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Circulating factors resulting from immune dysfunction have been proposed as one of the causes for increased risk of graft loss associated with recurrence of focal segmental glomerulosclerosis (FSGS) after kidney transplant. However, the precise identity of the circulating factors and their sources remain incompletely characterized.In vivostudies in mouse models have implicated a role for immature bone marrow cells in the development of FSGS. Using single-cell RNA sequencing we have profiled >50,000 cells from bone marrow of FSGS patients with or without recurrence after kidney transplant and controls including healthy individuals and patients with end-stage renal disease due to non-FSGS causes. Bone marrow mononuclear cells from patients with recurrence of FSGS after transplant display an inflammatory phenotype with activation of cytokine and interferon signaling in neutrophils, T cells and B cells. We also observe a dramatically depleted B cell population in R-FSGS patients. Conditioned media from BMNCs of R-FSGS patients have higher levels of pro-inflammatory cytokine MIP-1α/CCL3, reduced anti-inflammatory chemokine CCL22 and cause injury in a human podocyte cell culture model. Our studies provide evidence for the role of bone marrow cells in FSGS associated inflammatory milieu and elucidate the transcriptional changes associated with the disease.

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.