| Objective:Diabetic nephropathy(DN)is the most common cause of the end-stage renal disease(ESRD).Inflammation and complement activation play important roles in the pathogenesis and progression of DN.Recent studies have found that the levels of mannose-binding lectin(MBL)in blood and urine of patients with DN are significantly increased,and are correlated with the level of urinary protein.However,the role and mechanism of MBL in the progression of DN have not been elucidated.The aims of this study are as follows: 1.To explore the relationship between the prognosis of DN patients and MBL levels;2.To determine whether elevated MBL can promote the progress of DN;3.To explore the mechanism of MBL promoting the progression of DN,and to provide the basis for prognosis prediction and clinical intervention.Methods:Patients with type 2 DN diagnosed by renal biopsy in the First Affiliated Hospital of Medical College of Zhejiang University from August 2013 to September 2016 were included.The serum and urine MBL levels and single nucleotide polymorphism(SNP)sites of MBL were detected.According to the follow-up results,patients were divided into ESRD group(progressed into ESRD during follow-up)and non-ESRD group(did not progress into ESRD during follow-up).The relationship between the serum and urine MBL levels,MBL SNPs and DN prognosis were analyzed.Adeno-associated virus(AAV)-MBL1/2-sh RNA was injected into db/db mice via tail vein to knock down MBL1/2 expression.At 24 weeks,blood glucose,triglyceride,serum MBL1/2,and urine protein were measured.Also,the infiltration of macrophages in the kidney was determined by flow cytometry.Western blot(WB)and immunohistochemistry were used to assess inflammation and fibrosis.The same cohort with our previous study was included,the serum levels of C3,C4 and C5b-9 were detected.Besides,the relationship between the C3/C4 deposit in kidney and renal injury was analyzed.In vitro,MBL was administrated to intervene renal tubular epithelial cells(HK2),human mesangial cells(HMC),and macrophages(U937)to explore its damaging effect on cells.Furthermore,the co-culture system was used to determine the effect of macrophages on other cells mentioned before.Human primary macrophages were isolated using magnetic beads and cultured in the presence of M-CSF for 7 days.These cells were stimulated with MBL,and then RNA sequencing was performed to explore the underlying molecular mechanism of MBL promoting the injury of macrophages.Results:1.A total of 77 DN patients were included and followed up until August 31,2018,including 33 cases in ESRD group and 44 cases in non-ESRD group.The serum and urine MBL baseline levels in ESRD group were significantly higher than those who were not.Cox regression analysis indicated that the level of MBL in serum was an independent risk factor for ESRD(HR = 4.644,95% CI: 1.320-16.337,P = 0.017).The rs1800450 GA genotype was an independent protective factor for ESRD(HR =0.485,95% CI: 0.237-0.991,P = 0.047).2.In db/db mice,blood glucose,triglyceride,serum MBL1/2 and urine protein were augmented.The kidney tissue of db/db mice showed several pathological features,including increased glomerular mesangial matrix,significant tubulointerstitial injury,increased number of macrophages infiltration,and increased i NOS expression in macrophages.Immunohistochemistry and WB showed that the expression of FN andα-SMA in the kidney increased.After knocking down MBL levels,the blood glucose,triglyceride,urine protein,injuries in glomerulus and renal tubules,and the degree of renal inflammation and fibrosis were ameliorated in db/db mice.3.There are no significant differences in the C3,C4 or C5b-9 levels between the ESRD group and non-ESRD group.In addition,there are no differences in the renal function or the pathological parameters between the C3/C4 deposits group and non C3/C4 deposits group.In vitro,MBL did not cause inflammation and fibrosis in HK2 and HMC.When human macrophages were stimulated by MBL,NF-κB was activated and the release of inflammatory factors was increased significantly.Inflammatory and fibrotic responses in HK2 and HMC were triggered when co-cultured with macrophages in the presences of MBL.RNA sequencing analysis revealed that toll like receptor(TLR)signaling pathway and NF-κB pathway may play important roles.Further experiments found that MBL can bind to TLR4 of macrophages to promote NF-κB activation,and thus leading to cell injuries.Conclusion:The levels of MBL at baseline in serum and urine of DN patients with ESRD were significantly higher than that of non-ESRD patients.The rs1800450 GA genotype was an independent protective factor for ESRD progression.MBL contributes to the progression of diabetic nephropathy in diabetic nephropathy mice through binding to TLR4 of macrophages and activating the NF-κB pathway. |
