Mechanisms of Renal Cell Injury

肾细胞损伤的机制

• 批准号: 10013592
• 负责人: GOUTAM GHOSH CHOUDHURY
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10013592
• 关键词: 20 year old; Accounting; Address; Affect; Automobile Driving; Binding; CRISPR/Cas technology; Cell Membrane Permeability; Cells; Chromosome 10; Codon Nucleotides; Complex; Complications of Diabetes Mellitus; Data; Development; Diabetes Mellitus; Diabetic Nephropathy; Diabetic mouse; Disease; Dissociation; Down-Regulation; End stage renal failure; Epithelial Cells; Event; Exposure to; FRAP1 gene; Fibronectins; Glucose; Healthcare; Human; Hyperglycemia; Hypertrophy; Immune; Immunoblotting; Immunofluorescence Immunologic; Immunohistochemistry; Immunoprecipitation; Kidney; Kidney Diseases; Knock-out; Lead; Maps; Methods; Modality; Modeling; Molecular; Mus; PTEN gene; Pathogenesis; Pathologic; Pathology; Patients; Permeability; Phosphorylation; Phosphotransferases; Plasminogen Activator Inhibitor 1; Platelet Activation; Platelet-Derived Growth Factor B; Platelet-Derived Growth Factor beta Receptor; Process; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Recombinant Proteins; Recombinants; Regulation; Renal Tissue; Renal function; Reporting; Resources; Rodent; Rodent Model; Role; Signal Transduction; Small Interfering RNA; Streptozocin; Techniques; Testing; Therapeutic; Therapeutic Agents; Transfection; Transforming Growth Factors; Translations; Tricarboxylic Acids; Tubular formation; Type 2 diabetic; Tyrosine; United States; Variant; Veterans; age group; cell injury; cost; db/db mouse; demographics; diabetic; diabetic rat; falls; inhibitor/antagonist; kidney cell; kidney cortex; mesangial cell; military veteran; morphometry; novel; novel therapeutic intervention; pandemic disease; prevent; protein expression; response; small molecule; therapeutic protein; transfection/expression vector; type I diabetic

Pathologic manifestations of diabetic nephropathy (DN) include glomerular and tubular hypertrophy and matrix protein fibronectin expression. These changes occur concomitant with increased expression of TGFb (transforming growth factor-b) that contributes to the pathogenesis of human and experimental DN. We interrogate the molecular signaling events by which high glucose and TGFb drive the pathologies and provide new potential therapeutic strategies for complications of DN. We have reported that Akt kinase/mTORC1 (mechanistic target of rapamycin complex 1) axis contributes to renal hypertrophy and fibronectin expression in mesangial and proximal tubular epithelial (PTE) cells, and in kidneys of type 1 and type 2 diabetic mice. Recently, a novel longer translational variant of PTEN (phosphatase and tensin homolog deleted in chromosome 10), PTEN-Long, a negative regulator of Akt kinase, has been identified as a secretory and membrane permeable protein. Our preliminary data show markedly reduced levels of PTEN-Long along with increased Akt kinase activity in the kidney cortex of type 1 and type 2 diabetic mice and in high glucose- or TGFb-treated mesangial and PTE cells. Moreover, we show that high glucose and TGFb activate PDGFRb (platelet-derived growth factor-b). We find PDGFRb as a substrate for PTEN-Long; thus downregulation of PTEN-Long results in PDGFRb activation by high glucose and TGFb. Furthermore, in the kidney and in mesangial and PTE cells, we identify Akt-2 as the predominant isotype of Akt kinase that acts downstream of PTEN-Long/PDGFRb. Also, Akt-2 is activated in kidneys of type 1 and type 2 diabetic mice kidneys. We plan to exploit the intrinsic protective function of PTEN-long during the progression of DN. In this proposal, using cultured mesangial and PTE cells and renal tissues from diabetic OVE26 and db/db mice, we will test the hypothesis that hyperglycemia/TGFb-induced inappropriate downregulation of PTEN-Long results in PDGFRb/Akt-2 activation that contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the novel negative regulatory function of PTEN-Long, we will use it as a therapeutic agent for DN. In the first specific aim, we will determine the role of PTEN-Long in hypertrophy and, fibronectin and PAI-1 (plasminogen-activator inhibitor-1) expression. In the second aim, how PTEN-Long forces activation of PDGFRb to regulate mesangial and PTE cell hypertrophy, and matrix protein expression will be investigated. In specific aim 3, we will study the contribution of Akt-2 isotype to hypertrophy and fibronectin/PAI-1 expression in response to high glucose and TGFb in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, morphometry, immunohistochemistry, transfection of expression vectors and siRNAs, administration of recombinant therapeutic protein and small molecule compound will be employed. Our study will establish a network of interconnections involving PTEN- Long, PDGFRb and Akt-2 for driving DN.

糖尿病肾病（DN）的病理表现包括肾小球和肾小管肥大， 基质蛋白纤连蛋白表达。这些变化伴随着TGF β表达的增加而发生。 （转化生长因子-b），其有助于人类和实验性DN的发病机制。我们 询问高葡萄糖和TGF β驱动病理的分子信号传导事件， DN并发症的新的潜在治疗策略。我们已经报道Akt激酶/mTORC 1 （雷帕霉素复合物1的机制靶点）轴有助于肾肥大和纤连蛋白表达， 肾小球系膜和近端肾小管上皮（PTE）细胞，以及1型和2型糖尿病小鼠的肾脏中。 最近，一种新的更长的翻译变体的PTEN（磷酸酶和张力蛋白同源物删除， 第10号染色体），PTEN-Long，Akt激酶的负调节因子，已被鉴定为分泌型， 膜透性蛋白我们的初步数据显示，PTEN-Long沿着 在1型和2型糖尿病小鼠的肾皮质和高糖或高糖小鼠中，Akt激酶活性增加， TGF β处理的系膜和PTE细胞。此外，我们发现高糖和TGF β激活PDGFRb， （血小板衍生生长因子-b）。我们发现PDGFRb是PTEN-Long的底物，因此PDGFRb的表达下调。 PTEN-Long导致高葡萄糖和TGFb激活PDGFRb。此外，在肾脏和 在系膜细胞和PTE细胞中，我们鉴定Akt-2为Akt激酶的主要同种型，其作用于 PTEN-Long/PDGFR b.此外，Akt-2在1型和2型糖尿病小鼠肾脏中被激活。我们计划 探讨PTEN-long在DN进展过程中的内在保护作用。在本提案中，使用 我们将从糖尿病OVE 26和db/db小鼠培养的系膜和PTE细胞以及肾组织中，检测 假设高血糖/TGF β诱导的PTEN-Long的不适当下调导致 PDGFRb/Akt-2激活导致糖尿病肾脏肥大和基质扩张 疾病探索PTEN-Long的新的负调节功能，我们将使用它作为治疗剂， DN.在第一个具体目标中，我们将确定PTEN-Long在肥大中的作用，以及纤连蛋白和派-1在肥大中的作用。 （纤溶酶原激活物抑制剂-1）表达。在第二个目标中，PTEN-Long如何迫使激活 将研究PDGFRb调节肾小球系膜和PTE细胞肥大以及基质蛋白表达。 在具体目标3中，我们将研究Akt-2同种型对肥大和纤连蛋白/派-1表达的贡献 在肾小球系膜和PTE细胞以及糖尿病小鼠肾脏中对高葡萄糖和TGF β的响应。解决 这些目的是，包括免疫印迹，免疫沉淀，形态测定，免疫组织化学， 表达载体和siRNA的转染，重组治疗性蛋白和小分子的施用， 将使用分子化合物。我们的研究将建立一个涉及PTEN的互联网络， Long、PDGFRb和Akt-2用于驱动DN。