Determining the Pathogenic Mechanism Underlying Childhood ADPKD Caused by a Monoallelic Pathogenic Variant in NEK8

Background: While autosomal dominant polycystic kidney disease (ADPKD) typically manifests in adults, a fraction present early and rapidly progress to kidney failure during infancy and childhood. We recently identified a monoallelic variant in NEK8 (c.133C>T (p.Arg45Trp)), resulting in an enzymatic deficient serine/threonine kinase that resides in primary cilia. Unlike biallelic pathogenic NEK8 variants causing systemic ciliopathies, the p.Arg45Trp variant causes early-onset ADPKD with few extrarenal defects. Thus, NEK8R45W seems specific for renal cystogenesis and is a unique tool to dissect the pathogenic mechanism of PKD in children. Methods: Mouse renal epithelial cell lines null for NEK8 were generated using CRISPR/Cas9 and wild type (WT) or R45W human NEK8 constructs were reexpressed: Ciliary biogenesis, structure, and signaling were analyzed. Phospho-site and proximity-dependent proteomics were conducted using the cell lines to identify proteins that are differentially phosphorylated, deposited into cilia, or associated with NEK8 variants. We constructed a mouse model to study the physiological role of NEK8R45W. Results: Although Nek8-null cilia are shortened, NEK8R45W cilia retained comparable length and overall structural integrity to the WT cilia, but the PKD1/PKD2 proteins, PC1 and PC2, were significantly reduced in NEK8R45W cilia. Although the ciliary level of GPR161 remained unchanged, SAG induced less accumulation of Smoothened and Gli3 in NEK8R45W cilia than WT, indicating a weaker activation of the Hedgehog (Hh) signaling. Phospho-site proteomics results supported the defective kinase activity of NEK8R45W and revealed mis-regulated pathways in cells expressing NEK8R45W. Importantly, Nek8+/R45W mice showed aggressive renal cystogenesis within 3 weeks after birth, recapitulating the early-onset ADPKD phenotype in patients. Conclusion: We further study the characteristics of the monoallelic NEK8 p.Arg45Trp and determined that the kinase activity of NEK8 in cilia is necessary for the ciliary trafficking of the PC complex, activation of Hh signaling, and epithelial morphogenesis. These signaling pathways may collectively contribute to the establishment and maintenance of renal tubule structures. Understanding the crosstalk between these pathways via studying the role of NEK8R45W may reveal potential drug targets for the treatment of ADPKD in children. Funding: Other U.S. Government Support