NGFN-TRANSFER
Activation of neutrophil granulocytes through small plasma proteins of uremic patients
| Coordinator: | Dr. Horst-Dieter Lemke | |
| Institution: | EXcorLab GmbH, R&D, Industriecenter Obernburg, D-63784 Obernburg | |
| Homepage: | www.excorlab.de |
Hemodialysis is the gold standard for the treatment of state 5 kidney disease patients. Removal of uremic toxins by hemodialysis is rather non-specific and based on size-exclusion by membranes. Albumin (Mr: 69 kD) is believed to be the critical molecule which should not be removed by dialysis. Which of the more than 100 know uremic toxins are in particular involved in the pathogenesis of uremia is currently unknown.
In this subproject we wanted to purify small plasma proteins, e.g. β2-microglobulin (Mr=11.8 kD), myoglobin (Mr=18 kD) and retinol binding protein (Mr=23 kD) from hemofiltrates of uremic individuals (class 5), to test if these proteins activate neutrophils in vitro and to examine if these proteins contribute to the inflammatory state and vascular damage in these patients.
Plasma proteins were purified by ion exchange and gel chromatography. Up-regulation of adhesion receptors and oxidative burst activity of neutrophils was analysed by flow-cytometry. Release of myeloperoxidase (MPO), elastase und interleukin-1β (IL-1β) (from monocytes) was measured by ELISA. Up-regulation of the receptor VCAM on human primary endothelial cells (EC) by flow-cytometry and release of IL-8 (ELISA) were considered as parameters of EC activation. Cytotoxicity of the proteins isolated on EC and smooth muscle cells (SMC) was studied by a fluorescent viability dye.
The isolated proteins did not up-regulate adhesion receptors on neutrophils and EC. Oxidative burst activity increased slightly after incubation with cystatin c from hemofiltrate. Release of MPO and elastase reflected exactly the results of the oxidative burst assay. Plasma proteins from uremic patients led to a modest proliferation of EC and SMC.
All plasma proteins from hemofiltrate examined showed in all biological assays low activity, which means low toxicity. Therefore, these proteins are not a promising target for improved, more specific extracorporeal elimination.
In this subproject we wanted to purify small plasma proteins, e.g. β2-microglobulin (Mr=11.8 kD), myoglobin (Mr=18 kD) and retinol binding protein (Mr=23 kD) from hemofiltrates of uremic individuals (class 5), to test if these proteins activate neutrophils in vitro and to examine if these proteins contribute to the inflammatory state and vascular damage in these patients.
Plasma proteins were purified by ion exchange and gel chromatography. Up-regulation of adhesion receptors and oxidative burst activity of neutrophils was analysed by flow-cytometry. Release of myeloperoxidase (MPO), elastase und interleukin-1β (IL-1β) (from monocytes) was measured by ELISA. Up-regulation of the receptor VCAM on human primary endothelial cells (EC) by flow-cytometry and release of IL-8 (ELISA) were considered as parameters of EC activation. Cytotoxicity of the proteins isolated on EC and smooth muscle cells (SMC) was studied by a fluorescent viability dye.
The isolated proteins did not up-regulate adhesion receptors on neutrophils and EC. Oxidative burst activity increased slightly after incubation with cystatin c from hemofiltrate. Release of MPO and elastase reflected exactly the results of the oxidative burst assay. Plasma proteins from uremic patients led to a modest proliferation of EC and SMC.
All plasma proteins from hemofiltrate examined showed in all biological assays low activity, which means low toxicity. Therefore, these proteins are not a promising target for improved, more specific extracorporeal elimination.
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