Peti-Peterdi J, Chambrey R, Bebok Z, Biemesderfer D, St John P L, Abrahamson D R, Warnock D G, Bell P D
Department of Medicine, Division of Nephrology and Nephrology Research and Training Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Am J Physiol Renal Physiol. 2000 Mar;278(3):F452-63. doi: 10.1152/ajprenal.2000.278.3.F452.
Functional and immunohistochemical studies were performed to localize and identify Na(+)/H(+) exchanger (NHE) isoforms in macula densa cells. By using the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, intracellular pH (pH(i)) was measured with fluorescence microscopy by using 2',7'-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein. NHE activity was assayed by measuring the initial rate of Na(+)-dependent pH(i) recovery from an acid load imposed by prior lumen and bath Na(+) removal. Removal of Na(+) from the bath resulted in a significant, DIDS-insensitive, ethylisopropyl amiloride (EIPA)-inhibitable decrease in pH(i). This basolateral transporter showed very low affinity for EIPA and Hoechst 694 (IC(50) = 9.0 and 247 microM, respectively, consistent with NHE4). The recently reported apical NHE was more sensitive to inhibition by these drugs (IC(50) = 0.86 and 7.6 microM, respectively, consistent with NHE2). Increasing osmolality, a known activator of NHE4, greatly stimulated basolateral NHE. Immunohistochemical studies using antibodies against NHE1-4 peptides demonstrated expression of NHE2 along the apical and NHE4 along the basolateral, membrane, whereas NHE1 and NHE3 were not detected. These results suggest that macula densa cells functionally and immunologically express NHE2 at the apical membrane and NHE4 at the basolateral membrane. These two isoforms likely participate in Na(+) transport, pH(i), and cell volume regulation and may be involved in tubuloglomerular feedback signaling by these cells.
进行了功能和免疫组织化学研究，以定位和鉴定致密斑细胞中的钠氢交换体（NHE）亚型。使用从兔肾中分离出的带有附着肾小球的灌注厚升支制备物，通过荧光显微镜使用2',7'-双（2-羧乙基）-5-（和-6）羧基荧光素测量细胞内pH值（pH(i)）。通过测量在先前管腔和浴液中去除钠后施加酸负荷时钠依赖性pH(i)恢复的初始速率来测定NHE活性。从浴液中去除钠导致pH(i)显著下降，对二异丙基氨基磺酸钠（DIDS）不敏感，但可被乙基异丙基氨氯吡咪（EIPA）抑制。这种基底外侧转运体对EIPA和Hoechst 694的亲和力非常低（IC(50)分别为9.0和247 microM，与NHE4一致）。最近报道的顶端NHE对这些药物的抑制更敏感（IC(50)分别为0.86和7.6 microM，与NHE2一致）。增加渗透压是已知的NHE4激活剂，可极大地刺激基底外侧NHE。使用针对NHE1-4肽的抗体进行的免疫组织化学研究表明，NHE2沿顶端表达，NHE4沿基底外侧膜表达，而未检测到NHE1和NHE3。这些结果表明，致密斑细胞在功能和免疫方面在顶端膜表达NHE2，在基底外侧膜表达NHE4。这两种亚型可能参与钠转运、pH(i)和细胞体积调节，并可能参与这些细胞的肾小管-肾小球反馈信号传导。
