The Mechanism Of Chop Deficiency Preventing UUO-induced Renal Fibrosis Of Mice

Research backgroundRenal fibrosis, featured by glomerulosclerosis and tubulointerstitial fibrosis, is generally thought to be the final manifestation of a wide variety of chronic kidney diseases (CKDs). Given that renal fibrosis is an unavoidable consequence caused by the excessive accumulation of extracellular matrix virtually in the setting of patients with different types of CKDs, it serves as a pathological marker relevant to end-stage renal failure, a condition that requires dialysis or renal transplantation for maintaining the life of patients. There is compelling evidence that mesangial and fibroblast activation, tubular epithelial-tomesenchymal transition (EMT), inflammatory (monocyte, macrophage and T cell) infiltration and apoptosis are common cellular events leading to renal fibrosis. Past extensive studies have consistently demonstrated the essential role of TGF-β and its downstream Smad signaling had in the pathogenesis of renal fibrosis, but the causative factors that trigger TGF-β expression and the molecular mechanisms that initiate the above-described cellular events are yet to be fully addressed.Endoplasmic reticulum (ER) is crucial for protein biosynthesis, folding, trafficking and modification, and therefore, disturbances of ER homeostasis by extracellular stimuli such as oxidative stress would affect protein folding and cause ER stress. Given that ER stress-associated apoptosis modulates organ remodeling after insult, ER stress has been demonstrated with implications in the pathogenesis of cardiac and hepatic fibrosis. In contrast, the impact of ER stress in the pathoetiology of renal fibrosis, however, is not yet to be clearly elucidated. More recently, Chiang et al. provided feasible evidence suggesting the involvement of ER stress in renal apoptosis and fibrosis, and studies in albumin-overloaded renal tubular cells further revealed that suppression of oxidative stress attenuates EMT and reduces ER stress. Based on these observations, we first conducted studies in CKD patients to demonstrate the presence of ER stress during the course of renal fibrosis, unilateral ureteral obstruction (UUO) was then induced in mice deficient in CCAAT/enhancer-binding protein (C/EBP) homologous protein (Chop) to dissect the cellular and molecular events relevant to ER stress in renal fibrosis. It was noted that loss of Chop provided remarkable protection for mice against UUO-induced renal fibrosis. Altered Chop expression rendered tubular cells undergoing apoptosis and secondary necrosis along with Hmgb1 passive release, which then recruited immune cells such as macrophages into the damaged site along with active secretion of copious amount of Hmgb1. Extracellular Hmgb1 thus bound to TLR4, and by which, it activated MyD88-NFκB pathway to enhance IL-1β expression, which in turn promoted TGF-β/Smad2/3 and Pi3k/Akt signaling to exacerbate renal fibrosis.Methods and ResultsIn the early period, we collected renal biopsy from chronic kidney disease patients. Through the pathological staining, we observed that severe tubulointerstitial fibrosis and macrophages infiltration occurring in kidney of end stage renal disease patients. Meanwhile, we detected more expression of Chop mRNA in patients compared to normal persons.To discover the influence and mechanism of Chop in the renal fibrosis further, we established Chop gene knockout mice model. Unilateral ureteral obstruction (UUO) is a traditional model to induce animal renal fibrosis. Thus, we performed UUO surgery in Chop -/- mice and C57BL/6 mice to induce renal fibrosis separately. At the 14th day after UUO, we detected robust fibrosis in the renal suffered from surgery, abundant macrophages infiltration in renal interstitium, obviously renal tubular expansion and protein canal and barely tubular brush border from pathological staining analysis in C57BL/6 mice. The high expression of protein and mRNA from C57BAL mice indicated the pathological progress of renal fibrosis was associated with ER stress. Nevertheless, Chop deficiency reversed the phenotype of renal fibrosis in mice suffered from UUO surgery. Morphology images showed that the extent of ponding in pelvis was smaller in Chop -/- mice compared with C57BL/6 mice, besides the cortex of renal was better in knockout mice. Consist with the morphological phenotype, Chop deficiency prevented the development of renal fibrosis extremely, reduced the inflammation cells infiltration in renal interstitium, improved the degree of renal tubular expansion and preserved partial integrity of brush border. F4/80 is membrane marker of macrophage from mouse, therefore we analyzed the number of infiltrated macrophages in renal interstitium using F4/80 IHC staining. The visible number of macrophages was decreased in Chop -/- mice dramatically. CHOP is a apoptosis-related factor located at downstream of ER stress regulating signaling pathway, but Chop deficiency prohibited the expression of upstream signal pathway factors of ER stress due to positive feedback.The mainly function of CHOP induced by ER stress is modulating cell programmed death (apoptosis). Altered apoptosis is generally associated with secondary necrosis, from which HMGB1 passively released. Due to TUNEL is a kind of technic with high specificity, sensitive sensibility, directly visible and easily application for apoptotic detection, we employed TUNEL to test the number of apoptotic cells in kidney. The result of analyzed data implicated less apoptotic cells in Chop -/- mice. Subsequently, we used of western blotting and RT-PCR to test apoptosis-related protein, according to the TUNEL result, apoptosis-related protein was reduced after Chop deficiency. LDH, which indicates the source of necrosis, is passively released from necrotic cells. We detected the content of LDH in serum from UUO suffered mice. The result of LDH testing showed less necrosis in Chop-/-mice, with which we speculated that Chop deficiency prevented secondary necrosis. Extracellular Hmgbl can be passively released or positively secreted. Regardless of vivo experiment or vitro experiment, we detected decreased Hmgbl expression after Chop knockout. We also observed lots of extracellular Hmgbl in end stage renal disease patients. TLR2, TLR4 and RAGE are crucial membrane receptors of Hmgbl. We tested the three receptors with western blotting, and found that TLR2, TLR4 and RAGE were increased in kidney suffered UUO surgery, but only high expression of TLR4 was inhibited in Chop-/-mice. TLR4-regulated downstream factor MyD88 and NFκB were also repressed. HMGB1 that secreted to extracellular stimulates macrophage to secrete IL1β which promotes renal fibrosis through Pi3K/Aktsignal and TGFβ/Smad2/3 signal. Our results demonstrated that the signaling pathway associated protein was decreased.ConclusionIn this paper, we adopted ER stress as a point of contact, Chop as focus to research the mechanism of renal fibrosis. In kidney from end stage renal disease patient, we detected high expression of ER stress-related protein. To clarify the pathogenesis of renal fibrosis, we established mouse UUO model. In the investigate, we demonstrated that Chop represses Hmgbl/TLR4 signaling following UUO induction, leading to repressed NFκB transcriptional activity along with suppressed IL-1β production, which then reduces TGF-β1 production and Pi3K/Akt activity to attenuate the development of renal fibrosis.