Role of KLF15 in podocyte differentiation

足细胞分化丧失导致的足细胞损伤与许多肾脏疾病有关，如局灶节段性肾小球硬化(FSGS)和HIV相关性肾病(HIVAN)。FSGS和HIVAN是美国终末期肾脏疾病最突出的原因之一，特别是在非裔美国人中。已有研究表明，维甲酸(RA)通过刺激cAMP/PKA/CREB途径诱导足细胞分化。然而，介导RA对足细胞分化标志物影响的转录因子尚不清楚。基因芯片研究RA处理的人足细胞，以确定受RA高度调控的基因。计算分析显示，CREB靶向基因Krupper-like factor15(KLF15)在足细胞中被RA高度上调。KLF15不仅具有补肾作用，而且此前已被证明具有促进脂肪细胞分化的作用。首先，证实RA刺激条件永生化的小鼠和人足细胞KLF15的mRNA和蛋白表达显著增加。这一发现在培养的HIV感染足细胞以及从HIV转基因小鼠分离的肾小球中重复。此外，KLF15的过表达刺激了野生型和HIV感染的小鼠足细胞中突触素(足细胞分化标志)的表达。此外，染色质免疫沉淀研究表明，KLF15结合到RA处理的小鼠足细胞中neparin和podocin(狭缝隔膜蛋白)的启动子区域。尽管在基线状态下，KLF15-/-小鼠的蛋白尿增加了两倍，足细胞损伤最小，但与脂多糖治疗的野生型小鼠相比，脂多糖(一种足细胞损伤的小鼠模型)治疗的KLF15-/-小鼠的蛋白尿和足细胞减少显著增加。基于这些发现，假设KLF15是足细胞分化的关键调节因子。第一个目的是通过处理KLF15缺陷细胞并或不伴RA来确定KLF15在RA介导的足细胞分化中的作用。这些发现将在体内得到验证。第二个目标是确定KLF15在疾病状态中的作用。最初，KLF15在健康供者的肾活检组织中的表达将与FSGS和HIVAN患者进行比较。接下来，将在转基因小鼠模型(HIVAN)和阿霉素诱导的肾病(FSGS)小鼠模型中表征KLF15的作用。最后，将确定在这些疾病状态下调节KLF15的机制。通过鉴定KLF15是足细胞分化的重要转录调控因子，将对肾脏生理和疾病有新的认识，并将为维甲酸用于肾小球疾病的治疗提供进一步的证据。

Podocyte injury resulting from a loss of podocyte differentiation has been implicated in many kidney diseases such as Focal Segmental Glomerular Sclerosis (FSGS) and HIV Associated Nephropathy (HIVAN). FSGS and HIVAN are among the most prominent causes of end stage renal disease in the United States, especially in African Americans. It has been previously shown that Retinoic Acid (RA) induces podocyte differentiation via stimulation of cAMP/PKA/CREB pathway. However, the transcription factors mediating the effects of RA on podocyte differentiation markers are not known. Microarray gene expression studies in human podocytes treated with RA were done to identify genes highly regulated by RA. Computational analysis revealed a CREB-targeted gene called Krupper-Like Factor 15 (KLF15) was highly upregulated by RA in podocytes. KLF15 is not only kidney enriched, but has been previously shown to mediate adipocyte differentiation. First, it was confirmed that RA stimulated a significant increase in KLF15 mRNA and protein expression in conditionally immortalized mouse and human podocytes. This finding was replicated in cultured HIV infected podocytes as well as in glomeruli isolated from HIV transgenic mice. Furthermore, the over- expression of KLF15 stimulated the expression of synaptopodin (podocyte differentiation marker) in wild-type and HIV-infected murine podocytes. Additionally, Chromatin Immunoprecipitation studies revealed that KLF15 binds to the promoter region of nephrin and podocin (slit diaphragm proteins) in RA treated mouse podocytes. Although, KLF15-/- mice at baseline had only a two-fold increase in proteinuria with minimal podocyte injury, KLF15-/- mice treated with lipopolysaccharide (LPS), a murine model of podocyte injury, showed a significant increase in proteinuria and podocyte effacement as compared to LPS treated wild-type mice. Based on these findings, the hypothesis is that KLF15 is a key regulator of podocyte differentiation. The first aim is to determine the role of KLF15 in RA-mediated podocyte differentiation by treating KLF15 deficient cells with or without RA. These findings will be validated in vivo. The second aim is to characterize the role of KLF15 in diseased states. Initially, the expression of KLF15 in kidney biopsies of healthy donors will be compared to patients with FSGS and HIVAN. Next, the role of KLF15 will be characterized in murine models of HIVAN (transgenic murine model) and FSGS (Adriamycin induced nephropathy). Finally, the mechanism mediating the regulation of KLF15 in these diseased states will be determined. By identifying that KLF15 is an important transcriptional regulator of podocyte differentiation, new insight will be gained in kidney physiology and disease as well as further evidence will be provided for the use of Retinoic Acid in the treatment of glomerular disease.