Kidney Week

Abstract: FR-PO787

Shiga Toxin Downregulates the uPAR/uPA Fibrinolytic Pathway in Human Umbilical Vein Endothelial Cells and on Glomeruli of a Murine Model of Enterohemorrhagic Hemolytic Uremic Syndrome

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

• Mazzotta, Celestina, Massachusetts General Hospital, Boston, Massachusetts, United States

Celestina Mazzotta, PhD

• Ingelfinger, Julie R., Massachusetts General Hospital, Boston, Massachusetts, United States

Julie R. Ingelfinger, MD

• Davies, Julia, Massachusetts General Hospital, Boston, Massachusetts, United States

Julia Davies

• Stahl, Gregory L., Harvard Medical School, Boston, Massachusetts, United States

Gregory L. Stahl, MSc, PhD

• Grabowski, Eric F., Massachusetts General Hospital, Boston, Massachusetts, United States

Eric F. Grabowski, MD, MSc, PhD

Background

Coagulation and fibrinolysis ensures balanced hemostasis through the coordinate interaction of pro-coagulant/fibrinolytic factors and the activation of the complement systems. Enterohemorrhagic hemolytic uremic syndrome (eHUS), caused by Shiga toxin results in a prothrombotic microenvironment in the kidney, especially in the glomeruli, with platelet and fibrin deposition and activation of the lectin pathway of complement. We used human umbilical vein endothelial cell (HUVECs) and kidneys of our murine model of eHUS (MBL^+/+2mbl1^−/−mbl2^−/−) to study the expression of the fibrinolytic pathway: urokinase-type Plasminogen Activator (uPA) and urokinase Plasminogen Activator Receptor (uPAR).

Methods

Using immunofluorescence (IF) and western blot, uPA and uPAR were evaluated in HUVECs treated with TNF-α, Stx-1 or both, vs. control. Expression of uPA and uPAR was studied with IF in kidneys of mice treated with Stx-2, +/- anti-MBL2 antibody (3F8), anti-factor XIa antibody (3G3), or both, vs. untreated mice. In kidneys of our mouse model, colocalization of uPAR/PAI-1 and uPA/PAI-1 (Plasminogen Activator Inhibitor-1) was evaluated.

Results

Stx-1, TNF-α or both significantly reduced uPA and uPAR expression in HUVECs. The glomeruli of mice treated with Stx-2 showed decreased levels of uPA and uPAR vs mice treated with Stx-2 +3F8, or +3G3, or both. Endothelial cells in mouse glomeruli treated with Stx-2 expressed low levels of uPA and uPAR vs. untreated mice and vs. Stx-2+3F8, or +3G3 or both. The levels of PAI-1 in kidneys increased in mice treated with Stx-2 vs. Stx-2+3F8 or +3G3, or both. PAI-1/uPAR and PAI-1/uPA colocalization significantly increased in glomeruli of mice treated with Stx-2 compared to Stx-2+3F8 or +3G3, or both.

Conclusion

In the presence of Stx-1, TNF-α, or both, uPA and uPAR expression declined in HUVECs. The glomeruli of mice treated with Stx-2 showed reduced levels of uPA and uPAR which are restored with 3F8, 3G3 or both. Our findings provide evidence that the fibrinolytic pathway mediated by uPA/uPAR is affected in the presence of Stx, suggesting that the higher deposition of fibrin and the thrombotic events in the kidney might be generated by dysregulated fibrinolysis modulated by uPA/uPAR.

Funding

• NIDDK Support