The Role Of Type 2 Innate Lymphoid Cells On Pathogenesis Of Diabetic Kidney Disease

Objective: Diabetic kidney disease(DKD)is an serious chronic microvascular complications of diabetes mellitus,with glomerular hypertrophy,increased extracellular matrix,as well as small vascular hyalinization as the main pathological features.And the primary clinical features of diabetic nephropathy are microalbuminuria and massive albuminuria with renal impairment.DKD is the second major cause of end-stage renal disease in China,and it is also the primary cause of end-stage renal disease in western countries.However,the pathogenesis of DKD is not clear yet.The current research shows that DKD is a chronic low grade inflammatory disease.Immune factors,such as Th1/Th2 imbalance,play an important role in its development.Type 2 innate lymphoid cell(ILC2)belongs to the family of innate lymphocytes,which can express transcription factor ROR-α and GATA3.In addition,ILC2 can produce type 2 cytokines(IL-4,IL-5,IL-9,IL-13)mediating type 2 immune response.As the bridge of innate and adaptive immunity ILC2 plays an important role in inflammation remodeling repair of tissue.However,there is still little research on the role of ILC2 in DKD.Therefor,in order to study the effect of ILC2 on pathogenesis of DKD,we detected ILC2 and its secreted type 2 cytokines(IL-4,IL-5,IL-13)in the peripheral blood and stimulated human proximal renal tubular epithelial cells(HK-2)in vitro by these cytokines.Methods : 1.The proportion of ILC2 in peripheral blood mononuclear cells(PBMCs)of healthy controls(group NC),type 2 diabetic patients(group DM),early diabetic nephropathy(group DKD1)and late diabetic nephropathy(group DKD2)were detected by flow cytometry.2.The levels of plasma IL-4,IL-5 and IL-13 in group NC,group DM,group DKD1 and group DKD2 were detected by Enzyme-linked immunosorbent assay(ELISA).3.Cell experiment: HK-2 were cultured in vitro,IL-4,IL-13 and high glucose as the intervention factors simulating the role of ILC2 on diabetic kidney.We categorized as follows:(1)normal control group(group NC): DMEM/F12 culture medium;(2)different concentrations of IL-4 group: 1ng/ml IL-4 group,10ng/ml IL-4 group,100ng/ml IL-4 group;(3)different concentrations of IL-13: 1ng/ml IL-13 group,10ng/ml IL-13 group,100ng/ml IL-13 group;(4)high glucose group(group HG): DMEM/F12 culture medium containing 30mmol/L glucose;(5)IL-4+high glucose group(group HG+IL-4): 100ng/ml IL-4+30mmol/L glucose;(6)IL-13+high glucose group(group HG+IL-13): 1ng/ml IL-13+30mmol/L glucose.Methods to detect cell experiments:(1)Observe the effect of IL-4 on the fibrosis of HK-2 in intro:(1)ELISA detected the expression of transforming growth factor-β1(TGF-β1)and fibronectin(FN)in cell supernatant induced by different concentrations of IL-4(1ng/ml、10ng/ml、100ng/ml)for 24 h and 48 h.(2)And q-PCR detected the expression of TGF-β1 mRNA and FN mRNA.(2)Observe the effect of IL-13 on the fibrosis of HK-2 in intro:(1)ELISA detected the expression of TGF-β1 and FN in cell supernatant induced by different concentrations of IL-13(1ng/ml、10ng/ml、100ng/ml)for 24 h and 48 h.(2)And q-PCR detected the expression of TGF-β1 mRNA and FN mRNA.(3)Observe the effects of IL-4 and IL-13 on the fibrosis of HK-2 in high glucose: IL-4(100ng/ml)and IL-13(1ng/ml)were used to stimulate the HK-2 cultured in high glucose 48 h,respectively,and the level of TGF-β1 and FN in cell supernatant was detected by ELISA,and the expression of TGF-β1 mRNA and FN mRNA were detected by q-PCR.3.Statistical Analysis:All datas are indicated as mean±standard deviation(SD)and analyzed by one-way analysis of variance(ANOVA),followed by the LSD post hoc test for multiple comparisons,and variance homogeneity test used in group comparison(SPSS17.0 statistical software).P<0.05 was definited significant.Results:1.Compared to normal control group,the proportion of ILC2 in PBMCs of group DM and DKD were significantly increased,and the group DKD was higher than that of the group DM,and the proportion of ILC2 in group DKD2 was higher than that of the group DKD1(P﹤0.05).2.Compared with the normal control group,the levels of IL-4 and IL-13 in plasma of group DM and DKD were significantly augmented,and the group DKD was more obvious than that of the group DM,and it was highest in group DKD2(P﹤0.05).3.The expression of TGF-β1 mRNA and FN mRNA were significantly enhanced by IL-4 in a dose and time dependent manner compared with the normal control group(P﹤0.05).4.Compared with the normal control group,the expression of FN and TGF-β1 were up-regulated by IL-13(P﹤0.05).5.Compared with the high glucose group,the expressions of FN and TGF-β1 in IL-4+high glucose group and IL-13+high glucose group were significantly increased(P﹤0.05).Conclusion:1.The proportion of ILC2 and the levels of cytokines IL-4,IL-5,and IL13 in peripheral blood were increased in patients with diabetic nephropathy,and advanced diabetic nephropathy was higher than that of early diabetic nephropathy,suggesting that ILC2 is involved in the development of diabetic nephropathy.2.The type 2 cytokines,secreted by ILC2,IL-4 and IL-13,can enhance the expression of FN and TGF-β1 in HK-2 in vitro by cooperating with high glucose,suggesting that ILC2 may promote renal fibrosis in diabetic nephropathy by secreting lymphatic factors.