Proteome Analysis of Serum from Type 2 Diabetics with Nephropathy Hyun-Jung Kim, Eun-Hee Cho, Ji-Hye Yoo, Pan-Kyeom Kim, Jun-Seop Shin, Mi-Ryung Kim, and Chan-Wha Kim* School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea Received September 18, 2006
Diabetic nephropathy (DN) is a renal disease which develops as a consequence of diabetes mellitus. Microalbuminuria is the earliest clinical sign of DN. There are no specific diagnostic biomarkers for type 2 diabetics with nephropathy other than microalbuminuria and macroalbuminuria. However, microalbuminuria does not constitute a sole independent indicator for type 2 diabetics with nephropathy, and thus, another screening method, such as a biomarker assay, is required in order to diagnose it more correctly. Therefore, we have utilized two-dimensional electrophoresis (2-DE) to identify human serum protein markers for the more specific and accurate prediction of progressive nephropathy in type 2 diabetes patients, via comparisons of the serum proteome in three experimental groups: type 2 diabetes patients without microalbuminuria (DM, n ) 30), with microalbuminuria (MA, n ) 29), and with chronic renal failure (CRF, n ) 31). As a result, proteins which were differentially expressed with statistical significance (p < 0.05) in MA and CRF groups as compared to those in DM group were selected and identified by ESI-Q-TOF MS/MS. Among these identified proteins, two proteins which might be useful as diagnostic biomarkers of type 2 diabetics with nephropathy were verified by Western blotting: extracellular glutathione peroxidase (eGPx) and apolipoprotein (ApoE) were found to exhibit a progressive reduction in MA and CRF groups. Notably, eGPx was further verified by ELISA using DM (n ) 100) and MA (n ) 96) patient samples. Collectively, our results show that the two proteins identified in this study may constitute potential biomarkers for the diagnosis of type 2 diabetics with nephropathy. Keywords: Serum • Diabetic Nephropathy • 2-DE • eGPx • ApoE • ESI-Q-TOF MS/MS
Introduction Diabetic nephropathy (DN) is a renal disease which arises as the consequence of diabetes. It is the most frequent cause of end-stage renal disease (ESRD) worldwide, and currently accounts for approximately 40% of patients that require renal replacement therapy.1 DN is also one of the most significant long-term complications in terms of morbidity and mortality for individual diabetes patients. As compared to patients suffering from type 2 diabetes, a higher proportion of those with type 1 diabetes progress to nephropathy, primarily due to the fact that, in type 2 diabetes, death as the result of cardiovascular causes is more common than death from renal failure.2 In addition, the prevalence of type 2 diabetes has been predicted to double within the next 15 years,3 and the incidence of ESRD is also expected to continue rising, concomitantly with the progressively declining rate of mortality due to cardiovascular causes. The earliest putative diagnostic sign for the presence of diabetic renal damage is a stable increase in the albumin excretion rate, that is, microalbuminuria.4,5 Contrasted with patients suffering from type 1 diabetes, the presence of * Address for correspondence: Prof. Chan-Wha Kim, School of Life Sciences and Biotechnology, Korea University, 1, 5-ka, Anam-dong, Sungbukku, Seoul 136-701, Korea. Tel: +82-2-3290-3439. Fax: +82-2-3290-3957. E-mail:
[email protected]. 10.1021/pr060489g CCC: $37.00
2007 American Chemical Society
microalbuminuria in type 2 diabetics is seldom reversible,6 and progresses to macroalbuminuria (overt proteinuria) in 20-40% of patients.7,8 In 10-50% of patients evidencing proteinuria, chronic renal failure (CRF) develops, and ultimately necessitates dialysis or transplantation.9-11 Forty to 50% of type 2 diabetes patients with microalbuminuria eventually die as the result of cardiovascular disease;12,13 this rate of death is 3 times higher than the rate of death from cardiac causes in diabetes patients with no evidence of renal disease.14 Early identification of patients who are prone to the development of this devastating complication is therefore important, as it enables prompt medical intervention for the prevention of further disease progression,6,9 thus, saving enormous costs in the renal replacement therapy of patients who progress to chronic renal failure. However, there are several limitations of the use of microalbuminuria as a renal function index. Microalbuminuria in diabetic patients not only has been identified as a predictor of DN progression, but has also been recognized as a compelling independent risk factor for cardiovascular disease,15-17 along with other risk factors found in insulin resistance syndrome. It is, therefore, clearly desirable to identify additional protein markers that might augment the prediction of DN in early and progressive stages. Journal of Proteome Research 2007, 6, 735-743
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Published on Web 01/06/2007
research articles Recently, proteomics techniques have been applied to the study of DN, and proteomic investigations into diabetic renal disease have uncovered new mechanisms of DN pathology, in addition to potential urinary markers of DN.18 Therefore, we have utilized this technique to identify human serum protein markers for the more specific and accurate prediction of progressive nephropathy in patients with diabetes. Also, differentially expressed proteins were identified by ESI-MS/MS analysis, and the proteins which might be utilized as diagnostic biomarkers for type 2 DN were confirmed via Western blotting and ELISA.
Experimental Procedures Materials and Apparatus. For two-dimensional electrophoresis (2-DE) analyses, the IPGphor IEF system, and Ettan DALT II SDS system with 24 cm Immobiline DryStrips (pH 4-7) were obtained from GE Healthcare (Uppsala, Sweden). Urea, immobilized pH gradient (IPG) buffer (pH 4-7), and dithiothreitol (DTT) were acquired from GE Healthcare (Uppsala, Sweden). DryStrip cover fluid, CHAPS, Tris, glycine, acrylamide, piperazine diacrylamide (PDA), SDS, and ammonium persulfate were purchased from Bio-Rad (Hercules, CA). Iodoacetamide (IAA), TEMED, glycerol, bromophenol blue (BPB), silver nitrate, thiourea, acetone, and ammonium bicarbonate were all provided by the Sigma Chemical Co. (St. Louis, MO). The protease inhibitor cocktail (PIC) was acquired from Roche (Indianapolis, IN). All chemicals used in the 2-DE and ESI-Q-TOF MS/MS analyses were of either electrophoresis-grade or analytic-grade. All buffers were prepared using Milli-Q water. Subjects. Serum samples were taken from type 2 diabetic patients without microalbuminuria (DM, 13 males and 17 females), with microalbuminuria (MA, 19 males and 10 females), and suffering from chronic renal failure (CRF, 15 males and 16 females) with mean ages of 52, 54, and 58 years, respectively. Some patients with other complications such as retinopathy were excluded from MA and CRF groups to maximize specificity of study as much as possible. The protocols of this study were approved by the Korea University Hospital Institutional Review Board (IRB) with the informed consent of the patients. Serum parameters were measured in the clinical laboratory, and all demographic and clinical data are summarized in Table 1. Removal of High-Abundance Proteins. Highly abundant proteins (i.e., albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) were removed from crude serum in accordance with the standard LC protocol using the Agilent Multiple Affinity Removal System. In brief, crude serum samples were diluted five times with buffer A containing protease inhibitors (COMPLETE, Roche) and filtered through 0.22 µm spin filters by 2 min of spinning at 16 000g at room temperature. Sample injections were conducted with a 4.6 × 100 mm column at room temperature at a flow rate of 0.5 mL/min of Agilent buffer A on a Agilent HPLC system. The flow-through fractions were collected and centrifuged for 4 h at 3500 rpm using centrifugal devices (Centricon, 3 kDa cutoff, Amicon Millipore, Bedford) to remove salt and lipids. Then, acetone precipitation was conducted to concentrate proteins from the centrifuged sample, and the protein pellet was resolublized in sample buffer for the protein assay. The protein concentration was determined by the Bradford method.19 Isoelectric Focusing (IEF) and 2-DE. Immobiline DryStrips (24 cm, pH 4-7) were rehydrated with the samples, 40 µg of protein, in 450 µL of a solubilization solution containing 8 M 736
Journal of Proteome Research • Vol. 6, No. 2, 2007
Kim et al. Table 1. Clinical Data of Patients with Type 2 Diabetes and Diabetic Nephropathya value
variable
Mean age (years) Gender (M/F) Fasting blood sugar (mg/dL) HbA1c (%) Albumin excretion rate (mg/24 h) Serum creatinine (mg/dL) Blood urea nitrogen (mg/dL) Serum total cholesterol (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) Triglycerides (mg/dL) Hemoglobin (g/dL) Retinopathy (n)
normal range
normoalbuminuria (n ) 30)
microalbuminuria (n ) 29)
chronic renal failure (n ) 31)
