Translational Strategy to Evaluate Urinary Plasminogen as a Targetable Biomarker of Glomerular Diseases

Background: Given the heterogeneity of glomerular diseases and necessity of kidney biopsies for predictive purposes, non-invasive biomarkers defining mechanisms underlying progression are needed. Our previous work identified plasminogen as a cause of podocyte injury and established a correlation between plasminogenuria and renal dysfunction. Here, we propose a translational strategy to evaluate the plasminogen as a mediator of podocyte damage and the potential utility of plasminogen inactivation, through urokinase-type plasminogen activator (uPA) inhibition, as a protective strategy. Methods: To evaluate the predictive value of the basal plasminogen, we performed a multicenter cohort study that included 1010 patients from CureGN with biopsy-proven glomerular disease. The main predictor was urine plasminogen at baseline and Cox regression was used to examine ESKD progression. To test uPA inhibition as a protective strategy, we developed a podocyte surface uPA activity assay in immortalized human podocytes, followed by High-Content Image Analysis of the cytoskeleton and focal adhesions by TIRF-Microscopy of podocytes treated with/without human plasminogen and uPA inhibitors. Results: In the cohort of patients, adjusted Log2uPlasminogen/Cr was significantly associated with ESKD (HR per doubling Log2uPlasminogen/Cr 1.31 (95%CI 1.22-1.40), P < 0.001). Comparison of the predictive performance of models including Log2uPlasminogen/Cr, Log2UPCR or both markers showed plasminogen model superiority. In the cell-surface uPA activity assay, amiloride had a IC50 of 8824 nM (5964-12661), compared to Candidates A and B (amiloride derivatives with computationally predicted increased uPA inhibitory activity), whose IC50 were 334.0 nM (249-397) and 643 (385-955). Quantitative high-content image analysis showed that plasminogen treatment resulted in a reduction in cell area and focal adhesions in plasminogen treated cells. Partial rescue was achieved after amiloride cotreatment whereas Candidates A and B completely reversed the phenotype. Conclusion: Urinary plasmin(ogen) is independently associated with ESKD in patients with glomerular disease. Plasminogen inactivation by uPA inhibition may be a strategy to reduce podocyte damage. Patients most likely to respond to uPA inhibition could potentially be identified using our urinary biomarker approach. Funding: NIDDK Support - NIDDK Support, NIDDK Support