Hypoxia/re‐oxygenation increases the response of afferent arterioles to angiotensin II ‐ Investigation in mouse kidney slices

Abstract

Hypoxia/re‐oxygenation (H/R) plays an important role in the pathogenesis of acute kidney injury (AKI). However, the influence of H/R on cortical microvessel function, especially that of afferent arterioles, is sparsely investigated. We hypothesize that H/R increases the vasoreactivity to angiotensin II (Ang II), which may promote the development of kidney injury. To test the hypothesis, we applied an innovative in situ model of live kidney slices.Experiments were performed in acute kidney slices (200 μm) of C57BL/6 mice. The TUNEL assay was used to estimate viability of slices by determining the percentage of apoptotic cells. For functional experiments, kidney slices were situated in a chamber at 37°C. Bath solution was equilibrated with carbogen. The afferent arterioles were visualized by differential interference contrast applied to an inverted microscope. To characterize arteriolar reactivity in this model of kidney slices, Ang II, norepinephrine, endothelin‐1 and ATP were administered. For H/R experiments, the slices were transferred into a hypoxia chamber (1% O2) for 30, 60 or 90 minutes and re‐oxygenated for 10 or 20 minutes.H/R did not significantly influence the apoptotic cell portion in the slice compared to control conditions. Afferent arterioles responded to the bolus application of Ang II (10−7 mol/l) with a maximum constriction, to 44.5 ± 2.8 % of the initial diameter. Norepinephrine (10−5 mol/l), endothelin‐1 (10−5 mol/l) and ATP (10−5 mol/l) reduced the diameter under control conditions to 50.0 ± 2.2%, 45.3 ± 2.6% and 74.1 ± 1.8, respectively. Inhibition of the NO‐synthesis by L‐NAME (10−4 mol/l) significantly enhanced the vasoconstriction to Ang II (32.3 ± 2.3 %). H/R increased the Ang II response of afferent arterioles compared to control conditions. Longer periods of hypoxia were associated with greater arteriolar vasoconstriction to Ang II (30/20 vs. 90/20: 39.7 ± 4.1% vs. 25.9 ± 2.0 %.). Also, increasing the duration of re‐oxygenation resulted in increased vasoconstriction (90/10 vs. 90/20: 34.7 ± 3.5% vs. 25.9 ± 2.00%).TUNEL staining and functional testing with vasoactive substances indicates the usefulness of the in situ model for the investigation of afferent arteriolar function. The increased response to Ang II in H/R suggests this effect as one component in the multifactorial genesis of AKI.Support or Funding InformationGerman Research Foundation (DFG PA 479/10‐2)