Inhibition of HCN Channels Enhances Oxidative Stress and Autophagy of NRK-52E Cells Under NH4Cl Treatment

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the kidney participate in reabsorbing potassium (K⁺) and ammonium (NH₄⁺) 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 (NH₄Cl). NH₄Cl arrested autophagic flux and produced extracellular acidosis and, under this condition, mitoHCN3 and lysoHCN3 were up-regulated. NH₄Cl or/and ZD7288, a specific blocker of HCN channels, enhanced mROS. ZD7288 in NH₄Cl conditions at 24 h stimulated autophagy by reducing Beclin1, LC3BII, p62, and Parkin in an mROS- or Δψm independent pathway. Therefore, ZD7288 reverted NH₄Cl inhibited autophagy through lysoHCN3 inhibition. Oxidative stress induced by H₂O₂ 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 NH₄Cl treatment.