Role of RTN1A in the Progression of Diabetic Nephropathy

项目摘要 糖尿病肾病(DKD)仍然是美国终末期肾病(ESRD)的主要原因，治疗方案包括 有限的。因此，找出导致DKD进展的因素是至关重要的。通过分析 在具有不同疾病进展速度的小鼠模型中，我们发现了网状蛋白-1 (Rtn1)，一个编码内质网(ER)相关蛋白的基因，作为进展的危险因素 CKD。我们发现RTN1a在病变肾脏中的表达显著增加，并且呈负相关 在患有DKD的人中存在EGFR。在体外，我们发现rtn1a介导了肾脏的内质网应激和细胞凋亡。 肾小管上皮细胞(RTEC)。在活体中，我们发现Rtn1a表达的全球敲除减弱 链脲佐菌素(STZ)诱导的糖尿病小鼠蛋白尿和肾脏损伤。在上一个资助期内，我们 进一步证明在STZ诱导的糖尿病小鼠的RTEC中RTN1a的表达 导致肾功能下降和肾纤维化的发展，这在 STZ诱导的糖尿病小鼠。令人惊讶的是，RTN1A相互作用蛋白的筛选结果表明， 排名靠前的是线粒体蛋白，如己糖激酶1(HK1)、TOMM40和SLC25A12。 与此一致的是，最近的一项研究也证明了内质网线粒体中存在大量的RTN1A 联系地点。内质网-线粒体接触(EMC)，或也称为线粒体相关的内质网 (MAMS)，对多种细胞内事件具有多效性，包括线粒体损伤、钙 信号、内质网应激、细胞凋亡和自噬。然而，EMC与肾脏疾病的直接联系 目前还没有报道。我们的初步数据显示，在HK2细胞中过表达RTN1A可诱导 不仅是内质网应激，还有线粒体功能障碍。高糖增加内质网应激和线粒体 RTN1a过表达可进一步加重HK2细胞功能障碍。在体内，糖尿病小鼠 RTEC中RTN1A的过度表达增加了内质网应激和线粒体功能障碍的标志物。 这些数据表明，RTN1A可能在内质网应激和线粒体功能障碍中发挥关键作用 通过EMC。此外，我们还证实了RTN1A与HK1相互作用并诱导HK1在 RTEC和HK1是细胞凋亡和炎性小体激活的关键调节因子。根据这些数据，我们 假设RTN1A介导的EMC在诱导肾小管细胞损伤和进展中起关键作用 DKD的一部分。为了验证这一点，我们建议确定1)RTN1A在介导EMC在RTEC损伤中的作用 糖尿病状态；2)RTN1A介导的EMC在糖尿病状态下RTEC损伤中的机制；以及3)糖尿病状态下RTN1A介导的EMC的机制 将EMC作为治疗DKD的潜在干预措施。

Project Summary Diabetic kidney disease (DKD) remains as the leading cause of ESRD in USA and the treatment options are limited. Therefore, it is critical for us to identify contributing factors for the progression of DKD. By analyzing the transcriptomic datasets in mouse models with different rates of disease progression, we identified Reticulon-1 (Rtn1), a gene encoding an endoplasmic reticulum (ER)-associated protein, as a risk factor for progression of CKD. We found that RTN1A expression is markedly increased in the diseased kidney and inversely correlated with eGFR in human with DKD. In vitro, we showed that RTN1A mediates ER stress and apoptosis in renal tubular epithelial cells (RTEC). In vivo, we showed that a global knockdown of Rtn1a expression attenuated albuminuria and kidney injury in streptozotocin (STZ)-induced diabetic mice. During the last funding period, we further demonstrated that the induction of RTN1A expression in RTEC in the STZ-induced diabetic mice resulted in the decline of renal function and development of renal fibrosis, which are not typically observed in STZ-induced diabetic mice. Screening of RTN1A-interacting proteins surprisingly showed that the highly ranked proteins were mitochondrial proteins, such as hexokinase 1 (HK1), TOMM40, and SLC25A12. Consistent with this, a recent study also demonstrates a large amount of RTN1A in the ER-mitochondrial contact sites. ER-mitochondrial contact (EMC), or also referred as mitochondria-associated ER membranes (MAMs), have pleiotropic effects on a variety of intracellular events including mitochondrial damage, Ca2+ signaling, ER stress, apoptosis, and autophagy. However, the direct association of EMC with kidney disease has not been reported. Our preliminary data showed that overexpression of RTN1A in HK2 cells induced not only ER stress but also mitochondrial dysfunction. High glucose increased both ER stress and mitochondrial dysfunction in HK2 cells, which was further aggravated by RTN1A overexpression. In vivo, diabetic mice with RTN1A overexpression in RTEC had increased markers for both ER stress and mitochondrial dysfunction. These data suggest a critical role of RTN1A in mediating both ER stress and mitochondrial dysfunction likely through EMC. Also, we confirmed that RTN1A interacted with HK1 and induced the degradation of HK1 in RTEC and HK1 is a key regulator of apoptosis and inflammasome activation. Based on these data, we hypothesize that RTN1A-mediated EMC plays a critical role in inducing tubular cell injury and the progression of DKD. To test this, we propose to determine 1) the role of RTN1A in mediating EMC in RTEC injury in diabetic condition; 2) the mechanism of RTN1A-mediated EMC in RTEC injury in diabetic condition; and 3) the potential interventions to regulate EMC as a therapy for DKD.