Interaction Between Nitric Oxide And Superoxide In The Macula Densa In Aldosterone-induced Alterations Of Tubuloglomerular Feedback

OBJECTIVEHypertension, identified by the World Health Organization, as the leading cause of cardiovascular mortality. It increases the risk of ischemic heart disease, strokes, peripheral vascular disease, and other cardiovascular diseases, including heart failure, aortic aneurysms, diffuse atherosclerosis, and pulmonary embolism.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. In the kidney, it has been clearly shown that increased O2-activity induces vasoconstriction and enhances tubular sodium reabsorptive function. NO is characterized as a major vasodilator agent regulating basal vascular tone and it also inhibits renal tubular transport of sodium and thus plays an important role in overall excretory function of the kidney.PKC is a signal transduction protein that mediates rapid responses to steroid hormones. In several cell types, PKC has been reported to be an important mediator in aldosterone-induced signaling pathway. However, whether the PKC signaling pathway plays an important role in the prooxidant effect of aldosterone in MD cells is not known.The present study was designed to test the hypothesis that aldosterone stimulates O2-production in the MD which, in turn, scavenges NO and buffers the effect of NO on the TGF response. The role of PKC in aldosterone-stimulated O2-production was also studied.Part I Aldosterone stimulates nitrio oxide and superoxide production in macula densaMATERIALS AND METHODSMMDD1Cells cultureExperiments were undertaken in MMDD1cells, a renal epithelial cell line with properties of MD cells,(developed and kindly provided by Dr. J. Schnermann, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Similar to our previously reported protocols, MMDD1cells at passages21-25were cultured in100*20mm style dishes in DMEM nutrient mixture-Ham’s F-12(DMEM/F12. supplemented with10%fetal bovine serum.100U/ml penicillin, and100μg/ml streptomycin) and incubated in a humidified atmosphere of95%room air-5%CO2at37℃NO Measurement When cells reached70%-80%of confluence(100*20mm dish), they were loaded with10μmol/L4,5-diaminofluorescein diacetate (DAF-2DA), a cell-permeable fluorescent NO indicator, in0.5%dimethyl sulfoxide (DMSO) for30minutes. Then, they were transferred to a glass chamber mounted on the inverted microscope, with the temperature maintained at37℃. The rate of increase in intensity was calculated (increase of units/min) and NO was measured. Then aldosterone (10-8M) was added into bath for10min. Time control experiments were performed using the same protocol without aldosterone treatment.O2-MeasurementWe measured O2-production in the MMDD1cells using a lucigenin-enhanced chemiluminescence assay. Briefly, When MMDD1cells reached70%-80%of confluence,(100*20mm dish), they were washed by PBS twice, trypsinized from the dish and kept in12ml Krebs/Hepes buffer. NAD(P)H and lucigenin was added to each of the samples, after aldosterone (10-8M) was added into one sample and incubated for30min at37℃with oxygen bubbling, O2-was measured. Time control experiments were performed using the same protocol without aldosterone treatment.Isolation and microperfusion of juxtaglomerular apparatus (JGA):We used methods similar to those we described previously to isolate and microperfuse the afferent arteriole (Af-Art) and attached MD. Briefly, young male New Zealand white rabbits were anesthetized with ketamine. The kidneys were removed and sliced along the corticomedullary axis. A single superficial Af-Art and its intact glomerulus were microdissected together with adherent tubular segments consisting of portions of the thick ascending limb of the loop of Henle (TAL), MD, and the early distal tubule under a stereomicroscope. The microdissected complex was transferred to a temperature-regulated chamber mounted on an inverted microscope. Both the Af-Art and the end of either the distal tubule or thick ascending limb were cannulated with an array of glass pipettes. Microdissections and perfusions were completed within60minutes at8℃, and the samples were then transferred into the bath and gradually warmed to37℃for the rest of the experiment. A30-minute equilibration period was allowed before taking any measurements.Measurement of O2-with dihydroethidium in perfused MD:We used a O2-sensitive fluorescent dye, dihydroethidium, to detect O2-levels as we recently described. Once the TAL was perfused, MD cells were loaded with10μM dihydroethidium in0.1%DMSO plus0.1%pluronic acid from the lumen for30min then washed for10min. The loaded MD cells were exposed to excite dihydroethidium and oxyethidium, respectively. oxyethidium/dihydroethidium as an indicator of O2-production. Since we previously found that increased luminal NaCl and aldosterone induce NO release by the MD. we added the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) to the bath and lumen while measuring O2-to eliminate its reaction with NO as we reported previously.To determine the time course of aldosterone-induced O2-in the MD, we added aldosterone in the tubule and measured O2-generation in the MD every15min for75min in the presence of L-NAME. StatisticsData were analyzed as repeated measures when multiple samples were 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. T-tests were used for single comparisons. The changes were considered to be significant if p<0.05. Data are presented as mean±SEM.RESULTSAldosterone stimulates NO production in cultured MMDD1CellsIn basal conditions, the rate of NO generation was40.4±4.3U/min. After adding aldosterone for15minutes, the rate of NO production increased significantly to644.1±118.5U/min. In time control experiments only with vehicle, the rate of increase in NO generation was45.9±5.2U/min at basal and53.4±6.1U/min15minutes later. Aldosterone stimulates O2-production in cultured MMDD1cells In group without aldosterone treatment, O2-levels were1293±106RLU·s-1.105cells-1in cultured MMDD1cells. It increased to2349±222RLU·s-1.105cells-1in group with aldosterone treatment for30min.Aldosterone stimulates O2-production in isolated perfused MDWhen the MD was perfused with NaCl from10mmol/L to80mmol/L or stimulated with aldosterone (10-8or10-7mol/L) for15min, we did not detect any changes in O2-generation in the MD. The intensity of signal in the MD was not significantly different from the background. To eliminate the reaction with NO, we added L-NAME (10-4mol/L) to the bath and lumen while measuring O2-in the following experiments. When the MD was perfused with80mmol/L NaCl, O2-generation was9.4±1.5units/min. Then aldosterone (10-7mol/L) was added in the tubule for15min, O2-generation increased to17.2±1.3units/min. Aldosterone at10-8mol/L had no significant effect on O2-generation.To determine the time course of aldosterone-induced O2-in the MD, we added aldosterone (10-/mol/L) in the tubule and measured O2-generation in the MD every15min for75min in the presence of L-NAME. we found that O2-generation in the MD increased correspondingly over time.CONCLUSIONSAldosterone could stimulates NO and O2-production in cultured MMDD1cells, and aldosterone also could stimulates O2-production in isolated perfused MD.Part Ⅱ Interaction between nitric oxide and superoxide in the macula densa which induced by aldosterone could alter tubuloglomerular feedbackMATERIALS AND METHODSInteraction between nitric oxide and superoxide in the macula densa in aldosterone-induced alterations of tubuloglomerular feedbackThe microdissected complex was transferred to a temperature-regulated chamber mounted on an inverted microscope, after the30-minute equilibration period, the MD perfusate was switched from10to80mmol/L NaCl, and luminal diameter of the Af-Art was observed for5minutes. We used the average change in diameter of the Af-Art as our control TGF response. Then the MD perfusate was switched back to10mmol/L NaCl.To study the effect of aldosterone on regulation of the TGF response in vitro, aldosterone was added to the tubular perfusate for15min and a TGF response was measured.To confirm the effect of the O2-scavenger tempol on TGF response,the O2-scavenger tempol was added to the tubular perfusate for15min and a TGF response was measured. To determine if the effect of tempol on TGF was consistent, we repeat above protocol.To determine whether scavenging O2-in the MD had any effect on aldosterone-induced TGF inhibition, we measured TGF response in the presence of both tempol and aldosterone.Signaling pathway of aldosterone-induced O2-generation in MDTo determine whether aldosterone-induced O2-generation is mediated by PKC. we used a PKC inhibitor and repeated above protocols in isolated perfused MD. A PKC inhibitor, CC, was added to the tubular lumen for30min in80mmol/L NaCl tubular solution and measured the O2-generation. Then aldosterone (10-/mol/L) was added to the tubular lumen for15min, and measured the O2-generation.To determine whether aldosterone-induced O2-generation is mediated by PKCa, we used a PKCa inhibitor and repeated above protocols in isolated perfused MD. A PKCα specific inhibitor, Go, was added to the tubular lumen for30min in80mmol/L NaCl tubular solution and measured the O2-generation. Then aldosterone (10-/mol/L) was added to the tubular lumen for15min, and measured the O2-generation.StatisticsData were analyzed as repeated measures when multiple samples were 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. T-tests were used for single comparisons. The changes were considered to be significant if p<0.05. Data are presented as mean±SEM.RESULTSScavenging O2-in the MD further attenuates the effect of aldosterone on the TGF responseFirst, we confirmed the effect of aldosterone on TGF response. when NaCl concentration in tubular perfusate was increased from10to80mmol/L, the Af-Art diameter decreased from17.5±0.5to15.1±0.5μm, and the TGF response was2.4±0.3μm.After aldosterone (10-/mol/L) was added to the tubular perfusate for15min. when NaCl concentration in tubular perfusate was increased from10to80mmol/L,the Af-Art diameter decreased from17.6±0.5to16.1±0.6μm and the TGF response was1.4±0.2μmAfter tempol (10-4mol/L) was added in the tubular perfusate for15min and was present during the experiment. when NaCl concentration in tubular perfusate was increased from10to80mmol/L, the Af-Art diameter decreased from17.2±0.5to15.6±0.4μm, and the TGF response was1.5±0.2μm. To determine if the effect of tempol on TGF was consistent, we repeat above protocol. When we increased tubular NaCl concentration from10to80mmol/L again, the Af-Art diameter decreased from17.3±0.6to15.8±0.7μm and the TGF response was1.4±0.2μm.To determine whether scavenging O2-in the MD had any effect on aldosterone-induced TGF inhibition, we measured TGF response in the presence of both tempol and aldosterone. In the presence of tempol (10-4mol/L) in tubular perfusate, when NaCl concentration was increased from10to80mmol/L, the Af-Art diameter decreased from18.1±0.5to16.7±0.4μm and the TGF response was1.4±0.3μm. In the presence of both tempol (10-4mol/L) and aldosterone (10-7mol/L) in tubular perfusate, when NaCl concentration was increased from10to80mmol/L, the Af-Art diameter decreased from18.4±0.6to18.1±0.7μm and the TGF response was0.4±0.2μm.PKCa is the signaling pathway of aldosterone-induced O2-in the MDA PKC inhibitor. CC (10-/mol/L), was added to the tubular lumen for30min in80mmol/L NaCl tubular solution. O2-generation was7.3±1.1units/min. Then aldosterone (10-/mol/L) was added to the tubular lumen for15min, O2-generation was5.9±1.7units/minA PKCa specific inhibitor, Go (10-/mol/L), was added to the tubular lumen for30min in80mmol/L NaCl tubular solution. O2-generation was7.9±1.2units/min. Then aldosterone (10-7mol/L) was added to the tubular lumen for15min, O2-generation was6.3±1.5units/min.CONCLUSIONSAldosterone-induced increases of O2-buffers the effect of NO in the MD in control of TGF responsiveness; and PKCa is an important mediator of the aldosterone-induced O2-production in MD cells.SIGNIFICANCEThese studies will greatly enhance our understanding of the mechanism of aldosterone in TGF regulating,salt-water metabolism and hemodynamics of kidney. And clear the role of aldosterone in hypertension and the hapertension related organ damage.