The Protection And Mechanism Of N-acetylcysteine(NAC) To Renal Epithelial Tubular Cell Apoptosis Induced By Renal Ischemia/Reperfusion Injury In Rats

Background:Acute kidney injury (AKI) is a common and severe clinical problem. AKI involves sudden loss of renal function characterized by reduction in glomerular filtration rate along with rise in nitrogenous waste material in blood. Ischemia-reperfusion injury (IRI) is one of the leading causes of AKI. The renal transplantation, suprarenal aneurysms, contrast agent-induced nephropathy, and shock are the clinical conditions involving IRI that may lead to AKI. A primary factor in the initiation of the pathological response to reperfusion injury is the generation of high levels of reactive oxygen species (ROS). ROS-induced cell death has been reported in a wide variety of cells, including renal tubular epithelial cells (RTEs). The structural as well as functional damage of tubular epithelial cells contributes to interstitial inflammation, necrosis, and finally leading to renal dysfunction.The Nrf2pathway is one of the main adaptive responses activated under conditions of oxidative stress. Nrf2is a transcription factor responsible for the induction of phase II detoxification enzymes the primary role of which is to alleviate redox stress. Under normal conditions, Keap1binds to Nrf2, sequesters this transcription factor in the cytoplasm, and targets it for ubiquitination and proteasome degradation. Oxidative stress disrupts the Keapl-Nrf2complex, then Nrf2evades repression by Keapl and translocates to the nucleus, where it heterodimerizes with small Maf proteins and upregulates the expression of target genes at specific anti-oxidant response elements (ARE) within the regulatory regions of responsive genes.N-acetylcysteine (NAC) has been used as an antioxidant precursor to glutathione (y-glutamylcysteinylglycine; GSH) and previous report demonstrated that NAC could protect rat kidney tissue against reperfusion injury. However, the relationship between NAC-induced cytoprotection and signal transduction pathway has not been well elucidated, the goal of this study was to investigate whether NAC can protect the kidney against ischemic injury through Nrf2signaling pathway.Objective:1. To investigate the effect of N-acetylcysteine (NAC) on kidney function in Rats.2. To investigate the effect of NAC on cell apoptosis of ischemic injury.3. To investigate the effect of NAC on kidney cell apoptosis and possible mechanisms.Methods:1. Male Sprague-Dawley rats weighing200-260g were divided into the following groups:sham group; I/R group; I/R+saline-treated group; and I/R+NAC-treated group. Following induction of anesthesia (chloral hydrate,300mg/kg, intraperitoneally), bilateral clamping of kidney pedicles was performed using an atraumatic vascular clamp. During ischemia and reperfusion, rectal temperature was maintained at about37℃. The rats in the NAC-treated group and saline-treated group were pretreated with NAC (150mg/kg dissolved in0.9%saline, intraperitoneally) or an equal volume of saline for3days before ischemia. The sham operation was performed using the same surgical exposure procedures except for occlusion of the pedicles. Reperfusion was achieved by removing the clamp. The results of varying the duration of reperfusion were assessed by occluding the renal pedicles for45min and reperfusing the kidney for the indicated periods. Rats were sacrificed upon completion of ischemia/reperfusion.2. blood samples were collected, and analyzed for blood serum creatinine and blood urea nitrogen.3. Kidneys were immediately removed and rapidly frozen in liquid nitrogen or fixed in paraformaldehyde for TUNEL staining (TDT-mediated dUTP nick-end labeling method). 4. Using Western blot assay detect the protein expression of Nrf2and keapl.5. Using Western blot assay detect the protein expression of HO-1and NAPDH.Results:1. We foundd serum creatinine levels, an index of kidney function, increased to159u mol/L in the untreated group after24h of reperfusion, while in the treatment group, these levels declined to near normal level by24h of reperfusion. BUN levels in the untreated group increased to22mmol/L after24h of reperfusion. The BUN levels in the NAC treated group decreased to near normal level by24h of reperfusion.2. Malondialdehyde (MDA), the end product of lipid peroxidation, is a good marker of free radical-mediated damage and oxidative stress. MDA concentration in kidney tissue was measured, and observed to increase rapidly in the renal ischemia/reperfusion group compared to the sham group. In the NAC-treated group, the MDA level declined significantly compared to the renal ischemia/reperfusion group.3. We investigated the ability of NAC pretreatment to mediate protection against ischemia induced apoptotic cell death. Adult Sprague-Dawley rats were subjected to45min ischemia followed by24h reperfusion. After24h reperfusion, rats were perfusion-fixed with paraformaldehyde and TUNEL staining was employed to determine apoptosis of renal tubular epithelial cells. A significant number of TUNEL-positive cells were observed at24h reperfusion after ischemia, predominantly located at the distal tubules of the outer medulla, a few at the proximal tubules of the cortex, some shed into the renal tubular cave. Few TUNEL-positive cells were observed in the sham group. Administration of NAC significantly decreased TUNEL-positive cells as compared to the renal ischemia/reperfusion group, and vehicle-treated group did not show any protection.4. We examined the effects of NAC on the expression of Nrf2and downstream HO-1. Our result showed that pre-treatment of NAC resulted in a significant increase in Nrf2nuclear accumulation after3h, which was not observed in the ischemia/reperfusion group. Also, downstream gene HO-1was up-regulated by NAC. Meanwhile, The cleavage of pro-caspase3induced by ischemia/reperfusion were significantly suppressed by NAC. However, the same dose of saline had no effect on the expression of Nrf2, HO-1and pro-caspase3. Since it is well known that excessive ROS generation was responsible for activation of the P53, we further examined the effects of NAC on the expression of P53. Our result showed that NAC could inhibit the increased P53induced by renal ischemia/reperfusion.Conclusions:1. N-acetylcysteine (NAC) can inhibit the ubiquitination and proteasome degradation of Nrf2;2. N-acetylcysteine (NAC) can significant increase the Nrf2’s expression and upregulate the nuclear accumulation;3. N-acetylcysteine (NAC) can protect rat kidney tissue against ischemia-reperfusion injury by the activation of Nrf2-dependent antioxidant downstream gene expression.In summary we have demonstrated for the induction of Nrf2activation and Nrf2-dependent antioxidant gene expression in an in vivo model. We therefore postulate that specific activation of the Nrf2antioxidant pathway as a contributory mechanism to the ongoing and subsequent oxidant damage in IRI. We also present evidence for the involvement of NAC-specific activation of Nrf2. The results of this study support NAC preconditioning as a novel strategy to protect the kidney and other organs against IR injury, thus constituting an alternate preconditioning strategy with potential clinical application. Further investigation into Nrf2signaling pathways involved in this response may help our understanding of NAC preconditioning toward future therapeutic potential.