The Diagnostic Values Of VEGF-A₁₆₅b In Urinary Extracellular Vesicles For Diabetic Kidney Disease

Objectives:DKD is one of the main causes of CKD and ESRD in China.Diagnosis for early stage of DKD has great significance for DKD prevention.Extracellular vesicles（EVs）are nanoscale lipid bilayer structures secreted by cells,which carry a variety of biomolecules such as proteins,metabolites and nucleic acids.Detection of u EVs is convenient,non-invasive and highly stable.EVs in urine may represent an important source of novel biomarkers for kidney disease.VEGF-A₁₆₅b is one of the important anti-angiogenic isoform of VEGF-A,which is secreted form glomerular podocytes and tubular epithelial cells,and may play an important role in DKD.This study aims to investigate VEGF-A₁₆₅b loading in different sizes u EVs at different stages of DKD and its correlation with clinical characteristics,with aims to explore the value as a diagnostic biomarker for DKD.Methods:（1）A total of 96 subjects who received in-patient treatment and health examination in Zhongda Hospital affiliated to Southeast University were enrolled and divided into four groups:healthy control group and T2DM patients（n=24 for each group）.DKD was divided into DKD A2 and DKD A3 based on UACR according to KDIGO guideline（n=24 for each group）.（2）50m L morning urine was collected from each subject.The L-u EVs and S-u EVs were obtained by differential centrifugation.（3）The particle size distribution of L-u EVs and S-u EVs were observed by NTA,diameter and morphology were observed by TEM,and the markers of EVs were displayed by WB.（4）The level of VEGF-A₁₆₅b in each group of u EVs was analyzed by WB.（5）One-way ANOVA was performed to analyze the u EVs VEGF-A₁₆₅b expression in different stages of DKD.Pearson correlation analysis was applied to evaluate the correlation between u EVs VEGF-A₁₆₅b level and clinical parameters in DKD patients.ROC curve was constructed to analyze the diagnostic efficacy of u EVs VEGF-A₁₆₅b.（6）The renal biopsy tissue sections of DKD patients and STZ-induced DN rats were selected to confirm the expression of VEGF-A₁₆₅b.Results:（1）Analysis of clinical baseline data showed that there were no significant differences in age,sex,BMI and DBP between healthy control group,T2DM group and DKD group.Compared with healthy control,serum albumin and FPG in T2DM group were significantly different（p<0.05）.SBP,plasma albumin,BUN,Scr and FPG increased significantly in DKD group（p<0.05）.Compared with T2DM group,DKD group showed statistical differences in the duration of diabetes,BUN and Scr.（2）L-u EVs and S-u EVs were identified by NTA,TEM and WB,respectively.The peak particle size of L-u EVs was 222nm,and that of S-u EVs was 105nm.TEM images of L-u EVs and S-u EVs showed typical lipid bilayer vesicle structure.S-u EVs expressed exosome markers CD63 and Alix,both of L-u EVs and S-u EVs expressed AQP1 and Nephrin by WB analysis.（3）VEGF-A₁₆₅b increased with the progression of DKD in groups of healthy controls,T2DM and DKD A2,and A3,among which VEGF-A₁₆₅b levels of u EVs in DKD A3 were significantly increased（p<0.01）.（4）VEGF-A₁₆₅b increased with decreasing e GFR,which showed the highest level in DKD patients with e GFR<15ml/min/1.73m²（p<0.005）.（5）u EVs VEGF-A₁₆₅b increased with the increasing duration of diabetes.（6）The loading of VEGF-A₁₆₅b in average individual u EVs in DKD was significantly higher than T2DM.（7）The level of u EVs VEGF-A₁₆₅b was positively correlated with Cys C,BUN and MA,and negatively correlated with e GFR,and the correlation for L-u EVs was slightly better than S-u EVs.There was no correlation between u EVs VEGF-A₁₆₅b and urinary glucose,Hb A1C,CRP and diabetic retinopathy.（8）ROC curve was established to evaluate the ability of VEGF-A₁₆₅b to differentiate healthy control and DKD.It was found that the area under ROC curve（AUC）of S-u EVs and L-u EVs were 0.9453 and 0.9444,respectively.For differentiation of healthy control and DKD A2,the AUC of S-u EVs and L-u EVs were 0.8993 and 0.9115,respectively.For T2DM and DKD,the AUC of S-u EVs and L-u EVs were 0.8168 and 0.8472,respectively.For identifyting early DKD A2 from T2DM,the AUC of S-u EVs and L-u EVs were 0.7205 and 0.7969,respectively.（9）Immunohistochemical staining showed that the expression of VEGF-A₁₆₅b was significantly increased in kidney tissues of DKD patients and DN rats,especially in renal tubules.Conclusions:（1）VEGF-A₁₆₅b was found in both S-u EVs and L-u EVs that increased with the progression of DKD,which was closely related to e GFR,Cys C,BUN,MA and the duration of diabetes.（2）u EVs VEGF-A₁₆₅b showed efficient ability for the diagnosis of DKD and may become a new non-invasive biomarker for DKD.（3）L-u EVs VEGF-A₁₆₅b showed better diagnostic ability and correlation with clinic parameters compared to S-u EVs.The simplicity of the separation method for L-u EVs may make it easier for translation to clinic.（4）The expression of VEGF-A₁₆₅b in renal section of DKD patients and DN rats increased significantly.This suggests that u EVs reflects the expression of molecules changes in kidney,and VEGF-A₁₆₅b may be involved in the occurrence and development of DKD.Therfore,u EVs VEGF-A₁₆₅b may be a novel non-invasive biomarker for the diagnosis of DKD and may be involved in the development of DKD which deserves further studies.