Yang Li E, Maunsbach Arvid B, Leong Patrick K K, McDonough Alicia A
Dept. of Physiology and Biophysics, University of Southern California Keck School of Medicine, 1333 San Pablo Street, MMR 626, Los Angeles, CA 90089-9142, USA.
Am J Physiol Renal Physiol. 2004 Nov;287(5):F896-906. doi: 10.1152/ajprenal.00160.2004. Epub 2004 Jul 20.
We previously reported that Na(+)/H(+) exchanger type 3 (NHE3) and NaPi2 are acutely retracted from the proximal tubule (PT) microvilli (MV) during acute hypertension [high blood pressure (BP)] or parathyroid hormone (PTH) treatment. By subcellular membrane fractionation, NHE3 and NaPi2 show indistinguishable redistribution patterns out of light-density into heavy-density membranes in response to either treatment consistent with a retraction from the apical MV to the intermicrovillar cleft region. This study aimed to examine the redistribution of PT NHE3 vs. NaPi2 by confocal and electron microscopy during high BP and during PTH treatment to determine whether their respective destinations overlap or are distinct. High-BP protocol: systolic BP was increased 50-60 mmHg by increasing peripheral resistance for 20 min; PTH protocol: rats were infused with 6.6 microg/kg iv of PTH followed by 0.1 microg.kg(-1).min(-1) infusion for 1 h. For light microscopy, rats were infused with 25 mg of horseradish peroxidase (HRP) 10 min before kidney fixation. Kidney slices were dual labeled with either NHE3 or NaPi2 and either clathrin-coated vesicle adaptor protein AP2 or endosome marker HRP. The results demonstrate retraction of NHE3 from the MV to the base of MV during either high-BP or PTH treatment: NHE3 staining did not retract below the AP2-stained domain or to HRP-labeled endosomes in either model. In comparison, NaPi2 was retracted from MV to below the AP2-stained region in both models, a little colocalizing with HRP staining. At the electron microscopic level with immunogold labeling, during high BP NHE3 was concentrated in a distinct domain in the base of the MV while NaPi2 moved to endosomes. The results demonstrate that there are divergent routes of retraction of PT NHE3 and NaPi2 from the MV during acute hypertension or PTH treatment: NHE3 is not internalized but remains at the base of the MV while NaPi2 is internalized.
我们之前报道过，在急性高血压[高血压（BP）]或甲状旁腺激素（PTH）治疗期间，3型钠/氢交换体（NHE3）和钠-磷协同转运蛋白2（NaPi2）会从近端小管（PT）微绒毛（MV）上急性回缩。通过亚细胞膜分级分离，NHE3和NaPi2在两种处理下均呈现出从低密度膜向高密度膜的难以区分的重新分布模式，这与从顶端微绒毛向微绒毛间裂隙区域的回缩一致。本研究旨在通过共聚焦显微镜和电子显微镜检查高血压和PTH治疗期间PT的NHE3与NaPi2的重新分布情况，以确定它们各自的去向是重叠还是不同。高血压方案：通过增加外周阻力20分钟，使收缩压升高50 - 60 mmHg；PTH方案：给大鼠静脉注射6.6 μg/kg的PTH，随后以0.1 μg·kg⁻¹·min⁻¹的速度输注1小时。对于光学显微镜检查，在肾脏固定前10分钟给大鼠注射25 mg辣根过氧化物酶（HRP）。肾脏切片用NHE3或NaPi2以及网格蛋白包被囊泡衔接蛋白AP2或内体标记物HRP进行双重标记。结果表明，在高血压或PTH治疗期间，NHE3从微绒毛回缩至微绒毛基部：在两种模型中，NHE3染色均未回缩至AP2染色区域以下或HRP标记的内体。相比之下，在两种模型中，NaPi2均从微绒毛回缩至AP2染色区域以下，与HRP染色有少量共定位。在电子显微镜免疫金标记水平，高血压期间NHE3集中在微绒毛基部的一个独特区域，而NaPi2则移向内体。结果表明，在急性高血压或PTH治疗期间，PT的NHE3和NaPi2从微绒毛回缩的途径不同：NHE3未被内化，而是保留在微绒毛基部，而NaPi2被内化。
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