Proteinuria Severity Is Directly Correlated with Markers of Hypofibrinolysis in a Podocyte Specific Experimental Model of Nephrotic Syndrome

Introduction

Nephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury or dysfunction), hypoalbuminemia, and edema, and is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications.  However, anticoagulation is associated with a substantial risk for adverse bleeding events.  Recently published epidemiology studies suggest that proteinuria severity is directly correlated with thrombotic risk. However, further validation of this candidate biomarker for thrombotic risk requires appropriate validation and adequate pathophysiologic explanation. We have recently demonstrated that proteinuria severity is directly proportional to hypercoagulability (as assessed by ex vivo and in vivo markers of thrombotic capacity), in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats).  Thromboelastometry studies also suggested a resistance to fibrinolysis during rat NS.  Thus, the aim of the present study was to further delineate the relationship between proteinuria severity and hypofibrinolysis using a podocyte-specific rat model of NS. We hypothesized that hypofibrinolysis is directly proportional to severity of proteinuria.

Methods

Using a transgenic rat which expresses the human diphtheria toxin receptor (hDTR) on a podocyte specific promoter (podocin), we compared markers of global hemostasis (ROTEM) and fibrinolysis to proteinuria severity. A range of proteinuria severity was induced by a single I.P. injection of diphtheria toxin (0, 25, 50 & 75 ng/kg; n= 7-8/group). On Day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio. Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately analyzed with ROTEM (whole blood; intem) before being spun down to platelet poor plasma (PPP). Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Thrombin and plasmin generation assays are currently being performed.

 Results

There were significant differences (P<0.005) between the highest proteinuria hDTR rat group and controls, in both hypercoagulopathic (clot formation time, maximum clot firmness, and clot size (amplitude at 10 & 20 min)), and hypofibrinolytic (amount of lysis at 60 min; LI60) ROTEM parameters. Importantly, there was a significant negative correlation between proteinuria severity and LI60 (R²=0.362, P=0.02), suggesting that hypofibrinolysis is directly proportional to podocyte injury and therefore disease severity during NS. Preliminary results from the CLA (n=2 control & 2 high proteinuria) also suggest a marked impairment (~50% difference) in plasma clot lysis time in proteinuria rats.

Conclusions

These results demonstrate that proteinuria severity is directly proportional to both hypercoagulability and hypofibrinolytic capacity in a podocyte-specific rodent model of NS, thus confirming our recent findings in two other well-established animal models of NS in a third, more specific, experimental model of glomerular disease. Importantly, these data also strongly suggest a marked impairment in fibrinolysis during NS, which is directly correlated with proteinuria severity.  Therefore it appears that severe proteinuria is associated with both hypercoagulopathic and hypofibrinolytic defects in the coagulation system.  Future studies will delve into the molecular mechanisms involved in these defects.