Capasso Giovambattista, Rizzo Maria, Garavaglia Maria Lisa, Trepiccione Francesco, Zacchia Miriam, Mugione Alessandra, Ferrari Patrizia, Paulmichl Markus, Lang Florian, Loffing Johannes, Carrel Monique, Damiano Sara, Wagner Carsten A, Bianchi Giuseppe, Meyer Giuliano
Department of Internal Medicine, Chair of Nephrology, Faculty of Medicine, Second University of Napoli, Napoli, Italy.
Am J Physiol Renal Physiol. 2008 Aug;295(2):F556-67. doi: 10.1152/ajprenal.00340.2007. Epub 2008 May 14.
We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.
我们研究了哪些氯化钠转运体参与盐敏感性高血压的维持。对出生3个月后的米兰高血压（MHS）大鼠进行了研究。在MHS大鼠中，与正常血压品系（MNS）相比，通过对分离肾小管进行竞争性PCR定量分析，近端小管钠氢交换体3（NHE3）和钠钾氯共转运体2（NKCC2）的mRNA丰度没有变化，但远曲小管（DCT）中氯化钠共转运体（NCC）的mRNA丰度更高（119%，n = 5，P < 0.005）。蛋白质免疫印迹法证实了这些结果，结果显示：1）皮质中的NHE3和外髓质中的NKCC2没有变化；2）皮质中的NCC显著增加（52%，n = 6，P < 0.001）；3）α和β钠通道[上皮钠通道（ENaC）]在肾皮质中未受影响，在外髓质中略有减少，而γ-ENaC保持不变。Pendrin蛋白表达未受影响。免疫细胞化学研究显示MHS动物DCT细胞顶端膜上的NCC增加，清除实验表明MHS大鼠对苄氟噻嗪的敏感性更高（P < 0.001），这些研究都进一步证明了NCC的作用。通过对肾皮质进行蛋白质免疫印迹实验以及对从MNS和MHS动物分离的DCT原代培养细胞进行膜片钳研究，对肾脏特异性氯通道（ClC-K）进行了研究。MHS大鼠中ClC-K通道的电生理特性没有变化，但在膜片钳实验中，MNS大鼠每个膜片上的活性通道数量为0.60±0.21（n = 35），而MHS大鼠为2.17±0.59（n = 23）（P < 0.05）。数据表明，在盐敏感性高血压中，沿远曲小管的NCC和ClC-K均有强烈上调，这解释了高血压的持续存在。
