Pituitary adenylate cyclase-activating polypeptide (PACAP) in zebrafish models of nephrotic syndrome

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.

Nephrotic syndrome‐associated hypercoagulopathy is alleviated by both pioglitazone and glucocorticoid which target two different nuclear receptors

“…Current evidence suggests that platelets may play a role in VTE pathogenesis and it is well-known that NS may be associated with thrombocytosis (Kerlin et al, 2012a;Wolberg et al, 2015). Meanwhile there are conflicting data regarding the effects of NS on platelet function, which has previously been reported to be both enhanced and impaired during NS (Eneman et al, 2017;Eneman, Levtchenko, van den Heuvel, Van Geet, & Freson, 2016;Svetlov, Moskaleva, Pinelis, Daikhin, & Serebruany, 1999). Our plasma hypercoagulopathy data omit potential contributions from the platelet compartment, although our previously reported whole blood rotational thromboelastometry data correlate well with ETP, suggesting that methods incorporating cellular blood components do not enhance discrimination of hypercoagulopathy (Kerlin, Waller, et al, 2015).…”

Expression Patterns of PACAP and PAC1R Genes and Anorexigenic Action of PACAP1 and PACAP2 in Zebrafish

“…Various pathological condition models have been developed for zebrafish. It has been reported that administration of PACAP could inhibit adriamycin-induced kidney damage and hydrogen peroxide-induced damage of sensory hair cells in zebrafish (34, 35). The results of the distribution of PACAP and PACAP receptors in the zebrafish obtained in this study are also important knowledge for the pathophysiological study of PACAP in zebrafish.…”

Protective Effects of PACAP in Peripheral Organs

“…PACAP suppression experiments in zebrafish of Eneman et al revealed that nephrin depletion, a model of nephrotic syndrome is associated with adcyap1a and vip downregulation (76). Using adcyap1a and adcyap1b morpholinos the authors decribed more severe sequelae of nephrin depletion.…”

“…Furthermore, nephrotic fishes showed reduced protein expression of PACAP. PACAP morpholinos worsened the change in phenotype induced by adriamycin exposure, which could be attenuated by addition of human PACAP38 (76).…”