Angiotensin-(1-7) Formation Upon Fluid Flow Shear Stress in Human Podocytes

Abstract

Objective: Glomerular hyperfiltration associates with increased fluid flow shear stress (FFSS) on podocytes. This can lead to irreversible podocyte damage, which in turn negatively affects the permeability and integrity of the glomerular filtration barrier. AngiotensinII (AngII) is an important mediator in chronic kidney disease. The negative effects of AngII are at least partially opposed by the alternative signaling pathways within the renin-angiotensin system (RAS) consisting of angiotensin-converting enzyme 2 (ACE2), angiotensin-(1-7) (Ang-[1-7]) and the MAS1 receptor. This counteracting pathway may exert renoprotective effects and support anti-inflammatory processes. As the pathomechanisms and signaling pathways underlying podocyte damage induced by FFSS are not fully understood, our aim is to elucidate the potential renoprotective impact of the alternative RAS axis in human podocytes in the context of glomerular hyperfiltration. Design and method: Conditionally immortalized human podocytes (hPCs) were used for mechanistic studies. Cells seeded on collagen IV coated Culture Slips® were inserted in a Streamer® Shear Stress Device and exposed to FFSS of 1dyne/cm2 for 2h. Control cells were not exposed to FFSS. Afterwards, the formation rate of AngII to Ang-[1-7] was measured in whole cell lysates (1 experiment, n = 6 samples per condition) using spiked AngII as a substrate with and without addition of Z-Pro-Prolinal and MLN-4760. Additionally, gene expression analyses of the enzymes involved in Ang-[1-7] formation were performed. Results: Preliminary data show an increased AngII to Ang-[1-7] formation rate in hPCs following FFSS exposure compared to control. Ang-[1-7] formation under both conditions is reduced by addition of Z-Pro-Prolinal, whereas MLN-4760 has no effect. RNA-expression levels of prolylcarboxypeptidase and prolylendopeptidase are not significantly affected upon FFSS compared to control. In contrast, ACE2 is significantly upregulated by FFSS although expression levels are substantially lower under both conditions compared to prolylcarboxypeptidase and prolylendopeptidase. Conclusions: Our results suggest an elevated AngII to Ang-[1-7] formation rate upon FFSS in hPCs compared to no-flow control. Under both settings, Ang-[1-7] formation in hPCs seems to be mediated by prolylcarboxypeptidase and/or prolylendopeptidase but not ACE2. These results support further studies to confirm the potential role and mechanisms of Ang-[1-7] upregulation in response to glomerular hyperfiltration.