ORIGINAL ARTICLESApplication of Oxford classification,and overexpression of transforming growth factor-b1and immunoglobulins in immunoglobulin Anephropathy: correlation with World HealthOrganization classification of immunoglobulin Anephropathy in a Chinese patient cohort

HONGXUE MENG, LEI ZHANG, XIAOQIANG E, FEI YE, HUINING LI, CHANGSONG HAN,MITSUNORI YAMAKAWA, and XIAOMING JIN

YAMAGATA, JAPAN; AND HARBIN, PEOPLE’S REPUBLIC OF CHINA

From the Department of Path

University Faculty of Medicine,

Pathology, Harbin Medical Univer

China; Department of Orthopedic

Harbin Medical University, Harb

Department of Pathology, The F

Longjiang University of Chine

Republic of China; Heilongjiang

Infection and Immunity, Harbi

People’s Republic of China.

Conflicts of Interest: All authors hav

flicts of interest and have none to d

This work was supported by the He

Technology Innovation Team in Hig

tion and Immunity, Harbin Medica

8

Immunoglobulin A nephropathy (IgAN) is characterized by the qualitative abnor-mality of immunoglobulin A (IgA) in circulation and deposits of IgA in the renalmesangium. Transforming growth factor b1 (TGF-b1) plays a key role in fibrogenesisand the progression of renal damage. This study aimed to investigate the clinico-pathologic data on IgAN in northeastern China and the presence of TGF-b1, totalIgA, and secretory IgA in the glomeruli and sera, as well as changes in galactose-deficient IgA1 in the serum.We investigated theclinicopathologic data of 1050 casesof IgAN diagnosed in a single center over 13 years. We then assessed the concentra-tions of TGF-b1 and immunoglobulins in the serum of 100 patients with IgAN and 56healthy control subjects by enzyme-linked immunosorbent assay, and investigatedtheir presence in the glomeruli by immunofluorescence and reverse transcriptase-polymerase chain reaction. From our data, 76.17% of the IgAN cases belonged toclasses I and II according to the World Health Organization classification, represent-ing the early stage. Compared with other studies, we found significantly lower fre-quencies of segmental glomerulosclerosis (27.71%) but higher frequencies ofendocapillary proliferation (50.67%), anda similar proportion ofmesangial hypercel-lularity (68.48%) and tubular atrophy/interstitial fibrosis (moderate, 17.81%; severe,

ological Diagnostics, Yamagata

Yamagata, Japan; Department of

sity, Harbin, People’s Republic of

s, The First Affliated hospital of

in, People’s Republic of China;

irst Affiliated Hospital of Hei

se Medicine, Harbin, People’s

Provincial Key Laboratory for

n Medical University, Harbin,

e read the journal’s policy on con-

eclare.

ilongjiang Provincial Science and

her Education Institutes for Infec-

l University, China; Grant-in-Aid

for Scientific Research (c) (25460451) in Japan; and the Doctoral

Foundation of The First Affiliated Hospital of HarbinMedical Univer-

sity (2013B12).

Hongxue Meng, Lei Zhang, and Xiaoqiang E contributed equally to

this work.

Submitted for publication December 29, 2012; revision submitted

June 20, 2013; accepted for publication June 29, 2013.

Reprint requests: Xiaoming Jin, Department of Pathology, Basic Med-

ical Science College, Harbin Medical University, 157 Baojian Road,

Harbin, Nangang District, 150081, China; e-mail: k-moh@med.id.

yamagata-u.ac.jp.

1931-5244/$ - see front matter

� 2014 Mosby, Inc. All rights reserved.

http://dx.doi.org/10.1016/j.trsl.2013.06.007

AT A GLANCE COMM

Meng H, et al.

Background

ImmunoglobulinAnephro

ized by the qualitative abno

lin A in circulation and dep

A in the renal mesangium

system is needed for predic

Translational Significanc

Our results indicate that th

the Oxford classificatio

World Health Organizat

was an increased presence

factor b1 and immunoglo

glomeruli of IgAN, which

of pathologic classificatio

forming growth factor bcould be used as the bio

genic mechanisms, and ac

to the original Oxford Cla

Translational ResearchVolume 163, Number 1 Meng et al 9

1.52%) in the northeastern Chinese cohort. There was an increased presence of TGF-b1 and immunoglobulins in the serum and glomeruli of IgAN, which correlates withthe progression of pathologic classification. The pathologic variables of the Oxfordclassification correlated significantly with theWHOclassifications. TGF-b1 and immu-noglobulins could beusedas biomarkers of IgANpathogenicmechanisms, acting asimportant adjuncts to the original Oxford Classification. (Translational Research2014;163:8–18)

Abbreviations: BUN ¼ blood urea nitrogen; ESRD ¼ end-stage renal disease; FITC ¼ fluoresceinisothiocyanate; Gd-IgA1 ¼ galactose-deficient IgA1; HAA ¼ helix aspersa; IgA ¼ immunoglob-ulinA; IgAN¼ immunoglobulinAnephropathy; IgG¼ immunoglobulinG; IgM¼ immunoglobulinM;mRNA¼messenger RNA; SCr¼ serumcreatinine; SIgA¼ secretory IgA; TGF-b1¼ transforminggrowth factor b1; WHO ¼ World Health Organization

ENTARY

pathy (IgAN) is character-

rmality of immunoglobu-

osits of immunoglobulin

. An ideal classification

ting progression of IgAN

e

e pathologic variables of

n correlated well with

ion classifiations. There

of transforming growth

bulins in the serum and

correlated with progress

n, suggesting that trans-

1 and immunoglobulins

markers of IgAN patho-

t as an important adjunct

ssifiation.

Immunoglobulin A nephropathy (IgAN) has a high inci-dence in the Asian and the Pacific regions and accountsfor 50% of all primary glomerular disease in Japan,Singapore, and China. IgAN is also the most commonprimary glomerular disease among all races from Eu-rope, America, and Australia, with the exception ofblacks. The prognosis of this disease is good in the earlystages, but over the next 5 to 25 years, 9% to 50% of af-fected patients will develop irreversible end-stage renaldisease (ESRD).1,2 However, the precise pathogenesisand clinicopathologic features of IgAN are still unclear.In our practice, we have become aware of increased

numbers of patients with IgAN in recent years.3 Notably,77.28% of patients with IgAN belong to classes I and IIby the World Health Organization (WHO) grading

system, which represent the early stage. We need to de-termine the clinicopathologic characteristics of earlyIgAN to prevent or control its progression, reducingthe probability of ESRD. Several histologic classifica-tion systemshave been devised to predict the progressionof IgAN, such as the WHO grading classification.4-7

However, these classifications have not been widelyused, in part because the reproducibility of histologicvariables was not tested in those classifications. In2009, the Oxford classification was developed asa pathologic classification system for IgAN to predictthe risk of disease progression.8,9 However, there areseveral limitations to the Oxford classification ofIgAN. IgAN is a highly heterogeneous disease, withvariability in clinical patterns, pathologic features, andgeographic prevalence. The number of patients in theOxford study was relatively small (n 5 265), soevaluative studies using a larger number of cohortsfrom different geographic areas and of differentethnicities are needed. Although several validationstudies have been published, no registries havecollected data relating specifically to renal biopsy innortheastern China, which has a relatively fixedpopulation of more than 100 million people. The aimsof this retrospective study were to report theclinicopathologic data of IgAN and to compare theOxford and WHO classifications for patients innortheastern China.IgAN is characterized by a qualitative abnormality of

immunoglobulin A (IgA) in circulation and deposits ofIgA in the renal mesangium. Increased serum levels ofIgA, secretory IgA (SIgA), and galactose-deficientIgA1 (Gd-IgA1) have been reported in IgAN, andsome studies have suggested the deposition of IgA,SIgA, and Gd-IgA1 in the glomeruli in IgAN. However,no data have been presented concerning the presence ofserum and glomerular IgA, SIgA, and Gd-IgA1 in thesame IgAN cohort. It is not clear whether there isa change or correlation between the presence of theseimmunoglobulins in the glomeruli and serum in IgAN.

Table

I.Relationship

betw

eenclin

icalp

arametersandhistologic

classesofIgAN

Clin

icalp

arameters

Class

I,%

(n5

303)

Class

II,%

(n5

508)

Class

III,%

(n5

166)

Class

IV,%

(n5

53)

Class

V,%

(n5

20)

Total(n5

l050)

Pvalue

Male/female

160/143

235/273

93/73

22/31

6/14

516/534

0.4547

Age,y

30.586

8.30

31.716

8.50

32.106

7.98

31.626

7.14

26.256

5.25

31.576

8.29

0.3265

Hyp

ertension

19(6.27%)

42(8.27%)

27(16.27%)

11(20.75%)

6(30%)

105(10%)

0.0031

Proteinuria

33(10.89%)

64(12.60%)

23(13.86%)

7(13.21%)

5(25%)

132(12.57%)

0.001

Hematuria

30(23.76%)

101(19.88%)

16(9.64%)

4(7.55%)

0(0%)

151(14.38%)

0.932

Proteinuria

andhematuria

181(59.74%)

324(63.37%)

125(75.3%)

44(83%)

14(70%)

688(65.52%)

BUN

8(2.64%)

20(3.94%)

15(9.04%)

5(9.43%)

1(5%)

49(4.67%)

,0.0001

SCr

42(13.86%)

13(2.56%)

13(7.83%)

7(13.21%)

3(15%)

78(7.43%)

,0.0001

BUNandSCr

4(1.32%)

15(2.95%)

9(5.42%)

1(1.88%)

2(10%)

31(2.95%)

Abbreviations:BUN,bloodureanitrogen;IgAN,im

munoglobulin

Anephropathy;SC

r,se

rum

creatinine.

Therelationship

betw

eenclin

icalp

arametersandhistologic

classesofIgANwasanalyze

dbySp

earm

ancorrelationanalysis.Proteinuria

andhematuria

scoreswere

deriv

edasdesc

ribedin

Clin

-

icalP

arameters.Hypertension,theamountofproteinuria

,BUN,andSC

rsignificantlycorrelatedwithclassification.

Translational Research10 Meng et al January 2014

In a variety of glomerular diseases, transforminggrowth factor b1 (TGF-b1) has been demonstrated topromotepodocyte injury and the development of glomer-ulosclerosis. Its continuing production leads to the devel-opment of glomerular sclerosis and tubulointerstitialfibrosis, which are associated with the loss of renal func-tion and progression toward ESRD.10,11 The purpose ofour study was to determine the serum levels andglomerular deposition of TGF-b1 in a large cohort ofpatients by kidney biopsy and to evaluate the possiblerelationship of these parameters with the presence ofimmunoglobulins and pathologic classifications.In this work, we performed a retrospective study on

the clinicopathologic relevance of the pathologic classi-fications and compared the Oxford andWHO classifica-tions of 1050 cases of IgAN, determining theircharacteristics with the goal of providing new variablesfor understanding the pathogenesis of IgAN. In addi-tion, we collected serum and renal biopsy tissues from100 patients with IgAN. We then assessed the presenceof serum and glomerular TGF-b1, IgA, SIgA, and Gd-IgA1 in the same group of patients.

MATERIALS AND METHODS

Research subjects. A total of 1050 cases (20.19%) ofIgAN from 5200 renal biopsies, which were diagnosedbetween January 1998 and March 2010 in the Depart-ment of Pathology, Harbin Medical University, were en-rolled in this study. Indications for renal biopsy werenephrotic syndrome, acute renal failure, proteinuria,chronically reduced function, microhematuria, andothers. Renal disease diagnoses were based on tissuestudies using light, electron, and immunofluorescencemicroscopy, according to the WHO classification.12

Cases with Henoch–Sch€onlein purpura, systemic lupuserythematosus, liver cirrhosis, or other systemicdiseases were excluded. Of the selected patients, 516were male and 534 were female, with an average ageof 31.57 6 8.29 years (range, 10–72 years). Serumsamples from 100 patients with IgAN were collectedon the day of renal biopsy. Serum samples from 56healthy sex- and age-matched volunteers with negativeurinalysis results were collected as normal controls.We assessed the concentrations of IgA, SIgA, and Gd-

IgA1 in the serum of 100 patients with IgAN and 56healthy control subjects. Of the 100 patients withIgAN, there were 46 males and 54 females age 7 to66 years, with an average age of 34.11 6 11.15 years.The pathologic distribution of patients based on theWHO classification were 30 in class I, 30 in class II, 20in class III, 10 in class IV, and 10 in class V. The 56 con-trol subjects had an average age of 28.56 5.62 years.Wethen investigated the presence of renal glomerular IgA

Translational ResearchVolume 163, Number 1 Meng et al 11

and SIgA in the sampled tissues of these 100 patientswith IgAN. All biopsies had adequate tissue for diagno-sis, contained at least 6 glomeruli in the lightmicroscopysection, and underwent immunohistologic and electronmicroscopic analyses.

Clinical parameters. The clinical data of the 1050patients with IgAN, including gender, age, initial symp-toms (hypertension, proteinuria, and hematuria) andrenal function (blood urea nitrogen [BUN] and serumcreatinine [SCr]) were analyzed according to the patho-logic class of IgAN (Table I). Hypertension was definedas blood pressure greater than 130/90 mmHg. On thebasis of examining urine samples, the percentage ofpatients with increased hematuria (.5 erythrocytes/high-power field) or proteinuria (.30 mg/dL), orpatients with increased hematuria and proteinuria wereanalyzed in each class. We also analyzed thepercentage of patients with increased SCr levels(.178 mmol/L) or BUN levels (.9 mmol/L), orpatients with increased SCr and BUN levels in eachclass, as described previously.13

Histopathologic parameters of renal injury. All cases ofIgAN were categorized according to the WHO classifi-cation and the Oxford classification. The pathologicvariables of the Oxford classification and theWHO clas-sificationwere compared. At the time of biopsy, all casesof IgANhad been categorized into 5 classes according totheWHOclassification. One pathologist reviewed all re-nal biopsy slides and scored the pathologic variables ac-cording to the Oxford classification.8,9 The 4 pathologicvariables of the Oxford classification were scored asfollows: mesangial score less than or equal to 0.5 (M0)or greater than 0.5 (M1), segmental glomerulosclerosisabsent (S0) or present (S1), endocapillary hyper-cellularity absent (E0) or present (E1), and tubularatrophy/interstitial fibrosis less than or equal to 25%(T0), 26%–50% (T1), or more than 50% (T2).

Immunofluorescence of renal glomerular lesions. Forimmunofluorescence staining, unfixed renal biopsy tis-sue was embedded in optimal cutting temperature(O.C.T) compound (Sakura Tissue-Tek, 4583; SakuraFinetek USA, Inc., Torrance, Calif) and cut intosections on a freezing microtome. Subsequently, 5-mm-thick sections were placed on slides and stored at220�C until immunostaining. For indirect immuno-fluorescence, after fixation in cold acetone, tissueswere incubated sequentially with rabbit polyclonalantibodies to IgA (polyclonal rabbit antihuman IgA/fluorescein isothiocyanate (FITC), F0204; DakoCytomation, Inc., Carpentaria, Calif), IgM (polyclonalrabbit antihuman IgM/FITC, F0203; DakoCytomation), IgG (polyclonal rabbit antihuman IgG/FITC, F0202; Dako Cytomation), and C3 (polyclonalrabbit antihuman C3c/FITC, F0201; Dako Cyto-

mation). The immunofluorescence findings were notedas presence or absence of glomerular IgA, IgM, IgG,and C3 staining; more than trace when present wastaken as positive. Immunofluorescence staining of thesecretory component glycoprotein (mouse IgG1, SC-05; Abcam plc. 332, Cambridge Science, Cambridge,UK) was performed for the detection of SIgA, andmouse monoclonal antibody (mouse IgG2 b, ab49574;Abcam plc., Tokyo, Japan) was performed fordetection of TGF-b1, which was followed by testingwith the appropriate fluorescently labeled secondaryantibody (FITC-conjugated goat antimouse IgG; VectorLaboratories Inc., Burlingame, Calif). Evaluation ofimmunofluorescence staining was performed blindlyby two independent observers. Secretory IgA and TGF-b1 staining were scored as absent (0), mild (11),moderate (21), or positive (31). Global sclerosis-related changes in the intensity of the immunoglobulindeposition have been corrected.

Detection of serum TGF-b1, IgA, SIgA, and Gd-IgA1 byenzyme-linked immunosorbent assay. Human TGF-b1(SB100 B), IgA, and SIgA enzyme-linked immunosorbentassay kits purchased from R&D Systems (Minneapolis,Minn), Bethyl Laboratories (Montgomery, Tex) andEIAab & USCNLIFE (Wuhan EIAab Science Co., Ltd.,Wuhan, China), respectively, were used for the detectionof stock serum according to the manufacturers’ instruc-tions. The results were recorded at the net optical densityof 450 nm.Serum Gd-IgA1 was detected by specific biotinylated

helix aspersa (HAA) (Sigma-Aldrich, St. Louis, Mo),which binds specifically to the terminal N-acetylgalac-tosamine of IgA1, as reported previously.10 Briefly, thewells of the plates were coated with antihuman IgA(Dako, Glostrup, Denmark) in 100 mL 0.05 mol/Lbicarbonate buffer or bicarbonate buffer alone, half byhalf, and PH9 at 37�C for 1 hour. The other half of thewells were coated with a bicarbonate buffer alone toact as antigen-free wells. The plates were washed withphosphate buffered saline and Tween-20 3 times foreach step. Coated plates were blocked with phosphatebuffered saline and Tween-20 containing 2% bovine se-rum albumin at 37�C for 1 hour. After 3 washes, theplates were loaded with the test serum in duplicate ina blocking buffer. Gd-IgA1 isolated from the serum ofa patient with IgA1 myeloma was used to mimic thestandard Gd-IgA1 for quantitation. Every plate con-tained a blank control and IgA1 that had been digestedby neuraminidase and galactosidase as a positive con-trol. The serum were incubated overnight at 37�C, fol-lowed by washing. The plates were then treated withneuraminidase (10 mU/mL) to remove terminal sialicacid from O-linked N-acetylgalactosamine on IgA1and incubated at 37�C for 3 hours. HAA (1 mg/mL)

Fig 1. Distribution of pathologic variables by Oxford classification in

different cohorts. E1, endocapillary hypercellularity present; M1, me-

sangial score greater than 0.5; S1, segmental glomerulosclerosis pres-

ent; T1, tubular atrophy/interstitial fibrosis 26%–50%; T2, tubular

atrophy/interstitial fibrosis more than 50%.

Translational Research12 Meng et al January 2014

diluted to 1:200 in a blocking buffer was added into eachplate and incubated at 37�C for 3 hours, followed by40 minutes of incubation at 37�C with horseradishperoxidase-conjugated Avidin (Invitrogen Corporation,Carlsbad, Calif). Anti-IgA1 monoclonal antibody(B3506B4, LifeSpan BioSciences, Wash) in the block-ing bufferwas added to detect the serum IgA1concentra-tion. Both Gd-IgA1 and total IgA1 were developed with0.04% O-phenylenediamine in a 0.1 mol/L citrate phos-phate buffer (pH 5.0) and 0.3% H2O2 (volume per vol-ume). The reaction was stopped with 1 mol/L H2SO4.The absorbance was recorded as the net optical densityat 490 nm. The relative concentration of serum IgA1and the level of glycan were calculated as follows: Theabsorbance value of the blank control was regarded as0 and the positive control as 100%, and then the absor-bance value of each sample was calculated. The relativelectin binding per unit IgA1 (HAA/IgA1) was expressedas the relative concentration of lectin binding level overthe relative IgA1 concentration. Because the standardIgA1 myeloma protein is not entirely devoid of galac-tose, the expression of results in milligrams per millime-ter does not reflect precisely the concentration ofGd-IgA1 in the sera. Therefore, the results were ex-pressed in units per millimeter, and 1 unit of HAA/IgA1 was defined as 1 mg of this standard. The coeffi-cient of variance of the intra- and intertest variabilityof the HAA assay was controlled below 10%.

RNA extraction and reverse transcriptase-polymerasechain reaction analyses for expression of TGF-b1messenger RNA. After renal biopsy, the tissueswere sub-merged immediately in RNAlater RNA StabilizationReagent (R0901, SIGMA) and then stored at280�C un-til RNA purification. Total RNAwas extracted using theQIAshredder (catalog no. 79654; QIAGEN, Tokyo, Ja-pan) and RNeasy Mini Kit (catalog no. 74104; QIA-GEN), according to the manufacturer’s instructions.Complementary DNAwas synthesized using the Quan-tiTect Reverse Transcription Kit (catalog no. 205311;QIAGEN). The expression of messenger RNA(mRNA) was determined by reverse transcriptase-poly-merase chain reaction. The forward- and reverse-specific primer sequences size of the amplifiedfragments and annealing temperatures for TGF-b1were as follows: forward, 50-ACCAACTATTGCTT-CAGCTC-30; reverse, 50-TTATGCTGGTTGTACA-GGG-30; 197 bp; and 50�C; those for b-actinwere forward, 50-CAGAGCAAGAGAGGCATCCT-30;reverse, 50-ACGTACATGGCTGGGGTG-30; 227 bp;and 55�C. Polymerase chain reaction products weresubjected to 2% agarose gel electrophoresis andvisualized by ethidium bromide on an imager.

Statistical analyses. We analyzed the relationshipsamong clinical presentation; renal dysfunction; degree of

Oxford classification; TGF-b1 and SIgA deposition in theglomeruli; TGF-b1, IgA, SIgA, and Gd-IgA1 con-centrations in the serum; and WHO pathologic classesof patients with IgAN using Spearman’s correlationanalysis. With regard to the interactions of each factor,we also analyzed the relationship between these factorsand pathologic classes using multifactor logisticregression. TGF-b1, IgA, SIgA, and Gd-IgA1 concen-trations in the serum were compared between patientsand normal control subjects using the c2 test. Therelationship between TGF-b1, IgA, and SIgA depositionin the glomeruli and the level of serum TGF-b1, IgA,SIgA, and Gd-IgA1 were compared using Pearson’scorrelation analysis and Student’s t test (SAS InstituteInc., Cary, NC).

Ethics. Informed consent was obtained from all pa-tients in this study, and the protocols were approvedby the ethics committee of Harbin Medical University,China.

RESULTS

Comparison of theOxford andWHOclassifications. Thedistribution of patients based on the WHO classificationwas 303 (28.86%) in class I, 508 (48.38%) in class II, 166(15.81%) in class III, 53 (5.05%) in class IV, and 20(1.9%) in class V. The distribution of pathologicvariables by the Oxford classification was as follows:M0/M1, 331/719 (31.52%/68.48%); S0/S1, 759/291(72.29%/27.71%); E0/E1, 518/532 (49.33%/50.67%),and T0/T1/T2, 847/187/16 (80.67%/17.81%/1.52%;Fig 1). The portions of M1, S1, E1, T1, or T2 increasedsignificantly as the WHO class score increased (Fig 2).Using Spearman’s correlation analysis, the pathologicvariables of the Oxford classification correlated signi-ficantly with the WHO classes.Several registries have collected data relating to path-

ologic lesions according to the Oxford classifications inChinese (not including northeastern China), Indian,Korean, North American, and French cohorts.14-18

Compared with the Oxford cohort, the northeasternChinese cohort had a lower proportion of patients with

Fig 2. Distribution of pathologic variables by Oxford classification in World Health Organization (WHO) classi-

fication. (A) Distribution of M1 byWHO classification: 50.5% in I, 67.13% in II, 92.77% in III, 98.11% in IV, and

95% inV (P, 0.0001,R5 0.45). (B) Distribution of S1 byWHO classification: 19.14% in I, 26.38% in II, 36.75%

in III, 43.4% in IV, and 75% inV (P, 0.0001,R5 0.42). (C) Distribution of E1 byWHO classification: 33.99% in

I, 48.62% in II, 71.69% in III, 83.02% in IV, and 95% in V (P, 0.0001,R5 0.37). (D) Distribution of T1 byWHO

classification: 4.62% in I, 12.01% in II, 41.57% in III, 58.49% in IV, and 60% in V (P, 0.0001, R5 0.37). Dis-

tribution of T2 by WHO classification; 0.33% in I, 0.2% in II, 2.41% in III, 7.55% in IV, and 30% in V). The por-

tions of M1, S1, E1, T1, and T2 increased significantly as the WHO class score increased. E0/E1, endocapillary

hypercellularity absent or present; M0/M1, mesangial score less than or equal to 0.5 (M0) or greater than 0.5 (M1);

S0/S1, segmental glomerulosclerosis absent or present; T0/T1/T2, tubular atrophy/interstitial fibrosis less than or

equal to 25% (T0), 26%–50% (T1), or more than 50% (T2).

Table II. Relationship between clinical parameters and pathologic variables of IgAN

Clinicalparameters

Mesangial score

P value

Segmental glomerulosclerosis

P valueM0 (n 5 331) M1 (n 5 719) S0 (n 5 759) S1 (n 5 291)

Male/female 171/160 345/374 0.298 380/379 136/155 0.369Age, y 30.63 6 8.1 31.60 6 7.80 0.750 31.43 6 7.24 32.58 6 8.36 0.695Hypertension 22 (6.65%) 83 (1 1.54%) 0.019 66 (8.70%) 39 (13.4%) 0.031Proteinuria 26 (7.85%) 106 (14.74%) 0.002 82 (10.8%) 50 (17.18%) 0.007Hematuria 48 (14.5%) 103 (14.33%) 0.985 107 (14.10%) 44 (15.12%) 0.746Proteinuria and

hematuria184 (55.59%) 504 (70.10%) ,0.001 466 (61.40%) 222 (76.29%) ,0.001

BUN 12 (3.63%) 37 (5.15%) 0.353 31 (4.10%) 18 (6.19%) 0.200SCr 16 (4.83%) 62 (8.62%) 0.040 60 (7.91%) 18 (6.19%) 0.412BUN and SCr 7 (2.11%) 24 (3.34%) 0.373 19 (2.5%) 12 (4.12%) 0.236

Abbreviations: BUN, blood urea nitrogen; IgAN, immunoglobulin A nephropathy; M0, mesangial score less than or equal to 0.5; M1, me-

sangial score greater than 0.5; S0, segmental glomerulosclerosis absent; S1, segmental glomerulosclerosis present; SCr, serum creatinine.

Translational ResearchVolume 163, Number 1 Meng et al 13

mesangial hypercellularity (68.48%), but these data arehigher than a different Chinese registry that did notinclude the northeastern Chinese cohort. Comparedwith other studies, there were significantly lowerfrequencies of segmental glomerulosclerosis (27.71%)but higher frequencies of endocapillary proliferation(50.67%), as well as similar tubular atrophy/interstitial

fibrosis (moderate, 17.81%; severe, 1.52%) in thenortheastern Chinese cohort (Fig 1).

Relationship between clinical parameters andpathologic class of IgAN. Patients age 20–30 years hadthe highest incidence of IgAN (n 5 363, 34.57%),followed by patients age 30–40 years (n 5 288,27.43%; Table I). According to WHO classification,

Table III. Relationship between clinical parameters and pathologic variables of IgAN

Clinicalparameters

Endocapillary hypercellularity

P value

Tubular atrophy/interstitial fibrosis

P valueE0 (n 5 518) El (n 5 532) T0 (n 5 847) T1 (n 5 187) T2 (n 5 16)

Male/female 266/252 250/282 0.177 417/430 93/94 6/10 0.968Age, y 31.25 6 7.88 31.73 6 8.18 0.726 30.68 6 8.46 32.76 6 7.86 31.46 6 8.31 0.812Hypertension 25 (4.83%) 80 (15.04%) ,0.001 70 (8.26%) 24 (12.83%) 11 (68.75%) ,0.001Proteinuria 114 (22.01%) 18 (3.38%) ,0.001 103 (12.16%) 28 (14.97%) 1 (6.255%) 0.355Hematuria 142 (27.41%) 9 (1.69%) ,0.001 126 (14.88%) 25 (13.37%) 1 (6.255%) 0.521Proteinuria and

hematuria243 (46.91%) 445 (83.65%) ,0.001 504 (59.50%) 170 (90.90%) 14 (87.50%) ,0.001

BUN 38 (7.34%) 11 (2.07%) ,0.001 29 (3.42%) 19 (10.16%) 1 (6.255%) ,0.001SCr 64 (12.36%) 14 (2.63%) ,0.001 58 (6.85%) 15 (8.02%) 5 (31.25%) 0.001BUN and SCr 25 (4.83%) 6 (1.13%) ,0.001 22 (2.59%) 7 (3.74%) 3 (18.75%) 0.002

Abbreviations: BUN, blood urea nitrogen; E0/E1, endocapillary hypercellularity absent or present; IgAN, immunoglobulin A nephropathy; SCr,serum creatinine; T0/T1/T2, tubular atrophy/interstitial fibrosis less than or equal to 25% (T0), 26%–50% (T1), or more than 50% (T2).

Translational Research14 Meng et al January 2014

classes I and II were present at the highest frequency,with a proportion of 77.28% (811 of 1050). UsingSpearman’s correlation analysis, hypertension, theamounts of proteinuria, BUN levels, and SCr levelscorrelated significantly with pathologic classification.

Associations between clinical and pathologicvariables. As shown in Table II and Table III, segmentalglomerulosclerosis, endocapillary hypercellularity, andtubular atrophy/interstitial fibrosis scores were stronglyassociated with hypertension, and the endocapillaryhypercellularity and tubular atrophy/interstitial fibrosisscores were associated strongly with BUN and SCrlevels at biopsy. All histologic lesions (M1, E1, S1, T1,and T2) were associated individually with proteinuriaand hematuria, and there were more patients with pro-teinuria and hematuria for M1, E1, S1, T1, and T2 thanfor M0, E0, S0, and T0.

Relationship between immunoglobulin andcomplement deposition and pathologic variables. Allhistologic lesions (M1, E1, S1, T1, and T2) were asso-ciated individually with IgM and C3 deposition, andthere were more patients with IgM deposition for M1,E1, S1, T1, and T2 than for M0, E0, S0, and T0(Supplemental Tables I and II). Significantly, C3deposition reached 80.72% in 1050 cases of IgAN,and the percentages of C3-positive cases in each classwere 74.2%, 79.1%, 75.9%, 86.8%, and 90% (forclasses I through V, respectively), with a gradualupward trend in accordance with the WHO pathologicclassification.

Multifactor logistic regression analysis. With the multi-factor logistic regression analysis between each clinico-pathologic parameter and pathologic class, the factorsof hypertension (P , 0.05), the amount of proteinuria(P , 0.05), the level of SCr (P , 0.01), mesangial hy-percellularity (P, 0.0001), endocapillary proliferation(P 5 0.002), and tubular atrophy/interstitial fibrosis

(P, 0.05) correlated significantly with the WHO path-ologic classification.

Serum concentration of TGF-b1, IgA, SIgA, and Gd-IgA1in IgAN. The concentrations of TGF-b1, IgA, SIgA, andGd-IgA1 in patients’ serum were higher than thoseof normal control subjects (P , 0.001; Fig 3). Theconcentrations of TGF-b1, IgA, SIgA, and Gd-IgA1were higher in advanced stages (classes III, IV, and V)than in early stages (classes I and II; P , .05). Thelevel of TGF-b1 correlated with the levels of IgA(R 5 0.9, P , 0.01), SIgA (R 5 0.88, P , 0.01) andGd-IgA1 (R 5 0.86, P , 0.01). The levels of IgA(R 5 0.438, P , 0.05) and SIgA (R 5 0.559, P ,0.05) correlated with the degree of hematuria.

Glomerular deposition of TGF-b1, IgA, and SIgA inIgAN. Immunofluorescence staining for TGF-b1 andSIgAwas performed on renal biopsies from 100 patientswith IgAN (Fig 4). The presence of SIgA was detectedwith a monoclonal antibody against secretorycomponent, combined with double staining for IgA(Fig 5). Immunofluorescence staining for IgA wasperformed on diagnosis, and the data for these 100patients were extracted from the records. GlomerularTGF-b1 and SIgA positivity were observed ina mesangial pattern in 46 biopsies (46.00%) and 52biopsies (52.00%), respectively (Fig 4). TGF-b1, IgA,and SIgA staining in the glomeruli increased inaccordance with the increase of WHO pathologicclass. The concentrations of TGF-b1, IgA, and SIgAin the patients’ serum were correlated with theirglomerular deposition. For the clinical parameters, thestaining of IgA and SIgA correlated significantly withhypertension (R 5 0.301, P , 0.05).TGF-b1mRNAwas found in renal biopsy tissues, and

the expression of TGF-b1 mRNA in IgAN was higherthan that of control subjects. For the biopsy tissues ofpatients with IgAN, TGF-b1 mRNA was increased in

Fig 3. (A–D) Serum concentration of immunoglobulin (Ig) A (A), secretory IgA (SIgA) (B), galactose-deficient

IgA1 (C), and transforming growth factor b1 (TGF-b1) (D). The concentrations of IgA, SIgA, galactose-deficient

IgA1, and TGF-b1 in the patients’ serum were higher than those of normal control subjects (P , 0.0001).

Translational ResearchVolume 163, Number 1 Meng et al 15

accordance with increased WHO pathologic class ofIgAN (Fig 6).

DISCUSSION

IgAN, the most common primary glomeruli disease,usually has a poor prognosis at its end stages.19 An au-thoritative standard of the histopathologic grading sys-tem for prediction of renal prognosis of IgAN isneeded to determine the policies of treatment and pre-vention, and to control its progression.20,21 In 2009,the International IgA Nephropathy Networkdeveloped the Oxford classification, a new predictorof the clinical identification of IgAN.8,9 However,more large-scale and controlled studies in differentraces are needed to prove the overall superiority ofthe Oxford classification. Recently, several studies onthe validation of the Oxford classification for pediatricIgA nephropathy were published from China, India,Korea, North America, and France.14-18 Consequently,differences in the study populations have surfaced.Compared with other studies, there were significantlylower frequencies of segmental glomerulosclerosis(27.71%) but higher frequencies of endocapillaryproliferation (50.67%) in our cohort, which indicatesthat immunosuppressive therapy should be considered

as part of the therapeutic strategy in northeasternChina, because endocapillary proliferative lesions areresponsive to immunosuppressive therapy. Our studyshows that each pathologic variable of the Oxfordclassification correlates significantly with the WHOclassification, which demonstrates that the Oxfordclassification is a simple and effective method forroutine use.Most of the IgAN patients were of class I and class II,

and just under half the patients had hematuria (mainlymicroscopic hematuria) as an initial symptom in thisstudy. Patients age 20–30 years had the highest incidenceof IgAN. When the blood pressure of the patients withhematuria and proteinuria was increased, their renalfunction decreased. Hypertension is known as a factorthat can accelerate the deterioration of renal functionand has also been associated with an unfavorable prog-nosis for IgAN22,23; some patients have to receivedialysis treatment soon after diagnosis. Therefore, theprevention of hypertension is important in patientswith IgAN. In addition, most patients with IgAN showa simultaneous clinical presence of proteinuria andhematuria, with only a few patients showing hematuriaalone, especially in classes III, IV, or V. Our resultsshowed that the amount of proteinuria correlatedsignificantly with pathologic class of IgAN. This

Fig 4. The expression of secretory immunoglobulin A (SIgA; secretory component) in immunoglobulin A ne-

phropathy (IgAN) kidney by immunofluorescence. (A) Representative negative (0) expression of SIgA in renal

biopsy tissue from a patient with IgAN. (B–D) Representative mild (11) (B), moderate (21) (C), and intense

(31) (D) expressions of SIgA in renal biopsy tissue from patients with IgAN. Magnification 3460.

Translational Research16 Meng et al January 2014

information was consistent with previously publishedstudies, which have suggested that, if patients havesubstantial proteinuria, their pathologic changesinclude severe mesangial proliferation and usuallyfocal sclerosis, indicative of a poor prognosis.24 There-fore, the amount of proteinuria might be used as a nonin-vasive biomarker to evaluate kidney injury in IgAN.Changes in renal function (SCr and BUN) and IgAN re-nal failure are the result of various diseases. In this study,we analyzed patients with increased SCr and/or BUNlevels. The results suggested that SCr or BUN, orSCr and BUN, levels increased gradually with an in-crease in the pathologic class of IgAN. We also foundthat SCr levels can be increased in class I, which indi-cates the importance of understanding abnormal renalfunction and treating patients during the early stages ofIgAN. For patients with abnormal hematuria, hyperten-sion, proteinuria, and BUN and SCr levels, we can detectrenal damage earlier and carry out active and effective

treatment according to the symptoms to reduce the inci-dence of renal failure.Our immunofluorescent results showed that comple-

ment C3 deposition in IgAN reached 80.72% and hada high ratio from class I to II, which suggests that com-plement C3 is activated during the early stages of IgANand that it could form the membrane attack complex,which causes the perforation and dissolution of targetcells. At the same time, anaphylatoxin, which is pro-duced in a series of C3 activations, may damage tissuecells.25,26

IgAN is defined by the presence of IgA-dominant glo-merular deposits.27 The Oxford classification of IgANidentifies4histologic features that are independentpredic-tors of clinical outcome but do not include immunostain-ing patterns in the analysis.8,9 Similar studies applied tothe presence of glomerular immunofluorescence andserum immunoglobulin in native renal disease have beenlacking before our study. Here, we investigated the

Fig 5. The expression of secretory immunoglobulin A (SIgA; secretory component) in immunoglobulin A ne-

phropathy (IgAN) glomeruli by laser scanning confocal microscopy. (A) Immunofluorescent microscopy analysis

of IgA (green staining) in IgAN glomeruli. (B) Immunofluorescent microscopy analysis of serum creatinine (red

staining) in IgAN glomeruli. (C) Merger of IgA staining (A) and serum creatinine staining (B) shows SIgA ex-

pression in IgAN glomeruli. Magnification 3460.

Fig 6. Expression of transforming growth factor b1 (TGF-b1) mes-

senger RNA (mRNA) in immunoglobulin A nephropathy (IgAN)

and normal renal biopsy tissues. There were five patients with IgAN

(P1, diagnosed as World Health Organization [WHO] class I; P2, di-

agnosed as WHO class II; P3, class III; P4, class IV; P5, class V) in

the IgAN group and five normal control subjects (P6–P10). There

was a greater TGF-b1 mRNA expression in the IgAN group than in

the control group, as well as a greater TGF-b1 mRNA expression in

high grade (P4, P5) than in low grade of WHO pathological classifica-

tion (P3, P2, P1).

Translational ResearchVolume 163, Number 1 Meng et al 17

presence of total IgA and SIgA in the glomeruli and sera,as well as the change of Gd-IgA1 in serum. We concludethat there was an increased presence of immunoglobulinsin the serum and glomeruli of IgAN, correlating with theprogression of the pathologic classifications. IgA, SIgA,and Gd-IgA1 could be used as biomarkers of IgAN pro-gression. It is recommended that the location and intensityof immunoglobulin staining and the serum concentrationof immunoglobulins be included routinely in the renal bi-opsy report.An ideal classification system should be based on

pathogenic mechanisms and should thus suggest an ap-propriate therapeutic strategy. The classification ofIgAN should also be improved based on the biomarkersof pathogenic mechanisms, such as TGF-b1, which areessential for the induction of IgA class switching andthe progression of renal damage.11,28 Our data showan increased presence of TGF-b1 in the serum ofIgAN that correlated with the levels of IgA, SIgA,Gd-IgA1, and the progress of pathologic classification.TGF-b1 has an important role in IgA class switchingmechanisms by inducing immunoglobulin germline al-pha transcription and subsequent class switching re-

combination to IgA.29,30 Thus, elevated TGF-b1levels in the serum may correlate with the increasedexpression of serum IgA, SIgA, and Gd-IgA1 throughthe induction of IgA class switching. Our data showthat IgA and SIgA staining in the glomeruli increasedin accordance with the increase in WHO pathologicclass. IgA, SIgA, and Gd-IgA1 deposit in the glomeruliand contribute to IgAN progression. TGF-b1 proteinand mRNAwere found more frequently in the glomeruliof IgAN than in that of control subjects, and were in-creased in accordance with increased WHO pathologicclass of IgAN. Overexpression of TGF-b1 was reportedto activate salt water baths and Ras protein/extracellularsignal-regulated kinase signaling pathways in podo-cytes, initiating the development of glomerulosclerosis,stimulating mesangial matrix synthesis, and promo-ting podocyte proliferation.11,31-33 TGF-b1 andimmunoglobulins seem to be key factors in thepathogenesis of IgAN and correlate with the progressof IgAN. The same was found by monitoringquantitative immunoglobulins among patients withmonoclonal gammopathy.34 TGF-b1 and immunoglob-ulins could be used as biomarkers for IgAN pathogenicmechanisms and act as an important adjunct to the orig-inal Oxford classification.Continuous exposure to circulating immune com-

plexes containing IgA, aberrantly glycosylated IgA1,SIgA, and sequential immune responses seem to be es-sential to the disease progression of IgAN. The removalof elevated TGF-b1 and immune complexes from theserum and kidneys may be challenging, but it is consid-ered a fundamental approach in the treatment of IgAN.In conclusion, in our study, IgAN cases had certain

similarities but were not identical between the Oxfordand WHO classifications. Thus, geography, race, andpersonal differences in this disease should not be ig-nored. The pathologic variables of the Oxford classifica-tion correlated significantly with those of the WHO

Translational Research18 Meng et al January 2014

classifications. TGF-b1 and immunoglobulins seem tobe key factors in the pathogenesis of IgAN and are cor-related with markers of disease progression. TGF-b1and immunoglobulins could be used as biomarkers ofIgAN pathogenic mechanisms and act as an importantadjunct to the original Oxford classification.

The authors thank Elsevier Language Editing Services from Elsev-

ier Ltd. for excellent editing of this manuscript.

Supplementary Data

Supplementary data related to this article can befound at http://dx.doi.org/10.1016/j.trsl.2013.06.007.

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