Abstract: SA-PO487
The Impact of Uraemia on Oxidative Stress Signalling Pathways in the Presence of NPHP Mutations
Session Information
- Cystic Kidney Diseases: Basic/Translational
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1001 Genetic Diseases of the Kidneys: Cystic
Author
- Gupta, Shabarni, Macquarie University, Sydney, New South Wales, Australia
Group or Team Name
- Phillips and Atkin Laboratory, Faculty of Medicine and Health Sciences, Macquarie University
Background
Mutations in the Nek8 gene, encoding a member of the serine/threonine protein kinase family related to NIMA (never in mitosis, gene A), are associated with nephronophthisis (NPHP9); a juvenile form of autosomal recessive cystic kidney disease. With the onset of cystic disease due to the genetic mutation, deteriorating renal health leads to accumulation and circulation of several toxins acting as a double-edged sword. This study investigates any synergistic effect between mutation in the Nek8 gene and the uraemic toxin, indoxyl sulphate (IS) in driving an unfolded protein response (UPR) as an indicator of cellular stress.
Methods
Hek293 cells transiently expressing wild-type (WT) or H425Y mutant Nek8 found in human NPHP patients were used. UPR was investigated by monitoring expression of BiP and XBP1s using protein and RNA, respectively, from cells expressing WT and H425Y Nek8 alone, and in the presence of 5mM IS for 8hr (n = 3). Tunicamycin treated Hek293 cells were used as positive control (2.5µg/ml, 4hr). Data was analyzed using 2-way ANOVA.
Results
Overexpression of H425Y Nek8 did not result in elevating UPR markers (BiP and XBP1s) in comparison to WT. However, presence of IS, resulted in upregulation of BiP in WT by 1.45-fold and H425Y by 1.54-fold (calculated as mean ± SEM, p <0.05, n=3) in comparison to untreated WT. XBP1s was upregulated by 2-fold in treated WT and 3.1-fold in treated H425Y cells (calculated as mean ± SEM, p < 0.05, n=3), in comparison to WT untreated cells, such that this increased expression was significantly greater in H425Y Nek8 mutant cells.
Conclusion
Results from this study indicate that overexpression of the human variant of the Nek8 mutation, as a standalone, is unable to trigger the UPR pathway. However, in presence of uraemic toxin IS, expressions of both BiP and XBP1s in WT and H425Y cells are elevated. The greater elevation of XBP1s expression in treated H425Y compared to treated WT cells is consistent with the hypothesis that UPR responses triggered by uraemic toxins are potentiated in presence of the Nek8 mutation. Further studies examining additional markers of UPR and measures of cellular stress will further elucidate the impact of uraemic toxins and their interaction with PKD mutations and their potential impact on kidney function.
Funding
- Private Foundation Support