Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish.
Highlights
Nephrotic syndrome (NS) is a disease of the glomerular filtration barrier, characterized by severe proteinuria, hypoalbuminemia, edema, and hyperlipidemia [1]
We recently reported urinary losses of Pituitary adenylate cyclase-activating polypeptide (PACAP) and ceruloplasmin in NS children, which was more pronounced in children with the Congenital NS (CNS) of the Finnish type than in children with idiopathic NS (INS); platelet hyperaggregability during nephrotic state was present [9]
The first CNS zebrafish model was obtained by injection of Tg(cd41:EGFP) transgenic zebrafish with a splice site morpholino targeting nphs1 gene [21]
Summary
Nephrotic syndrome (NS) is a disease of the glomerular filtration barrier, characterized by severe proteinuria, hypoalbuminemia, edema, and hyperlipidemia [1]. Congenital NS (CNS) is a severe form of NS, manifesting within 3 months of life with profound edema and proteinuria being present intrauterine. NPHS1 mutations and subsequent disruption of the slit diaphragm cause CNS of the Finnish-type, a common and severe form of CNS with extremely pronounced proteinuria. Patients with NS have an increased risk of thromboembolic events, both deep venous and arterial thrombosis [6,7]. Elevated levels of prothrombotic factors such as factor V, factor VIII, von Willebrand factor and fibrinogen, and decreased levels of antithrombotic factors such as antithrombin III, protein C and protein S were described in NS patients and probably underlie the increased risk of deep venous thrombosis [6]. The fact that the risk of both venous and arterial thrombosis is elevated in NS, points to an additional role for blood platelets. Increased platelet counts and hyperaggregability were repeatedly observed in NS patients [8]
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