Failure of regulation results in an amplified oxidation burst by neutrophils in children with primary nephrotic syndrome - PubMed (original) (raw)

Comparative Study

Failure of regulation results in an amplified oxidation burst by neutrophils in children with primary nephrotic syndrome

R Bertelli et al. Clin Exp Immunol. 2010.

Abstract

The mechanism responsible for proteinuria in non-genetic idiopathic nephrotic syndrome (iNS) is unknown. Animal models suggest an effect of free radicals on podocytes, and indirect evidence in humans confirm this implication. We determined the oxidative burst by blood CD15+ polymorphonucleates (PMN) utilizing the 5-(and-6)-carboxy-2',7'-dichlorofluorescin diacetate (DCF-DA) fluorescence assay in 38 children with iNS. Results were compared with PMN from normal subjects and patients with renal pathologies considered traditionally to be models of oxidative stress [six anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis, seven post-infectious glomerulonephritis]. Radicals of oxygen (ROS) production was finally determined in a patient with immunodeficiency, polyendocrinopathy, enteropathy X-linked (IPEX) and in seven iNS children after treatment with Rituximab. Results demonstrated a 10-fold increase of ROS production by resting PMN in iNS compared to normal PMN. When PMN were separated from other cells, ROS increased significantly in all conditions while a near-normal production was restored by adding autologous cells and/or supernatants in controls, vasculitis and post-infectious glomerulonephritis but not in iNS. Results indicated that the oxidative burst was regulated by soluble factors and that this regulatory circuit was altered in iNS. PMN obtained from a child with IPEX produced 100 times more ROS during exacerbation of clinical symptoms and restored to a near normal-level in remission. Rituximab decreased ROS production by 60%. In conclusion, our study shows that oxidant production is increased in iNS for an imbalance between PMN and other blood cells. Regulatory T cells (Tregs) and CD20 are probably involved in this regulation. Overall, our observations reinforce the concept that oxidants deriving from PMN are implicated in iNS.

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Figures

Fig. 1

Fig. 1

Radicals of oxygen (ROS) production by resting polymorphonucleates (PMN). 5-(and-6)-carboxy-2′,7′-dichlorofluorescin diacetate (DCF-DA) fluorescence assay was performed with whole blood in which PMN (CD15+ cells) were analysed as separated gates by fluorescence activated cell sorter (FACS). Thirty-one normal subjects and 38 children with idiopathic nephrotic syndrome (iNS) were considered in this first set of experiments. Patients were evaluated in different clinical phases as defined by their urinary protein/urinary creatinine ratio (Pu/Cu) (remission Pu/Cu < 0·5; relapse Pu/Cu > 4; intermediate Pu/Cu 0·5–4). Six patients with renal vasculitis and five with other glomerulonephritis were evaluated in the same experimental conditions. Statistical analysis performed with parametric tests (one-way analysis of variance) showed significant difference in ROS production *P < 0·05; **P < 0·01.

Fig. 2

Fig. 2

Radicals of oxygen (ROS) production by polymorphonucleates (PMN) isolated from other cells. (a) 5-(and-6)-carboxy-2′,7′-dichlorofluorescin diacetate (DCF-DA) fluorescence assay performed utilizing whole blood as in Fig. 1. In other experiments, PMN (CD15+ cells) were first isolated by dextran sedimentation in various experimental conditions: comparison of PMN obtained from normal donors and from children with active idiopathic nephrotic syndrome (iNS); (b) comparison of isolated PMN obtained from normal subjects and from patients with iNS. DCF-DA analysis was performed with isolated PMN alone and/or in presence of autologous–heterologous cells and supernatants deriving from CD3+, CD11+ and CD20+ cells; (c) patients with vasculitis and post-streptococcal glomerulonephritis. Statistical analysis performed with parametric tests (one-way analysis of variance) showed significant difference in ROS production *P < 0·01.

Fig. 3

Fig. 3

Radicals of oxygen (ROS) production by polymorphonucleates (PMN) in an immunodeficiency, polyendocrinopathy, enteropathy X-linked (IPEX) child. 5-(and-6)-carboxy-2′,7′-dichlorofluorescin diacetate (DCF-DA) fluorescence assay performed with whole blood containing non-isolated PMN (CD15+ cells) in a child with immunodeficiency, polyendocrinopathy, enteropathy X-linked (IPEX) and his normal mother, carrier of the same mutation (p.Leu242Pro) of forkhead box P3 (FoxP3).

Fig. 4

Fig. 4

Radicals of oxygen (ROS) production after Rituximab. 5-(and-6)-carboxy-2′,7′-dichlorofluorescin diacetate (DCF-DA) fluorescence assay performed with isolated polymorphonucleates (PMN) (CD15+ cells) in patients with idiopathic nephrotic syndrome (iNS) after an in vivo treatment with anti-CD20 antibodies (Rituximab). Isolated PMN were studied alone and in the presence of autologous–heterologous cells and supernatants deriving from CD3+, CD11+ and CD20+ cells.

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