Guanidinylated Apolipoprotein C3 (ApoC3) Associates with Kidney and Vascular Injury
Authors: Schunk Stefan J. , Hermann Juliane , Sarakpi Tamim , Triem Sarah , Lellig Michaela , Hahm Eunsil , Zewinger Stephen , Schmit David , Becker Ellen , Möllmann Julia , Lehrke Michael , Kramann Rafael , Boor Peter , Lipp Peter , Laufs Ulrich , März Winfried , Reiser Jochen , Jankowski Joachim , Fliser Danilo , Speer Thimoteus , Jankowski Vera 
Published: September 2021

Significance Statement
Posttranslational guanidinylation of ApoC3, a major constituent of triglyceride-rich lipoproteins, enhances ApoC3′s proinflammatory properties in uremia. Guanidinylated ApoC3 (gApoC3) is generated by guanidine and urea, and accumulates significantly in the plasma of patients with CKD, and in the kidneys of mouse models of CKD. In humanized mice, gApoC3 promotes kidney tissue fibrosis and suppresses regeneration after vascular injury. Importantly, in a large observational trial examining the clinical relevance of this posttranslational modification in patients with CKD, higher plasma gApoC3 intensity was associated with adverse renal and cardiovascular outcomes. Therefore, gApoC3 represents a novel mediator of kidney injury and CKD-associated vascular disease and offers a potential treatment target to halt progression and to prevent vascular disease in patients with CKD.

Background
Coexistent CKD and cardiovascular diseases are highly prevalent in Western populations and account for substantial mortality. We recently found that apolipoprotein C-3 (ApoC3), a major constituent of triglyceride-rich lipoproteins, induces sterile systemic inflammation by activating the NOD-like receptor protein-3 (NLRP3) inflammasome in human monocytes via an alternative pathway.

Methods
To identify posttranslational modifications of ApoC3 in patients with CKD, we used mass spectrometry to analyze ApoC3 from such patients and from healthy individuals. We determined the effects of posttranslationally modified ApoC3 on monocyte inflammatory response in vitro, as well as in humanized mice subjected to unilateral ureter ligation (a kidney fibrosis model) and in a humanized mouse model for vascular injury and regeneration. Finally, we conducted a prospective observational trial of 543 patients with CKD to explore the association of posttranslationally modified ApoC3 with renal and cardiovascular events in such patients.

Results
We identified significant posttranslational guanidinylation of ApoC3 (gApoC3) in patients with CKD. We also found that mechanistically, guanidine and urea induce guanidinylation of ApoC3. A 2D-proteomic analysis revealed that gApoC3 accumulated in kidneys and plasma in a CKD mouse model (mice fed an adenine-rich diet). In addition, gApoC3 augmented the proinflammatory effects of ApoC3 in monocytes in vitro. In humanized mice, gApoC3 promoted kidney tissue fibrosis and impeded vascular regeneration. In CKD patients, higher gApoC3 plasma levels (as determined by mass spectrometry) were associated with increased mortality as well as with renal and cardiovascular events.

Conclusions
Guanidinylation of ApoC3 represents a novel pathogenic mechanism in CKD and CKD-associated vascular injury, pointing to gApoC3 as a potential therapeutic target.