Investigation of biomolecular alterations in Nek8 mutant models of Nephronophthisis
Creators: name: Gupta, Shabarni
Publisher: name: Macquarie University
Year: 2024
Subjects: classificationCode: 399999
subject: Other education not elsewhere classified

description: The dysfunction of primary cilia resulting from mutations in genes whose product localise to the primary cilium, basal body or the centrosome complex are termed ciliopathies (diseases of the cilia). These include conditions such as polycystic kidney disease (PKD), autosomal dominant tubulointerstitial kidney disease (ADTKD) and nephronophthisis (NPHP). While each of these ciliopathies have several distinguishing pathobiological characteristics, the overlap in the “cystic kidney” phenotype among these diseases suggest the existence of common precursors triggering pathways or a microenvironment that is conducive for cystogenesis. To delve into the nature of these biomolecular aberrations associated with cystic kidney diseases, this thesis employed mass spectrometry based proteomic and metabolomic profiling of cell lines and rodent models harbouring pathogenic mutant forms of the Nek8 gene. Nek8 is a member of the serine/threonine protein kinase family related to the highly conserved NIMA (never in mitosis, gene A) and mutations in Nek8 are responsible for NPHP type 9/NPHP9, a juvenile form of autosomal recessive cystic kidney disease seen in humans that leads to end stage renal disease (ESRD) within the first three decades of life. This thesis provides multi-faceted biomolecular insights into the pathobiology of Nek8 driven NPHP. In Study 1, a mass spectrometry based global proteome profile of cell line and animal model of Nek8 mutants enabled the identification of potentially pathogenic pathways and protein interaction networks involved in tumorigenesis and DNA damage repair pathway. Furthermore, the differential expression of the proteins SUGT1 and MIRO1 involved in cell proliferation, mitochondrial health and cytoskeletal stability were confirmed in Nek8 mutant models. Study 2 involving a multi-organ proteome profile (kidney, liver, brain and skeletal muscle from Lewis Polycystic Kidney (LPK) rodent model) enabled the identification of differential expression of proteins that may explain the renal specific nature of the cysts despite pervasive and ubiquitous germline mutations associated with NPHP. The in-silico analysis undertaken in this study, further predicted upstream factors that may regulate the expression of certain known pathogenic cystic genes. As a follow up to the findings from Study 2, Study 3 involved a dedicated kynurenine pathway (KP) metabolite profile of plasma, urine and kidney samples of Lewis and LPK animals. This study showcased the temporal differences in KP metabolites across 6-week and 12-week animals. While KP has been explored widely in neurological diseases, this study is the first to investigate the role of KP metabolites in the pathogenesis in cystic kidney diseases. As unfolded protein response (UPR) was one of the significantly dysregulated pathways in Nek8 mutants in study 1, this pilot study presented as an annexure to this thesis presents preliminary data demonstrating an accentuated UPR in response to dual triggers including the primary pathogenic mutation and secondary aetiological agents such as uraemic toxins. In conclusion, Nek8 as a pathogenic agent of NPHP is understudied and has very few canonical functions attributed to it. Using mass spectrometry-based proteomics and targeted metabolomics, this discovery-based thesis provides a snapshot of cellular pathways that may have causal roles in the development of cystic diseases. SUGT1, MIRO1 and KP metabolites are not only known biomarkers, but also therapeutic targets for cancers and neurodegenerative diseases. While their role in renal cystic pathology has never been explored before, this body of work provides a foundation for future studies targeting these proteins along with other identified putative candidates for exploration of their therapeutic potential in PKD.