MANF Inhibits LPS-induced Acute Kidney Injury Via Regulating Macrophage Differentiation

Acute Kidney Injury?AKI?is a clinically common complication characterized by tubular necrosis and renal dysfunction,which increases the incidence of chronic kidney disease and has a high mortality rate.Lipopolysaccharide?LPS?-induced spesis is the most common cause of AKI.Macrophages play an important role in the progress of AKI.Several studies have shown that mesencephalica strocyte-derived neurotrophic factor?MANF?has immunoregulatory effects in retina.However,how MANF affects renal macrophages in AKI is still unclear.In this study,we constructed LPS-induced AKI model in wild type?WT?and mono-macrophage-specific MANF knockout(M?MANF^-/-)mice.We found that M1 macrophages in M?MANF^-/-mice were increased significantly,whereas there was no significant change in M2 macrophages.Toll-like Receptors 4?TLR4?,p-p65,and tumor necrosis factor-??TNF-??were also increased in M?MANF^-/-mice of AKI.Our results indicate that MANF is involved in renal macrophages polarization in AKI by affecting TLR4 receptor and its downstream NF-?B signaling pathway in LPS-induced AKI.Objective To investigate the immune-regulator function of MANF on renal macrophages in LPS-induced AKI and explore the underlying mechanism.Methods WT and M?MANF^-/-mice were intraperitoneally injected with 10 mg/kg LPS for 3 days to induce AKI.The mice were sacrificed on the third day the kidney tissues and blood were collected.A part of the kidney tissues was dehydrated and embedded to make tissue sections;The rest was frozen in liquid nitrogen for the detection of m RNA and protein.Histological and pathological examination were performed by HE staining,PAS staining,immunohistochemical staining,and double labeled immunofluorescence.The levels of m RNA in tissues were detected by q PCR.The macrophages from peritoneal cavity were isolated and used for detections of signaling molecules or proteins in involved in TLR4/NF-kB pathway.Results1. Identification of MANF knockout and preparation of and AKI model in mice.The peritoneal macrophages were isolated from M?MANF^-/-mice.Flow Cytometry was used to detect MANF expression in the macrophages of MANF^-/-mice.The results showed that MANF-positive macrophages were very less?%?in MANF^-/-mice,compared with WT mice?%?,suggest that MANF was efficiently and specifically knocked out in macrophages.Then,we used LPS to induce AKI model in mice.HE staining and PAS staining was used to detect LPS-induced renal injury.The results showed that AKI model was successfully established.2. MANF knockout increased LPS-induced renal damage.The survival rate was reduced in the M?MANF^-/-mice after treated with LPS.HE staining and PAS staining showed that the kidney structure was more disordered in M?MANF^-/-mice than that in WT mice.Serum creatinine and urea nitrogen levels were increased in M?MANF^-/-mice,compared with WT mice.These results suggest that mono-macrophage-specific MANF knockout aggravated LPS-induced renal injury.3. MANF knockout promotes macrophage infiltration and polarization in LPS-induced AKI.The expressions of CCL2 and CD68 were detected by immunohistochemical assay with the corresponding antibodies.It was found that mono-macrophage-specific MANF knockout up-regulated CCL2 and CD68 expression in LPS-induced AKI.M1 macrophages in renal tissues were double labeled with anti-CD68?red?and anti-CCL2?green?antibodies.The M2 macrophages in renal tissues were double labeled with anti-CD68?red?and anti-CD206?green?antibodies.Mono-macrophage-specific MANF knockout promoted macrophages differentiation to M1 type,but not to M2 type.The m RNA levels of cytokines derived from M1 macrophages and M2 macrophages in kidney and peritoneal macrophages were detected by using q PCR.We found that mono-macrophage-specific MANF knockout up-regulated the cytokines derived from M1 macrophages in kidney and peritoneal macrophages,while the cytokines derived from M2 macrophages was not affected.4. MANF knockout promotes TLR4/NF-?B signaling pathway activation in LPS-induced AKI.Immunohistochemistry and q PCR showed that TLR4 level was increased in M?MANF^-/-mice than that in WT mice.The macrophages from peritoneal cavity were isolated and stimulated with LPS.RNA seq showed that NF-?B was up-regulated in peritoneal macrophages of M?MANF^-/-mice,compared with WT mice.Immunohistochemistry and q PCR showed that the levels of p-p65 and TNF-?were increased in M?MANF^-/-mice than that in WT mice.Conclusion1.Mono-macrophage-specific MANF knockout aggravates LPS-induced AKI and promotes renal macrophages to differentiate to M1 subtype.2.Mono-macrophage-specific MANF knockout activates TLR4/NF-?B signaling pathway in LPS-induced AKI.