Pathologic and Physiologic Importance of Aldo-keto reductase 1C enzymes in Glomerular Cells

肾小球细胞中醛酮还原酶 1C 的病理和生理重要性

• 批准号: 9053760
• 负责人: Aisha L. Siebert
• 金额: $ 4.86万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9053760
• 关键词: Affect; Affinity; Aldehyde Reductase; Aldehydes; American; Binding Sites; Biochemistry; Biology; Blood Circulation; Blood Glucose; Blood flow; Cells; Cellular biology; Chronic; Clinical Trials; Development; Diabetes Mellitus; Diabetic Nephropathy; Diffuse; Disease Outcome; Doctor of Philosophy; Drug Metabolic Detoxication; End stage renal failure; Environment; Enzymes; Equilibrium; Family; Financial compensation; Fructose; Functional disorder; Future; Genetic; Glucose; Goals; Homeostasis; Human; Hyperglycemia; Impairment; Individual; Insulin; Investigation; Isomerism; Kidney; Kidney Diseases; Kidney Failure; L-Iditol 2-Dehydrogenase; Lead; Life; Mannitol; Measurement; Measures; Mediating; Mentors; Metabolic; Metabolism; Molecular Biology; Morbidity - disease rate; NADH; NADP; Organ; Oxidative Stress; Oxidoreductase; Pathologic; Pathway interactions; Physiological; Process; Production; Protein Isoforms; Reagent; Renal function; Renal glomerular disease; Research Training; Risk; Role; Secondary to; Site; Sorbitol; Source; Stratification; Stress; Sugar Alcohols; Swelling; Testing; Tissues; Training; Translating; Treatment Cost; United States; Venoms; Wasp Venoms; career; cell injury; cell type; cofactor; diabetic; experience; extracellular; in vivo; inhibitor/antagonist; insight; kidney cell; knock-down; mesangial cell; mortality; novel; overexpression; podocyte; prevent; programs; public health relevance; research study; response; skills; sugar; tempol; therapeutic target

 DESCRIPTION (provided by applicant): Nephropathy secondary to Diabetes Mellitus (DM)-induced hyperglycemia is a leading cause of morbidity and mortality. Prolonged hyperglycemia activates the sorbitol pathway, resulting in osmotic damage, oxidative stress, impairment of renal function, and eventual kidney failure. The enzyme aldose reductase (AR) is believed to be the primary regulator of entry into this pathway by converting excess glucose to sorbitol and is a prime target for pharmacological therapy to preserve kidney function in diabetics. However, recent findings suggest that other aldo-keto reductases (AKRs) may also be important in synthesizing sorbitol. Many AR inhibitors have failed clinical trials due to lack of potency in human kidney and inability to target other AKR family enzymes, which serve redundant function in vivo. New strategies for developing more potent inhibitors with broader activity are needed. During our interrogation of the metabolic targets of Nasonia wasp venom in human cells, we identified non-canonical Aldo-keto reductase (AKR) enzymes (AKR1C* and 1D1) that may synthesize sorbitol more effectively and at lower substrate concentrations than the canonical enzyme AR. We hypothesize that non-canonical AKRs increase sorbitol accumulation, resulting in glomerular dysregulation in response to hyperglycemia. This proposal outlines an approach to study the role of non-canonical AKRs in sorbitol metabolism in human glomerular cells by investigating three primary aims: 1) Determine which AKR1C* sub-family (and AKR1D1) enzymes catalyze sorbitol synthesis in human glomerular cells by candidate AKR overexpression and knockdown. 2) Determine whether AKR1C* and 1D1 isoforms contribute to oxidative stress during sorbitol pathway activation by measuring site-specific ROS, and compensation by ROS scavengers (i.e. TEMPOL/Mito-TEMPOL). 3) Determine if AKR1C* sub-family (and AKR1D1) enzymes alter osmotic sensitivity in human glomerular cells by overexpressing candidate AKRs and measuring response to altered osmotic environment. The end result of this investigation will be identification of novel, non-canonical AKR regulators of te glucose to sorbitol conversion in human kidney cells. Identification of the enzymes affected will provide new insight into strategies for stratifying risk, blocking AR/AKR function in vivo, and preventing sorbitol accumulation and development of glomerular dysfunction. The formal mentoring program combines and integrates training and research opportunities in biomedical and basic biology highly tailored to the PD/PI's career goals, greatly enhancing the training potential of the applicant's dual MD/PhD program. A highly accomplished mentoring team with extensive mentoring experience and complementary skills in genetics, cell biology, molecular biology and biochemistry is in place.

 描述(由申请人提供)：糖尿病(DM)引起的高血糖继发肾病是发病率和死亡率的主要原因。长期的高血糖激活山梨醇途径，导致渗透损伤、氧化应激、肾功能损害，最终导致肾功能衰竭。醛糖还原酶(AR)被认为是通过将过量的葡萄糖转化为山梨醇而进入这一途径的主要调节因子，也是糖尿病患者保护肾功能的药物治疗的主要靶点。然而，最近的发现表明，其他醛酮还原酶(AKRs)在山梨醇的合成中也可能是重要的。由于在人类肾脏中缺乏效力，以及无法靶向其他在体内发挥多余功能的AKR家族酶，许多AR抑制剂都未能通过临床试验。需要新的策略来开发更有效、活性更广的抑制剂。在我们对黄蜂毒液在人类细胞中的代谢靶标的研究中，我们发现了非典型的醛酮还原酶(AKR)酶(AKR1C*和1D1)，它们可以更有效地合成山梨醇，而且底物浓度比典型的酶AR低。我们假设非典型性AKRs增加了山梨醇的积聚，导致肾小球对高血糖反应的失调。这项建议概述了一种研究非规范AKRs在人肾小球细胞山梨醇代谢中作用的方法，通过研究三个主要目标：1)通过候选AKR过表达和敲除，确定哪些AKR1C*亚家族(和AKR1D1)酶催化人肾小球细胞山梨醇的合成。2)通过检测特定部位的ROS和ROS清除剂(即temol/Mito-temol)的补偿，确定AKR1C*和1D1亚型是否参与山梨醇途径激活过程中的氧化应激。3)确定AKR1C*亚家族(和AKR1D1)酶是否通过过度表达候选AKRs和测量对改变的渗透环境的反应来改变人肾小球细胞的渗透敏感性。这项研究的最终结果将是确定在人类肾脏细胞中将TE葡萄糖转化为山梨醇的新的、非规范的AKR调节器。识别受影响的酶将为分层风险、阻断体内AR/AKR功能以及防止山梨醇积聚和肾小球功能障碍的发展提供新的见解。正式的指导计划结合并整合了高度符合PD/PI职业目标的生物医学和基础生物学方面的培训和研究机会，极大地增强了申请者的双MD/PHD计划的培训潜力。一支高度成功的指导团队已经到位，他们在遗传学、细胞生物学、分子生物学和生物化学方面拥有丰富的指导经验和互补的技能。