Establishment of a New Specific High Sensitivity Measurement System for Soluble Angiotensin Iv Receptor

Abstract

Objective: The renin angiotensin system plays a pivotal role in the development of cardiovascular and renal conditions. We had identified angiotensin IV receptor (AT4) as a hypertension/diabetes-specific gene through systemic transcriptome analysis followed by ontology based hierarchical clustering. Previously we have established a measurement system for soluble AT4 (sAT4) by polyclonal antibodies with 1 μg/ml sensitivity. Actual measurements for clinical subjects suggest the sAT4 measurement system would be useful for assessment of the pathophysiology of hypertension/obesity. Thus, we tried to establish a new specific high sensitivity measurement system for sAT4 with monoclonal antibodies. Design and method: As the antigen, we prepared a recombinant protein of AT4. Mice were immunized with the protein then spleen and lymph nodes were applied to make hybridomas with X63. We have screened the clones and selected candidate clones. Then we tested the best combination of the antibodies for the sandwich ELISA system and then optimized the reaction conditions. With this system, as a pilot study, we measured plasma concentrations of 114 subjects with life style related diseases and investigated relationships of sAT4 with various clinical and biochemical parameters by ANOVA. Results: Based on the standard curve for serial-step dilutions against reference molecules, the new measurement system for sAT4 showed 20 pg/ml sensitivity, 3–8 % intra-assay CV and about 10 % inter-assay CV without cross reactivities against 3 standard substrates. Actual measurements on clinical subjects revealed that the sAT4 values tended to be lower on hypertensive cases, obesity cases and dyslipidemic cases and that the sAT4 values were significantly low on CKD cases. Conclusions: Thus, we could establish a new specific high sensitivity measurement system for sAT4. The AT4 was originally identified from GLUT4 vesicles and thought to be involved in insulin sensitivity. It is also known to degrade angiotensin III to IV. Besides these findings, our data suggest the possibility that AT4 is involved in the pathophysiology of hypertension/obesity in a high sensitivity level for the first time. As perspectives we will assess the distribution of the concentrations and will measure the adequate numbers based on statistical power.