Abstract MP29: A Cloned Anti-aminopeptidase N Scfv Antibody Blocks Enzymatic Activity And Rescues A Low Sodium Phenotype In Proximal Tubule Cells Isolated From Inverse Salt Sensitive Participants

Abstract

Inverse Salt Sensitivity (ISS), defined as the paradoxical increase in blood pressure of individuals on a low sodium diet compared to a high sodium diet, may be associated with the increase in mortality and morbidity found in individuals on a low sodium diet. Our group has previously found that urine derived human renal proximal tubule cells (RPTC) isolated from ISS participants express higher Aminopeptidase N (APN) protein than cells isolated from salt resistant (SR) participants. An anti-Aminopeptidase N (APN) hybridoma was used construct a single chain variable fragment (scFv) into a bacterial expression system. The purified anti-APN scFv bound to live human renal proximal tubule cells, detected using an Alexa594 directly labelled anti-myc monoclonal antibody by immunofluorescent confocal microscopy. The anti-APN scFv was then tested to determine if it blocks APN enzymatic activity. We have previously shown that under low sodium conditions the amount of reduced-glutathione as determined by live cell fluorescence staining with the redox sensitive dye, monochlorobimane (mBCl), is decreased only in ISS cells under low sodium conditions at the two-hour time point. We hypothesized that the decreased mBCl signal may be due to the increased APN expression and activity altering the Ang II/Ang III ratio and reducing the Ang III activation of the AT 2 R. Low sodium reduced the mBCl signal in ISS (-29.2±4.3%, ISS vs SR, N=3 per group, p<0.05) and the addition of the anti-APN scFv at 1 ug/ml completely blocked the mBCl signal back to levels found in SR in normal salt conditions. In order to verify that the full rescue of this ISS specific response is due to enhanced Ang III – AT 2 R signaling, we next tried to block the effect of the anti-APN scFv by the addition of the AT 2 R antagonist, PD123319 (PD, 1 uM). Addition of PD alone to the ISS LS RPTCs did not significantly alter the mBCl signal, but when PD and anti-APN scFv are added to the LS treated ISS cells, there is complete reversal of the effect of anti-APN scFv alone (-38.1±3.9%, ISS anti-APN scFv + PD vs ISS anti-APN scFv, N=3 per group, p<0.05). The anti-APN scFv has potential therapeutic value by reducing APN enzymatic activity in ISS individuals and inducing the protective AT 2 R arm of the renin angiotensin system in low sodium conditions.