Urokinase plasminogen activator receptor and its soluble form in common biopsy-proven kidney diseases and in staging of diabetic nephropathy

    Urokinase plasminogen activator receptor and its soluble form in common biopsy-proven kidney diseases and in staging of diabetic nephropathy Chung-Ze Wu, Li-Chien Chang, Yuh-Feng Lin, Yi-Jen Hung, Dee Pei, Nain-Feng Chu, Jin-Shuen Chen PII: DOI: Reference:

S0009-9120(15)00262-3 doi: 10.1016/j.clinbiochem.2015.07.001 CLB 9068

To appear in:

Clinical Biochemistry

Received date: Revised date: Accepted date:

4 February 2015 9 June 2015 2 July 2015

Please cite this article as: Wu Chung-Ze, Chang Li-Chien, Lin Yuh-Feng, Hung Yi-Jen, Pei Dee, Chu Nain-Feng, Chen Jin-Shuen, Urokinase plasminogen activator receptor and its soluble form in common biopsy-proven kidney diseases and in staging of diabetic nephropathy, Clinical Biochemistry (2015), doi: 10.1016/j.clinbiochem.2015.07.001

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ACCEPTED MANUSCRIPT Urokinase plasminogen activator receptor and its soluble form in common

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biopsy-proven kidney diseases and in staging of diabetic nephropathy

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Chung-Ze Wu1,2, Li-Chien Chang3, Yuh-Feng Lin1,4, Yi-Jen Hung5, Dee Pei6, Nain-Feng Chu7,8, Jin-Shuen Chen*9

Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University,

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Taipei, Taiwan, R.O.C

Division of Endocrinology and Metabolism; Department of Internal Medicine, Shuang Ho

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Hospital, Taipei Medical University, Taipei, Taiwan, R.O.C School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, R.O.C

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Division of Nephrology, Department of Internal Medicine, Shuang Ho Hospital, Taipei

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Medical University, Taipei, Taiwan, R.O.C Division of Endocrinology and Metabolism, Department of Internal Medicine, Tri-service

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General Hospital, National Defense Medical Center, Taipei, Taiwan, R.O.C Department of Internal Medicine, Cardinal Tien Hospital, Xindian; Medical School,

Catholic Fu Jen University, Taipei, Taiwan, R.O.C 7

Superintendent, Taitung Hospital, Ministry of Health and Welfare, Taiwan, R.O.C

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School of Public Health, National Defense Medical Center, Taipei, Taiwan, R.O.C

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Division of Nephrology, Department of Internal Medicine, Tri-service General Hospital,

National Defense Medical Center, Taipei, Taiwan, R.O.C

Running title: uPAR in kidney diseases 1

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Address requests for reprint and correspondence to: Jin-Shuen Chen MD, PhD

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Division of Nephrology, Department of Internal Medicine, Tri-service, General Hospital,

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National Defense Medical Center; No 325, Sec 2, Chenggong Rd., Neihu District, Taipei

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City 114, Taiwan, R.O.C

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Tel: +886-2-87923311 Fax: +886-2-87927292

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E-mail:[email protected]

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Abbreviations:

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AEC, 3-amino-9-ethylcarbazole; ANOVA, analysis of variance; CKD, chronic kidney

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disease; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; CIN, chronic interstitial nephritis; DM, diabetes mellitus; DN, diabetic nephropathy; eGFR, estimated

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glomerular filtration rate; ELISA, Enzyme-linked immunosorbent assay; ESRD, end stage renal disease; FSGS, focal segmental glomerulosclerosis; HC, health control; hr, hour; IDMS, isotope dilution mass spectrometry; IgAN, immunoglobin A nephropathy; IHC, immunohistochemical; LN, lupus nephropathy; MacroP, macroproteinuria; MCD, minimal change disease; MicroP, microproteinuria; MN, membranous nephritis; NCRT, non-cancerous renal tissues; suPAR, soluble urokinase plasminogen activator receptor; uPAR, urokinase plasminogen activator receptor; UP/Cr, urine protein to creatinine ratio

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ACCEPTED MANUSCRIPT Abstract

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Objectives: Soluble urokinase plasminogen activator receptor (suPAR), derived from

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membrane bound uPAR, is associated with inflammatory diseases. In the present study, we

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explored the expression of uPAR/suPAR in common biopsy-proven kidney diseases and the relationship between suPAR and staging of type 2 diabetic nephropathy (DN).

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Design and Methods: Serum samples for suPAR and renal tissues for uPAR staining were

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investigated in various common kidney diseases. The levels of serum suPAR were measured

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characteristic (ROC) curve.

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and adequate cut-off values of different stage of DN were calculated by receiver operating

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Results: In our results, the expression of uPAR on renal tissues was pronounced in the majority of kidney diseases. Comparing of expression of uPAR among different kidney

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diseases, it was strongest in minimal change disease (MCD) and weakest in chronic interstitial nephritis. Serum suPAR in most kidney diseases, except of MCD, were significantly elevated and was highest in DN. As for DN and suPAR, we found that suPAR progressively increased with staging of DN. Moreover, suPAR was linearly and negatively related to estimated glomerular filtration rate and positively related to the amount of proteinuria. By ROC curve, the cut-off values of suPAR in DN for assessing development increased with the progression of the disease. Conclusions: We concluded that uPAR/suPAR is elevated in most kidney diseases and that 3

ACCEPTED MANUSCRIPT suPAR is a useful biomarker for assessing stages of DN.

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Keywords: soluble urokinase plasminogen activator receptor, diabetic nephropathy,

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proteinuria

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ACCEPTED MANUSCRIPT 1. Introduction

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Urokinase plasminogen activator receptor (uPAR) is a glycosyl-phosphatidyl-

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nositol-anchored receptor composed of three extracellular domains (D1, 2 and 3), expressed

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in various cells, including macrophage, neutrophils, endothelial cells, vascular smooth muscle cell, as well as neoplastic cells [1, 2]. uPAR may be shed from the cell surface by

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several protease as a soluble bioactive peptide in blood and body fluid, called soluble uPAR

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(suPAR), that has multiple biological properties [3]. Besides being associated with several cancers and metastasis [1], a large body of research shows that suPAR may be a valuable

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biomarker of sepsis, critical illness, and several inflammatory diseases for diagnosis and

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prognosis in a clinical setting [4-6]. Moreover, some studies indicated that suPAR is associated with cardiovascular disease, diabetes mellitus (DM) and the process of

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atherosclerosis.

As for uPAR in kidney diseases, uPAR is not expressed in a normal kidney [7], but de novo expression by glomerular and tubular epithelial cells and renal interstitial cells has been proved in diabetic nephropathy (DN) [8]. Moreover, mice were protected from endotoxin-induced proteinuria in absence of uPAR [9]. It is a reasonable assumption that the role of uPAR is important in proteinuric kidney disease. For suPAR in kidney diseases, it has been noted to be a pathogenic cause of focal segmental glomerulosclerosis (FSGS) [10]. Higher concentration of suPAR activates podocyte integrin, causing foot process effacement, 5

ACCEPTED MANUSCRIPT proteinuria and FSGS-like glomerulopathy. In patients with dialysis, suPAR levels have

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been noted to increase over healthy levels and to be correlated with increased fibrinolytic

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activity [11]. It is noteworthy that the level of suPAR elevated in non-smoking patients with

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type 2 DM in a recent study [12]. While there is a strong assumption of a link between

direct relationship between suPAR and DN.

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proteinuria kidney disease and DM, little empirical evidence has been found to establish a

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End stage renal disease (ESRD) is a global health crisis. However, to date, there are few effective strategies for common biopsy-proven kidney diseases, including nephrotic,

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nephritic, and tubulointerstitial kidney diseases. In addition, DN is a major cause of ESRD

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in Taiwan, and early diagnosis of DN development is the most effective strategy for preventing ESRD. DN has the characteristics of subclinical inflammation, declining renal

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function and progressive proteinuria. It is reasonably assumed that suPAR may be a useful biomarker for assessing development of DN. The purpose of this research is to explore the relationship between uPAR/suPAR and common biopsy-proven kidney diseases and compare the expression of uPAR/suPAR among common kidney diseases. We also assessed the levels and optimal cut-off values of suPAR in different stages of DN in the study.

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ACCEPTED MANUSCRIPT 2. Materials and Methods

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2.1. Subjects

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Two major groups of subjects were recruited. Subjects in the first group, who were

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health control (HC) and had type 2 DM, did not receive renal biopsy; subjects in the other groups had common biopsy-proven kidney diseases, including DN with macroproteinuria

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(MacroP). In the first group, all subjects were recruited from a division of nephrology in a

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medical teaching center in Taiwan. For the other group, subjects were collected by the Taiwan Renal Biomarker Consortium from major medical centers across Taiwan. Exclusion

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criteria included type 1 DM, malignancy, and acute infections, and the presence of other

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primary or secondary undermined renal diseases or pathologies. Pregnancy, acute or chronic viral infections, and any illicit drug use, liver cirrhosis or record of other chronic diseases

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were also exclusion criteria for all recruitments. All of enrolled subjects gave informed consent and the study protocol was reviewed and approved under the guidelines of the 1975 Declaration of Helsinki by the Institutional Review Boards (Approval No: 094-05-0031). The subjects at different stages of DN without renal biopsy were divided into groups according to renal function and amount of proteinuria. Blood and urine were collected in the morning after overnight fasting. The diagnosis of type 2 DM was based on the definition from the American Diabetes Association [13]. Creatinine was measured by kinetic modification of the Jaffe procedure (Beckman Coulter, Olympus AU 5800), traceable to an 7

ACCEPTED MANUSCRIPT isotope dilution mass spectrometry (IDMS). Renal function was evaluated by estimated

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glomerular filtration rate (eGFR) (calculated by Chronic Kidney Disease Epidemiology

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Collaboration (CKD-EPI), which has been found to be more precise for assessing renal

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function in patients with DM [14, 15]). The amount of proteinuria was assessed by urine protein to creatinine ratio (UP/Cr). Patients with type 2 DM and presenting with proteinuria

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(UP/Cr ≥ 0.03 mg/mg) were referred to as the DN group. Finally, there were five groups

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derived from subjects with type 2 DM, including DM without nephropathy, DN with microproteinuria (MicroP) (0.03 mg/mg ≤ UP/Cr < 0.3mg/mg), DN with MacroP (UP/Cr ≥

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0.3 mg/mg), DN with MacroP plus low eGFR (eGFR ≤60 ml/min, based on the consensus

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of DN in the American Diabetes Association [16]) and DN with ESRD. Exclusion criteria for the HC group were DM, any major significant medical diseases, presence of proteinuria,

ml/min.

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and abnormal renal function represented by serum creatinine > 1.2 mg/dl or eGFR < 60

As for the group with common biopsy-proven kidney diseases, subjects received ultrasound-guided percutaneous needle renal biopsy for confirming pathogenesis under local anesthesia. Study subjects according to the results of renal biopsy included idiopathic glomerular diseases (minimal change disease (MCD), membranous nephritis (MN), immunoglobin A nephropathy (IgAN), and FSGS), secondary glomerular diseases (DN with MacroP, lupus nephropathy (LN)) and chronic interstitial nephritis (CIN). Blood and urine 8

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samples were collected from the patients on the day of renal biopsy.

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2.2. Immunohistochemical (IHC) stain of kidney for uPAR

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Renal tissues from renal biopsy were fixed with paraformaldehyde. After rehydrating, the slices were incubated with 1:200 dilution of the primary antibody, rabbit anti-uPAR

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antibody (Santa Cruz, sc-10815, USA) in phosphate-buffered saline at 4C overnight. Then

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the slices were incubated with biotin conjugated anti-rabbit for 1 hour (hr) and washed with Tris-buffered saline containing 0.05% Tween 20 (pH 7.4). After incubating biotinylated

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secondary antibody, the tissue section was treated with an avidin-biotin-peroxidase. The

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reaction was visualized by use of 3-amino-9-ethylcarbazole (AEC) substrate chromogen following tissue counterstaining with hematoxylin. Images were taken under 100x, 200x

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and 400x magnification with a light microscope. As for the control group, non-cancerous renal tissues (NCRT) were harvested from cases of renal cancer after nephrectomy.

2.3. Enzyme-linked immunosorbent assay (ELISA) measurement of suPAR. Serum levels of suPAR were determined using a Human uPAR Quantikine ELISA Kit (R&D, DUP00, MN). Standards and samples were incubated for 2 hrs at room temperature. Two hundred µL of conjugate was added to each well, and then incubated 2 hrs at room temperature. After that, the kit was followed by 200 μL streptavidin horseradish peroxidase 9

ACCEPTED MANUSCRIPT and incubated for 30 minutes at room temperature. Fifty µL of Stop Solution was added to

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each well and read at 450 nm within 30 minutes. The 4000 pg/mL standard serves as the

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high standard. Calibrator Diluent solution serves as the zero standard (0 pg/mL). The

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minimum detectable dose of suPAR is typically less than 33 pg/mL. The intra-assay and

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inter-assay coefficients of variation were 4.1% and 5.1%, respectively.

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2.4. Statistical Analysis.

Analysis was performed using PASW Statistics version 18.0 statistical package for

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Windows (SPSS, Chicago, IL). One way-Analysis of Variance (ANOVA) with LSD’s test as

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a post hoc test was used for comparison of clinical demographics and suPAR in different groups. Pearson product-moment correlation coefficient was used to assess the linear

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association between eGFR, amount of proteinuria and suPAR in DN. Because of the close relation between eGFR and proteinuria, multivariate linear regression was applied for analysis of association of suPAR, eGFR and amount of proteinuria. Furthermore, for estimating the optimal cut-off point of suPAR in different stages of DN, the receiver operating characteristic (ROC) curve was applied. Area under ROC curve was assessed for statistical significance. The value of suPAR with adequate sensitivity and specificity was gained for predicting development of different stages of DN. All statistical data were

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ACCEPTED MANUSCRIPT expressed as two-sided, and p values less than 0.05 were considered to be statistically

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significant.

3. Results

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3.1. Serum suPAR levels and expression of uPAR on renal tissue in common biopsy-proven

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kidney diseases

The subjects’ serum suPAR levels, demographic and biochemistry data of HC and

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common biopsy-proven kidney diseases are listed in Table 1. In our cohort, the MCD

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patients were youngest. Subjects in all of the kidney disease groups showed significantly higher proteinuria compared with HC. The renal function represented by serum creatinine

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level was significantly higher in the DN with MacroP, CIN, IgAN, LN and FSGS groups compared with the HC group. Levels of suPAR significantly increased in the majority of kidney diseases, except MCD, in comparison with the HC. Moreover, the suPAR level in DN with MacroP was the highest among the common kidney disease subjects. Samples of kidney tissue for IHC staining of uPAR were from TSGH alone. At least 3 samples were tested for all diseases. The control specimen was from NCRT. The representative IHC stains of uPAR in common biopsy-proved kidney diseases are shown in Figure 1. The expression of uPAR on renal tissue increased significantly in the majority of 11

ACCEPTED MANUSCRIPT kidney diseases, and uPAR expression was strongest in MCD and weakest in CIN.

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Furthermore, the quantities of uPAR expression between glomerular and tubular regions in

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MCD, DN with MacroP and MN were similar. However, uPAR expression in the tubular

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region was more predominant than that in the glomerular region in CIN, IgAN, LN, and

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3.2. suPAR levels in different stages of DN

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FSGS.

The Demographic data, levels of renal function and proteinuria from different stages

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of DN are listed in Table 2. For further analysis, the suPAR levels were re-assessed in

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different stages of DN, as shown in Figure 2. It was interesting that with the severity of proteinuria and impaired eGFR, the levels of suPAR increased gradually with the advanced

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staging of DN. Also noteworthy is that serum suPAR significantly and rapidly increased as DN progressed beyond MacroP, the irreversible stage. Our results suggest that the remarkable increase of suPAR in type 2 DN may indicate the worsening of DN and so may have significant implications for clinical practice.

3.3. suPAR levels are associated with eGFR and amount of proteinuria in DN The relationship between suPAR, eGFR and the amount of proteinuria in patients with type 2 DN is depicted in Figure 3. Not surprisingly, suPAR has a strong negative 12

ACCEPTED MANUSCRIPT association with eGFR (r=-0.806, p<0.001) and a positive linear association with the

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amount of proteinuria (r=0.719, p<0.001) in patients with type 2 DN. Again, this association

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supports our above findings of elevated suPAR in advanced staging of type 2 DN. After

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adjusting for age and multivariate linear regression of eGFR and proteinuria, the suPAR levels are still significantly negatively related to eGFR (r=-0.579, p<0.001) and positively

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related to proteinuria (r=0.459, p<0.001). We suggest the more impaired the renal function

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and amount of proteinuria, the higher the levels of suPAR in type 2 DN.

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3.4. Cut-off values of suPAR in different stages of DN

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The cut-off points of different stages of type 2 DN were evaluated by ROC curve. The area under ROC curve showed the significance in suPAR at the different stages of DN. The

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results clearly showed cut-off values of suPAR increase gradually with the staging of type 2 DN (Table 3). Our optimal cut-off points of suPAR with simultaneous high sensitivity and specificity were DM without nephropathy: 2.435ng/ml, DN with MicroP: 2.865ng/ml, DN with MacroP: 3.255ng/ml, DN with MacroP+low eGFR: 3.505ng/ml, and DN with ESRD: 4.265ng/ml. By measuring suPAR in clinical practice, we might be able to detect worsening renal function earlier in patients with type 2 DN.

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ACCEPTED MANUSCRIPT 4. Discussion

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In the present study, we found higher suPAR levels in common biopsy-proven kidney

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diseases, especially in DN with MacroP. Up-regulated expression of uPAR in renal tissue

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was noted in most of the disease samples under investigation. Serum suPAR gradually increased with severity and staging of DN. In addition, levels of suPAR were linearly

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related to eGFR and to the amount of proteinuria in patients with various kidney diseases.

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We also assessed the adequate cut-off points of suPAR in different staging of DN for early diagnosis.

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A preponderance of research shows suPAR is associated with infection or

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inflammation and predicts the prognosis in different infective or inflammatory diseases. Our results found that suPAR was elevated in the majority of kidney diseases in the

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inflammatory status of nephritis through different mechanisms. Accordingly, elevated suPAR would be found in several kinds of nephritis, not only in FSGS. In addition, patients with type 2 DM are in pro-inflammatory status, which is associated with several inflammatory cytokines [17]. Haugaard SB, et al. indicated that the suPAR increased in non-smoking patients with new-onset type 2 DM, but attenuated in smoking patients [12]. Moreover, higher suPAR increased the risk and rate development of type 2 DM in non-smoking overweight persons [18, 19]. However, our results showed suPAR in type 2 DM without DN did not significantly increase as compared with healthy subjects. Some 14

ACCEPTED MANUSCRIPT factors, such as body weight, or smoking status, may influence our results. More studies are

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needed to clarify the role of these factors.

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Our results showed suPAR is inversely related to eGFR in patients with DN. Similar

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findings were found by several studies. Meijers B and his colleagues investigated patients with FSGS and non-FSGS glomerulopathy [20]. They found suPAR levels were negatively

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associated with eGFR in different kidney diseases, and indicated suPAR is not a suitable

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biomarker for FSGS. Similarly, Wada T et al. explored patients with FSGS, MCD, IgAN, and MN [21] and also found suPAR is inversely related to eGFR, which was consistent with

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our findings. The molecular weight of suPAR ranges from 20 to 50 kDa, which would pass

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through glomerular filtration. Thus, it is reasonably explained that plasma suPAR accumulates during impairment of renal function. However, it is noteworthy that our study

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confirmed the same findings in DN patients. Meanwhile, we noted the suPAR is positively related to the amount of proteinuria in DN. Our results were similar to previous studies. The possible explanation is that suPAR is associated with activation of podocyte β3 integrin, and causes foot process effacement [10]. However, some studies showed serum suPAR is not related to proteinuria. Sinha A et al. investigated 469 children with FSGS and other nephrotic syndrome [22]. He found serum suPAR is not related to amount of proteinuria, but urinary suPAR excretion is correlated to proteinuria. Contrarily, Bock ME et al. studied 99 children with idiopathic FSGS and 15

ACCEPTED MANUSCRIPT indicated that girls with nephrotic-range proteinuria had the lower suPAR levels [23].

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However, we found a positive relationship between suPAR and the amount of proteinuria in

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patients with type 2 DN. The different results and actual underalying pathophysiology are

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still unclear and need to be further explored.

The majority of DN cases have two important features: reduced eGFR and increased

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amount of proteinuria, which are associated with elevated suPAR. In our research, it is

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noteworthy that we assessed the adequate cut-off points for staging of DN by ROC curve. The cut-off points increased gradually with advanced staging of type 2 DN and showed high

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sensitivity and specificity. The values of suPAR could be applied in clinic practice to remind

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physicians that in advanced stages of type 2 DN as suPAR levels increase. Thus, suPAR might be a useful biomarker for evaluating stages of DN.

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There were some limitations in our study. First, type 2 DN and atherosclerosis are strongly associated with subclinical inflammation although we had excluded most patients with infection and inflammation. We did not test the levels of C-reactive protein or other inflammatory cytokines, which are also associated with suPAR and may result in some interfering factors. However, these factors do not strongly influence our results. Second, the number of study cases was limited, especially in the low eGFR group. We could not divide low eGFR into as chronic kidney disease (CKD), stages 3-5, by KDOQI guidelines, which may reduce the power of our statistical analysis. Nevertheless, our study determined that 16

ACCEPTED MANUSCRIPT suPAR increases in advanced stages of DN.

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In conclusion, suPAR is higher in common kidney diseases. Over-expression of uPAR

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is noted in kidney tissue, either glomerular or tubular region, of different kidney diseases.

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suPAR is associated with reduced eGFR and amount of proteinuria in DN, and increases

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with staging of DN. Thus, suPAR may be a biomarker for staging of DN in clinical practice.

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Acknowledgements

This work is supported by grants from the National Science Council (NSC

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101-2314-B-016-017 and NSC 102-2314-B-016-012), and the Tri-Service General Hospital

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(TSGH-C102-108), Taiwan, ROC. We deeply appreciate Professor Mary Goodwin's (English department, National Taiwan Normal University) efforts in the language support.

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All authors declared no competing interests.

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ACCEPTED MANUSCRIPT Figure Legends

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Figure 1 Immunohistochemical (IHC) stain of uPAR expression on renal tissue in

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non-cancerous renal tissues (NCRT) and common biopsy-proven renal diseases. At

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least 3 samples were tested by IHC for all diseases. As representative pictures show, expression of uPAR in renal tissue was elevated in the majority of kidney diseases,

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including minimal change disease (MCD), diabetic nephropathy with macroproteinuria (DN

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with MacroP), membranous nephritis (MN), chronic interstitial nephritis (CIN),

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glomerulosclerosis (FSGS).

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Immunoglobulin A nephropathy (IgAN), Lupus nephritis (LN) and focal segmental

Figure 2 suPAR in healthy control (HC) and different stages of type 2 diabetic

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nephropathy (DN). The suPAR levels elevated gradually with staging of type 2 DN. Significantly, levels of suPAR in DN with MacroP, DN with MacroP + low eGFR and DN with ESRD increased as compared with the HC group. * p < 0.05, *** p < 0.001

Figure 3 The relationship between suPAR and (a) renal function (estimated glomerular filtrating rate, eGFR), and (b) amount of proteinuria. The levels of suPAR were negatively linearly related to eGFR (r=-0.806, p < 0.001) and positively related to amount of proteinuria (UP/Cr: ratio of urine protein and creatinine) (r=0.719, p < 0.001). 22

AC

Fig. 1

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

23

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 2

24

AC

Fig. 3

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

25

ACCEPTED MANUSCRIPT

T

Table 1 Demographic data, biochemistry data, suPAR in common biopsy-proven kidney

IP

disease.

SC R

DN with HC

MCD

MN

(n=11)

(n=17)

(n=25)

(n=18)

(n=17)

33/4

4/7

8/9

14/11

2/16

13/4

5/9

CE P

10/12

(n=14)

TE

) Sex

FSGS

D

(n=37)

LN

MA

P (n=22

IgAN

NU

Macro

CIN

p

(M/F

AC

) Age

58.5±

25.54±

51.18± 54.36±1 51.00±1 36.72±1 37.83±1 55.47±1

<0.0

(year

17.2

8.88

9.23

6.29

9.58

3.20

8.05

6.05

01a

SCr

0.95±

0.86±0

1.86±1

0.73±0.

3.18±2.

1.86±2.

1.03±0.

4.61±6.

<0.0

(mg/

0.64

.22

.66

16

36

13

56

73

01b

0.15±

5.35±9

4.16±2

1.54±1.

1.29±1.

1.70±3.

4.82±5.

8.24±11

<0.0

s)

dl) UP/C

26

ACCEPTED MANUSCRIPT r

0.19

.23

.55

78

71

14

suPA

2.26±

2.24±0

4.38±1

3.11±0.

3.92±1.

2.93±1.

R

0.48

.91

.59

64

58

65

.78

01c

3.21±1.

3.83±1.

0.02

52

63

5d

77

T

(mg/

SC R

NU

(ng/

IP

mg)

MA

ml)

suPAR: Soluble urokinase plasminogen activator receptor; HC: Healthy control; MCD:

TE

D

Minimal change disease; DN: Diabetic nephropathy; MN: Membranous nephritis; CIN:

CE P

Chronic interstitial nephritis; IgAN: Immunoglobin A nephropathy; LN: Lupus nephropathy; FSGS: Focal segmental glomerulosclerosis; SCr: Serum creatinine; UP/Cr: ratio of urine

a

LSD’s test: FSGS>MN>DN with MacroP>CIN>LN> IgAN >MCD

b

c

AC

protein to urine creatinine

LSD’s test: FSGS>LN>MCD>MN

LSD’s test: DN with MacroP>CIN

d

LSD’s test: DN with MacroP>CIN>FSGS>MCD

27

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T

Table 2 Demographic data and biochemistry data in DM without nephropathy and different

DM

DN with

DN with

DN with

DN with

without

MicroP

MacroP

MacroP

ESRD

NU

nephropath

SC R

Variables

IP

stages of DN.

TE

(n=50)

D

MA

y

18/32

Age(years)

CE P

Sex(M/F)

AC

56.8±7.2

Creatinine(mg/dl)

+

low eGFR

(n=23)

(n=21)

(n=37)

(n=36)

6/17

7/14

13/24

18/18

59.3±11. 61.9±9.1

0.08 63.1±9.0

60.5±9.8

8 0.78±0.1

0.98±0.1

2.34±1.7

10.18±2.8

<0.001

4

7

2

5

a

0.64±0.13

eGFR(ml/min/1.73m

34.2±15. 103.2±9.1

2

p

87.5±9.5

71.0±7.6

)

UP/Cr(mg/mg)

0.21±0.1

2.35±3.0

<0.001 4.8±2.0

6

b

2.45±2.3

<0.001

0.12±0.20

1

6

6

DM: type 2 DM; eGFR: estimated glomerular filtrating rate; DN: diabetic nephropathy; 28

c

ACCEPTED MANUSCRIPT MicroP: microproteinuria; MacroP: macroproteinuria; ESRD: end stage renal disease LSD’s test: DM with ESRD> DN with MacroP +low eGFR> DN with MacroP> DN with

T

a

LSD’s test: DM without nephropathy> DN with MicroP> DN with MacroP> DN with

SC R

b

IP

MicroP> DM without nephropathy

MacroP +low eGFR> DN with ESRD

LSD’s test: DN with MacroP / DN with MacroP +low eGFR > DN with MicroP> DM

NU

c

AC

CE P

TE

D

MA

without nephropathy

29

ACCEPTED MANUSCRIPT

T

Table 3 Adequate cut-off points of suPAR levels on type 2 diabetes with different stages of

IP

SC R

DN

MacroP

with

2.865

3.255

3.505

4.265

TE

DN

0.923

0.939

0.943

0.98

76.0

83.8

87.2

87.7

97.2

76.2

88.7

87.2

83.5

80.4

DM without

DN

nephropathy

with

DN

DN with

with

NU

MacroP

+ low eGFR ESRD

Cut-off points of suPAR

D

2.435

MA

MicroP

(ng/ml)

Sensitivity (%)

AC

Specificity (%)

0.837

CE P

Area under ROC curve

30

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Highlights 1. Expression of uPAR on renal tissues among majority of kidney diseases increased. 2. Serum suPAR elevated in most common kidney diseases, except of MCD. 3. suPAR level was negatively related to eGFR and positively related to proteinuria. 4. suPAR progressively increased with staging of diabetic nephropathy.

31