Mitochondrial Recovery after Acute Kidney Injury Needs Ribonucleotide Reductase

急性肾损伤后的线粒体恢复需要核糖核苷酸还原酶

基本信息

批准号：
10396045
负责人：
MANJERI A VENKATACHALAM
金额：
$ 23.25万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10396045
关键词：
2&apos -Deoxythymidine Acute Renal Failure with Renal Papillary Necrosis Affect Apoptosis Atrophic Brain Bypass Catalytic Domain Cell Culture Techniques Cell Hypoxia Cells Chronic Kidney Failure Code Cultured Cells Cytosol DNA DNA Damage DNA Repair DNA biosynthesis Data Deoxycytidine Deoxyguanosine Development Dialysis procedure Disease Enzymes Failure Genes Growth Health Healthcare Hypoxia Impairment Incidence Injury Injury to Kidney Interphase Cell Investigation Kidney Kidney Diseases Kidney Failure Knockout Mice Lead Lesion LoxP-flanked allele Mediating Membrane Proteins Mitochondria Mitochondrial DNA Mitochondrial DNA depletion syndromes Modeling Molecular Morbidity - disease rate Mus Muscular Atrophy Mutation Normal Cell Nuclear Null Lymphocytes Oxidants Oxygen PINK1 gene Parkin Pathologic Pathology Pathway interactions Phenotype Process Production Proteins RRM1 gene RRM2 gene Recovery Regimen Regulation Reperfusion Injury Reporter Research Retina Ribonucleotide Reductase Ribonucleotide Reductase Subunit Risk Role Stress Superoxides Supplementation TP53 gene Therapeutic Therapeutic Agents Transgenes Transplantation Treatment Efficacy Tubular formation Undifferentiated cancer cell cell growth deoxyribonucleoside triphosphate efficacy testing improved in vivo insight molecular pathology mortality mouse model novel overexpression oxidant stress preservation prevent renal hypoxia repaired respiratory protein response tripolyphosphate

项目摘要

PROJECT SUMMARY/ABSTRACT Significance: Tubule atrophy underlies progression of acute kidney injury (AKI) to chronic kidney disease. The basis for tubule atrophy after AKI is unknown. We identified a pathology involving RRM2B, alternate regulatory subunit of ribonucleotide reductase (RNR) as a basis for tubule atrophy after AKI. Cell culture data suggest that molecular bypass of RNR by deoxynucleosides can produce deoxynucleoside triphosphates (dNTPs) for DNA synthesis and repair by oxygen independent salvage pathways after AKI to promote tubule recovery. RNR produces dNTPs. Its classical regulatory subunit RRM2 is inhibited by hypoxia, but the hypoxia tolerant RRM2B can substitute for RRM2 to maintain dNTPs during hypoxia to prevent nuclear and mitochondrial DNA damage and preserve cell integrity. RRM2B is induced by hypoxia in cultured cells. Hypoxia mediated increase of mitochondrial superoxide may induce RRM2B as a beneficial adaptation. A regulatory role for mitochondrial superoxide was suggested by our finding that MitoPQ, which increases superoxide selectively in mitochondria, markedly increased cellular RRM2B protein content. The benefits afforded by RRM2B in hypoxic cells would be abrogated if RRM2B is not available. In support, we showed that deletion of RRM2B from cultured tubule cells increases DNA damage during hypoxia, and prevents recovery during reoxygenation. Deoxynucleosides rescued such cells from DNA damage, decreased injury and promoted recovery. We showed also that tubule atrophy after AKI is accompanied by marked RRM2B depletion and DNA damage. Since recovering kidneys are hypoxic, RNR inhibition is expected. In such kidneys RRM2B loss in tubules (rather than an adaptive increase) will have deleterious consequences. Thus, RRM2B loss after AKI may be the cause for tubule atrophy. While the cause for RRM2B decline in tubules after AKi is unclear, its importance for recovery requires investigation. To this end, we have three Specific Aims. Aim 1. We will utilize mouse models of RRM2B deletion and overexpression in tubules to investigate its role in recovery from AKI. Aim 2. We will use cultured tubule cells with RRM2B deletion and overexpression, and deoxynucleoside supplementation, to investigate the role of RNR activity, RRM2B levels and salvage synthesis of dNTPs in cellular responses to hypoxia. To examine the role of mitochondrial oxidants in adaptation to stress, we will use Mito-PQ, a selective inducer of mitochondrial superoxide, to elucidate mechanisms of RRM2B regulation. Aim 3. We will test the efficacy of treatment with deoxynucleosides to promote recovery from ischemic AKI. Impact: The studies we propose will yield new insights into cellular mechanisms that determine successful or failed tubule recovery from AKI and possibly identify deoxynucleosides as novel useful therapeutic agents.

项目概要/摘要意义：肾小管萎缩是急性肾损伤 (AKI) 进展为慢性肾脏疾病的基础。这 AKI 后肾小管萎缩的基础尚不清楚。我们发现了一种涉及 RRM2B（替代监管）的病理学核糖核苷酸还原酶 (RNR) 亚基是 AKI 后肾小管萎缩的基础。细胞培养数据表明脱氧核苷对 RNR 的分子旁路可产生 DNA 脱氧核苷三磷酸 (dNTP) AKI 后通过不依赖于氧的挽救途径进行合成和修复，以促进肾小管恢复。 RNR 产生 dNTP。其经典调节亚基RRM2受到缺氧抑制，但耐缺氧 RRM2B可以替代RRM2在缺氧期间维持dNTPs以防止核和线粒体DNA 损伤并保持细胞完整性。 RRM2B 是由培养细胞缺氧诱导的。缺氧介导的增加线粒体超氧化物的增加可能会诱导 RRM2B 作为一种有益的适应。线粒体的调节作用我们的发现表明 MitoPQ 可以选择性地增加线粒体中的超氧化物，细胞RRM2B蛋白含量显着增加。如果 RRM2B 不可用，RRM2B 在缺氧细胞中提供的益处将被取消。为了支持，我们结果表明，从培养的肾小管细胞中删除 RRM2B 会增加缺氧期间的 DNA 损伤，并防止再氧合期间恢复。脱氧核苷可以使这些细胞免于 DNA 损伤，减少损伤并促进了康复。我们还发现 AKI 后的肾小管萎缩伴随着明显的 RRM2B 耗竭和DNA损伤。由于恢复中的肾脏处于缺氧状态，因此预计会出现 RNR 抑制。在这样的肾脏中 RRM2B 肾小管的损失（而不是适应性的增加）将产生有害的后果。因此，RRM2B 损失后 AKI 可能是肾小管萎缩的原因。虽然 AKi 后肾小管 RRM2B 下降的原因尚不清楚，但其对于恢复的重要性需要进行调查。为此，我们有三个具体目标。目标 1. 我们将利用 RRM2B 在肾小管中缺失和过度表达的小鼠模型来研究其在肾小管中的作用从 AKI 中恢复。目标 2. 我们将使用具有 RRM2B 缺失和过表达以及脱氧核苷的培养肾小管细胞补充，以研究 RNR 活性、RRM2B 水平和 dNTP 补救合成的作用细胞对缺氧的反应。为了检查线粒体氧化剂在适应应激中的作用，我们将使用 Mito-PQ 是线粒体超氧化物的选择性诱导剂，用于阐明 RRM2B 调节机制。目标 3. 我们将测试脱氧核苷治疗促进缺血性 AKI 恢复的功效。影响：我们提出的研究将对决定成功或失败的细胞机制产生新的见解。肾小管从 AKI 中恢复失败，并可能将脱氧核苷鉴定为新型有用的治疗剂。