Lu Ailing, Pallero Manuel A, Owusu Benjamin Y, Borovjagin Anton V, Lei Weiqi, Sanders Paul W, Murphy-Ullrich Joanne E
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL35294-0019, USA.
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294-0019, USA.
Matrix Biol Plus. 2020 Apr 3;8:100034. doi: 10.1016/j.mbplus.2020.100034. eCollection 2020 Nov.
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 () 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 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 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.
此前，我们实验室发现内质网（ER）和钙调节蛋白钙网蛋白（CRT）对胶原蛋白的转录、分泌以及组装进入细胞外基质（ECM）很重要，并且内质网CRT通过刺激内质网钙释放和NFAT激活，对TGF-β刺激I型胶原蛋白转录至关重要。糖尿病是终末期肾病的主要病因。TGF-β是糖尿病肾病发病机制中的关键因素。然而，钙网蛋白（）在糖尿病肾病纤维化中的作用尚未得到研究。在当前的工作中，我们使用体外和体内方法来评估内质网CRT在TGF-β和葡萄糖刺激肾小管细胞产生ECM以及在糖尿病小鼠中的作用。与对照细胞相比，在人近端肾小管细胞系（HK-2）中通过siRNA敲低显示，当受到TGF-β或高糖刺激时，可溶性胶原蛋白的诱导减少，同时纤连蛋白和IV型胶原蛋白转录水平也降低。与对照组相比，用链脲佐菌素诱导糖尿病并进行单侧肾切除以加速肾小管损伤的小鼠肾脏中，CRT蛋白增加。我们使用肾靶向超声递送Cre重组酶质粒，特异性敲低链脲佐菌素诱导糖尿病的单侧肾切除小鼠剩余肾脏中的CRT表达。这种方法使肾脏中CRT的表达降低了30 - 55%，主要是在肾小管上皮细胞中，并且在研究过程中持续存在。与注射生理盐水或接受超声并注射对照GFP质粒的糖尿病小鼠相比，CRT敲低的小鼠中通过尿白蛋白/肌酐比值测量的肾功能得到改善。肾脏的PAS染色以及I型和IV型胶原蛋白的免疫组化分析显示肾小球和肾小管间质纤维化减少。CRT敲低的糖尿病小鼠肾切片显示肾小管中核NFAT减少，用NFAT抑制剂11R-VIVIT治疗糖尿病小鼠可减少蛋白尿和肾纤维化。这些研究确定内质网CRT是糖尿病肾脏中TGF-β刺激的ECM产生的重要调节因子，可能是通过调节NFAT依赖的ECM转录来实现的。
