The RNA-binding protein QKI governs a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes.

AIMS: In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. METHODS AND RESULTS: We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. CONCLUSION: Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

Free p-cresyl sulfate shows the highest association with cardiovascular outcome in chronic kidney disease.

BACKGROUND: Several protein-bound uraemic toxins (PBUTs) have been associated with cardiovascular (CV) and all-cause mortality in chronic kidney disease (CKD) but the degree to which this is the case per individual PBUT and the pathophysiological mechanism have only partially been unraveled. METHODS: We compared the prognostic value of both total and free concentrations of five PBUTs [p-cresyl sulfate (pCS), p-cresyl glucuronide, indoxyl sulfate, indole acetic acid and hippuric acid] in a cohort of 523 patients with non-dialysis CKD Stages G1-G5. Patients were followed prospectively for the occurrence of a fatal or non-fatal CV event as the primary endpoint and a number of other major complications as secondary endpoints. In addition, association with and the prognostic value of nine markers of endothelial activation/damage was compared. RESULTS: After a median follow-up of 5.5 years, 149 patients developed the primary endpoint. In multivariate Cox regression models adjusted for age, sex, systolic blood pressure, diabetes mellitus and estimated glomerular filtration rate, and corrected for multiple testing, only free pCS was associated with the primary endpoint {hazard ratio [HR]1.39 [95% confidence interval (CI) 1.14-1.71]; P = 0.0014}. Free pCS also correlated with a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (r = -0.114, P < 0.05), angiopoietin-2 (ANGPT2) (r = 0.194, P < 0.001), matrix metallopeptidase 7 (MMP-7; (r = 0.238, P < 0.001) and syndecan 1 (r = 0.235, P < 0.001). Of these markers of endothelial activation/damage, ANGPT2 [HR 1.46 (95% CI 1.25-1.70); P < 0.0001] and MMP-7 [HR 1.31 (95% CI 1.08-1.59); P = 0.0056] were also predictive of the primary outcome. CONCLUSIONS: Among PBUTs, free pCS shows the highest association with CV outcome in non-dialysed patients with CKD. Two markers of endothelial activation/damage that were significantly correlated with free pCS, ANGPT2 and MMP-7 were also associated with CV outcome. The hypothesis that free pCS exerts its CV toxic effects by an adverse effect on endothelial function deserves further exploration.

The urinary proteomics classifier chronic kidney disease 273 predicts cardiovascular outcome in patients with chronic kidney disease.

BACKGROUND: The urinary proteomic classifier chronic kidney disease 273 (CKD273) is predictive for the development and progression of chronic kidney disease (CKD) and/or albuminuria in type 2 diabetes. This study evaluates its role in the prediction of cardiovascular (CV) events in patients with CKD Stages G1-G5. METHODS: We applied the CKD273 classifier in a cohort of 451 patients with CKD Stages G1-G5 followed prospectively for a median of 5.5 years. Primary endpoints were all-cause mortality, CV mortality and the composite of non-fatal and fatal CV events (CVEs). RESULTS: In multivariate Cox regression models adjusting for age, sex, prevalent diabetes and CV history, the CKD273 classifier at baseline was significantly associated with total mortality and time to fatal or non-fatal CVE, but not CV mortality. Because of a significant interaction between CKD273 and CV history (P = 0.018) and CKD stages (P = 0.002), a stratified analysis was performed. In the fully adjusted models, CKD273 classifier was a strong and independent predictor of fatal or non-fatal CVE only in the subgroup of patients with CKD Stages G1-G3b and without a history of CV disease. In those patients, the highest tertile of CKD273 was associated with a >10-fold increased risk as compared with the lowest tertile. CONCLUSIONS: The urinary CKD273 classifier provides additional independent information regarding the CV risk in patients with early CKD stage and a blank CV history. Determination of CKD273 scores on a random urine sample may improve the efficacy of intensified surveillance and preventive strategies by selecting patients who potentially will benefit most from early risk management.

Contribution of the uremic milieu to an increased pro-inflammatory monocytic phenotype in chronic kidney disease.

Intermediate (CD14++CD16+) monocytes have important pro-inflammatory and atherogenic features and are increased in patients with chronic kidney disease (CKD). The present study aims to elucidate the role of the uremic milieu and of platelet activation in monocyte differentiation. Monocyte subtypes were analyzed in CKD patients (n = 193) and healthy controls (n = 27). Blood from healthy controls (Ctrl; n = 8) and hemodialysis patients (HD; n = 8) was centrifuged, and plasma (pl) was exchanged between Ctrl and HD (Ctrlcells/HDpl and HDcells/Ctrlpl) or reconstituted as original (Ctrlsham and HDsham) and incubated for 24 h (T24). Monocyte differentiation and platelet aggregation to monocytes (MPA) was assessed by flow cytometry. Especially, a higher proportion of CD14++CD16+ monocytes was found in hemodialysis (HD) patients (p < 0.01). In plasma exchange experiments, Ctrl cells/HD pl T24 showed an increased percentage of CD14++CD16+ monocytes versus Ctrl sham (33.7% ± 15 vs. 15.7% ± 9.6; P < 0.005), comparable to the level of CD14++CD16+ monocytes in the HD sham condition. The percentage of CD14++CD16+ monocytes was lowered by suspending HD cells in Ctrl pl (18.4% ± 7.8 vs. 36.7% ± 15 in HD sham; P < 0.005) reaching the level of the Ctrl sham condition (15.7% ± 9.6). A mixture of uremic sulfates increased CD14++CD16+ monocytes compared to control (19.8 ± 9.6% vs. 15.8 ± 10.9%; P < 0.05), paralleled by a rise MPA. Blocking MPA by abciximab, a potential therapeutic strategy, or anti-CD62P did not inhibit differentiation towards the CD14++CD16+ monocytes. In conclusion, in the present cohort, CD14++CD16+ monocytes are especially increased in HD patients and this can at least in part be attributed to the presence of the uremic milieu, with uremic sulfates inducing a reversible shift towards pro-inflammatory CD14++CD16+ monocytes.

Serum levels of miR-126 and miR-223 and outcomes in chronic kidney disease patients.

Several microRNAs (miRNAs) have been linked to chronic kidney disease (CKD) mortality, cardiovascular (CV) complications and kidney disease progression. However, their association with clinical outcomes remains poorly evaluated. We used real-time qPCR to measure serum levels of miR-126 and miR-223 in a large cohort of 601 CKD patients (CKD stage G1 to G5 patients or on renal replacement therapy - CKD G5D) from Ghent University Hospital and 31 healthy controls. All-cause mortality and cardiovascular and renal events were registered as endpoints over a 6 year follow-up period. miR-126 levels were significantly lower from CKD stage G2 on, compared to controls. The serum levels of miR-223 were significantly lower from CKD stage G3B on. When considering overall mortality, patients with levels of either miR-126 or miR-223 below the median had a lower survival rate. Similar results were observed for CV and renal events. The observed link between the two miRNAs' seric levels and mortality, cardiovascular events or renal events in CKD appears to depend on eGFR. However, this does not preclude their potential role in the pathophysiology of CKD. In conclusion, CKD is associated with a decrease in circulating miR-223 and miR-126 levels.

Cardiovascular disease after transplantation: an emerging role of the immune system.

Cardiovascular disease (CVD) after transplantation remains a major concern. Little is known about what drives the increased cardiovascular risk in transplant recipients apart from traditional risk factors. The immune system is involved in the pathogenesis of hypertension, atherosclerosis, and coronary artery disease in the general population. Recently, inhibition of interleukin 1 - ß by canakinumab versus placebo decreased the incidence of cardiovascular events. Emerging evidence points to a role of adaptive cellular immunity in the development of CVD. Especially, expansion of pro-inflammatory and antiapoptotic cytotoxic CD4+ CD28null T cells is closely associated with incident CVD in various study populations including transplant recipients. The association of cytomegalovirus exposure with increased cardiovascular mortality might be explained by its capacity to upregulate these cytotoxic cells. Also, humoral immunity seems to be relevant for cardiovascular outcome in transplant recipients. Panel-reactive antibodies at baseline and donor-specific antibodies are independently associated with poor cardiovascular outcome after kidney transplantation. Cardiovascular effects of immunosuppressive drugs and statins do not only imply indirect positive or negative effects on traditional cardiovascular risk factors but also intrinsic immunological effects. How immunosuppressive drugs modify atherosclerosis largely remains elusive.

Increased urinary osmolyte excretion indicates chronic kidney disease severity and progression rate.

Background: Chronic kidney disease (CKD) is a recognized global health problem. While some CKD patients remain stable after initial diagnosis, others can rapidly progress towards end-stage renal disease (ESRD). This makes biomarkers capable of detecting progressive forms of CKD extremely valuable, especially in non-invasive biofluids such as urine. Screening for metabolite markers using non-targeted metabolomic techniques like nuclear magnetic resonance spectroscopy is increasingly applied to CKD research. Methods: A cohort of CKD patients (n = 227) with estimated glomerular filtration rates (eGFRs) ranging from 9.4-130 mL/min/1.73 m2 was evaluated and urine metabolite profiles were characterized in relation to declining eGFR. Nested in this cohort, a retrospective subset (n = 57) was investigated for prognostic metabolite markers of CKD progression, independent of baseline eGFR. A transcriptomic analysis of murine models of renal failure was performed to validate selected metabolomic findings. Results: General linear modeling revealed 11 urinary metabolites with significant associations to reduced eGFR. Linear modelling specifically showed that increased urine concentrations of betaine (P < 0.05) and myo-inositol (P < 0.05) are significant prognostic markers of CKD progression. Conclusions: Renal organic osmolytes, betaine and myo-inositol play a critical role in protecting renal cells from hyperosmotic stress. Kidney tissue transcriptomics of murine preclinical experimentation identified decreased expression of Slc6a12 and Slc5a11 mRNA in renal tissue consistent with defective tubular transport of these osmolytes. Imbalances in renal osmolyte regulation lead to increased renal cell damage and thus more progressive forms of CKD. Increases in renal osmolytes in urine could provide clinical diagnostic and prognostic information on CKD outcomes.

Biochemical and Clinical Impact of Organic Uremic Retention Solutes: A Comprehensive Update.

In this narrative review, the biological/biochemical impact (toxicity) of a large array of known individual uremic retention solutes and groups of solutes is summarized. We classified these compounds along their physico-chemical characteristics as small water-soluble compounds or groups, protein bound compounds and middle molecules. All but one solute (glomerulopressin) affected at least one mechanism with the potential to contribute to the uremic syndrome. In general, several mechanisms were influenced for each individual solute or group of solutes, with some impacting up to 7 different biological systems of the 11 considered. The inflammatory, cardio-vascular and fibrogenic systems were those most frequently affected and they are one by one major actors in the high morbidity and mortality of CKD but also the mechanisms that have most frequently been studied. A scoring system was built with the intention to classify the reviewed compounds according to the experimental evidence of their toxicity (number of systems affected) and overall experimental and clinical evidence. Among the highest globally scoring solutes were 3 small water-soluble compounds [asymmetric dimethylarginine (ADMA); trimethylamine-N-oxide (TMAO); uric acid], 6 protein bound compounds or groups of protein bound compounds [advanced glycation end products (AGEs); p-cresyl sulfate; indoxyl sulfate; indole acetic acid; the kynurenines; phenyl acetic acid;] and 3 middle molecules [ß2-microglobulin; ghrelin; parathyroid hormone). In general, more experimental data were provided for the protein bound molecules but for almost half of them clinical evidence was missing in spite of robust experimental data. The picture emanating is one of a complex disorder, where multiple factors contribute to a multisystem complication profile, so that it seems of not much use to pursue a decrease of concentration of a single compound.

Assessment of the association between increasing membrane pore size and endotoxin permeability using a novel experimental dialysis simulation set-up.

BACKGROUND: Membranes with increasing pore size are introduced to enhance removal of large uremic toxins with regular hemodialysis. These membranes might theoretically have higher permeability for bacterial degradation products. In this paper, permeability for bacterial degradation products of membranes of comparable composition with different pore size was investigated with a new in vitro set-up that represents clinical flow and pressure conditions. METHODS: Dialysis was simulated with an AK200 machine using a low-flux, high-flux, medium cut-off (MCO) or high cut-off (HCO) device (n = 6/type). A polyvinylpyrrolidone-solution (PVP) was recirculated at blood side. At dialysate side, a challenge solution containing a filtrated lysate of two water-borne bacteria (Pseudomonas aeruginosa and Pelomononas saccharophila) was infused in the dialysate flow (endotoxin ≥ 4EU/ml). Blood and dialysate flow were set at 400 and 500 ml/min for 60 min. PVP was sampled before (PVPpre) and after (PVPpost) the experiment and dialysate after 5 and 55 min. Limulus Amebocyte Lysate (LAL) test was performed. Additionally, samples were incubated with a THP-1 cell line (24 h) and IL-1ß levels were measured evaluating biological activity. RESULTS: The LAL-assay confirmed presence of 9.5 ± 7.4 EU/ml at dialysate side. For none of the devices the LAL activity in PVPpre vs. PVPpost was significantly different. Although more blood side PVP solutions had a detectable amount of endotoxin using a highly sensitive LAL assay in the more open vs traditional membranes, the permeability for endotoxins of the 4 tested dialysis membranes was not significantly different but the number of repeats is small. None of the PVP solutions induced IL-1ß in the THP-1 assay. CONCLUSIONS: A realisitic in vitro dialysis was developed to assess membrane translocation of bacterial products. LAL activity on the blood side after endotoxin exposure did not change for all membranes. Also, none of the PVPpost solutions induced IL-1ß in the THP-1 bio-assay.

Hereditary polycystic kidney disease is characterized by lymphopenia across all stages of kidney dysfunction: an observational study.

Background: Polycystic kidney disease (PKD) is characterized by urinary tract infections and extrarenal abnormalities such as an increased risk of cancer. As mutations in polycystin-1 and -2 are associated with decreased proliferation of immortalized lymphoblastoid cells in PKD, we investigated whether lymphopenia could be an unrecognized trait of PKD. Methods: We studied 700 kidney transplant recipients with (n = 126) or without PKD at the time of kidney transplantation between 1 January 2003 and 31 December 2014 at Ghent University Hospital. We also studied 204 patients with chronic kidney disease (CKD) with PKD and 204 matched CKD patients without PKD across comparable CKD strata with assessment between 1 January 1999 and 1 February 2016 at three renal outpatient clinics. We compared lymphocyte counts with multiple linear regression analysis to adjust for potential confounders. We analysed flow cytometric immunophenotyping data and other haematological parameters. Results: Lymphocyte counts were 264/µL [95% confidence interval (CI) 144-384] and 345/µL (95% CI 245-445) (both P < 0.001) lower in the end-stage kidney disease (ESKD) and CKD cohort, respectively, after adjustment for age, sex, ln(C-reactive protein) and estimated glomerular filtration rate (in the CKD cohort only). In particular, CD8+ T and B lymphocytes were significantly lower in transplant recipients with versus without PKD (P < 0.001 for both). Thrombocyte and monocyte counts were lower in patients with versus without PKD in both cohorts (P < 0.001 for all analyses except P = 0.01 for monocytes in the ESKD cohort). Conclusion: PKD is characterized by distinct cytopenias and especially lymphopenia, independent of kidney function. This finding has the potential to alter our therapeutic approach to patients with PKD.

Binding of bromocresol green and bromocresol purple to albumin in hemodialysis patients.

BACKGROUND: Colorimetric albumin assays based on binding to bromocresol purple (BCP) and bromocresol green (BCG) yield different results in chronic kidney disease. Altered dye binding of carbamylated albumin has been suggested as a cause. In the present study, a detailed analysis was carried out in which uremic toxins, acute phase proteins and Kt/V, a parameter describing hemodialysis efficiency, were compared with colorimetrically assayed (BCP and BCG) serum albumin. METHODS: Albumin was assayed using immunonephelometry on a BN II nephelometer and colorimetrically based on, respectively, BCP and BCG on a Modular P analyzer. Uremic toxins were assessed using high-performance liquid chromatography. Acute phase proteins (C-reactive protein and α1-acid glycoprotein) and plasma protein α2-macroglobulin were assayed nephelometrically. In parallel, Kt/V was calculated. RESULTS: Sixty-two serum specimens originating from hemodialysis patients were analyzed. Among the uremic toxins investigated, total para-cresyl sulfate (PCS) showed a significant positive correlation with the BCP/BCG ratio. The serum α1-acid glycoprotein concentration correlated negatively with the BCP/BCG ratio. The BCP/BCG ratio showed also a negative correlation with Kt/V. CONCLUSIONS: In renal insufficiency, the BCP/BCG ratio of serum albumin is affected by multiple factors: next to carbamylation, uremic toxins (total PCS) and α1-acid glycoprotein also play a role.

Prediction of Chronic Kidney Disease Stage 3 by CKD273, a Urinary Proteomic Biomarker.

INTRODUCTION: CKD273 is a urinary biomarker, which in advanced chronic kidney disease predicts further deterioration. We investigated whether CKD273 can also predict a decline of estimated glomerular filtration rate (eGFR) to <60 ml/min per 1.73 m2. METHODS: In analyses of 2087 individuals from 6 cohorts (46.4% women; 73.5% with diabetes; mean age, 46.1 years; eGFR ≥ 60 ml/min per 1.73 m2, 100%; urinary albumin excretion rate [UAE] ≥20 µg/min, 6.2%), we accounted for cohort, sex, age, mean arterial pressure, diabetes, and eGFR at baseline and expressed associations per 1-SD increment in urinary biomarkers. RESULTS: Over 5 (median) follow-up visits, eGFR decreased more with higher baseline CKD273 than UAE (1.64 vs. 0.82 ml/min per 1.73 m2; P < 0.0001). Over 4.6 years (median), 390 participants experienced a first renal endpoint (eGFR decrease by ≥10 to <60 ml/min per 1.73 m2), and 172 experienced an endpoint sustained over follow-up. The risk of a first and sustained renal endpoint increased with UAE (hazard ratio ≥ 1.23; P ≤ 0.043) and CKD273 (≥ 1.20; P ≤ 0.031). UAE (≥20 µg/min) and CKD273 (≥0.154) thresholds yielded sensitivities of 30% and 33% and specificities of 82% and 83% (P ≤ 0.0001 for difference between UAE and CKD273 in proportion of correctly classified individuals). As continuous markers, CKD273 (P = 0.039), but not UAE (P = 0.065), increased the integrated discrimination improvement, while both UAE and CKD273 improved the net reclassification index (P ≤ 0.0003), except for UAE per threshold (P = 0.086). DISCUSSION: In conclusion, while accounting for baseline eGFR, albuminuria, and covariables, CKD273 adds to the prediction of stage 3 chronic kidney disease, at which point intervention remains an achievable therapeutic target.

Spontaneous variability of pre-dialysis concentrations of uremic toxins over time in stable hemodialysis patients.

BACKGROUND AND AIM: Numerous outcome studies and interventional trials in hemodialysis (HD) patients are based on uremic toxin concentrations determined at one single or a limited number of time points. The reliability of these studies however entirely depends on how representative these cross-sectional concentrations are. We therefore investigated the variability of predialysis concentrations of uremic toxins over time. METHODS: Prospectively collected predialysis serum samples of the midweek session of week 0, 1, 2, 3, 4, 8, 12, and 16 were analyzed for a panel of uremic toxins in stable chronic HD patients (N = 18) while maintaining dialyzer type and dialysis mode during the study period. RESULTS: Concentrations of the analyzed uremic toxins varied substantially between individuals, but also within stable HD patients (intra-patient variability). For urea, creatinine, beta-2-microglobulin, and some protein-bound uremic toxins, Intra-class Correlation Coefficient (ICC) was higher than 0.7. However, for phosphorus, uric acid, symmetric and asymmetric dimethylarginine, and the protein-bound toxins hippuric acid and indoxyl sulfate, ICC values were below 0.7, implying a concentration variability within the individual patient even exceeding 65% of the observed inter-patient variability. CONCLUSION: Intra-patient variability may affect the interpretation of the association between a single concentration of certain uremic toxins and outcomes. When performing future outcome and interventional studies with uremic toxins other than described here, one should quantify their intra-patient variability and take into account that for solutes with a large intra-patient variability associations could be missed.

The Place of Large Pore Membranes in the Treatment Portfolio of Patients on Hemodialysis.

Cardiovascular disease is a major concern in patients with end-stage kidney disease (ESKD). Inflammation induced by retention of uremic toxins, of which a substantial fraction has a molecular weight in the middle molecular range, has been associated with increased cardiovascular risk. In an attempt to reduce inflammation and thus cardiovascular toxicity in patients with ESKD, hemodiafiltration (HDF) has been promoted to enhance the clearance of middle molecular weight substances during dialysis. However, HDF increases the technical complexity and costs, and requires ultrapure dialysis fluid. Also, HDF becomes less beneficial when it is not possible to achieve sufficient convective volume in all patients. Over the last years, membranes with larger pore sizes, such as medium cut-off and high cut-off, have been introduced. These membranes, applied in hemodialysis mode, appear to have removal rates of middle molecular weight molecules that are comparable to those achieved with HDF, and could thus obviate the need for HDF. However, ultrapure dialysis fluid might still be required if there is a risk of transmigration of contaminants from the dialysate side into the blood because of the increased pore size. This transmigration of pyrogens might upregulate the expression of cytokines and other pro-inflammatory factors, and thus completely neutralize the beneficial effects of higher clearance of middle molecules. This chapter will explore the existing evidence on permeability of membranes with large pores for bacterial degradation products, and based on this information we will try to define the position of these novel membranes among the spectrum of existing membranes.

Quantification of carbamylated albumin in serum based on capillary electrophoresis.

Protein carbamylation, a nonenzymatic posttranslational modification promoted during uremia, is linked to a poor prognosis. In the present study, carbamylation of serum albumin was assayed using the symmetry factor on a capillary electrophoresis instrument (Helena V8). The symmetry factor has been defined as the distance from the center line of the peak to the back slope, divided by the distance from the center line of the peak to the front slope, with all measurements made at 10% of the maximum peak height. Serum albumin, creatinine, and urea concentrations were assayed using routine methods, whereas uremic toxins were determined using HPLC. In vitro carbamylation induced a marked albumin peak asymmetry. Reference values for the albumin symmetry factor were 0.69-0.92. In kidney patients, albumin peak asymmetry corresponded to the chronic kidney disease stage (p < 0.0001). The symmetry factor correlated well with serum urea (r = -0.5595, p < 0.0001) and creatinine (r = -0.5986, p < 0.0001) concentrations. Several protein-bound uremic toxins showed a significant negative correlation with the symmetry factor. Morphology of the albumin fraction was not affected by presence of glycated albumin and protein-bound antibiotics. In conclusion, the presented method provides a simple, practical way for monitoring protein carbamylation.

Metabolic profiling of human plasma and urine in chronic kidney disease by hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry: a pilot study.

A typical characteristic of chronic kidney disease (CKD) is the progressive loss in renal function over a period of months or years with the concomitant accumulation of uremic retention solutes in the body. Known biomarkers for the kidney deterioration, such as serum creatinine or urinary albumin, do not allow effective early detection of CKD, which is essential towards disease management. In this work, a hydrophilic interaction liquid chromatography time-of-flight mass spectrometric (HILIC-TOF MS) platform was optimized allowing the search for novel uremic retention solutes and/or biomarkers of CKD. The HILIC-ESI-MS approach was used for the comparison of urine and plasma samples from CKD patients at stage 3 (n = 20), at stage 5 not yet receiving dialysis (n = 20) and from healthy controls (n = 20). Quality control samples were used to control and ensure the validity of the metabolomics approach. Subsequently the data were treated with the XCMS software for multivariate statistical analysis. In this way, differentiation could be achieved between the measured metabolite profile of the CKD patients versus the healthy controls. The approach allowed the elucidation of a number of metabolites that showed a significant up- and downregulation throughout the different stages of CKD. These compounds are cinnamoylglycine, glycoursodeoxycholic acid, 2-hydroxyethane sulfonate, and pregnenolone sulfate of which the identity was unambiguously confirmed via the use of authentic standards. The latter three are newly identified uremic retention solutes.

Response to Tsikas et al. Comments on Boelaert et al. Determination of Asymmetric and Symmetric Dimethylarginine in Serum from Patients with Chronic Kidney Disease: UPLC-MS/MS versus ELISA. Toxins 2016, 8, 149.

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Determination of Asymmetric and Symmetric Dimethylarginine in Serum from Patients with Chronic Kidney Disease: UPLC-MS/MS versus ELISA.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, and its structural isomer symmetric dimethylarginine (SDMA) are uremic toxins accumulating in chronic kidney disease (CKD) patients. The objective of this study was to develop and validate a robust UPLC-MS/MS method for the simultaneous determination of ADMA and SDMA in human serum. Chromatographic separation after butyl ester derivatization was achieved on an Acquity UPLC BEH C18 column, followed by tandem mass spectrometric detection. After validation, the applicability of the method was evaluated by the analysis of serum samples from 10 healthy controls and 77 CKD patients on hemodialysis (CKD5HD). Both ADMA (0.84 ± 0.19 µM vs. 0.52 ± 0.07 µM) and SDMA concentrations (2.06 ± 0.82 µM vs. 0.59 ± 0.13 µM) were significantly (p < 0.001) elevated in CKD5HD patients compared to healthy controls. In general, low degrees of protein binding were found for both ADMA and SDMA. In addition, an established commercially available ELISA kit was utilized on the same samples (n = 87) to compare values obtained both with ELISA and UPLC-MS/MS. Regression analysis between these two methods was significant (p < 0.0001) but moderate for both ADMA (R = 0.78) and SDMA (R = 0.72).

New insights in molecular mechanisms involved in chronic kidney disease using high-resolution plasma proteome analysis.

BACKGROUND: The reduced glomerular filtration rate in the advanced stages of chronic kidney disease (CKD) leads to plasma accumulation of uraemic retention solutes including proteins. It has been hypothesized that these changes may, at least in part, be responsible for CKD-associated morbidity and mortality. However, most studies focused on the role of individual proteins, while a holistic, large-scale, integrative approach may generate significant additional insight. METHODS: In a discovery study, we analysed the plasma proteome of patients with stage 2-3 CKD (n = 14) and stage 5 CKD with haemodialysis (HD) (n = 15), using high-resolution LC-MS/MS analysis. Selected results were validated in a cohort of 40 patients with different CKD stages with or without HD, using ELISA. RESULTS: Of a total of 2054 detected proteins, 127 displayed lower, while 206 displayed higher abundance in the plasma of patients on HD. Molecular pathway analysis confirmed the modification of known processes involved in CKD complications, including decreased haemostasis and increased inflammation, complement activation and vascular damage. In addition, we identified the plasma increase during CKD progression of lysozyme C and leucine-rich alpha-2 glycoprotein, two proteins related to vascular damage and heart failure. High level of leucine-rich alpha-2 glycoprotein was associated with higher mortality in stage 5 CKD patients on HD. CONCLUSIONS: This study provides for the first time a comprehensive assessment of CKD plasma proteome, contributing to new knowledge and potential markers of CKD. These results will serve as a basis for future studies investigating the relevance of these molecules in CKD associated morbidity and mortality.