The Mechanisms Of Notch Signaling On Regulating Myeloid Cells During Renal Fibrosis

?Background? There are 13–16% of the adult population living with CKD. The common pathogenetic pathway of chronic kidney disease(CKD) is renal fibrosis. The main pathogenetic change of renal fibrosis is excess deposition of extracellular matrices(ECM).Tubular epithelial cells act as early responders to injury and later as victims of fibrosis due to the loss of their regenerative abilities. Loss of interstitial capillary integrity that compromises oxygen delivery and leads to a vicious cascade of hypoxia–oxidant stress that accentuates injury and fibrosis. With the progress of renal fibrosis, people face the possible need of renal replacement therapy with dialysis and/or a kidney transplant at some point in the future. Unfortunately, the majority will never reach that point as CKD confers at least a fivefold increased risk of premature death due to accelerated cardiovascular disease.So it is important to research the mechanism of renal fibrosis. The deposition of ECM is a major feature of renal fibrosis.Myofibroblast is the main source of ECM,and fibroblasts,pericytes,endothelial cells,renal tubular epithelial cells also can turn into myofibroblasts under injury stimulus. The endothelial cells turn intomyofibroblasts in End MT, the renal tubular epithelia cells turn into myofibroblasts in EMT. What's more, the cells from bone marrow can also turn into myofibroblasts under injury stimulus. Beside myofibroblasts,the inflammatory cells also impact the renal fibrosis.Among the inflammatory cells, monocytes, macrophages, dendritic cells, granular leukocytes and lymphocytes have been thought have an important role in renal fibrosis. After kidney injury, the injured parenchyma will secret inflammatory mediators and chemokine to recruit the myeloid cells. There are some renal resident immune cells, which will be activated under injury stimulus. The research showed that both the inflammatory cells and kidney resident activated immune cells can take part in renal fibrosis. Notch signaling participates in cell development and differentiation. Notch signaling not only regulates the development of lymphocytes but also regulates the development ofmyeloid system, especially takes part in the activation of macrophage and DCs. Macrophages and dentritic cells(DCs) both from bone marrow con-progenitor cell under the stimulus of critical growth factor. DCs participate in kinds of kidney disease. Renal DCs belong to innate immune cells, they locate in renal interstitium, can response to endogenous and exogenous deleterious signaling to inspire immune reaction to antigen. which can inspire and regulate inflammation effect and protect kidney from infection. Notch signaling can regulate the activation of DCs. There is no report about Notch signaling regulate DCs in renal fibrosis. Macrophages have an important role in renal fibrosis, Lots of study demonstrated that macrophages not only can promote renal fibrosis but also can alleviate fibrosis, this is because of the heterogeneity of macrophages. Our team have confirmed that there are less macrophages accumulation in macrophage specific RBP-J knockout mice than control mice in UUO, and macrophage specific RBP-J deficiency attenuated kidney fibrosis, suggesting Notch signaling regulates macrophages participate in renal fibrosis.However, the mechanism is still unclear. Above all,we used CD11 C positive-specific knockout RBP-J mice and macrophage specific RBP-J knockout mice accompanied with UUO model to study their role andmechanism in renal fibrosis.?Aims? To invest the mechanismthat Notch signaling regulat myeloid cells in renal fibrosis, we used CD11 C positive-specific knockout RBP-J mice and macrophage specific RBP-J knockout mice accompanied with UUO model and in different level valued the degree of fibrosis.?Methods? 1. Bred three kinds thansgene mice:CD11C positive-specific knockout RBP-J mice( CD11c-cre /RBP-Jflox/flox) and macrophage specific RBP-J knockout mice(lyz2-cre/RBP-Jflox/flox). 2. Etablished UUO model and valued the degree of fibrosis(Masson, Picro-Sirius red,immunofluorescence and RT-PCR. 3. Using Immunofluorescence and FACS assay to detect inflammation in UUO model. 4. Obtainedprimary renal tubular epithelial cells.Used co-culture experiment to detect the influence of macrophages from different miceon EMT. 5. Used ACS assay to obtain macrophages from fibrosis kidney, extracted RNA from these macrophages and examined the expression of CCR2. 6. Constructing the monocyte chemokine vector. 7. Used transwell to test the influence of Notch on the ability of migration of macrophage. 8. Used CHIP to test the effect of Notch signaling on the expression of CCR2.?Results? 1. We successfully induced renal fibrosis mice model by UUO. 2. Renal fibrosis induced by UUO 2W was not affected by CD11 C positive-specific knockout Notch signaling mice. 3. We successfully obtained primary renal tubular epithelial cell.Co-culture experiments found that Macrophage-specific RBP-J deficiency can alleviate EMT and alleviate renal fibrosis.4. The expression of CCR2 was reduced in macrophage-specific RBP-J deficiency. 5. The migration of macrophage was reduced in macrophage-specific RBP-J deficiency, but the migration of macrophage was enhanced in macrophage over expressed NICD. 6. Notch signaling upregulate the expression of CCR2 indirectly.?Conclusions? 1. Renal fibrosis induced by UUO 2W was not affected by CD11 C positive-specific knockout Notch signaling mice. 2. Notch signaling hasapositive correlationwith the expression of CCR2, regulates the recruitment of macrophage;...