The role of PACAP in nephrotic syndrome.

An elevated risk for thromboembolic disease, including both deep venous and arterial thrombosis, leads to an increase in morbidity and mortality rates in nephrotic syndrome (NS) patients. Platelet alterations have repeatedly been observed in patients with NS. However, the mechanisms underlying these platelet alterations in NS remained unclear. Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently identified as an inhibitor of megakaryopoiesis and platelet aggregation, and inhibition of PACAP or its receptors stimulated platelet production and function. In this Ph.D. thesis, the possible role of plasma PACAP deficiency in increased platelet count and hyperreactivity in NS was studied. First, a small-scale study was performed in 4 patients with Finnish-type congenital NS (CNS) due to NPHS1 gene mutations. Our findings were then validated in a prospective, multicenter study, investigating a larger group of 24 patients with steroid-sensitive idiopathic NS (INS). We observed plasma PACAP deficiency due to excessive urinary excretion during the nephrotic state in both CNS and INS patients. Plasma PACAP deficiency was more pronounced in CNS patients. During the PACAP-deficient state, in vitro megakaryopoiesis was increased for CNS patients and this effect could be reversed by the addition of recombinant PACAP. Thrombocytosis was observed in all CNS and in some cases with INS during nephrotic state. We further found that NF-κB, a downstream target of PACAP, was reduced in platelet lysates of CNS patients. Platelet hyperaggregability was observed during nephrotic state in both CNS and INS. In INS patients, the addition of recombinant PACAP to patients’ platelets was investigated and resulted in decreased aggregation during nephrotic state. Platelet aggregation correlated inversely with plasma PACAP levels, but not with serum albumin levels. Our observations highly support a role for PACAP deficiency in upregulated megakaryopoiesis and increased platelet activation in NS. In order to further elucidate the role of PACAP deficiency in platelet alterations in NS, two zebrafish CNS models were studied: a nephrin depleted and an adriamycin exposed model, generated in Tg(cd41:EGFP) transgenic zebrafish. PACAP deficiency was confirmed in the adriamycin model, but not in the nephrin depleted fish embryos. Different from our findings in human CNS patients, we did not observe increased thrombocytes in those models. We concluded that none of both CNS zebrafish models were suitable for investigating the role of PACAP and ceruloplasmin deficiency in megakaryopoiesis in CNS. Furthermore, while a nephroprotective effect of PACAP has been demonstrated in a variety of renal disease models, little was known about the expression of PACAP and its receptors in the kidney. Moreover, no data were available about the role of PACAP in proteinuria related renal damage. In this Ph.D. thesis the expression of PACAP and its receptors was investigated in the healthy and nephrotic kidney, as well as the role of PACAP in proteinuria related proximal tubular damage in vitro. Both in healthy and nephrotic renal tissue, VPAC1 expression was detected in the proximal tubular epithelial cells and in the glomeruli. Furthermore, weak expression of PACAP was found in the proximal tubules of healthy renal tissue, while a much stronger signal was observed in the renal sections of children with NS, probably due to reuptake of filtered PACAP. We further demonstrated expression of a functional VPAC1 receptor in human proximal tubular epithelial HK-2 cells. Expression of VPAC2 was absent and PAC1 was very low in HK-2 cells, suggesting that VPAC1 is the main active PACAP receptor in HK-2 cells. We further investigated the effect of PACAP on albumin exposed HK-2 cells, mimicking proteinuria related cell damage. The addition of recombinant PACAP did not influence decreased cell viability or increased TGF-β1 expression due to albumin exposure.status: publishe

An incidental X-ray finding in a toddler

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Platelet abnormalities in nephrotic syndrome

Nephrotic syndrome (NS) is a common kidney disease associated with a significantly increased risk of thrombotic events. Alterations in plasma levels of pro- and anti-coagulant factors are involved in the pathophysiology of venous thrombosis in NS. However, 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 platelet hyperactivity have been observed in nephrotic children. Platelet hyperaggregability, increased release of active substances, and elevated surface expression of activation-dependent platelet markers have been documented. The mechanisms underlying those platelet alterations are multifactorial and are probably due to changes in plasma levels of platelet-interfering proteins and lipid changes, as a consequence of nephrosis. The causal relationship between platelet alterations seen in NS and the occurrence of thromboembolic phenomena remains unclear. Moreover, the efficiency of prophylactic treatment using antiplatelet agents for the prevention of thrombotic complications in nephrotic patients is also unknown. Thus, antiplatelet medication is currently not generally recommended for routine prophylactic therapy.status: publishe

Distribution and Function of PACAP and Its Receptors in the Healthy and Nephrotic Kidney

Plasma deficiency of pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated in children with nephrotic syndrome (NS). Previous studies have reported an important protective effect of PACAP on kidney proximal tubules. The aim of this study was to explore the expression of PACAP and its receptors PAC1, VPAC1 and VPAC2 in the healthy and nephrotic kidney and to determine if PACAP has an effect on renal proximal tubular cells exposed to albumin.status: publishe

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

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.status: publishe

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

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

Pituitary adenylate cyclase-activating polypeptide deficiency associated with increased platelet count and aggregability in nephrotic syndrome

The pituitary adenylate cyclase-activating polypeptide (PACAP) was recently identified as an inhibitor of megakaryopoiesis and platelet aggregability.status: publishe

Phenotype analysis, nephrin expression and evaluation of glomerular function in adriamycin exposed zebrafish.

<p>(A) Different categories of phenotype were defined in the adriamycin exposed embryos at 4 dpf: embryos with a normal phenotype, embryos with visible pericardial edema (white arrow) and dysmorphic or dead embryos. (B) 100 embryos per condition were live-screened at 4 dpf and assigned to a phenotype category. Increasing adriamycin concentrations in the culture medium were associated with an increasing percentage of embryos with pericardial edema. (C) qPCR was performed using total RNA from control and adriamycin exposed embryos at 4 dpf. A significantly decreased expression of nephrin (corrected for housekeeping gene <i>elfa</i>) was observed in the adriamycin exposed embryos compared to the control fish. The experiment was performed twice in triplicate. Bars represent means ± SD. * P<0.05 in comparison to condition without the addition of adriamycin. (D) Rhodamine-labeled 70 kDa dextran was injected in the cardiac venous sinus of 75 hpf old embryos. LEFT: A representative immunofluorescence picture of a control embryo immediately after injection shows the distribution of fluorescence through the vascular system of the zebrafish larva. A dose-dependent diminishing effect of adriamycin on fluorescence recorded in the fish eye 5 hours after injection was observed. Representative images of the eye from 0, 10 and 30 μM adriamycin treated embryos 5 hours after injection are shown. RIGHT: A diagrammatic representation shows the quantification of the mean fluorescence intensity ± SD recorded in the retinal vascular bed. * P < 0.05 in comparison to condition without the addition of adriamycin.</p