Ankyrin is the major oxidised protein in erythrocyte membranes from end-stage renal disease patients on chronic haemodialysis and oxidation is decreased by dialysis and vitamin C supplementation

Abstract

Chronically haemodialysed end-stage renal disease patients are at high risk of morbidity arising from complications of dialysis, the underlying pathology that has led to renal disease and the complex pathology of chronic kidney disease. Anaemia is commonplace and its origins are multifactorial, involving reduced renal erythropoietin production, accumulation of uremic toxins and an increase in erythrocyte fragility.

Oxidative damage is a common risk factor in renal disease and its co-morbidities and is known to cause erythrocyte fragility. Therefore, we have investigated the hypothesis that specific erythrocyte membrane proteins are more oxidised in end-stage renal disease patients and that vitamin C supplementation can ameliorate membrane protein oxidation.

Eleven patients and 15 control subjects were recruited to the study. Patients were supplemented with 2 × 500 mg vitamin C per day for 4 weeks. Erythrocyte membrane proteins were prepared pre- and post-vitamin C supplementation for determination of protein oxidation. Total protein carbonyls were reduced by vitamin C supplementation but not by dialysis when investigated by enzyme linked immunosorbent assay. Using a western blot to detect oxidised proteins, one protein band, later identified as containing ankyrin, was found to be oxidised in patients but not controls and was reduced significantly by 60% in all patients after dialysis and by 20% after vitamin C treatment pre-dialysis. Ankyrin oxidation analysis may be useful in a stratified medicines approach as a possible marker to identify requirements for intervention in dialysis patients.

Keywords::

• protein oxidation
• carbonyl
• red blood cells

Author contribution

TR designed the intervention study in collaboration with SD and NK. TR isolated membrane proteins. HRG designed the analysis of membrane protein oxidation study. TR, SJB and CRB analysed membrane protein oxidation. SJB prepared samples for mass spectrometry and CRB performed immunoprecipitation.

Acknowledgments

Authors gratefully acknowledge the cooperation and support of this study of Prof. Sasa Mitrev, as well as the cooperation of the staff of the Department of Haemodialysis and Laboratory for Biochemistry at Clinical Hospital in Stip. Maja Jancovska, Galaba Naumova and Viktorija Maksimova helped with the preparation of the samples and recruitment of the healthy volunteers, which are highly appreciated. The Orbitrap Velos mass spectrometer used in this research was obtained through the Birmingham Science City Translational Medicine: Experimental Medicine Network of Excellence project, with support from Advantage West Midlands (AWM).

Funding

This study was supported by the COST CM1001 Action, Grants No. a) COST-STSM-CM1001-250512-017498 and b) COST-STSM-CM1001-12598.

The authors declare no conflict of interests.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.