Thrombospondin1 antagonists and diabetic nephropathy

描述(申请人提供)：尽管加强了血糖控制和肾素-血管紧张素系统抑制剂的使用，但糖尿病肾病仍然是终末期肾病的主要原因。纤维化细胞因子转化生长因子-β是糖尿病肾病发病机制中的关键分子因子。糖尿病患者肾组织中转化生长因子-β的表达及活性增加。转化生长因子β是一种生物潜伏性分子，必须转化为活性形式才能诱导纤维化作用。这一激活步骤代表了转化生长因子-β生物活性的一个主要调节点。我们发现血栓反应蛋白1(TSP1)是糖尿病中转化生长因子β活化的分子调节因子。糖尿病肾病的介质如葡萄糖和血管紧张素II可增加TSP1蛋白的表达。体外研究和STZ诱导的糖尿病合并高血压大鼠模型显示，四氨基酸多肽(LSKL)拮抗TSP1依赖的转化生长因子-β的激活，阻断葡萄糖和血管紧张素Ⅱ刺激的转化生长因子-β的活性，细胞外基质的产生，预防和逆转心肌纤维化。在该模型和其他非糖尿病的肾和肝纤维化模型中，LSKL多肽在该模型和其他非糖尿病模型中是一种有效的拮抗转化生长因子依赖的纤维化的药物。LSKL多肽代表了治疗糖尿病肾病的一种有效的治疗策略：这种多肽具有独特的优势，只选择性地抑制由于TSP1介导的激活而导致的病理性的转化生长因子-β活性的增加。这将LSKL与其他抑制转化生长因子-β作用的策略区分开来，后者不区分动态平衡水平和病理性过度的转化生长因子-β活性。在这些研究中，我们将在一种新开发的1型糖尿病遗传小鼠模型(129/SvEv Ins2 Akita)中测试这一假说，即通过阻断转化生长因子-β的激活来改善肾功能和减轻肾脏纤维化。1型糖尿病有明显的蛋白尿和系膜硬化。在特定的目标1中，小鼠将每周接受三次ip。注射LSKL或对照(LSAL)多肽超过15周，将评估肾功能、形态和转化生长因子-β信号。具体目标2将确定同时针对血管紧张素II 1型受体和TSP1的联合治疗方法是否增加了益处。重要的是，这项建议将解决转化生长因子β的动态平衡功能是否因拮抗TSP1依赖的激活而受到损害。具体目标3将阐述阻断TSP1激活的转化生长因子β是否会在全身组织病理学、肿瘤发病率、免疫细胞图谱和皮肤伤口愈合方面对转化生长因子β的动态平衡功能产生有害影响。这些研究将有助于建立这种依赖于TSP1的转化生长因子-β激活的多肽拮抗剂作为糖尿病肾病的一种新的治疗方法。

DESCRIPTION (provided by applicant): Despite increased glycemic control and use of renin-angiotensin system inhibitors, diabetic nephropathy remains a leading cause of end stage renal disease. The fibrogenic cytokine, transforming growth factor-¿ (TGF-¿), is a key molecular factor in the pathogenesis of diabetic nephropathy. The expression of TGF-¿ and its activity are increased in diabetes. TGF-¿ is expressed as a biologically latent molecule that must be converted to its active form in order to induce fibrogenic effects. This activation step represents a major point of regulation of TGF-¿ bioactivity. We identified thrombospondin 1 (TSP1) as the molecular regulator of TGF-¿ activation in diabetes. TSP1 protein expression is increased by mediators of diabetic nephropathy such as glucose and angiotensin II. In vitro studies and a rat model of STZ-induced diabetes with hypertension showed that antagonism of TSP1-dependent TGF-¿ activation by a four amino acid peptide (LSKL) blocked glucose and angiotensin II stimulation of TGF-¿ activity, extracellular matrix production, and prevented and reversed myocardial fibrosis. The LSKL peptide when administered by intraperitoneal injection is an effective antagonist of TGF-¿ -dependent fibrosis in this model and in other non-diabetic models of renal and hepatic fibrosis. The LSKL peptide represents an effective therapeutic strategy for treatment of diabetic nephropathy: this peptide has the unique advantage of selectively inhibiting only the pathogenic increases in TGF-¿ activity due to TSP1-mediated activation. This distinguishes LSKL from other strategies for inhibiting TGF-¿ action, which do not discriminate between homeostatic levels and pathologic excesses of TGF-¿ activity. In these studies, we will test the hypothesis that administration of the LSKL peptide improves renal function and attenuates renal fibrosis by blocking activation of TGF- ¿ in a newly developed genetic mouse model (129/SvEv Ins2 Akita) of type 1 diabetes, which has significant proteinuria and mesangial sclerosis. In Specific Aim 1, mice will receive thrice weekly i.p. injections of LSKL or control (LSAL) peptide over a 15 week period and renal function, morphology, and TGF-¿ signaling will be evaluated. Specific Aim 2 will determine whether a combined therapeutic approach that targets both the angiotensin II type 1 receptor and TSP1 has increased benefit. Importantly, this proposal will address whether homeostatic functions of TGF-¿ are compromised by antagonism of TSP1-dependent activation. Specific Aim 3 will address whether blockade of TSP1-activated TGF-¿ has deleterious effects on homeostatic functions of TGF-¿ with respect to systemic histopathology, tumor incidence, immune cell profile, and dermal wound healing. These studies will help establish the utility of this peptide antagonist of TSP1-dependent TGF-¿ activation as a novel therapeutic for diabetic nephropathy.