The Significance And Changes Of AQP2 In The Graft Of Acute Rejection Rat Renal Transplantation Model

Aquaporins (AQPS) are a transmembrane protein family which is found recently. The water which is necessary to the cell metabolism is transported by the AQPs which have the high selectable ability to water crossing biological membranes specifically. By far, two ways of water transmembrane transportation are accepted, one way is diffusion and the other is by AQPs, and the second mode is the main effect on water trafficking through the biological membranes. The chief organ modulating water balance of the whole body is the kidney which expresses most subtypes of AQPs. The water dynamic equilibrium depends on the regulation of kidney collecting duct (CD). The regulation of water permeation in CD involves vasopressin receptors and AQP2. Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane is critical to water reabsorption in renal collecting ducts and its regulation maintains body water homeostasis.The ratio of rejection after renal transplantation is decreased significantly for the administration of new immunosuppressant and improvement of zygosity. However, acute rejection (AR) is both the main complication of renal transplantation and the critical risk factor that causes chronic rejection and dysfunction of the graft. Therefore, it is a future study field that detects AR exactly and promptly by invasive or non-invasive methods. For this reason, it might be helpful for both the diagnosis and therapy of AR and long term survival time of the graft that to have a deep study on the underlying cellular and molecular mechanism caused by AR in the graft.Renal transplantation in humans is often accompanied by different levels of water and sodium detention, and then the salt and water homeostasis are disturbed. The water homeostasis relates to the AQPs closely, and the collecting system is the key part of urine concentration. Among the AQPs family, AQP2 is the primary target for vasopressin regulation of collecting duct principal cells and involved in antidiuretic hormone-depending water permeability. Therefore, it becomes a heating research that to learn the changes of AQP2 and its significance in the graft of AR.The establishing of renal transplantation rat model50 pairs of male inbred strain Wistar rats were both the donors and receptors who were forbidden to food but accessed to water before the operation. The renal transplantation was operated in a sanitation but non-sterile environment with constant temperature of 25℃and relative humidity of 60%. The kidneys and vessels of the donor and the recipient were approached through an abdominal midline incision. The excision of the donor's left kidney was done after low-temperature perfusion in situ under the anesthesia by peritoneal injection of 10% chloral hydrate by applying microsurgical techniques. Recipient s kidney was removed also by applying microsurgical techniques. The left kidney of the donor was transferred immediately to the recipient. The transferred material included the ureter and the renal artery and the renal vein. The renal artery and ureter of the donor was sutured to the recipients'end-to-end. The renal veins were sutured with the aid of cuff bolster. Results showed that the graft turned cherry immediately after re-perfusion, the engorged renal artery impulse was good and urine was formed 3-5 min after blood current rebuilt. Total operation time was (100±20)min; heat ischemia time of the grafts did not exceed 10 sec and cold ischemia time did not exceed 40 min. The achievement ratio of transplantation exceeded 86%.The establishing of renal transplantation acute rejection rat model50 pairs of male inbred strain Wistar rats and 30 pairs out-bred strain SD rats were donors and receptors. Control group was normal male Wistar rats; syngeneic renal transplantation control group (sTX) was the Wistar rats acted as both donors and recipients; allogenenic renal transplantation (aTX) was the SD and Wistar rats acted as donors and recipients respectively. Immunodepression group(aTX+CsA) was aTX with the administration of ciclosporin A. Results showed that compared with the control group, 3, 5 and 7 d after TX, the semi-quantity scores of AR in aTX group were increased significantly(p<0.01); the semi-quantity scores of AR in sTX group were increased also, but no statistics difference (p>0.05). compared with the sTX group and aTX+CsA, 3, 5 and 7 d after TX, the semi-quantity scores of AR in aTX group were increased significantly(p<0.01).In conclusion, the kidneys with the Wistar rats acting as both donors and receipts could play the role of control to renal transplantation acute rejection rat models because of the lagging AR and low degree of AR; so the kidneys with SD and Wistar rats acting as donors and receipts respectively were the renal transplantation acute rejection rat models. The AR could be abated or suppressed by the support of CsA.The significance and changes of aquaporin 2(AQP2) in rat model in the graft of acute rejection rat renal transplantation modelBased on the establishment of the renal transplantation acute rejection rat models, RT-PCR technique was employed to assess the changes of AQP2 mRNA and immunohistochemistry and Western-blot immunoblotting of proteins techniques were applied to detect the expression of the protein of AQP2 in renal transplantation acute rejection rat models.The results of RT-PCR indicated that AQP2 mRNA in aTX group were decreased remarkably with the comparison to control group 3, 5 and 7 d after the TX (p<0.01).In aTX group, AQP2 mRNA expression was down regulated notably 5 and 7 d after TX with the comparison to that 3 d after TX (p<0.05). There were no significant changes of AQP2 mRNA expression in sTX and aTX+CsA groups with the comparison to that in control group 3, 5 and 7 d after TX (p>0.05).Results of immunohistochemistry and Western- blot immunoblotting of proteins showed that with the comparison to control group, the expression of AQP2 protein in aTX group was down regulated significantly 3, 5 and 7 d after TX (p<0.01). In aTX group, AQP2 protein expression was down regulated notably 5 and 7 d after TX with the comparison to that 3 d after TX (p<0.05). There were no significant changes of AQP2 protein expression in sTX and aTX+CsA groups with the comparison to that in control group 3, 5 and 7 d after TX (p>0.05).These results in short were: AQP2 expression in aTX group was decreased significantly compared with control group(p<0.01), and it's expression 5 and 7 d after TX was also decreased significantly compared with that 3 d after TX (p<0.05). in conclusion, AQP2 expression was decreased under the AR of renal transplantation and the downregulation was paralleled with the severe level of AR.In order to excluded the effects of ischemic-reperfusion and denervation on the down regulation of AQP2, inbred strain Wistar rats were employed for establishment of sTX group. There were no statistic significance on AQP2 expression in sTX group compared with control group (p>0.05). The results provided a proof that there was no relationship between the AQP2 downregulation and ischemic-reperfusion and denervation.Also, in order to prove that the AR caused the AQP2 expression depression, aTX+CsA group was established which showed that the depression of AQP2 expression was lessened or became normal accompanied by lessen AR or reversed AR by histomorphology. Some papers showed that to use CsA alone was not able to increase the AQP2 protein expression in kidney; therefore, it is AR after TX results in the downregulation of the AQP2 protein expression in grafts.To sum up, AQP2 plays a critical role on urine condensation. The expression of both mRNA and protein of AQP2 was decreased markedly when AR occurred. It explained that the condensation function of collecting system was impaired by AR which caused less urine reabsorbing, and it was helpful to abate the water retention. Ana Velic considered that reduced expression of AQP2 accompanied by reduced sodium ion channel of collecting duct epithelial cells which may be protective means to save energy, support recovery of the stressed organ and protect the transplant from further non-immunologic and immunologic damages as seen in the proximal tubule, benefit to the recovery of the function of the graft.