NO₂-AA improves mitochondrial function in ANG II stimulated HK-2 cells.

<p>(A)HK-2 cells (1x10⁶ cells, black line) were pre-incubated with vehicle, 5 or 10 μM NO₂-AA or 10 μM AA and then treated with 0.1 μM ANG II for 3 h.Oxygen consumption was recorded at 37°C in intact cells using high resolution respirometry (OROBOROS Oxygraph-2K). Arrows indicate steps in the titration regime, inducing the following respiratory states: Oligomycin, inhibition of ATP syntase; FCCP, maximal stimulation by uncoupling of oxidative phosphorylation, and antimycin A, inhibition of complex III. Respiratory control ratio (RCR) values(B), maximal respiratory rate (C) and spare respiratory capacity(D) were determined as explained in Methods section and plotted as the mean ± SEM, <i>n</i> = 4. * p<0.05 relative to control cells; # p<0.05 relative to ANG II-treated cells.</p

NO₂-AA spares SDH and ATPase in kidney mitochondria.

<p>Isolated mitochondria, enriched with either AA or NO₂-AA as described in methods, were exposed to peroxynitriteand both SDH(A) and ATPase(B) specific activities determined. Results are representative of three independent experiments and correspond to the mean ± SD, n = 3. Controls of NO₂-AA addition in the absence of peroxynitrite addition were included for both complexes activities. * p<0.05 data relative to control mitochondria; # p<0.05 relative to peroxynitrite-treated mitochondria.</p

Schematic representation of mitochondrial dysfunction modulation by NO₂-AA in a cellular model of kidney cells activated by ANG II.

<p>On the left of the diagram, the stimulation with ANG II in HK-2 increases the O₂^.- production as well as inducible NOS expression and formation of peroxynitrite. This highly oxidizing molecule produces a decrease in the activities (red line) of the respiratory chain complex SDH (complex II) and ATPase (complex V) as well as increases oxidation and nitration of mitochondrial proteins. Therefore, HK-2 exposed to ANG II exhibited mitochondrial dysfunction. The right side of the diagram represent the modulation of cell damage by the nitroalkene. In the presence of NO₂-AA a reduction of ANG II-induced HK-2 damage is produced, with lower extents of O₂^.- production as well as lower levels of inducible NOS expression leading to a decrease in peroxynitrite formation. The activities of the respiratory chain complex are restored and NO₂-AA also prevents oxidation and nitration of mitochondrial proteins. In summary, NO₂-AA modulates ANG II mediated oxidative damage improving mitochondrial function.</p

NO₂-AA decrease NOS expression in ANG II stimulated HK-2 cells.

<p>(A)Western blot of NOS expression in ANG II-treated HK-2 cells (1x10⁶ cells) pre-incubated 30 min with vehicle, Losartan or NO₂-AA was analyzed. (B)Densitometric analysis of the bands was performed and the % of the relative density of NOS to β-actin of the observed bands was plotted as the mean ± SD, n = 3. * express significant differences relative to either control or NO₂-AA treated cells (p<0.05).</p

Modulation of ANG II- mediated peroxynitrite production by NO₂-AA_.

<p>HK-2 cells (1x10⁶ cells) were treated as previously and exposed to 30 μM Fl-B for 3 h. The fluorescence of Fl-B was followed by flow cytometry. Representative histograms are shown for controls compared to the ANG II stimulated cells in the absence (A) or presence of 10 μM AA (B) or 10 μM NO₂-AA (C). (D) Quantitative analysis of the histograms was performed determining a M1 region that corresponded to the cell population that exhibits high fluorescence due to Fl-B oxidation by peroxynitrite. A control with the nitroalkene in the absence of stimulation with ANG II was included as a control. *, # express significant differences respect to control and ANG II-treated cells, respectively (p<0.05).</p