Hypoxia-reoxygenation enhances murine afferent arteriolar vasoconstriction by angiotensin II.

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.

Endothelial cell migration, adhesion and proliferation on different polymeric substrates.

BACKGROUND: The formation of a functionally-confluent endothelial cell (EC) monolayer affords proliferation of EC, which only happens in case of appropriate migratory activity. AIM OF THE STUDY: The migratory pathway of human umbilical endothelial cells (HUVEC) was investigated on different polymeric substrates. MATERIAL AND METHODS: Surface characterization of the polymers was performed by contact angle measurements and atomic force microscopy under wet conditions. 30,000 HUVEC per well were seeded on polytetrafluoroethylene (PTFE) (θadv = 119°±2°), on low-attachment plate LAP (θadv = 28°±2°) and on polystyrene based tissue culture plates (TCP, θadv = 22°±1°). HUVEC tracks (trajectories) were recorded by time lapse microscopy and the euclidean distance (straight line between starting and end point), the total distance and the velocities of HUVEC not leaving the vision field were determined. RESULTS: On PTFE, 42 HUVEC were in the vision field directly after seeding. The mean length of single migration steps (SML) was 6.1±5.2 µm, the mean velocity (MV) 0.40±0.3 µm·min-1 and the complete length of the trajectory (LT) was 710±440 µm. On TCP 82 HUVEC were in the vision field subsequent to seeding. The LT was 840±550 µm, the SML 6.1±5.2 µm and the MV 0.44±0.3 µm·min-1. The trajectories on LAP differed significantly in respect to SML (2.4±3.9 µm, p < 0.05), the MV (0.16±0.3 µm·min-1, p < 0.05) and the LT (410±300 µm, p < 0.05), compared to PTFE and TCP. Solely on TCP a nearly confluent EC monolayer developed after three days. While on TCP diffuse signals of vinculin were found over the whole basal cell surface organizing the binding of the cells by focal adhesions, on PTFE vinculin was merely arranged at the cell rims, and on the hydrophilic material (LAP) no focal adhesions were found. CONCLUSION: The study revealed that the wettability of polymers affected not only the initial adherence but also the migration of EC, which is of importance for the proliferation and ultimately the endothelialization of polymer-based biomaterials.

Hypoxia/Reoxygenation of Rat Renal Arteries Impairs Vasorelaxation via Modulation of Endothelium-Independent sGC/cGMP/PKG Signaling.

Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N2, 5% CO2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O2, 5% CO2). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels KCa3.1 and KCa2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.

Short-term hypoxia and vasa recta function in kidney slices.

BACKGROUND: Descending vasa recta (DVR) supply the inner part of outer renal medulla an area at risk for hypoxic damages. OBJECTIVE: We hypothesize increased vasoreactivity after hypoxia/re-oxygenation (H/R) in DVR, which might contribute to the reduced medullary perfusion after an ischemic event. METHODS: Live kidney slices (200µm) from SD rats were used for functional experiments. TUNEL assay and H&E staining were used to estimate slice viability. Kidney slices were treated with carbogen or hypoxia (1% O2) for 60 or 90 min and vasoreactivity to Ang II (10-7 M) was recorded by DIC microscopy after re-oxygenation with carbogen. Expression of NOS and NADPH enzymes mRNA were determined in iron-perfusion isolated VR. RESULTS: Percentage of apoptotic cells increased in control and H/R after 90 min in the medulla. Ang II- induced constriction of DVR was reduced after 90 min in control (compared to 60 min), but not after H/R. NOS enzymes mRNA expression levels decreased over 90 min hypoxia. CONCLUSIONS: Increased reactivity of DVR to Ang II after H/R compared to control (90 min) suggest a role of DVR in renal ischemia/reperfusion injury.