Novel Models of Diabetic Nephropathy

描述（由申请人提供）： 背景资料：在a）患有1型糖尿病的OVE 26小鼠、B）患有饮食诱导的肥胖症和胰岛素抵抗的C57 BI/6小鼠和c）患有2型糖尿病的db/db小鼠中，我们发现转录因子i）固醇调节元件结合蛋白1和2（SREBP-1和SREBP-2），和ii）碳水化合物反应元件结合蛋白（ChREBP），其导致甘油三酯和胆固醇的合成和积累增加。脂质蓄积与肾小球硬化、肾小管间质纤维化和蛋白尿的发生有关。我们还发现法尼醇X受体（FXR）在肾脏中高度表达，并且FXR及其靶酶的表达在糖尿病肾脏中降低。此外，我们已经确定FXR是SREBP-1和ChREBP表达以及氧化应激、晚期糖基化终产物（AGEs/AGEs）、促炎细胞因子和纤维化诱导生长因子的重要调节剂。 假设：基于这些发现，我们提出FXR在糖尿病肾病的发病机制中起重要作用。我们假设在1型（OVE 26）和2型（db/db）糖尿病小鼠模型中，FXR的缺失将显著增强糖尿病肾病，而FXR的过表达将减轻糖尿病肾病。 小鼠模型1：A）我们将产生FXR敲除小鼠，目前在C57 BI/6遗传背景下，在背景下：在a）具有1型糖尿病的OVE 26小鼠中，B）具有饮食诱导的肥胖和胰岛素FVB的C57 BI/6小鼠中，以及如果需要和DBA/2 J遗传背景，2种遗传背景已被证明对糖尿病肾病具有增加的易感性。B）然后，我们将FVB背景下的FXR KO小鼠与i）FVB背景下的OVE 26小鼠（1型糖尿病）或ii）FVB背景下的db/db小鼠（2型糖尿病）杂交，以确定FXR缺失是否加重和加速糖尿病肾病。C）我们将使用Lox-Cre方法在FVB背景下产生肾足细胞特异性FXR敲除小鼠（FXRf/f小鼠与Nphs 2Cre小鼠杂交）。D）然后我们将足细胞FXR KO小鼠与i）OVE 26小鼠或ii）db/db小鼠杂交。小鼠模型2：A）我们将在FVB背景中产生肾足细胞特异性条件和诱导型FXR转基因小鼠。B）然后我们将足细胞特异性FXR转基因小鼠与OVE 26或db/db小鼠杂交以确定FXR表达的增加是否减弱或预防糖尿病性肾病。 表型分析：在这些小鼠中，我们将确定a）糖尿病肾病的表现，包括肾小球滤过率、肾小球硬化、肾小管间质纤维化和蛋白尿，和B）介导糖尿病肾损伤的细胞和生化机制，包括脂质和碳水化合物代谢、炎症、纤维化、氧化应激和AGEs/β 2。

DESCRIPTION (provided by applicant): Background: In a) OVE26 mice with type 1 diabetes, b) C57BI/6 mice with diet induced obesity and insulin resistance, and c) db/db mice with type 2 diabetes mellitus, we have found increased renal expression of the transcriptional factors, i) the sterol regulatory element binding proteins 1 and 2 (SREBP-1 and SREBP-2), and ii) the carbohydrate response element binding protein (ChREBP), which result in increased synthesis and accumulation of triglyceride and cholesterol. The lipid accumulation is associated with development of robust glomerulosclerosis, tubulointerstitial fibrosis, and proteinuria. We have also found that the Farnesoid X Receptor (FXR) is highly expressed in the kidney and the expression of FXR and its target enzymes is decreased in the diabetic kidney. Furthermore we have determined that FXR is an important regulator of SREBP-1 and ChREBP expression as well as oxidative stress, advanced glycation end products (AGEs/RAGE), pro-inflammatory cytokines, and fibrosis inducing growth factors. Hypothesis: Based on these finding we propose that FXR plays an important role on the pathogenesis of diabetic nephropathy. We hypothesize that deletion of FXR will markedly enhance and overexpression of FXR will attenuate diabetic nephropathy in mouse models of type 1 (OVE26) and type 2 (db/db) diabetes. Mouse Model 1: A) We will generate FXR knockout mice, currently on the C57BI/6 genetic background, on Background: In a) OVE26 mice with type 1 diabetes, b) C57BI/6 mice with diet induced obesity and insulin the FVB, and if need be and DBA/2J genetic backgrounds, 2 genetic backgrounds that have been documented to have increased susceptibility to diabetic nephropathy. B) We will then cross breed FXR KO mice on FVB background with i) OVE26 mice (type 1 diabetes) on FVB background or ii) db/db mice (type 2 diabetes) on FVB background to determine if FXR deletion accentuates and accelerates diabetic nephropathy. C) We will generate renal podocyte specific FXR knockout mice on FVB background using the Lox-Cre approach (FXRf/f mice crossed with Nphs2 Cre mice). D) We will then crossbreed podocyte FXR KO mice with i) OVE26 mice or ii) db/db mice. Mouse Model 2: A) We will generate renal podocyte specific conditional and inducible FXR transgenic mice in the FVB Background. B) We will then crossbreed the podocyte specific FXR transgenic mice with OVE26 or db/db mice to determine if increased expression of FXR attenuates or prevents diabetic nephropathy. Phenotyping: In these mice we will determine a) the manifestation of diabetic nephropathy, including glomerular filtration rate, glomerulosclerosis, tubulointerstitial fibrosis, and proteinuria and b) the cellular and biochemical mechanisms that mediate diabetic renal injury including lipid and carbohydrate metabolism, inflammation, fibrosis, oxidative stress, and AGEs/RAGE.