Universität zu Köln

Baten, Bijan (2025). Ciliary and Nuclear Characterization of NPHP9 (NEK8) and NPHP10 (SDCCAG8) Proteins in the Context of Nephronophthisis. PhD thesis, Universität zu Köln.

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Abstract

Nephronophthisis (NPH) is an autosomal recessive kidney disease with renal cysts and fibrotic degeneration of the kidney as hallmarks. These changes lead to a progressive loss of kidney function. NPH is the most common monogenetic cause of end-stage kidney failure before the age of 30 with dialysis or kidney transplantation as the only therapeutic options. NPH belongs to the family of renal ciliopathies, as it is caused by pathogenetic variants in the NPHP genes (NPHP 1-20), which encode proteins localized to primary cilia. Cilia are antennae-like, sensory organelles present on the apical surface of tubular epithelial cells, from where they project into the tubular lumen. In the present work, we studied the function of two NPH proteins, Serologically defined colon cancer antigen 8 (SDCCAG8; NPHP10) and Never in mitosis gene A (NIMA)-related kinase-8 (NEK8; NPHP9) and aimed to understand how their loss might affect the signaling of tubular cells. First, we aimed to overexpress the two proteins SDCCAG8 and NEK8 with a Flag or GFP tag in mouse kidney cells using a single-copy integration system. Both confocal microscopy and immunoblotting could not confirm localization and expression. In the second approach, we used Crispr/Cas9-mediated genome editing to create knockout cell lines (KO) of the SDCCAG8 and NEK8 genes, respectively. Four cell lines with SDCCAG8-KO and one cell line with NEK8-KO were successfully established. These KOs were validated by DNA sequencing and quantitative polymerase chain reaction (qPCR). Moreover, we successfully generated interactomes for NEK8 and SDCCAG8 by transiently transfecting the KO cells with the proteins of interest. Moreover, we performed a kinase activity screen, through which numerous differences in kinase activities and signaling pathways could be identified between wild-type and NEK8- or SDCCAG8- deficient cells. Taken together, despite the fact that SDCCAG8 and NEK8 are very low-abundant proteins, their loss has significant effects on cellular signaling networks and pathways. Our findings might contribute to the understanding of signaling pathways and kinase activities altered in NPH. They could help to unravel not only the function of SDCCAG8 and NEK8 proteins in signaling networks but also the disease-causing pathophysiology underlying fibrosis, cyst formation, and cell death. A more detailed understanding of these mechanisms will be essential for establishing suitable future therapeutic strategies for kidney diseases.