Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been in- tegrated into the genome of HKC. Western blot indicates that the fusion proteins have been ex- pressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics ap- proach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for ex- pression and the trans-membrane currents are measured by using two-microelectrode volt- age-clamp technique. Na+ inward currents are present on cellular membrane of the injected oo- cytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lu- men membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane lo- calization signal of the SDCT1 protein maybe locate in the C-terminal sequence.
BAI XueyuanCHEN XiangmeiFEN ZheWU DiHOU KaiCHENG GenyangPENG Lixia
IgA肾病(immunoglobulin A nephropathy,IgAN)是最常见的原发性肾小球肾炎,也是引起终末期肾脏疾病的一个重要原因。其具体的发病机制尚不明确,许多研究证实其与遗传易感性有关,属于多基因病。连锁分析发现IgAN与染色体6q22-23和2q36等连锁,关联研究发现多个候选基因(如血管紧张素转换酶基因、Fcα受体基因、人类白细胞抗原基因等)与IgAN相关。近年来,随着分子遗传学和人类基因组计划的进展,遗传因素在IgAN中的致病作用越来越受到人们的关注。
