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      Eluate Derived by Extracorporal Antibody-Based Immunoadsorption Elevates the Cytosolic Ca 2+ Concentration in Podocytes via B 2 Kinin Receptors

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          Background/Aim: 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<sup>2+</sup> concentration ([Ca<sup>2+</sup>]<sub>i</sub>) 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<sup>2+</sup>]<sub>i</sub> levels concentration dependently (EC<sub>50</sub> 0.14 mg/ml; n = 3–19). 1 mg/ml eluate increased the [Ca<sup>2+</sup>]<sub>i</sub> 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<sup>2+</sup>]<sub>i</sub> consisted of an initial Ca<sup>2+</sup> peak followed by a Ca<sup>2+</sup> plateau which depended on the extracellular Ca<sup>2+</sup> concentration. The eluate-induced increase of [Ca<sup>2+</sup>]<sub>i</sub> was inhibited by the specific B<sub>2</sub> kinin receptor antagonist Hoe 140 in a concentration-dependent manner (IC<sub>50</sub> 2.47 nmol/l). In addition, prior repetitive application of bradykinin desensitized the effect of eluate on [Ca<sup>2+</sup>]<sub>i</sub>. 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<sup>2+</sup>]<sub>i</sub> increase in podocytes which again was inhibited by Hoe 140. Free kinins were detected in all eluate preparations. Conclusion: The procedure of antibody-based immunoadsorption leads to kinin in the eluate which elevates the [Ca<sup>2+</sup>]<sub>i</sub> level of podocytes via B<sub>2</sub> kinin receptors.

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          Most cited references 6

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          Congenital nephrotic syndrome in mice lacking CD2-associated protein.

          CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm.
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            Circulating factor associated with increased glomerular permeability to albumin in recurrent focal segmental glomerulosclerosis.

            Heavy proteinuria and progressive renal injury recur after transplantation in up to 40 percent of patients with renal failure caused by idiopathic focal segmental glomerulosclerosis. A circulating factor may be responsible for this recurrence. To determine whether patients with focal segmental glomerulosclerosis have a circulating factor capable of causing glomerular injury, we tested serum samples from 100 patients with the disorder in an in vitro assay of glomerular permeability to albumin. Of the 56 patients who had undergone renal transplantation, 33 had recurrences. Sixty-four patients, many of whom had undergone transplantation, were being treated with dialysis. Thirty-one patients with other renal diseases and nine normal subjects were also studied. The 33 patients with recurrent focal segmental glomerulosclerosis after transplantation had a higher mean (+/-SE) value for permeability to albumin (0.47+/-0.06) than the normal subjects (0.06+/-0.07) or the patients who did not have recurrences (0.14+/-0.06). After plasmapheresis in six patients with recurrences, the permeability was reduced (from 0.79+/-0.06 to 0.10+/-0.05, P = 0.008), and proteinuria was significantly decreased. Patients with corticosteroid-sensitive nephrotic syndrome or with membranous nephropathy after transplantation had low levels of serum activity. The circulating factor bound to protein A and hydrophobic-interaction columns and had an apparent molecular mass of about 50 kd. A circulating factor found in some patients with focal segmental glomerulosclerosis is associated with recurrent disease after renal transplantation and may be responsible for initiating the renal injury.
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              Effect of plasma protein adsorption on protein excretion in kidney-transplant recipients with recurrent nephrotic syndrome.

              Among patients with the idiopathic nephrotic syndrome who have focal and segmental glomerulosclerosis and undergo renal transplantation, 15 to 55 percent have recurrent nephrotic syndrome. The recurrence may be caused by a plasma factor or factors that increase glomerular permeability, because plasma exchange transiently decreases or abolishes proteinuria in some patients. We studied the effect on proteinuria of the removal of protein (mostly immunoglobulins) by adsorption onto protein A from the plasma of patients with recurrent nephrotic syndrome. Eight patients were treated with one to three cycles of two to seven 1-day sessions of protein adsorption, and the patients' urinary protein excretion was measured repeatedly. Their immunosuppressive regimens were not changed during the treatment. The adsorbed proteins were eluted from the protein A and injected into rats, and the urinary albumin excretion of the rats was measured. The protein-adsorption treatment consistently decreased urinary protein excretion by an average of 82 percent at the end of a cycle (P < 0.001). In one patient proteinuria disappeared, and in another urinary protein excretion remained below 2.5 g per day with repeated cycles of protein adsorption. In all but one patient the effect of adsorption was limited in time, with a return to the preadsorption level of protein excretion within a maximum of two months. The administration to rats of material eluted from the protein A increased urinary albumin excretion 2.9- to 4.6-fold (P < 0.001 and P = 0.005, respectively). Although protein A primarily binds immunoglobulins, the active fraction of the eluted proteins had a molecular weight below 100,000, indicating that immunoglobulin was not directly involved. Adsorption of plasma protein decreases urinary protein excretion in patients with recurrence of the nephrotic syndrome after renal transplantation. Studies of the adsorbed proteins should provide information about the mechanism of this disease.

                Author and article information

                Kidney Blood Press Res
                Kidney and Blood Pressure Research
                S. Karger AG
                26 February 2003
                : 25
                : 6
                : 384-393
                aDepartment of Medicine, Division of Nephrology and General Medicine, University Hospital Freiburg, Freiburg, and bDepartment of Clinical Chemistry and Clinical Biochemistry, University of Munich, Munich, Germany
                68697 Kidney Blood Press Res 2002;25:384–393
                © 2002 S. Karger AG, Basel

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                Page count
                Figures: 5, Tables: 1, References: 43, Pages: 10
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/68697
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