以前,我们的实验室显示内质网(ER)和钙调节蛋白,钙网蛋白(CRT),对胶原蛋白转录很重要,分泌,并组装到细胞外基质(ECM)中,ERCRT对于通过刺激ER钙释放和NFAT激活来刺激I型胶原蛋白转录的TGF-β至关重要。糖尿病是终末期肾病的主要原因。TGF-β是糖尿病肾病发病的关键因素。然而,钙网蛋白(Calr)在糖尿病肾病纤维化中的作用尚未被研究。在目前的工作中,我们使用体外和体内方法来评估ER-CRT在肾小管细胞和糖尿病小鼠中TGF-β和葡萄糖刺激的ECM产生中的作用。与对照细胞相比,在人近端肾小管细胞系(HK-2)中通过siRNA敲低CALR显示,当被TGF-β或高葡萄糖刺激时,可溶性胶原蛋白的诱导降低。以及纤连蛋白和胶原IV转录水平的降低。与对照组相比,用链脲佐菌素制成的糖尿病小鼠的肾脏中CRT蛋白增加,并进行单肾切除术以加速肾小管损伤。我们使用Cre重组酶质粒的肾脏靶向超声递送来特异性敲低未切除的患有链脲佐菌素诱导的糖尿病的Calrfl/fl小鼠的剩余肾脏中的CRT表达。这种方法减少了肾脏中的CRT表达,主要在肾小管上皮,30-55%,在研究过程中持续存在。与注射盐水或进行超声并注射对照GFP质粒的糖尿病小鼠相比,在CRT敲低的小鼠中通过尿白蛋白/肌酸酐比率测量的肾功能得到改善。肾脏的PAS染色和I型和IV型胶原蛋白的免疫组织化学分析显示肾小球和肾小管间质纤维化减少。来自CRT敲除的糖尿病小鼠的肾脏切片显示肾小管中核NFAT减少,并且用11R-VIVIT治疗糖尿病小鼠,NFAT抑制剂,减少蛋白尿和肾纤维化。这些研究确定ER-CRT是糖尿病肾脏中TGF-β刺激ECM产生的重要调节因子,可能通过调节NFAT依赖性ECM转录。
Previously, our lab showed that the endoplasmic reticulum (ER) and calcium regulatory protein, calreticulin (CRT), is important for collagen transcription, secretion, and assembly into the extracellular matrix (ECM) and that ER CRT is critical for TGF-β stimulation of type I collagen transcription through stimulation of ER calcium release and NFAT activation. Diabetes is the leading cause of end stage renal disease. TGF-β is a key factor in the pathogenesis of diabetic nephropathy. However, the role of calreticulin (Calr) in fibrosis of diabetic nephropathy has not been investigated. In current work, we used both in vitro and in vivo approaches to assess the role of ER CRT in TGF-β and glucose stimulated ECM production by renal tubule cells and in diabetic mice. Knockdown of CALR by siRNA in a human proximal tubular cell line (HK-2) showed reduced induction of soluble collagen when stimulated by TGF-β or high glucose as compared to control cells, as well as a reduction in fibronectin and collagen IV transcript levels. CRT protein is increased in kidneys of mice made diabetic with streptozotocin and subjected to uninephrectomy to accelerate renal tubular injury as compared to controls. We used renal-targeted ultrasound delivery of Cre-recombinase plasmid to knockdown specifically CRT expression in the remaining kidney of uninephrectomized Calr fl/fl mice with streptozotocin-induced diabetes. This approach reduced CRT expression in the kidney, primarily in the tubular epithelium, by 30-55%, which persisted over the course of the studies. Renal function as measured by the urinary albumin/creatinine ratio was improved in the mice with knockdown of CRT as compared to diabetic mice injected with saline or subjected to ultrasound and injected with control GFP plasmid. PAS staining of kidneys and immunohistochemical analyses of collagen types I and IV show reduced glomerular and tubulointerstitial fibrosis. Renal sections from diabetic mice with CRT knockdown showed reduced nuclear NFAT in renal tubules and treatment of diabetic mice with 11R-VIVIT, an NFAT inhibitor, reduced proteinuria and renal fibrosis. These studies identify ER CRT as an important regulator of TGF-β stimulated ECM production in the diabetic kidney, potentially through regulation of NFAT-dependent ECM transcription.