Authors’ Reply: Deciphering the Cell-Specific Specificity of the α-Klotho–Cellular Inhibitor of Apoptosis Protein 1 Axis in Autosomal Dominant Polycystic Kidney Disease

We appreciate the thoughtful Letter to the Editor from Ling et al.,1 for their generous comments on the significance of our study published in JASN2 and for their interest in the mechanisms underlying the internalization and anti-inflammatory effects of αKlotho in autosomal dominant polycystic kidney disease. Our study demonstrated that αKlotho suppresses NF-κB signaling through its interaction with cellular inhibitor of apoptosis protein 1 (cIAP1) in renal epithelial cells, leading to reduced expression of TNFα and monocyte chemoattractant protein-1, key mediators of macrophage recruitment and activation. Consistent with these findings, macrophage accumulation was decreased in kidneys with transgenic αKlotho or treatment with recombinant αKlotho. Whether αKlotho directly modulates immune cells independently of epithelial cell–derived signals remains unknown. The expression of cIAP1 in macrophage clusters was unchanged in kidneys from Pkd1-HOMO-KLTg mice as revealed by single-cell RNA sequencing analysis, supporting that αKlotho did not regulate cIAP1 at the transcriptional level. Although αKlotho has been reported to regulate macrophage polarization through indirect mechanisms,3 there is no evidence that αKlotho is internalized by macrophages. If αKlotho can be taken up by macrophages, it is conceivable that it may interact with and destabilize cIAP1 in those cells, like its action in renal epithelial cells, which can promote apoptosis of infiltrating macrophages and/or alter macrophage polarization through the modulation of NF-κB signaling. Soluble αKlotho primarily functions extracellularly by binding to target molecules, such as transient receptor potential vanilloid, renal outer medullary potassium channel, lipid rafts, and fibroblast growth factor receptor,4 but it can also exert intracellular effects after cellular internalization. Our study supports the intracellular entry of recombinant αKlotho into HEK293T cells and demonstrates its interaction with the caspase recruitment domain of cIAP1 (Figure 6F). In addition, transcytosis of soluble αKlotho by renal epithelial cells, where αKlotho is taken up from the basolateral side and released at the apical surface, has been previously reported.5 The mechanisms governing αKlotho entry remain to be elucidated, and the defining of those pathways will be critical for determining cell type–specific internalization, bioavailability, and potential toxicity of αKlotho-based therapeutics. In summary, our study uncovers the role and mechanisms of αKlotho by identifying the αKlotho–cIAP1 axis in renal epithelial cells and provides strong evidence supporting αKlotho as a therapeutic candidate for ADPKD, in addition to its potential roles in other age-related diseases. We highlight the need for further investigation of the direct effects of αKlotho on immune cells. Such studies will be important for understanding immunologic effects of αKlotho and optimizing dosing strategies in future clinical trials.