The Pathophysiology of Delayed Graft Function

移植物功能延迟的病理生理学

• 批准号: 9235127
• 负责人: Alkesh Harihar Jani
• 金额: --
• 依托单位: VA EASTERN COLORADO HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9235127
• 关键词: Affect; American; Antibodies; Apoptosis; Apoptosis Inhibitor; Apoptotic; Binding; CASP3 gene; Caring; Caspase Inhibitor; Cell Nucleus; Cells; Chronic Kidney Failure; Clinical; Creatinine; Cytoplasm; Data; Diabetes Mellitus; End stage renal failure; Epithelial Cells; Failure; Family suidae; Functional disorder; HMGB1 Protein; HTRA2 gene; Hour; Human; In Vitro; Inhibition of Apoptosis; Ischemia; Kidney; Kidney Transplantation; Lead; Ligands; Link; Mammals; Mediating; Mitochondria; Modeling; Mus; Necrosis; Phosphotransferases; Prevention; Protein-Serine-Threonine Kinases; Proteins; Protocols documentation; Public Health; Publishing; Recombinants; Risk Factors; Serum; Small Interfering RNA; TLR4 gene; Therapeutic; Transplantation; Tubular formation; Up-Regulation; Veterans; Waiting Lists; Wild Type Mouse; delayed graft function; experimental study; improved; in vivo; inhibitor/antagonist; interest; interstitial; mouse model; neutralizing antibody; novel; overexpression; prevent; public health relevance; treatment choice; tubular necrosis; x-linked inhibitor of apoptosis protein

 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.

 描述（由申请人提供）： 移植肾功能延迟恢复是指移植肾功能不能正常发挥的现象。DGF是一个严重的临床问题，可独立预测1年和5年肾移植存活率降低。> 24小时的长时间冷缺血（CI）是DGF的众所周知的风险因素，但DGF发生的机制尚不清楚。该建议试图确定长期CI如何易患DGF。我们已发表的数据表明，小鼠和猪肾脏长期CI增加半胱天冬酶-3和肾小管上皮细胞（RTEC）凋亡。我们已经开发了DGF的小鼠肾移植模型，其证明：（a）单独延长的CI导致RTEC细胞凋亡;（B）延长的CI随后肾移植导致DGF，表现为血清肌酐（sCr）增加，RTEC凋亡和肾小管坏死（ATN）;（c）没有CI的单独肾移植既不导致RTEC凋亡、ATN也不导致DGF。因此，我们建议证明的机制，RTEC细胞凋亡在长期CI易患ATN和DGF。 我们的初步数据表明，X连锁凋亡抑制因子（XIAP）蛋白在caspase-3介导的RTEC细胞凋亡在长期CI。我们的初步数据还表明，在体内和体外的小鼠RTEC的CI延长导致XIAP减少，细胞凋亡和高迁移率族蛋白1（HMGB 1），Toll样受体4（TLR 4）的配体的释放。HMGB 1与TLR 4的结合激活RIP激酶-1和-3，RIP激酶-1和-3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，长期CI导致RTEC细胞凋亡和HMGB 1从凋亡细胞核释放到细胞质，并进入间质空间。我们还假设，在移植经受长期CI的肾脏后：（a）存在TLR 4的增加的RTEC表达;（B）TLR 4被HMGB 1激活;（c）TLR 4随后激活RIP激酶-1和-3，导致其他RTEC和DGF的ATN。在具体目标1中，我们将确定XIAP的上调或抑制是否对RTEC凋亡、ATN和sCr有影响。在DGF小鼠模型中。XIAP抑制将通过使用针对XIAP或XIAP缺陷型小鼠的siRNA来实现。XIAP过表达将通过抑制HTRA 2来实现，HTRA 2是一种从破坏的线粒体释放的蛋白质，其阻止XIAP与半胱天冬酶-3的结合，从而引起RTEC凋亡。在具体目标2中，我们将确定HMGB 1的上调或抑制是否对ATN和sCr有影响。在DGF小鼠模型中。使用HMGB 1中和抗体实现HMGB 1抑制。将使用重组HMGB 1实现HMGB 1过表达。在具体目标3中，我们将确定TLR 4的上调或抑制是否对ATN和DGF小鼠模型有影响。sCr.in将在长时间CI后移植的野生型肾和移植到TLR 4缺陷的同基因受体中的野生型肾中检查TLR 4过表达。TLR 4抑制将通过以下方式实现：（a）针对TLR 4的抑制性抗体;（B）将TLR 4缺陷型肾移植到野生型同基因受体中。