| Part 1 Serum biomarker for incipient diabetic nephropathy identified by proteomicsOBJECTIVE To screen serum biomarker for incipient diabetic nephropathy (DN) by proteomics and investigate the association of the biomarker with the pathogenesis of DN.RESEARCH DESIGN AND METHODS Mixed serum from five male healthy control subjects and mixed serum from five male type 2 diabetic patients were used respectively assay and identify by a combination of one dimensional gel electrophoresis (1DE) and online electrospray tandem mass spectrometry (GeLC-MS/MS). Based on the results from the comparative proteomic analysis in diabetic patient and healthy control serum, serum from 72 patients with varied stages of DN(24 patients with normoalbuminuria, NA; 24 patients with microalbuminuria, MA; and 24 patients with clinical proteinuria, CP) and 24 healthy control subjects were collected for semi-quantitative validation of differential expression protein by Western blot. In addition, HbA1c, lipids, high sensitive C reactive protein, creatinine, and urinary albumin excretion rate (AER) were measured. Estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease Study equation.RESULTS1 Results from the proteomic analysis(1) Totally 3,010 proteins and 3,224 proteins were identified respectively from the non-diabetic subjects and the type 2 diabetic patients.(2) The differential protein patterns between healthy control and diabetic serum were particularly based on low-abundant proteins. In healthy control subjects, 13 proteins expression were upregulated. In diabetic patients, 29 proteins expression were upregulated, of which ficolin3 was significantly increased.(3) Compared with healthy control subjects, complement and coagulation system were significantly upregulated in the serum from diabetic patients by pathway-associated differential profiling analysis.2 Results from the validation analysis for ficolin3(1) Ficolin3 was found to be up-regulated in the serum of MA compared with that in the healthy controls and NA (2.13±1.34 vs. 0.89±0.43 or 2.13±1.34 vs. 1.42±0.88, P<0.01). Similarly, Ficolin3 in NA was more than that in the healthy controls (1.42±0.88 vs. 0.89±0.43, P<0.05).(2) Ficolin3 showed independent correlations with HbA1c, LDL-C, and urinary albumin excretion rate (R=0.263, 0.321, and 0.245, P<0.01, respectively) in MA. Moreover, logistic regression analyses revealed that a standardized (1-SD) increase in ficolin3 was associated with incipient DN (odds ratio= 2.37, P<0.05) in the diabetic patients, as was comparable with the odds ratios of increased HA1c for DN (odds ratio=2.01, P <0.05).(3) In clinical proteinuria patients, the serum ficolin3 levels decreased significantly when compared with those with MA, further, there were no significant differences between CP and the healthy controls or NA. Moreover, the serum ficolin3 levels only had significant positive correlation with eGFR, except for HbA1c, lipids, high sensitive C reactive protein and AER.(4) In NA or MA, there was no significance in the serum ficolin3 level between the patients with diabetic retinopathy and those without diabetic retinopathy.CONCLUSIONS(1) Proteomic analysis is a valuable tool for screening the serum biomarker for DN.(2) 13 proteins and 29 proteins were upregulated respectively in non-diabetic subjects and type 2 diabetic patients. Furthermore, complement and coagulation system were activated in the diabetes.(3) Ficolin3 may not only have great potential as a predictive biomarker of incipient DN, but also correlate with the advanced stage of DN. Part 2 Serum Pigment Epithelial–derived Factor is Associated with Nephropathy in Diabetic PatientsOBJECTIVE: Pigment epithelial-derived factor (PEDF) plays a key role in the progression of nephropathy in diabetic animals. The present study is aimed to illuminate the change of serum PEDF (sPEDF) in diabetic patients with nephropathy and the relationship between sPEDF and parameters associated with diabetic nephropathy (DN).RESEARCH DESIGN AND METHODS: Serum from 46 healthy control subjects, 166 type 2 diabetic patients at varied stages of DN (59 patients with normoalbuminuria, NA; 68 patients with microalbuminuria, MA; and 39 patients with clinical proteinuria, CP) were collected to measure sPEDF by ELISA. HbA1c, lipids, high sensitive C reactive protein (hs-CRP), creatinine, and urinary albumin excretion rate (AER) were measured. Estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease Study equation.RESULTS(1) The sPEDF level in DN was upregulated significantly compared with the healthy control subjects(P<0.01)and NA(P<0.05), and it was increased as DN advanced. (2) Pearson correlation tests demonstrated significant correlations between the sPEDF level and age (R=0.242, P<0.01), systolic blood pressure (R = 0.147, P < 0.05), diastolic blood pressure (R=0.224, P< 0.01), body mass index (R = 0.162, P < 0.01), triglyceride (TG) (R=0.189, P< 0.01), high density lipoprotein-cholesterol (HDL-c) (R=-0.154, P<0.05), hs-CRP (R=0.268, P<0.01), AER (R=0.359, P<0.01), and estimated glomerular filtration rate (eGFR)(R=-0.417, P<0.01). Furthermore, multiple stepwise regression analysis revealed that the sPEDF level had independent correlations with eGFR (R=-0.368, P < 0.01), TG (R=0.279, P<0.01), and AER (R=0.180, P<0.05).(3)The level of sPEDF in GFR less than 90ml?min-1?1.73m2 subgroup was significant higher than that in GFR more than 90ml?min-1?1.73m2 subgroup in each group.CONCLUSIONS: sPEDF level is evaluated in DN and sPEDF level associated with parameters of DN. Part 3 Effect of Angiotensin II Type 1 Receptor Blocker on Serum and Urine VEGF Level in Nephropathy in Type 2 PatientsObjective: Angiotensin II type 1 receptor blocker (ARB) can ameliorate the renal histopathologicol lesions through downrgulating their vascular endothelial growth factor (VEGF) expression in diabetic animal. The present study is performed to explore the implication of serum VEGF (sVEGF) and urine VEGF(uVEGF) level in the pathogenesis of diabetic nephropathy (DN) and elucidate the effect of ARB irbesartan on the VEGF expression in patients with DN.RESEARCH DESIGN AND METHODS: Samples from 46 healthy control subjects, 166 type 2 diabetic patients at varied stages of DN (59 patients with normoalbuminuria, NA; 68 patients with microalbuminuria, MA; and 39 patients with clinical proteinuria, CP) and 18 hypertensive patients with microalbuminuria who were treated with irbesartan for 6 months, were collected to measure sVEGF and uVEGF by ELISA. In addition, HbA1c, lipids, high sensitive C reactive protein (hs-CRP), creatinine, and urinary albumin excretion rate (AER) were measured. Estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease Study equation.RESULTS:(1) sVEGF level in the diabetic patients without nephropathy was found to be significantly increased compared with the healthy control subjects(P <0.05), however, there was no significant difference among MA, CP and the healthy control subjects(P >0.05. Pearson correlation tests indicated that sVEGF correlated with body mass index (R=0.194, P <0.01) and HbA1c (R=0.124, P<0.05), but did not with AER and eGFR (P>0.05).(2) The uVEGF level in the diabetic patients was upregulated significantly compared with the healthy control subjects(P <0.01), and it was increased as DN advanced. Pearson correlation tests demonstrated significant correlation between the uVEGF level and age (R = 0.344, P < 0.01), duration (R=0.249, P <0.01), systolic blood pressure (R = 0.354, P < 0.01), diastolic blood pressure (R=0.224, P < 0.01), HbA1c (R=0.215, P <0.01), high density lipoprotein-cholesterol (HDL-c) (R=-0.167, P < 0.05), hs-CRP (R = 0.359, P < 0.01), AER (R = 0.452, P < 0.01), and eGFR (R=-0.316, P<0.01). Furthermore, multiple stepwise regression analysis revealed that the uVEGF level had independent correlations with AER (R = 0.268, P<0.01), hs-CRP (R=0.2, P < 0.05), duration(R=0.167, P <0.05)and age (R=0.163, P <0.05).(3) The uVEGF level was significantly decreased after the irbesartan treatment (P<0.05), and the AER had concomitantly reduced in the remission group (n=12). In the non-remission group(n=6), the uVEGF level and AER remained unchanged (P>0.05). However, the sVEGF level had no significant difference in the two groups. Furthermore, the results demonstrated that the change in the uVEGF level correlated significantly with the change in AER before and after 6 months of irbesartan treatment after adjustment for the changes in the average blood pressure(R=0.57, P <0.01). CONCLUSIONS: uVEGF level but sVEGF is associated with the pathogenesis of DN. ARB reduces AER and concomitantly decreases uVEGF level. VEGF might represent an important effective target of therapeutic intervention for DN. Summary1 Proteomic analysis is a valuable tool for screening the serum biomarker for DN.2 13 proteins and 29 proteins were upregulated respectively in non-diabetic subjects and type 2 diabetic patients. Furthermore, complement and coagulation sysytem were activated in the diabetes.3 Ficolin3 may not only have great potential as a predictive biomarker of incipient DN, but also correlate with the development of DN.4 sPEDF level is evaluated in DN and sPEDF level associated with parameters of DN.5 uVEGF level but not sVEGF is associated with the pathogenesis of DN. ARB decreases uVEGF level and concomitantly reduces in AER. VEGF might represent an important effective target of therapeutic intervention for DN. |
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