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Abstract: FR-OR99

Hemizygous Loss of Vegfa in Cap Mesenchyme Is Associated with Thrombotic Microangiopathy and Metabolic Reprogramming of the Thick Ascending Limb (TAL) Epithelium

Session Information

Category: Glomerular Diseases

  • 1403 Podocyte Biology

Authors

  • Kaminski, Dorian, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
  • Deb, Dilip K., Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
  • Zhou, Yalu, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
  • Quaggin, Susan E., Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States
Background

Tonic secretion of VEGFA from nephron progenitors and their epithelial derivatives plays a key role in both development and homeostatic maintenance of kidney microvascular beds. Reduction of Vegfa production by podocytes or tubular epithelial cells results in glomerular endotheliosis and loss of peritubular capillaries, respectively. Here, we report a kidney epithelial-specific, hemizygous Vegfa knockout mouse model. Fifty per cent reduction of Vegfa in Six2-progenitor cells results in a thrombotic microangiopathy phenotype, with progressive loss of glomerular function after 4 weeks of age.

Methods

Vegfa floxed mice were generated using the CRISPR-Cas9 system and were crossed with transgenic Six2-eGFPCre mice to generate VegfAΔ/+;Six2-Cre mice, leading to excision of Vegfa from nephron progenitor cells of cap mesenchyme. Kidneys from 1, 2, and 4 week old mice were examined histologically using light and electron microscopy and single-cell RNAseq libraries were generated from knockout and control kidneys at 2 and 4 weeks which were analyzed in R using Seurat.

Results

VegfAΔ/+;Six2-Cre mice began to develop albuminuria at 2 weeks. On light microscopy, kidneys from VegfAΔ/+;Six2-Cre mice were indistinguishable from those of their control littermates at 1 week. Electron microscopy at 2 weeks showed evidence of glomerular injury, with subendothelial expansion and podocyte foot process effacement. At 4 weeks, VegfAΔ/+;Six2-Cre glomeruli exhibited florid thrombotic microangiopathy. In scRNAseq data, glomerular endothelial cell clusters from VegfAΔ/+;Six2-Cre mice exhibited decreased expression of transcripts encoding markers of glomerular identity. Analysis also identified a new population of thick ascending limb (newTAL) cells that were found only in knockout kidneys at both time points. Compared to other TAL clusters, newTAL cells expressed cytokeratin 19 as well as classic genes such as UMOD. Pathway analysis of differentially expressed genes in the newTAL showed downregulation several metabolic pathways with enrichment of growth factor signaling.

Conclusion

Vegfa gene dosage in renal epithelium is important for the maintenance of the glomerular endothelium and plays a role in the metabolic programming of the TAL.