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. 2011 Sep;301(3):F529-35.
doi: 10.1152/ajprenal.00596.2010. Epub 2011 Jan 26.

Aldosterone stimulates superoxide production in macula densa cells

Xiaolong Zhu  1 , R Davis Manning Jr, Deyin Lu, Celso E Gomez-Sanchez, Yiling Fu, Luis A Juncos, Ruisheng Liu
Affiliations

Aldosterone stimulates superoxide production in macula densa cells

Xiaolong Zhu et al. Am J Physiol Renal Physiol. 2011 Sep.

Abstract

Two major factors which regulate tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole are release of superoxide (O(2)(-)) and nitric oxide (NO) by macula densa (MD) cells. MD O(2)(-) inactivates NO; however, among the factors that increase MD O(2)(-) release, the role of aldosterone is unclear. We hypothesize that aldosterone activates the mineralocorticoid receptor (MR) on MD cells, resulting in increased O(2)(-) 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 O(2)(-) 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 O(2)(-) production to the same degree. These data suggest that aldosterone-stimulated MD O(2)(-) 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 O(2)(-) 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 O(2)(-) production by activating MR; and 3) aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O(2)(-). The resulting balance between O(2)(-) and NO in the MD is important in modulating TGF.

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Figures

Fig. 1.
Fig. 1.
Expression of mineralocorticoid receptor (MR) in MMDD1 cells. A: RT-PCR shows that the MMDD1 cells express the mRNA for the MR. Mouse myocytes were used as a positive control. B: Western blotting demonstrates the MR protein in MMDD1 cells.
Fig. 2.
Fig. 2.
Aldosterone (aldo) stimulates superoxide production via MR in MMDD1 cells. Aldosterone stimulates O2− significantly (n = 45, where n is the number of independent samples). Blocking MR with eplerenone (n = 45) blocked aldosterone-induced increased in O2−. Eplerenone (n = 48) had no effect on O2− in the absence of aldosterone compared with control group (n = 44). *P < 0.05 vs. untreated cells, which were used as a control.
Fig. 3.
Fig. 3.
mRNAs of cyclooxygenase-2 (COX-2) and NOX-2 and NOX-4, isoforms of NAD(P)H oxidase, are detected in MMDD1 cells. RT-PCR shows that COX-2, NOX-2, and NOX-4 are expressed in MMDD1 cells. The ladder was cut from the same gel film.
Fig. 4.
Fig. 4.
Dose-response of aldosterone in mRNAs of COX-2, NOX-2, and NOX-4. Aldosterone at 10−9 and 10−8 mol/l for 30 min enhances mRNAs of COX-2, NOX-2, and NOX-4, and aldosterone at 10−8 mol/l had the greater effect; n = 3 for each group. *P < 0.05 vs. control.
Fig. 5.
Fig. 5.
Aldosterone enhances protein levels of COX-2, NOX-2, and NOX-4 as measured by Western blotting. Aldosterone at 10−8 mol/l for 30 min increases protein levels of COX-2, NOX-2, and NOX-4 in MMDD1 cells. The amount of protein for the band was 40 ng. The bands were cut from the same gel films. GAPDH was used as a control for both NOX-2 and NOX-4.
Fig. 6.
Fig. 6.
Aldosterone-induced O2− production is blocked by COX-2 or NAD(P)H oxidase inhibition. Aldosterone (10−8 mol/l) stimulated O2− significantly in MMDDl cells (n = 61) compared with untreated control cells (n = 58). Blocking COX-2 with NS-398 (n = 60), NAD(P)H oxidase with apocynin (n = 61), or both COX-2 and NAD(P)H oxidase (n = 48) blocked aldosterone-induced O2−. *P < 0.05 vs. control. #P < 0.05 vs. aldosterone group.
Fig. 7.
Fig. 7.
Efficiency and specificity of COX-2 siRNA. A: effects of COX-2 small-interfering RNA (siRNA) or scrambled siRNA on COX-2 mRNA. COX-2 siRNA reduces COX-2 mRNA level significantly (n = 17) compared with the untreated control cells (n = 21). Scrambled siRNA (n = 6) had no effect on COX-2 mRNA level. B: knocking down COX-2 mRNA with COX-2 siRNA did not affect the NOX-2 and NOX-4 mRNAs (no. of experiments for each group: control = 9, NOX-2 = 9, NOX-4 = 12; COX-2 siRNA = 12, NOX-2 = 12, NOX-4 = 12). Scrambled siRNA (NOX-2 = 6, NOX-4 = 5) had no effect. *P < 0.05 vs. control.
Fig. 8.
Fig. 8.
COX-2 siRNA blocked aldosterone-induced increases in mRNAs of COX-2, NOX-2, and NOX-4. Aldosterone stimulated COX-2, NOX-2, and NOX-4 mRNA levels significantly (control = 7, COX-2 = 7, NOX-2 = 5, NOX-4 = 6; aldo+COX-2 = 5, aldo+NOX-2 = 5, aldo+NOX-4 = 4). Knocking down the COX-2 mRNA with COX-2 siRNA (COX-2 = 8, NOX-2 = 7, NOX-4 = 7) blocked the effect of aldosterone on COX-2, NOX-2, and NOX-4 mRNAs. *P < 0.05 vs. control.
Fig. 9.
Fig. 9.
COX-2 siRNA blocked aldosterone-induced increases in O2−. Knocking down COX-2 mRNA with COX-2 siRNA blocked the effect of aldosterone on O2−; n = 3. *P < 0.05 vs. untreated control cells.
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