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）： 高胰岛素血症是否有助于高胰岛素胰岛素中的肾脏损伤受体（IR）激活，例如，我们的新型试点数据显示（1）。蛋白质，蛋白尿和MTORC1以及增益，胰岛素抵抗，高胰岛素血症Butod Lucose WT小鼠中的合成酶（CBS）降低了CSE CBS的肾脏含量，但在KPTIRKO小鼠中却不会使HFD中的IR激活减少CSE作为肾脏的一部分。这些数据中的肾小管损伤表明CSE和CBS的非转录调节。通过减少氢的生成来造成肾脏损伤。 NAHS，H2S供体，在30umoles/l饮用水中进行3个月的研究：肾脏肥大，免疫组织化学，MRNA/蛋白质分析的基质蛋白。 。在AIM 1中进行研究。在缺乏H2S生产的小鼠中，IFD Indury进行了研究非转录调节的肾脏CSSPRESS，我们将通过在饮食中对NFD和HFD上的肾脏制剂进行肾脏制剂的失败而探索机制，类似于体内数据。 CBS通过蛋白酶体的蛋白酶体活性和蛋白质亚基蛋白Expr，我们将使用bortezomib，Mg132和sirna来测试ifagradadation。通过使用Oroquine或siRNA对ATG7进行溶酶体途径增强CSE CSE CSE CSE降解。