Kidney Week

Abstract: FR-PO298

A scRNAseq Approach to Define Early Events in Cyst Formation and Stepwise Transformation

Session Information

• Genetic Diseases of the Kidneys: Cystic - II
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1101 Genetic Diseases of the Kidneys: Cystic

Authors

• Clerici, Sara, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy

Sara Clerici,

• Spies, Daniel, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy

Daniel Spies,

• Boletta, Alessandra, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy

Alessandra Boletta,

Background

We have previously generated and described a mouse model carrying kidney-specific inactivation of the Tsc1 gene using a Cadherin16-Cre line (KspCre). The peculiarity of the Tsc1^f/f;KspCre model is that it develops a slowly progressive cystic kidney disease model, triggered by mTORC1-dependent downregulation of polycystin-1 (PC-1), which progressively transform into cystadenomas and carcinomas (Pema et al, Nat Comms, 2016; Drusian et al, Cell Reports, 2018). As they transform, the renal lesions lose the expression of tubule-specific markers.

Methods

In order to track all KO cells within the kidney, we generated mT/mG;Tsc1^f/f;KspCre mice. Indeed, only KspCre expressing cells would excise mTomato (mT) in favor of mGFP (mG), while the surrounding tissue ubiquitously expresses mTomato. Mutants were characterized through histological and immunofluorescent analyses. Single cell suspension from control and mutant kidney was obtained through mechanical and enzymatic dissociation with Miltenyi kits and validated by flow cytometry confirming viability and aggregates requirements for single-cell sequencing.

Results

Histological characterization of mT/mG;Tsc1^f/f;KspCre confirmed the progression from cysts (at P20) to cystadenomas and carcinomas (at P50 and P80) with the previously reported frequency. Furthermore, careful characterization of the mT/mG;Tsc1^f/f;KspCre revealed that cystic structure, papillae, cystadenomas and carcinomas displayed mG-positivity in all lesions of all stages of the phenotype. A dissociation protocol was set up obtaining 75% viability in both control and mutant kidneys. Flow cytometry confirmed the 25-30% mG⁺ cells expected in controls, which expanded to 65% in mutant kidneys, in line with the robust proliferation observed in these mutants. scRNAseq analysis of control mT/mG and mT/mG;Tsc1^f/f;KspCre kidneys is being set-up at different time points (P10, precystic; P20 cystic; P50 and P80 transformed).

Conclusion

We generated a mouse model allowing to separate and study Tsc1 mutant cells and the surrounding healthy tissue in a mouse model of progressive cysts and renal cell carcinoma (RCC) development. Single-cell sequencing analysis will help defining early versus late events in cystogenesis and in transformation towards RCC.

Funding

• Private Foundation Support