER: Chung JJ

PROVIDER: S-EPMC4518691 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Albumin-associated free fatty acids induce macropinocytosis in podocytes.

Chung Jun-Jae JJ Huber Tobias B TB Gödel Markus M Jarad George G Hartleben Björn B Kwoh Christopher C Keil Alexander A Karpitskiy Aleksey A Hu Jiancheng J Huh Christine J CJ Cella Marina M Gross Richard W RW Miner Jeffrey H JH Shaw Andrey S AS

The Journal of clinical investigation 20150427 6

Podocytes are specialized epithelial cells in the kidney glomerulus that play important structural and functional roles in maintaining the filtration barrier. Nephrotic syndrome results from a breakdown of the kidney filtration barrier and is associated with proteinuria, hyperlipidemia, and edema. Additionally, podocytes undergo changes in morphology and internalize plasma proteins in response to this disorder. Here, we used fluid-phase tracers in murine models and determined that podocytes acti ...[more]

PMID: 25915582

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Project description:IntroductionObesity during pregnancy increases the risk for maternal complications like gestational diabetes, preeclampsia, and maternal inflammation. Maternal obesity also increases the risk of childhood obesity, intrauterine growth restriction (IUGR) and diabetes to the offspring. Increased circulating free fatty acids (FFAs) in obesity due to adipose tissue lipolysis induces lipoapoptosis to hepatocytes, cholangiocytes, and pancreatic-β-cells. During the third trimester of human pregnancy, there is an increase in maternal lipolysis and release of FFAs into the circulation. It is currently unknown if increased FFAs during gestation as a result of maternal obesity cause placental cell lipoapoptosis. Increased exposure of FFAs during maternal obesity has been shown to result in placental lipotoxicity. The objective of the present study is to determine saturated FFA-induced trophoblast lipoapoptosis and also to test the protective role of monounsaturated fatty acids against FFA-induced trophoblast lipoapoptosis using in vitro cell culture model. Here, we hypothesize that saturated FFAs induce placental trophoblast lipoapoptosis, which was prevented by monounsaturated fatty acids.MethodsBiochemical and structural markers of apoptosis by characteristic nuclear morphological changes with DAPI staining, and caspase 3/7 activity was assessed. Cleaved PARP and cleaved caspase 3 were examined by western blot analysis.ResultsTreatment of trophoblast cell lines, JEG-3 and JAR cells with palmitate (PA) or stearate (SA) induces trophoblast lipoapoptosis as evidenced by a significant increase in apoptotic nuclear morphological changes and caspase 3/7 activity. We observed that saturated FFAs caused a concentration-dependent increase in placental trophoblast lipoapoptosis. We also observed that monounsaturated fatty acids like palmitoleate and oleate mitigates placental trophoblast lipoapoptosis caused due to PA exposure.ConclusionWe show that saturated FFAs induce trophoblast lipoapoptosis. Co-treatment of monounsaturated fatty acids like palmitoleate and oleate protects against FFA-induced trophoblast lipoapoptosis.

Project description:Increased levels of serum free fatty acids (FFAs) are closely associated with microvascular dysfunction. In our previous study, a coronary microvascular dysfunction (CMD) model was successfully established via lipid infusion to increase the levels of serum FFAs in mice. However, the underlying mechanisms remained poorly understood. Therefore, the aim of the present study was to explore the mechanism underlying FFA‑induced CMD. A CMD mouse model was established via lipid combined with heparin infusion for 6 h to increase the concentration of serum FFAs. Following the establishment of the model, the coronary flow reserve (CFR), extent of leukocyte activation and cardiac microvascular structures were assessed in the mice. Cardiac microvascular endothelial cells (CMECs) were treated with different concentrations of palmitic acid and cell viability was evaluated. Changes in the expression levels of AMP‑activated protein kinase (AMPK), Krüppel‑like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) were identified by immunohistochemical and western blot analyses. Experiments using AMPK activator, KLF2 overexpression plasmid, small interfering RNAs and nicorandil were subsequently designed to investigate the potential involvement of the AMPK/KLF2/eNOS signaling pathway. These experiments revealed that FFAs could induce CMD in mice, which was characterized by reduced CFR (1.89±0.37 vs. 2.74±0.30) and increased leukocyte adhesion (4,350±1,057.5 vs. 11.8±5.4 cells/mm2) compared with the control mice. CD11b expression and intracellular reactive oxygen species (ROS) levels were increased in CMD model mice compared with control mice. Serum TNF‑α and IL‑6 levels were higher in the model group than in the control group. Transmission electron microscopy revealed that CMECs in heart tissues of model mice were severely swollen. In addition, palmitic acid decreased CMEC viability and increased ROS production in a dose‑dependent manner. Notably, the AMPK/KLF2/eNOS signaling pathway was demonstrated to be suppressed by FFAs both in vivo and in vitro. Activation of this axis with AMPK activator, KLF2 overexpression plasmid or nicorandil restored the CFR in CMD model mice, inhibited oxidative stress and increased CMEC viability. Taken together, the results of the present study demonstrated that FFAs could induce CMD via inhibition of the AMPK/KLF2/eNOS signaling pathway, whereas activation of this pathway led to the alleviation of FFA‑induced CMD, which may be a therapeutic option for CMD.

Project description:Macropinocytosis is exploited by many pathogens for entry into cells. Coronaviruses (CoVs) such as severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV are important human pathogens; however, macropinocytosis during CoV infection has not been investigated. We demonstrate that the CoVs SARS CoV and murine hepatitis virus (MHV) induce macropinocytosis, which occurs late during infection, is continuous, and is not associated with virus entry. MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells. MHV-induced macropinocytosis requires fusogenic spike protein on the cell surface and is dependent on epidermal growth factor receptor activation. Inhibition of macropinocytosis reduces supernatant viral titers and syncytia but not intracellular virus titers. These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading. Our studies are the first to demonstrate virus use of macropinocytosis for a role other than entry and suggest a much broader potential exploitation of macropinocytosis in virus replication and host interactions. Importance: Coronaviruses (CoVs), including severe acute respiratory syndrome (SARS) CoV and Middle East respiratory syndrome CoV, are critical emerging human pathogens. Macropinocytosis is induced by many pathogens to enter host cells, but other functions for macropinocytosis in virus replication are unknown. In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion. Murine hepatitis virus macropinocytosis requires a fusogenic virus spike protein and signals through the epidermal growth factor receptor and the classical macropinocytosis pathway. These studies demonstrate CoV induction of macropinocytosis for a purpose other than entry and indicate that viruses likely exploit macropinocytosis at multiple steps in replication and pathogenesis.

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Albumin-associated free fatty acids induce macropinocytosis in podocytes.

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Albumin-associated free fatty acids induce macropinocytosis in podocytes.

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