| IntroductionIt is necessary to block renal circulation temporarily in some complicated renal operations such as nephropyelolithotomy and nephron sparing nephrectomy of renal tumors. How to attenuate renal ischemia-reperfusion injury is a highlight in nephroprotective studies. The unique progress of kidney repair after ischemia/reperfusion injury has been studied in the past few years. The cellular and molecular basis of repair process is elucidated in parallel with deeper insights on the mechanisms of injury. PI3K/Akt pathway has been shown to play a critical role in regulating mitogenic signaling, apoptosis, cell proliferation and survival in different systems. Several studies have reported that the PI3K/Akt pathway is involved in ischemic injury and repair of brain and heart. Recently, Andreucci et al reported that PI3K and Akt are activated after renal ischemic/reperfusion injury, which may affected epithelial cell fate in acute renal failure. However, it remains to clarify the PI3K/Akt pathway that are involved in renal ischemic injury and repair. The aim of our study was to examine the effects of PI3-kinase inhibitor wortmannin on the progressive renal injury, renal Skp2, p27 expression and cell proliferation that is induced by ischemia/reperfusion injury in mice, the changes of PI3K/Akt pathway, such as Akt, PDKl,GSK3-p and PTEN phosphorylation, to investigated the role of PI3K/Akt pathway involved in renal ischemia/reperfusion.Materials and Methods Animal protocolMale BALB/C mice, weighting 25-30g were used for all experiments. There were a total of 3 experimental groups. Sham-operative mice were examined 24 h day (n = 6),The control or wortmannin -treated IRI mice subjected to renal injury were similarly studied at 30, 90 minutes, 24, 48 hours(n = 6) following reperfusion. Wortmannin (1.14mg/kg) or its vehicle (0.1ml DMSO-EtOH) was given intraperitoneally at 4 hours before mice were subjected to surgery. The abdominal cavity was opened under anesthesia. Noncrushing vascular clamps were placed on both renal arteries and veins for completely blocking renal blood flow. The kidney was carefully visualized to ensure that the entire kidney blanched in response to the clamp. Thirty-five minutes later, the clamps were removed and blood flow returned to the kidneys. After removal of the clamp, reperfusion of the kidney was verified visually, and the abdominal wall was then sutured closed. The animals were allowed to regain consciousness and were given free access to food and water. The sham-operative animals underwent the same anesthesia and operation for 35 minutes. The kidneys were removed at 30, 90 minutes, 24, 48 hours following reperfusion in each group. The right renal tissue was immediately frozen in liquid nitrogen and then kept at -80℃ for assay, the left kidney was fixed with 4% neutral buffered paraformaldehyde for immunohistochemical analysis. Blood samples were also collected from main artery after reperfusion. Samples of serum were stored at -20℃ until measurement. Genera] methodsSerum creatinine values, serum BUN level were measured by Automatic Biochemistry Analyzer (Hitachi). Renal tissue fixed in 4% paraformaldehyde was processed by dehydration and embedded in paraffin. Sections were cut at 4 urn subjected to with hematoxylin and eosin-staining and periodic acid Schiff-staining for histological assessment. Examination and scoring of the sections wereperformed on a blinded basis. In the assessment of kidneys harvested after 24 h ,48h of reperfusion, the severity of renal damage in terms of morphological changes was scored with semiquantitative scale that was established by Paller . Reverse Transcription-PCR Analysis for Skp2Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD), and 2 μg of RNA from each sample were reverse transcribed using RevertAid? M-MiLV reverse Transcriptase (MBI Fermentas) in a total reaction volume of 20 μl. Two μl of reverse-transcription product (cDNA) was PCR amplified with AmpliTaq DNA polymerase (Promega) and 15 pmol e...
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