Next, samples were incubated with biotinylated anti-mice or anti-rabbit immunoglobulins for 15 min at 37C. hemorrhage in renal tissue after HRSS Rabbit Polyclonal to p90 RSK treatment compared to saline treatment. After I/R injury, BUN, Cr, Bcl-2, caspase-3, caspase-9, caspase-8, IL-6, and TNF- were all significantly increased, while Bax expression was decreased. HRSS remarkably reversed these changes. Moreover, BUN and Cr decreased more rapidly in the rats treated with HRSS compared to the rats treated with control saline solution. Conclusions: HRSS showed a protective effect in the prevention of renal injury and could promote renal function recovery after I/R injury in rats. HRSS might partially exert its role through an anti-apoptotic and anti-inflammatory action in kidney cells. = 30); (2) I/R rats receiving vehicle physiological saline solution treatment (I/R group, = 30); (3) I/R rats receiving HRSS treatment at 4 h intervals for a total of 108 h (long-term treatment group, = PF-06751979 30); (4) I/R rats receiving HRSS treatment for 24 h and then vehicle physiological saline solution treatment for 84 h (short-term treatment group, = 30). In the control group, rats received a sham operation, followed by a continuous intraperitoneal administration of 0.9% NaCl solution (1 ml/kg). In the I/R group, the right kidney was surgically removed, and the left renal artery was occluded. This operation was followed by intraperitoneal administration of 0.9% NaCl solution (1 ml/kg) at 4 h intervals. Left renal artery occlusion was performed for 45 PF-06751979 min under pentobarbital anesthesia. In the HRSS-I/R groups, the right kidney was surgically removed, and the left renal artery was occluded (Shingu et al., 2010; Wang et al., 2011). This was followed by intraperitoneal administration of HRSS (1 ml/kg) immediately after reperfusion at 4 h intervals. Occlusion of the left renal artery was performed as described above. Rats received saline solution or HRSS immediately after reperfusion at 4 h intervals for a total of 108 h. The animals were anesthetized by intraperitoneal injection of pentobarbital (Biomics Laibo, Beijing; 0.7 mg/kg) before any surgical treatments. Analysis of Renal Function Blood urea nitrogen and Cr were measured to evaluate renal function. Samples were analyzed using commercial kits (Sigma, St. Louis, MO, USA) and a COBAS Mira chemical analyzer (Roche, Basel, Switzerland). Histological Analyses Renal samples were embedded in paraffin, cut into 4 m sections and stained with hematoxylin and eosin (H&E). Samples evaluation was performed under light microscopy by experienced technicians blinded to the groups. These expert observers made all assessments. To assess tubular injury, the percentages of tubules in the outer medulla and corticomedullary junction with tubular cell necrosis, cytoplasmic vacuole formation, hemorrhage and tubular dilatation were measured. Semi-quantitative injury scores ranged from 0 to 5 (0: normal kidney; 1: 0C10% injury; 2: 11C25% injury; 3: 26C45% injury; 4: 46C75% injury; 5: 76C100% injury). Apoptosis Assay Kidneys were perfused with PBS (50 ml) using the transcardiac approach, followed by 4% phosphate-buffered formalin. Perfusion-fixed kidney tissues were further fixed overnight in a solution of 4% paraformaldehyde in PBS, embedded in paraffin and cut into 4 m serial sections. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed on paraffin embedded sections by using an cell death detection kit (Roche, Indianapolis, USA). According to standard protocols, the sections were PF-06751979 dewaxed and rehydrated by heating the slides at 60C. Then, these sections were incubated in a proteinase K working solution (20 mg/ml) for 15 min at room temperature. The slides were rinsed three times with PBS before the incubation in the TUNEL PF-06751979 reaction mixture for 1 h at 37C. The area around the sample was dried by filter paper, and Converter-AP was added to the samples for 1 h at 37C. After rinsing with PBS (5 min, three times), sections were stained with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) until they became dark. Four slide fields were randomly examined using a.