Research ArticleElevated Plasma 120572-Defensins (HNP1ndash3) Levels Correlated withIgA1 Glycosylation and Susceptibility to IgA Nephropathy

Yuan-yuan Qi1234 Xu-jie Zhou1234 Fa-juan Cheng5 and Hong Zhang1234

1Renal Division Peking University First Hospital Beijing 100034 China2Peking University Institute of Nephrology Beijing 100034 China3Key Laboratory of Renal Disease Ministry of Health of China Beijing 100034 China4Key Laboratory of Chronic Kidney Disease Prevention and Treatment Peking University Ministry of EducationBeijing 100034 China5Shandong Provincial Hospital Shandong University Jinan Shandong 250021 China

Correspondence should be addressed to Hong Zhang hongzhbjmueducn

Received 2 March 2016 Revised 11 June 2016 Accepted 22 June 2016

Academic Editor Hitoshi Suzuki

Copyright copy 2016 Yuan-yuan Qi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Aim IgA nephropathy (IgAN) is the most common form of glomerulonephritis Recent genome-wide association study (GWAS)suggested that DEFA locus (which encodes 120572-defensins) may play a key role in IgAN Methods The levels of 120572-defensins in 169IgAN patients and 83 healthy controls were tested by ELISA Results We observed that 120572-defensins human neutrophil peptides 1ndash3(HNP1ndash3) in IgAN patients were elevated compared with healthy controls The mean levels of 120572-defensins of 83 healthy controlsand 169 IgAN patients were 50 ngmL and 7842 ngmL When the results were adjusted to the mean levels of 120572-defensins of IgANpatients the percentage of individuals with high levels of 120572-defensins increased in IgAN patients (225) compared to healthycontrols (96) (119901 = 0013) The elevation of 120572-defensins in IgAN patients was independent of renal function or neutrophil countwhich were major sources of 120572-defensins in circulation More importantly negative correlation was observed between galactose-deficient IgA1and 120572-defensins Conclusion As 120572-defensin is a lectin-like peptide we speculated that it might be involved in IgAgalactose deficiencyThe data implied that patients with IgANhad higher plasma120572-defensins levels and high 120572-defensins correlatedwith IgA galactose deficiency further suggesting a pathogenic role of 120572-defensins in IgAN

1 Introduction

IgAN is the most common cause of end-stage renal dis-ease (ESRD) among Asian populations [1] The diagnostichallmark of IgAN is the predominance of deposition withimmune complex containing IgA in mesangial regions butthe pathogenetic factors remain obscure Multiple studieshave established that aberrantly glycosylated IgA1 plays animportant role in the pathogenesis of IgAN [2ndash6] In patientswith IgAN some circulating IgA1 are deficient of O-glycansFurthermore mesangial immune deposits eluted directlyfrom glomeruli of IgAN patients contained Gd-IgA1 [7 8]Our previous study discovered that the elevation of serumgalactose-deficient IgA1 is associated with a poor prognosisin IgAN [9] On the basis of the potential pathogenic roleof Gd-IgA1 in the development of IgAN the mechanism

of the glomerular mesangium deposition of IgA1 immunecomplexes remained to be an import issue Recent advancesimplicated that genetic factor especially in promoting theoverproduction of an aberrant form of IgA1 plays a keyrole in IgAN More recently several genome-wide associ-ation studies and candidate gene studies confirmed thatsingle nucleotide polymorphisms (SNPs) inDEFA locus werestrongly associated with IgAN susceptibility further support-ing the likely key role of mucosal immunity in IgAN [10ndash14]

HNP1ndash3 encoded by DEFA1 and DEFA3 are part of the120572-defensins family of peptides which in humans include sixknown members and are almost identical in sequences Theyare normally synthesized in neutrophil precursor cells andreleased by mature circulating neutrophils at inflammatorysites [15] 120572-defensins are generally considered to be directeffectors of innate antimicrobial immunity to kill andor

Hindawi Publishing CorporationDisease MarkersVolume 2016 Article ID 8123138 7 pageshttpdxdoiorg10115520168123138

2 Disease Markers

inactivate a particular spectrum of bacteria fungi or someenveloped viruses in vitro [15] In addition120572-defensinsmightalso be a component of the effector of adaptive immunitymediating B- and T-cell interactions linking innate andadaptive immunity [16]

In certain disease states plasma or local 120572-defensinswould depart from normal levels 120572-defensins levels wereelevated in plasma of SLE patients and could serve as amarker for disease activity [17 18] In patients with diabeticnephropathy HNP1ndash3 concentrations are increased and therewas an independent relationship between estimated GFR(CockcroftndashGault) and HNP1ndash3 [19] Serological studies ofdefensins would provide crucial insight in exploring thepathogenesis and the development of diseases As for IgAnephropathy the current progress is in genetic field alongwithmultiple SNP identified contributed to the IgAN suscep-tibility Since serological investigation is absent in IgAN weconducted a case-control study to check the plasma levels of120572-defensins in IgAN patients

2 Materials and Methods

21 Subjects In the present study 169 IgAN patients whoseplasma were available and were followed up at least 12months were enrolled The diagnosis of IgAN was provedby renal biopsy 83 healthy controls were blood donors withmatched gender and age with the IgAN patients Patientswith IgAN were confirmed by renal biopsy and those withsecondary IgAN or other comorbid renal diseases suchas systemic lupus erythematosus vasculitis or Henoch-Schonlein purpura were not included Clinical data includ-ing age (years) gender (malefemale ) systolic bloodpressure (SBP) (mmHg) diastolic blood pressure (DBP)(mmHg) neutrophil count (lowast109L) prodromal infection() elevated C-reactive protein (CRP) levels () ery-throcyte sedimentation rate (ESR) (mm1st h) triglycerides(TG) (mmolL) total cholesterol (TCHOL) (mmolL) high-density lipoprotein cholesterol (HDL) (mmolL) low-densitylipoprotein cholesterol (LDL) (mmolL) hyperlipidemia ()uric acid (120583molL) serum creatinine (Scr) (120583molL) estimateglomerular filtration rate (eGFR) (mLmin per 173m2) stage1 2 3 4 and 5 CKD () galactose-deficient IgA1 (Gd-IgA1) (UmL median IQR) total IgA (120583gmL medianIQR) plasma IgA1 (120583gmL median IQR) initial proteinuria(gday) urinary red blood cell (RBC) count (120583L) grosshematuria () 24-hour total urine protein (UTP) (gday)histological grading I II III IV andV () Oxford classifica-tion M () E () S () and T () angiotensin-convertingenzyme (ACE) inhibitors or angiotensin II receptor blockers(ARBs) () and prednisone () were collected at the timeof renal biopsy The composite end point was defined as50 decline from baseline eGFR or end-stage renal disease(ESRD) or death For the purpose of this study ESRD wasdefined as eGFR lt 15mLmin per 173m2 or need for renalreplacement therapy (such as hemodialysis peritoneal dialy-sis or renal transplantation)Themodification of diet in renaldisease formula was used to calculate the eGFR [20] At leasttwo independent pathologists graded and reviewed the renal

biopsy specimens and reached a consensus The histologicallesions were classified according to HASS grading (IndashV)from mild lesions to severe lesions [21] Hyperlipidemia wasassigned when any of the TG TCHOL and LDL indexes wasabove the normal ranges of the department of laboratory ofour hospital This study was approved by the medical ethicscommittee of Peking University First Hospital and writteninformed consent was obtained from all the patients

22 Detection of Plasma HNP1ndash3 IgA Plasma IgA1 and Gd-IgA1 by ELISA Plasma IgA IgA1 and Gd-IgA1 levels weredetected by ELISA as previously published [9] Briefly forGd-IgA1 25mgmL F(ab1015840)

2fragment of goat anti-human

IgA (Jackson ImmunoResearch Labs West Grove PA) wasincubated in high-binding MaxiSorp 96-well plates (Nalge-Nunc Rochester NY) and blocked with 1 bovine serumalbumin (Sigma Chemical Company St Louis MO) in PBS005 Tween 20 The standard curve was made by duplicatesof twofold dilutions of serum samples with a range from1 2000 to 1 16000 for Gd-IgA1 To quantitate Gd-IgA1 astandard consisting of a polymeric Gd-IgA1 protein isolatedfrom a patient with multiple myeloma was used and theterminal sialic acid from O-linked N-acetylgalactosamineon bound samples and the standard IgA1 myeloma proteinwere removed by incubation with 1mUwell neuraminidase(Roche Diagnostic Indianapolis IN) in 001molL acetatebuffer pH 5 for 3 h at 37∘C Then they were incubatedwith 100mL biotin-labeled HAA (1 500 dilution Sigma)for 3 h at 37∘C After washing they were incubated with100mL mouse anti-human monoclonal antibody (1 50000dilution Sigma) for 1 h at 37∘C followed by an incubationwithhorseradish peroxidase-ExtrAvidin (Sigma) for 1 h at 37∘C1molL sulfuric acid was used to stop the color reaction andthe absorbance was measured at 490 nm with an EL312 Bio-Kinetics microplate reader (Bio-Tek Instruments WinooskiVT) The determination of Gd-IgA1 concentration in thetested samples was calculated with the DeltaSoft II program(BioMetallics Princeton NJ) by interpolating the opticaldensities on calibration curves constructed using a Gd-IgA1myeloma protein Influenced by galactose in the standardIgA1 myeloma protein the concentration of Gd-IgA1 wasexpressed in UmL The HAA-ELISA was repeated with 108randomly selected patientsrsquo samples for intra-assay variabil-ity

Plasma 120572-defensin concentration of IgAN patients wasquantified by ELISA using a commercial human HNP1ndash3 ELISA Kit (Hycult Biotechnology BV Uden Nether-lands) with the measurable concentration ranges from 156to 10000 pgmL Briefly plasma samples were stored atminus80∘C after being separated by centrifugation at 1500 rpmfor 15min at room temperature and clarification centrifu-gation at 3000 rpm According to manufacturerrsquos instruc-tions samples and standards are incubated for 60min inmicrotiter wells coated with human HNP1ndash3 antibodiesAfter 4 times of thorough washing the second antibodycontaining biotinylated tracer antibody was added to capturehuman HNP1ndash3 for 60min and then washed again andincubated with treptavidin-peroxidase conjugate for 60min

Disease Markers 3

The reaction was stopped with 1molL sulfuric acid and theabsence was measured at 450 nm with an EL312 Bio-Kineticsmicroplate reader (Bio-Tek Instruments Winooski VT) Toassess the degree of intra-assay variability one sample hadbeen repeated 3 times to minimize the deviation

23 Statistical Analyses Means and standard deviations wereused to summarize statistics for normally distributed quan-titative variables We used median and IQR to summarizenonnormally distributed variables and ratios and percentagesfor categorical data Studentrsquos t-test (two groups) or analysisof variance (multiple groups)was used to compare differencesinmeans for continuous variables Differences in proportionswere tested by 1205942 test The association of plasma HNP1ndash3levels and the primary outcome were analyzed by Cox pro-portional hazards models We used natural log to transformHNP1ndash3 and Gd-IgA1 as it was highly skewed to the rightin this group of patients Statistical analysis was achievedwith the SPSS 160 software package (SPSS Inc ChicagoIL) A two-tailed 119901 value lt 005 was considered statisticallysignificant

3 Results

31 Patient Baseline Characteristics and Clinical Outcome Asis shown in Table 1 there were 86 (509) males and 83(491) females with mean age at the time of kidney biopsyof 327 plusmn 105 yearsThemedian proteinuria of IgAN patientson biopsy was 198plusmn190 g per 24 h (range 014ndash1372 g24 h)and median Scr was 91 120583molL (range 54ndash789120583molL) Theaverage eGFR was 8166 plusmn 2684mLmin per 173m2 (range630ndash16490mLmin per 173m2) Systolic blood pressurewas 124 plusmn 17mmHg and diastolic blood pressure was 80 plusmn12mmHg with 49 patients (29) being hypertensive atbaseline The distribution by Haas grades I II III IV andV was 101 0 343 42 and 136 respectively Themedian follow-up time was 48months (range 12ndash96monthsTable 1) 165 (976) patients receivedACE inhibitors orARBstherapy and 79 (467) received oral corticosteroids alone orcombined with other immunosuppressive agents during thefollow-up period In total 30 patients reached the compositeend point of 50 decline in eGFR ESRD or death

32 Levels of HNP1ndash3 in IgAN Patients We first set out todetermine the levels of theHNP1ndash3 in the plasma of our IgANpatients by ELISA The mean levels of plasma 120572-defensins83 healthy controls and 169 patients with IgA nephropathywere 50 ngmL and 7842 ngmL As can be shown in Figure 1some patients with IgAN showedmarked value of120572-defensinWhen the results were adjusted to the value of the meanlevel of 120572-defensin in IgAN patients it was shown that thepercentage of individuals with high levels of 120572-defensinsincreased in patients (225) comparing with that in healthycontrols (96) (119901 = 0013 OR 2719 95 CI 1206ndash6135)

33 The Levels of HNP1ndash3 Correlation with IgAN ClinicalCharacteristics and Prognosis To test the clinical manifes-tations differences between patients with high and low 120572-defensins levels the median 120572-defensins level of healthy

p = 00132000

1800

1600

1400

1000800600400200

100806040200

Plas

ma H

NP1

ndash3(n

gm

L)

7842ngmL

Healthy controls IgAN patients

Figure 1 Plasma 120572-defensins (HNP1ndash3) levels of IgAN patientsand healthy controls The line was mean levels of 120572-defensins for169 IgAN patients (7842 ngmL) defined as the cut-off for dividingpatients into two groups with high (above 7842 ngmL) and low(below 7842 ngmL) 120572-defensins levels

controls (7842 ngmL) was defined as the cut-off for dividingpatients into two groups Low plasma 120572-defensins levelswere defined as levels below 7842 ngmL High plasma 120572-defensins levels were defined as levels above 7842 ngmL131 (775) IgAN patients were assigned to the group withlow (below 7842 ngmL) 120572-defensins levels and 38 (225)IgAN patients were assigned to the group with high (above7842 ngmL)120572-defensins levelsThemain clinicalmanifesta-tions demonstrated that there were no differences in age sexblood pressure initial proteinuria and eGFR in two groupsof patients Serum creatinine and eGFR were not associatedwith the levels of 120572-defensins in IgANpatients likely suggest-ing no accumulation of 120572-defensins as the result of reducedglomerular filtration rate (119903 = 0039 119901 = 0614 and 119903 =minus0031 119901 = 0687 resp) (Figures 2(d) and 2(c)) In additionthere was no correlation between the neutrophil count andthe levels of 120572-defensins in IgAN patients (119903 = 0140 119901 =0093) (Figure 2(b))

In the group of patients with high 120572-defensins plasma lev-els Gd-IgA1 was significantly lower (median 2369847UmLand interquartile range 20310ndash35001UmL) than patientswith low 120572-defensins levels (median 31856UmL interquar-tile range 24280ndash40567UmL and 119901 = 0036) More impor-tantly we also found that low Gd-IgA1 was correlated withhigh 120572-defensins levels (119903 = minus0204 119901 = 0008) (Figure 2(a))Levels of plasma IgA or plasma IgA1 showed no significantbetween patients with high and low 120572-defensins (Table 1)

No association were observed in plasma HNP1ndash3 levelwith other clinical manifestations (such as SBP DBP prodro-mal infection gross hematuria 24-hour UTP Scr and eGFR)and renal outcome (Table 1)

4 Discussion

In this study we measured the levels of HNP1ndash3 in IgANpatients We found that the 120572-defensins in IgAN patientswere elevated compared with healthy controls which wasindependent of renal function and neutrophil count The

4 Disease Markers

Table 1 Clinical and laboratory data for IgAN patients with different plasma 120572-defensins levels

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

Age (years) 327 plusmn 105 325 plusmn 107 335 plusmn 98 0614

Gender (malefemale ) 86 (509)83(491)

67 (511)64(489)

19 (500)19(500) 0901

SBP (mmHg) 1239 plusmn 169 1248 plusmn 178 1207 plusmn 133 0184DBP (mmHg) 795 plusmn 132 800 plusmn 138 778 plusmn 107 0350Neutrophil count (lowast109L) 462 plusmn 200 449 plusmn 207 508 plusmn 171 0147

Prodromal infection () 127 (751)42(249)

98 (748)33(252)

29 (763)9(237) 0850

CRP () 112 (663)7 (41) 82 (626)5 (38) 30 (789)2 (53) 0918ESR (mm1st h) 181 plusmn 177 175 plusmn 169 198 plusmn 204 0514TG (mmolL) 193 plusmn 151 198 plusmn 160 179 plusmn 111 0499TCHOL (mmolL) 503 plusmn 150 498 plusmn 146 518 plusmn 1663 0457HDL (mmolL) 121 plusmn 057 123 plusmn 062 114 plusmn 033 0384LDL (mmolL) 289 plusmn 098 283 plusmn 090 309 plusmn 119 0153

Hyperlipidemia () 66 (391)103(609)

51 (389)80(611)

15 (395)23(605) 0952

Uric acid (120583molL) 3567 plusmn 1183 3615 plusmn 1190 3402 plusmn 1159 0330Scr (120583molL) 1048 plusmn 656 1064 plusmn 716 993 plusmn 391 0556eGFR (mLmin per 173m2)3 814 plusmn 278 809 plusmn 278 832 plusmn 282 0654

Stage 1 2 3 4 and 5 CKD ()50 (296) 67

(396) 34 (201)2 (12)

40 (305) 60(458) 30 (229)

1 (08)

10 (263) 7(184) 4 (105) 1

(26)0224

Gd-IgA1 (UmL medianIQR)

30386(23143ndash39602)

31856(24280ndash40567)

23698(20310ndash35001) 0036

Total IgA (120583gmL medianIQR)

26300(21535ndash33335)

26580(21670ndash33470)

25705(20293ndash33653) 0565

Plasma IgA1 (120583gmL medianIQR)

16964(13829ndash20757)

1643(1353ndash2064)

18756(14872ndash21646) 0318

Initial proteinuria (gday) 198 plusmn 190 189 plusmn 165 232 plusmn 258 0219Urinary RBC (120583L) 286 plusmn 537 318 plusmn 599 184 plusmn 219 0181

Gross hematuria () 129 (763)40(237)

99 (756)32(244) 30 (789)8 (211) 0667

24-hour UTP (gday) 114 plusmn 106 113 plusmn 108 116 plusmn 102 0886

Histological grading I II IIIIV V4 ()

17 (101) 0 (0)58 (343) 71

(420) 23 (136)

10 (76) 0 (0) 43(328) 58 (443)

20 (153)

7 (184) 0 (0) 15(395) 13 (342)

3 (79)0129

Oxford classification

M () 54 (320) 112(663)

38 (290)92(702)

16 (421)20(526) 0085

E () 60 (355) 106(627)

45 (344)85(649)

15 (395)21(553) 0436

S () 45 (266) 121(716)

36 (275)94(718) 9 (237)27 (711) 0748

T () 109 (645) 36(213) 21 (124)

86 (656) 28(214) 16 (122)

23 (605) 8(222) 5 (132) 0959

Disease Markers 5

Table 1 Continued

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

TherapyACE inhibitors or ARBs () 4 (24)165 (976) 3 (23)128 (977) 1 (26)37 (974) 0903

Prednisone () 79 (467)90(533)

65 (496)66(504)

14 (368)24(632) 0165

SBP systolic blood pressure DBP diastolic blood pressure CRP C-reactive protein ESR erythrocyte sedimentation rate TG triglycerides TCHOL totalcholesterol HDL high-density lipoprotein cholesterol LDL low-density lipoprotein cholesterol Scr serum creatinine eGFR estimate glomerular filtrationrate CKD chronic kidney disease 24-hour UTP 24-hour total urine protein Gd-IgA1 galactose-deficient IgA1 ACE angiotensin-converting enzyme ARBangiotensin II receptor blocker1Low plasma 120572-defensins levels were defined as levels below 7842 ngmL 2High plasma 120572-defensins levels were defined as levels above 7842 ngmL 3eGFRwas calculated based on MDRD formula modified population 4Histological grading was classified according to pathological proposed by Haas

r = minus0204p = 0008

2000

1500

1000

500

0

Gd-

IgA1

150010005000 2000

HNP1ndash3(a)

r = 0140p = 0093

15

10

5

0

Neu

troph

il co

unt

150010005000 2000

HNP1ndash3(b)

r = minus0031

p = 0687

eGFR

150010005000 2000

HNP1ndash3

200

150

100

50

0

(c)

r = 0039p = 0614

Scr

150010005000 2000

HNP1ndash3

1000

800

600

400

200

0

(d)

Figure 2 Two-tailed bivariate correlations between plasma levels of HNP1ndash3 (ngmL) and IgAN clinical ((a) Gd-IgA1 UmL (b) neutrophilcount lowast109L (c) eGFR mLmin per 173m2 (d) Scr 120583molL) characteristics

high level of plasma 120572-defensins might be due to neutrophilactivation instead of increased neutrophil numbers Moreimportantly we also found that low Gd-IgA1 was associatedwith high 120572-defensins levels On the basis of the potentialpathogenic role of Gd-IgA1 in the development of IgAN 120572-defensins may be involved in the pathogenesis of IgAN notjust through serological changes

The defensins of vertebrate animals comprised by 18ndash45 amino-acid residues are small cationic peptides includ-ing three subfamilies of defensins namely 120572- 120573- and 120579-defensins There are six human 120572-defensin peptides namelyHNP1ndash3 HNP4 and HD5-6 which shows consistency inamino acids sequences to some degree HNP1ndash3 and HD5have lectin-like affinity for glycosylated molecules that can

6 Disease Markers

bind carbohydrates in making glycolipids glycoproteins andproteoglycans [22ndash25] IgA1 are glycoproteins which consistof a hinge region (HR) with a high content of proline (Pro)serine (Ser) and threonine (Thr) between the first and secondheavy chain constant region domains Within the IgA1HR six O-linked glycan chains have been identified whichhave been localized to SerThr residues Thr225 Thr228Ser230 Ser232 Thr233 and Thr236 [26ndash30] IgA1 O-linkedglycans are composed of N-acetylgalactosamine (GalNAc)with a 1205731 3-linked Gal 1205722 3-linked Gal or 1205722 6-linkedGalNAc may be capable of sialic acid attachment [31ndash33]Glycosylation is the process that a carbohydrate such asa glycosyl donor is attached to a hydroxyl or a glycosylacceptor Gd-IgA1 is mainly represented as the deficiencyof galactose in hinge regions which may lead to increasedexposure of glycosyl in GalNAc 120572-defensin is a lectin-likepeptide which has carbohydrate-binding activity [25] Wehypothesized that 120572-defensin could bond to exposed glycosylin N-acetylgalactosamine of IgA1 molecular which mightcontribute to IgA1 aggregation and deposition in mesangialregions Our present study supported the hypothesis showingthat120572-defensinswere elevated in patientswith IgANandwerenegatively correlated with galactose deficiency of circulatingIgA1

In addition to their role in circulation 120572-defensins canalso play a role in local tissues 120572-defensins can inducethe secretion of inflammatory cytokines and inflamma-tory chemokines in lung epithelial cells promoting localinflammatory response Besides 120572-defensins can also facil-itate the migration of fibroblastsepithelial cell proliferationand increase extracellular matrix production [34] Since 120572-defensins have both inflammatory and fibrosis effects in otherorgans further study should be carried out in depth abouttheir effects on renal tissue Unfortunately no significantdifference as for M E S and T classification was observedbetween patients with the low and high 120572-defensins levelsThis negative finding might be due to certain cell lines usedin the inflammatory and fibrosis effects of 120572-defensins studieswithout evidences from renal resident cells Since the effect ofdifferent cell lines might not be the same the inflammatoryand fibrosis effects of renal resident cells should be furtherinvestigatedHowever the correlation between the levels of120572-defensins and Gd-IgA1 was relatively low And no significantassociation of clinical manifestations was observed betweenpatients with high and low levels of 120572-defensins Future morewidespread replications and functional assays were of specialinterest

In conclusion our study identified that the 120572-defensinsin IgAN patients were elevated which was independent ofrenal function and neutrophil count And we also foundthat high Gd-IgA1 was associated with low 120572-defensinslevels We speculate that 120572-defensins may bind Gd-IgA1 incirculation which finally deposit in the kidney promotinglocal inflammation and fibrosis

Abbreviations

IgAN IgA nephropathyGd-IgA1 Galactose-deficient IgA1

GWAS Genome-wide association studyHNP Human neutrophil peptideESRD End-stage renal diseaseSNPs Single nucleotide polymorphismseGFR Estimated glomerular filtration rateGalNAc N-AcetylgalactosamineACE Angiotensin-converting enzymeARB Angiotensin II receptor blockerCKD Chronic kidney diseaseDBP Diastolic blood pressureSBP Systolic blood pressureCRP C-reactive proteinESR Erythrocyte sedimentation rateTG TriglyceridesTCHOL Total cholesterolHDL High-density lipoprotein cholesterolLDL Low-density lipoprotein cholesterolScr Serum creatinine24-hour UTP 24-hour total urine protein

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Xu-jie Zhou and Hong Zhang designed the research Yuan-yuan Qi and Fa-juan Cheng performed the research Yuan-yuan Qi and Xu-jie Zhou analyzed the data and Yuan-yuanQi and Xu-jie Zhou wrote the paper

Acknowledgments

The authors thank their collaborators including Ali GGharavi fromDepartment of Medicine Columbia UniversityCollege of Physicians and Surgeons New York USA forassistance They also thank all the members of the laboratoryfor technical assistance and the patients and their familiesfor their cooperation and for giving consent to participatein this study This work was supported by Grants fromthe National Natural Science Foundation of China (no81570629) Beijing Natural Science Foundation (7152148)Chinese Society of Nephrology (15020030591) the MajorState Basic Research Development Program of China (973Program no 2012CB517700) the Research Fund of BeijingMunicipal Science and Technology for the Outstanding PhDProgram (no 20121000110) the Foundation of the Ministryof Education of China (no 20120001120008) the NaturalScience Fund of China to the Innovation Research Group(no 81021004) and National Key Technology RampD Program(2013BAI09B14)

Disease Markers 7

References

[1] M Levy and J Berger ldquoWorldwide perspective of IgA nephro-pathyrdquo American Journal of Kidney Diseases vol 12 no 5 pp340ndash347 1988

[2] A Amore P Cirina G Conti P Brusa L Peruzzi and RCoppo ldquoGlycosylation of circulating IgA in patients with IgAnephropathy modulates proliferation and apoptosis of mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 12no 9 pp 1862ndash1871 2001

[3] J Barratt and J Feehally ldquoIgA nephropathyrdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2088ndash20972005

[4] J Barratt J Feehally and A C Smith ldquoPathogenesis of IgAnephropathyrdquo Seminars in Nephrology vol 24 no 3 pp 197ndash217 2004

[5] A C Smith J F de Wolff K Molyneux J Feehally and JBarratt ldquoO-glycosylation of serum IgD in IgA nephropathyrdquoJournal of the American Society of Nephrology vol 17 no 4 pp1192ndash1199 2006

[6] H Suzuki Z Moldoveanu S Hall et al ldquoIgA1-secreting celllines from patients with IgA nephropathy produce aberrantlyglycosylated IgA1rdquo Journal of Clinical Investigation vol 118 no2 pp 629ndash639 2008

[7] J Novak B A Julian M Tomana and J Mestecky ldquoIgAglycosylation and IgA immune complexes in the pathogenesisof IgA nephropathyrdquo Seminars in Nephrology vol 28 no 1 pp78ndash87 2008

[8] A C Allen E M Bailey P E Brenchley K S Buck J Barrattand J Feehally ldquoMesangial Iga1 in IgA nephropathy exhibitsaberrant O-glycosylation observations in three patientsrdquo Kid-ney International vol 60 no 3 pp 969ndash973 2001

[9] N Zhao P Hou J Lv et al ldquoThe level of galactose-deficientIgA1 in the sera of patients with IgA nephropathy is associatedwith disease progressionrdquo Kidney International vol 82 no 7pp 790ndash796 2012

[10] A G Gharavi K Kiryluk M Choi et al ldquoGenome-wide asso-ciation study identifies susceptibility loci for IgA nephropathyrdquoNature Genetics vol 43 no 4 pp 321ndash327 2011

[11] C Yang W Jie Y Yanlong et al ldquoGenome-wide associationstudy identifies TNFSF13 as a susceptibility gene for IgA in aSouthChinese population in smokersrdquo Immunogenetics vol 64no 10 pp 747ndash753 2012

[12] K Kiryluk Y Li F Scolari et al ldquoDiscovery of new risk loci forIgAnephropathy implicates genes involved in immunity againstintestinal pathogensrdquo Nature Genetics vol 46 no 11 pp 1187ndash1196 2014

[13] X-Q Yu M Li H Zhang et al ldquoA genome-wide associationstudy in Han Chinese identifies multiple susceptibility loci forIgA nephropathyrdquo Nature Genetics vol 44 no 2 pp 178ndash1822012

[14] Y Y Qi X J Zhou F J Cheng et al ldquoDEFA gene variantsassociated with IgA nephropathy in a Chinese populationrdquoGenes amp Immunity vol 16 no 3 pp 231ndash237 2015

[15] R I Lehrer and W Lu ldquo120572-Defensins in human innate immu-nityrdquo Immunological Reviews vol 245 no 1 pp 84ndash112 2012

[16] D Yang A Biragyn L W Kwak and J J Oppenheim ldquoMam-malian defensins in immunity more than just microbicidalrdquoTrends in Immunology vol 23 no 6 pp 291ndash296 2002

[17] Z M Sthoeger S Bezalel N Chapnik I Asher and O FroyldquoHigh 120572-defensin levels in patients with systemic lupus erythe-matosusrdquo Immunology vol 127 no 1 pp 116ndash122 2009

[18] F-J Cheng X-J Zhou Y-F Zhao M-H Zhao and H ZhangldquoHuman neutrophil peptide 1ndash3 a component of the neutrophil

extracellular trap as a potential biomarker of lupus nephritisrdquoInternational Journal of Rheumatic Diseases vol 18 no 5 pp533ndash540 2015

[19] M Saraheimo C Forsblom K Pettersson-Fernholm A Flyvb-jerg P-H Groop and J Frystyk ldquoIncreased levels of 120572-defensin(-1 -2 and -3) in type 1 diabetic patients with nephropathyrdquoNephrology Dialysis Transplantation vol 23 no 3 pp 914ndash9182008

[20] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[21] M Haas ldquoHistologic subclassification of IgA nephropathya clinicopathologic study of 244 casesrdquo American Journal ofKidney Diseases vol 29 no 6 pp 829ndash842 1997

[22] R I Lehrer G Jung P Ruchala S Andre H J Gabius andW Lu ldquoMultivalent binding of carbohydrates by the human 120572-defensin HD5rdquo The Journal of Immunology vol 183 no 1 pp480ndash490 2009

[23] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[24] E Leikina H Delanoe-Ayari K Melikov et al ldquoCarbohydrate-bindingmolecules inhibit viral fusion and entry by crosslinkingmembrane glycoproteinsrdquoNature Immunology vol 6 no 10 pp995ndash1001 2005

[25] W Wang S M Owen D L Rudolph et al ldquoActivity of 120572- and120579-defensins against primary isolates of HIV-1rdquo The Journal ofImmunology vol 173 no 1 pp 515ndash520 2004

[26] Z Moldoveanu R J Wyatt J Y Lee et al ldquoPatients withIgA nephropathy have increased serum galactose-deficient IgA1levelsrdquo Kidney International vol 71 no 11 pp 1148ndash1154 2007

[27] M B Renfrow H J Cooper M Tomana et al ldquoDeterminationof aberrant O-glycosylation in the IgA1 hinge region by electroncapture dissociation fourier transform-ion cyclotron resonancemass spectrometryrdquo Journal of Biological Chemistry vol 280no 19 pp 19136ndash19145 2005

[28] K Takahashi S BWallH Suzuki et al ldquoClusteredO-glycans ofIgA1 defining macro- and microheterogeneity by use of elec-tron capturetransfer dissociationrdquoMolecular and Cellular Pro-teomics vol 9 no 11 pp 2545ndash2557 2010

[29] M M Gomes H Suzuki M T Brooks et al ldquoRecognitionof galactose-deficient O-glycans in the hinge region of IgA1by N-acetylgalactosamine-specific snail lectins a comparativebinding studyrdquoBiochemistry vol 49 no 27 pp 5671ndash5682 2010

[30] J S Moore R Kulhavy M Tomana et al ldquoReactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteinsrdquoMolecular Immunology vol 44 no 10 pp 2598ndash2604 2007

[31] S J Mellis and J U Baenziger ldquoStructures of the O-glyco-sidically linked oligosaccharides of human IgDrdquo Journal of Bio-logical Chemistry vol 258 no 19 pp 11557ndash11563 1983

[32] T SMattu R J Pleass A CWillis et al ldquoThe glycosylation andstructure of human serum IgA1 Fab and Fc regions and the roleof N-glycosylation on Fc120572 receptor interactionsrdquoThe Journal ofBiological Chemistry vol 273 no 4 pp 2260ndash2272 1998

[33] M C Field S Amatayakul-Chantler T W Rademacher PM Rudd and R A Dwek ldquoStructural analysis of the N-glycans from human immunoglobulin A1 comparison of nor-mal human serum immunoglobulin A1 with that isolated frompatients with rheumatoid arthritisrdquo Biochemical Journal vol299 no 1 pp 261ndash275 1994

[34] E Arnett and S Seveau ldquoThe multifaceted activities of mam-malian defensinsrdquo Current Pharmaceutical Design vol 17 no38 pp 4254ndash4269 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

inactivate a particular spectrum of bacteria fungi or someenveloped viruses in vitro [15] In addition120572-defensinsmightalso be a component of the effector of adaptive immunitymediating B- and T-cell interactions linking innate andadaptive immunity [16]

In certain disease states plasma or local 120572-defensinswould depart from normal levels 120572-defensins levels wereelevated in plasma of SLE patients and could serve as amarker for disease activity [17 18] In patients with diabeticnephropathy HNP1ndash3 concentrations are increased and therewas an independent relationship between estimated GFR(CockcroftndashGault) and HNP1ndash3 [19] Serological studies ofdefensins would provide crucial insight in exploring thepathogenesis and the development of diseases As for IgAnephropathy the current progress is in genetic field alongwithmultiple SNP identified contributed to the IgAN suscep-tibility Since serological investigation is absent in IgAN weconducted a case-control study to check the plasma levels of120572-defensins in IgAN patients

2 Materials and Methods

21 Subjects In the present study 169 IgAN patients whoseplasma were available and were followed up at least 12months were enrolled The diagnosis of IgAN was provedby renal biopsy 83 healthy controls were blood donors withmatched gender and age with the IgAN patients Patientswith IgAN were confirmed by renal biopsy and those withsecondary IgAN or other comorbid renal diseases suchas systemic lupus erythematosus vasculitis or Henoch-Schonlein purpura were not included Clinical data includ-ing age (years) gender (malefemale ) systolic bloodpressure (SBP) (mmHg) diastolic blood pressure (DBP)(mmHg) neutrophil count (lowast109L) prodromal infection() elevated C-reactive protein (CRP) levels () ery-throcyte sedimentation rate (ESR) (mm1st h) triglycerides(TG) (mmolL) total cholesterol (TCHOL) (mmolL) high-density lipoprotein cholesterol (HDL) (mmolL) low-densitylipoprotein cholesterol (LDL) (mmolL) hyperlipidemia ()uric acid (120583molL) serum creatinine (Scr) (120583molL) estimateglomerular filtration rate (eGFR) (mLmin per 173m2) stage1 2 3 4 and 5 CKD () galactose-deficient IgA1 (Gd-IgA1) (UmL median IQR) total IgA (120583gmL medianIQR) plasma IgA1 (120583gmL median IQR) initial proteinuria(gday) urinary red blood cell (RBC) count (120583L) grosshematuria () 24-hour total urine protein (UTP) (gday)histological grading I II III IV andV () Oxford classifica-tion M () E () S () and T () angiotensin-convertingenzyme (ACE) inhibitors or angiotensin II receptor blockers(ARBs) () and prednisone () were collected at the timeof renal biopsy The composite end point was defined as50 decline from baseline eGFR or end-stage renal disease(ESRD) or death For the purpose of this study ESRD wasdefined as eGFR lt 15mLmin per 173m2 or need for renalreplacement therapy (such as hemodialysis peritoneal dialy-sis or renal transplantation)Themodification of diet in renaldisease formula was used to calculate the eGFR [20] At leasttwo independent pathologists graded and reviewed the renal

biopsy specimens and reached a consensus The histologicallesions were classified according to HASS grading (IndashV)from mild lesions to severe lesions [21] Hyperlipidemia wasassigned when any of the TG TCHOL and LDL indexes wasabove the normal ranges of the department of laboratory ofour hospital This study was approved by the medical ethicscommittee of Peking University First Hospital and writteninformed consent was obtained from all the patients

22 Detection of Plasma HNP1ndash3 IgA Plasma IgA1 and Gd-IgA1 by ELISA Plasma IgA IgA1 and Gd-IgA1 levels weredetected by ELISA as previously published [9] Briefly forGd-IgA1 25mgmL F(ab1015840)

2fragment of goat anti-human

IgA (Jackson ImmunoResearch Labs West Grove PA) wasincubated in high-binding MaxiSorp 96-well plates (Nalge-Nunc Rochester NY) and blocked with 1 bovine serumalbumin (Sigma Chemical Company St Louis MO) in PBS005 Tween 20 The standard curve was made by duplicatesof twofold dilutions of serum samples with a range from1 2000 to 1 16000 for Gd-IgA1 To quantitate Gd-IgA1 astandard consisting of a polymeric Gd-IgA1 protein isolatedfrom a patient with multiple myeloma was used and theterminal sialic acid from O-linked N-acetylgalactosamineon bound samples and the standard IgA1 myeloma proteinwere removed by incubation with 1mUwell neuraminidase(Roche Diagnostic Indianapolis IN) in 001molL acetatebuffer pH 5 for 3 h at 37∘C Then they were incubatedwith 100mL biotin-labeled HAA (1 500 dilution Sigma)for 3 h at 37∘C After washing they were incubated with100mL mouse anti-human monoclonal antibody (1 50000dilution Sigma) for 1 h at 37∘C followed by an incubationwithhorseradish peroxidase-ExtrAvidin (Sigma) for 1 h at 37∘C1molL sulfuric acid was used to stop the color reaction andthe absorbance was measured at 490 nm with an EL312 Bio-Kinetics microplate reader (Bio-Tek Instruments WinooskiVT) The determination of Gd-IgA1 concentration in thetested samples was calculated with the DeltaSoft II program(BioMetallics Princeton NJ) by interpolating the opticaldensities on calibration curves constructed using a Gd-IgA1myeloma protein Influenced by galactose in the standardIgA1 myeloma protein the concentration of Gd-IgA1 wasexpressed in UmL The HAA-ELISA was repeated with 108randomly selected patientsrsquo samples for intra-assay variabil-ity

Plasma 120572-defensin concentration of IgAN patients wasquantified by ELISA using a commercial human HNP1ndash3 ELISA Kit (Hycult Biotechnology BV Uden Nether-lands) with the measurable concentration ranges from 156to 10000 pgmL Briefly plasma samples were stored atminus80∘C after being separated by centrifugation at 1500 rpmfor 15min at room temperature and clarification centrifu-gation at 3000 rpm According to manufacturerrsquos instruc-tions samples and standards are incubated for 60min inmicrotiter wells coated with human HNP1ndash3 antibodiesAfter 4 times of thorough washing the second antibodycontaining biotinylated tracer antibody was added to capturehuman HNP1ndash3 for 60min and then washed again andincubated with treptavidin-peroxidase conjugate for 60min

Disease Markers 3

The reaction was stopped with 1molL sulfuric acid and theabsence was measured at 450 nm with an EL312 Bio-Kineticsmicroplate reader (Bio-Tek Instruments Winooski VT) Toassess the degree of intra-assay variability one sample hadbeen repeated 3 times to minimize the deviation

23 Statistical Analyses Means and standard deviations wereused to summarize statistics for normally distributed quan-titative variables We used median and IQR to summarizenonnormally distributed variables and ratios and percentagesfor categorical data Studentrsquos t-test (two groups) or analysisof variance (multiple groups)was used to compare differencesinmeans for continuous variables Differences in proportionswere tested by 1205942 test The association of plasma HNP1ndash3levels and the primary outcome were analyzed by Cox pro-portional hazards models We used natural log to transformHNP1ndash3 and Gd-IgA1 as it was highly skewed to the rightin this group of patients Statistical analysis was achievedwith the SPSS 160 software package (SPSS Inc ChicagoIL) A two-tailed 119901 value lt 005 was considered statisticallysignificant

3 Results

31 Patient Baseline Characteristics and Clinical Outcome Asis shown in Table 1 there were 86 (509) males and 83(491) females with mean age at the time of kidney biopsyof 327 plusmn 105 yearsThemedian proteinuria of IgAN patientson biopsy was 198plusmn190 g per 24 h (range 014ndash1372 g24 h)and median Scr was 91 120583molL (range 54ndash789120583molL) Theaverage eGFR was 8166 plusmn 2684mLmin per 173m2 (range630ndash16490mLmin per 173m2) Systolic blood pressurewas 124 plusmn 17mmHg and diastolic blood pressure was 80 plusmn12mmHg with 49 patients (29) being hypertensive atbaseline The distribution by Haas grades I II III IV andV was 101 0 343 42 and 136 respectively Themedian follow-up time was 48months (range 12ndash96monthsTable 1) 165 (976) patients receivedACE inhibitors orARBstherapy and 79 (467) received oral corticosteroids alone orcombined with other immunosuppressive agents during thefollow-up period In total 30 patients reached the compositeend point of 50 decline in eGFR ESRD or death

32 Levels of HNP1ndash3 in IgAN Patients We first set out todetermine the levels of theHNP1ndash3 in the plasma of our IgANpatients by ELISA The mean levels of plasma 120572-defensins83 healthy controls and 169 patients with IgA nephropathywere 50 ngmL and 7842 ngmL As can be shown in Figure 1some patients with IgAN showedmarked value of120572-defensinWhen the results were adjusted to the value of the meanlevel of 120572-defensin in IgAN patients it was shown that thepercentage of individuals with high levels of 120572-defensinsincreased in patients (225) comparing with that in healthycontrols (96) (119901 = 0013 OR 2719 95 CI 1206ndash6135)

33 The Levels of HNP1ndash3 Correlation with IgAN ClinicalCharacteristics and Prognosis To test the clinical manifes-tations differences between patients with high and low 120572-defensins levels the median 120572-defensins level of healthy

p = 00132000

1800

1600

1400

1000800600400200

100806040200

Plas

ma H

NP1

ndash3(n

gm

L)

7842ngmL

Healthy controls IgAN patients

Figure 1 Plasma 120572-defensins (HNP1ndash3) levels of IgAN patientsand healthy controls The line was mean levels of 120572-defensins for169 IgAN patients (7842 ngmL) defined as the cut-off for dividingpatients into two groups with high (above 7842 ngmL) and low(below 7842 ngmL) 120572-defensins levels

controls (7842 ngmL) was defined as the cut-off for dividingpatients into two groups Low plasma 120572-defensins levelswere defined as levels below 7842 ngmL High plasma 120572-defensins levels were defined as levels above 7842 ngmL131 (775) IgAN patients were assigned to the group withlow (below 7842 ngmL) 120572-defensins levels and 38 (225)IgAN patients were assigned to the group with high (above7842 ngmL)120572-defensins levelsThemain clinicalmanifesta-tions demonstrated that there were no differences in age sexblood pressure initial proteinuria and eGFR in two groupsof patients Serum creatinine and eGFR were not associatedwith the levels of 120572-defensins in IgANpatients likely suggest-ing no accumulation of 120572-defensins as the result of reducedglomerular filtration rate (119903 = 0039 119901 = 0614 and 119903 =minus0031 119901 = 0687 resp) (Figures 2(d) and 2(c)) In additionthere was no correlation between the neutrophil count andthe levels of 120572-defensins in IgAN patients (119903 = 0140 119901 =0093) (Figure 2(b))

In the group of patients with high 120572-defensins plasma lev-els Gd-IgA1 was significantly lower (median 2369847UmLand interquartile range 20310ndash35001UmL) than patientswith low 120572-defensins levels (median 31856UmL interquar-tile range 24280ndash40567UmL and 119901 = 0036) More impor-tantly we also found that low Gd-IgA1 was correlated withhigh 120572-defensins levels (119903 = minus0204 119901 = 0008) (Figure 2(a))Levels of plasma IgA or plasma IgA1 showed no significantbetween patients with high and low 120572-defensins (Table 1)

No association were observed in plasma HNP1ndash3 levelwith other clinical manifestations (such as SBP DBP prodro-mal infection gross hematuria 24-hour UTP Scr and eGFR)and renal outcome (Table 1)

4 Discussion

In this study we measured the levels of HNP1ndash3 in IgANpatients We found that the 120572-defensins in IgAN patientswere elevated compared with healthy controls which wasindependent of renal function and neutrophil count The

4 Disease Markers

Table 1 Clinical and laboratory data for IgAN patients with different plasma 120572-defensins levels

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

Age (years) 327 plusmn 105 325 plusmn 107 335 plusmn 98 0614

Gender (malefemale ) 86 (509)83(491)

67 (511)64(489)

19 (500)19(500) 0901

SBP (mmHg) 1239 plusmn 169 1248 plusmn 178 1207 plusmn 133 0184DBP (mmHg) 795 plusmn 132 800 plusmn 138 778 plusmn 107 0350Neutrophil count (lowast109L) 462 plusmn 200 449 plusmn 207 508 plusmn 171 0147

Prodromal infection () 127 (751)42(249)

98 (748)33(252)

29 (763)9(237) 0850

CRP () 112 (663)7 (41) 82 (626)5 (38) 30 (789)2 (53) 0918ESR (mm1st h) 181 plusmn 177 175 plusmn 169 198 plusmn 204 0514TG (mmolL) 193 plusmn 151 198 plusmn 160 179 plusmn 111 0499TCHOL (mmolL) 503 plusmn 150 498 plusmn 146 518 plusmn 1663 0457HDL (mmolL) 121 plusmn 057 123 plusmn 062 114 plusmn 033 0384LDL (mmolL) 289 plusmn 098 283 plusmn 090 309 plusmn 119 0153

Hyperlipidemia () 66 (391)103(609)

51 (389)80(611)

15 (395)23(605) 0952

Uric acid (120583molL) 3567 plusmn 1183 3615 plusmn 1190 3402 plusmn 1159 0330Scr (120583molL) 1048 plusmn 656 1064 plusmn 716 993 plusmn 391 0556eGFR (mLmin per 173m2)3 814 plusmn 278 809 plusmn 278 832 plusmn 282 0654

Stage 1 2 3 4 and 5 CKD ()50 (296) 67

(396) 34 (201)2 (12)

40 (305) 60(458) 30 (229)

1 (08)

10 (263) 7(184) 4 (105) 1

(26)0224

Gd-IgA1 (UmL medianIQR)

30386(23143ndash39602)

31856(24280ndash40567)

23698(20310ndash35001) 0036

Total IgA (120583gmL medianIQR)

26300(21535ndash33335)

26580(21670ndash33470)

25705(20293ndash33653) 0565

Plasma IgA1 (120583gmL medianIQR)

16964(13829ndash20757)

1643(1353ndash2064)

18756(14872ndash21646) 0318

Initial proteinuria (gday) 198 plusmn 190 189 plusmn 165 232 plusmn 258 0219Urinary RBC (120583L) 286 plusmn 537 318 plusmn 599 184 plusmn 219 0181

Gross hematuria () 129 (763)40(237)

99 (756)32(244) 30 (789)8 (211) 0667

24-hour UTP (gday) 114 plusmn 106 113 plusmn 108 116 plusmn 102 0886

Histological grading I II IIIIV V4 ()

17 (101) 0 (0)58 (343) 71

(420) 23 (136)

10 (76) 0 (0) 43(328) 58 (443)

20 (153)

7 (184) 0 (0) 15(395) 13 (342)

3 (79)0129

Oxford classification

M () 54 (320) 112(663)

38 (290)92(702)

16 (421)20(526) 0085

E () 60 (355) 106(627)

45 (344)85(649)

15 (395)21(553) 0436

S () 45 (266) 121(716)

36 (275)94(718) 9 (237)27 (711) 0748

T () 109 (645) 36(213) 21 (124)

86 (656) 28(214) 16 (122)

23 (605) 8(222) 5 (132) 0959

Disease Markers 5

Table 1 Continued

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

TherapyACE inhibitors or ARBs () 4 (24)165 (976) 3 (23)128 (977) 1 (26)37 (974) 0903

Prednisone () 79 (467)90(533)

65 (496)66(504)

14 (368)24(632) 0165

SBP systolic blood pressure DBP diastolic blood pressure CRP C-reactive protein ESR erythrocyte sedimentation rate TG triglycerides TCHOL totalcholesterol HDL high-density lipoprotein cholesterol LDL low-density lipoprotein cholesterol Scr serum creatinine eGFR estimate glomerular filtrationrate CKD chronic kidney disease 24-hour UTP 24-hour total urine protein Gd-IgA1 galactose-deficient IgA1 ACE angiotensin-converting enzyme ARBangiotensin II receptor blocker1Low plasma 120572-defensins levels were defined as levels below 7842 ngmL 2High plasma 120572-defensins levels were defined as levels above 7842 ngmL 3eGFRwas calculated based on MDRD formula modified population 4Histological grading was classified according to pathological proposed by Haas

r = minus0204p = 0008

2000

1500

1000

500

0

Gd-

IgA1

150010005000 2000

HNP1ndash3(a)

r = 0140p = 0093

15

10

5

0

Neu

troph

il co

unt

150010005000 2000

HNP1ndash3(b)

r = minus0031

p = 0687

eGFR

150010005000 2000

HNP1ndash3

200

150

100

50

0

(c)

r = 0039p = 0614

Scr

150010005000 2000

HNP1ndash3

1000

800

600

400

200

0

(d)

Figure 2 Two-tailed bivariate correlations between plasma levels of HNP1ndash3 (ngmL) and IgAN clinical ((a) Gd-IgA1 UmL (b) neutrophilcount lowast109L (c) eGFR mLmin per 173m2 (d) Scr 120583molL) characteristics

high level of plasma 120572-defensins might be due to neutrophilactivation instead of increased neutrophil numbers Moreimportantly we also found that low Gd-IgA1 was associatedwith high 120572-defensins levels On the basis of the potentialpathogenic role of Gd-IgA1 in the development of IgAN 120572-defensins may be involved in the pathogenesis of IgAN notjust through serological changes

The defensins of vertebrate animals comprised by 18ndash45 amino-acid residues are small cationic peptides includ-ing three subfamilies of defensins namely 120572- 120573- and 120579-defensins There are six human 120572-defensin peptides namelyHNP1ndash3 HNP4 and HD5-6 which shows consistency inamino acids sequences to some degree HNP1ndash3 and HD5have lectin-like affinity for glycosylated molecules that can

6 Disease Markers

bind carbohydrates in making glycolipids glycoproteins andproteoglycans [22ndash25] IgA1 are glycoproteins which consistof a hinge region (HR) with a high content of proline (Pro)serine (Ser) and threonine (Thr) between the first and secondheavy chain constant region domains Within the IgA1HR six O-linked glycan chains have been identified whichhave been localized to SerThr residues Thr225 Thr228Ser230 Ser232 Thr233 and Thr236 [26ndash30] IgA1 O-linkedglycans are composed of N-acetylgalactosamine (GalNAc)with a 1205731 3-linked Gal 1205722 3-linked Gal or 1205722 6-linkedGalNAc may be capable of sialic acid attachment [31ndash33]Glycosylation is the process that a carbohydrate such asa glycosyl donor is attached to a hydroxyl or a glycosylacceptor Gd-IgA1 is mainly represented as the deficiencyof galactose in hinge regions which may lead to increasedexposure of glycosyl in GalNAc 120572-defensin is a lectin-likepeptide which has carbohydrate-binding activity [25] Wehypothesized that 120572-defensin could bond to exposed glycosylin N-acetylgalactosamine of IgA1 molecular which mightcontribute to IgA1 aggregation and deposition in mesangialregions Our present study supported the hypothesis showingthat120572-defensinswere elevated in patientswith IgANandwerenegatively correlated with galactose deficiency of circulatingIgA1

In addition to their role in circulation 120572-defensins canalso play a role in local tissues 120572-defensins can inducethe secretion of inflammatory cytokines and inflamma-tory chemokines in lung epithelial cells promoting localinflammatory response Besides 120572-defensins can also facil-itate the migration of fibroblastsepithelial cell proliferationand increase extracellular matrix production [34] Since 120572-defensins have both inflammatory and fibrosis effects in otherorgans further study should be carried out in depth abouttheir effects on renal tissue Unfortunately no significantdifference as for M E S and T classification was observedbetween patients with the low and high 120572-defensins levelsThis negative finding might be due to certain cell lines usedin the inflammatory and fibrosis effects of 120572-defensins studieswithout evidences from renal resident cells Since the effect ofdifferent cell lines might not be the same the inflammatoryand fibrosis effects of renal resident cells should be furtherinvestigatedHowever the correlation between the levels of120572-defensins and Gd-IgA1 was relatively low And no significantassociation of clinical manifestations was observed betweenpatients with high and low levels of 120572-defensins Future morewidespread replications and functional assays were of specialinterest

In conclusion our study identified that the 120572-defensinsin IgAN patients were elevated which was independent ofrenal function and neutrophil count And we also foundthat high Gd-IgA1 was associated with low 120572-defensinslevels We speculate that 120572-defensins may bind Gd-IgA1 incirculation which finally deposit in the kidney promotinglocal inflammation and fibrosis

Abbreviations

IgAN IgA nephropathyGd-IgA1 Galactose-deficient IgA1

GWAS Genome-wide association studyHNP Human neutrophil peptideESRD End-stage renal diseaseSNPs Single nucleotide polymorphismseGFR Estimated glomerular filtration rateGalNAc N-AcetylgalactosamineACE Angiotensin-converting enzymeARB Angiotensin II receptor blockerCKD Chronic kidney diseaseDBP Diastolic blood pressureSBP Systolic blood pressureCRP C-reactive proteinESR Erythrocyte sedimentation rateTG TriglyceridesTCHOL Total cholesterolHDL High-density lipoprotein cholesterolLDL Low-density lipoprotein cholesterolScr Serum creatinine24-hour UTP 24-hour total urine protein

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Xu-jie Zhou and Hong Zhang designed the research Yuan-yuan Qi and Fa-juan Cheng performed the research Yuan-yuan Qi and Xu-jie Zhou analyzed the data and Yuan-yuanQi and Xu-jie Zhou wrote the paper

Acknowledgments

The authors thank their collaborators including Ali GGharavi fromDepartment of Medicine Columbia UniversityCollege of Physicians and Surgeons New York USA forassistance They also thank all the members of the laboratoryfor technical assistance and the patients and their familiesfor their cooperation and for giving consent to participatein this study This work was supported by Grants fromthe National Natural Science Foundation of China (no81570629) Beijing Natural Science Foundation (7152148)Chinese Society of Nephrology (15020030591) the MajorState Basic Research Development Program of China (973Program no 2012CB517700) the Research Fund of BeijingMunicipal Science and Technology for the Outstanding PhDProgram (no 20121000110) the Foundation of the Ministryof Education of China (no 20120001120008) the NaturalScience Fund of China to the Innovation Research Group(no 81021004) and National Key Technology RampD Program(2013BAI09B14)

Disease Markers 7

References

[1] M Levy and J Berger ldquoWorldwide perspective of IgA nephro-pathyrdquo American Journal of Kidney Diseases vol 12 no 5 pp340ndash347 1988

[2] A Amore P Cirina G Conti P Brusa L Peruzzi and RCoppo ldquoGlycosylation of circulating IgA in patients with IgAnephropathy modulates proliferation and apoptosis of mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 12no 9 pp 1862ndash1871 2001

[3] J Barratt and J Feehally ldquoIgA nephropathyrdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2088ndash20972005

[4] J Barratt J Feehally and A C Smith ldquoPathogenesis of IgAnephropathyrdquo Seminars in Nephrology vol 24 no 3 pp 197ndash217 2004

[5] A C Smith J F de Wolff K Molyneux J Feehally and JBarratt ldquoO-glycosylation of serum IgD in IgA nephropathyrdquoJournal of the American Society of Nephrology vol 17 no 4 pp1192ndash1199 2006

[6] H Suzuki Z Moldoveanu S Hall et al ldquoIgA1-secreting celllines from patients with IgA nephropathy produce aberrantlyglycosylated IgA1rdquo Journal of Clinical Investigation vol 118 no2 pp 629ndash639 2008

[7] J Novak B A Julian M Tomana and J Mestecky ldquoIgAglycosylation and IgA immune complexes in the pathogenesisof IgA nephropathyrdquo Seminars in Nephrology vol 28 no 1 pp78ndash87 2008

[8] A C Allen E M Bailey P E Brenchley K S Buck J Barrattand J Feehally ldquoMesangial Iga1 in IgA nephropathy exhibitsaberrant O-glycosylation observations in three patientsrdquo Kid-ney International vol 60 no 3 pp 969ndash973 2001

[9] N Zhao P Hou J Lv et al ldquoThe level of galactose-deficientIgA1 in the sera of patients with IgA nephropathy is associatedwith disease progressionrdquo Kidney International vol 82 no 7pp 790ndash796 2012

[10] A G Gharavi K Kiryluk M Choi et al ldquoGenome-wide asso-ciation study identifies susceptibility loci for IgA nephropathyrdquoNature Genetics vol 43 no 4 pp 321ndash327 2011

[11] C Yang W Jie Y Yanlong et al ldquoGenome-wide associationstudy identifies TNFSF13 as a susceptibility gene for IgA in aSouthChinese population in smokersrdquo Immunogenetics vol 64no 10 pp 747ndash753 2012

[12] K Kiryluk Y Li F Scolari et al ldquoDiscovery of new risk loci forIgAnephropathy implicates genes involved in immunity againstintestinal pathogensrdquo Nature Genetics vol 46 no 11 pp 1187ndash1196 2014

[13] X-Q Yu M Li H Zhang et al ldquoA genome-wide associationstudy in Han Chinese identifies multiple susceptibility loci forIgA nephropathyrdquo Nature Genetics vol 44 no 2 pp 178ndash1822012

[14] Y Y Qi X J Zhou F J Cheng et al ldquoDEFA gene variantsassociated with IgA nephropathy in a Chinese populationrdquoGenes amp Immunity vol 16 no 3 pp 231ndash237 2015

[15] R I Lehrer and W Lu ldquo120572-Defensins in human innate immu-nityrdquo Immunological Reviews vol 245 no 1 pp 84ndash112 2012

[16] D Yang A Biragyn L W Kwak and J J Oppenheim ldquoMam-malian defensins in immunity more than just microbicidalrdquoTrends in Immunology vol 23 no 6 pp 291ndash296 2002

[17] Z M Sthoeger S Bezalel N Chapnik I Asher and O FroyldquoHigh 120572-defensin levels in patients with systemic lupus erythe-matosusrdquo Immunology vol 127 no 1 pp 116ndash122 2009

[18] F-J Cheng X-J Zhou Y-F Zhao M-H Zhao and H ZhangldquoHuman neutrophil peptide 1ndash3 a component of the neutrophil

extracellular trap as a potential biomarker of lupus nephritisrdquoInternational Journal of Rheumatic Diseases vol 18 no 5 pp533ndash540 2015

[19] M Saraheimo C Forsblom K Pettersson-Fernholm A Flyvb-jerg P-H Groop and J Frystyk ldquoIncreased levels of 120572-defensin(-1 -2 and -3) in type 1 diabetic patients with nephropathyrdquoNephrology Dialysis Transplantation vol 23 no 3 pp 914ndash9182008

[20] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[21] M Haas ldquoHistologic subclassification of IgA nephropathya clinicopathologic study of 244 casesrdquo American Journal ofKidney Diseases vol 29 no 6 pp 829ndash842 1997

[22] R I Lehrer G Jung P Ruchala S Andre H J Gabius andW Lu ldquoMultivalent binding of carbohydrates by the human 120572-defensin HD5rdquo The Journal of Immunology vol 183 no 1 pp480ndash490 2009

[23] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[24] E Leikina H Delanoe-Ayari K Melikov et al ldquoCarbohydrate-bindingmolecules inhibit viral fusion and entry by crosslinkingmembrane glycoproteinsrdquoNature Immunology vol 6 no 10 pp995ndash1001 2005

[25] W Wang S M Owen D L Rudolph et al ldquoActivity of 120572- and120579-defensins against primary isolates of HIV-1rdquo The Journal ofImmunology vol 173 no 1 pp 515ndash520 2004

[26] Z Moldoveanu R J Wyatt J Y Lee et al ldquoPatients withIgA nephropathy have increased serum galactose-deficient IgA1levelsrdquo Kidney International vol 71 no 11 pp 1148ndash1154 2007

[27] M B Renfrow H J Cooper M Tomana et al ldquoDeterminationof aberrant O-glycosylation in the IgA1 hinge region by electroncapture dissociation fourier transform-ion cyclotron resonancemass spectrometryrdquo Journal of Biological Chemistry vol 280no 19 pp 19136ndash19145 2005

[28] K Takahashi S BWallH Suzuki et al ldquoClusteredO-glycans ofIgA1 defining macro- and microheterogeneity by use of elec-tron capturetransfer dissociationrdquoMolecular and Cellular Pro-teomics vol 9 no 11 pp 2545ndash2557 2010

[29] M M Gomes H Suzuki M T Brooks et al ldquoRecognitionof galactose-deficient O-glycans in the hinge region of IgA1by N-acetylgalactosamine-specific snail lectins a comparativebinding studyrdquoBiochemistry vol 49 no 27 pp 5671ndash5682 2010

[30] J S Moore R Kulhavy M Tomana et al ldquoReactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteinsrdquoMolecular Immunology vol 44 no 10 pp 2598ndash2604 2007

[31] S J Mellis and J U Baenziger ldquoStructures of the O-glyco-sidically linked oligosaccharides of human IgDrdquo Journal of Bio-logical Chemistry vol 258 no 19 pp 11557ndash11563 1983

[32] T SMattu R J Pleass A CWillis et al ldquoThe glycosylation andstructure of human serum IgA1 Fab and Fc regions and the roleof N-glycosylation on Fc120572 receptor interactionsrdquoThe Journal ofBiological Chemistry vol 273 no 4 pp 2260ndash2272 1998

[33] M C Field S Amatayakul-Chantler T W Rademacher PM Rudd and R A Dwek ldquoStructural analysis of the N-glycans from human immunoglobulin A1 comparison of nor-mal human serum immunoglobulin A1 with that isolated frompatients with rheumatoid arthritisrdquo Biochemical Journal vol299 no 1 pp 261ndash275 1994

[34] E Arnett and S Seveau ldquoThe multifaceted activities of mam-malian defensinsrdquo Current Pharmaceutical Design vol 17 no38 pp 4254ndash4269 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 3

The reaction was stopped with 1molL sulfuric acid and theabsence was measured at 450 nm with an EL312 Bio-Kineticsmicroplate reader (Bio-Tek Instruments Winooski VT) Toassess the degree of intra-assay variability one sample hadbeen repeated 3 times to minimize the deviation

23 Statistical Analyses Means and standard deviations wereused to summarize statistics for normally distributed quan-titative variables We used median and IQR to summarizenonnormally distributed variables and ratios and percentagesfor categorical data Studentrsquos t-test (two groups) or analysisof variance (multiple groups)was used to compare differencesinmeans for continuous variables Differences in proportionswere tested by 1205942 test The association of plasma HNP1ndash3levels and the primary outcome were analyzed by Cox pro-portional hazards models We used natural log to transformHNP1ndash3 and Gd-IgA1 as it was highly skewed to the rightin this group of patients Statistical analysis was achievedwith the SPSS 160 software package (SPSS Inc ChicagoIL) A two-tailed 119901 value lt 005 was considered statisticallysignificant

3 Results

31 Patient Baseline Characteristics and Clinical Outcome Asis shown in Table 1 there were 86 (509) males and 83(491) females with mean age at the time of kidney biopsyof 327 plusmn 105 yearsThemedian proteinuria of IgAN patientson biopsy was 198plusmn190 g per 24 h (range 014ndash1372 g24 h)and median Scr was 91 120583molL (range 54ndash789120583molL) Theaverage eGFR was 8166 plusmn 2684mLmin per 173m2 (range630ndash16490mLmin per 173m2) Systolic blood pressurewas 124 plusmn 17mmHg and diastolic blood pressure was 80 plusmn12mmHg with 49 patients (29) being hypertensive atbaseline The distribution by Haas grades I II III IV andV was 101 0 343 42 and 136 respectively Themedian follow-up time was 48months (range 12ndash96monthsTable 1) 165 (976) patients receivedACE inhibitors orARBstherapy and 79 (467) received oral corticosteroids alone orcombined with other immunosuppressive agents during thefollow-up period In total 30 patients reached the compositeend point of 50 decline in eGFR ESRD or death

32 Levels of HNP1ndash3 in IgAN Patients We first set out todetermine the levels of theHNP1ndash3 in the plasma of our IgANpatients by ELISA The mean levels of plasma 120572-defensins83 healthy controls and 169 patients with IgA nephropathywere 50 ngmL and 7842 ngmL As can be shown in Figure 1some patients with IgAN showedmarked value of120572-defensinWhen the results were adjusted to the value of the meanlevel of 120572-defensin in IgAN patients it was shown that thepercentage of individuals with high levels of 120572-defensinsincreased in patients (225) comparing with that in healthycontrols (96) (119901 = 0013 OR 2719 95 CI 1206ndash6135)

33 The Levels of HNP1ndash3 Correlation with IgAN ClinicalCharacteristics and Prognosis To test the clinical manifes-tations differences between patients with high and low 120572-defensins levels the median 120572-defensins level of healthy

p = 00132000

1800

1600

1400

1000800600400200

100806040200

Plas

ma H

NP1

ndash3(n

gm

L)

7842ngmL

Healthy controls IgAN patients

Figure 1 Plasma 120572-defensins (HNP1ndash3) levels of IgAN patientsand healthy controls The line was mean levels of 120572-defensins for169 IgAN patients (7842 ngmL) defined as the cut-off for dividingpatients into two groups with high (above 7842 ngmL) and low(below 7842 ngmL) 120572-defensins levels

controls (7842 ngmL) was defined as the cut-off for dividingpatients into two groups Low plasma 120572-defensins levelswere defined as levels below 7842 ngmL High plasma 120572-defensins levels were defined as levels above 7842 ngmL131 (775) IgAN patients were assigned to the group withlow (below 7842 ngmL) 120572-defensins levels and 38 (225)IgAN patients were assigned to the group with high (above7842 ngmL)120572-defensins levelsThemain clinicalmanifesta-tions demonstrated that there were no differences in age sexblood pressure initial proteinuria and eGFR in two groupsof patients Serum creatinine and eGFR were not associatedwith the levels of 120572-defensins in IgANpatients likely suggest-ing no accumulation of 120572-defensins as the result of reducedglomerular filtration rate (119903 = 0039 119901 = 0614 and 119903 =minus0031 119901 = 0687 resp) (Figures 2(d) and 2(c)) In additionthere was no correlation between the neutrophil count andthe levels of 120572-defensins in IgAN patients (119903 = 0140 119901 =0093) (Figure 2(b))

In the group of patients with high 120572-defensins plasma lev-els Gd-IgA1 was significantly lower (median 2369847UmLand interquartile range 20310ndash35001UmL) than patientswith low 120572-defensins levels (median 31856UmL interquar-tile range 24280ndash40567UmL and 119901 = 0036) More impor-tantly we also found that low Gd-IgA1 was correlated withhigh 120572-defensins levels (119903 = minus0204 119901 = 0008) (Figure 2(a))Levels of plasma IgA or plasma IgA1 showed no significantbetween patients with high and low 120572-defensins (Table 1)

No association were observed in plasma HNP1ndash3 levelwith other clinical manifestations (such as SBP DBP prodro-mal infection gross hematuria 24-hour UTP Scr and eGFR)and renal outcome (Table 1)

4 Discussion

In this study we measured the levels of HNP1ndash3 in IgANpatients We found that the 120572-defensins in IgAN patientswere elevated compared with healthy controls which wasindependent of renal function and neutrophil count The

4 Disease Markers

Table 1 Clinical and laboratory data for IgAN patients with different plasma 120572-defensins levels

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

Age (years) 327 plusmn 105 325 plusmn 107 335 plusmn 98 0614

Gender (malefemale ) 86 (509)83(491)

67 (511)64(489)

19 (500)19(500) 0901

SBP (mmHg) 1239 plusmn 169 1248 plusmn 178 1207 plusmn 133 0184DBP (mmHg) 795 plusmn 132 800 plusmn 138 778 plusmn 107 0350Neutrophil count (lowast109L) 462 plusmn 200 449 plusmn 207 508 plusmn 171 0147

Prodromal infection () 127 (751)42(249)

98 (748)33(252)

29 (763)9(237) 0850

CRP () 112 (663)7 (41) 82 (626)5 (38) 30 (789)2 (53) 0918ESR (mm1st h) 181 plusmn 177 175 plusmn 169 198 plusmn 204 0514TG (mmolL) 193 plusmn 151 198 plusmn 160 179 plusmn 111 0499TCHOL (mmolL) 503 plusmn 150 498 plusmn 146 518 plusmn 1663 0457HDL (mmolL) 121 plusmn 057 123 plusmn 062 114 plusmn 033 0384LDL (mmolL) 289 plusmn 098 283 plusmn 090 309 plusmn 119 0153

Hyperlipidemia () 66 (391)103(609)

51 (389)80(611)

15 (395)23(605) 0952

Uric acid (120583molL) 3567 plusmn 1183 3615 plusmn 1190 3402 plusmn 1159 0330Scr (120583molL) 1048 plusmn 656 1064 plusmn 716 993 plusmn 391 0556eGFR (mLmin per 173m2)3 814 plusmn 278 809 plusmn 278 832 plusmn 282 0654

Stage 1 2 3 4 and 5 CKD ()50 (296) 67

(396) 34 (201)2 (12)

40 (305) 60(458) 30 (229)

1 (08)

10 (263) 7(184) 4 (105) 1

(26)0224

Gd-IgA1 (UmL medianIQR)

30386(23143ndash39602)

31856(24280ndash40567)

23698(20310ndash35001) 0036

Total IgA (120583gmL medianIQR)

26300(21535ndash33335)

26580(21670ndash33470)

25705(20293ndash33653) 0565

Plasma IgA1 (120583gmL medianIQR)

16964(13829ndash20757)

1643(1353ndash2064)

18756(14872ndash21646) 0318

Initial proteinuria (gday) 198 plusmn 190 189 plusmn 165 232 plusmn 258 0219Urinary RBC (120583L) 286 plusmn 537 318 plusmn 599 184 plusmn 219 0181

Gross hematuria () 129 (763)40(237)

99 (756)32(244) 30 (789)8 (211) 0667

24-hour UTP (gday) 114 plusmn 106 113 plusmn 108 116 plusmn 102 0886

Histological grading I II IIIIV V4 ()

17 (101) 0 (0)58 (343) 71

(420) 23 (136)

10 (76) 0 (0) 43(328) 58 (443)

20 (153)

7 (184) 0 (0) 15(395) 13 (342)

3 (79)0129

Oxford classification

M () 54 (320) 112(663)

38 (290)92(702)

16 (421)20(526) 0085

E () 60 (355) 106(627)

45 (344)85(649)

15 (395)21(553) 0436

S () 45 (266) 121(716)

36 (275)94(718) 9 (237)27 (711) 0748

T () 109 (645) 36(213) 21 (124)

86 (656) 28(214) 16 (122)

23 (605) 8(222) 5 (132) 0959

Disease Markers 5

Table 1 Continued

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

TherapyACE inhibitors or ARBs () 4 (24)165 (976) 3 (23)128 (977) 1 (26)37 (974) 0903

Prednisone () 79 (467)90(533)

65 (496)66(504)

14 (368)24(632) 0165

SBP systolic blood pressure DBP diastolic blood pressure CRP C-reactive protein ESR erythrocyte sedimentation rate TG triglycerides TCHOL totalcholesterol HDL high-density lipoprotein cholesterol LDL low-density lipoprotein cholesterol Scr serum creatinine eGFR estimate glomerular filtrationrate CKD chronic kidney disease 24-hour UTP 24-hour total urine protein Gd-IgA1 galactose-deficient IgA1 ACE angiotensin-converting enzyme ARBangiotensin II receptor blocker1Low plasma 120572-defensins levels were defined as levels below 7842 ngmL 2High plasma 120572-defensins levels were defined as levels above 7842 ngmL 3eGFRwas calculated based on MDRD formula modified population 4Histological grading was classified according to pathological proposed by Haas

r = minus0204p = 0008

2000

1500

1000

500

0

Gd-

IgA1

150010005000 2000

HNP1ndash3(a)

r = 0140p = 0093

15

10

5

0

Neu

troph

il co

unt

150010005000 2000

HNP1ndash3(b)

r = minus0031

p = 0687

eGFR

150010005000 2000

HNP1ndash3

200

150

100

50

0

(c)

r = 0039p = 0614

Scr

150010005000 2000

HNP1ndash3

1000

800

600

400

200

0

(d)

Figure 2 Two-tailed bivariate correlations between plasma levels of HNP1ndash3 (ngmL) and IgAN clinical ((a) Gd-IgA1 UmL (b) neutrophilcount lowast109L (c) eGFR mLmin per 173m2 (d) Scr 120583molL) characteristics

high level of plasma 120572-defensins might be due to neutrophilactivation instead of increased neutrophil numbers Moreimportantly we also found that low Gd-IgA1 was associatedwith high 120572-defensins levels On the basis of the potentialpathogenic role of Gd-IgA1 in the development of IgAN 120572-defensins may be involved in the pathogenesis of IgAN notjust through serological changes

The defensins of vertebrate animals comprised by 18ndash45 amino-acid residues are small cationic peptides includ-ing three subfamilies of defensins namely 120572- 120573- and 120579-defensins There are six human 120572-defensin peptides namelyHNP1ndash3 HNP4 and HD5-6 which shows consistency inamino acids sequences to some degree HNP1ndash3 and HD5have lectin-like affinity for glycosylated molecules that can

6 Disease Markers

bind carbohydrates in making glycolipids glycoproteins andproteoglycans [22ndash25] IgA1 are glycoproteins which consistof a hinge region (HR) with a high content of proline (Pro)serine (Ser) and threonine (Thr) between the first and secondheavy chain constant region domains Within the IgA1HR six O-linked glycan chains have been identified whichhave been localized to SerThr residues Thr225 Thr228Ser230 Ser232 Thr233 and Thr236 [26ndash30] IgA1 O-linkedglycans are composed of N-acetylgalactosamine (GalNAc)with a 1205731 3-linked Gal 1205722 3-linked Gal or 1205722 6-linkedGalNAc may be capable of sialic acid attachment [31ndash33]Glycosylation is the process that a carbohydrate such asa glycosyl donor is attached to a hydroxyl or a glycosylacceptor Gd-IgA1 is mainly represented as the deficiencyof galactose in hinge regions which may lead to increasedexposure of glycosyl in GalNAc 120572-defensin is a lectin-likepeptide which has carbohydrate-binding activity [25] Wehypothesized that 120572-defensin could bond to exposed glycosylin N-acetylgalactosamine of IgA1 molecular which mightcontribute to IgA1 aggregation and deposition in mesangialregions Our present study supported the hypothesis showingthat120572-defensinswere elevated in patientswith IgANandwerenegatively correlated with galactose deficiency of circulatingIgA1

In addition to their role in circulation 120572-defensins canalso play a role in local tissues 120572-defensins can inducethe secretion of inflammatory cytokines and inflamma-tory chemokines in lung epithelial cells promoting localinflammatory response Besides 120572-defensins can also facil-itate the migration of fibroblastsepithelial cell proliferationand increase extracellular matrix production [34] Since 120572-defensins have both inflammatory and fibrosis effects in otherorgans further study should be carried out in depth abouttheir effects on renal tissue Unfortunately no significantdifference as for M E S and T classification was observedbetween patients with the low and high 120572-defensins levelsThis negative finding might be due to certain cell lines usedin the inflammatory and fibrosis effects of 120572-defensins studieswithout evidences from renal resident cells Since the effect ofdifferent cell lines might not be the same the inflammatoryand fibrosis effects of renal resident cells should be furtherinvestigatedHowever the correlation between the levels of120572-defensins and Gd-IgA1 was relatively low And no significantassociation of clinical manifestations was observed betweenpatients with high and low levels of 120572-defensins Future morewidespread replications and functional assays were of specialinterest

In conclusion our study identified that the 120572-defensinsin IgAN patients were elevated which was independent ofrenal function and neutrophil count And we also foundthat high Gd-IgA1 was associated with low 120572-defensinslevels We speculate that 120572-defensins may bind Gd-IgA1 incirculation which finally deposit in the kidney promotinglocal inflammation and fibrosis

Abbreviations

IgAN IgA nephropathyGd-IgA1 Galactose-deficient IgA1

GWAS Genome-wide association studyHNP Human neutrophil peptideESRD End-stage renal diseaseSNPs Single nucleotide polymorphismseGFR Estimated glomerular filtration rateGalNAc N-AcetylgalactosamineACE Angiotensin-converting enzymeARB Angiotensin II receptor blockerCKD Chronic kidney diseaseDBP Diastolic blood pressureSBP Systolic blood pressureCRP C-reactive proteinESR Erythrocyte sedimentation rateTG TriglyceridesTCHOL Total cholesterolHDL High-density lipoprotein cholesterolLDL Low-density lipoprotein cholesterolScr Serum creatinine24-hour UTP 24-hour total urine protein

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Xu-jie Zhou and Hong Zhang designed the research Yuan-yuan Qi and Fa-juan Cheng performed the research Yuan-yuan Qi and Xu-jie Zhou analyzed the data and Yuan-yuanQi and Xu-jie Zhou wrote the paper

Acknowledgments

The authors thank their collaborators including Ali GGharavi fromDepartment of Medicine Columbia UniversityCollege of Physicians and Surgeons New York USA forassistance They also thank all the members of the laboratoryfor technical assistance and the patients and their familiesfor their cooperation and for giving consent to participatein this study This work was supported by Grants fromthe National Natural Science Foundation of China (no81570629) Beijing Natural Science Foundation (7152148)Chinese Society of Nephrology (15020030591) the MajorState Basic Research Development Program of China (973Program no 2012CB517700) the Research Fund of BeijingMunicipal Science and Technology for the Outstanding PhDProgram (no 20121000110) the Foundation of the Ministryof Education of China (no 20120001120008) the NaturalScience Fund of China to the Innovation Research Group(no 81021004) and National Key Technology RampD Program(2013BAI09B14)

Disease Markers 7

References

[1] M Levy and J Berger ldquoWorldwide perspective of IgA nephro-pathyrdquo American Journal of Kidney Diseases vol 12 no 5 pp340ndash347 1988

[2] A Amore P Cirina G Conti P Brusa L Peruzzi and RCoppo ldquoGlycosylation of circulating IgA in patients with IgAnephropathy modulates proliferation and apoptosis of mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 12no 9 pp 1862ndash1871 2001

[3] J Barratt and J Feehally ldquoIgA nephropathyrdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2088ndash20972005

[4] J Barratt J Feehally and A C Smith ldquoPathogenesis of IgAnephropathyrdquo Seminars in Nephrology vol 24 no 3 pp 197ndash217 2004

[5] A C Smith J F de Wolff K Molyneux J Feehally and JBarratt ldquoO-glycosylation of serum IgD in IgA nephropathyrdquoJournal of the American Society of Nephrology vol 17 no 4 pp1192ndash1199 2006

[6] H Suzuki Z Moldoveanu S Hall et al ldquoIgA1-secreting celllines from patients with IgA nephropathy produce aberrantlyglycosylated IgA1rdquo Journal of Clinical Investigation vol 118 no2 pp 629ndash639 2008

[7] J Novak B A Julian M Tomana and J Mestecky ldquoIgAglycosylation and IgA immune complexes in the pathogenesisof IgA nephropathyrdquo Seminars in Nephrology vol 28 no 1 pp78ndash87 2008

[8] A C Allen E M Bailey P E Brenchley K S Buck J Barrattand J Feehally ldquoMesangial Iga1 in IgA nephropathy exhibitsaberrant O-glycosylation observations in three patientsrdquo Kid-ney International vol 60 no 3 pp 969ndash973 2001

[9] N Zhao P Hou J Lv et al ldquoThe level of galactose-deficientIgA1 in the sera of patients with IgA nephropathy is associatedwith disease progressionrdquo Kidney International vol 82 no 7pp 790ndash796 2012

[10] A G Gharavi K Kiryluk M Choi et al ldquoGenome-wide asso-ciation study identifies susceptibility loci for IgA nephropathyrdquoNature Genetics vol 43 no 4 pp 321ndash327 2011

[11] C Yang W Jie Y Yanlong et al ldquoGenome-wide associationstudy identifies TNFSF13 as a susceptibility gene for IgA in aSouthChinese population in smokersrdquo Immunogenetics vol 64no 10 pp 747ndash753 2012

[12] K Kiryluk Y Li F Scolari et al ldquoDiscovery of new risk loci forIgAnephropathy implicates genes involved in immunity againstintestinal pathogensrdquo Nature Genetics vol 46 no 11 pp 1187ndash1196 2014

[13] X-Q Yu M Li H Zhang et al ldquoA genome-wide associationstudy in Han Chinese identifies multiple susceptibility loci forIgA nephropathyrdquo Nature Genetics vol 44 no 2 pp 178ndash1822012

[14] Y Y Qi X J Zhou F J Cheng et al ldquoDEFA gene variantsassociated with IgA nephropathy in a Chinese populationrdquoGenes amp Immunity vol 16 no 3 pp 231ndash237 2015

[15] R I Lehrer and W Lu ldquo120572-Defensins in human innate immu-nityrdquo Immunological Reviews vol 245 no 1 pp 84ndash112 2012

[16] D Yang A Biragyn L W Kwak and J J Oppenheim ldquoMam-malian defensins in immunity more than just microbicidalrdquoTrends in Immunology vol 23 no 6 pp 291ndash296 2002

[17] Z M Sthoeger S Bezalel N Chapnik I Asher and O FroyldquoHigh 120572-defensin levels in patients with systemic lupus erythe-matosusrdquo Immunology vol 127 no 1 pp 116ndash122 2009

[18] F-J Cheng X-J Zhou Y-F Zhao M-H Zhao and H ZhangldquoHuman neutrophil peptide 1ndash3 a component of the neutrophil

extracellular trap as a potential biomarker of lupus nephritisrdquoInternational Journal of Rheumatic Diseases vol 18 no 5 pp533ndash540 2015

[19] M Saraheimo C Forsblom K Pettersson-Fernholm A Flyvb-jerg P-H Groop and J Frystyk ldquoIncreased levels of 120572-defensin(-1 -2 and -3) in type 1 diabetic patients with nephropathyrdquoNephrology Dialysis Transplantation vol 23 no 3 pp 914ndash9182008

[20] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[21] M Haas ldquoHistologic subclassification of IgA nephropathya clinicopathologic study of 244 casesrdquo American Journal ofKidney Diseases vol 29 no 6 pp 829ndash842 1997

[22] R I Lehrer G Jung P Ruchala S Andre H J Gabius andW Lu ldquoMultivalent binding of carbohydrates by the human 120572-defensin HD5rdquo The Journal of Immunology vol 183 no 1 pp480ndash490 2009

[23] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[24] E Leikina H Delanoe-Ayari K Melikov et al ldquoCarbohydrate-bindingmolecules inhibit viral fusion and entry by crosslinkingmembrane glycoproteinsrdquoNature Immunology vol 6 no 10 pp995ndash1001 2005

[25] W Wang S M Owen D L Rudolph et al ldquoActivity of 120572- and120579-defensins against primary isolates of HIV-1rdquo The Journal ofImmunology vol 173 no 1 pp 515ndash520 2004

[26] Z Moldoveanu R J Wyatt J Y Lee et al ldquoPatients withIgA nephropathy have increased serum galactose-deficient IgA1levelsrdquo Kidney International vol 71 no 11 pp 1148ndash1154 2007

[27] M B Renfrow H J Cooper M Tomana et al ldquoDeterminationof aberrant O-glycosylation in the IgA1 hinge region by electroncapture dissociation fourier transform-ion cyclotron resonancemass spectrometryrdquo Journal of Biological Chemistry vol 280no 19 pp 19136ndash19145 2005

[28] K Takahashi S BWallH Suzuki et al ldquoClusteredO-glycans ofIgA1 defining macro- and microheterogeneity by use of elec-tron capturetransfer dissociationrdquoMolecular and Cellular Pro-teomics vol 9 no 11 pp 2545ndash2557 2010

[29] M M Gomes H Suzuki M T Brooks et al ldquoRecognitionof galactose-deficient O-glycans in the hinge region of IgA1by N-acetylgalactosamine-specific snail lectins a comparativebinding studyrdquoBiochemistry vol 49 no 27 pp 5671ndash5682 2010

[30] J S Moore R Kulhavy M Tomana et al ldquoReactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteinsrdquoMolecular Immunology vol 44 no 10 pp 2598ndash2604 2007

[31] S J Mellis and J U Baenziger ldquoStructures of the O-glyco-sidically linked oligosaccharides of human IgDrdquo Journal of Bio-logical Chemistry vol 258 no 19 pp 11557ndash11563 1983

[32] T SMattu R J Pleass A CWillis et al ldquoThe glycosylation andstructure of human serum IgA1 Fab and Fc regions and the roleof N-glycosylation on Fc120572 receptor interactionsrdquoThe Journal ofBiological Chemistry vol 273 no 4 pp 2260ndash2272 1998

[33] M C Field S Amatayakul-Chantler T W Rademacher PM Rudd and R A Dwek ldquoStructural analysis of the N-glycans from human immunoglobulin A1 comparison of nor-mal human serum immunoglobulin A1 with that isolated frompatients with rheumatoid arthritisrdquo Biochemical Journal vol299 no 1 pp 261ndash275 1994

[34] E Arnett and S Seveau ldquoThe multifaceted activities of mam-malian defensinsrdquo Current Pharmaceutical Design vol 17 no38 pp 4254ndash4269 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Disease Markers

Table 1 Clinical and laboratory data for IgAN patients with different plasma 120572-defensins levels

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

Age (years) 327 plusmn 105 325 plusmn 107 335 plusmn 98 0614

Gender (malefemale ) 86 (509)83(491)

67 (511)64(489)

19 (500)19(500) 0901

SBP (mmHg) 1239 plusmn 169 1248 plusmn 178 1207 plusmn 133 0184DBP (mmHg) 795 plusmn 132 800 plusmn 138 778 plusmn 107 0350Neutrophil count (lowast109L) 462 plusmn 200 449 plusmn 207 508 plusmn 171 0147

Prodromal infection () 127 (751)42(249)

98 (748)33(252)

29 (763)9(237) 0850

CRP () 112 (663)7 (41) 82 (626)5 (38) 30 (789)2 (53) 0918ESR (mm1st h) 181 plusmn 177 175 plusmn 169 198 plusmn 204 0514TG (mmolL) 193 plusmn 151 198 plusmn 160 179 plusmn 111 0499TCHOL (mmolL) 503 plusmn 150 498 plusmn 146 518 plusmn 1663 0457HDL (mmolL) 121 plusmn 057 123 plusmn 062 114 plusmn 033 0384LDL (mmolL) 289 plusmn 098 283 plusmn 090 309 plusmn 119 0153

Hyperlipidemia () 66 (391)103(609)

51 (389)80(611)

15 (395)23(605) 0952

Uric acid (120583molL) 3567 plusmn 1183 3615 plusmn 1190 3402 plusmn 1159 0330Scr (120583molL) 1048 plusmn 656 1064 plusmn 716 993 plusmn 391 0556eGFR (mLmin per 173m2)3 814 plusmn 278 809 plusmn 278 832 plusmn 282 0654

Stage 1 2 3 4 and 5 CKD ()50 (296) 67

(396) 34 (201)2 (12)

40 (305) 60(458) 30 (229)

1 (08)

10 (263) 7(184) 4 (105) 1

(26)0224

Gd-IgA1 (UmL medianIQR)

30386(23143ndash39602)

31856(24280ndash40567)

23698(20310ndash35001) 0036

Total IgA (120583gmL medianIQR)

26300(21535ndash33335)

26580(21670ndash33470)

25705(20293ndash33653) 0565

Plasma IgA1 (120583gmL medianIQR)

16964(13829ndash20757)

1643(1353ndash2064)

18756(14872ndash21646) 0318

Initial proteinuria (gday) 198 plusmn 190 189 plusmn 165 232 plusmn 258 0219Urinary RBC (120583L) 286 plusmn 537 318 plusmn 599 184 plusmn 219 0181

Gross hematuria () 129 (763)40(237)

99 (756)32(244) 30 (789)8 (211) 0667

24-hour UTP (gday) 114 plusmn 106 113 plusmn 108 116 plusmn 102 0886

Histological grading I II IIIIV V4 ()

17 (101) 0 (0)58 (343) 71

(420) 23 (136)

10 (76) 0 (0) 43(328) 58 (443)

20 (153)

7 (184) 0 (0) 15(395) 13 (342)

3 (79)0129

Oxford classification

M () 54 (320) 112(663)

38 (290)92(702)

16 (421)20(526) 0085

E () 60 (355) 106(627)

45 (344)85(649)

15 (395)21(553) 0436

S () 45 (266) 121(716)

36 (275)94(718) 9 (237)27 (711) 0748

T () 109 (645) 36(213) 21 (124)

86 (656) 28(214) 16 (122)

23 (605) 8(222) 5 (132) 0959

Disease Markers 5

Table 1 Continued

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

TherapyACE inhibitors or ARBs () 4 (24)165 (976) 3 (23)128 (977) 1 (26)37 (974) 0903

Prednisone () 79 (467)90(533)

65 (496)66(504)

14 (368)24(632) 0165

SBP systolic blood pressure DBP diastolic blood pressure CRP C-reactive protein ESR erythrocyte sedimentation rate TG triglycerides TCHOL totalcholesterol HDL high-density lipoprotein cholesterol LDL low-density lipoprotein cholesterol Scr serum creatinine eGFR estimate glomerular filtrationrate CKD chronic kidney disease 24-hour UTP 24-hour total urine protein Gd-IgA1 galactose-deficient IgA1 ACE angiotensin-converting enzyme ARBangiotensin II receptor blocker1Low plasma 120572-defensins levels were defined as levels below 7842 ngmL 2High plasma 120572-defensins levels were defined as levels above 7842 ngmL 3eGFRwas calculated based on MDRD formula modified population 4Histological grading was classified according to pathological proposed by Haas

r = minus0204p = 0008

2000

1500

1000

500

0

Gd-

IgA1

150010005000 2000

HNP1ndash3(a)

r = 0140p = 0093

15

10

5

0

Neu

troph

il co

unt

150010005000 2000

HNP1ndash3(b)

r = minus0031

p = 0687

eGFR

150010005000 2000

HNP1ndash3

200

150

100

50

0

(c)

r = 0039p = 0614

Scr

150010005000 2000

HNP1ndash3

1000

800

600

400

200

0

(d)

Figure 2 Two-tailed bivariate correlations between plasma levels of HNP1ndash3 (ngmL) and IgAN clinical ((a) Gd-IgA1 UmL (b) neutrophilcount lowast109L (c) eGFR mLmin per 173m2 (d) Scr 120583molL) characteristics

high level of plasma 120572-defensins might be due to neutrophilactivation instead of increased neutrophil numbers Moreimportantly we also found that low Gd-IgA1 was associatedwith high 120572-defensins levels On the basis of the potentialpathogenic role of Gd-IgA1 in the development of IgAN 120572-defensins may be involved in the pathogenesis of IgAN notjust through serological changes

The defensins of vertebrate animals comprised by 18ndash45 amino-acid residues are small cationic peptides includ-ing three subfamilies of defensins namely 120572- 120573- and 120579-defensins There are six human 120572-defensin peptides namelyHNP1ndash3 HNP4 and HD5-6 which shows consistency inamino acids sequences to some degree HNP1ndash3 and HD5have lectin-like affinity for glycosylated molecules that can

6 Disease Markers

bind carbohydrates in making glycolipids glycoproteins andproteoglycans [22ndash25] IgA1 are glycoproteins which consistof a hinge region (HR) with a high content of proline (Pro)serine (Ser) and threonine (Thr) between the first and secondheavy chain constant region domains Within the IgA1HR six O-linked glycan chains have been identified whichhave been localized to SerThr residues Thr225 Thr228Ser230 Ser232 Thr233 and Thr236 [26ndash30] IgA1 O-linkedglycans are composed of N-acetylgalactosamine (GalNAc)with a 1205731 3-linked Gal 1205722 3-linked Gal or 1205722 6-linkedGalNAc may be capable of sialic acid attachment [31ndash33]Glycosylation is the process that a carbohydrate such asa glycosyl donor is attached to a hydroxyl or a glycosylacceptor Gd-IgA1 is mainly represented as the deficiencyof galactose in hinge regions which may lead to increasedexposure of glycosyl in GalNAc 120572-defensin is a lectin-likepeptide which has carbohydrate-binding activity [25] Wehypothesized that 120572-defensin could bond to exposed glycosylin N-acetylgalactosamine of IgA1 molecular which mightcontribute to IgA1 aggregation and deposition in mesangialregions Our present study supported the hypothesis showingthat120572-defensinswere elevated in patientswith IgANandwerenegatively correlated with galactose deficiency of circulatingIgA1

In addition to their role in circulation 120572-defensins canalso play a role in local tissues 120572-defensins can inducethe secretion of inflammatory cytokines and inflamma-tory chemokines in lung epithelial cells promoting localinflammatory response Besides 120572-defensins can also facil-itate the migration of fibroblastsepithelial cell proliferationand increase extracellular matrix production [34] Since 120572-defensins have both inflammatory and fibrosis effects in otherorgans further study should be carried out in depth abouttheir effects on renal tissue Unfortunately no significantdifference as for M E S and T classification was observedbetween patients with the low and high 120572-defensins levelsThis negative finding might be due to certain cell lines usedin the inflammatory and fibrosis effects of 120572-defensins studieswithout evidences from renal resident cells Since the effect ofdifferent cell lines might not be the same the inflammatoryand fibrosis effects of renal resident cells should be furtherinvestigatedHowever the correlation between the levels of120572-defensins and Gd-IgA1 was relatively low And no significantassociation of clinical manifestations was observed betweenpatients with high and low levels of 120572-defensins Future morewidespread replications and functional assays were of specialinterest

In conclusion our study identified that the 120572-defensinsin IgAN patients were elevated which was independent ofrenal function and neutrophil count And we also foundthat high Gd-IgA1 was associated with low 120572-defensinslevels We speculate that 120572-defensins may bind Gd-IgA1 incirculation which finally deposit in the kidney promotinglocal inflammation and fibrosis

Abbreviations

IgAN IgA nephropathyGd-IgA1 Galactose-deficient IgA1

GWAS Genome-wide association studyHNP Human neutrophil peptideESRD End-stage renal diseaseSNPs Single nucleotide polymorphismseGFR Estimated glomerular filtration rateGalNAc N-AcetylgalactosamineACE Angiotensin-converting enzymeARB Angiotensin II receptor blockerCKD Chronic kidney diseaseDBP Diastolic blood pressureSBP Systolic blood pressureCRP C-reactive proteinESR Erythrocyte sedimentation rateTG TriglyceridesTCHOL Total cholesterolHDL High-density lipoprotein cholesterolLDL Low-density lipoprotein cholesterolScr Serum creatinine24-hour UTP 24-hour total urine protein

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Xu-jie Zhou and Hong Zhang designed the research Yuan-yuan Qi and Fa-juan Cheng performed the research Yuan-yuan Qi and Xu-jie Zhou analyzed the data and Yuan-yuanQi and Xu-jie Zhou wrote the paper

Acknowledgments

The authors thank their collaborators including Ali GGharavi fromDepartment of Medicine Columbia UniversityCollege of Physicians and Surgeons New York USA forassistance They also thank all the members of the laboratoryfor technical assistance and the patients and their familiesfor their cooperation and for giving consent to participatein this study This work was supported by Grants fromthe National Natural Science Foundation of China (no81570629) Beijing Natural Science Foundation (7152148)Chinese Society of Nephrology (15020030591) the MajorState Basic Research Development Program of China (973Program no 2012CB517700) the Research Fund of BeijingMunicipal Science and Technology for the Outstanding PhDProgram (no 20121000110) the Foundation of the Ministryof Education of China (no 20120001120008) the NaturalScience Fund of China to the Innovation Research Group(no 81021004) and National Key Technology RampD Program(2013BAI09B14)

Disease Markers 7

References

[1] M Levy and J Berger ldquoWorldwide perspective of IgA nephro-pathyrdquo American Journal of Kidney Diseases vol 12 no 5 pp340ndash347 1988

[2] A Amore P Cirina G Conti P Brusa L Peruzzi and RCoppo ldquoGlycosylation of circulating IgA in patients with IgAnephropathy modulates proliferation and apoptosis of mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 12no 9 pp 1862ndash1871 2001

[3] J Barratt and J Feehally ldquoIgA nephropathyrdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2088ndash20972005

[4] J Barratt J Feehally and A C Smith ldquoPathogenesis of IgAnephropathyrdquo Seminars in Nephrology vol 24 no 3 pp 197ndash217 2004

[5] A C Smith J F de Wolff K Molyneux J Feehally and JBarratt ldquoO-glycosylation of serum IgD in IgA nephropathyrdquoJournal of the American Society of Nephrology vol 17 no 4 pp1192ndash1199 2006

[6] H Suzuki Z Moldoveanu S Hall et al ldquoIgA1-secreting celllines from patients with IgA nephropathy produce aberrantlyglycosylated IgA1rdquo Journal of Clinical Investigation vol 118 no2 pp 629ndash639 2008

[7] J Novak B A Julian M Tomana and J Mestecky ldquoIgAglycosylation and IgA immune complexes in the pathogenesisof IgA nephropathyrdquo Seminars in Nephrology vol 28 no 1 pp78ndash87 2008

[8] A C Allen E M Bailey P E Brenchley K S Buck J Barrattand J Feehally ldquoMesangial Iga1 in IgA nephropathy exhibitsaberrant O-glycosylation observations in three patientsrdquo Kid-ney International vol 60 no 3 pp 969ndash973 2001

[9] N Zhao P Hou J Lv et al ldquoThe level of galactose-deficientIgA1 in the sera of patients with IgA nephropathy is associatedwith disease progressionrdquo Kidney International vol 82 no 7pp 790ndash796 2012

[10] A G Gharavi K Kiryluk M Choi et al ldquoGenome-wide asso-ciation study identifies susceptibility loci for IgA nephropathyrdquoNature Genetics vol 43 no 4 pp 321ndash327 2011

[11] C Yang W Jie Y Yanlong et al ldquoGenome-wide associationstudy identifies TNFSF13 as a susceptibility gene for IgA in aSouthChinese population in smokersrdquo Immunogenetics vol 64no 10 pp 747ndash753 2012

[12] K Kiryluk Y Li F Scolari et al ldquoDiscovery of new risk loci forIgAnephropathy implicates genes involved in immunity againstintestinal pathogensrdquo Nature Genetics vol 46 no 11 pp 1187ndash1196 2014

[13] X-Q Yu M Li H Zhang et al ldquoA genome-wide associationstudy in Han Chinese identifies multiple susceptibility loci forIgA nephropathyrdquo Nature Genetics vol 44 no 2 pp 178ndash1822012

[14] Y Y Qi X J Zhou F J Cheng et al ldquoDEFA gene variantsassociated with IgA nephropathy in a Chinese populationrdquoGenes amp Immunity vol 16 no 3 pp 231ndash237 2015

[15] R I Lehrer and W Lu ldquo120572-Defensins in human innate immu-nityrdquo Immunological Reviews vol 245 no 1 pp 84ndash112 2012

[16] D Yang A Biragyn L W Kwak and J J Oppenheim ldquoMam-malian defensins in immunity more than just microbicidalrdquoTrends in Immunology vol 23 no 6 pp 291ndash296 2002

[17] Z M Sthoeger S Bezalel N Chapnik I Asher and O FroyldquoHigh 120572-defensin levels in patients with systemic lupus erythe-matosusrdquo Immunology vol 127 no 1 pp 116ndash122 2009

[18] F-J Cheng X-J Zhou Y-F Zhao M-H Zhao and H ZhangldquoHuman neutrophil peptide 1ndash3 a component of the neutrophil

extracellular trap as a potential biomarker of lupus nephritisrdquoInternational Journal of Rheumatic Diseases vol 18 no 5 pp533ndash540 2015

[19] M Saraheimo C Forsblom K Pettersson-Fernholm A Flyvb-jerg P-H Groop and J Frystyk ldquoIncreased levels of 120572-defensin(-1 -2 and -3) in type 1 diabetic patients with nephropathyrdquoNephrology Dialysis Transplantation vol 23 no 3 pp 914ndash9182008

[20] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[21] M Haas ldquoHistologic subclassification of IgA nephropathya clinicopathologic study of 244 casesrdquo American Journal ofKidney Diseases vol 29 no 6 pp 829ndash842 1997

[22] R I Lehrer G Jung P Ruchala S Andre H J Gabius andW Lu ldquoMultivalent binding of carbohydrates by the human 120572-defensin HD5rdquo The Journal of Immunology vol 183 no 1 pp480ndash490 2009

[23] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[24] E Leikina H Delanoe-Ayari K Melikov et al ldquoCarbohydrate-bindingmolecules inhibit viral fusion and entry by crosslinkingmembrane glycoproteinsrdquoNature Immunology vol 6 no 10 pp995ndash1001 2005

[25] W Wang S M Owen D L Rudolph et al ldquoActivity of 120572- and120579-defensins against primary isolates of HIV-1rdquo The Journal ofImmunology vol 173 no 1 pp 515ndash520 2004

[26] Z Moldoveanu R J Wyatt J Y Lee et al ldquoPatients withIgA nephropathy have increased serum galactose-deficient IgA1levelsrdquo Kidney International vol 71 no 11 pp 1148ndash1154 2007

[27] M B Renfrow H J Cooper M Tomana et al ldquoDeterminationof aberrant O-glycosylation in the IgA1 hinge region by electroncapture dissociation fourier transform-ion cyclotron resonancemass spectrometryrdquo Journal of Biological Chemistry vol 280no 19 pp 19136ndash19145 2005

[28] K Takahashi S BWallH Suzuki et al ldquoClusteredO-glycans ofIgA1 defining macro- and microheterogeneity by use of elec-tron capturetransfer dissociationrdquoMolecular and Cellular Pro-teomics vol 9 no 11 pp 2545ndash2557 2010

[29] M M Gomes H Suzuki M T Brooks et al ldquoRecognitionof galactose-deficient O-glycans in the hinge region of IgA1by N-acetylgalactosamine-specific snail lectins a comparativebinding studyrdquoBiochemistry vol 49 no 27 pp 5671ndash5682 2010

[30] J S Moore R Kulhavy M Tomana et al ldquoReactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteinsrdquoMolecular Immunology vol 44 no 10 pp 2598ndash2604 2007

[31] S J Mellis and J U Baenziger ldquoStructures of the O-glyco-sidically linked oligosaccharides of human IgDrdquo Journal of Bio-logical Chemistry vol 258 no 19 pp 11557ndash11563 1983

[32] T SMattu R J Pleass A CWillis et al ldquoThe glycosylation andstructure of human serum IgA1 Fab and Fc regions and the roleof N-glycosylation on Fc120572 receptor interactionsrdquoThe Journal ofBiological Chemistry vol 273 no 4 pp 2260ndash2272 1998

[33] M C Field S Amatayakul-Chantler T W Rademacher PM Rudd and R A Dwek ldquoStructural analysis of the N-glycans from human immunoglobulin A1 comparison of nor-mal human serum immunoglobulin A1 with that isolated frompatients with rheumatoid arthritisrdquo Biochemical Journal vol299 no 1 pp 261ndash275 1994

[34] E Arnett and S Seveau ldquoThe multifaceted activities of mam-malian defensinsrdquo Current Pharmaceutical Design vol 17 no38 pp 4254ndash4269 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 5

Table 1 Continued

All patientsPatients with

low 120572-defensinsplasma levels1

Patients withhigh120572-defensinsplasma levels2

P value

TherapyACE inhibitors or ARBs () 4 (24)165 (976) 3 (23)128 (977) 1 (26)37 (974) 0903

Prednisone () 79 (467)90(533)

65 (496)66(504)

14 (368)24(632) 0165

SBP systolic blood pressure DBP diastolic blood pressure CRP C-reactive protein ESR erythrocyte sedimentation rate TG triglycerides TCHOL totalcholesterol HDL high-density lipoprotein cholesterol LDL low-density lipoprotein cholesterol Scr serum creatinine eGFR estimate glomerular filtrationrate CKD chronic kidney disease 24-hour UTP 24-hour total urine protein Gd-IgA1 galactose-deficient IgA1 ACE angiotensin-converting enzyme ARBangiotensin II receptor blocker1Low plasma 120572-defensins levels were defined as levels below 7842 ngmL 2High plasma 120572-defensins levels were defined as levels above 7842 ngmL 3eGFRwas calculated based on MDRD formula modified population 4Histological grading was classified according to pathological proposed by Haas

r = minus0204p = 0008

2000

1500

1000

500

0

Gd-

IgA1

150010005000 2000

HNP1ndash3(a)

r = 0140p = 0093

15

10

5

0

Neu

troph

il co

unt

150010005000 2000

HNP1ndash3(b)

r = minus0031

p = 0687

eGFR

150010005000 2000

HNP1ndash3

200

150

100

50

0

(c)

r = 0039p = 0614

Scr

150010005000 2000

HNP1ndash3

1000

800

600

400

200

0

(d)

Figure 2 Two-tailed bivariate correlations between plasma levels of HNP1ndash3 (ngmL) and IgAN clinical ((a) Gd-IgA1 UmL (b) neutrophilcount lowast109L (c) eGFR mLmin per 173m2 (d) Scr 120583molL) characteristics

high level of plasma 120572-defensins might be due to neutrophilactivation instead of increased neutrophil numbers Moreimportantly we also found that low Gd-IgA1 was associatedwith high 120572-defensins levels On the basis of the potentialpathogenic role of Gd-IgA1 in the development of IgAN 120572-defensins may be involved in the pathogenesis of IgAN notjust through serological changes

The defensins of vertebrate animals comprised by 18ndash45 amino-acid residues are small cationic peptides includ-ing three subfamilies of defensins namely 120572- 120573- and 120579-defensins There are six human 120572-defensin peptides namelyHNP1ndash3 HNP4 and HD5-6 which shows consistency inamino acids sequences to some degree HNP1ndash3 and HD5have lectin-like affinity for glycosylated molecules that can

6 Disease Markers

bind carbohydrates in making glycolipids glycoproteins andproteoglycans [22ndash25] IgA1 are glycoproteins which consistof a hinge region (HR) with a high content of proline (Pro)serine (Ser) and threonine (Thr) between the first and secondheavy chain constant region domains Within the IgA1HR six O-linked glycan chains have been identified whichhave been localized to SerThr residues Thr225 Thr228Ser230 Ser232 Thr233 and Thr236 [26ndash30] IgA1 O-linkedglycans are composed of N-acetylgalactosamine (GalNAc)with a 1205731 3-linked Gal 1205722 3-linked Gal or 1205722 6-linkedGalNAc may be capable of sialic acid attachment [31ndash33]Glycosylation is the process that a carbohydrate such asa glycosyl donor is attached to a hydroxyl or a glycosylacceptor Gd-IgA1 is mainly represented as the deficiencyof galactose in hinge regions which may lead to increasedexposure of glycosyl in GalNAc 120572-defensin is a lectin-likepeptide which has carbohydrate-binding activity [25] Wehypothesized that 120572-defensin could bond to exposed glycosylin N-acetylgalactosamine of IgA1 molecular which mightcontribute to IgA1 aggregation and deposition in mesangialregions Our present study supported the hypothesis showingthat120572-defensinswere elevated in patientswith IgANandwerenegatively correlated with galactose deficiency of circulatingIgA1

In addition to their role in circulation 120572-defensins canalso play a role in local tissues 120572-defensins can inducethe secretion of inflammatory cytokines and inflamma-tory chemokines in lung epithelial cells promoting localinflammatory response Besides 120572-defensins can also facil-itate the migration of fibroblastsepithelial cell proliferationand increase extracellular matrix production [34] Since 120572-defensins have both inflammatory and fibrosis effects in otherorgans further study should be carried out in depth abouttheir effects on renal tissue Unfortunately no significantdifference as for M E S and T classification was observedbetween patients with the low and high 120572-defensins levelsThis negative finding might be due to certain cell lines usedin the inflammatory and fibrosis effects of 120572-defensins studieswithout evidences from renal resident cells Since the effect ofdifferent cell lines might not be the same the inflammatoryand fibrosis effects of renal resident cells should be furtherinvestigatedHowever the correlation between the levels of120572-defensins and Gd-IgA1 was relatively low And no significantassociation of clinical manifestations was observed betweenpatients with high and low levels of 120572-defensins Future morewidespread replications and functional assays were of specialinterest

In conclusion our study identified that the 120572-defensinsin IgAN patients were elevated which was independent ofrenal function and neutrophil count And we also foundthat high Gd-IgA1 was associated with low 120572-defensinslevels We speculate that 120572-defensins may bind Gd-IgA1 incirculation which finally deposit in the kidney promotinglocal inflammation and fibrosis

Abbreviations

IgAN IgA nephropathyGd-IgA1 Galactose-deficient IgA1

GWAS Genome-wide association studyHNP Human neutrophil peptideESRD End-stage renal diseaseSNPs Single nucleotide polymorphismseGFR Estimated glomerular filtration rateGalNAc N-AcetylgalactosamineACE Angiotensin-converting enzymeARB Angiotensin II receptor blockerCKD Chronic kidney diseaseDBP Diastolic blood pressureSBP Systolic blood pressureCRP C-reactive proteinESR Erythrocyte sedimentation rateTG TriglyceridesTCHOL Total cholesterolHDL High-density lipoprotein cholesterolLDL Low-density lipoprotein cholesterolScr Serum creatinine24-hour UTP 24-hour total urine protein

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Xu-jie Zhou and Hong Zhang designed the research Yuan-yuan Qi and Fa-juan Cheng performed the research Yuan-yuan Qi and Xu-jie Zhou analyzed the data and Yuan-yuanQi and Xu-jie Zhou wrote the paper

Acknowledgments

The authors thank their collaborators including Ali GGharavi fromDepartment of Medicine Columbia UniversityCollege of Physicians and Surgeons New York USA forassistance They also thank all the members of the laboratoryfor technical assistance and the patients and their familiesfor their cooperation and for giving consent to participatein this study This work was supported by Grants fromthe National Natural Science Foundation of China (no81570629) Beijing Natural Science Foundation (7152148)Chinese Society of Nephrology (15020030591) the MajorState Basic Research Development Program of China (973Program no 2012CB517700) the Research Fund of BeijingMunicipal Science and Technology for the Outstanding PhDProgram (no 20121000110) the Foundation of the Ministryof Education of China (no 20120001120008) the NaturalScience Fund of China to the Innovation Research Group(no 81021004) and National Key Technology RampD Program(2013BAI09B14)

Disease Markers 7

References

[1] M Levy and J Berger ldquoWorldwide perspective of IgA nephro-pathyrdquo American Journal of Kidney Diseases vol 12 no 5 pp340ndash347 1988

[2] A Amore P Cirina G Conti P Brusa L Peruzzi and RCoppo ldquoGlycosylation of circulating IgA in patients with IgAnephropathy modulates proliferation and apoptosis of mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 12no 9 pp 1862ndash1871 2001

[3] J Barratt and J Feehally ldquoIgA nephropathyrdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2088ndash20972005

[4] J Barratt J Feehally and A C Smith ldquoPathogenesis of IgAnephropathyrdquo Seminars in Nephrology vol 24 no 3 pp 197ndash217 2004

[5] A C Smith J F de Wolff K Molyneux J Feehally and JBarratt ldquoO-glycosylation of serum IgD in IgA nephropathyrdquoJournal of the American Society of Nephrology vol 17 no 4 pp1192ndash1199 2006

[6] H Suzuki Z Moldoveanu S Hall et al ldquoIgA1-secreting celllines from patients with IgA nephropathy produce aberrantlyglycosylated IgA1rdquo Journal of Clinical Investigation vol 118 no2 pp 629ndash639 2008

[7] J Novak B A Julian M Tomana and J Mestecky ldquoIgAglycosylation and IgA immune complexes in the pathogenesisof IgA nephropathyrdquo Seminars in Nephrology vol 28 no 1 pp78ndash87 2008

[8] A C Allen E M Bailey P E Brenchley K S Buck J Barrattand J Feehally ldquoMesangial Iga1 in IgA nephropathy exhibitsaberrant O-glycosylation observations in three patientsrdquo Kid-ney International vol 60 no 3 pp 969ndash973 2001

[9] N Zhao P Hou J Lv et al ldquoThe level of galactose-deficientIgA1 in the sera of patients with IgA nephropathy is associatedwith disease progressionrdquo Kidney International vol 82 no 7pp 790ndash796 2012

[10] A G Gharavi K Kiryluk M Choi et al ldquoGenome-wide asso-ciation study identifies susceptibility loci for IgA nephropathyrdquoNature Genetics vol 43 no 4 pp 321ndash327 2011

[11] C Yang W Jie Y Yanlong et al ldquoGenome-wide associationstudy identifies TNFSF13 as a susceptibility gene for IgA in aSouthChinese population in smokersrdquo Immunogenetics vol 64no 10 pp 747ndash753 2012

[12] K Kiryluk Y Li F Scolari et al ldquoDiscovery of new risk loci forIgAnephropathy implicates genes involved in immunity againstintestinal pathogensrdquo Nature Genetics vol 46 no 11 pp 1187ndash1196 2014

[13] X-Q Yu M Li H Zhang et al ldquoA genome-wide associationstudy in Han Chinese identifies multiple susceptibility loci forIgA nephropathyrdquo Nature Genetics vol 44 no 2 pp 178ndash1822012

[14] Y Y Qi X J Zhou F J Cheng et al ldquoDEFA gene variantsassociated with IgA nephropathy in a Chinese populationrdquoGenes amp Immunity vol 16 no 3 pp 231ndash237 2015

[15] R I Lehrer and W Lu ldquo120572-Defensins in human innate immu-nityrdquo Immunological Reviews vol 245 no 1 pp 84ndash112 2012

[16] D Yang A Biragyn L W Kwak and J J Oppenheim ldquoMam-malian defensins in immunity more than just microbicidalrdquoTrends in Immunology vol 23 no 6 pp 291ndash296 2002

[17] Z M Sthoeger S Bezalel N Chapnik I Asher and O FroyldquoHigh 120572-defensin levels in patients with systemic lupus erythe-matosusrdquo Immunology vol 127 no 1 pp 116ndash122 2009

[18] F-J Cheng X-J Zhou Y-F Zhao M-H Zhao and H ZhangldquoHuman neutrophil peptide 1ndash3 a component of the neutrophil

extracellular trap as a potential biomarker of lupus nephritisrdquoInternational Journal of Rheumatic Diseases vol 18 no 5 pp533ndash540 2015

[19] M Saraheimo C Forsblom K Pettersson-Fernholm A Flyvb-jerg P-H Groop and J Frystyk ldquoIncreased levels of 120572-defensin(-1 -2 and -3) in type 1 diabetic patients with nephropathyrdquoNephrology Dialysis Transplantation vol 23 no 3 pp 914ndash9182008

[20] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[21] M Haas ldquoHistologic subclassification of IgA nephropathya clinicopathologic study of 244 casesrdquo American Journal ofKidney Diseases vol 29 no 6 pp 829ndash842 1997

[22] R I Lehrer G Jung P Ruchala S Andre H J Gabius andW Lu ldquoMultivalent binding of carbohydrates by the human 120572-defensin HD5rdquo The Journal of Immunology vol 183 no 1 pp480ndash490 2009

[23] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[24] E Leikina H Delanoe-Ayari K Melikov et al ldquoCarbohydrate-bindingmolecules inhibit viral fusion and entry by crosslinkingmembrane glycoproteinsrdquoNature Immunology vol 6 no 10 pp995ndash1001 2005

[25] W Wang S M Owen D L Rudolph et al ldquoActivity of 120572- and120579-defensins against primary isolates of HIV-1rdquo The Journal ofImmunology vol 173 no 1 pp 515ndash520 2004

[26] Z Moldoveanu R J Wyatt J Y Lee et al ldquoPatients withIgA nephropathy have increased serum galactose-deficient IgA1levelsrdquo Kidney International vol 71 no 11 pp 1148ndash1154 2007

[27] M B Renfrow H J Cooper M Tomana et al ldquoDeterminationof aberrant O-glycosylation in the IgA1 hinge region by electroncapture dissociation fourier transform-ion cyclotron resonancemass spectrometryrdquo Journal of Biological Chemistry vol 280no 19 pp 19136ndash19145 2005

[28] K Takahashi S BWallH Suzuki et al ldquoClusteredO-glycans ofIgA1 defining macro- and microheterogeneity by use of elec-tron capturetransfer dissociationrdquoMolecular and Cellular Pro-teomics vol 9 no 11 pp 2545ndash2557 2010

[29] M M Gomes H Suzuki M T Brooks et al ldquoRecognitionof galactose-deficient O-glycans in the hinge region of IgA1by N-acetylgalactosamine-specific snail lectins a comparativebinding studyrdquoBiochemistry vol 49 no 27 pp 5671ndash5682 2010

[30] J S Moore R Kulhavy M Tomana et al ldquoReactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteinsrdquoMolecular Immunology vol 44 no 10 pp 2598ndash2604 2007

[31] S J Mellis and J U Baenziger ldquoStructures of the O-glyco-sidically linked oligosaccharides of human IgDrdquo Journal of Bio-logical Chemistry vol 258 no 19 pp 11557ndash11563 1983

[32] T SMattu R J Pleass A CWillis et al ldquoThe glycosylation andstructure of human serum IgA1 Fab and Fc regions and the roleof N-glycosylation on Fc120572 receptor interactionsrdquoThe Journal ofBiological Chemistry vol 273 no 4 pp 2260ndash2272 1998

[33] M C Field S Amatayakul-Chantler T W Rademacher PM Rudd and R A Dwek ldquoStructural analysis of the N-glycans from human immunoglobulin A1 comparison of nor-mal human serum immunoglobulin A1 with that isolated frompatients with rheumatoid arthritisrdquo Biochemical Journal vol299 no 1 pp 261ndash275 1994

[34] E Arnett and S Seveau ldquoThe multifaceted activities of mam-malian defensinsrdquo Current Pharmaceutical Design vol 17 no38 pp 4254ndash4269 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Disease Markers

bind carbohydrates in making glycolipids glycoproteins andproteoglycans [22ndash25] IgA1 are glycoproteins which consistof a hinge region (HR) with a high content of proline (Pro)serine (Ser) and threonine (Thr) between the first and secondheavy chain constant region domains Within the IgA1HR six O-linked glycan chains have been identified whichhave been localized to SerThr residues Thr225 Thr228Ser230 Ser232 Thr233 and Thr236 [26ndash30] IgA1 O-linkedglycans are composed of N-acetylgalactosamine (GalNAc)with a 1205731 3-linked Gal 1205722 3-linked Gal or 1205722 6-linkedGalNAc may be capable of sialic acid attachment [31ndash33]Glycosylation is the process that a carbohydrate such asa glycosyl donor is attached to a hydroxyl or a glycosylacceptor Gd-IgA1 is mainly represented as the deficiencyof galactose in hinge regions which may lead to increasedexposure of glycosyl in GalNAc 120572-defensin is a lectin-likepeptide which has carbohydrate-binding activity [25] Wehypothesized that 120572-defensin could bond to exposed glycosylin N-acetylgalactosamine of IgA1 molecular which mightcontribute to IgA1 aggregation and deposition in mesangialregions Our present study supported the hypothesis showingthat120572-defensinswere elevated in patientswith IgANandwerenegatively correlated with galactose deficiency of circulatingIgA1

In addition to their role in circulation 120572-defensins canalso play a role in local tissues 120572-defensins can inducethe secretion of inflammatory cytokines and inflamma-tory chemokines in lung epithelial cells promoting localinflammatory response Besides 120572-defensins can also facil-itate the migration of fibroblastsepithelial cell proliferationand increase extracellular matrix production [34] Since 120572-defensins have both inflammatory and fibrosis effects in otherorgans further study should be carried out in depth abouttheir effects on renal tissue Unfortunately no significantdifference as for M E S and T classification was observedbetween patients with the low and high 120572-defensins levelsThis negative finding might be due to certain cell lines usedin the inflammatory and fibrosis effects of 120572-defensins studieswithout evidences from renal resident cells Since the effect ofdifferent cell lines might not be the same the inflammatoryand fibrosis effects of renal resident cells should be furtherinvestigatedHowever the correlation between the levels of120572-defensins and Gd-IgA1 was relatively low And no significantassociation of clinical manifestations was observed betweenpatients with high and low levels of 120572-defensins Future morewidespread replications and functional assays were of specialinterest

In conclusion our study identified that the 120572-defensinsin IgAN patients were elevated which was independent ofrenal function and neutrophil count And we also foundthat high Gd-IgA1 was associated with low 120572-defensinslevels We speculate that 120572-defensins may bind Gd-IgA1 incirculation which finally deposit in the kidney promotinglocal inflammation and fibrosis

Abbreviations

IgAN IgA nephropathyGd-IgA1 Galactose-deficient IgA1

GWAS Genome-wide association studyHNP Human neutrophil peptideESRD End-stage renal diseaseSNPs Single nucleotide polymorphismseGFR Estimated glomerular filtration rateGalNAc N-AcetylgalactosamineACE Angiotensin-converting enzymeARB Angiotensin II receptor blockerCKD Chronic kidney diseaseDBP Diastolic blood pressureSBP Systolic blood pressureCRP C-reactive proteinESR Erythrocyte sedimentation rateTG TriglyceridesTCHOL Total cholesterolHDL High-density lipoprotein cholesterolLDL Low-density lipoprotein cholesterolScr Serum creatinine24-hour UTP 24-hour total urine protein

Disclosure

The funders had no role in study design data collection andanalysis decision to publish or preparation of the paper

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Xu-jie Zhou and Hong Zhang designed the research Yuan-yuan Qi and Fa-juan Cheng performed the research Yuan-yuan Qi and Xu-jie Zhou analyzed the data and Yuan-yuanQi and Xu-jie Zhou wrote the paper

Acknowledgments

The authors thank their collaborators including Ali GGharavi fromDepartment of Medicine Columbia UniversityCollege of Physicians and Surgeons New York USA forassistance They also thank all the members of the laboratoryfor technical assistance and the patients and their familiesfor their cooperation and for giving consent to participatein this study This work was supported by Grants fromthe National Natural Science Foundation of China (no81570629) Beijing Natural Science Foundation (7152148)Chinese Society of Nephrology (15020030591) the MajorState Basic Research Development Program of China (973Program no 2012CB517700) the Research Fund of BeijingMunicipal Science and Technology for the Outstanding PhDProgram (no 20121000110) the Foundation of the Ministryof Education of China (no 20120001120008) the NaturalScience Fund of China to the Innovation Research Group(no 81021004) and National Key Technology RampD Program(2013BAI09B14)

Disease Markers 7

References

[1] M Levy and J Berger ldquoWorldwide perspective of IgA nephro-pathyrdquo American Journal of Kidney Diseases vol 12 no 5 pp340ndash347 1988

[2] A Amore P Cirina G Conti P Brusa L Peruzzi and RCoppo ldquoGlycosylation of circulating IgA in patients with IgAnephropathy modulates proliferation and apoptosis of mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 12no 9 pp 1862ndash1871 2001

[3] J Barratt and J Feehally ldquoIgA nephropathyrdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2088ndash20972005

[4] J Barratt J Feehally and A C Smith ldquoPathogenesis of IgAnephropathyrdquo Seminars in Nephrology vol 24 no 3 pp 197ndash217 2004

[5] A C Smith J F de Wolff K Molyneux J Feehally and JBarratt ldquoO-glycosylation of serum IgD in IgA nephropathyrdquoJournal of the American Society of Nephrology vol 17 no 4 pp1192ndash1199 2006

[6] H Suzuki Z Moldoveanu S Hall et al ldquoIgA1-secreting celllines from patients with IgA nephropathy produce aberrantlyglycosylated IgA1rdquo Journal of Clinical Investigation vol 118 no2 pp 629ndash639 2008

[7] J Novak B A Julian M Tomana and J Mestecky ldquoIgAglycosylation and IgA immune complexes in the pathogenesisof IgA nephropathyrdquo Seminars in Nephrology vol 28 no 1 pp78ndash87 2008

[8] A C Allen E M Bailey P E Brenchley K S Buck J Barrattand J Feehally ldquoMesangial Iga1 in IgA nephropathy exhibitsaberrant O-glycosylation observations in three patientsrdquo Kid-ney International vol 60 no 3 pp 969ndash973 2001

[9] N Zhao P Hou J Lv et al ldquoThe level of galactose-deficientIgA1 in the sera of patients with IgA nephropathy is associatedwith disease progressionrdquo Kidney International vol 82 no 7pp 790ndash796 2012

[10] A G Gharavi K Kiryluk M Choi et al ldquoGenome-wide asso-ciation study identifies susceptibility loci for IgA nephropathyrdquoNature Genetics vol 43 no 4 pp 321ndash327 2011

[11] C Yang W Jie Y Yanlong et al ldquoGenome-wide associationstudy identifies TNFSF13 as a susceptibility gene for IgA in aSouthChinese population in smokersrdquo Immunogenetics vol 64no 10 pp 747ndash753 2012

[12] K Kiryluk Y Li F Scolari et al ldquoDiscovery of new risk loci forIgAnephropathy implicates genes involved in immunity againstintestinal pathogensrdquo Nature Genetics vol 46 no 11 pp 1187ndash1196 2014

[13] X-Q Yu M Li H Zhang et al ldquoA genome-wide associationstudy in Han Chinese identifies multiple susceptibility loci forIgA nephropathyrdquo Nature Genetics vol 44 no 2 pp 178ndash1822012

[14] Y Y Qi X J Zhou F J Cheng et al ldquoDEFA gene variantsassociated with IgA nephropathy in a Chinese populationrdquoGenes amp Immunity vol 16 no 3 pp 231ndash237 2015

[15] R I Lehrer and W Lu ldquo120572-Defensins in human innate immu-nityrdquo Immunological Reviews vol 245 no 1 pp 84ndash112 2012

[16] D Yang A Biragyn L W Kwak and J J Oppenheim ldquoMam-malian defensins in immunity more than just microbicidalrdquoTrends in Immunology vol 23 no 6 pp 291ndash296 2002

[17] Z M Sthoeger S Bezalel N Chapnik I Asher and O FroyldquoHigh 120572-defensin levels in patients with systemic lupus erythe-matosusrdquo Immunology vol 127 no 1 pp 116ndash122 2009

[18] F-J Cheng X-J Zhou Y-F Zhao M-H Zhao and H ZhangldquoHuman neutrophil peptide 1ndash3 a component of the neutrophil

extracellular trap as a potential biomarker of lupus nephritisrdquoInternational Journal of Rheumatic Diseases vol 18 no 5 pp533ndash540 2015

[19] M Saraheimo C Forsblom K Pettersson-Fernholm A Flyvb-jerg P-H Groop and J Frystyk ldquoIncreased levels of 120572-defensin(-1 -2 and -3) in type 1 diabetic patients with nephropathyrdquoNephrology Dialysis Transplantation vol 23 no 3 pp 914ndash9182008

[20] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[21] M Haas ldquoHistologic subclassification of IgA nephropathya clinicopathologic study of 244 casesrdquo American Journal ofKidney Diseases vol 29 no 6 pp 829ndash842 1997

[22] R I Lehrer G Jung P Ruchala S Andre H J Gabius andW Lu ldquoMultivalent binding of carbohydrates by the human 120572-defensin HD5rdquo The Journal of Immunology vol 183 no 1 pp480ndash490 2009

[23] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[24] E Leikina H Delanoe-Ayari K Melikov et al ldquoCarbohydrate-bindingmolecules inhibit viral fusion and entry by crosslinkingmembrane glycoproteinsrdquoNature Immunology vol 6 no 10 pp995ndash1001 2005

[25] W Wang S M Owen D L Rudolph et al ldquoActivity of 120572- and120579-defensins against primary isolates of HIV-1rdquo The Journal ofImmunology vol 173 no 1 pp 515ndash520 2004

[26] Z Moldoveanu R J Wyatt J Y Lee et al ldquoPatients withIgA nephropathy have increased serum galactose-deficient IgA1levelsrdquo Kidney International vol 71 no 11 pp 1148ndash1154 2007

[27] M B Renfrow H J Cooper M Tomana et al ldquoDeterminationof aberrant O-glycosylation in the IgA1 hinge region by electroncapture dissociation fourier transform-ion cyclotron resonancemass spectrometryrdquo Journal of Biological Chemistry vol 280no 19 pp 19136ndash19145 2005

[28] K Takahashi S BWallH Suzuki et al ldquoClusteredO-glycans ofIgA1 defining macro- and microheterogeneity by use of elec-tron capturetransfer dissociationrdquoMolecular and Cellular Pro-teomics vol 9 no 11 pp 2545ndash2557 2010

[29] M M Gomes H Suzuki M T Brooks et al ldquoRecognitionof galactose-deficient O-glycans in the hinge region of IgA1by N-acetylgalactosamine-specific snail lectins a comparativebinding studyrdquoBiochemistry vol 49 no 27 pp 5671ndash5682 2010

[30] J S Moore R Kulhavy M Tomana et al ldquoReactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteinsrdquoMolecular Immunology vol 44 no 10 pp 2598ndash2604 2007

[31] S J Mellis and J U Baenziger ldquoStructures of the O-glyco-sidically linked oligosaccharides of human IgDrdquo Journal of Bio-logical Chemistry vol 258 no 19 pp 11557ndash11563 1983

[32] T SMattu R J Pleass A CWillis et al ldquoThe glycosylation andstructure of human serum IgA1 Fab and Fc regions and the roleof N-glycosylation on Fc120572 receptor interactionsrdquoThe Journal ofBiological Chemistry vol 273 no 4 pp 2260ndash2272 1998

[33] M C Field S Amatayakul-Chantler T W Rademacher PM Rudd and R A Dwek ldquoStructural analysis of the N-glycans from human immunoglobulin A1 comparison of nor-mal human serum immunoglobulin A1 with that isolated frompatients with rheumatoid arthritisrdquo Biochemical Journal vol299 no 1 pp 261ndash275 1994

[34] E Arnett and S Seveau ldquoThe multifaceted activities of mam-malian defensinsrdquo Current Pharmaceutical Design vol 17 no38 pp 4254ndash4269 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 7

References

[1] M Levy and J Berger ldquoWorldwide perspective of IgA nephro-pathyrdquo American Journal of Kidney Diseases vol 12 no 5 pp340ndash347 1988

[2] A Amore P Cirina G Conti P Brusa L Peruzzi and RCoppo ldquoGlycosylation of circulating IgA in patients with IgAnephropathy modulates proliferation and apoptosis of mesan-gial cellsrdquo Journal of the American Society of Nephrology vol 12no 9 pp 1862ndash1871 2001

[3] J Barratt and J Feehally ldquoIgA nephropathyrdquo Journal of theAmerican Society of Nephrology vol 16 no 7 pp 2088ndash20972005

[4] J Barratt J Feehally and A C Smith ldquoPathogenesis of IgAnephropathyrdquo Seminars in Nephrology vol 24 no 3 pp 197ndash217 2004

[5] A C Smith J F de Wolff K Molyneux J Feehally and JBarratt ldquoO-glycosylation of serum IgD in IgA nephropathyrdquoJournal of the American Society of Nephrology vol 17 no 4 pp1192ndash1199 2006

[6] H Suzuki Z Moldoveanu S Hall et al ldquoIgA1-secreting celllines from patients with IgA nephropathy produce aberrantlyglycosylated IgA1rdquo Journal of Clinical Investigation vol 118 no2 pp 629ndash639 2008

[7] J Novak B A Julian M Tomana and J Mestecky ldquoIgAglycosylation and IgA immune complexes in the pathogenesisof IgA nephropathyrdquo Seminars in Nephrology vol 28 no 1 pp78ndash87 2008

[8] A C Allen E M Bailey P E Brenchley K S Buck J Barrattand J Feehally ldquoMesangial Iga1 in IgA nephropathy exhibitsaberrant O-glycosylation observations in three patientsrdquo Kid-ney International vol 60 no 3 pp 969ndash973 2001

[9] N Zhao P Hou J Lv et al ldquoThe level of galactose-deficientIgA1 in the sera of patients with IgA nephropathy is associatedwith disease progressionrdquo Kidney International vol 82 no 7pp 790ndash796 2012

[10] A G Gharavi K Kiryluk M Choi et al ldquoGenome-wide asso-ciation study identifies susceptibility loci for IgA nephropathyrdquoNature Genetics vol 43 no 4 pp 321ndash327 2011

[11] C Yang W Jie Y Yanlong et al ldquoGenome-wide associationstudy identifies TNFSF13 as a susceptibility gene for IgA in aSouthChinese population in smokersrdquo Immunogenetics vol 64no 10 pp 747ndash753 2012

[12] K Kiryluk Y Li F Scolari et al ldquoDiscovery of new risk loci forIgAnephropathy implicates genes involved in immunity againstintestinal pathogensrdquo Nature Genetics vol 46 no 11 pp 1187ndash1196 2014

[13] X-Q Yu M Li H Zhang et al ldquoA genome-wide associationstudy in Han Chinese identifies multiple susceptibility loci forIgA nephropathyrdquo Nature Genetics vol 44 no 2 pp 178ndash1822012

[14] Y Y Qi X J Zhou F J Cheng et al ldquoDEFA gene variantsassociated with IgA nephropathy in a Chinese populationrdquoGenes amp Immunity vol 16 no 3 pp 231ndash237 2015

[15] R I Lehrer and W Lu ldquo120572-Defensins in human innate immu-nityrdquo Immunological Reviews vol 245 no 1 pp 84ndash112 2012

[16] D Yang A Biragyn L W Kwak and J J Oppenheim ldquoMam-malian defensins in immunity more than just microbicidalrdquoTrends in Immunology vol 23 no 6 pp 291ndash296 2002

[17] Z M Sthoeger S Bezalel N Chapnik I Asher and O FroyldquoHigh 120572-defensin levels in patients with systemic lupus erythe-matosusrdquo Immunology vol 127 no 1 pp 116ndash122 2009

[18] F-J Cheng X-J Zhou Y-F Zhao M-H Zhao and H ZhangldquoHuman neutrophil peptide 1ndash3 a component of the neutrophil

extracellular trap as a potential biomarker of lupus nephritisrdquoInternational Journal of Rheumatic Diseases vol 18 no 5 pp533ndash540 2015

[19] M Saraheimo C Forsblom K Pettersson-Fernholm A Flyvb-jerg P-H Groop and J Frystyk ldquoIncreased levels of 120572-defensin(-1 -2 and -3) in type 1 diabetic patients with nephropathyrdquoNephrology Dialysis Transplantation vol 23 no 3 pp 914ndash9182008

[20] A S Levey J P Bosch J B Lewis T Greene N Rogers and DRoth ldquoAmore accuratemethod to estimate glomerular filtrationrate from serum creatinine a new prediction equationrdquo Annalsof Internal Medicine vol 130 no 6 pp 461ndash470 1999

[21] M Haas ldquoHistologic subclassification of IgA nephropathya clinicopathologic study of 244 casesrdquo American Journal ofKidney Diseases vol 29 no 6 pp 829ndash842 1997

[22] R I Lehrer G Jung P Ruchala S Andre H J Gabius andW Lu ldquoMultivalent binding of carbohydrates by the human 120572-defensin HD5rdquo The Journal of Immunology vol 183 no 1 pp480ndash490 2009

[23] R I Lehrer ldquoPrimate defensinsrdquo Nature Reviews Microbiologyvol 2 no 9 pp 727ndash738 2004

[24] E Leikina H Delanoe-Ayari K Melikov et al ldquoCarbohydrate-bindingmolecules inhibit viral fusion and entry by crosslinkingmembrane glycoproteinsrdquoNature Immunology vol 6 no 10 pp995ndash1001 2005

[25] W Wang S M Owen D L Rudolph et al ldquoActivity of 120572- and120579-defensins against primary isolates of HIV-1rdquo The Journal ofImmunology vol 173 no 1 pp 515ndash520 2004

[26] Z Moldoveanu R J Wyatt J Y Lee et al ldquoPatients withIgA nephropathy have increased serum galactose-deficient IgA1levelsrdquo Kidney International vol 71 no 11 pp 1148ndash1154 2007

[27] M B Renfrow H J Cooper M Tomana et al ldquoDeterminationof aberrant O-glycosylation in the IgA1 hinge region by electroncapture dissociation fourier transform-ion cyclotron resonancemass spectrometryrdquo Journal of Biological Chemistry vol 280no 19 pp 19136ndash19145 2005

[28] K Takahashi S BWallH Suzuki et al ldquoClusteredO-glycans ofIgA1 defining macro- and microheterogeneity by use of elec-tron capturetransfer dissociationrdquoMolecular and Cellular Pro-teomics vol 9 no 11 pp 2545ndash2557 2010

[29] M M Gomes H Suzuki M T Brooks et al ldquoRecognitionof galactose-deficient O-glycans in the hinge region of IgA1by N-acetylgalactosamine-specific snail lectins a comparativebinding studyrdquoBiochemistry vol 49 no 27 pp 5671ndash5682 2010

[30] J S Moore R Kulhavy M Tomana et al ldquoReactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteinsrdquoMolecular Immunology vol 44 no 10 pp 2598ndash2604 2007

[31] S J Mellis and J U Baenziger ldquoStructures of the O-glyco-sidically linked oligosaccharides of human IgDrdquo Journal of Bio-logical Chemistry vol 258 no 19 pp 11557ndash11563 1983

[32] T SMattu R J Pleass A CWillis et al ldquoThe glycosylation andstructure of human serum IgA1 Fab and Fc regions and the roleof N-glycosylation on Fc120572 receptor interactionsrdquoThe Journal ofBiological Chemistry vol 273 no 4 pp 2260ndash2272 1998

[33] M C Field S Amatayakul-Chantler T W Rademacher PM Rudd and R A Dwek ldquoStructural analysis of the N-glycans from human immunoglobulin A1 comparison of nor-mal human serum immunoglobulin A1 with that isolated frompatients with rheumatoid arthritisrdquo Biochemical Journal vol299 no 1 pp 261ndash275 1994

[34] E Arnett and S Seveau ldquoThe multifaceted activities of mam-malian defensinsrdquo Current Pharmaceutical Design vol 17 no38 pp 4254ndash4269 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom