| BACKGROUNDAcute renal failure is one of clinical critical diseases and its incidence is related to factors such as hypoxia-ischemia. Since the clinical condition is often seen in neonates with hypoxia which will cause re-distribution of blood and renal ischemia injury,there is the occurrence of renal dysfunction.The dysfunction of urine concentration ability is contributed to renal failure.It has been confirmed that the transmembrane transport of water is related to AQPs and AQP1-4 are involved in urine concentration in renal tissue. Meanwhile, sodium-potassium adenosine triphosphatase is the main driver of exchanges of water and electrolyte in a kidney. On the basis, we intend to make neonatal rats unilaterally nephrectomized and subjected to 30min renal pedicle occlusion followed by different reperfusion including 0.5h,2h,4h,6h and build an animal model of neonatal rats with acute renal failure caused by ischemia/reperfusion injury,.This experiment aimed to understand changes of the expressions of AQP1-4 and the Na +-K +-ATPase activity in the kidney under condions of ischemia followed by reperfusion. ObjectivesTo duplicate the model of neonatal rats with ischemia-reperfusion injury (IRI), to observe dynamically ultrastructural changes of renal tissue and to explore the pathological changes of the expressions of AQP1,2,3,4 and Na +-K +-ATP activity in renal tissue as well as approaching the relations of them initially.Methods1. Duplication of IRI model36 newborn SD rats (age 7 days, weight 19.6±2.90g) were randomly divided into control group(group C,n=6) and experiment groups(group 0h,0.5h,2h,4h,6h,n=6) with male and female informal. There are 30 rats with unilateral renal ligation in experiment groups, 6 with only false operation (sham) treatment in control group. To product experimental neonatal rats, they were fixed in the operating table following intraperitoneal injection of sumianxin anesthesia, local disinfection (bromogeramine ) after the abdominal hair gone, abdominal incision along the ventral longitudinal midline.We first exposed the right kidney,isolated the right renal pedicle and ureter and ligated them by1-0 silk.Then we exposed the left kidney and isolated it, used non-invasive clamp to make 30min renal pedicle occlusion, slacken the clamp to restore renal perfusion, when the kidney indicated successful reperfusion by the bright red color to pale or dark red, and then into a bright red again; finally closed abdominal cavity with layer-by-layer suture after observating no surgical field bleeding and oozing. Experiment groups were divided into 5 sub-groups,according to reflow by 0h, 0.5h, 2h, 4h, 6h.Sham group were treated equally, such as anesthesia and opening abdominal cavity ,but the renal capsules of two kidneies were only exposed .2.Biochemical indices①Using transmission electron microscopy,to observe ultrastructural changes in renal tissue, reflecting the characteristics of cell damage of the renal tissue after ischemia/reperfusion injury.②To determinate Cr in serum, indicating the level of renal injury.③Using immunohistochemistry and RT-PCR,to detect the expressions of AQP1-4 in each samples.④By determination of phosphorus ,to judge Na +-K +-ATPase activity in each samples.Results11. From electronic microscope,it showed the microvilli of renal tubular cells in renal tissue injuried decreased, and part of renal tubular had the normal cell structure, while accompanied with the existence of apoptotic cells. Apoptotic cells had the performance of rarefaction, edema, vacuole formation of endochylema, as well as karyopyknosis.Some mitochondria of apoptotic cells swelled up, when its cristae blurred, broken and some areas of transparent matrix emerged, as well as mitochondrial space cavitated. Following the subsequent observation,the mount of apoptotic cells gradually increased and mitochondria cavitation became more obvious.2. In neonatal rats with ischemia-reperfusion injury (IRI), the blood serum of Cr gradually rose which suggested progressive renal injury.3. As to experiment groups, the expressions of AQP1-4 in renal tissue after ischemia-reperfusion was significantly lower than the control group, with statistically significant difference (P<0.01).There were also differences among subsequent timepoints and phase changes of both the expressions of AQP1-4 and ultrastructure of renal tubular cells were corrisponding;4. Na+-K+-ATPase activity were also constantly becoming lower after ischemia. The correlation analysis of expressions of AQP1-4 and Na+-K+-ATPase activity indicted strong relevance (R=0.79～0.92), and the greatest to AQP1. Conclusion1.Through the treatment of unilateral renal pedicle clamping and following blood restoration accompanied by contralateral renal pedicle ligation,the model of neonatal rats with IRI was set up successfully.Renal function of them had obviously descended after 30min ischemic injury and continued to fall after reperfusion.2. It was for ultrastructural changes of renal tissues with IRI that tubular apoptotic cells increased and mitochondria became cavitating gradually with the prolong of reperfusion. At the same time,the expressions of AQP1-4 in renal tissues had generally reduced, which proved the existence of the destruction of urine concentration ability. The Na +-K +-ATP activity in renal tissues also decreased after ischemia. There were strong correlations between expressions of AQP1-4 and Na +-K +-ATP activity. Not only this phenomenon confirmed the reabsorption of water and electrolyte is synchronous, but suggest the reasons for the changes of expressions of AQPs would be likely related to metabolic disorders and inflammatory mediators caused by IRI and the exact mechanism remains further study.
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