Urinary Angiostatin - A Novel Putative Marker of Renal Pathology Chronicity in Lupus Nephritis

• DOI: 10.1074/mcp.m112.021667
• Publication Date: 2013-01-24T01:42:57Z
• Authors: Tianfu Wu; Yong Du; Jie Han; Sandeep Singh; Chun Xie; Yuyuan Guo; Xin J. Zhou; Chul Ahn; Ramesh Saxena; Chandra Mohan
• Keywords: Angiostatin

There is a critical need to identify biomarkers for Systemic Lupus Erythematosus (SLE) which has high prevalence of renal failure. When urine from patients with lupus nephritis was recently screened the levels ∼280 molecules using an exploratory array-based proteomic platform, elevated angiostatin were noted. Angiostatin bioactive fragment plasminogen, and been known have modulatory function in angiogenesis inflammation. The significant elevation urinary next validated independent cohort SLE (n = 100) ELISA. Among SLE, significantly increased active compared inactive correlating well SLEDAI disease activity index SLICC score (r 0.66, p < 0.0001). ROC curve analysis further confirmed that had capacity discriminate those disease. Patients Class IV exhibited highest angiostatin. Immunohistochemistry staining localized expression tubular cells these patients. Finally, when paired urine-kidney samples procured concurrently LN examined, correlated strongly pathology chronicity index, but not index. Given changes forebode poor patient survival, emerges as novel noninvasive marker SLE. Longitudinal studies are progress assess disease-predictive potential erythematosus 1The abbreviations used are:SLEsystemic erythematosusESRDend stage diseaseCKDchronic kidney diseaseSLICCsystemic international collaborating clinicseGFRestimated glomerular filtration rate. chronic autoimmune capable causing devastating clinical manifestations such Approximately 60% present (1Cameron J.S. nephritis.J. Am. Soc. Nephrol. 1999; 10: 413-424Crossref PubMed Google Scholar); these, about 10–15% will eventually end-stage (ESRD) Scholar). It early diagnosis can offer better opportunity control progression. Therefore, biomarker diagnosing highly desirable clinic. Currently, biopsy still gold standard because despite its invasive nature, this method allows accurate detection severity pathology. Besides risk infection other complications associated needle biopsy, retrieving tissue might also limitations acquiring representative specimens reflective real pathological changes. Serum relatively less invasive; however, serological measurement anti-dsDNA, C3, C4, protein markers does necessarily correlate systemic end clinics estimated Urine emerged recent years proven effective reflecting nephritis. Potential include IL-6 (2Iwano M. Dohi K. Hirata E. Kurumatani N. Horii Y. Shiiki H. Fukatsu A. Matsuda T. Hirano Kishimoto Urinary nephritis.Clin. 1993; 40: 16-21PubMed Scholar), IL-18 (3Migliorini P. Anzilotti C. Pratesi F. Quattroni Bargagna Dinarello C.A. Boraschi D. inhibitor IL-18BP erythematosus.Eur. Cytokine Netw. 2010; 21: 264-271PubMed MCP-1 (4Marks S.D. Shah V. 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Rovin B.H. Michaelson nephritis: multicenter study.Arthritis Res. Ther. 2009; 11: R143Crossref (146) 10Liu Z.C. Q.L. Li X.Z. Yang J.H. Ao X. Veeraragoo Zuo X.X. Elevation tumor necrosis factor-like weak inducer apoptosis peripheral blood mononuclear erythematosus.Cytokine. 2011; 53: 295-300Crossref (18) Indeed may be by far best source screening diseases several reasons. First, easily obtained noninvasive. Second, direct product kidney, reflection function. Nevertheless, ideal monitoring (LN) remains elusive. A limited number reported date. Initial indications approach open up new avenues discovery Mosley et al. (2006) identified unique mass spectral patterns utilizing SELDI-TOF spectrometry could (11Mosley Tam F.W. Edwards R.J. Crozier Pusey C.D. Lightstone profiles distinguish between 45: 1497-1504Crossref (87) However, proteins represented spectra yet identified. By similar technology, Zhang (2008) hepcidin (12Zhang Jin Wu Nadasdy G. Harris Green-Church Nagaraja Birmingham D.J. 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Folkman Angiostatin: mediates suppression metastases Lewis lung carcinoma.Cell. 1994; 79: 315-328Abstract (3163) 20O'Reilly circulating suppresses growth.Cold Spring Harb. Symp. Quant. Biol. 59: 471-482Crossref (183) addition second array based indicated nephritis, described below. Thus, designed whether indicative cross-sectional design. recruited at Parkland St. Paul University Hospitals Texas Southwestern Medical Center Dallas. All patient-related procedures performed strictly following institution-approved IRB protocols. Five pilot array, Validation orthogonal method. Gender age-matched healthy volunteers draw collection, controls. CKD inclusion criterion all who fulfilled ACR classification criteria exclusion failure children Detailed information pertaining studied, their demographics, co-morbidities medications, listed Table biofluids processed detailed elsewhere Scholar).Table IDemographics characteristics validation studies. creatinine proteinuria/urine ratio: controls: 1.85 ± 0.22 mg/dL 2.73 0.84; Healthy 0.72 0.15 0.4 0.11No.100Female, no. (%)86 (86)Age, mean +/− S.E., years35.8 1.1Race, African American/Hispanic/Caucasian, no.45/42/13SLEDAI, median (interquartile)10 (4–16)Renal SLEDAI, (interquartile)4 (4–8)No. 0 (%)20 (20)Protein:creatinine ratio, mg/mg, S.E.2.0 0.3Serum Cr, mg/dl, S.E.1.5 0.1Comorbidities, (%)Diabetes Mellitus10 (10)Hypertension75 (75)Dyslipidemia58 (58)Cardiovascular disease13 (13)Anemia67 (67)Antiphospholipid syndrome2 (2)Venous thromboembolism8 (8)Others50 (50)Current (%)Prednisone71 (71)Mycophenolic acid36 (36)Cyclophosphamide7 (7)Azathioprine/MTX13 (13)Cyclosporine/Tacrolimus3 (3)Hydroxychloroquine47 (47)Angiotensin blocking agents49 Open table tab sample collection: Midstream clean-catch collected. requested first cleanse urethral area towelette, then void portion stream into toilet. midstream collected clean BD Vacutainer Brand specimen cup (Franklin Lakes, NJ USA). urinalysis on samples, remaining centrifuged 10,000 × g 2 min 4 °C. supernatant divided 1-ml aliquots frozen −80 °C storage. Each aliquot retrieved thawed only once assays study. Whole tubes (Cat #: 367812). Tubes incubated undisturbed room temperature 20 min, 3000 rpm 10 (serum) 200-μl initial screen comprised three individuals (mean age, 35; females, Hispanic), five 40.5; Hispanic one American female, male; [SLEDAI] ≥16 renal-related [rSLEDAI] ≥8). These diluted 5-fold buffer (1% bovine albumin phosphate-buffered saline) hybridized glass slide arrays interrogate level 274 different conducted RayBio® Human Antibody Array G-Series 4000 (Cat# AAH-CYT-G4000–8), consists 8 subarrays per subarray. Briefly, monoclonal antibodies against various cytokines (or mediators) printed slides bait capture corresponding or serum, mixture biotinylated secondary antibodies, detected Cy3 labeled streptavidin. analyte assayed duplicate. scanned GenePix 4000B scanner (Molecular Devices). Signals acquired transformed digits Genepix software. Positive Control spots (POS1, POS2, POS3) standardized amounts IgGs directly onto array. variables being equal, intensities should same each normalization results samples). Also Negative (NEG) consisting alone (used dilute array). presence analytes marked signal exceeded two deviations above background intensity. PMT 33% gain setting 550 (Dallas, TX) aliquoted prior storage Only assay avoid freeze/thaw cycles. measured precoated ELISA kit Raybiotech Inc. (Norcross, GA). Cayman Chemical (Ann Arbor, MI). normalized creatinine. (Systemic International Collaborating Clinics) computed Petri M (21Petri Kasitanon Lee Link Magder Bae S.C. Hanly J.G. D.A. Nived O. Sturfelt van Vollenhoven Wallace Alarcon G.S. Adu Avila-Casado Bernatsky S.R. Bruce I.N. Clarke A.E. Contreras Fine D.M. Gladman D.D. Gordon Kalunian K.C. Madaio M.P. Sanchez-Guerrero Steinsson Balow J.E. Buyon J.P. Ginzler E.M. Khamashta M.A. Urowitz M.B. Dooley Merrill J.T. Ramsey-Goldman Font Tumlin Stoll Zoma activity/response exercise: development response index.Arthritis 58: 1784-1788Crossref (52) calculating score, spot protein/creatinine ratios estimates daily (22Petri Singh comparison agreement rating response.Arthritis 1789-1795Crossref (17) Scholar): proteinuria 0.5–1 g/day (3 points), >1–3 (5 >3 (11 points). factored red count >10/high-power field white (1 point). Estimated Glomerular Filtration Rate (eGFR) calculated eGFR calculator National Kidney Foundation (http://www.kidney.org). Renal biopsies stages UT Center. Totally, 15 evaluated immunohistochemistry: controls, n 3; II, 4; IV, 8. 10% buffered, formalin-fixed, paraffin-embedded μm sections prepared. After dewax rehydration, microwaved mmol/L citrate (pH 6.0). Rabbit anti pAb (Abcam Inc, Cambridge, MA, USA, ab2904) 1:400 dilutions overnight incubation Then stained biotin-free immunoenzymatic antigen system (EXPOSE mouse/Rabbit specific HRP/DAB IHC kit, Abcam counterstained hematoxylin visualize nuclei. isotype IgG ab27478) dilution control. quantified counting positive tubules random fields slide. fragments Western blot. denatured containing 25 mm Tris base, 190 glycine 0.1% SDS, pH8.3 loaded SDS-PAGE. Proteins transferred polyvinylidene difluoride membrane rabbit polyclonal antibody (reactive human) Abcam, Catalog # 2904. procedure blot (23Wu Qin Kurepa Z. Kumar K.R. Liu Kanta Satterthwaite A.B. Davis L.S. Shared signaling networks B isolated genetically distinct mouse models lupus.J. Clin. Invest. 117: 2186-2196Crossref (80) Data plotted analyzed GraphPad Prism 5 (GraphPad, San Diego, CA) Medcalc software (Mariakerke, Belgium). t test where normality passed; otherwise, nonparametric Mann-Whitney analyze data. Likewise, Pearson Spearman correlation analyses. order antibody-based utilized scan 5) controls 3). screened, almost orders magnitude (p 0.0011) (Fig. 1). Hence, rest focuses molecule. To determine validate data shown Fig. 1. group included. Clearly, 0.0001), 2A). urinalysis, there increase 2C). Not surprisingly, difference overall did reach significance indicating involved types besides Receiver Operating Curve 2B) (AUC 0.93), 0.56). determining we asked reflect severity. We (SLEDAI ≤ 2, 0), > 0; "lupus nephritis" "LN"). Within group, distinguished subgroups: past history without Compared among subgroup 2D). More importantly, 0.0001) 2D), damage damage. analysis, AUC values 0.75 (Active versus HC, 0.95; Active inactive, 0.83), 2E). no healthy, groups 2F). clearly demonstrate subjects. Next, wanted ask lupus. showed positively 0.36, 0.0002, 3A), 0.52, 0.0001, 3B), 0.68, 3C), negatively −0.15, 0.07, 3D). suggest serve activity. comparison, either 3E) components 3F). examined ratio 0.28, 0.003), rSLEDAI 0.39, 0.002), 0.51, although invasive, prognostication. end, and, if so, how it predict degree For