Abstract: FR-PO597
Defining Precystic vs. Cystic Alterations in Pkd1 Renal Mutants
Session Information
- Cystic Kidney Diseases: Mechanisms and Models
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Cystic
Authors
- Spies, Daniel, IRCCS Ospedale San Raffaele, Milano, MI, Italy
- Clerici, Sara, IRCCS Ospedale San Raffaele, Milano, MI, Italy
- Chiaravalli, Marco, IRCCS Ospedale San Raffaele, Milano, MI, Italy
- Wang, Gangqi, Leids Universitair Medisch Centrum, Leiden, Zuid-Holland, Netherlands
- Cassina, Laura, IRCCS Ospedale San Raffaele, Milano, MI, Italy
- Rabelink, Ton J., Leids Universitair Medisch Centrum, Leiden, Zuid-Holland, Netherlands
- Boletta, Alessandra, IRCCS Ospedale San Raffaele, Milano, MI, Italy
Group or Team Name
- Unit of Cystic Kidneys Disorders - Alessandra Boletta Lab.
Background
ADPKD is among the most prevalent monogenic diseases. Multiple relevant orthologous animal models were generated over the years and helped defining mechanism of disease progression and identifying therapies able to retard end-stage-kidney-disease. We have previously reported that PKD tissue is characterized by a profound metabolic reprogramming. However, the initial stages of renal cystogenesis and whether metabolic re-programming is an early or late event in disease evolution is unknown. Identification of the cellular alterations occurring at the onset of cystogenesis would provide important information on mechanism of disease. Here we present a strategy aimed at identifying the earliest events occurring immediately after inactivation of the Pkd1 gene
Methods
Inducible Cre lines were used to inactivate the Pdkd1 gene at different time-points. Characterization was done by IHC and IF analysis. For spatial metabolomic tracing 350µm thick kidney sections were incubated with either 13C labeled Glucose, Glutamine or Linoeleate before being sectioned into 10µm thick slices and subjected to MALDI-MS followed by multiplexed IF staining
Results
We developed a tamoxifen-inducible Pkd1 mouse model carrying a fluorescent switch cassette (mT/mG) that allows identification of cells in which the Cre recombinase has been activated to study the initiating events in cystogenesis. Induction by 3 low dose injections of Tamoxifen resulted in cyst formation within two weeks. 5 days after the last injection we identified renal tubules completely green, but not cystic, while 8 days post injection green cells started to form cysts. mKspCrePkd1fl/fl;mT/mG kidneys were collected at 5 or 8 days post inactivation, and subjected either to spatial metabolic tracing by incubation with 13C labeled Glucose, Glutamine or Linoeleate followed by MALDI- MS, or subjected to scRNAseq. Data are being analyzed and the preliminary results will be presented
Conclusion
We have generated and characterized an inducible Pkd1 mouse model that enables the study of two time windows: cells are Pkd1 mutated but not yet cystic and cells are inactivated and cystic, in both cases carrying a GFP cassette for tracking. Our combined analysis of scRNAseq and spatial metabolic tracing will provide the first glimpse on changes of likely causative and consequential events in cyst initiation
Funding
- Private Foundation Support