A Nephritogenic Rat Monoclonal Antibody to Mouse Amlnopeptidase A. Induction of Massive Albuminuria after a Single Intravenous Injection. (1)
The hydrolase dipeptidyl peptidase IV (DPP IV) is one of these antigens that has been reported to be involved in the induction of an immune complex glomerulonephritis. Injection of monoclonal or polyclonal antisera against DPP IV into rats or mice results in an immediate, transient glomerular binding, but induces only a mild proteinuria of short duration.
The following is preparation of brush border (BB) suspensions from Mouse Kidneys. An enriched suspension of mouse renal BB membrane vesicles from proximal tubular epithelial cells was prepared from BALB/c kidneys according to the method of Malathi et al. using a 2-ram Tris-HCl buffer, pH 7.2, containing 50 mM mannitol, the protease inhibitors EDTA (20 mM), PMSF (1 mM), benzamidine (1 mM), Trasylol (10 U/ml), and 0.02% NaN3. To obtain a BB preparation enriched in integral membrane proteins that served as immunogen for the production of mAb, the crude BB fraction was solubilized with the non-ionic detergent Triton-X-114 (2%) in 50 mM Tris-HC1 at 4C for 30 min and subjected to Triton X-114 phase separation at 37C for 20 min followed by centrifugation at 300xg for 5 min. In the immunoprecipitation procedure a BB fraction solubilized with 2% Triton X-100 in 50 mM Tris-HC1 at pH 7.2, without the protease inhibitors, was used. For the fluorimetric enzyme assay BB membrane vesicles were solubilized with 1% Doc in 50 mM Tris-HCl at pH 8.5, also without protease inhibitors for 30 min at 4C and subsequently centrifuged at 100,000g for 1 h at 4C. The supernatant was then dialyzed overnight at 40C against 0.1% Doc.
A male Lou rat was immunized intraperitoneally with 1 ml of the detergent phase of the Triton X-114 extract of mouse BB and boosted by three intraperitoneal injections. 3 d after the last booster injection, spleen cells from the Lou rat were fused with SP 2/0 mouse myeloma cells by the procedure of KoShler and Milstein. Eight monoclonal antibodies (mAbs) were obtained. ASD-2, 3, and 4 which induced albuminuria, recognized 140 kDa protein in BB on SDS-PAGE with immunoprecipitated proteins with the mAbs.
BALB/c mice were injected intravenously with increasing (0.5-8 mg) amounts of ASD-4, an albuminuria could be induced at day I with doses >1 mg of mAb, which was massive with a dose of 8 mg. The albuminuria remained high and had not returned to normal values 16 days after the injection of the mAb. Mice injected with doses >8 mg became severely ill at the end of their contingent in the metabolic cages at day 1 and most did not survive. Isotype mAb with ASD-4, 8 mg, failed to induce albuminuria.
DPP IV is a possible antigen which has about 140 kDa. However, with several rat mAbs against mouse DPP IV produced, an immediate homogeneous binding to the glomerular capillary wall showed no redistribution during 8 days after intravenous injection. Furthermore, albuminuria was not induced, even when doses up to 15 mg of mAb were injected. At least two possible mechanisms may be operating in this model during the first 16 days after injection of the mAb that act either alone or synergistically. The first mechanism may be hemodynamically determined by the inhibitory effect of the injected mAb on the binding of angiotensin II to APA and its subsequent inactivation, leading to a prolonged and enhanced effect of this pressure peptide on the glomerular circulation and the traffic of proteins across the glomerular capillary wall. Of short duration, most probably due to the rapid inactivation of angiotensin II. Although the results of the three nephritogenic mAbs that can inhibit the APA activity on BB vesicles are in line with this mechanism, the data obtained with two of the five non nephritogenic mAbs that also could reduce enzymatic activity do not support the hypothesis that blocking of the active site on APA alone is responsible for the induction of the albuminuria. In fact, In the glomerulus, binding of angiotensin II to its receptor on mesangial cells induces contraction of these cells influencing the glomerular capillary surface area available for filtration and consequently the glomerular capillary uhrafiltration coefficient (Kf). At this moment, it is thought that APA on glomerular epithelial cells may bind and rapidly inactivate angiotensin II delivered to the glomerulus and then filtered via the capillary wall.
NOTE: An antibody has to inhibit APA activity to show proteinuria. The epitope is important.