Deciphering the Chronology of Cystogenesis and Metabolic Reprogramming
- Genetic Diseases: Cystic - Therapeutic Investigations and Prognosis
November 02, 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
- Spies, Daniel, Universita Vita Salute San Raffaele, Milano, Lombardia, Italy
- Clerici, Sara, Universita Vita Salute San Raffaele, Milano, Lombardia, Italy
- Cassina, Laura, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
- Chiaravalli, Marco, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
- Boletta, Alessandra, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
Group or Team Name
- Unit of Cystic Kidneys Disorders - Alessandra Boletta Lab.
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. However, the initial stages of renal cystogenesis have been poorly defined. Identification of the cellular alterations occurring at the onset of cystogenesis would provide important information on mechanism of disease and possibly help designing better therapies targeting initiating events and being more effective.
Mutant kidneys have been characterized by IHC and IF analysis. Single cell dissociation was performed pairing mechanical and enzymatic approaches. PARSE single cell combinatorial barcoding RNA sequencing was performed on 10k cells from P54 control kidneys, with a depth of 50k reads/cell
We have developed a tamoxifen-inducible Pkd1 animal model (TmKspCrePkd1fl/fl) 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 full penetrance (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. A pilot scRNA-seq of WT cells confirmed the capability to capture and distinguish induced from non-induced cells as well as the higher sequencing depth and reduced contamination of the combinatorial scRNA-seq approach.
Our inducible Pkd1 KO model enables the determination and characterization of both pre-cystic and cystic time windows, as well as the selection of low abundant pre-cystic mutant cells due to our colorSwitch reporter cassette. Comparison of mutant cells involved in cyst initiation and expansion will permit the exact determination of new mechanisms as well as therapeutic windows that allow tailoring of treatments to the corresponding metabolic stage of each cell-type. The experimental analysis is ongoing.