A Novel Tool to Study Glomerular Endothelial Cell Biology

Background: Glomerular endothelial cells (GEnCs) are integral to the filtration barrier, and their dysfunction is implicated in a wide range of kidney diseases. However, progress in understanding GEnC physiology has been limited by the lack of genetic tools. Existing endothelial promoters are pan-endothelial, limiting their utility for GEnC-targeted studies. To overcome this, we sought to develop a transgenic mouse with a GEnC-specific promoter to enable selective gene modulation. Methods: Using CRISPR-Cas9, we generated a transgenic mouse in which a reverse tetracycline-controlled transactivator (rtTA)-P2A cassette was inserted upstream of the ATG start codon of the GEnC-specific gene Eps15 homology domain-containing 3 (Ehd3). Ehd3-rtTA mice were bred with TRE-GFP mice and fed doxycycline (DOX) for two weeks before organs were harvested and analyzed with immunofluorescence (IF) for GFP and Isolectin B4. We also generated GEnC-specific Klf4 knockout (Ehd3-rtTa;TRE-Cre;Klf4flox/flox (Klf4DGenc)) and control (Klf4+/+) mice and performed rt-PCR to validate the utility of the promoter. Results: We identified Ehd3, an endocytic transport gene, as being specific to GEnCs by analysis of previously reported sequencing studies and confirmed EHD3 expression in human and mouse kidney samples to be exclusively in GEnCs by IF. In Ehd3-rtTA;TRE-GFP DOX+ mice, GFP expression in the kidney was largely limited to GEnCs with occasional expression in peritubular capillaries, and no expression in larger vessels and the medullary compartment. Outside of the kidney, GFP expression was present in a subset of endothelial cells in the liver and spleen. No GFP expression was detected in DOX- mice. As the transcription factor Kruppel-like Factor 4 (Klf4) is expressed across all renal endothelial cell compartments, we generated GEnC-specific Klf4 knockout (Klf4DGenc) and control (Klf4+/+) mice. After 2 weeks of DOX, we observed significantly decreased Klf4 expression in GEnCs in Klf4DGenc mice as compared with Klf4+/+ mice, with no differences in Klf4 expression in non-glomerular renal endothelial cells. Conclusion: We demonstrated that the novel Ehd3 promoter offers significantly greater specificity for GEnCs compared to currently available endothelial promoters. This enables selective and inducible modulation of gene expression in GEnCs, facilitating more precise studies of these highly specialized cells. Funding: Veterans Affairs Support, Private Foundation Support