Effect Of Adlsterone On The Oxygen Stress Induced Hypertension And The Underlying Mechanism

Part I Aldosterone stimulates superoxide production in macula densa cellsOBJECTIVEIn recent years, the role of excessive oxidative stress in the development of cardiovascular disease including hypertension has been highlighted. In patients with myocardial infarction or heart failure, aldosterone has been shown to promote cardiac inflammation. This is accompanied by increases in myocardial oxidative stress and release of inflammatory markers that include cyclooxygenase-2 (COX-2). Aldosterone also causes nitric oxide (NO)-mediated vasodilation and superoxide (O2-) release due to activation of NAD(P)H oxidase in cerebral and mesenteric arterioles. In cultured aortic endothelial cells, aldosterone has also been shown to induce O2-generation by activating NAD(P)H oxidase. Today there is no doubt that the correction of the oxidant/antioxidant imbalance in patients with chronic renal failure is an important approach for the reduction of the risk of those patients to develop cardiovascular disorders.Tubuloglomerular glomerular feedback (TGF) is a critical mechanism for regulation of renal hemodynamics, NaCl excretion and blood pressure. Increasing tubular flow to the macula densa (MD) initiates a TGF signal causing constriction of the afferent arteriole thus decreasing GFR. NO released from the MD modulates the TGF response, and O2- release from the MD will inactivate NO. Recently, we found that NOX-2 and NOX-4 isoforms of NAD(P)H oxidase are expressed in the rat MD and MMDD1 cells. Although the role of aldosterone as a prooxidant and a proinflammatory agent has been established in the heart and in blood vessels, the prooxidant role of aldosterone in the MD is poorly understood. In addition, aldosterone activates mineralocorticoid receptors (MR), which have been found throughout the body but of particular interest to this paper, in the distal tubules, connecting tubules, and collecting ducts of the kidney. The presence of MR in the MD cells has not been reported. Our hypothesis is that aldosterone increases O2-production from MD cells acting through MR, and this is accompanied by increases in COX-2 production and NAD(P)H oxidase via NOX-2 and NOX-4 production. These studies will greatly enhance our understanding of the role of aldosterone in regulating TGF and renal hemodynamics.MATERIALS AND METHODSMMDD1 CellsWe used MMDD1 cells, a renal epithelial cell line with properties of macula densa cells. These cells were derived from SV40 transgenic mice and have been shown to express well-known macula densa markers, eg, COX-2, nNOS, ROMK (Renal Outer Medullary Potassium channel), and NKCC2(Na+-K+-2Cl-co-transporter).Measurement of O2- with LucigeninWe measured O2- production in the MMDD1 cells using a lucigenin-enhanced chemiluminescence assay. Briefly, MMDD1 cells (lOcm-dish) were washed by PBS 2 times, trypsinized from the dish and kept in 12 ml Krebs/Hepes buffer. The Krebs/Hepes buffer was evenly divided into following five groups with different antagonists:1). non-treated; 2). aldosterone 10-8 mol/L; 3). aldosterone 10-8 mol/L and NS398 10-6 mol/L; 4).aldosterone 10-8 mol/L and apocynin 10-5 mol/L; 5). aldosterone 10-8 mol/L, NS398 10-6 mol/L and apocynin 10-5 mol/L. Then lucigenin (5×10-6 mol/L) was added to each of the samples which were incubated for 30 min at 37℃with oxygen bubbling. From each group, a 0.5 ml sample was transferred into a 1.6-mL polypropylene 8×50 mm tubes (Evergreen Scientific), and then using a Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany), O2- was measured following the manufacturer's instructions. Luminescence was measured for 10 sec with a delay of 5 sec.RT-PCR for MMDD1 CellsTotal RNA from the MMDD1 cells and mouse heart cells (as a positive control for the MR receptor) was extracted with an RNeasy Mini kit following the manufacturer's instructions. Oneμg of total RNA was reverse transcribed for 1 hour at 42℃using 1μL random primer 3μg/μL and the Ambion RETROscript kit following the manufacturer's instructions. In the protocol of RT-PCR for COX-2, NOX-2 and NOX-4, measured by specific subunit for each NOX isoform, the resultant RT product was then amplified by PCR by adding 1μL of the RT reaction,1μmol/L of the gene-specific primers,2μL 10×complete PCR buffer,1μL dNTP mix 2.5mmol/L each,0.2μL Super Taq 5U/μL,(1.2μL glycerol 50% only for NOX-2 and NOX-4), and adding nuclear-free water to achieve a volume of 20μL. The mixed samples were heated to 94℃for 3 min and cycled at 94℃for 20 sec,59.4℃(COX-2),56.6℃(NOX-2),52.9℃(NOX-4) for 30 sec, and 72℃for 30 sec for 40 cycles. Final extension was for 8 min at 72℃. In the protocol for RT-PCR for the MR receptor, the mixed samples using the described primers and using Titanium Taq polymerase were heated to 95℃for 2 min and cycled at 95℃for 40 sec, annealing temperature 58℃for 40 sec, and 72℃for 55 sec for 40 cycles. Final extension was for 8 min at 72℃. The amplified products of RT-PCR were run on 1.4%(0.5% for MR receptor) agarose gels containing ethidium bromide 10 mg/mL and visualized under UV light.β-actin, as a housekeeping gene, was set up as an internal loading control.Real-time PCR Real-time PCR was used to quantify mRNA level of the COX-2, NOX-2 and NOX-4 responses to the aldosterone. Total RNA was isolated using the RNeasy Micro kit, and complementary DNA synthesis was carried out as described in the method for RT-PCR. Real-time PCR was performed in a C1000TM Thermal Cycler real-time PCR machine. The (3-actin was used as a housekeeping gene.Preparations for siRNACOX-2 siRNA was used a pre-designed product from Ambion. siRNA transfection was performed using a siPORTTM Amine Transfection Agent according to the manufacturer's instructions. Scrambled siRNA (Invitrogen) were synthesized and used as negative controls. At 24 hours before transfection, MMDD1 cells were transferred onto 6-well plates (2×105 cells per well) with antibiotic-free medium. The cells were transfected with 10 nmol/L COX-2 siRNA duplex using Amine Transfection Agent for 18 hours in medium devoid of antibiotics. This procedure does not affect cell viability, measured with calcein as we described previously. The MMDD1 cells were washed once with PBS and grown in complete medium. Gene silencing was monitored by measuring RNA after incubation for 24 hours. We added aldosterone (10-8 mol/L) into each cell culture well and incubated at 37℃for 30 min in a cell incubator before harvesting the MMDD1 cells.Western blot for MMDD1 cellsMMDD1 cells proteins were extracted with RIPA buffer plus a protease inhibitor cocktail. Protein concentration was measured using a Nanodrop instrument. Cells were homogenized in RIPA buffer, centrifuged and protein measured using a Nanodrop spectrophotometer measuring absorption at 280 nm. Proteins extracted from MMDD1 cells in the amount of 100,50 and 20μg were separated by 8% polyacrylamide gel electrophoresis and transferred to PVDF membranes, respectively. After the transfer, membranes were blocked at room temperature for 1h with 1% fat-free dry milk in TTBS. The membranes were blocked and probed with the following primary antibodies, respectively:1) MR--mixture of rMR 1-18 clone 1D5 (1:50 dilution) and MRN 365 clone 2D6 (1:100 dilution, overnight incubation); 2) COX-2 antibody (1:500 dilution); NOX-2 antibody (1:1000 dilution); 3) NOX-4 antibody (HRP)(1:1000 dilution);4) GAPDH (1:5000)(4℃, overnight incubation), then followed by the secondary antibody (1:10,000 dilution) (1 h incubation, room temperature). The bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) and captured with a VersaDoc image analysis system (Bio-Rad).StatisticsData were analyzed as repeated measures over time or compared to a common control. We tested only the effects of interest, using analysis of variance (ANOVA) for repeated measures and a post-hoc Fisher LSD test. The changes were considered to be significant if P< 0.05. Data are presented as mean±SEM.RESULTS1. Mineralocorticoid receptors exist on the MMDD1 cellsRT-PCR results for the MR on MMDD1 cells. We used mouse myocardial cells as a positive control. Western blot results of MR protein compared to the positive control. The results for the first time demonstrate the existence of mRNA and protein for MR in MMDD1 cells.2. Aldosterone acts through the MR to produce superoxideWe measured O2- levels in aldosterone-treated MMDD1 cells with and without the MR receptor inhibitor (eplerenone 10-5 mol/L) to confirm that the effect of aldosterone is mediated by MR. Eplerenone completely prevented aldosterone-induced increases in O2- in the MMDD1 cells. In the presence of MR antagonist eplerenone (10-5 mol/L), aldosterone-induced O2- production was blocked. Eplerenone itself had no significant effect on O2- levels in MMDD1 cells.3. COX-2, NOX-2 and NOX-4 are expressed in MMDD1 cellsBecause the MMDD1 cell line may have some subtle differences with macula densa cells isolated from the in vivo, we made sure the MMDD1 cells expressed COX-2, NOX-2 and NOX-4. The representative blot depicted in COX-2, NOX-2 and NOX-4 are expressed in the MMDD1 cells, and this result also verifies the efficacy of all of our primers to detect COX-2, NOX-2 and NOX-4.4. Aldosterone enhances COX-2, NOX-2 and NOX-4 mRNA and protein expressionsWe used different aldosterone concentrations (1 nmol/L and 10 nmol/L,) to stimulate the MMDD1 cells for 30 min. The result shows that exposing MMDD1 cells to aldosterone at different concentration enhances COX-2, NOX-2 and NOX-4. Aldosterone at 10-8 mol/L had the greater effect. Therefore, we used this concentration of aldosterone in the following experiment.We measured protein levels of COX-2, NOX-2 and NOX-4 with specific antibodies in MMDD1 cells stimulated with aldosterone (10-8 mol/L) for 30 min. The result in shows that aldosterone significantly increased protein levels of COX-2, NOX-2 and NOX-4.5. COX-2, NOX-2 and NOX-4 are a major source of aldosterone-induced O2-We next identified the major sources of aldosterone-induced O2 in MMDD1 cells. For this, we measured O2- levels in aldosterone-treated cells (10-8 mol/L) in the presence of the following inhibitors:NS-398 (10-6 mol/L) (inhibits COX-2), apocynin (10-5 mol/L) (inhibits NOX) or in the presence of both NS-398 (10-6 mol/L) and apocynin (10-5 mol/L). Apocynin, NS398 or both NS398 and apocynin completely prevented aldosterone-induced increases in O2- in the MMDD1 cells. In the presence of either NS-398 or apocynin, aldosterone-induced increases in O2- were blocked. These data suggested that COX-2, NOX-2 and NOX-4 were the primary sources of O2- produced by the MMDD1 cells during aldosterone stimulation.6. A siRNA knocked down COX-2 in the MMDD1 cellsTo study the function of COX-2 in aldosterone-induced O2- generation, we used a siRNA to silence COX-2. COX-2 siRNA significantly reduced its target COX-2 mRNA. Scrambled siRNA had no significant effect on COX-2 mRNA. Therefore, the results show that the COX-2 siRNA effectively knocked down the COX-2 mRNA. Next, we determined if this siRNA had any effect on NOX-2 or NOX-4 mRNA levels. COX-2 siRNA did not affect the NOX-2 mRNA expression or the NOX-4 mRNA expression. The scrambled siRNA had no effect on the NOX-2 mRNA expression or the NOX-4 mRNA expression. These data demonstrate the efficiency and specificity of COX-2 siRNA.7. COX-2 siRNA blocked the effect of aldosterone on COX-2, NOX-2 and NOX-4 mRNAs and O2- generation.To study the role and their interactions between COX-2 and NOX-2 or NOX-4 in aldosterone-induced increases, we knocked down COX-2 and measured NOX-2 and NOX-4 levels. Aldosterone (10"8 mol/L) stimulated COX-2, NOX-2 and NOX-4 mRNA expression. In the MMDD1 cells treated with COX-2 siRNA, aldosterone-induced increases in NOX-2 and NOX-4 mRNAs were blocked, COX-2 mRNA was reduced.To study the role of COX-2 in aldosterone-induced O2- generation, we knocked down COX-2 and measured O2-. Aldosterone (10-8 mol/L) enhances O2-.In the MMDD1 cells treated with COX-2 siRNA, aldosterone-induced increases in O2-generation were blocked.CONCLUSIONSThese data indicate a novel signaling pathway for aldosterone-induced O2-generation in the MD cells. Aldosterone stimulates COX-2, which further activates NOX-2 and NOX-4 and generates O2-.Part II The prooxidant effect of aldosterone in macula densa cells is mediated by PKCaOBJECTIVEAldosterone activates mineralocorticoid receptors (MR) in the colon and renal epithelial cells, especially the principal cells and intercalated cells of the late distal tubule, collecting tubules and collecting ducts, and an increase in electrolyte flux occurs. However, aldosterone also causes cardiac inflammation and increases in myocardial oxidative stress. We have recently studied the mechanisms by which aldosterone increases O2- production in MMDD1 cells, a renal epithelial cell line with properties of macula densa cells. This prooxidant effect occurred because of increases in COX-2 and NAD(P)H oxidase. However, the signaling method by which aldosterone effects changes in these prooxidants is unknown.PKC is a signal transduction protein that mediates rapid responses to steroid hormones. A recent study has found that aldosterone triggers both early and late increases in sodium transport in renal cortical collecting duct cells, and the PKCa signaling pathway mediated the effects of aldosterone on the collecting duct cells. However, whether the PKCa signaling pathway plays an important role in the aldosterone prooxidant effect on renal MD cells is not known. We hypothesize that aldosterone increases O2- production in MD cells by activating PKCa which stimulates NAD(P)H oxidase via NOX-2 and NOX-4 production. Our results indicate that MD PKCa and NAD(P)H oxidase are important mediators of the aldosterone prooxidant effect on MD cells.METHODSThe method followed that of part I.RESULTS1. NAD(P)H oxidase inhibition decreased aldosterone-stimulated O2- production.To confirm that aldosterone stimulates O2- production in MMDD1 cells, we exposed MMDD1 cells to 10 nM aldosterone for 30 min and measured O2- production. Aldosterone increased O2- production from a control value of 1293±106 RLU·s-1·105 cells-1 to 2349±222 RLU·s-1·105 cells-1.To test if the NAD(P)H oxidase system is involved in mediating the increase in O2- production in MMDD1 cells during exposure to aldosterone, we added the NAD(P)H oxidase inhibitor apocynin. Aldosterone response in the presence of apocynin was markedly reduced.2. General inhibition of PKC attenuated the aldosterone prooxidant effectTo determine if PKC is involved in aldosterone-induced O2- production, we exposed the cells to 10-7 M chelerythrine chloride (CC), a non-selective PKC inhibitor in the media. Aldosterone increased O2- production from a control value of 1184±54 RLU·s-1·105 cells-1 to 1982±138 RLU·s-1·105 cells-1. Addition of CC markedly blunted the aldosterone-induced O2- production. This suggests that one or more of the isoforms of PKC is a mediator of the prooxidant effect of aldosterone.3. Specific inhibition of PKCa attenuated the aldosterone prooxidant effectTo determine if PKCa is involved in aldosterone-induced O2- production, we exposed the cells to PKCa specific inhibitor,10-7 M Go6976 (Go). Aldosterone increased O2- production in MMDD1 cells. Addition of the specific PKCa inhibitor, Go, nearly completely inhibited any effect of aldosterone on O2- production. This suggests an important role of PKCa in the aldosterone prooxidant response.4. PKCa siRNA inhibited the aldosterone prooxidant effectPKCa siRNA was used to test the hypothesis that PKCa is a mediator of aldosterone-stimulated O2- production. PKCa siRNA markedly decreased PKCa mRNA of MMDD1 cells as determined with real-time PCR. This indicates that our PKCa siRNA was a suitable inhibitor of PKCa expression.To provide further evidence that PKCa is involved in aldosterone-induced O2-production, we exposed the cells to PKCa siRNA. Aldosterone caused a large increase in MMDD1 O2- production. Addition of a scrambled siRNA did not inhibit the aldosterone-induced O2- production. However, addition of a specific siRNA for PKCa markedly inhibited O2- production in aldosterone-treated cells.5. PKCa siRNA inhibited the increase in NOX-2 and NOX-4 proteinTo determine if PKCa enhanced NOX-2 and NOX-4 protein expression, we exposed MMDD1 cells to aldosterone with and without PKCa siRNA. Aldosterone increased NOX-2 and NOX-4 protein. Addition of PKCa siRNA completely inhibited the aldosterone-induced increase in NOX-2 and NOX-4 protein. These data suggest that the aldosterone-induced increases in NOX-2 and NOX-4 protein were mediated by increases in PKCa.CONCLUSIONSAldosterone-induced increases in MD O2- production are mediated by PKCa, PKCa stimulates MD NOX-2 and NOX-4 which generate O2-. The resulting balance between O2- and NO in the MD is important in regulating TGF.