López-González Zinaeli, Escobar Laura I, León-Aparicio Daniel, Mejía-Peralta Abirán Fernando, Padilla-Flores Teresa, Larre Isabel, Salvador Carolina, Medina-Campos Omar Noel, Pedraza-Chaverri José, de la Fuente-Granada Marisol
Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510, Mexico.
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510, Mexico.
Int J Mol Sci. 2025 Sep 21;26(18):9227. doi: 10.3390/ijms26189227.
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the kidney participate in reabsorbing potassium (K) and ammonium (NH) in the nephron, contributing to the acid-base balance. Acidosis is a metabolic condition of renal tubular acidosis and chronic kidney disease. Acidosis stimulates the production of mitochondrial reactive oxygen species (mROS), activating protective mechanisms dependent on mitochondrial membrane potential (Δψm) such as autophagy. The HCN3 channel is expressed in the plasma membrane, mitochondria (mitoHCN3), and lysosomes (lysoHCN3) of the rat proximal tubule. In this work we aimed to investigate the role of HCN3 in autophagy, mROS production, and Δψm in cultured rat proximal tubule cells (NRK-52E) exposed to ammonium chloride (NHCl). NHCl arrested autophagic flux and produced extracellular acidosis and, under this condition, mitoHCN3 and lysoHCN3 were up-regulated. NHCl or/and ZD7288, a specific blocker of HCN channels, enhanced mROS. ZD7288 in NHCl conditions at 24 h stimulated autophagy by reducing Beclin1, LC3BII, p62, and Parkin in an mROS- or Δψm independent pathway. Therefore, ZD7288 reverted NHCl inhibited autophagy through lysoHCN3 inhibition. Oxidative stress induced by HO up-regulated mitoHCN3 expression, while Tiron had the opposite effect. In conclusion, inhibition of mito- and lysoHCN3 channels by ZD7288 can protect against mitochondrial oxidative stress and stimulate the lysosome-autophagy pathway in response to NHCl treatment.
肾脏中的超极化激活环核苷酸门控（HCN）通道参与肾单位中钾（K）和铵（NH）的重吸收，有助于酸碱平衡。酸中毒是肾小管酸中毒和慢性肾脏病的一种代谢状况。酸中毒刺激线粒体活性氧（mROS）的产生，激活依赖线粒体膜电位（Δψm）的保护机制，如自噬。HCN3通道在大鼠近端小管的质膜、线粒体（mitoHCN3）和溶酶体（lysoHCN3）中表达。在这项研究中，我们旨在探究HCN3在氯化铵（NH4Cl）处理的大鼠近端小管细胞（NRK-52E）的自噬、mROS产生及Δψm中的作用。NH4Cl阻断自噬流并导致细胞外酸中毒，在此条件下，mitoHCN3和lysoHCN3上调。NH4Cl或/和HCN通道特异性阻滞剂ZD7288增强mROS。在NH4Cl条件下，24小时的ZD7288通过一条不依赖mROS或Δψm的途径降低Beclin1、LC3BII、p62和Parkin从而刺激自噬。因此，ZD7288通过抑制lysoHCN3逆转了NH4Cl对自噬的抑制作用。过氧化氢诱导的氧化应激上调mitoHCN3表达，而钛铁试剂则有相反作用。总之，ZD7288对mitoHCN3和lysoHCN3通道的抑制可抵御线粒体氧化应激，并在NH4Cl处理时刺激溶酶体-自噬途径。
