| Summary: | 碩士 === 中興大學 === 生命科學系所 === 99 === Na+,K+-ATPase (NKA) is a widely existence and important transporter in animal cells. NKA uptake two of K+ and extrusion of Na+ in providing the driving force to trigger the secondary active transporters by using the energy of the hydrolysis of ATP. Cells regulated NAK function to adapt the environmental osmotic changes. The mammalian kidney was the key organ in osmotic homeostasis and proximal tubule was the one of major tissue. This study used human renal proximal tubule cells HK-2 and microarray screened the up-regulated genes which correlate to NKA function upon hypertonic challenge. FXYD2 protein was specificity in kidney, and it could effect on sodium/potassium affinity of NKA. To confirm the microarray data, FXYD2 isoform 3 was markedly expressed. There were still no reporters to discuss detail function of isoform 3 in osmotic regulation. Therefore, FXYD2 isoform 3 was the target of this study with functional assay. In this study, the mRNA and protein abundance of FXYD2 isoform 3 were significantly increased upon hypertonic challenge. In addition, the localization of FXYD2 isoform 3 was distributed on cell membrane as NKA α1 subunit. Moreover, we obtained FXYD2 isoform 3 indeed had interaction with NKA α1 subunit. Accordingly, it was suggested that FXYD2 isoforms3 played the role in regulation of NKA activity. Therefore, the potential role of FXYD2 isoform 3 will be illustrated during hypertonic challenge by using the RNAi-A (knockdown isoform 1, 2 and 3) and RNAi-B (knockdown isoform 1 and 2) knockdown cells. FXYD2 knockdown cells did not affect NKA protein expression level, but NKA activity was affected upon hypertonic stress. NKA activity high to low was RNAi-B > RNAi-A > wild type. FXYD2 isoform 1and 2 played the repressor role, and isoform 3 played an enhancer role in NKA activity regulation. On the other hand, some studies indicated TonEBP was a transcription factor and it was higher expression upon hypertonic challenge. TonEBP modulated its downstream genes to protect cells could adapt the effects of hypertonic challenge. Hence, modulation mechanism of TonEBP was important when cells were faced hypertonic environment. In TonEBP knockdown cells, FXYD2 isoform 3 expression was regulated by TonEBP and result in affecting NKA activity. Take together, this study was the first reporter to demonstrate that hypertonicity promoted FXYD2 isoform 3 expression through TonEBP-dependent regulation, and then enhanced NKA activity in HK-2 cells.
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