RAS‐mediated nitric oxide signaling in podocytes

Abstract

In the kidney, glomerular cells can release nitric oxide (NO) and regulate microvascular hemodynamics in concert with the renin‐angiotensin system (RAS). It has been shown that inducible nitric oxide synthase (iNOS) expression plays a pathophysiological role in glomerulonephritis, and NO deficiency in the glomeruli leads to podocyte damage and oxidative stress in glomerular cells. However, the functional effects of NO in podocytes and whether they express other NOS subunits are not well‐established. Our results suggest that NO signaling in podocytes is triggered by Ang II stimulation via Angiotensin II type 2 receptor (AT2R) since AT2R antagonist (PD123319) inhibited Ang II‐mediated NO production. To detect how different components of the RAS may modify NO production and intracellular Ca2+ signaling, we treated a conditionally immortalized human podocyte cell line with a variety of angiotensin metabolite peptides and measured changes in their NO production, intracellular Ca2+ flux, and cell volume. Understanding these fundamental pathways in podocytes is essential for discovering new therapeutical approaches to treat diseases of the glomerular filtration barrier (GFB). Our experiments determined that of the Ang metabolites tested, Ang III had the most pronounced effect on NO production. In contrast, Ang 1‐7 and Ang 1‐9 produce only small increases in NO, while Ang IV (and other peptides mainly targeting AT1R or the β‐arrestin pathway) do not produce any NO response. We also examined changes in cell volume using Scanning Ion Conductance Microscopy and found that NO production is associated with increase in podocyte cell volume. In contrast, Ca2+ influx without NO generation results in podocyte cell shrinkage. Lastly, we repeated these experiments with nNOS and iNOS specific inhibitors (Nω‐Propyl‐L‐arginine hydrochloride and L‐NIL, respectively), and determined that in normal podocyte cells, nNOS signaling is predominant compared to iNOS (70% and 30% of total response respectively, p<0.0001). In summary, these results demonstrate that nNOS‐ and iNOS‐mediated biosynthesis can be triggered by AT2R activation. This NO signaling can modulate podocyte cell motility, potentially affecting the integrity of the GFB. Additional experiments are needed to determine whether overproduction of NO and iNOS‐mediated pathway activation can cause pathophysiological changes in podocytes.