Protein Microarrays Discover Angiotensinogen and PRKRIP1 as Novel Targets for Autoantibodies in Chronic Renal Disease (2010)

Biomarkers for early detection of chronic kidney disease are needed, as millions of patients suffer from chronic diseases predisposing them to kidney failure. Protein microarrays may also hold utility in the discovery of auto-antibodies in other conditions not commonly considered auto-immune diseases. We hypothesized that proteins are released as a consequence of damage at a cellular level during end-organ damage from renal injury, not otherwise recognized as self-antigens, and an adaptive humoral immune response to these proteins might be detected in the blood, as a noninvasive tracker of this injury. The resultant antibodies (Ab) detected in the blood would serve as effective biomarkers for occult renal injury, enabling earlier clinical detection of chronic kidney disease than currently possible, because of the redundancy of the serum creatinine as a biomarker for early kidney injury. To screen for novel autoantibodies in chronic kidney disease, 24 protein microarrays were used to compare serum Ab from patients with chronic kidney disease against matched controls. From a panel of 38 antigens with increased Ab binding, four were validated in 71 individuals, with (n = 50) and without (n = 21) renal insufficiency. Significant elevations in the titer of novel auto-Ab were noted against angiotensinogen and PRKRIP1 in renal insufficiency. Current validation is underway to evaluate if these auto-Ab can provide means to follow the evolution of chronic kidney disease in patients with early stages of renal insufficiency, and if these rising titers of these auto-Ab correlate with the rate of progression of chronic kidney disease. Biomarkers for early detection of chronic kidney disease are needed, as millions of patients suffer from chronic diseases predisposing them to kidney failure. Protein microarrays may also hold utility in the discovery of auto-antibodies in other conditions not commonly considered auto-immune diseases. We hypothesized that proteins are released as a consequence of damage at a cellular level during end-organ damage from renal injury, not otherwise recognized as self-antigens, and an adaptive humoral immune response to these proteins might be detected in the blood, as a noninvasive tracker of this injury. The resultant antibodies (Ab) detected in the blood would serve as effective biomarkers for occult renal injury, enabling earlier clinical detection of chronic kidney disease than currently possible, because of the redundancy of the serum creatinine as a biomarker for early kidney injury. To screen for novel autoantibodies in chronic kidney disease, 24 protein microarrays were used to compare serum Ab from patients with chronic kidney disease against matched controls. From a panel of 38 antigens with increased Ab binding, four were validated in 71 individuals, with (n = 50) and without (n = 21) renal insufficiency. Significant elevations in the titer of novel auto-Ab were noted against angiotensinogen and PRKRIP1 in renal insufficiency. Current validation is underway to evaluate if these auto-Ab can provide means to follow the evolution of chronic kidney disease in patients with early stages of renal insufficiency, and if these rising titers of these auto-Ab correlate with the rate of progression of chronic kidney disease. Renal injury affects nearly a half-million patients in the United States and over a million people world-wide. Millions of additional patients diagnosed with chronic kidney disease (CKD) 1The abbreviations used are:CKDchronic kidney diseaseESRDend-stage renal diseaseELISAenzyme-linked immunoabsorbent assayAGTangiotensinogenACEangiotensin converting enzyme. are at highest risk for progression to end-stage renal disease (ESRD), and those individuals diagnosed with diabetes or hypertension are at high risk for developing this disease. The consequences of development of ESRD are severe, especially when progression occurs without early diagnosis. Early detection and initiation of potentially kidney-sparing therapies, such as angiotensin converting enzyme inhibitors and angiotensin II receptor blockers, can offer significant benefit to patients with renal injury (1.Lewis E.J. Hunsicker L.G. Clarke W.R. Berl T. Pohl M.A. Lewis J.B. Ritz E. Atkins R.C. Rohde R. Raz I. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.N. Engl. J. 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Outcome predictability of biomarkers of protein-energy wasting and inflammation in moderate and advanced chronic kidney disease.Am. J. Clin. Nutr. 2009; 90: 407-414Crossref PubMed Scopus (131) Google Scholar). Neutrophil gelatinase-associated lipocalin has been shown to be an early marker of progression from CKD to ESRD (8.Malyszko J. Malyszko J.S. Bachorzewska-Gajewska H. Poniatowski B. Dobrzycki S. Mysliwiec M. Neutrophil gelatinase-associated lipocalin is a new and sensitive marker of kidney function in chronic kidney disease patients and renal allograft recipients.Transplant Proc. 2009; 41: 158-161Crossref PubMed Scopus (81) Google Scholar, 9.Bolignano D. Lacquaniti A. Coppolino G. Donato V. Campo S. Fazio M.R. Nicocia G. Buemi M. Neutrophil gelatinase-associated lipocalin (NGAL) and progression of chronic kidney disease.Clin. J. Am. Soc. Nephrol. 2009; 4: 337-344Crossref PubMed Scopus (420) Google Scholar). However, besides osteoprotegerin, few of these markers have demonstrated utility as candidates that predict worsening of kidney function toward ESRD in patients without CKD. A chronic inflammatory state in renal injury and ESRD has been well described. C-reactive protein level, a broad marker of inflammation, is a strong predictor of mortality (10.Snaedal S. Heimburger O. Qureshi A.R. Danielsson A. Wikstrom B. Fellstrom B. Fehrman-Ekholm I. Carrero J.J. Alvestrand A. Stenvinkel P. Barany P. Comorbidity and acute clinical events as determinants of C-reactive protein variation in hemodialysis patients: implications for patient survival.Am. J. Kidney Dis. 2009; 53: 1024-1033Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), whereas specific immune dysregulation, including dysfunction of regulatory T cells, has been shown to be present in ESRD (11.Meier P. Golshayan D. Blanc E. Pascual M. Burnier M. Oxidized LDL modulates apoptosis of regulatory T cells in patients with ESRD.J. Am. Soc. Nephrol. 2009; 20: 1368-1384Crossref PubMed Scopus (45) Google Scholar). Levels of the inflammatory mediator long pentraxin 3 have been shown to be increased in the serum of patients with ESRD (12.Yilmaz M.I. Axelsson J. Sonmez A. Carrero J.J. Saglam M. Eyileten T. Caglar K. Kirkpantur A. Celik T. Oguz Y. Vural A. Yenicesu M. Lindholm B. Stenvinkel P. Effect of renin angiotensin system blockade on pentraxin 3 levels in type-2 diabetic patients with proteinuria.Clin. J. Am. Soc. Nephrol. 2009; 4: 535-541Crossref PubMed Scopus (59) Google Scholar). Variants in genes coding for immune factors, including IL-10, have been associated with susceptibility to ESRD (13.Manchanda P.K. Singh R. Mittal R.D. Cytokine (IL-10 -1082 and -819) and chemokine receptor (CCR2 and CCR5) gene polymorphism in North Indian patients with end-stage renal disease.DNA Cell Biol. 2009; 28: 177-183Crossref PubMed Scopus (11) Google Scholar). Given these studies, we hypothesized that we may be able to identify novel autoantibody biomarkers for CKD as it is likely that intracellular proteins are exposed as a consequence of damage at a cellular level from end-organ damage from chronic kidney disease. Given the increased inflammatory state, we hypothesized that some of these released proteins might not normally be exposed to B cells undergoing development and are not otherwise recognized as self-antigens. We suspected that some of these proteins could be recognized as antigenic, yielding an adaptive humoral immune response that could be detected in the blood in organ injury. To test our hypotheses and to screen for these putative auto-Ab responses, we used protein microarrays to screen sera from patients with CKD and control individuals. Protein microarrays have previously been used to identify tumor antigens (14.Hudson M.E. Pozdnyakova I. Haines K. Mor G. Snyder M. Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 17494-17499Crossref PubMed Scopus (232) Google Scholar), auto-antibody targets in rheumatologic and autoimmune disease (15.Robinson W.H. DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H. E.N. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses.Nat. Med. 2002; 8: 295-301Crossref PubMed Scopus (643) Google Scholar), allo-antibodies in solid-organ transplantation (16.Li L. Wadia P. Chen R. Kambham N. Naesens M. Sigdel T.K. Miklos D.B. Sarwal M.M. Butte A.J. Identifying compartment-specific non-HLA targets after renal transplantation by integrating transcriptome and “antibodyome” measures.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 4148-4153Crossref PubMed Scopus (92) Google Scholar), and potential markers for graft-versus-host disease. Rather than expecting a role for autoimmunity in renal injury, we suspected that the humoral immune response could serve as an amplifier for abnormal proteins exposed in the end-organ damage. We therefore used protein microarrays to detect the presence of auto-Ab in the sera of patients with established renal insufficiency. In a first-stage analysis, we compared the serum antibodies from 17 patients with CKD with an estimated glomerular filtration rate < 30 ml/min/1.73m2 (17.Schwartz G.J. Haycock G.B. Edelmann Jr., C.M. Spitzer A. A simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine.Pediatrics. 1976; 58: 259-263Crossref PubMed Google Scholar)(CKD stages 4 and 5) against seven age-matched healthy control individuals with normal renal function. We identified 38 candidate antigens showing significantly higher antibody binding levels in patients with CKD as compared with controls. We then tested four of these antibodies (anti-AGT, Anti-PRKR1P1, anti-ATG7, and anti-DUSP26; p < in a using independent sera from patients with CKD and healthy age-matched controls. We < and < were significantly higher in patients with independent of of for Ab of CKD or diagnosis. from the for these and for from patients and A of blood from the patients a and at for at 30 The serum from the by at using a and at from 17 patients with CKD and were of renal in these patients to nephropathy (n = (n = and of (n = of the patients at the of of patients were from seven age-matched healthy were The protein used for serum with potential proteins in with N-terminal expressed in and conditions cellular protein derived from the were at to 4 for for 1 at 4 with of and at were at were then for with patient serum in of serum and at by with for and then for with were then with for and then to for 1 to The and were used to were at with a of a of and a of The were from a on the by the of protein Given that were from conditions not otherwise considered as on the protein microarrays were as The protein microarrays from to whereas microarrays and The the on the protein microarrays were significantly with with a of = in by the for and the the and the of and the of the at the and the with over of using were were from the on the protein for showing a in from control to CKD patient of or showing an test p with a in healthy the in those and with a in renal the in those significant were matched to using S.M. L. Sigdel T.K. Wadia Miklos D.B. Butte A.J. Sarwal M.M. Protein microarrays identify antibodies to protein that are associated with a risk of allograft in renal Int. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar), yielding 38 proteins were from these 38 on on these proteins and for validation in independent CKD patient and control To estimate a discovery we the CKD and control for of the and using the four of the the to estimate the discovery rate at or a of we the from the 17 CKD patients and control with and 17 CKD the we for showing a in the of higher or an test p with a in the in those and with a in the other the in those proteins these control with significant of these proteins with the 38 proteins discovered of the we tested the at that the seven control patients normally using the test of as in R. A of a p the of normal would not be the of on is our would that the of a of individuals could be as normally We significant in and of the control and renal protein To be in our were without or of when protein such that by the of the of our the p were were in assay validation for the candidate antibodies on 71 independent serum from patients with CKD and healthy and with of CKD. The validation CKD were from a of of CKD patients in at over the past to CKD in that of CKD. the CKD were from patients with or with with with 2 with 2 with antibody and 1 with type 1 antibody disease with an renal and enzyme assay to detect serum binding to PRKRIP1 and We previously T. O. N. T. in 2005; PubMed Scopus Google Scholar). In proteins to were from A to the to be to be on to the the were with protein in 30 and at 4 were using and the with the protein binding by in for 1 at the serum with in were in the for 1 at The were with and in The by using for at with a To control for binding, with proteins were as controls. The by the control used to test p were from the using J. is a novel protein that protein R.J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). To screen for putative auto-antibodies in patients with renal insufficiency, we used protein microarrays to serum antibodies from 17 patients with CKD and control individuals. We identified 38 proteins on of the or that demonstrated a significant in antibody binding levels as detected by antibody and to four of these angiotensinogen protein protein 1 and identified 38 proteins an in serum antibody binding in patients with ESRD as compared with We validation for and the were p protein binding type receptor and of of 1 protein protein associated with of protein 1 binding protein protein factor 3 protein 4 B in a new antibodies demonstrated a significant from a of in control to a of in renal test p = is a well and is to be expressed in the as well as and of the our candidate for PRKRIP1 is also to be well expressed in kidney and is by Smith J. G. K.A. Jr., renal injury and glomerular B in Nephrol. 2005; PubMed Scopus Google Scholar). can a role in glomerular injury in of M. S. T. J. S. I. J. N. Y. Y. E. K. 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We that were significantly higher in CKD than control in level, test p = autoantibodies were able to CKD from control with an the the of showing the of and antibodies in from patients with ESRD from control the are and We also autoantibodies were significantly higher in CKD than control in level, test p = and levels were the with chronic renal injury p of in the when and were using In the independent for from patients with significant in and antibodies by test p and p and or by p and significant in and antibodies on of when of renal injury were considered p and or when those 38 from four were considered and p and significant in and Ab on in the test p = and a patient on an test p and patients were on angiotensin converting enzyme significant in antibody antibody level significantly with or blood at the of that specific to and PRKRIP1 are not likely to be by renal or and to the of kidney injury in other candidates for and not significant in levels in CKD compared with test p = and the = and the of using protein microarrays to serum auto-antibodies at increased levels in renal from kidney injury. the serum antibody of patients with CKD healthy we and validated auto-antibodies in a disease not normally considered as an autoimmune and we antibodies significantly higher in the serum of patients with chronic renal injury. 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Cell Levels of for or and of Renal Scholar), to our auto-Ab have been in chronic renal injury. are to in our is on on a of in additional may especially for in our demonstrated a toward we the of Ab are of kidney we that these auto-Ab are because of increased of serum angiotensinogen or angiotensin from of can we the specific of these we the potential antigens may be it likely that PRKRIP1 and antigens are present in the organ undergoing damage. We these auto-Ab are angiotensinogen or angiotensin as in antibodies against these antigens has been II a novel in the of Biol. 2008; 8: PubMed Scopus Google Scholar). 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Early detection of organ injury a significant and for with resultant patient and We for on the We the of patients at