The Effect Of Carbon Monoxide And Endoplasmic Reticulum Stress Protein CHOP On Cyclosporine A-induced Injury Of Glomerular Endothelial Cells

Transplantation is the treatment of choice for patients with end-stage organ failure including patients with stage 5 chronic kidney disease. However, most of the grafts eventually lose their function because of chronic rejection, even with advanced use of multiple, complementary immunosuppressive reagents. Cyclosporine A (CsA) is one of the most commonly used immunosuppressive reagents. While preventing allograft immune rejection, CsA may injure many renal cells. However, if CsA can cause injury and dysfunction in glomerulus endothelial cells, a major lesion of transplant loss, is unknown. Heme oxygenase-1 (HO-1) is an inducible enzyme synthesized and localized in endoplasmic reticulum. It protects cells from injury by presenting anti-inflammatory, anti-oxidative and anti-apoptotic properties through its catalyzed products such as carbon monoxide (CO). Although HO-1 induction has been found beneficial in preventing transplant rejection, if it is protective for the injury of glomerulus endothelial cells is not known. Endoplasmic reticulum stress (ERs) is a subcellular protective response. Moderate ERs promotes cell repair. Severe ERs induces apoptosis for severe damaged cells that can not be repaired. However, if glomerulus endothelial cells have ERs and its roles in renal physiology and pathophysiology is unknown.Objective This study aimed to observe the effects of CsA on biological properties and ERs in glomerulus endothelial cells, the effects of HO-1 and CO on CsA-induced glomerulus endothelial cell injury and dysfunction as well as the role of ERs proteins.Methods Primary glomerulus endothelial cells were isolated by cell adherent and growth selection assay. 25μmol/L CsA were used to induce cell injury and ERs of the cells. Hemin and CORM-2 were used for representatives for HO-1 inducers and HO-1 catalyzed products, respectively. The expressions of HO-1 and ERs proteins (CHOP and ATF-6) were evaluated by Western bolting. Cell apoptosis, proliferation and migration were measured by annexin-V and PI double staining, MTT degradation and cell monolayer scratch assays, respectively.Results1. Cell identification: The isolated cells were factorⅧ-related antigen positive. The cell monolayer gave classical"cobblestone"appearance and form tube-like structure if not been splitted for over 1 week after confluence.2. CsA injured glomerulus endothelial cells and induced ERs protein expression: CsA promoted apoptosis and inhibited cell proliferation and migration of the cells. At 8h after treatment, HO-1 and CHOP expression in CsA -treated cells increased significantly compared to untreated cells. CsA did not affect the amounts and activation of CHOP upstream ERs protein ATF-6.3. Hemin did not prevent glomerulus endothelial cells from CsA-induced injury: Hemin increased HO-1 protein and mRNA expressions significantly. However, its induction capability is less strong than CsA. Although hemin decreased CsA-induced CHOP expression, it did not prevent CsA-induced apoptosis, proliferation and migration inhibitions.4. CO prevented the cells from CsA-indcued injury: Compared to untreated control, CO-releasing molecule CORM-2 did not affect biological properties and protein expressions of the cells other than decreasing cell migration. However, it blocked CsA-induced cell proliferation inhibition, apoptosis and CHOP expression although it did not change cell migration, ATF-6 expression and activation of CsA-treated cells.5. CHOP mediated CsA-induced apoptosis: Glomerulus endothelial cells did not express CHOP in normal condition. CHOP-siRNA inhibited CsA-induced CHOP expression and decreased apoptosis rate of the cells to normal level while other biological properties and protein expressions remained unchanged.Conclusion We found pharmacological dose of CsA inhibited cell functions and induced cell apoptosis of glomerulus endothelial cells. The CsA-increased expression of CHOP was the main mechanism of CsA-induced cell apoptosis. HO-1 inducer hemin was not protective for CsA-induced injury in glomerulus endothelial cells. However, HO-1 catalyzed product CO prevented the cells from CsA-induced injury. The different may be due to well-known oxidative property of hemin. Our results not only revealed a new mechanism of CsA-induced renal toxicity but also showed HO-1 catalyzed product CO may be a better candidate for preventing CsA side effect than HO-1 inducer.