Ahn K Y, Kone B C
DCI Laboratory of Molecular Biology in Nephrology, University of Florida College of Medicine, Gainesville 32610.
Am J Physiol. 1995 Jan;268(1 Pt 2):F99-109. doi: 10.1152/ajprenal.1995.268.1.F99.
The distribution of transcripts encoding the gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) alpha-subunit in the normal rat kidney was studied by reverse transcription-polymerase chain reaction (RT-PCR), combined with DNA sequence analysis and renal microdissection, and by nonradioactive in situ hybridization of fixed kidney sections using highly specific molecular probes. RT-PCR products corresponding to the gastric H(+)-K(+)-ATPase alpha-subunit were detected in the cortex, outer and inner medulla, and in isolated cortical (CCD) and inner medullary collecting ducts (IMCD). With digoxigenin-labeled cRNAs derived from the 5' and 3' ends of the gastric H(+)-K(+)-ATPase alpha-subunit cDNA, specific hybridization signal was detected prominently in all the cells of the connecting segment and CCD, the intercalated cells of the outer medullary collecting duct, the IMCD, and the renal pelvic epithelium lining the secondary pouches. Weak labeling was noted in the S3 segment of the proximal tubule, the distal convoluted tubule, and the cortical thick ascending limb of Henle. Hybridization with the sense probes produced no cellular labeling. These data provide the first direct demonstration for the expression and cellular distribution of mRNA encoding the gastric H(+)-K(+)-ATPase alpha-subunit in the normal, potassium-replete kidney, and they provide essential tools for the molecular analysis of renal acid base and potassium transport under physiological and pathophysiological conditions.
采用逆转录-聚合酶链反应（RT-PCR），结合DNA序列分析和肾显微切割技术，并使用高特异性分子探针，对固定肾切片进行非放射性原位杂交，研究了正常大鼠肾脏中编码胃H(+)-K(+)-三磷酸腺苷酶（ATP酶）α亚基的转录本分布。在皮质、外髓和内髓以及分离的皮质集合管（CCD）和内髓集合管（IMCD）中检测到了与胃H(+)-K(+)-ATP酶α亚基相对应的RT-PCR产物。用源自胃H(+)-K(+)-ATP酶α亚基cDNA 5'和3'端的地高辛标记cRNA，在连接段和CCD的所有细胞、外髓集合管的闰细胞、IMCD以及次级肾盏内衬的肾盂上皮细胞中均显著检测到特异性杂交信号。在近端小管的S3段、远曲小管和亨氏袢皮质厚升支中观察到弱标记。与正义探针杂交未产生细胞标记。这些数据首次直接证明了在正常、钾充足的肾脏中编码胃H(+)-K(+)-ATP酶α亚基的mRNA的表达和细胞分布，为生理和病理生理条件下肾脏酸碱和钾转运的分子分析提供了重要工具。
