Dataset Information

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K_ATP channel activation.

ABSTRACT:

Purpose

The expression of aquaporin-8 (AQP8), which plays a crucial role in the maintenance of the cellular fluid and electrolyte balance, was shown to be increased in RPE cells under hyperosmotic conditions. The aim of the present study was to investigate the mechanisms of hyperosmotic AQP8 gene expression and the localization of AQP8 in cultured human RPE cells.

Methods

Hyperosmolarity was produced with the addition of 100 mM NaCl or 200 mM sucrose. Hypoxia was induced by cell culture in a 0.2% O₂ atmosphere or the addition of the hypoxia mimetic CoCl₂. Oxidative stress was induced by the addition of H₂O₂. Gene expression was determined with real-time RT-PCR analysis. AQP8 protein localization and secretion of VEGF were evaluated with immunocytochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA).

Results

Immunocytochemical and western blot data suggest that the AQP8 protein is mainly located in the mitochondria. Extracellular hyperosmolarity, hypoxia, and oxidative stress induced increases in AQP8 gene expression. Hyperosmotic AQP8 gene expression was reduced by inhibitors of the p38 MAPK and PI3K signal transduction pathways, and by JAK2 and PLA₂ inhibitors, and was in part mediated by the transcriptional activity of CREB. Hyperosmotic AQP8 gene expression was also reduced by autocrine/paracrine interleukin-1 signaling, the sulfonylureas glibenclamide and glipizide, which are known inhibitors of K_ATP channel activation, and a pannexin-blocking peptide. The K_ATP channel opener pinacidil increased the expression of AQP8 under control conditions. The cells contained Kir6.1 and SUR2B gene transcripts and displayed Kir6.1 immunoreactivity. siRNA-mediated knockdown of AQP8 caused increases in hypoxic VEGF gene expression and secretion and decreased cell viability under control, hyperosmotic, and hypoxic conditions.

Conclusions

The data indicate that hyperosmotic expression of AQP8 in RPE cells is dependent on the activation of K_ATP channels. The data suggest that AQP8 activity decreases the hypoxic VEGF expression and improves the viability of RPE cells which may have impact for ischemic retinal diseases like diabetic retinopathy and age-related macular degeneration.

SUBMITTER: Schnabel B

PROVIDER: S-EPMC7803296 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K<sub>ATP</sub> channel activation.

Schnabel Benjamin B Kuhrt Heidrun H Wiedemann Peter P Bringmann Andreas A Hollborn Margrit M

Molecular vision 20201230

<h4>Purpose</h4>The expression of aquaporin-8 (<i>AQP8</i>), which plays a crucial role in the maintenance of the cellular fluid and electrolyte balance, was shown to be increased in RPE cells under hyperosmotic conditions. The aim of the present study was to investigate the mechanisms of hyperosmotic <i>AQP8</i> gene expression and the localization of AQP8 in cultured human RPE cells.<h4>Methods</h4>Hyperosmolarity was produced with the addition of 100 mM NaCl or 200 mM sucrose. Hypoxia was ind ...[more]

PMID: 33456300

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Project description:BACKGROUND:Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. METHODOLOGY/PRINCIPAL FINDINGS:Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-?1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-?1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38?/? MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38?/? MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-?1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-?1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes. CONCLUSION:Hyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-?1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.

Project description:PurposeHigh intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells.MethodsHuman RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA.ResultsExtracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.ConclusionsHyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and edema via the stimulation of VEGF production in RPE. The downregulation of AQP5 under hypoxic conditions may prevent the resolution of edema.

Project description:BackgroundProliferative vitreoretinopathy (PVR) is a severe blinding complication of retinal detachment surgery. Increasing evidence demonstrate that PVR is associated with oxidative stress. Scutellarin is a natural flavone compound that has been reported to have anti-oxidative activity. However, the effect of scutellarin on PVR remains unknown. In the current study, we assessed the effect of scutellarin on hydrogen peroxide (H2O2)-induced oxidative injury in human retinal pigment epithelium cells (ARPE-19).MethodsARPE-19 cells were pretreated with different concentrations of scutellarin for 2 h, and then challenged with H2O2 (1 mM) for 24 h. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) and glutathione (GSH) activity were measured to assess the level of oxidative stress. Flow cytometry was performed to detect the apoptosis rate of ARPE-19 cells. Expression levels of bcl-2, bax, cleaved-caspase-3, p-JAK2, JAK2, p-STAT3, and STAT3 were measured using western blot.ResultsOur results revealed that scutellarin improved the cell viability of H2O2-induced ARPE-19 cells. Scutellarin alleviated the H2O2-induced oxidative stress in ARPE-19 cells, which was illustrated by reduced levels of ROS and MDA, accompanied by increased SOD activity and GSH level. The increased apoptosis rate of ARPE-19 cells caused by H2O2 induction was significantly decreased after scutellarin treatment. H2O2 treatment resulted in significant increase in bax expression and decrease in bcl-2 expression, while the changes in the expressions of bax and bcl-2 were reversed by scutellarin treatment. In addition, scutellarin promoted the activation of JAK2/STAT3 signaling pathway in H2O2-induced ARPE-19 cells. Suppression of JAK2/STAT3 signaling pathway abolished the protective effects of scutellarin on H2O2-induced ARPE-19 cells.ConclusionThese findings suggested that scutellarin was capable for alleviating H2O2-induced oxidative damage in ARPE-19 cells, which might be ascribed to the activation of JAK2/STAT3 signaling pathway.

Dataset Information

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K_ATP channel activation.

Purpose

Methods

Results

Conclusions

Publications

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K<sub>ATP</sub> channel activation.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on KATP channel activation.

Purpose

Methods

Results

Conclusions

Publications

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K<sub>ATP</sub> channel activation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K_ATP channel activation.