Inhibition of urokinase-plasminogen reduces intratubular C3a and C5a generation in vivo in mice

IntroductionProteinuria leads to progressive kidney injury, but the mechanisms are not well known. Aberrantly filtered plasminogen is activated by urokinase-type plasminogen activator (uPA) in tubular fluid. We hypothesized that that uPA-plasminogen activate co-filtered complement in the tubular fluid, and that activation can be inhibited by targeting uPA.MethodsPurified C3 and C5, plasminogen, urokinase and urine from healthy humans were used for in vitro/ex vivo reactions. In vitro activation was assessed by immunoblotting or ELISA. Urine and plasma samples from nephrotic patients, kidney transplant recipients with or without albuminuria, before and after treatment with high dose amiloride, and urine from podocin deleted mice with proteinuria treated with amiloride or vehicle or inhibiting monoclonal anti-uPA antibodies or isotype control were assessed.ResultsuPA-plasminogen activated C3 and C5 and generated C3a and C5a which could be inhibited by amiloride and aprotinin in vitro. uPA or plasminogen alone did not activate complement. Urine from healthy controls generated C3a from C3 when exogenous plasminogen was added. In nephrotic urine C3a correlated to albumin in a log-log manner (R2 = 0.90, n = 10). Amiloride did not reduce C3a excretion in kidney transplant recipients with albuminuria (p = 0.083). In mice, C3a and C5a 24 h urine excretion was reduced after treatment with amiloride (t-test, p