Soluble Urokinase Receptor Modulates T Cell Dynamics via Interacting with CD4

Background: High levels of circulating soluble urokinase receptor (suPAR) have been implicated in both acute and chronic kidney injuries. We recently reported that, suPAR links dysregulated immune response and sepsis-induced acute kidney injury (AKI). Transgenic mouse model over-expressing suPAR, exhibited increased T cell infiltration in the kidneys in the cecal slurry model. Whether and how suPAR regulates T cell dynamics remains unknown. Methods: In this study, we performed a structure-based in-silico protein-protein interaction (PPI) analysis to profile suPAR interactome. We applied AlphaFold3 analysis for accurate structure prediction of potential biomolecular interactions. To validate these interactions in a cellular system, we employed HEK 293T cell co-transfections expressing both human suPAR and its interacting partners, followed by co-immunoprecipitation (Co-IP) and western blot. To examine if suPAR affects T cell migration and proliferation, we isolated T cells from mouse spleen tissues and incubated with different concentration of suPAR. Further, we examined the immune cell infiltration by immunostaining in a kidney inflammation model induced by high dose of folic acid (FA) injection. Results: Proteome widePPI analysis suggests that suPAR interacts with CD4 with potentially high binding affinity. AlphaFold3 predicts that the positively charged loop in the first domain of CD4 binds the negatively charged suPAR loop in Domain 2 and 3. Co-IP assay demonstrated that CD4 interacts with suPAR. Trans-well assay showed suPAR accelerates the CD4+ T cells migration in a concentration dependent pattern. Though, no significant changes were observed in the T cell proliferation after incubation with suPAR. Consistent with our previous findings in cecal slurry models, CD4+ T cell infiltration was readily observed in the kidney tissues of FA treated mice that over-express suPAR. In contrast, no or minimal number of CD4+ T cells were detected in wild type mice or mice deficient in suPAR/uPAR expression. Conclusion: Collectively, these findings demonstrate that suPAR interacts biophysically with the CD4 protein, influencing CD4+ T cell migration and/or infiltration. Further studies are warranted to explore the implication of suPAR in T cell dysregulation in patients with relevant kidney disease. Funding: NIDDK Support