Two major factors which regulate tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole are release of superoxide (O2−) and nitric oxide (NO) by macula densa (MD) cells. MD O2− inactivates NO; however, among the factors that increase MD O2− release, the role of aldosterone is unclear. We hypothesize that aldosterone activates the mineralocorticoid receptor (MR) on MD cells, resulting in increased O2− production due to upregulation of cyclooxygenase-1 (COX-2) and NOX-2, and NOX-4, isoforms of NAD(P)H oxidase. Studies were performed on MMDD1 cells, a renal epithelial cell line with properties of MD cells. RT-PCR and Western blotting confirmed the expression of MR. Aldosterone (10−8 mol/l for 30 min) doubled MMDD1 cell O2− production, and this was completely blocked by MR inhibition with 10−5 mol/l eplerenone. RT-PCR, real-time PCR, and Western blotting demonstrated aldosterone-induced increases in COX-2, NOX-2, and NOX-4 expression. Inhibition of COX-2 (NS398), NADPH oxidase (apocynin), or a combination blocked aldosterone-induced O2− production to the same degree. These data suggest that aldosterone-stimulated MD O2− production is mediated by COX-2 and NADPH oxidase. Next, COX-2 small-interfering RNA (siRNA) specifically decreased COX-2 mRNA without affecting NOX-2 or NOX-4 mRNAs. In the presence of the COX-2 siRNA, the aldosterone-induced increases in COX-2, NOX-2, and NOX-4 mRNAs and O2− production were completely blocked, suggesting that COX-2 causes increased expression of NOX-2 and NOX-4. In conclusion 1) MD cells express MR; 2) aldosterone increases O2− production by activating MR; and 3) aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O2−. The resulting balance between O2− and NO in the MD is important in modulating TGF.
Effect of cyclosporin-A on the blood–retinal barrier permeability in streptozotocin-induced diabetes
