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如局灶节段性肾小球硬化（FSGS）和HIV相关肾病（HIVAN）有关。FSGS和HIVAN是美国终末期肾病的最主要原因之一，尤其是在非洲裔美国人中。先前已经表明，视黄酸（RA）通过刺激cAMP/PKA/CREB途径诱导足细胞分化。然而，调节RA对足细胞分化标志物影响的转录因子尚不清楚。微阵列基因表达的研究，在人类足细胞与RA处理，以确定基因高度调节RA。计算分析显示，CREB靶向基因Krupper样因子15（KLF 15）在足细胞中被RA高度上调。KLF 15不仅富含肾脏，而且先前已显示介导脂肪细胞分化。首先，证实RA刺激条件永生化小鼠和人足细胞中KLF 15 mRNA和蛋白表达的显著增加。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得到了复制。此外，KLF 15的过表达刺激野生型和HIV感染的鼠足细胞中突触足蛋白（足细胞分化标志物）的表达。此外，染色质免疫沉淀研究显示，KLF 15结合到RA处理的小鼠足细胞中nephrin和podocin（狭缝隔膜蛋白）的启动子区域。尽管KLF 15-/-小鼠在基线时蛋白尿仅增加两倍，足细胞损伤最小，但与LPS处理的野生型小鼠相比，脂多糖（LPS）处理的KLF 15-/-小鼠（足细胞损伤的小鼠模型）显示蛋白尿和足细胞消失显著增加。基于这些发现，假设KLF 15是足细胞分化的关键调节因子。第一个目的是通过用或不用RA处理KLF 15缺陷细胞来确定KLF 15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。第二个目的是描述KLF 15在疾病状态中的作用。首先，将健康供体肾活检组织中KLF 15的表达与FSGS和HIVAN患者进行比较。接下来，将在HIVAN（转基因鼠模型）和FSGS（阿霉素诱导的肾病）的鼠模型中表征KLF 15的作用。最后，将确定在这些疾病状态下介导KLF 15调节的机制。通过鉴定KLF 15是足细胞分化的重要转录调节因子，将在肾脏生理学和疾病方面获得新的见解，并将为维甲酸治疗肾小球疾病提供进一步的证据。

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.