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

Similar Datasets

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:PURPOSE: High 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. METHODS: Human 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. RESULTS: Extracellular 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-1? 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-?B, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia. CONCLUSIONS: Hyperosmolarity 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:PurposeVariants of complement factor genes, hypoxia and oxidative stress of the outer retina, and systemic hypertension affect the risk of age-related macular degeneration. Hypertension often results from the high intake of dietary salt that increases extracellular osmolarity. We determined the effects of extracellular hyperosmolarity, hypoxia, and oxidative stress on the expression of complement genes in cultured (dedifferentiated) human RPE cells and investigated the effects of C9 siRNA and C9 protein on RPE cells.MethodsHyperosmolarity was induced by adding 100 mM NaCl or sucrose to the culture medium. Hypoxia was induced by culturing cells in 1% O2 or by adding the hypoxia mimetic CoCl2. Oxidative stress was induced by adding H2O2. Gene and protein expression levels were determined with real-time RT-PCR, western blot, and ELISA analyses. The expression of the nuclear factor of activated T cell 5 (NFAT5) and complement factor (C9) was knocked down with siRNA.ResultsExtracellular hyperosmolarity, hypoxia, and oxidative stress strongly increased the transcription of the C9 gene, while the expression of the C3, C5, CFH, and CFB genes was moderately altered or not altered at all. Hyperosmolarity also induced a moderate increase in the cytosolic C9 protein level. The hyperosmotic C9 gene expression was reduced by inhibitors of the p38 MAPK, ERK1/2, JNK, and PI3K signal transduction pathways and of the transcription factors STAT3 and NFAT5. The hypoxic C9 gene expression was reduced by a STAT3 inhibitor. The knockdown of C9 with siRNA decreased the hypoxic vascular endothelial growth factor (VEGF) and NLRP3 gene expression, the hypoxic secretion of VEGF, and the hyperosmotic expression of the NLRP3 gene. Exogenous C9 protein inhibited the hyperosmotic expression of the C9 gene, the hypoxic and hyperosmotic VEGF gene expression, and the hyperosmotic expression of the NLRP3 gene. Both C9 siRNA and C9 protein inhibited inflammasome activation under hyperosmotic conditions, as indicated by the decrease in the cytosolic level of mature IL-1β.ConclusionsThe expression of the C9 gene in cultured RPE cells is highly induced by extracellular hyperosmolarity, hypoxia, and oxidative stress. The data may support the assumption that C9 gene expression may stimulate the expression of inflammatory (NLRP3) and angiogenic growth factors (VEGF) in RPE cells. Extracellular C9 protein may attenuate this effect, in part via negative regulation of the C9 mRNA level.

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.

Similar Datasets

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.