Maskey Dipak, Liao Tang-Dong, Potter D'Anna L, Ortiz Pablo A
Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan, United States.
Department of Physiology, Integrative Bioscience Center, Wayne State University, Detroit, Michigan, United States.
Am J Physiol Renal Physiol. 2024 Dec 1;327(6):F1026-F1036. doi: 10.1152/ajprenal.00119.2024. Epub 2024 Oct 24.
In the kidney, the thick ascending limb (TAL) of the loop of Henle plays a vital role in NaCl homeostasis and blood pressure regulation. In human and animal models of salt-sensitive hypertension, NaCl reabsorption via the apical Na/K/2Cl cotransporter (NKCC2) is abnormally increased in the TAL. We showed that NaCl reabsorption is controlled by the presence of NKCC2 at the apical surface of TALs. However, the molecular mechanisms that maintain the steady-state levels of NKCC2 at the apical surface are not clearly understood. Here, we report that NKCC2 interacts with the F-actin cross-linking protein actinin-4 (ACTN4). We find that ACTN4 is expressed in TALs by Western blot and immunofluorescence microscopy. ACTN4 immunoprecipitated with NKCC2 and recombinant glutathione--transferase (GST)-ACTN4 pulled down NKCC2 from TAL lysates. ACTN4 is involved in endocytosis in other cells. Therefore, we hypothesized that ACTN4 binds apical NKCC2 and regulates its trafficking. To study the role of ACTN4 in NKCC2 surface expression, we silenced ACTN4 in vivo via shRNA or CRISPR/Cas9 system to decrease ACTN4 expression in TALs. We observed that silencing ACTN4 in vivo via shRNA or CRISPR/Cas9 system increased the amount of NKCC2 at the apical surface of TALs. Consistent with an increase in surface NKCC2, bumetanide-induced diuresis and natriuresis were enhanced by 35% after silencing of ACTN4 in vivo (AV-NKCC2-Cas9: 3,841 ± 709 vs. AAV-gRNA-ACTN4: 5,546 ± 622 µmol Na/8 h, = 5, < 0.05). We conclude that ACTN4 binds NKCC2 to regulate its surface expression. Selective depletion of ACTN4 in TALs using shRNA or CRISPR/Cas9 enhances surface NKCC2 and TAL-NaCl reabsorption, indicating that regulation of the ACTN4-NKCC2 interaction is important for renal NaCl reabsorption and could be related to hypertension. ACTN4 function and dysfunction in glomerular podocytes have been extensively studied. However, the function of ACTN4 in the nephron has not been studied. Our paper shows for the first time that ACTN4, in the nephron, regulates NaCl reabsorption in part by affecting NKCC2 surface expression. Protein-protein interactions between ACTN4 and NKCC2 seem to mediate NKCC2 endocytosis in TALs. When ACTN4 was silenced in the TAL in vivo using CRISPR/Cas9 or shRNAs, surface NKCC2 and NaCl reabsorption increased.
在肾脏中，亨氏袢的厚壁升支（TAL）在氯化钠稳态和血压调节中起着至关重要的作用。在盐敏感性高血压的人类和动物模型中，通过顶端钠/钾/2氯协同转运蛋白（NKCC2）进行的氯化钠重吸收在TAL中异常增加。我们发现，氯化钠重吸收受TAL顶端表面NKCC2的存在控制。然而，维持NKCC2在顶端表面稳态水平的分子机制尚不清楚。在此，我们报告NKCC2与F-肌动蛋白交联蛋白辅肌动蛋白-4（ACTN4）相互作用。我们通过蛋白质印迹法和免疫荧光显微镜发现ACTN4在TAL中表达。ACTN4与NKCC2免疫共沉淀，重组谷胱甘肽-S-转移酶（GST）-ACTN4从TAL裂解物中下拉NKCC2。ACTN4参与其他细胞的内吞作用。因此，我们推测ACTN4结合顶端NKCC2并调节其转运。为了研究ACTN4在NKCC2表面表达中的作用，我们通过短发夹RNA（shRNA）或CRISPR/Cas9系统在体内使ACTN4沉默，以降低TAL中ACTN4的表达。我们观察到，通过shRNA或CRISPR/Cas9系统在体内使ACTN4沉默会增加TAL顶端表面NKCC2的量。与表面NKCC2的增加一致，在体内使ACTN4沉默后，布美他尼诱导的利尿和利钠作用增强了35%（腺相关病毒-NKCC2- Cas9：3841±709 vs.腺相关病毒-引导RNA-ACTN4：5546±622 μmol钠/8小时，t = 5，P < 0.05）。我们得出结论，ACTN4结合NKCC2以调节其表面表达。使用shRNA或CRISPR/Cas9选择性地消耗TAL中的ACTN4可增强表面NKCC2和TAL-氯化钠重吸收，表明ACTN4-NKCC2相互作用的调节对于肾脏氯化钠重吸收很重要，并且可能与高血压有关。ACTN4在肾小球足细胞中的功能和功能障碍已得到广泛研究。然而，ACTN4在肾单位中的功能尚未得到研究。我们的论文首次表明，在肾单位中，ACTN4部分通过影响NKCC2表面表达来调节氯化钠重吸收。ACTN4与NKCC2之间的蛋白质-蛋白质相互作用似乎介导了TAL中的NKCC2内吞作用。当使用CRISPR/Cas9或shRNAs在体内使TAL中的ACTN4沉默时，表面NKCC2和氯化钠重吸收增加。
