Unsupervised analysis of combined lipid and coagulation data reveals coagulopathy subtypes among dialysis patients

Hemodialysis (HD) and peritoneal dialysis (PD) are the primary means of managing end stage renal disease (ESRD). However, these treatment modalities are associated with the onset of coagulation abnormalities. Effective management of coagulation risk among these patients requires the identification of surrogate markers that provide an early indication of the coagulation abnormalities. The role of sphingolipids in the manifestation and prediction of coagulation abnormalities among dialysis patients have never been investigated. Herein, we report the first instance of an in depth investigation into the sphingolipid changes among ESRD patients undergoing HD and PD. The results reveal distinct differences in terms of perturbations to specific sphingolipid biosynthetic pathways that are highly dependent on the treatment modality. Our studies also demonstrated strong correlation between specific sphingolipids and coagulation parameters, such as HexCer(d18:1/26:0) and maximal amplitude (MA), SM(d18:1/24:1) and tissue factor pathway inhibitor, and sphingosine 1-phosphate d18:1 and FX (Spearman ρ of 0.93, 0.89, and −0.89, respectively). Furthermore, our study revealed the potential for using HexCer(d18:1/22:0), HexCer(d18:1/24:0), and HexCer(d18:1/26:0) (r2 = 0.71, 0.82, and 0.63, respectively) and coagulation parameter MA (r2 = 0.7) for successful diagnosis of differential coagulopathies among ESRD patients undergoing HD, providing an opportunity toward personalized disease management. Hemodialysis (HD) and peritoneal dialysis (PD) are the primary means of managing end stage renal disease (ESRD). However, these treatment modalities are associated with the onset of coagulation abnormalities. Effective management of coagulation risk among these patients requires the identification of surrogate markers that provide an early indication of the coagulation abnormalities. The role of sphingolipids in the manifestation and prediction of coagulation abnormalities among dialysis patients have never been investigated. Herein, we report the first instance of an in depth investigation into the sphingolipid changes among ESRD patients undergoing HD and PD. The results reveal distinct differences in terms of perturbations to specific sphingolipid biosynthetic pathways that are highly dependent on the treatment modality. Our studies also demonstrated strong correlation between specific sphingolipids and coagulation parameters, such as HexCer(d18:1/26:0) and maximal amplitude (MA), SM(d18:1/24:1) and tissue factor pathway inhibitor, and sphingosine 1-phosphate d18:1 and FX (Spearman ρ of 0.93, 0.89, and −0.89, respectively). Furthermore, our study revealed the potential for using HexCer(d18:1/22:0), HexCer(d18:1/24:0), and HexCer(d18:1/26:0) (r2 = 0.71, 0.82, and 0.63, respectively) and coagulation parameter MA (r2 = 0.7) for successful diagnosis of differential coagulopathies among ESRD patients undergoing HD, providing an opportunity toward personalized disease management. Dialysis is the primary treatment for patients with end stage renal disease (ESRD). While this treatment modality has made significant advances over the past years, ERSD patients still have unacceptably high rates of mortality (1.Kidney Disease Statistics for the United States. Accessed March 8, 2016, at http://www.niddk.nih.gov/health-information/health-statistics/Pages/kidney-disease-statistics-united-states.aspx.Google Scholar). Indeed, CVD is the primary cause of mortality among patients with ESRD, accounting for about 45% of all deaths (2.Hage F.G. Venkataraman R. Zoghbi G.J. Perry G.J. DeMattos A.M. Iskandrian A.E. The scope of coronary heart disease in patients with chronic kidney disease.J. Am. Coll. Cardiol. 2009; 53: 2129-2140Crossref PubMed Scopus (181) Google Scholar). Complicating this is the fact that ESRD patients present paradoxical pathology of coagulation; a pro-coagulant state is observed while they are also at significant risk of increased bleeding (3.Hughes S. Szeki I. Nash M.J. Thachil J. Anticoagulation in chronic kidney disease patients–the practical aspects.Clin. Kidney J. 2014; 7: 442-449Crossref PubMed Scopus (75) Google Scholar). The pro-coagulant profile is a prevalent problem on the arteriovenous graft site (4.Palomar R. Morales P. Rodrigo E. Castañeda O. Fernández-Fresnedo G. Gómez-Alamillo C. Arias M. Venous graft thrombosis in patients on peritoneal dialysis before transplantation.Transplant. Proc. 2007; 39: 2128-2130Crossref PubMed Scopus (16) Google Scholar) leading to complications in performing the hemodialysis (HD) treatment and places these patients at risk of general vascular thrombosis. Therefore, the hemostatic balance in ESRD patients receiving dialysis modalities is often a guessing game. Thus, bleeding abnormalities associated with ESRD are difficult to assess due to both the overlapping pro-coagulant and pro-bleeding profiles (5.Hedges S.J. Dehoney S.B. Hooper J.S. Amanzadeh J. Busti A.J. Evidence-based treatment recommendations for uremic bleeding.Nat. Clin. Pract. Nephrol. 2007; 3: 138-153Crossref PubMed Scopus (179) Google Scholar). We have previously investigated the coagulopathy associated with ESRD in a single-center cross-sectional study of HD and peritoneal dialysis (PD) patients (6.Brophy D.F. Carl D.E. Mohammed B.M. Song J. Martin E.J. Bostic J.L. Gehr T.W.B. Differences in coagulation between hemodialysis and peritoneal dialysis.Perit. Dial. Int. 2014; 34: 33-40Crossref PubMed Scopus (11) Google Scholar). This study identified a pro-coagulant phenotype among the dialysis patients. Furthermore, this pro-coagulant profile was observed to be more prominent among the PD patients. However, the coagulation parameters used in this study were unable to identify significant determinants of the pro-coagulant risk among the HD population because of the inclusion of subjects with high and low thrombotic potentials. This suggests that coagulation parameters alone are insufficient to determine the pro-coagulant risk among the study population and that additional data are required. In terms of the identification of coagulopathies, lipids have been relatively understudied despite the fact that many lipids are involved in coagulation cascades (7.Kim J-A. Kim J-E. Song S.H. Kim H.K. Influence of blood lipids on global coagulation test results.Ann. Lab. Med. 2015; 35: 15-21Crossref PubMed Scopus (40) Google Scholar). Furthermore, to date, no studies have investigated the role of sphingolipids, specifically, with respect to the coagulation potential of ESRD patients undergoing dialysis. Given that the coagulation parameters alone were insufficient to discriminate the pro-coagulant potential among the cohort of dialysis patients in our previous study, and the fact that the lipids have a close association with coagulation, led us to investigate the variability of the lipidome and especially the sphingolipidome as an additional orthogonal measurement to delineate between pro-coagulant dialysis subpopulations. The data demonstrate a clear distinction in the sphingolipid profiles of the different coagulation phenotypes among the dialysis patients and the feasibility of using a limited subset of combined sphingolipids and coagulation parameters as a potential biomarker for identifying patient subpopulations susceptible to clotting events. A single-center cross-sectional pilot study was designed to characterize differences in biochemical, cellular, and functional coagulation parameters in patients receiving maintenance HD and PD. Fifty subjects were enrolled in the study, including: 10 healthy volunteers who served as a baseline reference; 20 subjects receiving thrice-weekly maintenance HD; and 20 subjects receiving maintenance continuous cycling PD. The continuous cycling PD regimen consisted of four 2 h exchanges nightly and one 6 h dialysis exchange daily. Each HD patient received thrice-weekly 4 h high-flux HD sessions using a Fresenius Optiflux 180 dialyzer (Fresenius Medical Care North America, Waltham, MA). Of the 20 HD patients, 18 had arteriovenous grafts and 2 had tunneled central venous catheters because of multiple arteriovenous graft failures. Only patients that had a full dataset for coagulation parameters and lipidomic variables were used in the statistical analysis strategy, resulting in 9 healthy volunteers, 19 HD patients, and 17 PD patients. All subjects received recombinant human erythropoietin as a standard-of-care anemia treatment. Subjects were excluded if they had any recent trauma or surgery (<7 days), active bleeding or a known bleeding disorder (for example, von Willebrand disease, hemophilia), active thrombosis or known thrombotic tendency (for example, antithrombin III, protein C, or protein S deficiency), cirrhosis or other liver abnormality, active cancer, thrombocytopenia (platelets <100 × 109/l), or concurrent use of fish oil or antiplatelet or antithrombotic medications. The VCU Institutional Review Board approved the study before subject enrollment, and the study itself was conducted in accordance with the Declaration of Helsinki. All subjects provided written informed consent before study commencement. Upon enrollment of subjects into the study, demographics, laboratory chemistry parameters, and coagulation parameters were recorded. Blood (approximately 25 ml) was collected through a 15-gauge needle into a syringe: 5 ml was injected into each of four 3.2% sodium citrate tubes, and 5 ml was injected into a serum separator tube. In HD patients, the blood samples were drawn immediately before dialysis to avoid interference with heparin administration. Sodium citrate tubes were treated with 180 μl heparinase before sample processing to avoid potential heparin contamination. All blood samples were assayed within 2 h of collection for coagulation parameters. 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