| Background and Objective: The hNaDC3 (human NaVdicarboxylate cotransporter 3)mediates the transport of Krebs cycle intermediates, such as succinate, citrate and a-ketoglutarate, across the plasma membrane of many epithelial cells. It not only is involved in energy utilization as well as in the synthesis of amino acid and glucocorticosteroid, but also plays an important role in the formation of renal calcium-containing stones. It is well known that in patients who suffer from starvation, chronic metabolic acidosis, chronic renal insufficiency, distal renal tubular acidosis and nephrolithiasis, the reabsorption of urinary citrate is increased and the excretion is decreased in kidney. Recently, it is reported that NaDC may be a key factor in the regulating process of the life-span of mammalian animals. Therefore, it is valuable to elucidate the biological function of hNaDC3 and its relationship to renal diseases. This study was designed to construct the fusion gene of hNaDC3 and green fluorescent protein(GFP), and study the localization of the fusion protein in renal proximal tubule epithelia. Meanwhile, designed to construct two recombinant eukaryotic expression vectors of hNaDC3, pcDNA3-hNaDC3 encoding sense mRNA and pcDNA3-AhNaDC3 encoding antisense mRNA, and investigate the effect of sense and antisense hNaDC3 on mitochondrial membrane potential ( ) and energy metabolism of the cell in human proximal tubular epithelial cells(HKC).Methods: The hNaDCS full-length cDNA containing the signal peptide was amplified by reverse transcription and polymerase chain reaction (RT-PCR) from total RNA of human normal renal tissues, and then cloned into pGEM-T vector. After DNA sequencing, the hNaDC3 was inserted into mammalian expression plasmid pEGFP-C3 successfully. The fused gene was transfected into LLC-PK1 cell with lipofectAMINE. Meanwhile, capped cRNA for microinjection was synthesized in vitro from linearized plasmid DNA, and injected into oocytes. Then two recombinant eukaryotic expression vectors, pcDNA3-hNaDC3 encoding sense mRNA and pcDNA3-AhNaDC3 encoding antisense mRNA were constructed by using DNA recombining techniques. These two vectors were then transfected into HKC with LipofectAMINE. RT-PCR, Northern blotting and Western blotting were used to detect the integration and expression of hNaDCS. Fluorescent JC-1 in combination with confocal fluorescence microscopy and flow cyometry was used to detect the changes of in normal HKC, pcDNAS transfected-, pcDNA3-hNaDC3 transfected- and pcDNA3-AhNaDC3 transfected-HKC.Resu I ts: (1) Confocal fluorescence microscopy showed that green fluorescence spread over the whole cell of LLC-PK1 transfected with pEGFP-C3. After transfection with pEGFP-C3-hNaDC3, fluorescence distributed in the cytoplasm and on the plasma membrane on first day, located mainly on the plasma membrane on fifth day. After 2 days, cRNA-injected oocytes had a distinct plasma membrane-associated flurescence while water-injected oocytes showed negative. (2) Both sense and antisense hNaDC3 were successfully integrated into HKC and expressed the sense and antisense hNaDC3 mRNA. Compared with control, HKC transfected with pcDNA3-hNaDC3 had a low , JC-1 monomer were formed in the inner membrane of mitochondria and emitted green fluorescence, while HKC transfected with pcDNA3-AhNaDC3 had a little high , JC-1 monomer were associated to aggregates, which emitted red fluorescence.Conclusions: It is suggested that the hNaDCS protein is produced in cytoplasm, localizes on the plasma membrane. Overexpression of hNaDCS induced by sense hNaDCS caused decrease of , in contrast, suppression of hNaDCS expression by antisense hNaDCS caused increase of . It is suggested that NaDCS might be involved in energy metabolism of the cell by causing the decrease of mitochondria! membrane potential.
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