Post-translational modifications in kidney diseases and associated cardiovascular risk.

Patients with chronic kidney disease (CKD) are at an increased cardiovascular risk compared with the general population, which is driven, at least in part, by mechanisms that are uniquely associated with kidney disease. In CKD, increased levels of oxidative stress and uraemic retention solutes, including urea and advanced glycation end products, enhance non-enzymatic post-translational modification events, such as protein oxidation, glycation, carbamylation and guanidinylation. Alterations in enzymatic post-translational modifications such as glycosylation, ubiquitination, acetylation and methylation are also detected in CKD. Post-translational modifications can alter the structure and function of proteins and lipoprotein particles, thereby affecting cellular processes. In CKD, evidence suggests that post-translationally modified proteins can contribute to inflammation, oxidative stress and fibrosis, and induce vascular damage or prothrombotic effects, which might contribute to CKD progression and/or increase cardiovascular risk in patients with CKD. Consequently, post-translational protein modifications prevalent in CKD might be useful as diagnostic biomarkers and indicators of disease activity that could be used to guide and evaluate therapeutic interventions, in addition to providing potential novel therapeutic targets.

Lineage tracing reveals transient phenotypic adaptation of tubular cells during acute kidney injury.

Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease.

Increased levels of a mycophenolic acid metabolite in patients with kidney failure negatively affect cardiomyocyte health.

Chronic kidney disease (CKD) significantly increases cardiovascular risk and mortality, and the accumulation of uremic toxins in the circulation upon kidney failure contributes to this increased risk. We thus performed a screening for potential novel mediators of reduced cardiovascular health starting from dialysate obtained after hemodialysis of patients with CKD. The dialysate was gradually fractionated to increased purity using orthogonal chromatography steps, with each fraction screened for a potential negative impact on the metabolic activity of cardiomyocytes using a high-throughput MTT-assay, until ultimately a highly purified fraction with strong effects on cardiomyocyte health was retained. Mass spectrometry and nuclear magnetic resonance identified the metabolite mycophenolic acid-ß-glucuronide (MPA-G) as a responsible substance. MPA-G is the main metabolite from the immunosuppressive agent MPA that is supplied in the form of mycophenolate mofetil (MMF) to patients in preparation for and after transplantation or for treatment of autoimmune and non-transplant kidney diseases. The adverse effect of MPA-G on cardiomyocytes was confirmed in vitro, reducing the overall metabolic activity and cellular respiration while increasing mitochondrial reactive oxygen species production in cardiomyocytes at concentrations detected in MMF-treated patients with failing kidney function. This study draws attention to the potential adverse effects of long-term high MMF dosing, specifically in patients with severely reduced kidney function already displaying a highly increased cardiovascular risk.

Cardiovascular Consequences of Uremic Metabolites: an Overview of the Involved Signaling Pathways.

The crosstalk of the heart with distant organs such as the lung, liver, gut, and kidney has been intensively approached lately. The kidney is involved in (1) the production of systemic relevant products, such as renin, as part of the most essential vasoregulatory system of the human body, and (2) in the clearance of metabolites with systemic and organ effects. Metabolic residue accumulation during kidney dysfunction is known to determine cardiovascular pathologies such as endothelial activation/dysfunction, atherosclerosis, cardiomyocyte apoptosis, cardiac fibrosis, and vascular and valvular calcification, leading to hypertension, arrhythmias, myocardial infarction, and cardiomyopathies. However, this review offers an overview of the uremic metabolites and details their signaling pathways involved in cardiorenal syndrome and the development of heart failure. A holistic view of the metabolites, but more importantly, an exhaustive crosstalk of their known signaling pathways, is important for depicting new therapeutic strategies in the cardiovascular field.

Multiple urinary peptides display distinct sex-specific distribution.

Previous studies have established the association of sex with gene and protein expression. This study investigated the association of sex with the abundance of endogenous urinary peptides, using capillary electrophoresis-coupled to mass spectrometry (CE-MS) datasets from 2008 healthy individuals and patients with type II diabetes, divided in one discovery and two validation cohorts. Statistical analysis using the Mann-Whitney test, adjusted for multiple testing, revealed 143 sex-associated peptides in the discovery cohort. Of these, 90 peptides were associated with sex in at least one of the validation cohorts and showed agreement in their regulation trends across all cohorts. The 90 sex-associated peptides were fragments of 29 parental proteins. Comparison with previously published transcriptomics data demonstrated that the genes encoding 16 of these parental proteins had sex-biased expression. The 143 sex-associated peptides were combined into a support vector machine-based classifier that could discriminate males from females in two independent sets of healthy individuals and patients with type II diabetes, with an AUC of 89% and 81%, respectively. Collectively, the urinary peptidome contains multiple sex-associated differences, which may enable a better understanding of sex-biased molecular mechanisms and the development of more accurate diagnostic, prognostic, or predictive classifiers for each individual sex.

Exploratory Study Analyzing the Urinary Peptidome of T2DM Patients Suggests Changes in ECM but Also Inflammatory and Metabolic Pathways Following GLP-1R Agonist Treatment.

Type II diabetes mellitus (T2DM) accounts for approximately 90% of all diabetes mellitus cases in the world. Glucagon-like peptide-1 receptor (GLP-1R) agonists have established an increased capability to target directly or indirectly six core defects associated with T2DM, while the underlying molecular mechanisms of these pharmacological effects are not fully known. This exploratory study was conducted to analyze the effect of treatment with GLP-1R agonists on the urinary peptidome of T2DM patients. Urine samples of thirty-two T2DM patients from the PROVALID study ("A Prospective Cohort Study in Patients with T2DM for Validation of Biomarkers") collected pre- and post-treatment with GLP-1R agonist drugs were analyzed by CE-MS. In total, 70 urinary peptides were significantly affected by GLP-1R agonist treatment, generated from 26 different proteins. The downregulation of MMP proteases, based on the concordant downregulation of urinary collagen peptides, was highlighted. Treatment also resulted in the downregulation of peptides from SERPINA1, APOC3, CD99, CPSF6, CRNN, SERPINA6, HBA2, MB, VGF, PIGR, and TTR, many of which were previously found to be associated with increased insulin resistance and inflammation. The findings indicate potential molecular mechanisms of GLP-1R agonists in the context of the management of T2DM and the prevention or delaying of the progression of its associated diseases.

Intermediate filament network perturbation in the C. elegans intestine causes systemic dysfunctions.

Intermediate filaments (IFs) are major components of the metazoan cytoskeleton. A long-standing debate concerns the question whether IF network organization only reflects or also determines cell and tissue function. Using Caenorhabditis elegans, we have recently described mutants of the mitogen-activated protein kinase (MAPK) SMA-5 which perturb the organization of the intestinal IF cytoskeleton resulting in luminal widening and cytoplasmic invaginations. Besides these structural phenotypes, systemic dysfunctions were also observed. We now identify the IF polypeptide IFB-2 as a highly efficient suppressor of both the structural and functional deficiencies of mutant sma-5 animals by removing the aberrant IF network. Mechanistically, perturbed IF network morphogenesis is linked to hyperphosphorylation of multiple sites throughout the entire IFB-2 molecule. The rescuing capability is IF isotype-specific and not restricted to sma-5 mutants but extends to mutants that disrupt the function of the cytoskeletal linker IFO-1 and the IF-associated protein BBLN-1. The findings provide strong evidence for adverse consequences of the deranged IF networks with implications for diseases that are characterized by altered IF network organization.

DNA binding protein YB-1 is a part of the neutrophil extracellular trap mediation of kidney damage and cross-organ effects.

Open-heart surgery is associated with high morbidity, with acute kidney injury (AKI) being one of the most commonly observed postoperative complications. Following open-heart surgery, in an observational study we found significantly higher numbers of blood neutrophils in a group of 13 patients with AKI compared to 25 patients without AKI (AKI: 12.9±5.4 ×109 cells/L; non-AKI: 10.1±2. 9 ×109 cells/L). Elevated serum levels of neutrophil extracellular trap (NETs) components, such as dsDNA, histone 3, and DNA binding protein Y-box protein (YB)-1, were found within the first 24 hours in patients who later developed AKI. We could demonstrate that NET formation and hypoxia triggered the release of YB-1, which was subsequently shown to act as a mediator of kidney tubular damage. Experimentally, in two models of AKI mimicking kidney hypoperfusion during cardiac surgery (bilateral ischemia/reperfusion (I/R) and systemic lipopolysaccharide (LPS) administration), a neutralizing YB-1 antibody was administered to mice. In both models, prophylactic YB-1 antibody administration significantly reduced the tubular damage (damage score range 1-4, the LPS model: non-specific IgG control, 0.92±0.23; anti-YB-1 0.65±0.18; and in the I/R model: non-specific IgG control 2.42±0.23; anti-YB-1 1.86±0.44). Even in a therapeutic, delayed treatment model, antagonism of YB-1 ameliorated AKI (damage score, non-specific IgG control 3.03±0.31; anti-YB-1 2.58±0.18). Thus, blocking extracellular YB-1 reduced the effects induced by hypoxia and NET formation in the kidney and significantly limited AKI, suggesting that YB-1 is part of the NET formation process and an integral mediator of cross-organ effects.

Glycosylation Analysis of Urinary Peptidome Highlights IGF2 Glycopeptides in Association with CKD.

Chronic kidney disease (CKD) is prevalent in 10% of world's adult population. The role of protein glycosylation in causal mechanisms of CKD progression is largely unknown. The aim of this study was to identify urinary O-linked glycopeptides in association to CKD for better characterization of CKD molecular manifestations. Urine samples from eight CKD and two healthy subjects were analyzed by CE-MS/MS and glycopeptides were identified by a specific software followed by manual inspection of the spectra. Distribution of the identified glycopeptides and their correlation with Age, eGFR and Albuminuria were evaluated in 3810 existing datasets. In total, 17 O-linked glycopeptides from 7 different proteins were identified, derived primarily from Insulin-like growth factor-II (IGF2). Glycosylation occurred at the surface exposed IGF2 Threonine 96 position. Three glycopeptides (DVStPPTVLPDNFPRYPVGKF, DVStPPTVLPDNFPRYPVG and DVStPPTVLPDNFPRYP) exhibited positive correlation with Age. The IGF2 glycopeptide (tPPTVLPDNFPRYP) showed a strong negative association with eGFR. These results suggest that with aging and deteriorating kidney function, alterations in IGF2 proteoforms take place, which may reflect changes in mature IGF2 protein. Further experiments corroborated this hypothesis as IGF2 increased plasma levels were observed in CKD patients. Protease predictions, considering also available transcriptomics data, suggest activation of cathepsin S with CKD, meriting further investigation.

NRF2/Itaconate Axis Regulates Metabolism and Inflammatory Properties of T Cells in Children with JIA.

BACKGROUND: CD4+ T cells critically contribute to the initiation and perturbation of inflammation. When CD4+ T cells enter inflamed tissues, they adapt to hypoxia and oxidative stress conditions, and to a reduction in nutrients. We aimed to investigate how this distinct environment regulates T cell responses within the inflamed joints of patients with childhood rheumatism (JIA) by analyzing the behavior of NRF2-the key regulator of the anti-oxidative stress response-and its signaling pathways. METHODS: Flow cytometry and quantitative RT-PCR were used to perform metabolic profiling of T cells and to measure the production of inflammatory cytokines. Loss of function analyses were carried out by means of siRNA transfection experiments. NRF2 activation was induced by treatment with 4-octyl-Itaconate (4-OI). RESULTS: Flow cytometry analyses revealed a high metabolic status in CD4+ T cells taken from synovial fluid (SF) with greater mitochondrial mass, and increased glucose and fatty acid uptake. This resulted in a heightened oxidative status of SF CD4+ T cells. Despite raised ROS levels, expression of NRF2 and its target gene NQO1 were lower in CD4+ T cells from SF than in those from blood. Indeed, NRF2 activation of CD4+ T cells downregulated oxidative stress markers, altered the metabolic phenotype and reduced secretion of IFN-γ. CONCLUSION: NRF2 could be a potential regulator in CD4+ T cells during chronic inflammation and could instigate a drift toward disease progression or regression, depending on the inflammatory environment.

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Stimulation of the EP3 receptor causes lung oedema by activation of TRPC6 in pulmonary endothelial cells.

BACKGROUND: Prostaglandin E2 (PGE2) increases pulmonary vascular permeability by activation of the PGE2 receptor 3 (EP3), which may explain adverse pulmonary effects of the EP1/EP3 receptor agonist sulprostone in patients. In addition, PGE2 contributes to pulmonary oedema in response to platelet-activating factor (PAF). PAF increases endothelial permeability by recruiting the cation channel transient receptor potential canonical 6 (TRPC6) to endothelial caveolae via acid sphingomyelinase (ASMase). Yet, the roles of PGE2 and EP3 in this pathway are unknown. We hypothesised that EP3 receptor activation may increase pulmonary vascular permeability by activation of TRPC6, and thus, synergise with ASMase-mediated TRPC6 recruitment in PAF-induced lung oedema. METHODS: In isolated lungs, we measured increases in endothelial calcium (ΔCa2+) or lung weight (Δweight), and endothelial caveolar TRPC6 abundance as well as phosphorylation. RESULTS: PAF-induced ΔCa2+ and Δweight were attenuated in EP3-deficient mice. Sulprostone replicated PAF-induced ΔCa2+ and Δweight which were blocked by pharmacological/genetic inhibition of TRPC6, ASMase or Src-family kinases (SrcFK). PAF, but not sulprostone, increased TRPC6 abundance in endothelial caveolae. Immunoprecipitation revealed PAF- and sulprostone-induced tyrosine-phosphorylation of TRPC6 that was prevented by inhibition of phospholipase C (PLC) or SrcFK. PLC inhibition also blocked sulprostone-induced ΔCa2+ and Δweight, as did inhibition of SrcFK or inhibitory G-protein (Gi) signalling. CONCLUSIONS: EP3 activation triggers pulmonary oedema via Gi-dependent activation of PLC and subsequent SrcFK-dependent tyrosine phosphorylation of TRPC6. In PAF-induced lung oedema, this TRPC6 activation coincides with ASMase-dependent caveolar recruitment of TRPC6, resulting in rapid endothelial Ca2+ influx and barrier failure.

Identification and characterization of post-translational modifications: Clinical implications.

Post-translational modifications (PTMs) generate marginally modified isoforms of native peptides, proteins and lipoproteins thereby regulating protein functions, molecular interactions, and localization. With a key role in functional proteomics, post-translational modifications are recently also associated with the onsets and progressions of various diseases, such as cancer, cardiovascular, renal, and metabolic diseases. With the impact of post-translational modifications becoming increasingly clear, its reliable detection and quantification remain a major obstacle in the translation of these novel pathological markers into clinical diagnosis. While current antibody-based clinical diagnostics struggle to detect and quantify these marginal protein and lipoprotein alterations, state-of-the-art mass spectrometric, proteomic approaches provide the mass accuracy and resolving power necessary to isolate, identify and quantify novel and pathological post-translational modifications; however clinical translation of mass spectrometric applications are still facing major challenges. Here we review the status quo of the clinical translation of mass-spectrometric applications as novel diagnostic tools for the identification and quantification of post-translational modifications and focus on the emerging role of mass spectrometric methods in the clinical assessment of PTMs in disease states.

Irreversible post-translational modifications - Emerging cardiovascular risk factors.

Despite the introduction of lipid-lowering drugs, antihypertensives, antiplatelet and anticoagulation therapies for primary prevention of cardiovascular and heart diseases (CVD), it remains the number one cause of death globally, raising the question for novel/further essential factors besides traditional risk factors such as cholesterol, blood pressure and coagulation. With continuous identification and characterization of non-enzymatic post-translationally modified isoforms of proteins and lipoproteins, it is becoming increasingly clear that irreversible non-enzymatic post-translational modifications (nPTMs) alter the biological functions of native proteins and lipoproteins thereby transforming innate serum components into CVD mediators. In particular renal insufficiency and metabolic imbalance are major contributors to the systemically increased concentration of reactive metabolites and thus increased frequency of nPTMs, promoting multi-morbid disease development centering around cardiovascular disease. nPTMs are significantly involved in the onset and progression of cardiovascular disease and represent a significant and novel risk factor. These insights represent potentially new avenues for risk assessment, prevention and therapy. This review chapter summarizes all forms of nPTMs found in CKD and under metabolic imbalance and discusses the biochemical connections between molecular alterations and the pathological impact on increased cardiovascular risk, novel nPTM-associated non-traditional cardiovascular risk factors, and clinical implication of nPTM in cardiovascular disease.

Carbamylated sortilin associates with cardiovascular calcification in patients with chronic kidney disease.

Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular risk factor in the general population. Patients with chronic kidney disease (CKD) are highly susceptible to develop cardiovascular complications such as calcification. However, specific CKD-induced posttranslational protein modifications of sortilin and their link to cardiovascular calcification remain unknown. To investigate this, we examined two independent CKD cohorts for carbamylation of circulating sortilin and detected increased carbamylated sortilin lysine residues in the extracellular domain of sortilin with kidney function decline using targeted mass spectrometry. Structure analysis predicted altered ligand binding by carbamylated sortilin, which was verified by binding studies using surface plasmon resonance measurement, showing an increased affinity of interleukin 6 to in vitro carbamylated sortilin. Further, carbamylated sortilin increased vascular calcification in vitro and ex vivo that was accelerated by interleukin 6. Imaging by mass spectrometry of human calcified arteries revealed in situ carbamylated sortilin. In patients with CKD, sortilin carbamylation was associated with coronary artery calcification, independent of age and kidney function. Moreover, patients with carbamylated sortilin displayed significantly faster progression of coronary artery calcification than patients without sortilin carbamylation. Thus, carbamylated sortilin may be a risk factor for cardiovascular calcification and may contribute to elevated cardiovascular complications in patients with CKD.

Altered vitamin K biodistribution and metabolism in experimental and human chronic kidney disease.

Chronic kidney disease (CKD) is accompanied with extensive cardiovascular calcification, in part correlating with functional vitamin K deficiency. Here, we sought to determine causes for vitamin K deficiency beyond reduced dietary intake. Initially, vitamin K uptake and distribution into circulating lipoproteins after a single administration of vitamin K1 plus K2 (menaquinone 4 and menaquinone 7, respectively) was determined in patients on dialysis therapy and healthy individuals. The patients incorporated very little menaquinone 7 but more menaquinone 4 into high density lipoprotein (HDL) and low-density lipoprotein particles than did healthy individuals. In contrast to healthy persons, HDL particles from the patients could not be spiked with menaquinone 7 in vitro and HDL uptake was diminished in osteoblasts. A reduced carboxylation activity (low vitamin K activity) of uremic HDL particles spiked with menaquinone 7 vs. that of controls was confirmed in a bioassay using human primary vascular smooth muscle cells. Kidney menaquinone 4 tissue levels were reduced in 5/6-nephrectomized versus sham-operated C57BL/6 mice after four weeks of a vitamin K rich diet. From the analyzed enzymes involved in vitamin K metabolism, kidney HMG-CoA reductase protein was reduced in both rats and patients with CKD. In a trial on the efficacy and safety of atorvastatin in 1051 patients with type 2 diabetes receiving dialysis therapy, no pronounced vitamin K deficiency was noted. However, the highest levels of PIVKA-II (biomarker of subclinical vitamin K deficiency) were noted when a statin was combined with a proton pump inhibitor. Thus, profound disturbances in lipoprotein mediated vitamin K transport and metabolism in uremia suggest that menaquinone 7 supplementation to patients on dialysis therapy has reduced efficacy.

Reproducibility Evaluation of Urinary Peptide Detection Using CE-MS.

In recent years, capillary electrophoresis coupled to mass spectrometry (CE-MS) has been increasingly applied in clinical research especially in the context of chronic and age-associated diseases, such as chronic kidney disease, heart failure and cancer. Biomarkers identified using this technique are already used for diagnosis, prognosis and monitoring of these complex diseases, as well as patient stratification in clinical trials. CE-MS allows for a comprehensive assessment of small molecular weight proteins and peptides (<20 kDa) through the combination of the high resolution and reproducibility of CE and the distinct sensitivity of MS, in a high-throughput system. In this study we assessed CE-MS analytical performance with regards to its inter- and intra-day reproducibility, variability and efficiency in peptide detection, along with a characterization of the urinary peptidome content. To this end, CE-MS performance was evaluated based on 72 measurements of a standard urine sample (60 for inter- and 12 for intra-day assessment) analyzed during the second quarter of 2021. Analysis was performed per run, per peptide, as well as at the level of biomarker panels. The obtained datasets showed high correlation between the different runs, low variation of the ten highest average individual log2 signal intensities (coefficient of variation, CV < 10%) and very low variation of biomarker panels applied (CV close to 1%). The findings of the study support the analytical performance of CE-MS, underlining its value for clinical application.

Prevention of vascular calcification by the endogenous chromogranin A-derived mediator that inhibits osteogenic transdifferentiation.

The adrenal glands participate in cardiovascular (CV) physiology and the pathophysiology of CV diseases through their effects on sodium and water metabolism, vascular tone and cardiac function. In the present study, we identified a new adrenal compound controlling mesenchymal cell differentiation that regulates osteoblastic differentiation in the context of vascular calcification. This peptide was named the "calcification blocking factor" (CBF) due to its protective effect against vascular calcification and is released from chromogranin A via enzymatic cleavage by calpain 1 and kallikrein. CBF reduced the calcium content of cells and thoracic aortic rings under calcifying culture conditions, as well as in aortas from animals treated with vitamin D and nicotine (VDN animals). Furthermore, CBF prevented vascular smooth muscle cell (VSMC) transdifferentiation into osteoblast-like cells within the vascular wall via the sodium-dependent phosphate transporter PIT-1 and by inhibition of NF-κB activation and the subsequent BMP2/p-SMAD pathway. Pulse pressure, a marker of arterial stiffness, was significantly decreased in VDN animals treated with CBF. In line with our preclinical data, CBF concentration is significantly reduced in diseases characterized by increased calcification, as shown in patients with chronic kidney disease. In preparation for clinical translation, the active site of the native 19-AS long native CBF was identified as EGQEEEED. In conclusion, we have identified the new peptide CBF, which is secreted from the adrenal glands and might prevent vascular calcification by inhibition of osteogenic transdifferentiation. The anti-calcific effects of CBF and short active site may therefore promote the development of new tools for the prevention and/or treatment of vascular calcification.

Guanidinylated Apolipoprotein C3 (ApoC3) Associates with Kidney and Vascular Injury.

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.