Background: Thrombosis is a potentially life-threatening nephrotic syndrome (NS) complication. We have previously demonstrated that hypercoagulopathy is proportional to NS severity in rat models and that pioglitazone (Pio) reduces proteinuria both independently and in combination with methylprednisolone (MP), a glucocorticoid (GC). However, the effect of these treatments on NS-associated hypercoagulopathy remains unknown. We thus sought to determine the ability of Pio and GC to alleviate NS-associated hypercoagulopathy. Methods: Puromycin aminonucleoside-induced rat NS was treated with sham, Lowor High-dose MP, Pio, or combination (Pio + Low-MP) and plasma was collected at day 11. Plasma samples were collected from children with steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) upon presentation and after 7 weeks of GC therapy. Plasma endogenous thrombin potential (ETP), antithrombin (AT) activity, and albumin (Alb) were measured using thrombin generation, amidolytic, and colorimetric assays, respectively. Results: In a rat model of NS, both High-MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (p < .05). Proteinuria (p = .005) and hypoalbuminemia (p < .001) were correlated with ETP. In childhood NS, while ETP was not different at presentation, GC therapy improved proteinuria, hypoalbuminemia, and ETP in children with SSNS (p < .001) but not SRNS (p = .330). Conclusions: Both Pio and GC diminish proteinuria and significantly alleviate hypercoagulopathy. Both Pio and MP improved hypercoagulopathy in rats, and successful GC therapy (SSNS) also improved hypercoagulopathy in childhood NS. These data suggest that even a partial reduction in proteinuria may reduce NS-associated thrombotic risk. How to cite this article: Waller AP, Agrawal S, Wolfgang KJ, et al. Nephrotic syndrome-associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors.

Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome.

doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

Introduction Nephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury), hypoalbuminemia, and edema. Importantly, NS is associated with a complex acquired hypercoagulopathy and a high incidence (~25%) of life-threatening thrombotic complications. Both hypercoagulopathy and hypofibrinolysis are described contributors to NS-related VTE risk. However, the mechanisms underlying the latter are poorly understood. We previously showed NS disease severity is directly proportional to both hypercoagulopathy and fibrinolytic resistance There is evidence that fibrin clot structural density contributes to clot stability and has been observed in the presence of both increased plasma thrombin generation and fibrinogen levels, both of which we have previously demonstrated in NS. Thus the aim of the present study was to investigate the mechanistic relationship between fibrin clot structure and fibrinolysis using two rodent models of NS and a cohort of human NS patients. We hypothesized that hypofibrinolysis arises from increased fibrin network density in a manner directly proportional to NS disease severity. Methods Using two well-established rat models of NS, transgenic diphtheria toxin receptor (DTR) and puromycin aminonucleoside (PAN), we compared fibrinolytic markers to disease severity. A range of severity was induced by a single injection of diphtheria toxin (0-75 ng/kg IP) or PAN (0-150 mg/kg IV). On day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio (uPr:Cr). Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor and spun down to platelet poor plasma (PPP). Samples were also collected from a local cohort of pediatric and adult NS patients (n=23), along with the corresponding clinical lab data for each patient. Plasma clot lysis assay (CLA) was performed using urokinase (50 IU) +/- plasminogen (2.4 uM), on clots initiated with high (20 nM) or low (5 nM) thrombin. Clot fibrin network structure was visualized/assessed by laser scanning confocal microscopy using fluorescently-labeled fibrinogen as a tracer. Fibrinolytic markers in plasma were measured by ELISA. Results Hypofibrinolysis: Previous findings of a hypofibrinolytic defect was confirmed with the CLA, such that plasma clot lysis at 60 min was significantly negatively correlated with proteinuria (R2=0.196; P=0.007 & R2=0.214; P=0.010) and significantly positively correlated with hypoalbuminemia (R2=0.310; P<0.001 & R2=0.240; P=0.006), in the DTR & PAN models, respectively. Additionally, plasma clot lysis by CLA was decreased in NS patients with uPrCr ≥2 (n=16) vs. <2 mg/mg (n=7) (96.1 vs 55.2 %, respectively; P=0.041). Similar results were found when the assay was repeated using high or low thrombin concentrations or increased UK (200 IU), with and without the addition of physiologic amounts of plasminogen. When the assay was performed in the absence of UK (0 IU), lysis at 60 min was drastically reduced (~17%) with no difference between groups. Mechanisms of Hypofibrinolysis: Fibrin network density increased with disease severity such that it was positively correlated with proteinuria (P=0.022) and negatively correlated with hypoalbuminemia (P=0.01) in our DTR rat model, with similar results seen in our human samples (Figure). As expected, fibrin network density was negatively correlated with plasma clot lysis (P=0.04), while plasma fibrinogen concentration (P=0.017), and thrombin generation (P=0.047) were positively correlated with fibrin density. There was no correlation with plasma uPA, PAI-1, a2AP, tPA, TAFI, or plasminogen. Conclusions These data suggest that nephrotic plasma forms thrombi with a denser fibrin network that is resistant to fibrinolysis, in a manner that is proportional to disease severity. The significant correlation between thrombin generation and fibrin network density suggest that plasma thrombotic potential may be a key mechanism contributing to the altered clot structure and impaired clot lysis of NS. Current studies are exploring the mechanisms underlying and in vivo significance of fibrinolytic resistance in our rat NS models. Disclosures No relevant conflicts of interest to declare.

Background Nephrotic syndrome is associated with an acquired hypercoagulopathy that is thought to drive its predisposition for venous thromboembolism. Previous studies have suggested that urinary antithrombin (AT) loss leading to acquired AT deficiency is the primary mechanism underlying this hypercoagulopathy, but this hypothesis has not been directly tested. The objectives of this study were to test the influence of AT levels on hypercoagulopathy in nephrotic syndrome patient samples and perform meta-analyses to evaluate the likelihood of AT deficiency in patients with nephrotic syndrome.Methods Samples from three independent nephrotic syndrome cohorts were analyzed. AT antigen and activity assays were performed using ELISA and amidolytic assays, respectively. Plasma thrombin generation, albumin, and urine protein-to-creatinine ratios were determined using established methods. Meta-analyses were performed by combining these new data with previously published data.Results AT levels were not consistently related to either plasma albumin or proteinuria. AT was quantitatively related to hypercoagulopathy in adult nephrotic syndrome, whereas AT activity was inconsistently associated with hypercoagulopathy in childhood nephrotic syndrome. Notably, hypercoagulopathy did not differ between patients with normal AT levels and those with levels below the threshold used to define clinical AT deficiency (,70%). Moreover, ex vivo AT supplementation did not significantly alter hypercoagulopathy in AT-deficient plasma samples. The meta-analyses demonstrated that AT deficiency was not a uniform feature of nephrotic syndrome and was more common in children than adults.Conclusions These data suggest that AT deficiency plays only a limited role in the mechanisms underlying the acquired hypercoagulopathy of nephrotic syndrome. Moreover, AT deficiency was not present in all patients with nephrotic syndrome and was more likely in children than adults despite the higher risk for venous thromboembolism in adults than children.