Eluate derived by extracorporal antibody-based immunoadsorption elevates the cytosolic Ca2+ concentration in podocytes via B2 kinin receptors.
OBJECTIVE
Patients with idiopathic focal segmental glomerulosclerosis (FSGS) often develop a recurrence of the disease after kidney transplantation. In a number of FSGS patients, plasmapheresis and immunoadsorption procedures have been shown to transiently reduce proteinuria and are thought to do this by eliminating a circulating factor. Direct cellular effects of eluates from immunoadsorption procedures on podocytes, the primary target of injury in FSGS, have not yet been reported.


METHODS
Eluates were derived from antibody-based immunoadsorption of a patient suffering from primary FSGS, a patient with systemic lupus erythematosus, and a healthy volunteer. The cytosolic free Ca(2+) concentration ([Ca(2+)](i)) of differentiated podocytes was measured by single-cell fura-2 microfluorescence measurements. Free and total immunoreactive kinin levels were measured by radioimmunoassay.


RESULTS
FSGS eluates increased the [Ca(2+)](i) levels concentration dependently (EC(50) 0.14 mg/ml; n = 3-19). 1 mg/ml eluate increased the [Ca(2+)](i) values reversibly from 82 +/- 12 to 1,462 +/- 370 nmol/l, and then they returned back to 100 +/- 16 nmol/l (n = 19). The eluate-induced increase of [Ca(2+)](i) consisted of an initial Ca(2+) peak followed by a Ca(2+) plateau which depended on the extracellular Ca(2+) concentration. The eluate-induced increase of [Ca(2+)](i) was inhibited by the specific B(2) kinin receptor antagonist Hoe 140 in a concentration-dependent manner (IC(50) 2.47 nmol/l). In addition, prior repetitive application of bradykinin desensitized the effect of eluate on [Ca(2+)](i). A colonic epithelial cell line not reacting to bradykinin did not respond to eluate either (n = 6). Similar to FSGS eluates, the eluate preparations of both the systemic lupus patient and the healthy volunteer led to a biphasic, concentration-dependent [Ca(2+)](i) increase in podocytes which again was inhibited by Hoe 140. Free kinins were detected in all eluate preparations.


CONCLUSIONS
The procedure of antibody-based immunoadsorption leads to kinin in the eluate which elevates the [Ca(2+)](i) level of podocytes via B(2) kinin receptors.
Fischer
Karl-Georg
KG
Department of Medicine, Division of Nephrology and General Medicine, University Hospital Freiburg, Freiburg, Germany. fischer@med1.ukl.uni-freiburg.de
Huber
Tobias Bruno
TB
Henger
Anna
A
Fink
Edwin
E
Schwertfeger
Eckhard
E
Rump
Lars Christian
LC
Pavenstädt
Hermann
H
D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005260:Female; D005923:Glomerulosclerosis, Focal Segmental / Q000276:immunology / Q000378:metabolism* / Q000601:surgery; D019073:HT29 Cells; D006801:Humans; D007163:Immunosorbent Techniques; D007678:Kidney Glomerulus / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D016030:Kidney Transplantation; D007705:Kinins / Q000378:metabolism; D008181:Lupus Nephritis / Q000276:immunology / Q000378:metabolism*; D008297:Male; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000378:metabolism*
D007705:Kinins; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D001920:Bradykinin; D002118:Calcium