reperfusion injury - blood

THROMBOSIS AND HEMOSTASIS

Fibrinogen �–derived B�15-42 peptide protects against kidney ischemia/reperfusion injuryAparna Krishnamoorthy,1 Amrendra Kumar Ajay,1 Dana Hoffmann,1 Tae-Min Kim,2 Victoria Ramirez,1,3

Gabriela Campanholle,1 Norma A. Bobadilla,4 Sushrut S. Waikar,1 and Vishal S. Vaidya1,5

1Renal Division, Department of Medicine, Brigham and Women’s Hospital and 2Center for Biomedical Informatics, Harvard Medical School, Boston, MA;3Departamento de Nefrología y Metabolismo Mineral, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico; 4Instituto deInvestigaciones Biomedicas, Universidad Nacional Autonoma de Mexico and Instituto Nacional de Ciencias Medicas y Nutricion, Salvador Zubiran, Mexico City,Mexico; and 5Department of Environmental Health, Harvard School of Public Health, Boston, MA

Ischemia/reperfusion (I/R) injury in thekidney is a major cause of acute kidneyinjury (AKI) in humans and is associatedwith significantly high mortality. To iden-tify genes that modulate kidney injuryand repair, we conducted genome-wideexpression analysis in the rat kidneysafter I/R and found that the mRNA levelsof fibrinogen (Fg)�, Fg�, and Fg� chainssignificantly increase in the kidney andremain elevated throughout the regenera-tion process. Cellular characterization ofFg� and Fg� chain immunoreactive pro-

teins shows a predominant expression inrenal tubular cells and the localization ofimmunoreactive Fg� chain protein is pri-marily in the renal interstitium in healthyand regenerating kidney. We also showthat urinary excretion of Fg is massivelyincreased after kidney damage and iscapable of distinguishing human patientswith acute or chronic kidney injury(n � 25) from healthy volunteers (n � 25)with high sensitivity and specificity (areaunder the receiver operating characteris-tic of 0.98). Furthermore, we demonstrate

that Fg�-derived B�15-42 peptide adminis-tration protects mice from I/R-inducedkidney injury by aiding in epithelial cellproliferation and tissue repair. Given thatkidney regeneration is a major determi-nant of outcome for patients with kidneydamage, these results provide new oppor-tunities for the use of Fg in diagnosis,prevention, and therapeutic interventionsin kidney disease. (Blood. 2011;118(7):1934-1942)

Introduction

Kidney disease is a major public health concern receiving increasedglobal attention owing to the significantly increased prevalence and highmortality rates.1,2 Renal ischemia/reperfusion (I/R) accounts for asignificant number of acute kidney injury (AKI) cases in humans.Studies suggest that damage to the renal microvascular architecture anddeterioration of the angiogenic response constitutes the early steps in thecomplex multiple pathways involved in both early and chronic ischemicrenal injury.3 Restoration of blood flow to the site of injured tissue iscrucial for developing a successful repair response that involves thesurviving dedifferentiated cells spreading over the denuded basementmembrane, undergoing mitogenesis and ultimately redifferentiating tore-establish and restore functional integrity of the nephron.4,5 Althoughthese processes are well described at the pathologic level, very little isknown about the cellular and molecular mechanisms of action of bloodproteins within the kidney and their contribution to pathogenesis of renaldisease.

Fibrinogen (Fg), a 340-kDa dimeric blood protein, is made up of2 sets of 3 different polypeptide chains, Fg�, Fg�, and Fg�, that span alength of 50 kb on chromosome 4 in region q28.6 Although the primarysite for Fg synthesis is shown to be liver, extrahepatic synthesis of Fg byepithelial cells of intestine,7 cervix,8 and lung9,10 has been reported,suggesting that it may function in other structural and functionalcapacities. Furthermore, endogenous expression of Fg� and Fg� chainmRNA has been shown in the normal rat kidneys, levels of whichsignificantly increase at 2 hours after the onset of brain death injury,11,12

potentially restoring hemostasis by supporting extracellular matrix andwound repair processes after injury.12 In addition to its major role inblood clotting and circulation via interaction with platelets,13 Fg hasbeen recognized as an important regulator of inflammation,14 woundhealing,15 angiogenesis,16 and neoplasia17 via its action on a wide rangeof cellular receptors such as integrins, intracellular adhesion molecule-1,and vascular endothelial cadherin.18

In this study, we identify Fg�, Fg�, and Fg� chain mRNA to besignificantly up-regulated in the kidney after bilateral renal I/R injury.We provide evidence suggesting the ability of urinary Fg to serve as atranslational, noninvasive, and sensitive biomarker for early detection ofAKI. We also demonstrate a therapeutic potential of Fg-derived B�15-42

peptide that elicits � 50% protection from bilateral I/R injury byincreasing renal tissue repair and decreasing apoptosis. These resultsprovide novel insight into the role of Fg as a diagnostic biomarker andtherapeutic candidate in kidney disease and suggest that the presence ofFg in the kidney could serve as a protective mechanism against ischemicinjury to facilitate tubular epithelial cell proliferation and tissue repair.

Methods

Peptides and proteins

Endotoxin-free B�15-42 (GHRPLDKKREEAPSLRPAPPPISGGGYR) andrandom peptide (DRGAPAHRPPRGPISGRSTPEKEKLLPG) were custom

Submitted February 18, 2011; accepted June 6, 2011. Prepublished online asBlood First Edition paper, June 17, 2011; DOI 10.1182/blood-2011-02-338061.

The online version of the article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2011 by The American Society of Hematology

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synthesized (Invitrogen) with 95% modification and N-terminal aminegroup addition and free acid modification.

Animals

Male Wistar rats (280-320 g) and male C57Bl/6 mice (22-25 g) werepurchased from Harlan and Charles River Laboratories, respectively. Theanimals were maintained in central animal facility over wood chips free ofany known chemical contaminants under conditions of 21 � 1°C and50%-80% relative humidity at all times in an alternating 12-hour light-darkcycle. Animals were fed with commercial rodent chow (Teklad rodent diet7012), given water ad libitum, and were acclimated for 1 week before use.All animal maintenance and treatment protocols were in compliance withthe “Guide for Care and Use of Laboratory Animals” as adopted andpromulgated by the National Institutes of Health and were approved by theHarvard Medical School Animal Care and Use Committees.

Selection of participants

Critically ill patients in the intensive care unit with elevated serumcreatinine (SCr; � 1.5 mg/dL) were recruited. Causes of AKI or chronickidney disease (CKD) were obtained by detailed chart review including thetreating nephrologist’s consultation note and evaluation of laboratory databy a coauthor not involved in the patients’ care (S.S.W.). Healthy volunteerswere recruited from the staff at Brigham and Women’s Hospital. Healthyvolunteers were excluded if they reported a recent hospitalization, diagnosisof CKD, or treatment with nephrotoxic medications (nonsteroidal anti-inflammatory drugs were allowed). All participants were patients oremployees (healthy volunteers) of Brigham and Women’s Hospital, atertiary care teaching hospital. The Institutional Review Board approved theprotocols for recruitment and sample collection.

Human kidney biopsy sections

Human kidney biopsy sections were obtained through Brigham andWomen’s Hospital’s pathology service core, classified as patient withoutevidence of acute tubular injury (ATI): 58-year-old female diagnosed withrenal oncocytoma and patient with ATI: 78-year-old male diagnosed withactive glomerulonephritis with diffuse proliferative and crescentic patternof injury, active interstitial nephritis, active tubular injury involving focaltubular atrophy, and interstitial fibrosis (30%).

Experimental design

Rat studies. For whole genome expression profiling studies, 9 male Wistarrats underwent I/R surgery, and 3 rats underwent sham surgery simulatingI/R. To perform I/R surgery, the rats were anesthetized using pentobarbitalsodium (30 mg/kg ip), and renal ischemia was induced by nontraumaticvascular clamps over the pedicles for 20 minutes as described previ-ously.19,20 On release of the clamps, the incision was closed in 2 layers with2-0 sutures. The sham rats underwent anesthesia and a laparotomy only andwere killed after 24 hours. The rats in I/R group were further divided insubgroups of 3 rats each and killed after 6, 24, and 120 hours of reperfusion.To confirm the results of gene expression analysis, 20 male Wistar ratsunderwent 20 minutes bilateral I/R surgery and 5 rats underwent shamsurgery as described above and were killed at 6, 24, 72 and 120 hours afterreperfusion (n � 5/time point).

Mouse studies. Forty male C57Bl/6 wild-type mice were anesthetizedusing pentobarbital sodium (30 mg/kg ip) and subjected to 27 minutes ofbilateral renal I/R surgery by the retroperitoneal approach. Sham surgerywas performed with exposure of both kidneys but without induction ofischemia. Immediately on the start of reperfusion, 3.6 mg/kg B�15-42 orrandom peptide were administered intravenously to the mice via tail vein.One milliliter of warm saline (37°C) was injected intraperitoneally 3 hoursafter surgery for volume supplementation. Mice (n � 5-10/group) in therespective groups (sham or I/R-administered B�15-42 or random peptide)were killed at 24 and 48 hours after reperfusion using overdose ofpentobarbital (180 mg/kg ip).

Blood and urine analysis

At sacrifice, blood was collected from dorsal aorta in heparinized tubes. SCrconcentrations were measured using a Creatinine Analyzer II (BeckmanCoulter). Blood urea nitrogen (BUN) was measured spectrophotometricallyat 340 nm using a commercially available kit (Thermo Fisher Scientific) asdescribed previously.21

Urines were collected by placing animals in individual metabolic cages,and 1 mL of RNAlater (for rats and 40 �l for mice; Ambion) was added to thetubes to preserve RNA. Urinary kidney injury molecule-1 (Kim-1 in rats andKIM-1 in humans) was measured using previously established Luminex-basedassays.20,22 Urinary Kim-1 in mice was measured using a Luminex-based assayin Dr J. V. Bonventre’s laboratory (Brigham and Woman’s Hospital, HarvardMedical School). Urinary N-acetyl-�-D-glucosaminidase (NAG; Roche Diagnos-tics) was measured spectrophotometrically according to the manufacturer’sprotocols. Urinary creatinine concentration was used to normalize biomarkermeasurements to account for the influence of urinary dilution on biomarkerconcentrations. Fg in mouse, rat, and human urines was measured usingcommercially available species-specific Luminex-based assay kit from Millipore.

Whole genome expression profiling and hierarchical clustering

For genome-wide expression analysis, RatRef-12 bead array (Illumina) wasused that contains � 22 523 50-mer oligonucleotide probes primarily basedon National Center for Biotechnology Information RefSeq Release16 database. Gene expression and hybridization array data set has beensubmitted to the National Center for Biotechnology Information Geneexpression omnibus (accession:GSE27274; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token�drkjxksyckeqcrq&acc�GSE27274). Total RNAwas extracted from 30 mg of frozen tissue samples using TRIzol reagent(Invitrogen) according to the manufacturer’s instructions. Integrity of theisolated total RNA was determined by 1% agarose gel electrophoresis, andthe RNA concentration was measured by ultraviolet light absorbance at260 nm using a Nanodrop 2000C spectrophotometer (Thermo FisherScientific). Aliquots of RNA were converted into double-stranded cRNAand biotinylated using the Illumina TotalPrep RNA amplification kit(Ambion). The cRNA samples were then labeled with streptavidin-cyanin3 (Cy3) and hybridized onto RatRef-12 Expression BeadChip (Illumina).The image was scanned using a BeadArray reader (Illumina), and the datawere analyzed by BeadStudio software (Version 3.3.7; Illumina). Fornonredundant 22 523 symbols, the intensity profiles were quantile normal-ized. We used median absolute deviation (MAD) to select highly variablegenes (MAD � 0.4; n � 1571) for subsequent analysis. Hierarchicalclustering was performed using 1 Pearson correlation coefficient asdistance with average linkage option.

Real-time RT-PCR

The isolated RNA was treated with QuantiTect Reverse Transcription kit(QIAGEN Sciences). Real-time PCR of the tissue samples was performedwith QuantiFast SYBR Green (QIAGEN Sciences) using CFX96 RT-PCRinstrument (Bio-Rad Laboratories).21 Primers were designed to amplify a120- to 150-base pair fragment with the following cycle conditions: 95°Cfor 3 minutes and then the following steps were repeated 40 times: 95°C for30 seconds, 55°C for 30 seconds. Forward and reverse primer sequences forrat- and mouse-specific genes were designed using MacVector software(MacVector) and are listed in Table 1.

Immunofluorescence staining

Kidney tissues were perfused with cold PBS before harvesting and thenfixed in formalin for 16 hours and embedded in paraffin. The sectionsincubated overnight at 4°C in rabbit monoclonal anti-Fg� (Epitomics),rabbit polyclonal anti-Fg� (ProteinTech Group), rabbit polyclonal anti-Fg�(ProteinTech Group), anti–rat Fg (Nordic Immunologic Laboratories),anti–human fibrinogen (Sigma-Aldrich), and rabbit monoclonal anti-Ki67(Vector Laboratories). The primary antibody was detected using goatanti–rabbit Cy3–labeled (red) and donkey anti–goat Cy3–labeled secondaryantibodies (Jackson ImmunoResearch Laboratories). 4,6-Diamidino-2-phenylindole (Sigma-Aldrich) was used for nuclear staining (blue). The

FIBRINOGEN IN KIDNEY DAMAGE 1935BLOOD, 18 AUGUST 2011 � VOLUME 118, NUMBER 7




tissue sections were mounted using ProLong Gold antifade reagent(Invitrogen). The images were captured by Nikon DS-QiMc cameraattached to Nikon eclipse 90i flourescence microscope using oil immersionobjectives (100 magnification for rats and 60 magnification for miceand humans) by Nikon NIS elements AR Version 3.2 software.

TUNEL assay

Apoptosis was measured in kidney tissues by TUNEL assay using the InSitu Cell Death detection kit (Roche Applied Science) according to themanufacturer’s instructions.21

In vitro experimental design

The renal tubular epithelial cell line LLC-PK1 established from pig renalcortex was obtained from ATCC and maintained in DMEM containing 10%FBS. Two thousand and five hundred LLC-PK1 cells were plated in a96-well plate for 24 hours in DMEM and 10% FBS at which time theyformed a 50% confluent monolayer in the well. They were pretreated with6�M B�15-42 or random peptide for 6 hours and were immersed in 100 �Lof mineral oil on top of DMEM without any serum for 6 hours. This oilimmersion simulates in vivo ischemic conditions by restricting cellularexposure to oxygen and nutrients as well as limiting metabolite washout.23

After 6 hours, the mineral oil was removed, and cells were incubated with6�M B�15-42 or random peptide for 48 hours in serum-free conditions.5-Bromo-2�-deoxyuridine (BrdU) was measured as an index of cellproliferation by incubating cells with BrdU for 2 hours before harvesting,and the absorbance was quantified using a spectrophotometer (Millipore) at450 nm as per the manufacturer’s instructions. Absorbance obtained fromuntreated cells was taken as 100% (n � 6 wells/group), and the experimentwas repeated twice.

Statistics

Data are expressed as average � standard error. Statistical difference(P .05) as calculated by 1-way ANOVA or Student t test. P .05 isconsidered significant and is represented by a single asterisk (*) whereapplicable. All graphs were generated by Prism software (GraphPadSoftware). Diagnostic performance (ie, the ability of a urinary biomarker toidentify kidney injury) was assessed by evaluating sensitivity and specific-ity using the receiver operating characteristic (ROC) curve. The area underthe ROC curve and 95% confidence interval were calculated using thenonparametric method.22 The area under the ROC curve for a diagnostic testranges from 0.5 (no better than chance alone) to 1.0 (perfect test, equivalentto the standard). Statistical analyses were performed with MedCalc forWindows (Version 11.5; Mariakerke).

Results

mRNA expression of �, �, and � chains of Fg increases in thekidney after I/R injury

In the quest to identify early genes modulating kidney injury andrepair process, we conducted gene expression profiling in thecortex and medulla of rat kidney after 20 minutes of bilateral renalI/R. This reversible model of kidney injury results in elevatedkidney dysfunction (measured by SCr and BUN; supplementalFigure 1A, available on the Blood Web site; see the SupplementalMaterials link at the top of the online article) and proximal tubularinjury (measured by Kim-1 mRNA levels and histopathologicfindings characterized by proximal tubular necrosis and apoptosis;supplemental Figure 1B-C) at 24 hours of reperfusion followed byrecovery at 120 hours. We selected highly variable genes(MAD � 0.4; n � 1571) and performed hierarchical clustering toinvestigate their coexpression pattern during kidney regenerationafter ischemic injury (Figure 1A). The selected genes include thepreviously identified candidate genes lipocalin-2 (LCN2),24 clus-terin (CLU),25 tissue inhibitor of metalloproteinase-1 (TIMP1),26

Figure 1. Fg (Fg�, Fg�, and Fg� chains) is significantly up-regulated in kidneycortex and medulla of male Wistar rats after 20 minutes of bilateral renal I/Rinjury compared with sham surgery. (A) A heatmap shows the expression levels ofthe most variable 1571 genes that were selected from 22 523 transcripts (see “Wholegenome expression profiling and hierarchical clustering”). Coexpressed genes aregrouped by hierarchical clustering. The samples are presented in order of time afterischemic injury (0, 6, 24, and 120 hours; triplicate for each). (B) Real-time PCRanalysis in kidney. (C) Liver tissues for Fg�, Fg�, and Fg� chains, normalized using ahousekeeping gene (Gapdh) and fold change determined over sham group (n � 5/group). *P .05 as determined by 1-way ANOVA in comparison with sham rats.

Table 1. Real-time PCR primers used for the quantification of mRNAexpression levels in the study

Gene F/R Sequence

CD68 F TCTTTCTCCAGCTGTTCACC

R ATGATGAGAGGCAGCAAGAG

Gapdh F TCCGCCCCTTCTGCCGATG

R CACGGAAGGCCATGCCAGTGA

ICAM-1 F TGTTTTGCTCCCTGGAAGGC

R AGTCACTGCTGTTTGTGCTCTCC

IL-1� F ACCTGTCCTGTGTAATGAAAGACG

R TGGGTATTGCTTGGGATCC

IL-6 F CAAGAGACTTCCATCCAGTTGCC

R CATTTCCACGATTTCCCAGAGAAC

IL-10 F CAGCCTTGCAGAAAAGAGAG

R GGAAGTGGGTGCAGTTATTG

Mouse Fg� chain F TGTGGAGAGACATCAGAGTCAATG

R CGTCAATCAACCCTTTCATCC

Mouse Fg� chain F CTATGGCTGCTGCTGCTATTG

R GGCTCTTCCTTTCTCCTGTCAAC

Mouse Fg� chain F TGTGGCTACCAGAGATAACTGTTG

R ATGTCTTCCAGCGTTCGGAG

Mouse Kim-1 F GGAGATACCTGGAGTAATCACACTG

R TAGCCACGGTGCTCACAAGG

Rat Fg� chain F AGCAGCCCAGCGACAAGAAAAG

R GCAGTCAGAACCATCATCCGAAG

Rat Fg� chain F TAGCCTAAGACCTGCCCCTC

R ACCGTGAACACAGCCTCCTG

Rat Fg� chain F GGACAATGACAACGACAAGTTCG

R ATACCAGCGGGTTTTCCAGG

Rat Kim-1 F TATTTGGGGGAACAGGTTGC

R CAAGTCACTCTGGTTAGCCGTG

TNF� F AGAAGAGGCACTCCCCCAAAAG

R TTCAGTAGACAGAAGAGCGTGGTG

F indicates forward; and R, reverse.

1936 KRISHNAMOORTHY et al BLOOD, 18 AUGUST 2011 � VOLUME 118, NUMBER 7




and kidney injury molecule-1 (Kim-1).20 Although the up-regulation of LCN2 and CLU were more dominant in renal medullacompared with cortex, TIMP1 and Kim-1 were up-regulated both incortex and medulla at 24 hours after ischemic injury. We observed alocal cluster of genes that includes Fg� and Fg� chains (Figure 1Aarrow) whose expression pattern is similar to Kim-1, ie, clearup-regulation after 24 hours of ischemic injury both in cortex andmedulla. The probe for Fg� chain is absent in the RatRef-12 chipfrom Illumina. Therefore, we further evaluated the expressionprofile of Fg�, Fg�, and Fg� chains by real-time PCR in kidney(cortex and medulla; Figure 1B); liver (Figure 1C); and lung,spleen, and heart (supplemental Figure 2) over time, and we foundthat there was a modest elevation (3-fold) of Fg� and Fg� chains inthe liver at early time points (Figure 1C), whereas there was nosignificant change in the mRNA levels of any of the chains in lung,spleen, and heart (supplemental Figure 2). The medulla showedsignificantly higher expression of all 3 chains compared withcortex with the highest up-regulation after 72 hours of reperfusion(Fg� chain, 14-fold; Fg� chain, 50-fold; and Fg� chain, 10-fold;Figure 1B).

Immunoreactivity of Fg�, Fg�, and Fg� chains in the kidney

Immunostaining, to evaluate the cellular expression profile of Fgwhole protein and Fg�, Fg�, and Fg� chains, revealed immunore-active protein for all 3 chains and Fg whole molecule in renaltubular cells. Positive staining was observed using the anti-Fg�chain and anti-Fg whole molecule antibodies in the interstitialspaces, indicative of extracellular Fg. In sham kidneys (Figure2Ai), the Fg� chain is expressed with fine granular cytoplasmicreactivity and more pronounced expression at the peak of injury by24 hours (Figure 2Aii). Fg� chain immunoreactive protein, asdetected by the monoclonal antibody, continued to be expressed inthe proximal tubular epithelial cells and in the glomeruli through-

out the time course of injury (Figure 2Aiii). Fg� chain immunore-active molecules in the uninjured kidneys, assessed by a polyclonalantibody against Fg� chain, showed focal reactivity in the renalinterstitium (Figure 2Aiv). At the peak of injury by 24 hours, Fg�immunoreactivity distinctly outlined the peritubular capillaries(Figure 2Av), and a small proportion of tubular epithelial cellsexpressed the Fg� chain immunoreactive component in theircytoplasm. By 72 hours, intense, irregular and coarse distal tubularstaining (Figures 2Avi) and a distinct luminal outline alongproximal tubules featured. The Fg� chain staining, assessed by apolyclonal antibody against Fg� chain, was primarily located in thedistal tubules and collecting ducts with a diffuse cytoplasmicdistribution that gravitated along the basolateral side in uninjuredkidneys (Figure 2Avii). At the peak of injury by 24 hours, the Fg�chain immunoreactive protein stained in a coarse granular pattern,distributed centrally in the cytoplasm in the distal tubules andcollecting ducts (Figure 2Aviii). By 24 hours, the Fg� chain in thecortex was confined toward the apical side of the proximal tubules,whereas in the medulla, the cellular debris of injured S3 segmentsnonspecifically stained for the Fg� chain as well. By 72 hours, Fg�chain immunoreactive proteins showed a mixed pattern thatresembled sham and 24-hour injured kidneys in the staining anddistribution patterns (Figure 2Aix). The expression of Fg wholemolecule was identified in a linear pattern along the apical surfaceof epithelial cells (Figures 2Ax-xii) as well as along the glomerularbasement.

A consistent pattern of tubulo-interstitial expression of Fg andits chains was observed in human kidney biopsy sections obtainedfrom patients pathologically diagnosed with ATI compared withpatient without evidence of ATI (Figure 2Bi-viii). All 3 chainsalong with Fg were present in the interstitium in both ATI andnon-ATI patients in addition to which Fg� chain (Figure 2Bvi) andFg (Figure 2Bviii) was predominantly expressed on the apical sideof the tubules in the ATI patient.

Increased urinary levels of Fg in rats and humans serve as apotential biomarker for AKI

We hypothesized that if Fg was secreted into the urine on injury,then urinary Fg may serve as a biomarker for kidney injury. After20 minutes of bilateral renal I/R injury in rats, we observed� 100-fold increase in urinary Fg concentration as early as 6 hours(Figure 3Aa) that remained higher than baseline until day5 (� 4-fold) after reperfusion, correlating with proximal tubularnecrosis as assessed by histopathologic injury (supplementalFigure 1C) and elevated urinary NAG (Figure 3Aii) as well asurinary Kim-1 (Figure 3Aiii). There was no increase in plasma Fg(supplemental Figure 3) levels after sham or kidney I/R injurycompared with rats that did not undergo sham or I/R surgery. Toevaluate the performance of urinary Fg in distinguishing healthyvolunteers against patients with CKD and/or AKI, urinary Fg wasmeasured in 25 patients admitted to the intensive care unit withabnormal serum creatinine (� 1.5 mg/dL) with established kidneydamage from a variety of causes and 25 healthy volunteers. We alsocompared urinary Fg against 2 other well-studied AKI or CKDbiomarkers, NAG and KIM-1. Demographic and clinical data areshown in Table 2. Median urinary concentration of Fg wassignificantly higher in patients with AKI and CKD than in healthyvolunteers (P .001; Figure 3B) and corresponded with theincreased levels of urinary NAG and KIM-1 (supplemental Figure4). The diagnostic ability of urinary Fg to distinguish betweenpatients with AKI or CKD versus patients without kidney injurywas 0.98 as calculated by area under the ROC curve (Figure 3C).

Figure 2. Fg (immunoreactive Fg�, Fg�, and Fg� chains) protein is expressedin the kidney of rats and humans. (A) Representative formalin-fixed paraffin-embedded kidney tissue sections of sham male Wistar rats and those from rats thathad undergone 20 minutes of bilateral ischemia reperfusion stained for immunoreac-tive proteins to Fg� (i-iii), Fg� (iv-vi), Fg� (vii-ix), and Fg (x-xii) after 24 and 72 hoursof reperfusion and compared with healthy (sham) kidneys. Bar represents 50�m. (B)Representative formalin-fixed human-biopsied kidney sections of AKI and non-AKIpatients, stained for immunoreactive molecules recognizing Fg� (i-ii), Fg� (iii-iv), Fg�(v-vi), and Fg (vii-viii). Bar represents 50 �m. Arrowheads indicate respectiveimmunoreactive molecules. Asterisks in panels Bvii and Bix mark tubules with similarexpression pattern along the basolateral side of respective tubules.





Fibrinogen B�15-42 protects the kidney against I/R injury

Given that in our model Fg� chain was the highest up regulatedgene after kidney injury among the 3 chains (Figure 1B) and thefact that exogenous B�15-42 peptide administration has been shownto protect against myocardial I/R injury27-29 and lung injury,30 wenext evaluated the therapeutic potential of B�15-42 peptide in renalI/R injury. B�15-42 or random peptide (3.6 mg/kg) was administeredintravenously 1 minute after reperfusion after 27 minutes of bilat-eral renal I/R injury in C57Bl/6 mice (n � 5-10/group). A signifi-cant reduction in the vascular congestion (outlined by white dots inFigure 4A) was observed. Approximately 50% reduction in kidneydysfunction (measured by SCr and BUN) and kidney proximaltubular injury (measured by urinary levels of Kim-1 and Fg; Figure4B) and a significant decrease in proximal tubular damage in theouter stripe of outer medulla (histopathological evaluation of H&Estained kidney sections; Figure 4C) were recorded at 24 hours afterI/R injury in the mice administered B�15-42 peptide compared withrandom peptide. The kidney injury and dysfunction parametersdecreased by 48 hours, suggesting the onset of a complete struc-tural and functional recovery in both groups.

Decreased apoptosis and increased tissue repair in the kidneysof B�15-42–treated mice compared with mice treated withrandom peptide after I/R injury

To elucidate the mechanism of B�15-42–induced protection in I/Rmice, we measured candidate markers of inflammation, leukocyteinfiltration, apoptosis, and proliferation in kidney tissues over time.There was no difference in mRNA levels of inflammatory cyto-kines (IL-1�, IL-6, IL-10, TNF�, and intracellular adhesionmolecule-1 [ICAM-1]) or macrophage marker (CD68) between theB�15-42 and random peptide treatment groups (supplemental Figure5). Similarly, leukocyte infiltration (measured by myeloperoxidasestaining) also seemed to be similar between the 2 groups (supple-mental Figure 6). The number of TUNEL-positive apoptotic cellsin the renal medulla was similar at 24 hours (Figure 5A,C).However, at 48 hours there was a significant decrease in apoptosis(P .05) in the mice administered B�15-42 compared with randompeptide (Figure 5B,D). Interestingly, a significant number of cellsseemed to be in a proliferative state (Ki67 positive) in the renalmedulla at 48 hours after administration of the B�15-42 peptidecompared with random peptide administration (Figure 5G). In vitroexperiments using proximal tubular epithelial cells (LLC-PK1)mimicked the in vivo findings in demonstrating a protective effectof B�15-42 from hypoxic injury by stimulating renal epithelial cellproliferation (supplemental Figure 7), suggesting that the B�15-42

peptide promotes an efficient resolution of ischemic injury byinducing rapid tissue regeneration response, thereby decreasing thenecrosis and apoptosis in the kidney.

Discussion

Although it has been recognized that progressive kidney disease ischaracterized by gradual deterioration of the renal endothelium,which correlates with the development of tubulo-interstitial injury,fibrosis, and glomerulosclerosis,3 there has been little effort tocharacterize the regulatory role of blood proteins in pathophysiol-ogy of AKI. Here, we show that (1) in whole genome expressionprofiling studies Fg� and Fg� chains are among the highlyup-regulated genes after 24 hours of ischemic injury both in kidneycortex and medulla; (2) Fg could serve as an effective safety andefficacy biomarker for kidney injury not only because of themarked increase in urinary Fg after kidney damage (Figure 3) butalso because of their reduced levels on B�15-42 peptide mediatedprotection from kidney injury (Figure 5), demonstrating responsive-ness to both injury and recovery; and (3) Fg-derived B�15-42

administration protects mice from I/R-induced kidney injury byaiding kidney tissue repair, thereby demonstrating for the first timeits therapeutic potential in AKI. These findings not only highlightthe important role of Fg in renal tissue injury and repair but alsooffer a therapeutic strategy to enhance kidney regeneration asopposed to simply preventing further injury or deleterious inflam-mation in the damaged tissue.

The presence of Fg�, Fg�, and Fg� chain transcripts in thekidney at baseline11 as well as its up-regulation after nephrotoxic-ity31 or brain death–induced vascular endothelial activation inkidneys12,32 has been observed previously. Here, we characterizedthe cellular expression patterns of Fg and its individual chains inthe renal tubule after injury. Because we used polyclonal antibodiesfor immunostaining of the Fg� and Fg� chain, respectively, there isno way to distinguish between the intact fibrinogen versus interme-diates such as �/�, �/�, or the half-molecule of one each �/�/�

Figure 3. A significant increase in urinary Fg after kidney injury in rats andhumans. (A) Urinary Fg was compared with tubular injury biomarkers NAG andKim-1 in rats subjected to 20 minutes of bilateral renal I/R injury (n � 5/group).*P .05 as determined by Student t test in comparison with sham rats. (B) UrinaryFg was measured in a human cross-sectional study with clinically establishedmultifactorial AKI and/or CKD (n � 25) versus healthy volunteers (n � 25). Magentaline and corresponding number marked by arrow indicate a threshold cutoff value at95% specificity. (C) ROC comparing the sensitivity and specificity of urinary Fg, NAG,and KIM-1 to distinguish patients with AKI and/or CKD from healthy volunteers.

Table 2. Demographic and clinical characteristics of humansubjects

AKI or CKD(n � 25)

Healthyvolunteers*

(n � 25)

Mean age,† years � SD 64.8 � 19.5 35.6 � 10.7

Female,‡ % 64 68

Black,§ % 20 16

Cause of elevated sCr AKI from shock or sepsis (72%),

obstruction (4%), multifactorial

(12%), prerenal (4%), CKD (8%)

Mean (SD) peak sCr 4.5 (4.7) mg/dL

*Healthy volunteers were excluded if they reported a diagnosis of CKD; sCR wasnot measured.

†P .001.‡P � .73.§P � .68.





polypeptide chains. However, we found distinct expression pat-terns of Fg�, Fg�, and Fg� chains in the renal tubular epithelialcells, glomeruli, and interstitium at baseline and during theregeneration in the injured kidney. The increased Fg expressionafter injury can potentially be a consequence of plasma leakagebecause of organ damage, as seen after spinal cord injury,33 butthe observation of detectable transcript levels of Fg�, Fg�, andFg� chains (Figure 1) and corresponding immunoreactivity ofall 3 chains as well as whole Fg molecule in sham rats and inpatient without any evidence of tubular injury (Figure 2)suggests that the protein could be potentially synthesized andassembled in the kidney.

Plasma Fg has been associated with vascular disease innumerous epidemiologic studies.34 Although we did not find anyincrease in plasma Fg levels after I/R injury (supplemental Figure3), urinary Fg levels increased massively as early as 6 hours afterI/R injury that decreased over time but remained modestly elevatedduring the resolution phase of injury (Figure 3). In 1974, Naish etal35 reported higher levels of urinary fibrin degradation products(FDPs) in patients with glomerulonephritis. Subsequently, urinaryFDPs were shown to be able to make a diagnosis of 25 of 26 acuterejection episodes at least 24 hours before deterioration in renalfunction and the elevation of FDPs preceded the rise in NAG in 9 of11 patients.36 Consistent with these published reports, we show in across-sectional study of individuals with and without kidneydamage that urinary Fg performed very well in differentiatingbetween patients with and without AKI/CKD, with an area underthe ROC of 0.98. The urine samples were obtained well after thediagnosis of kidney injury was made; therefore, this study does notaddress the issue of early diagnosis before a rise in SCr. However, it

does suggest that the sensitivity and specificity of urinary Fg wascomparable with the other more advanced biomarkers of AKIsuch as NAG and KIM-1,19,20,22,37 and further warrants aninvestigation to evaluate the comparative efficacy and temporalpattern of urinary Fg excretion with other AKI/CKD biomarkersin longitudinal studies. Urinary fragments of Fg� (ROC of 0.85)and Fg� chains (ROC of 0.74) were found to distinguish patientswith malignant versus benign ovarian cancer38 and plasmalevels of Fg�� chain (a splice variant of Fg� chain) associatedwith several traditional cardiovascular risk factors in Framing-ham Offspring cohort.39 The current assay is a sandwichELISA-based Luminex assay using 2 polyclonal antibodiesagainst Fg protein; therefore, it will be interesting to use a moretargeted approach such as liquid chromatography-multiple reac-tion monitoring/mass spectrometry to identify whether there is apredominant excretion of Fg� chain polypeptides in the urineafter kidney injury that would correlate with the highest Fg�chain mRNA levels in the kidney.

We tested the therapeutic efficacy of Fg� chain–derived peptide(B�15-42) in mice subjected to bilateral renal I/R injury and foundthat B�15-42 substantially reduces acute tubular injury. Others haveinvestigated the role of Fg during kidney damage using theMalayan pit viper venom ancrod and found that it providedremarkable protection in experimental allergic glomerulonephritisin rabbits.40 This provides an indirect evidence for protectiverole of B�15-42 because ancrod reduces plasma levels of Fg andsimultaneously increases FDPs such as the bioactive polypep-tide (B�15-42).41 B�15-42 is a naturally occurring, 28-amino acidproduct cleaved from fibrin fragments, and at suprapharmaco-logic doses it has shown to protect from myocardial infarction28

Figure 4. B�15-42 peptide protects mice from renal I/R injury. Male C57Bl/6 mice were subjected to 27 minutes of bilateral renal I/R injury or sham surgery and 3.6 mg/kgB�15-42 peptide or random peptide was administered intravenously 1 minute after reperfusion. (A) The medullary congestion after ischemia was significantly less in the B�15-42

peptide–administered mice compared with mice administered random peptide. Outline of vascular congestion traced by white dots. (B) SCr and BUN as indicators of renaldysfunction and urinary levels of Fg and Kim-1 as indicators of kidney injury were measured at 24 and 48 hours in all the groups (n � 5/group of sham, n � 10/group at24 hours, and n � 5/ group at 48 hours). *P .05 as determined by 1-way ANOVA in comparison with sham mice. (C) Representative H&E stained kidney sections comparingsham, B�15-42 and random peptide–administered groups of mice at 24 and 48 hours after ischemia. Bar represents 100 �m (20 magnification).





and acute lung injury30,42 in animal models. In a multicenteredphase IIa clinical trial with B�15-42 peptide administration,successful protective effects were seen in patients with acutemyocardial infarction, whose endothelial barrier integrity hadnot been compromised.27,29 The peptide also has been shown tobe vasculoprotective in models of vascular leak in a Fyn-dependent pathway.42 B�15-42 has been shown to mediate plateletspreading, proliferation, capillary tube formation, and vonWillebrand factor release, and it has a binding site for hepa-rin.42,43 B�15-42 also has low-affinity interactions with vascularendothelial-cadherin, efficiently disrupting the interaction be-tween Fg with its receptors vascular endothelial-cadherin onendothelial cells, thereby stabilizing endothelial barriers that inturn elicits beneficial anti-inflammatory properties.28,42

Here, we show a unique mechanism of B�15-42 peptide–mediated protection in vivo and in vitro that results in increasedproliferation of renal tubular epithelial cells resulting in de-creased necrosis and apoptosis after damage (Figure 5). The factthat among the 3 chains of Fg, Fg� chain transcript levelsincrease the highest (� 50-fold) at 72 hours (Figure 1B), thepeak of regeneration in this model,44 further underscores the

finding that Fg is up-regulated in the kidney as a protectivemechanism to aid in regeneration. This result would be consis-tent with the studies conducted thus far, suggesting that Fgseems to be most important in tissue repair when cells mustinfiltrate and organize fluid-filled areas such as internal deadspace created by injury.15 In a cutaneous wound healing model,Fg deficient (Fg/) mice revealed an abnormal pattern of tissuerepair, including misguided epithelium, delayed wound closure,reduced tensile strength, and diminished ability to organize adead space.15 Fg has been shown to have multiple bindingpartners, such as �3-integrin45 and ICAM-1,46 regulating thetissue repair process in neurons and lung epithelial cells.47

Given that both �3-integrin and ICAM-1 expression on renalepithelial cells increases after kidney injury,48-50 it remains to beinvestigated whether Fg or the B�15-42 peptide aids in renalepithelial proliferation and extracellular remodeling via one ofthe known ligands and pathways. Kidney regeneration, after anepisode of AKI, is a major determinant of outcome for patientswith AKI and therefore the use of B�15-42 peptide offers a noveltherapy to improve the rate or effectiveness of the tissue repairprocess after ischemic kidney damage.

Figure 5. B�15-42 peptide aids in the resolution of injury by decreasing necrosis/apoptosis and inducing rapid tissue regeneration. Paraffin-embedded kidneys of micesubjected to 27 minutes of bilateral I/R that were administered either B�15-42 or random peptides were compared at 24 and 48 hours for number of apoptotic cells (green; A) byTUNEL assay (bar represents 100 �m, 40 magnification). Arrowheads indicate TUNEL-positive nuclei. (B) Number of proliferative cells determined by Ki67-positive stainingcells (red; bar represents 50 �m, 60 magnification). The numbers of positive staining TUNEL and Ki67 nuclei are represented graphically on the right of respectivephotomicrographs. Arrowheads indicate Ki67 positively stained nuclei. *P .05 as determined by Student t test between the 2 groups within the same time point.





In summary, our study provides evidence that Fg may functionas a key molecular link between tubulo-vascular damage andregeneration in the kidney and provides new opportunities for theuse of Fg in diagnosis, prevention, and therapeutic interventions inkidney disease.

Acknowledgments

The authors thank Dr Vanesa Bijol, MD, a pathologist in therenal division of the Brigham and Women’s Hospital, for helpwith unbiased inferences and interpretations of immunostainedsections of kidney.

V.S.V.’s laboratory is supported by the National Institute ofEnvironmental Health Sciences Pathway to Independence awardprogram (ES16723).

Authorship

Contribution: A.K., A.K.A., and V.S.V. designed research; A.K., A.K.A,D.H., T.-M.K., V.R., and G.C. performed research; N.A.B. and S.S.W.contributed biospecimens from rodents and humans; T.-M.K. providedbioinformatics support and data analysis; A.K., A.K.A., D.H., S.S.W.,and V.S.V. analyzed data; and A.K. and V.S.V. wrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Vishal S. Vaidya, Laboratory of Kidney Toxi-cology and Regeneration, Renal Division, Department of Medi-cine, Brigham and Women’s Hospital, Harvard Medical School,Department of Environmental Health, Harvard School of PublicHealth, Harvard Institutes of Medicine, Rm 562, 77 Avenue LouisPasteur, Boston, MA 02115; e-mail: [email protected].

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online June 17, 2011 originally publisheddoi:10.1182/blood-2011-02-338061

2011 118: 1934-1942

Gabriela Campanholle, Norma A. Bobadilla, Sushrut S. Waikar and Vishal S. VaidyaAparna Krishnamoorthy, Amrendra Kumar Ajay, Dana Hoffmann, Tae-Min Kim, Victoria Ramirez, reperfusion injury

peptide protects against kidney ischemia/15-42βderived B−βFibrinogen

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