New Markers And Pathogenesis Of Diabetic Nephropathy

Objective:To investigate the general clinical characteristics of Diabetic Kidney Disease(DKD)in inpatients with type 2 diabetes mellitus(T2DM),and to explore the association between urinary Smad3(usmad3)levels andglomerular filtration rate in patients withT2DM.On the basis of this,to further study the pathophysiology of DKD as to explore the response of the kidney and podocyte under endoplasmic reticulum stress and the effect of IRE1αon podocyte injury in diabetic nephropathy.Methods:(1)Clinical research:Determination of clinical and related biochemical indexes include age,blood pressure,body mass index,blood glucose,blood lipid,albumin-to-creatinine ratio glomerular filtration rate,glycosylated hemoglobin and so on.Retinal lesions were assessed by fundus photography.The estimatedglomerular filtration rate(eGFR)was assessed by MDRD formula.Albuminuria is defined by ACR.DKD is defined according to the latest NKF KDOQI classification definition that is macroalbuminuria or microalbuminuria accompanied by diabetic retinopathy(2012).The level of usmad3 was determined by enzyme-linked immunosorbent assay.(2)Basic research:To determine the response changes of IRE1αand other endoplasmic reticulum stress signal in diabetic nephropathy;to obtain glomerular podocyte specific knockout mice(PKO mice)by use of conditional gene knockout mice strategy,the phenotype of mice was observed in STZ-induced diabetes mellitus.Finally,the mechanism of IRE1αin podocyte injury was investigated by using lentiviral vector to knock down IRE1αin podocytes.Results:Clinical research:(1)The prevalence of DKD was 12.03%,the prevalence of CKD was 27.1%,the prevalence of albuminuria was 25.2%,and the prevalence of microalbuminuria and macroalbuminuria was 18.9%and 6.3%,respectively.The prevalence of normal albuminuria with renal insufficiency ratio was 1.9%,albuminuria with eGFR>90 was 13.9%.(2)The usmad3 levels were significantly higher than the control group.(3)Pearson’s correlation analysis suggested that the usmad3 level was significantly correlated with age,systolic blood pressure,fasting plasma glucose,insulin,C-peptide,glycated haemoglobin,andestimated glomerular filtration rate(eGFR).(4)A multiple linear stepwise regression analysis revealed that usmad3levels in patients with T2DM and an eGFR≥90 ml/min/1.73 m~2 were independently and positively correlated with eGFR,whereas in patients with T2DM and eGFR<90ml/min/1.73 m~2,the levels were independently and negatively correlated with eGFR.Basic research:(1)Western blot analysis showed that HG-treated podocytes can significantly upregulate the expression of bip and p-IRE1 and significantly upregulate the expression of cleaved-caspase3 and CHOP compared with the control group.(2)In the diabetic db/db mouse model,western immunoblotting showed that the markers of endoplasmic reticulum stress,Bip,P-IRE1,P-Eif2,cleaved-ATF6,were all activated in the case of diabetic nephropathy,consistent with in vitro levels.(3)Type 1 diabetes mellitus was induced by STZ.After 3 months of feeding,in STZ-induced type 1 diabetes mellitus,PKO mice showed more pronounced albuminuria compared to STZ-treated control mice.(4)Transmission electron microscopy analysis showed that podocyte foot process effacement and GBM thickening increased significantly in PKO mice.(5)Immunostaining forWT1,podocyte-specific marker,showed a significant reduction in the number of podocytes in PKO mice.(6)In vitro,using the PLKO.1 lentiviral vector system,we successfully constructed the stable cells for knockdown of IRE1αin podocyte,TUNEL staining showed the apoptotic levels of IRE1αknockout cells were significantly increased compared with the control cells.Conclusion:The prevalence of DKD in patients with T2DM is high,and urinary albuminuria has some shortcomings in the early diagnosis of diabetic nephropathy.The usmad3 levelwas significantly correlated with the biphasic changes in the GFRin patients with T2DM,and may serve as a new biomarker for early screening DKD,which is important for early diagnosis of DKD.In addition,we find endoplasmic reticulum protein IRE1αplays an important role in the damage of podocytes in the stress state of diabetic nephropathy,and its mechanism may be due to the increase of podocyte apoptosis.