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Nfat5 is involved in the hyperosmotic regulation of Tmem184b, which could be involved in ibuprofen transport in renal MDCK I cells

ABSTRACT: Purpose: To investigate the involvement of Nfat5 in the regulation of an ibuprofen transporter Method: Reverse transfection with siRNA against Nfat5 was used to knockdown Nfat5 in MDCK I cells. The uptake of both radiolabelled taurine and ibuprofen was measured in MDCK I cells, first treated with siRNA against Nfat5 for 24 h and afterwards cultivated with raffinose-supplemented normal growth medium (500 mOsm) for 24 h. The knock down of NFat5 was investigated by qpCR and western blotting. A transcriptome analysis of MDCK I cells treated with siRNA against Nfat5 was performed. Validation of the transcriptome analysis was perform with qPCR and radiolabelled uptake of ibuprofen. Results: The siRNA transfection resulted in a knock down of Nfat5, and the uptake of both taurine and ibuprofen was significant decreased in the transfected MDCK I cells. The transcriptome analysis of MDCK I cells treated with siRNA against Nfat5 revealed genes regulated by Nfat5 during hyperosmotic exposure. qPCR and the transciptome analysis showed an involvement of Nfat5 in the hyperosmotic regulation of Tmem184 and uptake in Tmem184b-siRNA treated MDCK I cells showed that Tmem184b might be involved in uptake of ibuprofen. Conclusion: The regulation of Tmem184b was found regulated by Nfat5 and could be a potential possible candidate for the gene product responsible for the uptake of ibuprofen in MDCK I cells

ORGANISM(S): Canis lupus familiaris

PROVIDER: GSE122074 | GEO | 2019/05/10

REPOSITORIES: GEO

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Project description:Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using E. coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above ~1.0 Os kg-1 causes a dose-dependent K+ leak from the cell, resulting in a substantial decrease in cytosolic K+ content and a concurrent accumulation of Na+ in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K+ uptake. Ion flux data is consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K+ uptake to efflux. Microarray experiments reveal that about 40% of up-regulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic- and non-ionic osmotica The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting the importance of voltage-gated K+ transporters for bacterial adaptation to hyperosmotic stress. Experiment Overall Design: Two biological replicates per treatment with microarray analysis using the Affymetrix GeneChip E. coli Genome array. Treatments used included: Experiment Overall Design: Control - E. coli Frag1, grown to early stationary growth phase in a mineral salts medium with 0.1% glucose at 25 C Experiment Overall Design: Sucrose hyperosmotic treatment - 1.25 M sucrose added to control culture Experiment Overall Design: for 10 min. Experiment Overall Design: NaCl hyperosmotic treatment - 1.37 M NaCl added to control culture Experiment Overall Design: For microarray data comparisons the sucrose and NaCl hyperosmotic treatment data was compared to the no treatment control data separately. The sucrose to control and NaCL to control comparison data tables are linked below.

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Nfat5 is involved in the hyperosmotic regulation of Tmem184b, which could be involved in ibuprofen transport in renal MDCK I cells

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