Angiotensinogen and C3 compete for renin-induced complement activation.

Area of Science:

• Biochemistry
• Immunology
• Renal Physiology

Background:

• Renin's ability to cleave complement protein C3 into C3a and C3b has been previously reported but also contested.
• Concerns were raised regarding potential trypsin contamination in recombinant renin preparations, which could explain observed C3 cleavage.
• Endogenous renin production by cells has also been linked to C3 deposition.

Purpose of the Study:

• To definitively investigate the cleavage of C3 by recombinant renin, addressing prior controversies.
• To examine the competitive inhibition of C3 cleavage by angiotensinogen, renin's primary substrate.
• To confirm the absence of trypsin contamination in recombinant renin preparations.

Main Methods:

• Mass spectrometry with endopeptidase LysC digestion to analyze recombinant renin for trypsin.
• Immunoblotting to detect C3b formation following incubation with recombinant renin.
• Enzyme-linked immunosorbent assay (ELISA) to quantify C3a generation and angiotensin I production.

Main Results:

• Mass spectrometry confirmed the absence of trypsin in the recombinant renin used.
• Recombinant renin demonstrated C3 cleavage to C3b, consistent across different protocols.
• C3a generation was rapid (within 1 min) and inhibited by aliskiren, a specific renin inhibitor.
• Angiotensinogen competed with C3 for renin, indicating it is a preferred substrate, while C3 did not inhibit angiotensinogen cleavage.

Conclusions:

• Renin directly cleaves complement protein C3, independent of trypsin contamination.
• Angiotensinogen is the preferred substrate for renin, but C3 cleavage can occur.
• The renin-C3 interaction may be functionally relevant in the kidney, especially under conditions of substrate depletion.