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）： 在高胰岛素血症胰岛素抵抗状态下，高胰岛素血症是否通过胰岛素受体（IR）激活直接导致肾损伤，例如，肥胖是未知的。我们的新的初步数据显示：（1）在C57 BL 6小鼠中，高脂饮食（HFD）导致肾皮质IR激活，基质蛋白增加，白蛋白尿和mTORC 1激活沿着体重增加，胰岛素抵抗，高胰岛素血症，但血糖正常。我们已经产生了肾近端肾小管IR敲除小鼠（KPTIRKO），其中HFD诱导的IR活化，基质增加，白蛋白尿和mTORC 1信号转导减少。(2)硫化氢（H2S）通常在肾脏中由胱硫醚γ裂解酶（CSE）和胱硫醚β合成酶（CBS）合成。在WT小鼠中，HFD降低了CSE和CBS的肾含量，但在KPTIRKO小鼠中没有，这表明HFD中的IR活化降低了CBS和CSE作为肾损伤的一部分。(3)HFD降低肾CSE和CBS含量，但不降低其mRNA;体外胰岛素降低近端肾小管上皮（MCT）细胞CSE和CBS蛋白，但不降低mRNA。这些数据表明胰岛素对CSE和CBS的非转录调节。虽然我们的数据表明IR在HFD相关肾损伤中的作用，但减少H2S生成的要求尚不清楚。假设：在高脂饮食诱导的高胰岛素血症胰岛素抵抗状态下，肾胰岛素受体激活通过减少硫化氢的产生而导致肾损伤。目标1.测试H2S给药是否改善HFD诱导的小鼠肾损伤。3-将1个月大的雄性和雌性C57 BL 6小鼠随机分配至正常脂肪饮食（NFD）或HFD，持续4个月。一旦确定肾损伤（蛋白尿），我们将以30 umoles/l饮用水的浓度给予氢硫化钠（NaHS）（一种H2S供体）3个月。将进行以下研究：结构变化：肾肥大、免疫组织化学、基质蛋白的mRNA/蛋白分析。功能变化：白蛋白尿、FITC菊粉清除。信号研究：IR激活，控制蛋白质合成的信号反应。目标2. (A)测试增加的H2S产生是否可防止HFD诱导的肾损伤。我们将在近端小管中过表达人CBS的雄性和雌性3月龄WT和转基因小鼠（PThCBS tg，由我们产生）随机分配至NFD或HFD，持续4个月。研究将按照目标1进行。(B)测试HFD诱导的肾损伤是否在H2S产生缺陷的小鼠中放大。我们将我们拥有的雄性和雌性3月龄WT和CSEKO小鼠随机分配至NFD或HFD 4个月。将按照目标1进行结构、功能和信号传导研究。目标3。探讨胰岛素降低CSE、CBS表达的机制。(A)体内研究：初步数据显示，HFD通过非转录调节降低肾CSE和CBS表达。我们将通过对C57 BL 6小鼠的肾脏制备物进行多核糖体测定来探索这是否是由于在4个月的饮食中NFD和HFD不能翻译它们的mRNA。将测量溶酶体和蛋白酶体途径蛋白表达的变化。(B)体外研究：高胰岛素通过非转录机制抑制MCT细胞中的CSE和CBS蛋白，与体内数据相似。在MCT细胞中使用1和10 nM胰岛素作为高胰岛素水平，我们将探索这是否是由于（i）多核糖体测定的mRNA翻译失败，和/或（ii）这是否是由于通过脉冲追踪测量放射性标记的CSE和CBS的降解而导致的降解增加。我们将通过测量蛋白酶体活性和蛋白酶体亚单位蛋白质和mRNA表达来测试降解增强是否是通过蛋白酶体机制。我们将使用硼替佐米、MG 132和siRNA用于蛋白酶体亚基。最近的研究表明mTORC 1通过蛋白酶体途径促进蛋白质降解。我们将探索mTOR在CSE和CBS降解中的作用。我们还将通过采用氯喹或针对Atg 7的siRNA来测试高胰岛素是否刺激溶酶体途径以增加CSE和CBS降解。