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）： 摘要 移植物功能延迟（DGF）是指移植后肾脏无法达到最佳功能，这是一个严重的临床问题，可独立预测移植肾的 1 年和 5 年存活率降低，超过 24 小时的长期冷缺血（CI）。 DGF 是众所周知的危险因素，但 DGF 发生的机制尚不清楚。我们发表的数据表明，长期 CI 会导致 DGF 的发生。经受延长的 CI 会增加 caspase-3 和肾小管上皮细胞 (RTEC) 的凋亡。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)； ATN，也不是 DGF。因此，我们建议证明延长 CI 期间 RTEC 凋亡易发生 ATN 和 DGF 的机制，我们的初步数据表明 X 连锁凋亡抑制剂 (XIAP) 参与 caspase-3 介导的 RTEC 凋亡。我们的初步数据还表明，体内和体外小鼠 RTEC 的 CI 延长会导致 XIAP 减少、细胞凋亡和高迁移率组蛋白 1 (HMGB1)（Toll 样配体）的释放。 TLR4 受体 4 (TLR4) 的 HMGB1 结合激活 RIP 激酶 - 1 和 -3，它们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，在肾移植之前，延长的 CI 会导致 RTEC 细胞凋亡和 RTEC 凋亡。我们还发现，在接受长期 CI 的肾脏移植后，HMGB1 从凋亡细胞核释放到细胞质，并进入间质空间：(a) 增加。 TLR4 的 RTEC 表达；(b) TLR4 被 HMGB1 激活；(c) TLR4 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 是否上调或抑制。通过使用针对 XIAP 或 XIAP 的 siRNA 可以实现对 DGF 小鼠模型中 RTEC 细胞凋亡、ATN 和 sCr 的影响。使用 HMGB1 过表达可实现对 DGF 小鼠模型中 ATN 和 sCr 的影响。在具体目标 3 中，我们将确定 TLR4 的上调或抑制是否对 ATN 和 sCr 产生影响。将在延长 CI 和野生型肾移植后的 DGF 小鼠模型中检查 TLR4 过度表达。移植到TLR4缺陷的同系受者的肾脏将通过以下方式实现：(a)针对TLR4的抑制性抗体；(b)移植TLR4缺陷的肾。将肾脏转化为野生型同基因受体。