Aldosterone stimulates superoxide production in macula densa cells

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.

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Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E2

AJP Renal Physiology ◽

10.1152/ajprenal.00287.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. F152-F159 ◽

Cited By ~ 58

Author(s):

Alex Paliege ◽

Diane Mizel ◽

Carmen Medina ◽

Anita Pasumarthy ◽

Yuning G. Huang ◽

...

Keyword(s):

Plasma Renin Activity ◽

Mrna Expression ◽

Real Time ◽

Plasma Renin ◽

Macula Densa ◽

Renin Secretion ◽

Renin Activity ◽

Rt Pcr ◽

Cox 2 ◽

Nnos Expression

It is well established that cyclooxygenase-2 (COX-2) and the neuronal form of nitric oxide synthase (nNOS) are coexpressed in macula densa cells and that the expression of both enzymes is stimulated in a number of high-renin states. To further explore the role of nNOS and COX-2 in renin secretion, we determined plasma renin activity in mice deficient in nNOS or COX-2. Plasma renin activity was significantly reduced in nNOS −/− mice on a mixed genetic background and in COX-2 −/− mice on either BALB/c or C57/BL6 congenic backgrounds. In additional studies, we accumulated evidence to show an inhibitory influence of PGE2 on nNOS expression. In a cultured macula densa cell line, PGE2 significantly reduced nNOS mRNA expression, as quantified by real-time RT-PCR. In COX-2 −/− mice, nNOS mRNA expression in the kidney, determined by real-time RT-PCR, was upregulated throughout the postnatal periods, ranging from postnatal day ( PND) 3 to PND 60. The induction of nNOS protein expression and NOS activity in COX-2 −/− mice was localized to macula densa cells using immunohistochemistry and NADPH-diaphorase staining methods, respectively. Therefore, these findings reveal that the absence of either COX-2 or nNOS is associated with suppressed renin secretion. Furthermore, the inhibitory effect of PGE2 on nNOS mRNA expression indicates a novel interaction between NO and prostaglandin-mediated pathways of renin regulation.

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NOX2 is the primary source of angiotensin II-induced superoxide in the macula densa

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00762.2009 ◽

2010 ◽

Vol 298 (3) ◽

pp. R707-R712 ◽

Cited By ~ 32

Author(s):

Yiling Fu ◽

Rui Zhang ◽

Deyin Lu ◽

Haifeng Liu ◽

Kiran Chandrashekar ◽

...

Keyword(s):

Angiotensin Ii ◽

Nadph Oxidase ◽

Primary Source ◽

Oxidase Inhibitor ◽

Macula Densa ◽

Tubuloglomerular Feedback ◽

Ang Ii ◽

Xanthine Oxidase Inhibitor ◽

Si Rna ◽

Full Activation

Macula densa (MD)-mediated regulation of renal hemodynamics via tubuloglomerular feedback is regulated by interactions between factors such as superoxide (O2−) and angiotensin II (ANG II). We have reported that NaCl-induced O2−in the MD is produced by the NOX2 isoform of NADPH oxidase (NOX); however, the source of ANG II-induced O2−in MD is unknown. Thus we determined the pathways by which ANG II increased O2−in the MD by measuring O2−in ANG II-treated MMDD1 cells, a MD-like cell line. ANG II caused MMDD1 O2−levels to increase by more than twofold ( P < 0.01). This increase was blocked by losartan (AT1receptor blocker) but not PD-123319 (AT2receptor antagonist). Apocynin (a NOX inhibitor) decreased O2−by 86% ( P < 0.01), whereas oxypurinol (a xanthine oxidase inhibitor) and NS-398 (a cyclooxygenase-2 inhibitor) had no significant effect. The NOX-dependent increase in O2−was due to the NOX2 isoform; a short interfering (si)RNA against NOX2 blunted ANG II-induced increases in O2−, whereas the NOX4/siRNA did not. Finally, we found that inhibiting the Rac1 subunit of NOX blunted ANG II-induced O2−production in NOX4/siRNA-treated cells but did not further decrease it in NOX2/siRNA-treated cells. Our results indicate that ANG II stimulates O2−production in the MD primarily via AT1-dependent activation of NOX2. Rac1 is required for the full activation of NOX2. This pathway may be an important component of ANG II enhancement of tubuloglomerular feedback.

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GTPase-Rac enhances depolarization-induced superoxide production by the macula densa during tubuloglomerular feedback

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00622.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. R453-R458 ◽

Cited By ~ 7

Author(s):

Ruisheng Liu ◽

Luis A. Juncos

Keyword(s):

Membrane Potential ◽

Nadph Oxidase ◽

Channel Blocker ◽

Apical Membrane ◽

Primary Source ◽

Macula Densa ◽

Tubuloglomerular Feedback ◽

Nadph Oxidase Activity ◽

Gtp Binding ◽

Apical Side

Superoxide (O2− ) enhances tubuloglomerular feedback (TGF) by scavenging nitric oxide at the macula densa (MD). The primary source of O2− in the MD during TGF is NADPH oxidase, which is activated by membrane depolarization. While Rac, a small GTP-binding protein, has been shown to enhance NADPH oxidase activity, its role in O2− generation by the MD is unknown. We hypothesized that depolarization of the MD leads to translocation of Rac to the apical membrane, and its activation, in turn, augments O2− generation during TGF. We tested this by measuring membrane potential and increased O2− levels during TGF responses in isolated, perfused tubules containing the intact MD plaque. Switching tubular NaCl from 10 to 80 mM, which induces TGF, depolarized membrane potential by 28.4 ± 4.5% from control ( P < 0.05) and O2− levels from 124 ± 19 to 361 ± 27 U/min. This NaCl-induced depolarization and O2− generation were blocked by a Cl− channel blocker, 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB; 10−6 M). Inhibition of Rac blunted NaCl-induced O2− generation by 47%. When the NaCl content of the MD perfusate was increased from 10 to 80 mM, immunointensity of Rac on the apical side increased from 32 ± 3.1 to 46 ± 2.5% of the total immunofluorescence in the MD, indicating that high NaCl induces the translocation of Rac to the apical membrane. This NaCl-induced Rac translocation was blocked by a Cl− channel blocker, NPPB, indicating that depolarization of the MD induced Rac translocation. In conclusion, we found that depolarization of the MD during TGF leads to translocation of Rac to the apical membrane, which enhances O2− generation by the MD.

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p22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00976.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. H1685-H1689 ◽

Cited By ~ 15

Author(s):

Pouneh Nouri ◽

Pritmohinder Gill ◽

Min Li ◽

Christopher S. Wilcox ◽

William J. Welch

Keyword(s):

Angiotensin Ii ◽

Nadph Oxidase ◽

Blood Pressure Response ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Distal Tubule ◽

Macula Densa ◽

Tubuloglomerular Feedback ◽

Ang Ii ◽

Renal Vasoconstriction ◽

Single Nephron

Angiotensin II (ANG II) infusion increases renal superoxide (O2−) and enhances renal vasoconstriction via macula densa (MD) regulation of tubuloglomerular feedback, but the mechanism is unclear. We targeted the p22 phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) with small-interfering RNA (siRNA) to reduce NADPH oxidase activity and blood pressure response to ANG II in rats. We compared single nephron glomerular filtration rate (SNGFR) in samples collected from the proximal tubule (PT), which interrupts delivery to the MD, and from the distal tubule (DT), which maintains delivery to the MD, to assess MD regulation of GFR. SNGFR was measured in control and ANG II-infused rats (200 ng·kg−1·min−1 for 7 days) 2 days after intravenous injection of vehicle or siRNA directed to p22 phox to test the hypothesis that p22 phox mediates MD regulation of SNGFR during ANG II. The regulation of SNGFR by MD, determined by PT SNGFR-DT SNGFR, was not altered by siRNA in control rats (control + vehicle, 13 ± 1, n = 8; control + siRNA, 12 ± 2 nl/min, n = 8; not significant) but was reduced by siRNA in ANG II-treated rats (ANG II + vehicle, 13 ± 2, n = 7; ANG II + siRNA, 7 ± 1 nl/min, n = 8; P < 0.05). We conclude that p22 phox and NADPH oxidase regulate the SNGFR during ANG II infusion via MD-dependent mechanisms.

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4-(Phenylsulfanyl) Butan-2-One Attenuates the Inflammatory Response Induced by Amyloid-β Oligomers in Retinal Pigment Epithelium Cells

Marine Drugs ◽

10.3390/md19010001 ◽

2020 ◽

Vol 19 (1) ◽

pp. 1

Author(s):

Peeraporn Varinthra ◽

Shun-Ping Huang ◽

Supin Chompoopong ◽

Zhi-Hong Wen ◽

Ingrid Y. Liu

Keyword(s):

Retinal Pigment Epithelium ◽

Inflammatory Response ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Amyloid Β ◽

Age Related Macular Degeneration ◽

Epithelial Cell Line ◽

Age Related ◽

Tnf Α ◽

Cox 2

Age-related macular degeneration (AMD) is a progressive eye disease that causes irreversible impairment of central vision, and effective treatment is not yet available. Extracellular accumulation of amyloid-beta (Aβ) in drusen that lie under the retinal pigment epithelium (RPE) has been reported as one of the early signs of AMD and was found in more than 60% of Alzheimer’s disease (AD) patients. Extracellular deposition of Aβ can induce the expression of inflammatory cytokines such as IL-1β, TNF-α, COX-2, and iNOS in RPE cells. Thus, finding a compound that can effectively reduce the inflammatory response may help the treatment of AMD. In this research, we investigated the anti-inflammatory effect of the coral-derived compound 4-(phenylsulfanyl) butan-2-one (4-PSB-2) on Aβ1-42 oligomer (oAβ1-42) added to the human adult retinal pigment epithelial cell line (ARPE-19). Our results demonstrated that 4-PSB-2 can decrease the elevated expressions of TNF-α, COX-2, and iNOS via NF-κB signaling in ARPE-19 cells treated with oAβ1-42 without causing any cytotoxicity or notable side effects. This study suggests that 4-PSB-2 is a promising drug candidate for attenuation of AMD.

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Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

Alternatives to Laboratory Animals ◽

10.1177/026119299302100212 ◽

1993 ◽

Vol 21 (2) ◽

pp. 191-195 ◽

Cited By ~ 1

Author(s):

Knut-Jan Andersen ◽

Erik Ilsø Christensen ◽

Hogne Vik

Keyword(s):

Epithelial Cells ◽

Three Dimensional ◽

Toxicity Testing ◽

Renal Tissue ◽

Epithelial Cell Line ◽

Multicellular Spheroids ◽

Renal Epithelial Cell ◽

Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

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Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

Alternatives to Laboratory Animals ◽

10.1177/026119299202000206 ◽

1992 ◽

Vol 20 (2) ◽

pp. 218-221

Author(s):

Henning F. Bjerregaard

Keyword(s):

Epithelial Cell ◽

Cell Line ◽

South African ◽

Mdck Cells ◽

Epithelial Cell Line ◽

Renal Epithelial Cell ◽

Electrophysiological Measurements ◽

Renal Epithelial Cell Line ◽

Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

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Lipopolysaccharide activates innate immune responses in murine intestinal myofibroblasts through multiple signaling pathways

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00022.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. G601-G611 ◽

Cited By ~ 65

Author(s):

Kristen L. W. Walton ◽

Lisa Holt ◽

R. Balfour Sartor

Keyword(s):

Immune Responses ◽

Signaling Pathways ◽

P38 Mapk ◽

Western Blotting ◽

Pi3 Kinase ◽

Innate Immune ◽

Innate Immune Responses ◽

Fold Induction ◽

Cox 2 ◽

Multiple Signaling

Myofibroblasts (MF) play an important role in intestinal wound healing. A compromised epithelial barrier exposes intestinal subepithelial MF to luminal bacterial products. However, responses of murine intestinal MF to bacterial adjuvants and potential roles of intestinal MF in innate immune responses are not well defined. Our aims in this study were to determine innate immune responses and intracellular signaling pathways of intestinal MF exposed to LPS, a prototypic Toll-like receptor (TLR) ligand. Expression of TLR4 in primary murine intestinal MF cultures was confirmed by RT-PCR and Western blotting. LPS-induced secretion of prostaglandin E2 (PGE2), interleukin (IL)-6, and keratinocyte-derived chemokines (KC) was measured by ELISA. Intracellular responses to LPS were assessed by Western blotting for NF-κB p65, Iκ-Bα, Akt, p38 MAP kinase, and cyclooxygenase-2 (COX-2). LPS induced rapid phosphorylation of NF-κB p65, Akt, and p38 MAPK and degradation of Iκ-Bα. LPS induced expression of COX-2 and secretion of PGE2 (2.0 ± 0.8-fold induction vs. unstimulated cells), IL-6 (6.6 ± 0.4-fold induction), and KC (12.5 ± 0.4-fold induction). Inhibition of phosphoinositide-3 (PI3)-kinase, p38 MAPK, or NF-κB pathways reduced LPS-induced PGE2, IL-6, and KC secretion. These studies show that primary murine intestinal MF respond to LPS, evidenced by activation of NF-κB, PI3-kinase, and MAPK signaling pathways and secretion of proinflammatory molecules. Inhibition of these pathways attenuated LPS-dependent PGE2, IL-6, and KC production, indicating that LPS activates MF by multiple signaling pathways. These data support the hypothesis that MF are a component of the innate immune system and may exert paracrine effects on adjacent epithelial and immune cells by responding to luminal bacterial adjuvants.

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