New Insights in Mechanisms of Renal Injury

肾损伤机制的新​​见解

• 批准号: 9269456
• 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/9269456
• 关键词: Albuminuria; Area; Biological Assay; Blood Glucose; Bortezomib; Chloroquine; Chronic Kidney Failure; Cystathionine; Data; Diet; Dominant-Negative Mutation; Epithelial Cells; 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; Reducing diet; Renal function; Research; Risk; Role; Signal Transduction; Sirolimus; Small Interfering RNA; Sodium; Testing; Transgenic Mice; Translating; Tubular formation; Veterans; Weight Gain; drinking water; in vivo; insight; mRNA Expression; male; multicatalytic endopeptidase complex; novel; novel therapeutics; overexpression; protein degradation; protein expression; public health relevance

 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（例如肥胖）中，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尚不清楚。我们的新试验数据显示 (1) 在 C57BL6 小鼠中，高脂肪饮食 (HFD) 导致肾皮质 IR 激活、基质蛋白增加、白蛋白尿和 mTORC1 激活，并伴随体重增加、胰岛素抵抗、高胰岛素血症，但血糖正常。我们培育了肾近端肾小管 IR 敲除小鼠 (KPTIRKO)，其中 HFD 诱导的 IR 激活、基质增量、白蛋白尿和 mTORC1 信号传导均减少。 (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 合酶 (CBS) 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，这表明 HFD 中的 IR 激活会降低 CBS 和 CSE，这是肾损伤的一部分。 (3) HFD降低肾脏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) 细胞中的 CSE 和 CBS 蛋白，但不降低 mRNA。这些数据表明胰岛素对 CSE 和 CBS 进行非转录调节。尽管我们的数据表明 IR 在 HFD 相关肾损伤中发挥作用，但减少 H2S 生成的必要性尚不清楚。假设：在高脂肪饮食引起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 给药是否可以改善 HFD 诱导的小鼠肾损伤。 3 个月大的雄性和雌性 C57BL6 小鼠将被随机分配至正常脂肪饮食 (NFD) 或 HFD，为期 4 个月。一旦肾损伤（白蛋白尿）确定，我们将按 30umole/l 饮用水施用硫氢化钠 (NaHS)（一种 H2S 供体），持续 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 菊糖清除率。信号研究：IR 激活、控制蛋白质合成的信号反应。目标 2. (A) 测试增加 H2S 产量是否可以预防 HFD 引起的肾损伤。我们将 3 个月大的雄性和雌性 WT 小鼠和在近曲小管中过表达人 CBS 的转基因小鼠（PThCBS tg，由我们生成）随机分组为 NFD 或 HFD，为期 4 个月。研究将按目标 1 进行。(B) 测试 HFD 诱导的肾损伤是否在缺乏 H2S 产生的小鼠中放大。我们将我们拥有的 3 个月大的雄性和雌性 WT 和 CSEKO 小鼠随机分配至 NFD 或 HFD 组，为期 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. 探索胰岛素诱导的 CSE、CBS 表达减少的机制。 (A) 体内研究：初步数据表明，HFD 通过非转录调节降低了肾脏 CSE 和 CBS 的表达。我们将通过对饮食 4 个月的 NFD 和 HFD 的 C57BL6 小鼠的肾脏制剂进行多聚体测定，探讨这是否是由于未能翻译其 mRNA 所致。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：高胰岛素在体外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，我们将探讨这是否是由于 (i) 通过多核糖体测定无法翻译 mRNA，和/或 (ii) 通过脉冲追踪测量放射性标记的 CSE 和 CBS 的降解而导致降解增加。我们将通过测量蛋白酶体活性以及蛋白酶体亚基蛋白和 mRNA 表达来测试增强的降解是否是通过蛋白酶体机制实现的。我们将使用硼替佐米、MG132 和 siRNA 作为蛋白酶体亚基。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会通过使用氯喹或针对 Atg7 的 siRNA 刺激溶酶体途径来增强 CSE 和 CBS 降解。