Bernardo A A, Kear F T, Santos A V, Ma J, Steplock D, Robey R B, Weinman E J
Section of Nephrology, Department of Medicine, College of Medicine, University of Illinois-Chicago, Chicago, Illinois 60612-7315, USA.
J Clin Invest. 1999 Jul;104(2):195-201. doi: 10.1172/JCI5344.
In the renal proximal tubule, the activities of the basolateral Na(+)/HCO(3)(-) cotransporter (NBC) and the apical Na(+)/H(+) exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na(+)/H(+) exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10(-8)M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF-expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 microM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby basolateral NBC and apical NHE3 activities may be coordinately regulated in renal proximal tubule cells.
在肾近端小管中，基底外侧Na⁺/HCO₃⁻共转运体（NBC）和顶端Na⁺/H⁺交换体（NHE3）的活性始终呈平行变化，这表明它们受到协同调节。蛋白激酶A（PKA）介导的对NHE3的抑制作用是由一种含PDZ基序的蛋白——Na⁺/H⁺交换体调节因子（NHE-RF）介导的。鉴于这些转运体在蛋白激酶A（PKA）激活后受到共同抑制，我们试图确定NHE-RF在PKA调节的NBC活性中是否也发挥作用。使用针对兔NHE-RF的抗肽抗体进行的肾皮质免疫印迹分析表明，这种调节因子存在于刷状缘膜（BBM）和基底外侧膜（BLM）中。通过重组实验，我们发现用胰蛋白酶对去垢剂溶解的兔肾BLM制剂进行有限消化后，NBC活性不受PKA激活的影响。将这些经胰蛋白酶处理的制剂与对应于野生型兔NHE-RF的重组蛋白共同重组，以浓度依赖的方式恢复了PKA对NBC活性的抑制作用。10⁻⁸M的NHE-RF可使NBC活性抑制60%；在无PKA的情况下未观察到这种效应。用热变性的NHE-RF重组也未能减弱NBC活性。为了进一步确定NHE-RF在NBC调节中的生理作用，对缺乏可检测到的内源性NHE-RF表达的肾上皮细胞系B-SC-1进行基因工程改造，使其稳定表达NHE-RF转基因。表达NHE-RF的B-SC-1细胞（B-SC-RF）的NBC活性基础水平明显低于野生型对照。用10μM的福斯可林处理后，B-SC-RF细胞中NBC活性的抑制作用增强，这与NHE-RF在介导PKA对NBC活性的抑制作用中的假定作用一致。这些发现不仅表明NHE-RF参与了PKA调节的NBC活性，还提供了一种独特的分子机制，通过该机制，基底外侧NBC和顶端NHE3的活性在肾近端小管细胞中可能受到协同调节。
