The Role And The Ralative Mechanism Of Sig-1R In Cisplatin-induced Injury In Renal Proximal Tubular Cells

Cisplatin is one of the most widely used and most potent antitumor agent, whose antineoplastic effect is dose dependent. However, its use is mainly limited by severe side effects in normal tissues, such as neurotoxicity, ototoxicity, especially nephrotoxicity. In recent years, studies have shown that the major molecular mechanism of cisplatin nephrotoxicity is associated with complicated cellular signaling pathways leading to tubular cell injury, apoptosis,and death induced by cisplatin, including endoplasmic reticulum (ER) stress and mitochondrial dysfunction. But the exactly cellular and molecular mechanism of cisplatin nephrotoxicity is not completely understood.The endoplasmic reticulum (ER) stress occurs when its functions have been disturbed by all kinds of pathological conditions, leading to the accumulation of unfolded and misfolded proteins in the ER lumen, and the disruption of Ca2+homeostasis. ER stress is associated with many types of kidney disease, such as membranous nephropathy, congenital nephrotic syndrome, and ischemic acute tubular injury. Meanwhile, the role of ER stress in cisplatin induced kidney injury has also been reported. Cisplatin induces the production of ER chaperone glucose regulated protein (GRP78) and the activation of caspase-12.Sigma-1Receptor (Sig-1R) is one of the major ER chaperone proteins, and is resided at the ER-mitochondrion contact called the mitochondrion-associated ER membrane(MAM). Sig-1R regulates ER-mitochondrial interorganellar Ca2+signaling and cell survival by stabilizing the conformation of type-3IP3receptors (IP3R) at MAM. A study has shown that in cultured cortical neurons from embryonic rats, activation of Sig-1R can ameliorate Ca2+dysregulation and cell apoptosis associated with ischemia in cortical neurons. BHDP protects liver against the deleterious effects of ischemia-reperfusion and suggest that Sig-1R plays an important role in the protective effect. Silencing of Sig-IR in human lens cells induces significant cell death occurred with an increased expression of caspase-3. In addition, overexpression of Sig-1R can suppresses ER stress.Mitochondria is the power plant of cells, where ATP synthesis, electron transfer, oxidative phosphorylation are happened. Recently, more and more attentions have been paid on the mitochondrial dysfunction in renal disease. In multiple kidney disease models, abnormalities of mitochondrial ultramicrostructure, mutations of mitochondrial DNA and downregulation of mitochondrial DNA copy numbers may be associated with podocyte injury. It has been proved that mitochondrial dysfunction is also associated with cisplatin induced acute kidney injury. Cisplatin induced the production of ROS, activation of proapoptotic protein such as Bax and Bak.The ER and mitochondria are the most important intracellular organelles. Physical interactions between these two compartments have been observed for a long time. The function of close communication between ER and mitochondria includes cell death pathways and intracellular calcium homeostasis. It has been known that the Ca2+signaling from the ER into mitochondria is important for the generation of ATP and the opening of mitochondrial permeablity transition pore (MPTP). Sig-1R ensures proper Ca2signaling from the ER into mitochondria.Part I ER stress and mitochondrial dysfunction mediate cisplatin-induced renal proximal tubular cell injuryObjective:To explore the effect of ER stress and Mitochondrial dysfunction in cisplatin-induced renal proximal tubular cell injury.Methods:Mice renal proximal tubular cells were cultured in vitro, and treated with cisplatin at the concentration of0,5,10,20μmol/L respectively and at different time points of0,6,12,24,48h respectively. The expressions of glucose regulated protein78(GRP78), glucose regulated protein94(GRP94), CCAAT/enhancer-binding protein homologous protein (CHOP), mitochondrial DNA (mtDNA) copy numbers were examined by real-time PCR. The expressions of epithelial cadherin (E-cadherin) were examined by western blot. Apoptosis of renal proximal tubular cells was assessed by annexin V/flow cytometry detection. The concentration of cytoplasm Ca2+was measured by Fluo-3fluorescence.The viability of renal proximal tubular cells was assessed by Cell Counting Kit (CCK-8).The production of cellular reactive oxygen species (ROS) was examined by DCFDA fluorescence. Mitochondrial membrane potential (MMP) was determined by JC-1fluorescence.Results:(1) Cisplatin dose-dependently and time-dependently inhibited proximal tubular cell viability, increased cell apoptosis, and decreased the expression of E-cadherin. Cell apoptosis was increased by249.7%, while E-cadherin expression was decreased by38.1%at24h in Cisplatin-treated proximal tubular cells.(2) Cisplatin dose-dependently increased the concentration of cytoplasm Ca2+. Cisplatin dose-dependently and time-dependently increased the expression of GRP78、GRP94、 CHOP. GRP78、GRP94expression were upregulated by55.2%and54.2%at12h, while CHOP expression were upregulated by118.7%at24h in Cisplatin-treated proximal tubular cells.(3) Cisplatin induced the production of ROS in dose-dependent. Cisplatin induced the reduction of MMP, mtDNA copy numbers in dose-dependent and time-dependent manner. Cisplatin induced decreases in mtDNA copy numbers and MMP after12h of treatment(decreased by33.0%and29.9%).Conclusion:ER stress and mitochondrial dysfunction were the early event in cisplatin-induced renal proximal tubular cell injury, and ER stress and mitochondrial dysfunction mediate cisplatin-induced renal proximal tubular cell injury. Part Ⅱ Sig-1R prevents renal proximal tubular cell injury induced by cisplatin via ameliorating ER stressObjective:To explore the effect of Sig-1R on renal proximal tubular cell injury and ER stress induced by cisplatin.Methods:Mice renal proximal tubular cells were cultured in vitro, transfected transiently with pcDNA3.0-Sig-1R or Sig-1R siRNA vector, than treated with cisplatin (10μmol/L) or Saline,and were divided into four groups:Saline group, Cisplatin group, Saline+pcDNA3.0-Sig-1R or Sig-1R siRNA group、Cisplatin+pcDNA3.0-Sig-1R or Sig-1R siRNA group. The expressions of GRP78, GRP94, CHOP were examined by real-time PCR. The expressions of E-cadherin, GRP78, GRP94, CHOP, caspase-12were examined by western blot. Apoptosis of renal proximal tubular cells was assessed by annexin V/flow cytometry detection and Hoechst33258staining. The concentration of cytoplasm Ca2+was measured by Fluo-3fluorescence.The viability of renal proximal tubular cells was assessed by Cell Counting Kit (CCK-8). Caspase-3activities in renal proximal tubular cells were assessed by caspase activity assay kits. In vivo, WT mice and Sig-1R transgenic mice were received a single intraperitoneal injection of20mg/kg cisplatin or Saline. And all the mice were divided into four groups:WT Saline group, WT Cisplatin group, Sig-1R transgenic Saline group, Sig-1R transgenic Cisplatin grou. Mice were sacrificed72hours after cisplatin treatment. The proteinuria was evaluated by urine protein/urine creatinine. The serum creatinine, blood urea nitrogen was evaluated. Histology of kidney was tested by HemateinEosin (HE) and Periodic acid Schiff (PAS) staining. Renal proximal tubular cell injury and ER stress were detected.Results:(1) Sig-1R expression decreased significantly after transfection with Sig-1R siRNA.(2) Downregulation of Sig-1R by siRNA inhibited proximal tubular cell viability by35.2%(P<0.05), increased cell apoptosis by80.4%(P<0.05) and increased the activation of Caspase-3, decreased the expression of E-cadherin, increased the concentration of cytoplasm Ca2+, the expression of GRP78、GRP94、 CHOP, and the activation of Caspase-12, while compared with Cisplatin group.(3) Sig-1R mRNA and protein expression increased significantly after transfection with pcDNA3.0-Sig-1R.(4) Overexpression of Sig-1R prevented renal proximal tubular cell injury induced by cisplatin, and inhibited the expression of GRP78、GRP94、 CHOP, the concentration of cytoplasm Ca2+, and the activation of Caspase-12.(5)In vivo, overexpression of Sig-1R reduced proteinuria, serum creatinine, blood urea nitrogen by67.7%,51.7%and75.4%(both P<0.05), decreased the expression of NGAL and IL-18by65.9%and37.4%(both P<0.05), ameliorated the renal histological damage, and inhibited renal proximal tubular cell injury and ER stress induced by cisplatin.Conclusion:Sig-1R prevents renal proximal tubular cell injury induced by cisplatin via ameliorating ER stress. Part Ⅲ Sig-1R prevents renal proximal tubular cell injury induced by cisplatin via ameliorating mitochondrial dysfunctionObjective:To explore the effect of Sig-1R on renal proximal tubular cell injury and mitochondrial dysfunction induced by cisplatin.Methods:Mice renal proximal tubular cells were cultured in vitro, transfected transiently with pcDNA3.0-Sig-1R or Sig-1R siRNA vector, than treated with cisplatin (10μmol/L) or Saline,and were divided into four groups:Saline group, Cisplatin group, Saline+pcDNA3.0-Sig-1R or Sig-1R siRNA group、Cisplatin+ pcDNA3.0-Sig-1R or Sig-1R siRNA group. The expression of peroxisome proliferator-activated receptor-y coactivator-la (PGC-la), mitochondrial transcription factor A (TFAM) were examined by real-time PCR and western blot. The opening of mPTP was observed by Calcein/AM fluorescence. mtDNA copy numbers were determined by real-time PCR. The production of cellular ROS was examined by DCFDA fluorescence. MMP was determined by JC-1fluorescence. In vivo, WT mice and Sig-1R transgenic mice were received a single intraperitoneal injection of20mg/kg cisplatin or Saline. And all the mice were divided into four groups:WT Saline group, WT Cisplatin group, Sig-1R transgenic Saline group, Sig-1R transgenic Cisplatin grou. Mice were sacrificed72hours after cisplatin treatment. Mitochondrial dysfunction was detected.Results:(1) Compared with Cisplatin group, the expression of PGC-la, TFAM were significantly decreased by49.7%and42.3%(both P<0.05), the production of ROS was increased by27.9%(P<0.05), and the mitochondrial membrane potential (MMP) and mtDNA copy number were significantly decreased17.5%and29.3%(both P<0.05) in Cisplatin+Sig-1R siRNA group.(2) Overexpression of Sig-1R ameliorated mitochondrial dysfunction:the expression of PGC-la, TFAM was increased, the production of ROS was decreased, and the MMP, mtDNA copy number were increased.(3) In vivo, overexpression of Sig-1R increased the expression of PGC-la, TFAM, blocked the ROS production, MMP, and mtDNA copy numbers.Conclusion:Sig-1R prevents renal proximal tubular cell injury induced by cisplatin via balancing the concentration of Ca2+, ameliorating mitochondrial dysfunction.