Mechanisms of Renal Cell Injury

肾细胞损伤的机制

基本信息

批准号：
10554236
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
项目类别：
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/10554236
关键词：
20 year old AKT inhibition 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 Immunofluorescence Immunologic Immunohistochemistry Immunoprecipitation Kidney Kidney Diseases Knock-out Maps Methods Modality Modeling Molecular Mus PTEN gene Pathogenesis Pathologic Pathology Patients Permeability Persons Phosphorylation Phosphotransferases Plasminogen Activator Inhibitor 1 Platelet Activation Platelet-Derived Growth Factor Platelet-Derived Growth Factor Receptor Process Protein Dephosphorylation 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 Phosphorylation United States Variant Veterans Western Blotting age group cell injury cost db/db mouse demographics diabetic diabetic rat falls inhibitor 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

项目摘要

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.

糖尿病性肾病（DN）的病理表现包括肾小球和管状肥大，基质蛋白纤连蛋白表达。这些变化随着TGFB表达的增加而发生（转化生长因子-B）有助于人类和实验DN的发病机理。我们询问高葡萄糖和TGFB驱动病理并提供的分子信号传导事件 DN并发症的新潜在治疗策略。我们报道了Akt激酶/MTORC1 （雷帕霉素复合物的机械靶标1）轴有助于肾肥大和纤连蛋白表达肾小球和近端管状上皮（PTE）细胞，以及1型和2型糖尿病小鼠的肾脏。最近，一种新型PTEN的更长的翻译变体（磷酸酶和Tensin同源物中删除染色体10），pten-long是Akt激酶的负调节剂，已被确定为分泌物，膜可渗透蛋白。我们的初步数据显示，PTEN-LONG的水平显着降低增加了1型和2型糖尿病小鼠的肾脏皮质中的Akt激酶活性以及高葡萄糖或高葡萄糖或 TGFB处理的膜和PTE细胞。此外，我们表明高葡萄糖和TGFB激活PDGFRB （血小板衍生的生长因子-B）。我们发现PDGFRB是PTEN-long的底物。因此下调长葡萄糖和TGFB导致PTEN-LONG导致PDGFRB激活。此外，在肾脏和肾小球和PTE细胞，我们将Akt-2识别为Akt激酶的主要同种型，它作用于下游 pten-long/pdgfrb。同样，AKT-2在1型和2型糖尿病小鼠肾脏的肾脏中被激活。我们计划在DN的进展过程中利用PTEN-LONG的固有保护功能。在此提案中，使用来自糖尿病OVE26和DB/DB小鼠的培养的肾小球和PTE细胞以及肾脏组织，我们将测试假设高血糖/TGFB诱导的不适当的pten长下调导致 PDGFRB/AKT-2激活有助于肾脏肥大和糖尿病肾脏的基质扩张疾病。探测PTEN-LONG的新型负调控功能，我们将其用作治疗剂 Dn。在第一个特定目的中，我们将确定pten-long在肥大和纤连蛋白和pai-1中的作用（纤溶酶原激活剂抑制剂1）表达。在第二个目标中，PTEN长的力如何激活 PDGFRB调节肾小球和PTE细胞肥大，将研究基质蛋白表达。在特定目标3中，我们将研究Akt-2同种型对肥大和纤连蛋白/PAI-1表达的贡献响应于肾小球和PTE细胞以及糖尿病小鼠肾脏中的高葡萄糖和TGFB。解决这些目的，包括免疫印迹，免疫沉淀，形态计量学，免疫组织化学的技术，表达载体和siRNA的转染，重组治疗蛋白的给药和小分子化合物将被使用。我们的研究将建立一个涉及PTEN-的互连网络长，PDGFRB和AKT-2用于驾驶DN。