Localization of Renin Angiotensin Peptidases In Mouse Kidney Using Dual Immunofluorescence and Microscopy

Abstract

The kidney renin angiotensin system (RAS) plays a pivotal role in the regulation of blood pressure. There is emerging evidence that the two main Ang peptide mediators, angiotensin (Ang) II and Ang-(1-7), have counter-regulatory roles. While Ang II functions as a potent vasoconstrictor, the vasodilator Ang-(1-7) has been found to protect against renal damage and cardiovascular disease. Ang II is converted to Ang-(1-7) by the renal peptidases: Ang converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP) and prolyl endopeptidase (PEP). Using dual immunofluorescence microscopy, we determined the expression pattern of ACE2, PCP and PEP in mouse kidney. The first step was to optimize the staining methodology. Factors adjusted were chemical constituents, incubation period and antisera dilutions. Following optimization, fixed kidney sections (20 µm) were incubated in primary antisera mixtures overnight (1:50-1:100 diluted in PBS containing 0.3% tritonX). Immunoreactive sites were revealed on day 2 with secondary antisera (diluted 1:100 in PBS containing 0.3% tritonX). ACE2, PCP and PEP were localized in renal tubules. In dual immunofluorescent preparations, we tested the combination of PCP+ACE2 and PEP+ACE2. PCP and PEP expression were co-localized with ACE2. As expected ACE2 was not seen in kidney tissue from ACE2 knockout mice. PCP and PEP expression remained unchanged in ACE2 KO. This approach will aid in our understanding of the role and site of Ang II processing to Ang-(1-7) in various renal pathologies. Supported by NIH R01HL093567 and 1R25GM090122.