Borschewski Aljona, Himmerkus Nina, Boldt Christin, Blankenstein Katharina I, McCormick James A, Lazelle Rebecca, Willnow Thomas E, Jankowski Vera, Plain Allein, Bleich Markus, Ellison David H, Bachmann Sebastian, Mutig Kerim
Department of Anatomy, Charité-Universitätsmedizin Berlin, Berlin, Germany;
Institute of Physiology, Kiel University, Kiel, Germany;
J Am Soc Nephrol. 2016 Jan;27(1):107-19. doi: 10.1681/ASN.2014070728. Epub 2015 May 12.
The furosemide-sensitive Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Aβ isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Aβ in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Aβ. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aβ and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aβ and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.
速尿敏感的钠-钾-2氯协同转运蛋白（NKCC2）对于肾脏髓袢升支粗段（TAL）中的氯化钠重吸收至关重要，并驱动尿液浓缩机制。NKCC2的活性受N端磷酸化和去磷酸化调节。已鉴定出激活NKCC2的丝氨酸-苏氨酸激酶，但对使NKCC2失活的磷酸酶了解较少。钙调神经磷酸酶的抑制已被证明可刺激TAL和远曲小管中的转运。在此，我们鉴定出NKCC2是钙调神经磷酸酶Aβ亚型的靶标。小鼠短期给予环孢素可增加磷酸化NKCC2的丰度，用环孢素处理分离的TAL可增加NKCC2的氯离子亲和力和转运活性。由于具有A型重复序列的分选相关受体（SORLA）可能影响NKCC2的磷酸调节，我们使用SORLA基因敲除小鼠来测试SORLA是否参与钙调神经磷酸酶依赖性的NKCC2调节。与同窝对照相比,SORLA缺陷小鼠在TAL细胞顶端区域显示出更多的钙调神经磷酸酶Aβ，而NKCC2的磷酸化和活性则较低。相反，在培养细胞中过表达SORLA可降低内源性钙调神经磷酸酶Aβ的丰度。环孢素给药可迅速使SORLA缺陷小鼠中磷酸化NKCC2的丰度恢复正常，并且大鼠肾脏提取物中的结合试验进一步证实了钙调神经磷酸酶Aβ与SORLA之间的功能相互作用。总之，我们已经表明钙调神经磷酸酶Aβ和SORLA是NKCC2磷酸调节的关键组成部分。这些结果可能对使用钙调神经磷酸酶抑制剂的免疫抑制治疗具有临床意义。
