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The Function and Mechanism of Endoplasmic Reticulum Stress Pre-treatment on the Improvement of Renal Tolerance Toward Ischemia-reperfusion Injury

Author: JiZhiYong
Tutor: HeZuoNi
School: Third Military Medical University
Course: Internal Medicine
Keywords: Acute kidney injury ischemia-reperfusion endoplasmic reticulum stress pre-treatment inflammation
CLC: R692.5
Type: Master's thesis
Year: 2010
Downloads: 19
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Abstract

Acute kidney injury (AKI) is a common clinical syndrome as well as a kind of complication in critically-ill patients, if the AKI develops into severe acute renal failure (ARF), the mortality rate will reach up to 50%, and 10% -14% of patients with acute renal failure need for lifelong renal replacement therapy since the renal function cannot be restored. Ischemia-reperfusion injury is the one of the causes of acute kidney failure, which often occurs after the kidney transplantation, cardiopulmonary bypass surgery and the context of trauma, hemorrhagic shock and sepsis etc. Currently, there is no other effective method except renal replacement treatment. So the illustration of ischemic AKI mechanism and the exploration of effective preventive and treatment measures have scientific significance in reducing the incidence and mortality of AKI.Essentially, the response of cells to ischemia, hypoxia, and oxidative stress, inflammatory and other damaging factors is a kind of stress response. Cell stress is an important reason for cell dysfunction or death. Endoplasmic reticulum is the largest cell organelle and undertakes the most important physiological functions, including post-translational modification, folding, assembly of newly synthesized secretory proteins and membrane protein. The normal endoplasmic reticulum function is the most basic criteria in cells’survival and implementation of the physiological functions. It is confirmed that once the cell is in the condition of unfolded protein aggregation, protein overload and intracellular calcium homeostasis imbalance caused by factors such as lacking of oxygen, sugar and energy shortage in the endoplasmic reticulum, the endoplasmic reticulum stress (ERS) mechanism will be activated. Appropriate ERS will, by the signal of PERK (PKR-like ER kinase) / eIf2 (eukaryotic translation initiation factor 2), IRE1α(inositol requiring kinase 1) / XBP1 (X-box binding protein-1) and ATF6 (mRNA encoding transcription factors) , slow the process of new protein synthesis and inhibit gene transcription; increase the expression of endoplasmic reticulum glucose-regulated protein -78 (Glucose-regulated protein 78, GRP-78) and heat shock protein -70 and other molecules to facilitate the proper folding, modification and transportation of protein, thus to maintain the stable condition in the endoplasmic reticulum to make the cells from the damage of all kinds of stress. When the ERS is too strong or the duration is too long and a steady-state within the ER cannot be re-established, then we shall achieve the goal of apoptosis through activating the downstream effects of channels, including ATF4/CHOP (also known as growth arrest and DNA damage inducible gene 153, GADD153), ATF6/CHOP, Caspase-12 and Ask1/JNK. Furthermore, excessive ERS can also activate NF-κB and infalmmatary caspase signaling pathway to induce non-infectious inflammation. It is confirmed that excessive ER stress is an important mechanism of acute ischemic heart and brain tissue damage. In the kidney, ischemia and reperfusion will make the apoptosis of renal tubular epithelial by means of increasing ATF4/CHOP. It is considered that ERS is the most critical aspect in the response mechanism of cells. It will regulate the expression of protection endoplasmic reticulum chaperone and effective molecules of endoplasmic reticulum and enhance the tissue damage tolerance. It has been found that the pre-handling of low doses of inducer in endoplasmic reticulum can be expressed by increasing renal endoplasmic reticulum chaperones Glucose-regulated protein 78, GRP78 and heat shock protein etc. and significantly reduce the renal ischemia reperfusion injury, which indicates that the endoplasmic reticulum stress pre-handling can give full play to the kidney through increasing ER chaperone. So whether the pretreatment of the endoplasmic reticulum can be through inhibiting the excessive endoplasmic reticulum stress to protect kidney from ischemia-reperfusion injury?Renal tubular epithelial cell apoptosis and inflammatory response is an important pathophysiological basis of acute ischemic renal injury. It is confirmed that acute ischemia-reperfusion starting over-endoplasmic reticulum stress is an important mechanism of a tubular epithelial cell apoptosis, but the function and mechanism of endoplasmic reticulum stress in acute ischemic renal injury is not clear. IL-1βis an early inflammatory response generated by pro-inflammatory cytokines, with the important biological function of inflammatory reaction with amplification cascade as well as a necessary role in the occurrence and development of acute ischemic renal injury. Studies show that under the condition of endotoxin lung injury, acute pancreatitis and colitis and other diseases, excessive ER stress will activate inflammation and IL-1β-converted enzyme (caspase-1) by up-regulating the expression of caspase-CHOP so as to promote IL Activation-1βprecursor to the mature IL-1β. Our previous study found that acute ischemia can up-regulate the expression of CHOP and renal IL-1βlevels, while inhibiting CHOP expression can reduce IL-1βproduction, which indicated that the increased expression of CHOP may be an important mechanism of inflammation of acute ischemic renal injury. This research will further study renal CHOP of acute ischemia-reperfusion and caspase expression and the laws of inflammatory changes in the IL-1β. It will also observe the effects of neomycin pretreatment on CHOP and renal inflammatory response, which will provide a scientific basis for the stress pre- treatment and prevention of acute ischemic renal injury in endoplasmic reticulum.Empirical Methods1. The effects of acute ischemia and reperfusion on renal expression of CHOP and its downstream molecules.The renal pedicle clamping ischemia-reperfusion, I / R injury model is adapted. We take an artery clip without damages and clamp the bilateral renal for 40min and release bilateral pedicle artery reperfusion in order to maintain the body temperature of insulation blanket. Then, we will test Cr, BUN level in the blood samples at the intervals of 1, 6, 12, 24 h respectively. The PAS staining renal pathology will change and Renal tissue injury was measured by PAS stain.The expression of GRP78, CHOP, caspase 11, caspase 1, IL-1βprotein in the ischemic renal tissue was detected by immunohistochemistry2. The function and mechanism of endoplasmic reticulum stress pre-treatment on the improvement of renal tolerance toward ischemia-reperfusion injuryThe renal pedicle clamping ischemia-reperfusion, I / R injury model is adapted. After a-40mins’renal pedicle clamping, we will open the bilateral artery and reperfusion for 12h. Rats were randomly divided into 5 groups: control group, I/R12h group, TM group, TM + I/R12h group, DMSO + I/R12h group. The Control group will only receive laparotomy without clamping renal pedicle. TM pre-treatment will receive medicine intraperitoneally 24h before operation (0.6mg/kg). DMSO + I/R12h group will be given the same dose of DMSO (1ml 2% DMSO, without TM) 24h before the procedure. We will test the Cr, BUN level after taking samples of serum and evaluate the PAS staining of renal tissue injury in the renal tissue. What’s more, Western blot will be used to detect the protein expression of kidney GRP78, CHOP, caspase 11, caspase 1, IL-1β. The immunohistochemistry will test the expression of renal tissue GRP78.Ⅱ. Results1. The renal CHOP and inflammatory caspase expression after acute ischemia-reperfusion and the changing laws of IL-β1.1 The changes of renal function and renal pathologyRenal function: compared with the control group, serum creatinine I/R1h has no significant change when, I/R6h begins to increase, 12h at its peak, 24h begin to decline. Pathological changes: under the light microscope, tissue damages induced by renal ischemia-reperfusion are mainly embodied through necrosis of renal tubular epithelial cells and the cell tube formation, exposed basement membrane, interstitial edema, mononuclear cell infiltration. As for the lesion, the medulla is heavier than the cortex; the junction part is the most important leather cord. The changes in injury severity and is related with ischemia-reperfusion time, namely, the junction of renal tubular epithelial cell swelled, vacuolar degenerated, and occasionally renal tubular epithelial cell abscised in terms of I / R 1 h; as for I / R 6h, the inter- parts of tubular epithelial cells exfoliate and form a cell tube; I / R 12 lesions most seriously, necrotic tubular epithelial cells form a large cell tube, basement membrane exposed, some tubular open, interstitial edema and mononuclear cells infiltration; as for I/R24h, there still exist necrotic tubular lumen in the epithelial cells, but the tube is reduced and so is the interstitial edema.1.2 The effects of acute ischemia and reperfusion on renal CHOP, inflammatory caspase expression and IL-βGRP78 expression: I / R began to increase after 1h, 12h peak, 24h begin to decline, but still higher level.CHOP expression: the control group CHOP reveals the basic level expression, when I/R is at1h, it begins to increase, at 6h significantly increase, at12h reaches peak, at 24h it begin to decline. Caspase-11 expression: the control group of caspase-11’s precursors has a basic level of expression. When I/R is at 1h, there is no obvious increase, when I/R is at 6h, it significantly increases, when I/R is at12h, it reaches the peak, when 24h, it begins to reduce; activated caspase-11 begins to increase when it is 6h, 12h at the peak, 24h begins to reduce. Caspase-1 expression: the precursor of control group, caspase-1’s expression has a basic level. When I/R is at 1h, it begins to increase, at 6h significantly increase, at 24h it begins to reduce; activated caspase-1 begins to increase when I/R is at1h, and at 12h reaches the peak, at 24h it begins to reduce.IL-βexpression:the precursor of control group, the expression of IL-βhas a basic level. When I/R is at 1h, it begins to increase, at 6h significantly increase, at 24h it begins to reduce; activated IL-βbegins to increase when I/R is at1h, and at 12h reaches the peak, at 24h it begins to reduce.2. The function and mechanism of endoplasmic reticulum stress pre-treatment on the improvement of renal tolerance toward ischemia-reperfusion injury2.1 protection of tunicamycin pre-treatment on ischemia-reperfusion renal injuryCompared with the control group, the level of low-dose tunicamycin serum creatinine has no significant difference, neither obvious pathological changes occur in renal light microscopy, which indicated that a low dose of tunicamycin has no obvious effects on kidney structure and function. In the I/R12h, the serum creatinine level increases significantly; in the light microscope, there is a large number of loss of renal tubular epithelial cells, exposed basement membrane, tubular dilatation, lumen loss of cells and cell debris, interstitial edema and mononuclear cell infiltration and tubulointerstitial. The Tunicamycin pretreatment serum creatinine level was significantly lower than I/R12h group, renal pathological changes were significantly reduced, which indicated tunicamycin pretreatment can significantly increase the renal tolerance to ischemia-reperfusion injury.2.2 The effects of tunicamycin pretreatment on ischemia-reperfusion renal CHOP, inflammatory caspase and IL-βGRP78 expression: compared with the control group, low-dose tunicamycin can induce GRP78 expression in renal tissue, the expression of I/R12h GRP78 in renal tissue is even more significant, tunicamycin pretreatment renal ischemia-reperfusion significantly increases the expression of GRP78 than I/R12h. CHOP expression: compared with the control group, low dose of tunicamycin has no effect on the expression of renal tissue CHOP; after I/R12h, the kidney tissue CHOP significantly increases; tunicamycin pretreatment can significantly reduce CHOP renal ischemia-reperfusion .Caspase -11 expression: compared with the control group, low dose of tunicamycin has no significant effects on the expression and activation of caspase-11; when I/R reaches 12h, the expression and activation of kidney after caspase-11 is significantly increased; the tunicamycin pretreatment can significantly inhibit renal ischemia-reperfusion and activation of caspase-11 expression.Caspase -1 expression: compared with the control group, low dose of tunicamycin has no significant effects on the expression and activation of caspase-1; when I/R reaches 12h, the expression and activation of kidney after caspase-1 is significantly increased; the tunicamycin pretreatment can significantly inhibit renal ischemia-reperfusion and activation of caspase-1 expression.IL-βexpression: compared with the control group, low dose of tunicamycin has no significant effects on the expression and activation of IL-β; when I/R reaches 12h, the expression and activation of kidney after IL-βis significantly increased; the tunicamycin pretreatment can significantly inhibit renal ischemia-reperfusion and activation of IL-βexpression.Ⅲ. Conclusion1. Acute ischemia and reperfusion may cause excessive renal endoplasmic reticulum stress and CHOP up-regulated. The increase of CHOP expression precedes caspase-11 and caspase-1 expression; the activation changes of caspase-1 and the maturity of IL-1βlevel is consistent with each other, which will indicate ischemia-reperfusion and starting over-ERS and increase the expression of CHOP. CHOP could regulate the inflammatory caspase to promote renal IL-1βproduction, which may be an important mechanism of acute ischemic renal injury inflammation.2. endoplasmic reticulum stress pre-treatment could increase I / R GRP78 expression in renal tissue significantly and inhibit CHOP expression, as well as activate caspase-11, caspase-1. The mature IL-1βis significantly reduced; renal injury is significantly reduced, suggesting that the endoplasmic reticulum stress pretreatment can protect the ischemia-reperfusion against renal injury and its effects may involve the inflammatory responses related up-regulated endoplasmic reticulum chaperones and inhibition of endoplasmic reticulum excessive stress

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