Disease Severity Correlates with Thrombotic Capacity in Experimental Nephrotic Syndrome
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BASIC RESEARCH www.jasn.org Disease Severity Correlates with Thrombotic Capacity in Experimental Nephrotic Syndrome † † Bryce A. Kerlin,* Amanda P. Waller,* Ruchika Sharma,* Melinda A. Chanley,* ‡ Marvin T. Nieman, and William E. Smoyer*§ *Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s, Columbus, Ohio; Divisions of †Hematology/Oncology/Blood & Marrow Transplantation and §Nephrology, Department of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio; and ‡Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio ABSTRACT Thrombotic disease, a major life–threatening complication of nephrotic syndrome, has been associated with proteinuria and hypoalbuminemia severity. However, it is not fully understood how disease severity correlates with severity of the acquired hypercoagulopathy of nephrotic syndrome. Without this knowl- edge, the utility of proteinuria and/or hypoalbuminemia as biomarkers of thrombotic risk remains limited. Here, we show that two well established ex vivo hypercoagulopathy assays, thrombin generation and rotational thromboelastometry, are highly correlated with proteinuria and hypoalbuminemia in the puro- mycin aminonucleoside and adriamycin rat models of nephrotic syndrome. Notably, in the puromycin aminonucleoside model, hyperfibrinogenemia and antithrombin deficiency were also correlated with pro- teinuria severity, consistent with reports in human nephrotic syndrome. Importantly, although coagulation was not spontaneously activated in vivo with increasing proteinuria, vascular injury induced a more robust thrombotic response in nephrotic animals. In conclusion, hypercoagulopathy is highly correlated with nephrotic disease severity, but overt thrombosis may require an initiating insult, such as vascular injury. Our results suggest that proteinuria and/or hypoalbuminemia could be developed as clinically meaningful surrogate biomarkers of hypercoagulopathy to identify patients with nephrotic syndrome at highest risk for thrombotic disease and potentially target them for anticoagulant pharmacoprophylaxis. J Am Soc Nephrol 26: 3009–3019, 2015. doi: 10.1681/ASN.2014111097 Nephrotic syndrome (NS), one of the most com- dynamics of coagulation system activation are not mon forms of glomerular disease, is characterized well understood. by massive proteinuria, hypoalbuminemia, edema, Thromboembolic disease is a major life–threatening and hyperlipidemia.1,2 As recently reviewed, it is well complication of NS, occurring in an estimated known that nephrotic-range proteinuria is associated 26.7% of adults and 2.9% of children with NS.3,7 with an acquired complex hypercoagulopathy.3–6 In fact, compared with other kidney diseases, NS The hypercoagulable state is associated with ac- carries the highest risk of thrombosis, many epi- quired deficiencies of key hemostatic regulatory sodes of which may be preventable.8,9 The acquired elements, such as antithrombin (AT) and free Pro- tein S, and elevated levels of high molecular weight procoagulant factors (including factors V and Received November 12, 2014. Accepted February 8, 2015. VIII,vWf,andfibrinogen). The predicted overall Published online ahead of print. Publication date available at effect of these coagulation derangements is an en- www.jasn.org. hanced capacity for thrombin generation (conver- Correspondence: Dr.BryceA.Kerlin,TheResearchInstituteat sion of prothrombin to thrombin), which is the Nationwide Children’s, 700 Children’s Drive, W325, Columbus, key effector enzyme of the hemostatic system.3–5 OH 43205. Email: [email protected] However, the effects of these derangements on the Copyright © 2015 by the American Society of Nephrology J Am Soc Nephrol 26: 3009–3019, 2015 ISSN : 1046-6673/2612-3009 3009 BASIC RESEARCH www.jasn.org blood coagulation derangements are believed to be a major divided into quartiles (first interquartile range [IQR] =1.37– contributor to NS–associated thrombotic risk. Although 4.20; second IQR =6.65–44.17; third IQR =56.70–98.32; fourth thrombosis could potentially be prevented with prophylactic IQR =99.54–116.60 mg/mg protein-to-creatinine), and saline- anticoagulation, this approach remains controversial be- injected rats were labeled as controls for the remaining analy- cause of the treatment-associated risk of bleeding compli- ses. Thus, peak thrombin (Figure 1E) and ETP (Figure 1F) were cations.10–12 Recent epidemiologic studies have revealed that significantly greater in the third and fourth quartiles of pro- both proteinuria and serum albumin may be associated with teinuria severity compared with both those in the first quartile the likelihood of thrombosis, with higher proteinuria and of proteinuria (P,0.05) and control rats (P,0.05). Hence, lower serum albumin correlating with increased thrombotic these data indicate that maximal enzymatic activity of thrombin risk, suggesting that disease severity may be directly correlated (peak thrombin) is weakly but significantly correlated with pro- with the severity of coagulation system derangement.13–18 teinuria, whereas ETP, which indicates total thrombin exposure Thus, determining the relationship between hypercoagulopathy over time, is more strongly correlated. and these markers of disease severity may lead to the identifica- tion of clinically meaningful urine protein and/or serum albumin PAN-Nephrotic Rats Exhibit Differential Derangements levels beyond which prophylaxis with anticoagulants may be in Whole–Blood Rotational Thromboelastometry warranted. Parameters That Correlate with Proteinuria Severity Importantly, derangements of individual coagulation sys- Tofurtherassessthedynamicsofnephroticcoagulationresponses tem components similar to those found in human NS have in the presence of all blood components, whole–blood rotational been previously shown in both the puromycin aminonucleo- thromboelastometry (ROTEM) was assessed.30 This assay is side (PAN) and adriamycin (ADR) –induced rat nephrosis used to assess the viscoelastic properties of blood clot formation models.19–21 However, these investigations have not previ- over time in whole blood, thus including the effects of cellular ously addressed the critically important collective effects of blood components on coagulation. Similarly to TGA, ROTEM these subcomponent variations on overall hemostatic poten- has been shown to correlate with hypercoagulability and throm- tial and their effects on in vivo thrombus formation. The aim botic risk.31–36 Correspondingly, PAN-nephrotic rats also ex- of this study was, thus, to determine the relationship between hibited differential proteinuria–dependent hypercoagulopathy NS severity and the dynamics of coagulation system activa- derangements in ROTEM parameters (Figure 2A). Clot forma- tion. We hypothesized that both ex vivo and in vivo coagulation tion time (CFT) (Figure 2B) was significantly decreased, whereas activation would be enhanced in the setting of nephrosis. a-angle (Figure 2C), amplitude at 10 (A10) (Figure 2D) and 20 min (A20) (Figure 2E), maximum clot firmness (MCF) (Figure 2F), and residual clot firmness after urokinase-induced fibrino- RESULTS lysis at 60 min (lysis index at 60 min; LI60) (Figure 2G) were all significantly increased in the rats exhibiting the highest levels of Proteinuria Severity in the PAN–Induced Rat Nephrosis proteinuria (third and fourth quartiles; P,0.05 for each param- Model Is Dose Dependent and Directly Proportional to eter). Furthermore, significant linear correlations were found Thrombin Generation between proteinuria and all measured ROTEM parameters (Fig- We first established that proteinuria is dose dependent in the ure 2, B–G, regression formulas, Table 1). Specifically, the extent PAN–induced rat nephrosis model. As expected, at 9 days of proteinuria was significantly negatively correlated with CFT post-PAN injection, rats displayed a dose-dependent escala- (R2=0.57; P,0.001) and positively correlated with the remain- tion of proteinuria (Figure 1A).21–23 We then assessed throm- ing parameters (a-angle, A10, A20, MCF, and LI60 at R2=0.65, bin generation in the platelet-poor plasma (PPP) collected R2=0.71, R2=0.57, R2=0.60, and R2=0.74, respectively; P,0.001 from these rats using a thrombin generation assay (TGA). for each parameter), indicating that the onset of clot formation TGA is a well established method used to assess the overall occurs earlier (CFT), proceeds more rapidly (a-angle), results in capacity of the coagulation cascade to convert prothrombin to higher viscoelastic strength (A10, A20, and MCF), and is more thrombin.24–29 Two TGA parameters, peak thrombin and the resistant to fibrinolysis (LI60) in a manner that is directly pro- area under the TGA curve (also known as endogenous throm- portional to proteinuria severity. bin potential [ETP]), are known to correlate with the likeli- hood of thrombotic disease. TGA displayed an escalating Hypercoagulability Is Inversely Proportional to Plasma dose–dependent effect in PAN-treated rats (Figure 1B). Pro- Albumin in the PAN–Induced Rat Nephrosis Model teinuria severity directly correlated with these TGA parame- As expected, plasma albumin decreased with increasing PAN ters, such that a significant positive linear correlation was doses (Figure 1A), and thus, plasma albumin both decreased found between proteinuria level and both peak thrombin successively with each quartile of increasing proteinuria sever- (R2=0.29; P,0.002) (Figure 1C) and ETP (R2=0.87; P,0.001) ity (Figure 3A) and exhibited