High glucose level and angiotensin II type 1 receptor stimulation synergistically amplify oxidative stress in renal mesangial cells

Figures

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  Figure 1. Fluorescence intensity of ethidium derived due to the oxidation of DHE by intracellular superoxide (Protocol 1). The fluorescence intensity reflects the levels of intracellular superoxide. The dihydroethidium (DHE) administered in the buffer penetrates cell membrane and is oxidized by superoxide anions to form ethidium (Eth), which emits red fluorescence (excitation filter: 480 nm wavelength; emission filter: 605 nm wavelength). All living cells, which were attached to the bottom of cover glass, were examined for the fluorescence emission after every 10 s. We estimated that the intracellular superoxide level was the highest after pre-conditioning with 13.5 mM glucose + Ang II. Abbreviations: Ang II, angiotensin II; DHE, dihydroethidium; Glu, glucose; mM, mmoL/L; RNH, angiotensin II type 1 receptor antagonist; s, seconds.

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  Figure 2. Chronological changes in levels of intracellular ethidium under different pre-conditionings (Protocol 1). The slope of intracellular ethidium signal from each living mesangial cell reflects the level of intracellular superoxide after each pre-conditioning (above: 5.5 mM glucose; below: 13.5 mM glucose). Ethidium signal was measured every 10 s after administering DHE. The signal slopes were significantly different in the presence of Ang II stimulation after pre-conditioning with 13.5 mM glucose, but were not different after pre-conditioning with 5.5 mM glucose. The error bar of each plot shows standard error of the measured ethidium signal. Abbreviations: Ang II, angiotensin II; DHE, dihydroethidium; n.s., not significant; RNH, angiotensin II type 1 receptor antagonist.

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  Figure 3. Chronological changes in levels of intracellular activated CM-H2DCFDA before and after angiotensin II stimulation (Protocol 2). In this protocol, acute response of H2O2 level against angiotensin II stimulation in mesangial cells was evaluated. The vehicle buffer containing only inactivated CM-H2DCFDA without angiotensin II did not increase the activated CM-H2DCFDA levels in any of the samples pre-conditioned with glucose. Once 10−7 M angiotensin II was added to the experimental buffer, intracellular superoxide level was rapidly enhanced within several minutes after changing the buffer at every glucose level, especially under high glucose levels (13.5 mM and 27.8 mM). The error bar of each plot shows standard error. Abbreviations: Ang II, angiotensin II; Glu, glucose; mM, mmoL/L; PEG-Cat; polyethylene glycol catalase; s, second.

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  Figure 4. Chronological changes in levels of intracellular activated CM-H2DCFDA in the presence of angiotensin II receptor antagonists (Protocol 2). Acute response of H2O2 levels against angiotensin II stimulation in mesangial cells was significantly enhanced by administration of angiotensin II type 2 receptor antagonist (PD), but was significantly reduced by administration of angiotensin II type 1 receptor antagonist (RNH). Based on this result, angiotensin II-induced generation of oxidative stress in mesangial cells was suggested to be mediated by angiotensin II type 1 receptor. The error bar of each plot shows standard error. Abbreviations: Ang II, angiotensin II; M, mol/l; PD, angiotensin II type 2 receptor antagonist; RNH, angiotensin II type 1 receptor antagonist; s, second.