Hao Shoujin, Lasaracina Anna Pia, Epps Jarred, Ferreri Nicholas R
Department of Pharmacology, New York Medical College, Valhalla, New York, United States.
Am J Physiol Renal Physiol. 2025 Apr 1;328(4):F489-F500. doi: 10.1152/ajprenal.00251.2024. Epub 2025 Mar 10.
We previously demonstrated that tumor necrosis factor-alpha (TNF) inhibits Na-K-2Cl cotransporter (NKCC2) phosphorylation in the thick ascending limb (TAL); however, the underlying mechanism remains unclear. We tested the hypothesis that the induction of calcineurin (CN) activity and the expression of CN isoforms contribute to the mechanism by which TNF inhibits phospho-NKCC2 (pNKCC2) expression. CN activity increased by approximately twofold in primary cultures of medullary (m)TAL cells challenged with mouse recombinant TNF. In contrast, silencing TNF production in mTAL cells using lentivirus U6-TNF-ex4 reduced CN activity. pNKCC2 expression decreased in mTAL cells challenged with TNF, whereas inhibition of CN activity with cyclosporine A (CsA) increased pNKCC2 expression. Although mTAL cells express both the calcineurin A subunit (CNA) α and β isoforms, only CNA β isoform mRNA increased after mTAL cells were challenged with TNF. In vivo, both TNF and CNA β expression increased in outer medulla (OM) from mice given 1% NaCl in the drinking water for 7 days and intrarenal lentivirus silencing of TNF selectively reduced expression of CNA β. Intrarenal injection of a lentivirus that specifically silenced CNA β (U6-CNAβ-ex6) increased pNKCC2 expression and attenuated the inhibitory effects of TNF on pNKCC2 expression in freshly isolated TAL tubules. Collectively, the study is the first to demonstrate that TNF increases CN activity and specifically induces β-isoform expression in the kidney. Since NKCC2 is a known target of the CNA β isoform, these findings suggest that a CN-dependent signaling pathway involving this isoform contributes to the mechanism by which TNF inhibits pNKCC2 expression. The beneficial immunosuppressive effects of CsA are tempered by renal side effects including reduction of GFR, proximal tubule damage, reduced urinary concentration, fibrosis and hypertension. As chronic administration of CN inhibitors frequently induce hypertension and renal nephropathy in humans, understanding the molecular mechanisms by which CN isoforms regulate the activity of renal transporters may provide the framework for developing new drugs that more selectively modulate the diverse functions of CN.
我们之前证明肿瘤坏死因子-α（TNF）可抑制厚壁升支（TAL）中的钠-钾-2氯协同转运蛋白（NKCC2）磷酸化；然而，其潜在机制仍不清楚。我们检验了以下假说：钙调神经磷酸酶（CN）活性的诱导及CN亚型的表达参与了TNF抑制磷酸化NKCC2（pNKCC2）表达的机制。在用小鼠重组TNF刺激的髓质（m）TAL细胞原代培养物中，CN活性增加了约两倍。相反，使用慢病毒U6-TNF-ex4沉默mTAL细胞中的TNF产生可降低CN活性。在用TNF刺激的mTAL细胞中，pNKCC2表达降低，而用环孢素A（CsA）抑制CN活性则增加了pNKCC2表达。尽管mTAL细胞同时表达钙调神经磷酸酶A亚基（CNA）α和β亚型，但在用TNF刺激mTAL细胞后，只有CNA β亚型的mRNA增加。在体内，给饮用1%氯化钠溶液7天的小鼠的外髓质（OM）中，TNF和CNA β表达均增加，而肾内慢病毒沉默TNF可选择性降低CNA β的表达。肾内注射特异性沉默CNA β的慢病毒（U6-CNAβ-ex6）可增加pNKCC2表达，并减弱TNF对新鲜分离的TAL小管中pNKCC2表达的抑制作用。总体而言，该研究首次证明TNF可增加肾脏中的CN活性并特异性诱导β亚型表达。由于NKCC2是CNA β亚型的已知靶点，这些发现表明涉及该亚型的CN依赖性信号通路参与了TNF抑制pNKCC2表达的机制。CsA有益的免疫抑制作用受到包括肾小球滤过率降低、近端小管损伤、尿浓缩功能降低、纤维化和高血压等肾脏副作用的影响。由于长期使用CN抑制剂在人类中经常会诱发高血压和肾病，了解CN亚型调节肾转运蛋白活性的分子机制可能为开发更具选择性地调节CN多种功能的新药提供框架。
