Levin, Michael; (1991) The role of cationic proteins and membrane charge in the steroid responsive nephrotic syndrome. Doctoral thesis (Ph.D), University of London, Institute of Child Health.

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Abstract

Over the past two decades evidence has accumulated which suggests that the basic defect responsible for the proteinuria of the Steroid Responsive Nephrotic Syndrome (SRNS) is a loss or reduction of the fixed negative charge present on all layers of the glomerular capillary wall. This thesis describes a program of work which was undertaken to confirm the presence of abnormal charge on cells and membranes in SRNS, and to elucidate the mechanisms responsible. In initial studies, platelets from children with SRNS were found to aggregate spontaneously in vitro. The pattern of aggregation was similar to that induced by neutralisation of platelet surface negative charge by polycations such as poly-lysine. This suggested that the abnormal platelet aggregation in SRNS might be due to a reduction of platelet negative charge, and that the same process responsible for reducing the negative charge on the glomerulus might also be responsible for reducing the platelet negative charge. As blood cells are more accessible for study than renal tissue, it seemed possible that they might be a useful model with which to study the mechanism responsible for reducing cell and membrane surface charge in SRNS. A novel method was developed to measure surface negative charge on red blood cells and platelets based on the binding of the cationic dye, Alcian blue 8 GX, to negatively charged groups on cell surfaces. The method was shown to be specific for charged groups on the cell surface, as the Alcian blue binding could be competitively inhibited by polycations and largely abolished following desialation of the cells by treatment with neuraminidase. Normal ranges for red blood cell and platelet surface charge were established in healthy individuals ranging from infancy to old age. The method was then used to compare red blood cell and platelet surface charge in healthy children, those with SRNS and those with other renal 'diseases. Surface negative charge on red blood cells and platelets from children with SRNS was found to be significantly reduced compared to healthy children and those with other renal diseases. However, the sialic acid content of the red blood cell membrane was normal, suggesting that the charged groups were masked or neutralized rather than quantitatively deficient. The following hypothesis was proposed to explain these findings: The reduction of glomerular capillary wall negative charge which is believed to underlie the proteinuria of SRNS , and the reduction of platelet and red blood cell negative charge might be due to the presence of a substance which binds to and reduces the negative charge on cells and membrane.s In order to confirm the existence of the proposed charge neutralizing substance, a method for detection of charge neutralizing substances was devised based on inhibition of the binding of the cationic dye Alcian blue to the anionic glycosaminoglycan (GAG) heparin. The method was shown to detect highly cationic proteins such as protamine and poly-1- lysine but not less highly charged proteins or heparin binding proteins such as lysozyme, beta thromboglobulin, or platelet Factor IV. Using this method, cationic fractions of plasma and urine from SRNS children prepared by DEAE Sephacel ion exchange chromatography were found to contain a charge neutralizing factor which was not detected in similar fractions of normal plasma and urine. Fractionation of SRNS or normal plasma and urine by heparin sepharose chromatography confirmed the presence of a charge neutralizing factor in SRNS but not in normal plasma or urine. Proteolytic digestion of the fractions containing the charge neutralizing factor abolished its activity, establishing that it is a protein. A procedure was devised to partially purify the charge neutralizing protein from SRNS plasma or urine. After initial dialysis and (for urine) concentration by ultrafiltration and lyophilization, SRNS urine or plasma was batch absorbed at pH 9 to QAE Sephadex. The active charge neutralizing factor appeared in the unabsorbed fraction and was then processed on a QAE Sephadex column, again eluting in the unbound fraction. The activity was further purified by gel filtration on Sephadex G-150 and finally by fast pressurized liquid chromatography (FPLC) on the cation binding medium Mono-S. from which it eluted as a single peak. The partially purified charge neutralizing protein was injected into rabbits and an antibody prepared which inhibited the activity of the protein. Although further studies are required to confirm a pathogenic role for this highly cationic protein in SRNS, it is likely that this protein would bind to and neutralize the charge of the glomerular capillary wall, and thus be responsible for the abnormal charge selectivity of SRNS. In view of the precipitation of relapses of SRNS by infection and allergic stimuli and the beneficial effects of immunosuppressive treatment, a likely origin for the protein is some cell of the immune system. The basic defect in SRNS may be uncontrolled or excessive release of a highly cationic protein from some immunologically active cell.

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