New Insights in Mechanisms of Renal Injury

 描述(由申请人提供)： 目前尚不清楚高胰岛素血症是否通过胰岛素受体(IR)在高胰岛素抵抗状态(如肥胖)的激活而直接导致肾脏损伤。我们新的实验数据显示：(1)在C57BL6小鼠中，高脂饮食(HFD)导致肾脏皮质IR激活，基质蛋白增加，蛋白尿增加，mTORC1激活，同时体重增加，胰岛素抵抗，高胰岛素血症，但血糖正常。我们已经建立了肾近端小管IR基因敲除小鼠(KPTIRKO)，在KPTIRKO中，HFD诱导的IR激活、基质增加、蛋白尿和mTORC1信号转导减少。(2)硫化氢(H_2S)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降低了肾脏CSE和CBS的含量，但在KPTIRKO小鼠中没有，这表明HFD的IR激活减少了CBS和CSE，这是肾脏损伤的一部分。(3)HFD可降低肾小管上皮细胞CSE和CBS的含量，但不影响其mRNA的表达，胰岛素可降低近端肾小管上皮细胞CSE和CBS蛋白的表达，但不影响其mRNA的表达。这些数据表明胰岛素对CSE和CBS的非转录调控。虽然我们的数据表明IR在HFD相关的肾损伤中起作用，但减少硫化氢生成的要求尚不清楚。假设：在高脂饮食诱导的高胰岛素抵抗状态下，肾脏胰岛素受体的激活通过减少硫化氢的生成而导致肾脏损伤。目的1.检测硫化氢对高脂饲料所致小鼠肾损伤的影响。3月龄雄性和雌性C57BL6小鼠随机分为正常脂肪饮食(NFD)和高脂饮食(HFD)组，为期4个月。一旦确定肾脏损伤(蛋白尿)，我们将给予硫化氢供体硫化钠(NaHS)，按30umoles/L饮水，连续3个月。主要研究内容如下：结构变化：肾脏肥大、免疫组织化学、基质蛋白的mRNA/蛋白分析。功能变化：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。目的2.(A)测试增加的H_2S产生是否对HFD诱导的肾损伤有保护作用。我们将3月龄WT和近端小管高表达人CBS的转基因小鼠(PThCBS TG)随机分为NFD组和HFD组，为期4个月。研究将按照目标1进行。(B)测试硫化氢产生不足的小鼠是否扩大了HFD引起的肾脏损伤。我们将3个月大的雄性和雌性WT和CSEKO小鼠随机分为NFD或HFD组，为期4个月。结构、功能和信号的研究将与目标1相同。目的3.探讨胰岛素诱导CSE、CBS表达降低的机制。(A)体内研究：初步数据显示，HFD通过非转录调控降低了肾脏CSE和CBS的表达。我们将通过对饮食4个月的NFD和HFD的C57BL6小鼠的肾脏标本进行多聚体分析来探索这是否由于未能翻译它们的mRNA所致。将测量溶酶体和蛋白酶体途径蛋白表达的变化。(B)体外实验：高胰岛素抑制MCT细胞CSE和CBS蛋白表达，其作用机制与体内实验结果相似。使用1和10 nM的胰岛素作为MCT细胞中的高胰岛素，我们将探索这是否由于(I)多聚体分析无法翻译mRNA，和/或(Ii)是否由于通过脉冲追踪法测量放射性标记的CSE和CBS的降解而增加降解所致。我们将通过测量蛋白酶体活性和蛋白酶体亚单位蛋白和mRNA的表达来测试是否通过蛋白酶体机制来增强降解。我们将使用Bortezomib、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。我们还将通过使用氯喹或针对ATG7的siRNA来测试高胰岛素是否刺激溶酶体途径来促进CSE和CBS的降解。

 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.