The Pathophysiology of Delayed Graft Function

 描述(由申请人提供)： 摘要移植肾功能延迟(DGF)是指移植后肾功能不能达到最佳状态。DGF是一个严重的临床问题，它独立地预测肾移植1年和5年存活率的降低。24小时持续冷缺血(CI)是DGF的一个众所周知的危险因素，但DGF发生的机制尚不清楚。这项建议试图确定长时间CI对DGF的易感性。我们已发表的数据表明，持续脑缺血的小鼠和猪肾脏增加了caspase-3和肾小管上皮细胞(RTEC)的凋亡。我们建立了一种小鼠肾移植DGF模型，该模型显示：(A)单独延长CI可导致RTEC凋亡；(B)延长CI可导致肾移植后出现DGF，表现为血肌酐(Scr)、RTEC凋亡和肾小管坏死(ATN)；(C)单独肾移植不含CI可导致RTEC凋亡、ATN和DGF。因此，我们建议证明延长脑梗塞时RTEC凋亡易患ATN和DGF的机制。我们的初步数据表明，X连锁的凋亡抑制蛋白(XIAP)参与了caspase-3介导的脑缺血后RTEC的凋亡。我们的初步数据还表明，在体内和体外，延长小鼠RTEC的CI会导致XIAP减少、细胞凋亡和Toll样受体4(TLR4)的配体高迁移率族蛋白1(HMGB1)的释放。HMGB1结合TLR4激活RIP-1和-3，这是细胞程序性坏死所必需的丝氨酸/苏氨酸蛋白激酶。因此，我们的总体假设是，在肾移植前，长时间的CI导致RTEC凋亡，HMGB1从凋亡的细胞核释放到细胞质，并进入间质。我们还假设，在长时间CI的肾脏移植后：(A)TLR4的RTEC表达增加；(B)TLR4被HMGB1激活；(C)TLR4随后激活RIP-1和-3，导致其他RTEC和DGF的ATN。在特定的目标1中，我们将确定XIAP的上调或抑制是否对RTEC的凋亡、ATN和Scr产生影响。在DGF的小鼠模型中。通过使用针对XIAP或XIAP缺陷小鼠的siRNA，可以实现对XIAP的抑制。XIAP的过度表达将通过抑制HTRA2实现，HTRA2是一种从破坏的线粒体中释放的蛋白质，它阻止XIAP与caspase-3的结合，从而导致RTEC凋亡。在具体目标2中，我们将确定HMGB1的上调或抑制是否会对ATN和Scr产生影响。在DGF的小鼠模型中。使用HMGB1中和抗体可以抑制HMGB1。利用重组HMGB1实现HMGB1的过表达。在特定的目标3中，我们将确定TLR4的上调或抑制是否对DGF小鼠模型的ATN和sCR有影响。TLR4的过度表达将在长期CI后移植的野生型肾脏和TLR4缺乏的同基因受体移植的野生型肾脏中检测到。TLR4的抑制将通过：(A)针对TLR4的抑制性抗体；(B)将TLR4缺陷的肾脏移植到野生型同基因受体中。

 DESCRIPTION (provided by applicant): Abstract Delayed Graft Function (DGF) refers to failure of a kidney to function optimally after transplantation. DGF is a serious clinical problem that independently predicts reduced 1- and 5- year kidney transplant survival. Prolonged Cold Ischemia (CI) of > 24 hours is a well-known risk factor for DGF, but the mechanism by which DGF occurs is not known. This proposal endeavors to determine how prolonged CI predisposes to DGF. Our published data demonstrate that mouse and porcine kidneys subjected to prolonged CI have increased caspase-3 and renal tubular epithelial cell (RTEC) apoptosis. We have developed a mouse kidney transplant model of DGF that demonstrates: (a) Prolonged CI alone results in RTEC apoptosis; (b) Prolonged CI followed by kidney transplant results in DGF, manifested by increased serum creatinine (sCr.), RTEC apoptosis and tubular necrosis (ATN); (c) Kidney transplant alone without CI results in neither RTEC apoptosis, ATN, nor DGF. Thus we propose to demonstrate the mechanisms by which RTEC apoptosis during prolonged CI predisposes to ATN and DGF. Our preliminary data implicate X-linked inhibitor of apoptosis (XIAP) protein in caspase-3 mediated RTEC apoptosis during prolonged CI. Our preliminary data also demonstrate that prolonged CI of mouse RTEC in vivo and in vitro results in decreased XIAP, apoptosis, and release of high-mobility group protein 1 (HMGB1), a ligand of Toll-like receptor 4 (TLR4). HMGB1 binding of TLR4 activates RIP kinases - 1 and -3, which are serine/threonine protein kinases essential for cellular programmed necrosis. Thus, our overall hypothesis is that before kidney transplant, prolonged CI leads to RTEC apoptosis and release of HMGB1 from apoptotic nuclei to the cytoplasm, and into the interstitial space. We also hypothesize that after transplant of kidneys subjected to prolonged CI: (a) there is increased RTEC expression of TLR4; (b) TLR4 is activated by HMGB1; (c) TLR4 subsequently activates RIP kinases - 1 and -3 leading to ATN of other RTECs and DGF. In Specific Aim 1, we will determine whether upregulation or inhibition of XIAP has an effect on RTEC apoptosis, ATN, and sCr. in a mouse model of DGF. XIAP inhibition will be achieved by using either siRNA against XIAP or XIAP deficient mice. XIAP overexpression will be achieved by inhibition of HTRA2, a protein released from disrupted mitochondria that prevents the association of XIAP with caspase-3, thus causing RTEC apoptosis. In Specific Aim 2, we will determine whether upregulation or inhibition of HMGB1 has an effect on ATN and sCr. in a mouse model of DGF. HMGB1 inhibition will be achieved using HMGB1 neutralizing antibody. HMGB1 overexpression will be achieved using recombinant HMGB1. In Specific Aim 3 we will determine whether upregulation or inhibition of TLR4 has an effect on ATN and sCr.in a mouse model of DGF. TLR4 overexpression will be examined in wild-type kidneys transplanted after prolonged CI, and wild-type kidneys transplanted into TLR4 deficient syngeneic recipients. TLR4 inhibition will be achieved by; (a) an inhibitory antibody against TLR4; (b) transplanting TLR4 deficient kidneys into wild-type syngeneic recipients.