Total and Phosphoproteomic Analyses in Kidneys from an Authentic Mouse Model of Human Autosomal Dominant Polycystic Kidney Disease
- Genetic Diseases: Cystic - Basic
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Cystic
- Ghazanfari, Davoud, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Mohammadi, Ario, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Zhou, Jing, Brigham and Women's Hospital, Boston, Massachusetts, United States
ADPKD (Autosomal Dominant Polycystic Kidney Disease) is a genetic disorder characterized by the development of multiple cysts in the kidneys. The disease is caused by mutations in PKD1 and PKD2, encoding PC1 and PC2 respectively. To date, PC1 and PC2 have been implicated in modulating a number of cellular events such as Ca2+ signaling, mTOR, cyclic AMP, Wnt, PCP, and STAT3 pathways. How the polycystins modulate these pathways remains elusive, however. While transcriptome studies provide essential insights into gene expression patterns, proteomic studies offer a more comprehensive understanding of the functional proteome, including protein abundance, alternative splicing and posttranslational modifications. Integrating both transcriptome and proteome data can provide a more holistic view of biological systems and diseases, complementing each other's strengths and limitations.
Mice homozygous with an inducible mutation in Pkd1 gene were generated by intraperitoneal injection of tamoxifen. Kidneys from Pkd1 knockout mice and controls were isolated at different stages and subjected to multiplexed proteomics and phosphoproteomics. Various bioinformatic techniques, RT-PCR, immunostaining, and western blotting were used for analysis and validation.
To determine the cystogenic total and phosphoproteome, we performed multiplexed quantitative mass spectrometry analysis in Pkd1 knockout mouse kidneys at pre-/early disease and disease stages. Several thousands of proteins were quantified and hundreds of phosphorylation events were measured. MS/MS data were searched against a Uniprot mouse database with both the forward and reverse sequences using the SEQUEST algorithm, MS3 data was used for quantification. Phosphopeptides were processed separately but taken through a similar software pipeline with an additional phosphorylation site localization step using the Ascore algorithm. Several significant pathways were identified. A specific selection of proteins identified in the total and phosphoproteomic studies are being validated and further analyzed.
We have identified several pathways that are changed in an authentic mouse model of human ADPKD and determined the sequence of change of specific pathways in relation to disease development.
- Other NIH Support