Thrombospondin1 antagonists and diabetic nephropathy

描述（由申请人提供）：尽管加强血糖控制和使用肾素-血管紧张素系统抑制剂，糖尿病肾病仍然是终末期肾脏疾病的主要原因。纤维化细胞因子转化生长因子-¿（TGF-¿）是糖尿病肾病发病的关键分子因子。TGF-¿在糖尿病中的表达及活性升高。TGF-¿是一种生物潜伏分子，必须将其转化为活性形式才能诱导成纤维作用。这一激活步骤是TGF-¿生物活性调控的重要环节。我们发现血栓反应蛋白1 （TSP1）是糖尿病中TGF-¿激活的分子调节剂。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诱导的高血压糖尿病大鼠模型表明，四氨基酸肽（LSKL）拮抗tsp1依赖性TGF-¿激活，可阻断葡萄糖和血管紧张素II刺激TGF-¿活性、细胞外基质生成，预防和逆转心肌纤维化。腹腔注射LSKL肽在该模型和其他非糖尿病性肾纤维化和肝纤维化模型中是TGF-依赖性纤维化的有效拮抗剂。LSKL肽是治疗糖尿病肾病的一种有效的治疗策略：该肽具有选择性地抑制由于tsp1介导的激活而导致的TGF-¿活性的致病性增加的独特优势。这将LSKL与其他抑制TGF-¿作用的策略区分开来，这些策略不区分TGF-¿活性的稳态水平和病理性过度。在这些研究中，我们将在新开发的1型糖尿病遗传小鼠模型（129/SvEv Ins2 Akita）中验证LSKL肽通过阻断TGF-¿的激活来改善肾功能和减轻肾纤维化的假设，该模型有明显的蛋白尿和肾小球系膜硬化。在Specific Aim 1中，小鼠每周接受三次LSKL或对照（LSAL）肽的腹腔注射，持续15周，并评估肾功能、形态学和TGF-¿信号。特异性目的2将确定同时靶向血管紧张素II型1受体和TSP1的联合治疗方法是否会增加获益。重要的是，该提案将解决TGF-¿的稳态功能是否会受到tsp1依赖性激活的拮抗。特异性目的3将探讨阻断tsp1激活的TGF-¿是否会对TGF-¿在系统组织病理学、肿瘤发生率、免疫细胞谱和皮肤伤口愈合方面的稳态功能产生有害影响。这些研究将有助于确立这种肽拮抗剂对tsp1依赖性TGF-¿激活的效用，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.