Isolating and characterization of polymorphonuclear leukocyte (PMNL) modulating factors from patients with renal failure

The high risk of bacterial infections is one of the mayor factors causing the increased incidence of morbidity and mortality among patients with chronic renal failure (CFR) undergoing hemodialysis (HD) or peritoneal dialysis (PD) treatment. An impairment of the unspecific immune defense system, especially of polymorphonuclear leukocytes (PMNLs) is primary responsible for this situation. PMNLs are cells of the first-line immune defense; furthermore, they are the main immune cells in the peritoneal cavity during bacterial infections. PMNLs from uremic patients show disturbances in their essential functions such as chemotaxis, phagocytosis, and oxidative metabolism. Uremic toxins, circulating plasma factors that accumulate in the uremic sera, play a crucial role in inhibiting PMNL-functions. Recently, a number of PMNL-inhibiting factors from CRF patients has been isolated and characterized, partly by the applicant. PMNLs from patients with acute renal failure (ARF) show disturbed functions as well. Due to their increased activation they contribute to tissue injury. PMNL-activation is further enhanced by superimposed sepsis. The removal of PMNLs from the site of infection is important for the normal resolution of inflammation and the control of inflammatory tissue injury. Apoptosis, a specific mode of self destruction, permits the recognition of intact senescent PMNLs. Evidence for the existence of PMNL-apoptosis modulating factors in patients with renal failure comes from experiments of the applicant. It could be shown that PD effluents as well as ultrafiltrates obtained from CFR patients undergoing HD treatment and from ARF patients are able to inhibit the spontaneous apoptosis of PMNLs from healthy donors. Furthermore, we demonstrated that factors previously shown to inhibit essential PMNL-functions (free immunoglobulin light chains) inhibit PMNL apoptosis in a concentration dependent manner. The main goal of this study is to isolate and characterize further PMNL-modulating protein factors from patients with renal failure. For the first set of experiments, we plan to use the following starting material: a) peritoneal effluent from a PD patient; b) ultrafiltrate from a CRF patient undergoing HD; c) ultrafiltrate from an AFR patient without sepsis; d) ultrafiltrate from an AFR patient with sepsis. The following separation methods will be used: 1) ion exchange chromatography; 2) gel filtration; 3) reversed phase chromatography; 4) chromatofocusing. For the first chromatographic separation it is planed to use anion exchange. Different protein peaks that usually still contain a mixture of proteins will be obtained. The biological activity of each peak will be tested. The following functional assays will be used to test the effect of the protein(s) on PMNLs from healthy donors: chemotaxis, hexose uptake, oxidative metabolism, phagocytosis and intracellular killing, intracellular calcium, and the modulation of spontaneous apoptosis (investigating characteristic DNA strand breaks and changes in morphology, membrane symmetry and the activity of proteases). The active peaks will be characterized on a molecular level: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) will determine the molecular weight of the proteins in the different peaks. Isoelectric focusing (IEF) will determine the pi of the proteins in the peak. Based on the results obtained it will be decided individually which separation method will be used next for each set of proteins. By applying the proteins from active peaks to further chromatographic columns, a set of purified proteins will be obtained. Each protein will modulate one or more essential PMNL functions and/or apoptosis and will be further characterized on a molecular level. To test if the results obtained are representative for the respective group of patients, a total of six isolations per group will be performed. The isolation and detailed characterization of PMNL-modulating factors is important for future diagnosis and therapy. This project will lead to the identification of a number of new protein factors that influence PMNL- functions. This will be the basis of future research: Studies on the mechanisms of the identified factors, developing assays for the determination of the concentration of the factors in the sera of patients with kidney failure, and studies on the selective removal of the identified uremic toxins from the sera of this group of patients.

From patients with kidney failure, we isolated and characterized factors that contribute to their disturbed immune function. The knowledge of these substances the prerequisite for the treatment of those patients. Uremic patients, i.e. patients with an impaired kidney function, have a high risk of bacterial infections and cardiovascular complications and as a consequence an increased incidence of morbidity and mortality. One main reason is the disturbance of essential functions of PMNL, cells of the unspecific immune defense. Uremic toxins are thought to play a major role in inhibiting PMNL functions. They are circulating plasma factors accumulating in the serum of uremic patients mainly as a result of reduced excretion. The purification and characterization of these factors is a prerequisite for future applications in diagnosis and therapy - such as the selective removal of uremic toxins from patients with kidney failure. Therefore, the goal of this project was the identification of new PMNL modulating factors from uremic patients. There are no reports on PMNL modulating factors in patients with acute renal failure that also show a disturbed immune defense. Therefore, part of the project focused on uremic toxins from this group of patients. The importance of the programmed cell death ("apoptosis") of PMNL in the resolution of inflammation and the modulation of PMNL activities became obvious within the last few years. Therefore, we investigated also PMNL apoptosis in this project. From ultrafiltrates obtained during hemodialysis treatment of patients with kidney failure, we purified proteins and tested their effects on PMNL functions. From ultrafiltrates of patients with acute renal failure, we purified and characterized basically three groups of proteins that influence PMNL functions: Free light chains of immunoglobulins, modified forms of ß2-microglobulin and retinol binding protein (RBP). Whereas the effects of free light chains of immunoglobulins and of modified forms of ß2-microglobulin isolated from patients with chronic renal failure on PMNL functions have already been described, there are no reports about the effect of RBP on PMNL. RBP is a protein that transports vitamin A (retinol) from the liver to peripheral organs. We could show that isolated RBP inhibits the directed movement (chemotaxis), the uptake of bacteria (phagocytosis) and the production of reactive oxygen (used to kill microorganisms) of PMNL. Furthermore, RBP increases the rate of the apoptotic cell death of PMNL. Our experiments with further RBP preparations and with commercially available RBP showed that the effects of RBP are partly dependent on its content of vitamin A. This work describes for the first time the influence of RBP on PMNL functions and the purification of a PMNL modulating protein, RBP, from the ultrafiltrate of ARF patients.