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Kidney Week

Abstract: TH-PO401

Chronology of Cyst Epithelium Transformation to Renal Cell Carcinoma Through scRNA-seq

Session Information

Category: Genetic Diseases of the Kidneys

  • 1201 Genetic Diseases of the Kidneys: Cystic

Authors

  • Clerici, Sara, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
  • Spies, Daniel, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
  • Boletta, Alessandra, IRCCS Ospedale San Raffaele, Milano, Lombardia, Italy
Background

We have described a mouse model suitable for studying the progression of renal cell carcinoma (RCC). Upon deletion of a single gene (Tsc1) by a kidney-specific Cre (KspCre) in specific segments of the nephron, the mTORC1-dependent downregulation of polycystin-1 initially triggers the establishment of an overt cystic phenotype. Mutated epithelial cells then transform into cancerous lesions. The slow progression of the phenotype allows to dissect between mechanisms driving cyst formation, and the ones supporting epithelium malignant transformation. However, mutant cells lose epithelial markers, making it difficult to follow the next steps of transformation.

Methods

Our RCC model (Tsc1;kKO) was crossed with mTmG strain, ubiquitously expressing membrane Tomato (mT) marker that recombines to mGFP (mG) upon KspCre expression, allowing to discriminate mutated cells over time. Mutant kidneys were characterized by histological and immunofluorescence analysis. Kidney single cell suspension was characterized by flow cytometry. Chromium 10x single cell RNA sequencing (scRNA-seq) was performed on 20 000 cells from P80 mutant and ctrl kidneys.

Results

mTmG;Tsc1;kKO mutants were comparable to our previous RCC model, with the epithelium lining the cysts progressively transforming to papillae (P20), cystadenomas (P50), and carcinomas (P80). All the structures identified were mG+, allowing following the mutated cells during transformation. Indeed, flow cytometric analysis confirmed the expansion of the mG+ population at P80, consistent with the reported prominent proliferation of Tsc1 KO cells. scRNA-seq identified clusters of cells from all the segments of the nephron, both in mutant and ctrl kidneys. Cells deriving from the distal tubule and collecting duct were identified by Egfp transcript. This uniquely defined the Tsc1 KO cells and their expansion in the mutated kidney. Interestingly, we identified sub-clusters of mG+ cells with a proliferative and de-differentiated profile, and we observed the expansion of different immune populations in mutated kidneys, potentially supporting the RCC progression.

Conclusion

We generated a model of RCC through which we can dissect between driving events of cystogenesis and mechanisms supporting epithelial transformation to cancerous lesions. scRNA-seq represents a valuable approach to deciphering key mechanisms and new populations in RCC.

Funding

  • Private Foundation Support