Balen Daniela, Ljubojevic Marija, Breljak Davorka, Brzica Hrvoje, Zlender Vilim, Koepsell Hermann, Sabolic Ivan
Molecular Toxicology, Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10001 Zagreb, Croatia.
Am J Physiol Cell Physiol. 2008 Aug;295(2):C475-89. doi: 10.1152/ajpcell.00180.2008. Epub 2008 Jun 4.
Previously, we characterized localization of Na(+)-glucose cotransporter SGLT1 (Slc5a1) in the rat kidney using a polyclonal antibody against the synthetic COOH-terminal peptide of the rat protein (Sabolić I, Skarica M, Gorboulev V, Ljubojević M, Balen D, Herak-Kramberger CM, Koepsell H. Am J Physiol Renal Physiol 290: 913-926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, we have presently generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolated membranes and tissue cryosections from male (M) and female (F) rats exhibited 1) in kidneys and small intestine, labeling of a major protein band of approximately 75 kDa; 2) in kidneys of adult animals, localization of rSGLT1 to the proximal tubule (PT) brush-border membrane (S1 < S2 < S3) and intracellular organelles (S1 > S2 > S3), with zonal (cortex < outer stripe) and sex differences (M < F) in the protein expression, which correlated well with the tissue expression of its mRNA in RT-PCR studies; 3) in kidneys of castrated adult M rats, upregulation of the protein expression; 4) in kidneys of prepubertal rats, weak and sex-independent labeling of the 75-kDa protein band and immunostaining intensity; 5) in small intestine, sex-independent regional differences in protein abundance (jejunum > duodenum = ileum); and 6) thus far unrecognized localization of the transporter in cortical thick ascending limbs of Henle and macula densa in kidney, bile ducts in liver, enteroendocrine cells and myenteric plexus in the small intestine, and initial ducts in the submandibular gland. Our improved rSGLT1-ab may be used to identify novel sites of SGLT1 localization and thus unravel additional physiological functions of this transporter in rat organs.
此前，我们使用针对大鼠蛋白合成羧基末端肽的多克隆抗体，对大鼠肾脏中钠 - 葡萄糖共转运蛋白SGLT1（Slc5a1）的定位进行了表征（萨博利奇I，斯卡里察M，戈尔布列夫V，柳博耶维奇M，巴伦D，赫拉科 - 克拉姆贝格尔CM，克普塞尔H。《美国生理学杂志：肾脏生理学》290：913 - 926，2006）。然而，该抗体在免疫化学研究中出现了一些假阳性反应。通过使用缩短的肽进行免疫，我们目前制备了一种改进的、更具特异性的抗大鼠SGLT1抗体（rSGLT1 - ab），该抗体在对雄性（M）和雌性（F）大鼠的分离膜和组织冰冻切片进行免疫化学研究时显示：1）在肾脏和小肠中，标记出一条约75 kDa的主要蛋白条带；2）在成年动物的肾脏中，rSGLT1定位于近端小管（PT）刷状缘膜（S1 < S2 < S3）和细胞内细胞器（S1 > S2 > S3），在蛋白表达上存在区域（皮质 < 外带）和性别差异（M < F），这与RT - PCR研究中其mRNA的组织表达情况良好相关；3）在去势成年雄性大鼠的肾脏中，蛋白表达上调；4）在青春期前大鼠的肾脏中，75 kDa蛋白条带的标记较弱且与性别无关，免疫染色强度也与性别无关；5）在小肠中，蛋白丰度存在与性别无关的区域差异（空肠 > 十二指肠 = 回肠）；6）迄今为止未被认识到的该转运蛋白在肾脏髓袢升支粗段和致密斑、肝脏胆管、小肠肠内分泌细胞和肌间神经丛以及下颌下腺初始导管中的定位。我们改进的rSGLT1 - ab可用于识别SGLT1定位的新位点，从而揭示该转运蛋白在大鼠器官中的其他生理功能。
