New Insights in Mechanisms of Renal Injury

肾损伤机制的新​​见解

• 批准号: 9137229
• 负责人: BALAKUNTALAM S KASINATH
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9137229
• 关键词: Albuminuria; Area; Biological Assay; Blood Glucose; Bortezomib; Cells; Chloroquine; Chronic Kidney Failure; Cystathionine; Data; Diet; Dominant-Negative Mutation; Epithelial; FRAP1 gene; Failure; Fasting; Fatty acid glycerol esters; Female; Fluorescein-5-isothiocyanate; Generations; Genetic Translation; High Fat Diet; Human; Hydrogen Sulfide; Hyperinsulinism; Hypertrophy; Immunohistochemistry; In Vitro; Injury; Injury to Kidney; Insulin; Insulin Receptor; Insulin Resistance; Inulin; Kidney; Knockout Mice; Lead; Lyase; MG132; Measures; Mediating; Messenger RNA; Metabolic Diseases; Mus; Obesity; Pathway interactions; Physiologic pulse; Pilot Projects; Polyribosomes; Preparation; Production; Protein Analysis; Protein Biosynthesis; Protein Subunits; Proteins; Radiolabeled; Randomized; Reaction; Receptor Activation; Regulation; Renal function; Research; Risk; Role; Signal Transduction; Sirolimus; Small Interfering RNA; Sodium; Testing; Transgenic Mice; Translating; Tubular formation; Veterans; Weight Gain; drinking water; feeding; in vivo; insight; mRNA Expression; male; multicatalytic endopeptidase complex; novel; novel therapeutics; overexpression; protein degradation; protein expression; public health relevance; radiotracer

 DESCRIPTION (provided by applicant): Whether hyperinsulinemia directly contributes to kidney injury by insulin receptor (IR) activation in hyperinsulinemic insulin resistant states, e.g., obesity is not known. Our novel pilot data show (1) In C57BL6 mice, high fat diet (HFD) led to renal cortical IR activation, increase in matrix protein, albuminuria, and mTORC1 activation along with weight gain, insulin resistance, hyperinsulinemia but normal blood glucose. We have generated the kidney proximal tubular IR knock out mice (KPTIRKO) in which HFD-induced IR activation, matrix increment, albuminuria and mTORC1 signaling were reduced. (2) Hydrogen sulfide (H2S) is normally synthesized in the kidney by cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). In WT mice HFD reduced the renal content of CSE and CBS but not in KPTIRKO mice suggesting that IR activation in HFD reduces CBS and CSE as a part of renal injury. (3) HFD decreased renal CSE and CBS content but not their mRNA; in vitro insulin decreased CSE and CBS protein but not mRNA in proximal tubular epithelial (MCT) cells. These data suggest non-transcriptional regulation of CSE and CBS by insulin. Although our data suggest a role for IR in HFD-associated kidney injury, the requirement of reduced H2S generation is not known. Hypothesis: In hyperinsulinemic insulin resistant state induced by high fat diet, renal insulin receptor activation contributes to kidney injury by reduced generation of hydrogen sulfide. Aim 1. To test if H2S administration ameliorates HFD-induced kidney injury in mice. 3-month old male and female C57BL6 mice will be randomized to normal fat diet (NFD) or HFD for 4 months. Once the kidney injury (albuminuria) is established, we will administer sodium hydrosulfide (NaHS), a H2S donor, at 30umoles/l drinking water for 3 months. The following studies will be done: Structural changes: renal hypertrophy, immunohistochemistry, mRNA/protein analysis of matrix proteins. Functional changes: albuminuria, FITC inulin clearance. Signaling studies: IR activation, signaling reactions that control protein synthesis. Aim 2. (A) To test if augmented H2S production protects against HFD-induced kidney injury. We will randomize male and female 3-month old WT and transgenic mice overexpressing human CBS in the proximal tubule (PThCBS tg, generated by us) to NFD or HFD for 4 months. Studies will be done as in Aim 1. (B) To test if HFD-induced kidney injury is amplified in mice deficient in H2S production. We will randomize male and female 3-month old WT and CSEKO mice that are in our possession to NFD or HFD for 4 months. Structural, functional and signaling studies will be done as in Aim 1. Aim 3. To explore the mechanism of insulin-induced reduction in CSE, CBS expression. (A) In vivo studies: Pilot data show that HFD reduced the renal CSE and CBS expression by non-transcriptional regulation. We will explore if this is due to failure to translate their mRNA by performing Polysome assay on renal preparations from C57BL6 mice on NFD and HFD at 4 months of diet. Changes in the expression lysosomal and proteasomal pathway proteins will be measured. (B) In vitro studies: High insulin inhibited CSE and CBS protein in MCT cells in vitro by non-transcriptional mechanism, similar to in vivo data. Employing 1 and 10 nM insulin as high insulin in MCT cells, we will explore if this is due to (i) failure of translation of mRNA by Polysome assay, and/or, (ii) if this is due to increase in degradation by measuring the degradation of radiolabeled CSE and CBS by pulse chase. We will test if augmented degradation is via proteasomal mechanism by measuring proteasomal activity and proteasomal subunit protein and mRNA expression. We will employ bortezomib, MG132 and siRNA for proteasomal subunits. Recent studies show that mTORC1 stimulates protein degradation by proteasome pathway. We will explore role of mTOR in CSE and CBS degradation. We will also test if high insulin stimulates lysosomal pathway to augment CSE and CBS degradation by employing chloroquine or siRNA against Atg7.

 描述（由申请人提供）： 高胰岛素血症是否直接导致高胰岛素胰岛素耐药态中胰岛素受体（IR）激活，例如肥胖尚不清楚。我们的新型试点数据显示（1）在C57BL6小鼠中，高脂肪饮食（HFD）导致肾皮质IR激活，基质蛋白，蛋白蛋白和MTORC1激活的增加以及体重增加，胰岛素耐药性，高胰岛素高胰岛素血症，但血糖正常。我们已经产生了肾脏近端IR敲除小鼠（kptirko），其中HFD诱导的IR激活，基质增量，蛋白蛋白尿素和MTORC1信号降低。 （2）硫化氢（H2S）通常由胱淀粉γ裂解酶（CSE）和胱硫醚β合酶（CBS）在肾脏中合成。在WT小鼠中，HFD降低了CSE和CBS的肾脏含量，但在Kptirko小鼠中却没有降低HFD中的IR激活会减少CBS和CSE作为肾脏损伤的一部分。 （3）HFD降低了肾脏CSE和CBS含量，而不是其mRNA；体外胰岛素降低了CSE和CBS蛋白，而不是近端管状上皮（MCT）细胞中的mRNA。这些数据表明胰岛素对CSE和CBS的非转录调节。尽管我们的数据表明IR在与HFD相关的肾脏损伤中的作用，但尚不清楚H2S生成减少的要求。假设：在高脂饮食诱导的高胰岛素胰岛素耐药态中，肾胰岛素受体的激活通过减少硫化氢的产生而导致肾脏损伤。目标1。测试H2S给药是否可以改善小鼠HFD诱导的肾脏损伤。 3个月大的雄性和雌性C57BL6小鼠将被随机分为正常脂肪饮食（NFD）或HFD 4个月。一旦建立了肾脏损伤（蛋白尿），我们将在30umoles/l饮用水3个月内管理H2S供体钠（NAHS）。将进行以下研究：结构变化：肾脏肥大，免疫组织化学，基质蛋白的mRNA/蛋白质分析。功能变化：蛋白尿，FITC染色蛋白清除率。信号研究：IR激活，控制蛋白质合成的信号反应。目标2。（a）测试增强的H2S产量是否可以防止HFD诱导的肾脏损伤。我们将在代理管中过表达人CB（我们生成的PTHCBS TG）中过表达人CB的男性和雌性3个月的男性和女性和转基因小鼠至NFD或HFD 4个月。研究将与AIM 1一样进行研究。我们将随机分配给NFD或HFD的男性和女性3个月大的WT和CSEKO小鼠4个月。结构，功能和信号转导研究将与AIM 1一样进行。目的3。探索胰岛素诱导的CSE，CBS表达减少的机理。 （a）体内研究：试验数据表明，HFD通过非转录调节降低了肾脏CSE和CBS的表达。我们将探讨这是否是由于未能通过对NFD上C57BL6小鼠的肾脏制剂和HFD的肾脏制剂进行多质体分析而无法翻译其mRNA的。将测量表达溶酶体和蛋白酶体途径蛋白的变化。 （b）体外研究：高胰岛素通过非转录机制在体外抑制MCT细胞中的CSE和CBS蛋白，类似于体内数据。在MCT细胞中，使用1和10 nm胰岛素作为高胰岛素，我们将探讨这是否是由于（i）通过多质体测定法对mRNA翻译的失败，并且/或（ii）如果这是由于测量降解而通过脉搏CBS降解而导致的降解。我们将通过测量蛋白酶体活性和蛋白酶体亚基蛋白和mRNA表达来测试增强降解是否通过蛋白酶体机理。我们将使用硼替佐米，MG132和siRNA用于蛋白酶体亚基。最近的研究表明，MTORC1通过蛋白酶体途径刺激蛋白质降解。我们将探讨MTOR在CSE和CBS降解中的作用。我们还将测试高胰岛素是否刺激通过使用氯喹或siRNA抗ATG7来刺激增加CSE和CBS降解的溶酶体途径。