Smoyer, W. E. | Search bioRxiv

bioRxiv2023

Prothrombin Knockdown Protects Podocytes and Reduces Proteinuria in Glomerular Disease

Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.

physiology↗

bioRxiv2020

Nephrotic Syndrome-Associated Hypercoagulopathy is Alleviated by Nuclear Receptor Agonist Therapy with both Pioglitazone and Glucocorticoids

BackgroundThrombosis 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. MethodsPuromycin aminonucleoside-induced rat NS was treated with sham, Low- or 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. ResultsIn a rat model of NS, both High-MP and Pio improved proteinuria and corrected hypoalbuminemia, ETP and AT activity (P<0.05). Proteinuria (P=0.005) and hypoalbuminemia (P<0.001) 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<0.001) but not SRNS (P=0.330). ConclusionsBoth 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. SIGNIFICANCE STATEMENTNephrotic syndrome (NS) is characterized by massive proteinuria and is complicated by a complex, acquired hypercoagulopathy that markedly increases the risk for potentially life-threatening venous thromboembolism (VTE). This study demonstrates a strong correlation between proteinuria reduction and improvement of an established VTE-risk biomarker, in both a well-established animal model and in childhood NS before and after steroid treatment. We show that nuclear receptor agonists with known disparate mechanisms of action successfully reduce proteinuria and simultaneously improve NS-associated hypercoagulopathy. These data suggest that complete or partial proteinuria reduction by any therapeutic modality may concurrently reduce NS-associated thrombotic risk.

pathology↗