Immunomodulatory Role Of Interleukin 33 In The Chronic Course Of Diabetic Nephropathy

Background:Diabetic Nephropathy(DN)has become the major cause of chronic kidney disease and also end-stage renal disease.Proteinuria is the main feature,accompanied by a progressive decline in glomerular filtration rate.However,there is still a lack of thorough understanding of the mechanism and effective therapy of DN.Interlukin 33(IL-33)is a member of the interleukin 1 family.It is released by endothelial cells after tissue injury to further activate the innate immune system.IL-33 is an early alarmin for inflammatory damage and could regulate many immunne components including Innate Lymphoid Cells(ILC2s).And it has also shown a relationship with fibrosis in multiple organs such as lung and liver.Inflammation plays a crucial role throughout the whole course of DN,and glomerulosclerosis and interstitial fibrosis are risk factors independently associated to the renal outcome of DN.There is little research on the role of IL-33 in chronic kidney diseases such as DN.regarding its relationship with inflammation and fibrosis,we hypothesize that IL-33 could exaggerate the progression of DN by mediating inflammation and fibrosis.Methods:1)Retrospectively included 21 patients with diabetic nephropathy diagnosed by renal biopsy in Peking Union Medical College Hospital.lncluding 5 cases of IgAN,and 5 cases of membranous nephropathy as the disease control group.Urine and kidney specimens were collected before biopsy.6 patients undergoing radical resection of solitary renal cancer were included,and the adjacent kidney tissues were collected as normal kidney tissue;5 cases of gender and age-matched healthy controls were included,and urine was collected.In order to compare the IL-33 expression among DN patients,other chronic kidney disease patients,and normal controls,enzyme-linked immunosorbent assay was used to measure IL-33 levels in serum and urine,semi-quantitative assessment of renal IL-33 were evaluated by immunohistochemistry.2)db/db mice and db/m mice(divided into db/db disease group and db/m control group)of different weeks of age were used as animal model to evaluate and confirm the IL-33 expression in DN condition.Enzyme-linked immunosorbent assay,immunohistochemistry of IL-33 and western blotting were performed to evaluate the kidney and urine IL-33 expression at different stages of DN;3)IL-33KO mouse STZ combined with unilateral nephrectomy(divided into IL-33 knockout C57BI/6J mice+STZ+unilateral nephrectomy group(IL-33KO+STZ+Unx group),wild-type C57BI/6J mice+STZ+unilateral nephrectomy group(WT+STZ+Unx group)and IL-33 knockout C57BI/6J mice+Sodium citrate+sham operation group(control group))were used as animal model to explore the regulatory effect of endogenous IL-33.MASSON staining,PAS staining,H&E staining,and biochemical indicators such as creatinine,albumin/creatinine ratio,and blood glucose to evaluate the effect of IL-33 knockout on the phenotype of DN.4)In order to further explore the mechanism of IL-33 in DN,the regulation of the downstream ILC2 by IL-33 and its relationship with fibrosis were discussed.the level of ILC2 in the kidneys of db/db mice and patients with diabetic nephropathy and also the expression pattern of endogenous IL33 in disease mode will be assessed based on immunofluorescence and flow cytometry.Results:1)Based on the immunohistochemistry,the DN group showed a significantly higher level of IL-33 in kidney than that in the normal kidney tissue(P<0.0001).However,the increase of IL-33 is not disease-specific in DN,and the level was positively related to the degree of kidney fibrosis(Spearman's ?=0.072,P=0.007).IgAN has a similar increase in renal tissue,which suggested that IL-33 may be more related to common injury induced during kidney disease.There was no difference in serum IL-33 between the disease group and the healthy control group(P=0.407),but the IL-33/Cr level of patients in the DN group was increased in urine(P=0.017).2)In the 16 weeks old db/db mice with early stage DN,IL-33 was increased in the kidney compared with the control db/m mice(P=0.011),which was earlier than the decrease of renal function and kidney pathological changes.The level of IL-33 in the kidney of 39 weeks old db/db mice was higher than that of 24 weeks old db/db mice(P=0.04).At the same time,the level of IL-33 in the urine of the db/db group was higher than that of the control group(P=0.030).And the level of IL-33 in the urine of the 39 weeks old db/db mice also presented with a higher but not significant tendency than that of the 24 weeks old db/db mice(P=0.335).3)In the IL-33 knockout mouse model of STZ-induced diabetes combined with unilateral nephrectomy,the 32 weeks old IL-33KO+STZ+Unx group had lower blood glucose level(P<0.001),reduced urinary albumin/creatinine level than(P=0.021)WT+STZ+Unx group.And overall survival was also prolonged with a not significant difference(P=0.060).Staining of renal tissue showed sever tubulointerstitial fibrosis,inflammatory cell infiltration and glomerular mesangial expansion WT+STZ+Unx group,all of which were significantly attenuated in IL-33KO+STZ+Unx group.4)IL-33 staining in human kidney indicates that the expression pattern of IL-33 positive cells in DN and IgAN patients is different from that in normal kidney tissue.The former is mainly expressed in renal interstitium with inflammation and fibrosis while the latter is mainly expressed in endothelial cells in large vascular or glomeruli.And further immunofluorescence staining suggested that IL-33 is expressed mainly by fibroblasts in disease condition in kidney.5)Quantification by flow cytometry shows that the renal ILC2 of db/db mice is extremely low with a level of 0.26-0.39%(percentage of lymphocytes),and the proportion of ILC2 in db/db group was lower than that in db/m group during different weeks of age(P=0.015 in 39 weeks).Immunofluorescence staining failed to locate ILC2 in patients with DN and normal kidney tissues due to the low proportion.Conclusion:IL-33 is involved throughout the course of DN.The increase of IL-33 can be seen in kidney and urine at the early stage of DN,which was earlier than the decrease of renal function and kidney pathological changes.suggesting that it may play as an early warning factor in the disease.And the expression of IL-33 increases with the progression of DN.Knockout of IL-33 can improve the renal damage in DN.In the disease condition,the endogenous IL-33 in the kidney is mainly secreted by fibroblasts rather than endothelial cells,supporting the argument that IL-33 may participate in aggravating diabetic nephropathy by mediating process of fibrosis.Based on our findings,IL-33 may have the potential to be a target for further mechanism research and treatment of DN.Background:The kidney is the most commonly involved organ in monoclonal immunoglobulin deposition disease(MIDD),and can progress to end-stage renal disease(ESRD).Few studies have evaluated associations between irreversible chronic histopathologic lesions and clinical features,and their prognostic value on renal outcomes in MIDD.Methods:A series of patients with MIDD and 20 with renal light chain amyloidosis(AL)proven by renal biopsy between January 2001 and December 2018 were retrospectively reviewed.The degree of chronic changes,including glomerulosclerosis,interstitial fibrosis,and arteriosclerosis were semiquantitatively scored,and overall chronic lesions were graded based on a grading system proposed in 2017.The associations between histopathologic lesions and clinical manifestations,and their correlation with risk of progression to ESRD were investigated.Results:MIDD patients showed more severe renal insufficiency(serum creatinine:193.0(117.3-356.25)vs.81.5(68.5-140.29),P<0.001),but milder hypoalbuminemia than AL patients(MIDD vs.AL,33.0±6.4 vs.24.9±7.6,P=0.001)at baseline.MIDD patients presented more significantly overt ischemia-related injury,showing global glomerulosclerosis and more severe tendency toward interstitial fibrosis.A significantly higher degree of overall chronic changes was also observed in MIDD compared with AL,and was independently associated with baseline estimated glomerular filtration rate(? coefficient(95%confidence interval):-4.618(-8.238-0.999),P=0.017).Additionally,overt interstitial fibrosis predicted the increased risk of progressing to ESRD in MIDD.Conclusion:The degree of overall chronic changes and overt interstitial fibrosis provides information on both baseline manifestations and renal survival.Careful grading and evaluation of chronic changes in MIDD may contribute to guiding treatment and assessing renal outcomes.