Kidney Week

Abstract: FR-PO786

Glomerular Endothelial Plasmalemma Vesicle-Associated Protein (PLVAP) Is Re-expressed in Thrombotic Microangiopathy and Associated with Endothelial Cell Dedifferentiation and Increased Permeability

Session Information

• Glomerular Diseases: Mechanisms and Podocyte Biology
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

• Estrada, Chelsea C., Northport VA Medical Center, Northport, New York, United States

Chelsea C. Estrada, DO

• Wilson, Craig, Stony Brook University, Stony Brook, New York, United States

Craig Wilson, MS, MSc

• Ahmed, Sumaya, Stony Brook University, Stony Brook, New York, United States

Sumaya Ahmed

• Gujarati, Nehaben A., Stony Brook University, Stony Brook, New York, United States

Nehaben A. Gujarati, PhD

• Mallipattu, Sandeep K., Stony Brook University, Stony Brook, New York, United States

Sandeep K. Mallipattu, MD, FASN

Background

GEnC injury is a common feature across the wide spectrum of glomerular diseases and is the inciting event that drives downstream glomerular changes and eventual chronic kidney disease in both Diabetic Kidney Disease (DKD) and Thrombotic Microangiopathy (TMA). We recently reported the development of subacute TMA and GEnC injury in mice with the combined double knockout of endothelial Klf4 and Nos3 (DKO), compared with wild type mice (Klf4^fl/fl) or single endothelial knockout of either Klf4 (Klf4^ΔEC) or eNOS (Nos3^-/-), however the mechanism(s) of GEnCs response to injury are poorly understood.

Methods

Single-cell RNAseq libraries were generated from frozen kidney cortex from 12-wk old male mice in each group (Klf4^fl/fl, Nos3^-/-, Klf4^ΔEC, DKO), which were analyzed in R using Seurat. The differentially expressed genes (DEG) were further examined with ClusterProfiler. Immunofluorescence (IF) for PLVAP was performed in the four groups. Immortalized murine GEnCs with stable Plvap overexpression were generated with lentiviral delivery and gene expression was measured with rt-PCR. Proliferation was measured with the MTS assay and monolayer permeability was measured with a transwell system.

Results

Using SnRNAseq, pathway analysis of the upregulated DEGs of the endothelial cluster revealed perturbation in pathways involved in angiogenesis, permeability, focal adhesion and cytoskeletal organization in DKO mice. We identified Plasmalemmal Vesicle like-protein (Plvap), a component of fenestral diaphragms and previously shown to be re-expressed in injured GEnCs in DKD, as upregulated specifically in the GEnC cluster in TMA, which was confirmed using IF. Overexpression of Plvap (Plvap-ORF) in immortalized GEnCs resulted in increased proliferation at 37 degrees compared with control cells (EV), as well as loss of the mature differentiation marker Ehd3. GEnC monolayers with Plvap-ORF had increased permeability compared with EV cells, as well as increased expression of Cav-1 and Vcam1, both associated with EC permeability.

Conclusion

Plvap is upregulated in injured GEnCs in TMA and is associated with increased permeability and endothelial cell dedifferentiation. Whether this is a driver versus response to GEnC injury is yet to be determined and the focus of future studies.

Funding

• Veterans Affairs Support