Pihakaski-Maunsbach Kaarina, Vorum Henrik, Honoré Bent, Tokonabe Shigeki, Frøkiaer Jørgen, Garty Haim, Karlish Steven J D, Maunsbach Arvid B
The Water and Salt Research Ctr, Institute of Anatomy Univ. of Aarhus, DK-8000 Aarhus, Denmark.
Am J Physiol Renal Physiol. 2006 Nov;291(5):F1033-44. doi: 10.1152/ajprenal.00086.2006. Epub 2006 Jun 6.
The gamma-subunit of Na-K-ATPase (FXYD2) and corticosteroid hormone-induced factor (CHIF; FXYD4) are considered pump regulators in kidney tubules. The aim of this study was to expand the information about their locations in the kidney medulla and to evaluate their importance for electrolyte excretion in an animal model. The cellular and subcellular locations and abundances of gamma and CHIF in the medulla of control and sodium-depleted rats were analyzed by immunofluorescence and immunoelectron microscopy and semiquantitative Western blotting. The results showed that antibodies against the gamma-subunit COOH terminus and splice variant gamma(a), but not splice variant gamma(b), labeled intercalated cells, but not principal cells, in the initial part of the inner medullary collecting duct (IMCD1). In subsequent segments (IMCD2 and IMCD3), all principal cells exhibited distinct basolateral labeling for both the gamma-subunit COOH terminus, splice variant gamma(a), and CHIF. Splice variant gamma(b) was abundant in the inner stripe of the outer medulla but absent in the inner medulla (IM). Double labeling by high-resolution immunoelectron microscopy showed close structural association between CHIF and the Na-K-ATPase alpha(1)-subunit in basolateral membranes. The present observations provide new information about the cellular and subcellular locations of gamma and CHIF in the renal medulla and show a new gamma variant in the IM. Extensive NaCl depletion did not induce significant changes in the locations or abundances of the gamma-subunit COOH terminus and CHIF in different kidney zones. We conclude that the unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during NaCl depletion.
钠钾ATP酶的γ亚基（FXYD2）和皮质类固醇激素诱导因子（CHIF；FXYD4）被认为是肾小管中的泵调节因子。本研究的目的是扩展关于它们在肾髓质中位置的信息，并在动物模型中评估它们对电解质排泄的重要性。通过免疫荧光、免疫电子显微镜和半定量蛋白质免疫印迹法分析了对照大鼠和缺钠大鼠髓质中γ亚基和CHIF的细胞及亚细胞定位和丰度。结果显示，针对γ亚基COOH末端和剪接变体γ(a)的抗体标记了内髓集合管起始段（IMCD1）的闰细胞，而非主细胞，但针对剪接变体γ(b)的抗体则未标记。在随后的节段（IMCD2和IMCD3）中，所有主细胞的γ亚基COOH末端、剪接变体γ(a)和CHIF均在基底外侧呈现明显标记。剪接变体γ(b)在外髓质内带丰富，但在内髓质（IM）中缺失。高分辨率免疫电子显微镜双重标记显示，CHIF与基底外侧膜中的钠钾ATP酶α(1)亚基存在紧密的结构关联。本研究结果提供了关于γ亚基和CHIF在肾髓质中细胞及亚细胞定位的新信息，并在内髓质中发现了一种新的γ变体。大量氯化钠缺失并未导致不同肾区γ亚基COOH末端和CHIF的定位或丰度发生显著变化。我们得出结论，这两种FXYD蛋白水平未变表明它们不是氯化钠缺失期间尿液电解质组成的主要决定因素。
