Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran’s effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO2− quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.
Background Diabetic nephropathy is a common diabetes mellitus complication associated with hypertension, proteinuria, and excretion of urinary plasmin that activates the epithelial sodium channel, ENaC, in vitro . Here we hypothesized that the deletion of plasminogen and amiloride treatment protect against hypertension in diabetes mellitus. Methods and Results Male plasminogen knockout (plasminogen‐deficient [Plg −/− ]) and wild‐type mice were rendered diabetic with streptozotocin. Arterial blood pressure was recorded continuously by indwelling catheters before and during 10 days of angiotensin II infusion (ANGII; 30–60 ng/kg per minute). The effect of amiloride infusion (2 mg/kg per day, 4 days) was tested in wild‐type, diabetic ANGII‐treated mice. Streptozotocin increased plasma and urine glucose concentrations and 24‐hour urine albumin and plasminogen excretion. Diabetic Plg −/− mice displayed larger baseline albuminuria and absence of urine plasminogen. Baseline mean arterial blood pressure did not differ between groups. Although ANGII elevated blood pressure in wild‐type, diabetic wild‐type, and Plg −/− control mice, ANGII did not change blood pressure in diabetic Plg −/− mice. Compared with ANGII infusion alone, wild‐type ANGII‐infused diabetic mice showed blood pressure reduction upon amiloride treatment. There was no difference in plasma renin, ANGII, aldosterone, tissue prorenin receptor, renal inflammation, and fibrosis between groups. Urine from wild‐type mice evoked larger amiloride‐sensitive current than urine from Plg −/− mice with or without diabetes mellitus. Full‐length γ‐ENaC and α‐ENaC subunit abundances were not changed in kidney homogenates, but the 70 kDa γ‐ENaC cleavage product was increased in diabetic versus nondiabetic mice. Conclusions Plasmin promotes hypertension in diabetes mellitus with albuminuria likely through the epithelial sodium channel.
There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated.Note that this technique is suitable for single dosing strategies.
Background T‐lymphocytes are important for angiotensin II (ANGII)‐hypertension in mice. T‐helper 17 cells produce the pro‐inflammatory cytokine interleukin‐17 (IL‐17). Plasma IL‐17A is increased in both hypertensive animals and humans. IL‐17A KO mice are protected against ANGII‐induced hypertension. Anti‐IL‐17 treatment of hypertensive mice decreases blood pressure. There is a lack of data directly documenting a hypertensive effect of IL‐17A. The objective of this study was to investigate the direct effect of IL‐17A on blood pressure in mice. It was hypothesised that continuous i.v. infusion of IL‐17A in mice will increase blood pressure and amplify ANGII‐induced hypertension. Methods A model of continuous IL‐17A i.v. infusion and blood pressure measurement was used by implantation of indwelling catheters in the femoral vein and artery. Femoral artery catheter was used for continuous blood pressure recording every 5 min and to collect undisturbed arterial blood in live non‐stressed mice. After a 5‐day recovery time upon operation, baseline blood pressure was measured for 3 days. Then, mice received IL‐17A at 0.1, 1.0 and 10 ug/day for 2, 2 and 4 days respectively. In another experiment, mice were given ANGII (60ng/kg/min) and IL‐17A (1 ug/day) or saline for 9 days. In an acute bolus experiment, mice received 10, 20, 40 and 80 ug IL‐17A in one bolus i.v. infusion with 10 min. interval. Continuous blood pressure measurements were obtained and EDTA blood was collected before and after infusion from all mice. Plasma IL‐17A levels was assessed before and after infusion using ELISA. Results IL‐17A dosis‐step‐up experiments with concentrations 0.1, 1.0 and 10 ug/day did not cause any blood pressure increase, instead blood pressure decreased significantly from 106 (102‐112) mmHg at baseline to 104 (99‐110) mmHg, 102 (98‐108) mmHg and 99 (95‐103) mmHg during IL‐17A infusion of dose 1, 2 and 3 respectively. Plasma IL‐17A levels of these mice after experiment increased significantly up to 2500 times compared to baseline levels from 0.6 (0.5‐1.1) pg/ml to 2523 (0.5‐8252) pg/ml. Also, IL‐17A did not accentuate ANGII‐induced hypertension. Instead, blood pressure was reduced in mice receiving ANGII with IL‐17A compared to ANGII and saline treated mice from 136 (130‐139) mmHg to 124 (121‐126) mmHg. Plasma IL‐17A was significantly higher in IL‐17A and ANGII treated mice compared to saline and ANGII treated mice. ANGII did not elevate plasma IL‐17A levels compared to baseline levels. In the dose‐step‐up experiment heart rate was significantly reduced from 661 (637‐585) BPM at baseline to 631 (604‐652) BPM at the highest dose given. Conclusion In summary, despite up to 2500 times increase in circulating IL‐17A from 2 different vendors and prolonged infusion for up to 9 days, no increase in blood pressure was observed during IL‐17 infusion alone, and IL‐17A did not accentuate ANGII effects neither. Instead a blood pressure reducing effect was observed. These data suggest that in complex models with inflammation, IL‐17A is likely to ...
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