Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria

Area of Science:

• Nephrology
• Immunology
• Biochemistry

Background:

• Proteinuria is linked to tubular inflammation and progressive kidney injury.
• Aberrantly filtered plasminogen is activated by urokinase-type plasminogen activator (uPA) in proteinuria.
• The hypothesis is that plasmin activates complement in tubular fluid, generating anaphylatoxins, and that amiloride inhibits this process.

Purpose of the Study:

• To investigate the role of the uPA-plasminogen cascade in complement activation within the tubular fluid during proteinuria.
• To determine if amiloride can attenuate this complement activation in vitro and in vivo.
• To explore the potential of targeting this pathway for renoprotection.

Main Methods:

• In vitro / ex vivo studies using purified complement factors, plasminogen, and urokinase with human urine.
• Analysis of urine and plasma from proteinuric mice (podocin KO) and patients with diabetic nephropathy treated with amiloride or anti-uPA antibodies.
• Assessment of complement activation via SDS-PAGE, immunoblotting, ELISA, and measurement of urinary C3dg and sC5b-9.

Main Results:

• uPA and plasminogen generated C3a and C5a in vitro, which was inhibited by amiloride.
• In podocin KO mice, amiloride and anti-uPA antibodies reduced urinary C3a and C5a excretion.
• Amiloride treatment in patients with diabetic nephropathy significantly reduced urinary C3dg and sC5b-9 levels.

Conclusions:

• The uPA-plasmin cascade generates anaphylatoxins in tubular fluid during proteinuria, activating complement.
• This pathway links proteinuria to intratubular inflammation and is sensitive to amiloride.
• Amiloride may offer renoprotective benefits beyond its known effects on natriuresis and blood pressure.