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We tested the hypothesis that hypoxia-reoxygenation (H/R) augments vasoreactivity to angiotensin II (ANG II). In particular, we compared an in situ live kidney slice model with isolated afferent arterioles (C57Bl6 mice) to assess the impact of tubules on microvessel response. Hematoxylin and eosin staining was used to estimate slice viability. Arterioles in the slices were located by differential interference contrast microscopy, and responses to vasoactive substances were assessed. Cytosolic calcium transients and NADPH oxidase (NOX) mRNA expression were studied in isolated afferent arterioles. SOD activity was measured in live slices. Both experimental models were subjected to control and H/R treatment (60 min). Slices were further analyzed after 30-, 60-, and 90-min hypoxia followed by 10- or 20-min reoxygenation (H/R). H/R resulted in enhanced necrotic tissue damage compared with control conditions. To characterize the slice model, we applied ANG II (10-7 M), norepinephrine (NE; 10-5 M), endothelin-1 (ET-1; 10-7 M), and ATP (10-4 M), reducing the initial diameter to 44.5 ± 2.8, 50.0 ± 2.2, 45.3 ± 2.6, and 74.1 ± 1.8%, respectively. H/R significantly increased the ANG II response compared with control in live slices and in isolated afferent arterioles, although calcium transients remained similar. TEMPOL incubation prevented the H/R effect on ANG II responses. H/R significantly increased NOX2 mRNA expression in isolated arterioles. SOD activity was significantly decreased after H/R. Enhanced arteriolar responses after H/R occurred independently from the surrounding tissue, indicating no influence of tubules on vascular function in this model. The mechanism of increased ANG II response after H/R might be increased oxidative stress and increased calcium sensitivity of the contractile apparatus. [ABSTRACT FROM AUTHOR]

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 μM 14,15- EET for 30 min reduced phosphorylation of NKCC2 at the AVPsensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis. [ABSTRACT FROM AUTHOR]

Females are naturally protected against ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) in various clinical and experimental settings. However, the underlying mechanisms are unknown. We hypothesized that female protection may be conferred by enhanced production of cytochrome P450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) that promote vasodilation as well as antiinflammatory and antiapoptotic pathways in the kidney. To test this hypothesis, we first analyzed the renal CYP-eicosanoid profile by liquid chromatography tandem mass spectrometry in male and female Lewis rats. Ischemia was induced through 45 min of left renal vessel clamping after right nephrectomy (n=6-8 per group). In non-ischemic controls, male and female kidneys stored almost identical amounts of EETs as well as 20-hydroxyeicosatetraenoic acid (20-HETE), both predominantly esterified into phospholipids, under basal non-ischemic conditions. 45 min of ischemia induced a massive release of EETs from membrane stores in females but not males. The free renal EET-levels reached 70.2±20.1 in females compared to only 4.6±1.3 ng/g in males. After ischemia, the ratio of free EETs to free 20-HETE was about 1:1 in females and 1:3 in males. Next, we proved the functional importance of EETs in renal protection by pretreating males with a synthetic EET-agonist (12-HUDE) and females with a selective EET-antagonist (14,15-EEZE-mSI). As analyzed two days after reperfusion, the EET-agonist protected males against loss of creatinine clearance (1.03±0.18 vs. 0.26±0.02 ml/min, p<0.01 vs. vehicle, compared to 1.28±0.06 ml/min in sham control). Females were rendered susceptible to I/R-injury by the EET-antagonist (creatinine clearance: 0.25±0.05 vs. 0.67±0.04; p<0.01 vs. vehicle, compared to 0.81±0.04 ml/min in sham control). Changes in inflammatory cell infiltration and tubular apoptosis paralleled these effects on renal function. Our results indicate that female rats are protected against renal I/R-injury by enhanced ischemia-induced EET-release and demonstrate that renal protection can be transferred to males using synthetic EET-agonists. [ABSTRACT FROM AUTHOR]