The heme oxygenase-1 genotype is a risk factor to renal impairment of IgA nephropathy at diagnosis, which is a strong predictor of mortality.

Abstract

The induction of heme oxygenase-1 (HO-1) ameliorates oxidative stress and inflammatory process, which play important roles in IgA nephropathy. We hypothesized length polymorphism in the promoter region of the HO-1 gene, which is related to the level of gene transcription, is associated with disease severity of IgA nephropathy. The subjects comprised 916 patients with IgA nephropathy and gene data. Renal impairment was defined as an estimated glomerular filtration rate less than 60 mL/min/1.73 m(2) at diagnosis. The short (S: <23), medium (M: 23-28), and long (L: >28) (GT) repeats in the HO-1 gene was determined. The frequencies of S/S, S/M, M/M, S/L, L/M, and L/L genotypes were 7.2%, 6.9%, 3.1%, 30.8%, 22.7%, and 29.4%, respectively. The baseline characteristics were not different. In the S/S genotypic group, the renal impairment rate was 18.2%, which was lower than 32.2% in the group with M/M, L/M, or L/L genotype. The odds ratio of renal impairment in S/S genotype, compared to that in M/M, L/M, or L/L genotype, was 0.216 (95% confidence interval, 0.060-0.774, p=0.019). The HO-1 gene promoter length polymorphism was related to the renal impairment of IgA nephropathy at diagnosis, which is an important risk factor for mortality in IgA nephropathy patients.

Full text

Page 1

INTRODUCTION
Heme oxygenase (HO) is the rate-limiting enzyme for the
breakdown of heme to generate carbon monoxide, iron, and
biliverdin. Biliverdin is rapidly converted to bilirubin by
biliverdin reductase (1). The induction of HO-1, the inducible
isoform of HO, is an important endogenous mechanism for
cytoprotection and mediates the beneficial effects of HO,
such as antioxidant, anti-inflammatory, anti-proliferative,
anti-apoptotic, and immunomodulatory effects (1). These
effects may be mediated through the products of heme degra-
dation(2-6) and regulation of MCP-1 (7), p38 mitogen acti-
vated protein (MAP) kinase (8), cell-cycle regulators (9, 10),
and T-cell- and natural killer cell-mediated cytotoxicity (11).
Experimental and clinical evidences support the impor-
tant protective role of HO-1 in several renal diseases. Induc-
tion of HO-1 is an adaptive and beneficial response to renal
injury secondary to rhabdomyolysis (12), ischemia-reperfu-
sion injury (13, 14), nephrotoxin (15), glomerulonephritis
(16), and renal transplant rejection (17). Human patients
with HO-1 deficiency showed iron deposition in the proxi-
mal tubule and pathologic change in the kidney, as well as
growth retardation, anemia, leukocytosis, lymphadenopa-
thy, and increased sensitivity to oxidative injury (18).
S30
Ho Jun Chin*
Tae Woo Lee
Hyung Jin Yoon
Suhnggwon Kim
Jun-Young Do‖, Jong-Won Park‖,
Kyung-Woo Yoon‖, Young-Tai Shin¶,
Kang Wook Lee¶, Ki-Ryang Na¶,
Dae Ryong Cha**, Young Sun Kang**, and
The Progressive REnal disease and Medical
Informatics and gEnomics Research (PRE-
MIER) members
,�, Hyun Jin Cho*,
�, Ki Young Na*,
�, Dong-Wan Chae*
�, Un Sil Jeon
,�,
�,
1
Department of Internal Medicine*, Seoul National
University Bundang Hospital, Seongnam; Renal Research
Institute
Seoul; Clinical Research Institute
University Hospital, Seoul; Biotechnology Center, Pohang
University of Science and Technology
Department of Internal Medicine‖; YeungnamUniversity
College of Medicine, Daegu; Department of Internal
Medicine¶, Chungnam National University College of
Medicine, Daejeon; Department of Internal Medicine,
Korea University Ansan Hospital**, Ansan, Korea
�, Seoul National University College of Medicine,
�, Seoul National
�,Pohang;
1Members are listed in Appendix.
Address for correspondence
Hyung Jin Yoon, M.D.
Department of Internal Medicine, Seoul National
University College of Medicine, 300 Gumi-dong,
Bundang-gu, Seongnam 463-707, Korea
Tel : +82.31-787-7025, Fax : +82.31-787-4052
E-mail : mednep@snubh.org
*This study was supported by the Collaborative
Research Grant of Korean Society of Nephrology.
J Korean Med Sci 2009; 24 (Suppl 1): S30-7
ISSN 1011-8934
DOI: 10.3346/jkms.2009.24.S1.S30
Copyright � The Korean Academy
of Medical Sciences
The Heme Oxygenase-1 Genotype is a Risk Factor to Renal Impairment
of IgA Nephropathy at Diagnosis, Which is a Strong Predictor of
Mortality
The induction of heme oxygenase-1 (HO-1) ameliorates oxidative stress and inflam-
matory process, which play important roles in IgA nephropathy. We hypothesized
length polymorphism in the promoter region of the HO-1 gene, which is related to
the level of gene transcription, is associated with disease severity of IgA nephropa-
thy. The subjects comprised 916 patients with IgA nephropathy and gene data.
Renal impairment was defined as an estimated glomerular filtration rate less than
60 mL/min/1.73 m2at diagnosis. The short (S: <23), medium (M: 23-28), and long
(L: >28) (GT) repeats in the HO-1 gene was determined. The frequencies of S/S,
S/M, M/M, S/L, L/M, and L/L genotypes were 7.2%, 6.9%, 3.1%, 30.8%, 22.7%,
and 29.4%, respectively. The baseline characteristics were not different. In the S/S
genotypic group, the renal impairment rate was 18.2%, which was lower than 32.2%
in the group with M/M, L/M, or L/L genotype. The odds ratio of renal impairment in
S/S genotype, compared to that in M/M, L/M, or L/L genotype, was 0.216 (95%
confidence interval, 0.060-0.774, p=0.019). The HO-1 gene promoter length poly-
morphism was related to the renal impairment of IgA nephropathy at diagnosis,
which is an important risk factor for mortality in IgA nephropathy patients.
Key Words : Heme Oxygenase; Glomerulonephritis, IGA; Renal Insufficiency
Received : 2 September 2008
Accepted : 3 November 2008

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Heme Oxygenase-1 and IgA Nephropathy S31
A (GT)n repeat region located between -198 and -258 of
the human HO-1 gene promoter is associated with up-reg-
ulation of HO-1 (19-21). The promoter activity of the HO-
1 gene was up-regulated by H2O2 exposure in genes with
(GT)16 or (GT)20, but not in genes with (GT)29 or (GT)38 (19).
The reporter gene expression driven by the HO-1 gene pro-
moter carrying (GT)22 was about four- and eight-fold higher
than that directed by promoters with (GT)26 and (GT)30,
respectively (20). Length polymorphism of this region cor-
relates with susceptibility in several diseases, such as coro-
nary artery disease (20, 22), ischemic cerebrovascular events
(19), lung cancer (23), and renal allograft dysfunction (17,
24), although, in some reports, length polymorphism is not
associated with disease conditions (25).
In IgA nephropathy, which is the most common type of
primary glomerulonephritis worldwide (26), oxidative stress
plays a role in the development and progression of nephropa-
thy (27, 28). HO-1 immunoreactivity in the kidney of pati-
ents with IgA nephropathy was increased compared that in
controls (27). The advanced oxidation protein products were
the independent risk factor to renal progression of IgA ne-
phropathy (27). We hypothesized that length polymorphism
in the HO-1 gene promoter region will be associated with
disease severity of IgA nephropathy at diagnosis.
MATERIALS AND METHODS
Study subjects
This study was approved by the Institutional Review Boards
at the participating hospitals before the data were gathered.
Informed consent was obtained from all patients. The subjects
were enrolled in the Progressive REnal disease and Medical
Informatics and gEnomics Research (PREMIER) study which
was sponsored by the Korean Society of Nephrology. In the
PREMIER study, 34 hospitals and clinics in Korea participat-
ed and shared the clinical data and genomic DNA extracted
from the peripheral blood of patients with primary and sec-
ondary glomerulonephritis diagnosed by renal biopsy in each
institute. There were 968 patients aged 18 yr or more with
IgA nephropathy, which was diagnosed by the pathologic find-
ings of prominent deposition of IgA antibodies in the me-
sangium detected by immunofluorescence staining (26), and
who agreed to collect DNA sample, from May 1986 to April
2007. Of the 968 patients, the serum creatinine values at
the time of renal biopsy were available for 916 subjects.
Clinical data
The participating researchers had selected the candidate
patients and one qualified nurse, who visited every partic-
ipated institution, input the clinical data into the formatted
database on the website (http://www.gn.or.kr) at the time of
renal biopsy and during follow-up visits. During the follow-
up period, we gathered the data on age, gender, body weight,
height, smoking habit, history of diabetes mellitus, current
hypertension, blood pressure, total bilirubin, alanine amino-
transaminase, aspartate aminotransferase, serum uric acid,
serum protein, serum albumin, serum cholesterol, hemoglobin,
serum creatinine, proteinuria by dipstick test, urine RBC
measured by microscopic examination of urine, medication
of angiotensin-converting enzyme inhibitors, angiotensin II
type I receptor blockers, other hypertensive agents, any kind
of steroid and HMG-CoA reductase inhibitors. Current hyper-
tension was defined as systolic blood pressure of 140 mmHg
or more, diastolic blood pressure of 90 mmHg or more, or
taking anti-hypertensive medication. Smoking habit was
categorized as current smoker or current non-smoker. We
calculated the estimated glomerular filtration rate (eGFR)
by the modified modification of diet in renal disease (MDRD)
equation (29). The renal impairment of IgA nephropathy at
biopsy was defined as eGFR less than 60 mL/min/1.73 m2.
The mortality and primary cause of death were obtained
from the database of the Korean National Statistical Office
(http://www.nso.go.kr). The mortality data until December
2006 available on this database were searched based on the
unique identifier. All study subjects were searched in the
national mortality database except 26 patients who were
enrolled after January 2007 and 5 patients whose identifiers
were not matched to the data of the Korean National Statis-
tical Office. The primary cause of mortality was provided as
a 4-digit, ICD-10 code, which was recorded by the physi-
cians. There were 11 deaths during a median follow-up peri-
od of 22.0 months (range 0.1-247 months).
HO-1 genotype assessment
Genomic DNA was isolated from whole blood samples
using the a QIAamp blood kit (Qiagen, Valencia, CA, U.S.A.)
Fig. 1. Frequency distribution of number of (GT)n repeats in all IgA
patients.
Frequency (%)
35
30
25
20
15
10
5
0
141516181920212223242526272829
31
30
323334353738
39
42
41
40
Number of (GT)n repeats

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S32H.J. Chin, H.J. Cho, T.W. Lee, et al.
according to the manufacturer’s protocol. Polymerase chain
reaction (PCR) amplifications of the HO-1 (GT)n repeat length
polymorphism were performed as described previously (30).
The HO-1 gene 5′ -flanking region containing a poly (GT)
n repeat was amplified by PCR with a 6-FAM-labeled sense
primer (5′ -AGAGCCTGCAGCTTCTCAGA-3′ ) and an
unlabeled antisense primer (5′ -ACAAAGTCTCCGGA-
TAGGAC-3′ ), which were designed on the basis of the pub-
lished sequence (31). Thirty PCR cycles of 94℃ for 30 sec,
57℃ for 30 sec and 72℃ for 30 sec were conducted. For
fragment analysis, 1 μ L of the PCR product was mixed with
9 μ L of HiDi-Formamide (Applied Biosystems, Foster City,
CA, U.S.A.) and 0.5 μ L of the Genscan 500 LIZ size standard
(Applied Biosystems) in 384-well plates. After a denatura-
tion and cooling step, the fragments were analyzed on the
ABI 3730XL sequencing system (Applied Biosystems). Each
repeat number was calculated with 2 cloned alleles (20, 22)
as size markers. For data analysis, we applied GeneMapper
version 3.7 software (Applied Biosystems).
Statistical analysis
The SPSS (SPSS version 12.0, Chicago, IL, U.S.A.) package
was used for statistical analysis. Differences in proportions
among the subject groups were compared by chi-square test.
Group differences for continuous variables were assessed by
the Student t-test or one-way ANOVA test according to the
number of groups. To determine whether the genotype group
was associated independently with the renal impairment of
IgA nephropathy at the time of renal biopsy, we used multi-
ple logistic regression analysis, adjusted for age, gender, and
univariate risk factors, for the renal impairment. We repeat-
ed the analyses after stratification by gender, age, hyperten-
sion, and proteinuria 3+ or more by dipstick test, which are
well-known risk factors of renal impairment. We compared
the cumulative incidence of mortality by log-rank test. We
used Cox’s hazard proportional model to determine the rela-
tionship between the HO-1 genotype group and mortality.
Two-sided p values are reported, with the level of statistical
significance set at 0.05. All data are shown as mean±stan-
dard deviation or frequency per observation.
RESULTS
Allele frequencies at the polymorphic locus
The number of (GT)n repeats ranged from 14-42 and exhib-
*Allele S, (GT)n repeats less than 23 in HO-1 promoter region; Allele M, (GT)n repeats 23-28 in HO-1 promoter region; Allele L, (GT)n repeats 29 or
more in HO-1 promoter region; hematuria, ≥5 RBCs/HPF under light microscopy, proteinuria measured by urine dipstick test; ACEI, angiotensin con-
verting enzyme inhibitor; ARB, angiotensin II type I receptor blocker.
Completeness
of data (%)
Genotype groups of HO-1 promoter region*
S/S
(n=66)
S/M, S/L
(n=345)
M/M, L/M, L/L
(n=505)
p value
Age (yr)
Gender (female %)
Habit of smoking (yes %)
History of diabetes mellitus (%)
Hypertension (%)
Body mass index (kg/m2)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Total bilirubin (mg/dL)
Alanine aminotransaminase (U/L)
Aspartate aminotransferase (U/L)
Uric acid (mg/dL)
Protein (g/dL)
Albumin (g/dL)
Cholesterol (mg/dL)
Hemoglobin (g/dL)
Creatinine (mg/dL)
Proteinuria 3+or more* (%)
Hematuria* (%)
Medication (%)
ACEI or ARB*
Steroids
HMG-CoA reductase inhibitor
100.0
100.0
92.7
94.1
90.4
83.2
92.0
92.1
93.8
96.6
93.2
86.7
95.3
95.6
91.5
97.7
100.0
91.7
92.1
33.1±12.8
37.9
21.9
3.1
35.5
24.0±3.9
125±15
78±10
0.65±0.38
18±10
20±6
6.0±1.9
6.6±0.7
3.8±0.6
182±53
13.3±2.1
1.1±0.5
25.0
95.2
36.6±13.8
43.5
15.9
3.4
42.0
23.2±3.2
125±17
78±12
0.67±0.35
20±17
20±8
6.0±1.8
6.6±0.8
3.9±0.6
191±75
13.0±2.0
1.3±1.3
17.9
88.5
36.1±13.4
46.3
12.9
2.5
41.8
23.6±3.8
125±19
76±13
0.65±0.35
20±18
22±17
6.2±1.9
6.6±0.9
3.8±0.7
190±63
12.9±2.0
1.3±1.0
23.0
90.9
0.158
0.368
0.126
0.777
0.613
0.167
0.942
0.641
0.778
0.685
0.324
0.498
0.696
0.530
0.682
0.395
0.308
0.171
0.221
100.0
100.0
100.0
60.6
10.6
12.1
59.7
11.9
17.4
55.0
11.9
16.0
0.340
0.953
0.556
Table 1. Characteristics of patients with IgA nephropathy

Page 4

Heme Oxygenase-1 and IgA Nephropathy S33
ited a bimodal distribution with one peak located at 22 GT
repeats and the other at 29 GT repeats (Fig. 1). Because the
inducibility of the HO-1 gene promoter was positively cor-
related with the number of (GT) repeats (19, 20), we divid-
ed allelic repeats into 3 subclasses with short, medium, and
long (GT)n repeats: short repeats with less than 23 (GT)n
were designated as allele class S (short), medium repeats with
23 to 28 (GT)n as allele class M (medium), and long repeats
with 29 or more (GT)n as allele class L (long). We had 6 geno-
types, S/S, S/M, M/M, S/L, L/M, and L/L, with percentage
frequencies (absolute frequencies) of 7.2% (66), 6.9% (63),
3.1% (28), 30.8% (282), 22.7% (208), and 29.4% (269),
respectively. The genotypes were categorized into 3 groups:
group 1 with S/S genotype, group 2 with S/M and S/L geno-
types, and group 3 with M/M, L/M, and L/L genotypes.
Clinical characteristics
There were no differences in any of the clinical parameters,
including medications, among the three genotype groups
(Table 1). Group 1 showed no difference in clinical parame-
ters from either of the other two groups. The bilirubin level
was not related to the HO-1 genotypes.
Allele S, (GT)n repeats less than 23 in HO-1 promoter region; Allele M, (GT)n repeats 23-28 in HO-1 promoter region; Allele L, (GT)n repeats 29 or more
in HO-1 promoter region.
Model 1-3: adjusted with gender and univariate factors to the presence of eGFR less than 60 mL/min/1.73 m2, such as age, diabetes mellitus, hyper-
tension, systolic blood pressure, diastolic blood pressure, serum cholesterol, serum uric acid, serum albumin, serum bilirubin, hemoglobin, protein-
uria 3+ or more by dipstick test, and genotype of HO-1 promoter region.
*, compared to M/M, L/M, L/L genotypes (group 3);
L/M, and L/L genotypes (group 3).
OR, odds ratio; C.I., confidence interval.
�, compared to S/M, S/L, M/M, L/M, and L/L genotypes (group 2 and group 3);
�, compared to M/M,
VariablesB OR [95% C.I.]p value Wald
Model 1
HO-1 promoter genotypes*
S/S
S/M, S/L
Age (yr)
Hypertension (yes)
Uric acid (mg/dL)
Hemoglobin (g/L)
Urine protein by dipstick (≥3+)
Model 2
HO-1 promoter genotypes (S/S)
Age (yr)
Hypertension (yes)
Uric acid (mg/dL)
Hemoglobin (g/L)
Urine protein by dipstick (≥3+)
Model 3
HO-1 promoter genotypes
(S/S, M/S, L/S)
Age (yr)
Hypertension (yes)
Uric acid (mg/dL)
Hemoglobin (g/L)
Urine protein by dipstick (≥3+)
--
0.035
0.019
0.153
<0.001
0.001
<0.001
<0.001
0.017
-
-1.531
-0.379
0.074
0.822
0.649
-0.570
0.686
5.543
2.044
47.764
10.501
68.237
62.240
5.672
0.216 [0.060-0.774]
0.685 [0.408-1.151]
1.077 [1.054-1.100]
2.275 [1.384-3.739]
1.914 [1.641-2.233]
0.566 [0.491-0.652]
1.985 [1.129-3.489]
�
-1.389
0.072
0.836
0.648
-0.566
0.732
4.676
46.503
10.919
68.336
62.007
6.562
0.031
<0.001
0.001
<0.001
<0.001
0.010
0.249 [0.071-0.878]
1.075 [1.053-1.098]
2.307 [1.405-3.788]
1.912 [1.640-2.230]
0.568 [0.493-0.654]
2.079 [1.187-3.638]
-0.5154.069
0.044
0.598 [0.362-0.985]
�
0.075
0.813
0.641
-0.563
0.641
49.650
10.383
67.213
62.375
5.063
<0.001
0.001
<0.001
<0.001
0.024
1.078 [1.055-1.100]
2.254 [1.375-3.695]
1.898 [1.628-2.212]
0.569 [0.495-0.655]
1.899 [1.086-3.320]
Table 2. Multivariate logistic regression model for the presence of eGFR less than 60 mL/min/1.73 m2at diagnosis
Frequency of renal impairment (%)
40
30
20
10
0
S/SS/M, S/L
Group 2
M/M, L/M, L/L
Group 3 Group 1
Genotypes of HO-1 promotor region
12/66*
p=0.038
Fig. 2. Frequency of eGFR less than 60 mL/min/1.73 m2accord-
ing to genotypes of the HO-1 promoter region.
*, compared between genotype group 1 and group 3; p=0.021.
94/354
162/505*

Page 5

S34H.J. Chin, H.J. Cho, T.W. Lee, et al.
The HO-1 genotype and renal impairment of IgA
nephropathy at the time of renal biopsy
The prevalence of renal impairment differed among the
three genotypic groups. In group 1, the renal impairment
rate of 18.2% was lower than 32.2% in group 3 (Fig. 2). The
univariate factors related to the renal impairment of IgA
nephropathy at the time of renal biopsy were age, diabetes
mellitus, hypertension, blood pressures, serum cholesterol,
serum uric acid, serum albumin, serum bilirubin, hemoglobin,
proteinuria 3+ or more by dipstick urine test, and genotype
of the HO-1 gene. In multiple logistic regression analysis
adjusted for these factors and gender, the HO-1 genotype
was an independent factor for renal impairment at diagnosis
(Table 2). The risk of renal impairment in patients with sin-
gle S allele (group 2) was not significantly different from that
of group 3 patients but the odds ratio of renal impairment
in group 1 was 0.216-fold lower than that in group 3. The
odds ratio of impaired renal function in S/S genotype group
was 0.249 compared to the other genotypes (group 2 and
group 3) and, in S allele group (group 1 and group 2), was
0.598 compared to the other genotypes (group 3).
The HO-1 genotype and renal impairment in subgroups
stratified by risk factors
When we stratified patients by gender, age with the crite-
rion of 50 yr, hypertension, and proteinuria 3+ or more by
dipstick urine test, which are related to the renal impairment
in IgA nephropathy, the rate of renal impairment in group 1
was lower than that in the other groups (group 2 and group
3), especially in subgroups of men, subjects aged 50 yr or
more, and normotensive subjects (Table 3). In subjects with
proteinuria by dipstick test less than 3+, group 1 tended to
have a lower frequency of renal impairment than the other
groups, although the difference was not statistically signifi-
cant. When we stratified the female group by age, the fre-
quency of renal impairment of group 1 was lower than that
of the other groups in females aged 50 yr or more (0/3 vs.
44/72, p=0.019).
The risk factors for mortality in IgA nephropathy
There were no deaths among the 64 patients of group 1
whereas there were 11 deaths in the 821 patients of the other
groups (p>0.05). All deaths occurred in patients with the L
allelic group (11/732). The causes of death were renal failure,
nephrotic syndrome, sepsis, heart failure, and subdural hem-
orrhage. Among univariate risk factors related to mortality,
diabetes mellitus, eGFR, and body mass index (BMI) were
independent risk factors (Table 4). Three of 24 patients with
diabetes mellitus died, as did 8 of the 807 patients without
diabetes mellitus (p=0.003). There was no mortality in 624
patients with eGFR 60 mL/min/1.73 m2or more, 6 deaths
out of 196 patients with eGFR 30-59 mL/min/1.73 m2, and
5 deaths among 65 patients with eGFR less than 30 mL/
min/1.73 m2 (p<0.001). The subgroups with BMI less than
18.5, 18.5-24.9, 25.0-29.9, and 30.0 kg/m2or more showed
mortality rates of 7.5% (3/40), 0.8% (4/474), 1.0% (2/210),
and 0.0% (0/23), respectively. The patients with BMI less
than 18.5 kg/m2showed significantly higher mortality than
those with BMI 18.5 kg/m2or more (p=0.011).
DISCUSSION
In this study, we demonstrated that HO-1 gene promoter
polymorphism was related to the prevalence of renal impair-
ment at diagnosis, which is an important risk factor for mor-
tality of IgA nephropathy. HO-1 messenger RNA expression
and enzyme activity are greater when the (GT) repeat length
in the HO-1 gene promoter region is short but the cut-off
point of (GT) repeat number related to HO-1 inducibility
Genotype
S/S
Genotype
others*
p value
In males
In females
In younger group aged less
than 50 yr
In older group aged 50 yr
or more
In normotensive patients
In hypertensive patients
In patients with proteinuria less
than 3+ by dipstick
In patients with proteinuria 3+
or more by dipstick
6 (14.6)
6 (24.0)
10 (17.5)
132 (28.3)
124 (32.3)
168 (23.8)
0.045
0.377
0.281
2 (22.2)88 (60.7)0.022
2 (5.0)
8 (36.4)
6 (13.3)
74 (16.6)
153 (47.7)
155 (25.1)
0.029
0.304
0.059
5 (33.3)75 (46.0)0.339
Table 3. The frequency of eGFR less than 60 mL/min/1.73 m2of
genotypes of the HO-1 promoter region in each subgroup
Number, n (%); Genotype S/S (group 1), Patients who had (GT)n repeats
less than 23 in both alleles of HO-1 promoter region.
*, Genotype S/M, S/L, M/M, L/M, L/L (group 2 and group 3).
VariablesB Waldp valueRR [95% C.I.]
Diabetes mellitus
(presence)
eGFR (mL/min/1.73 m2)
4.424 10.925
0.001
83.411
[6.053-1,149.387]
0.956
[0.930-0.982]
0.579
[0.409-0.821]
-0.045 10.469
0.001
Body mass index (kg/m2) -0.5469.422
0.002
Table 4. The Cox hazard proportional analysis for death in IgA
nephropathy
Model: adjusted with age, gender, diabetes mellitus, hypertension, body
mass index, systolic blood pressure, diastolic blood pressure, serum
cholesterol, serum albumin, hemoglobin, hematuria, estimated GFR,
and genotype of HO-1 promoter region.
RR, relative risk to death; C.I., confidence interval.

Page 6

Heme Oxygenase-1 and IgA Nephropathy S35
has not been defined absolutely. The cut-off criterion of (GT)
repeats of the short allele for HO-1 gene length polymor-
phism has most commonly been <25 (17, 25, 32, 33), but
has also included <28 (24), <27 (21-23), and <23 (19, 20).
With the transient-transfection assay with HO-1 gene pro-
moter regions containing various numbers of (GT) repeats,
the promoter activity of the HO-1 gene was up-regulated
in genes with 16 or 20 (GT) repeats but was not in genes
with 29 or 38 (GT) repeats (19). The reporter gene expres-
sion driven by the HO-1 gene promoter carrying 22 (GT)
repeats was about four- and eight-fold greater than that direct-
ed by promoters with 26 and 30 (GT) repeats, respectively
(20). Therefore, we grouped the HO-1 gene length polymor-
phism genotypes into 3 groups with the criteria of less than
23, 23 to 28, and 29 or more (GT) repeats.
The short allele of the HO-1 promoter gene was related
to the lower rate of renal impairment defined by the estimat-
ed GFR less than 60 mL/min/1.73 m2as calculated with
the modified MDRD equation. Although the MDRD equa-
tion was not fully verified to estimate kidney function in
the Korean population, we believe that our results are accept-
able because the risk of renal impairment defined by the cal-
culatedcreatinine clearance less than 60 mL/min by the Cock-
crauft-Gault equation (34) was also 0.073 (95% confidence
interval, 0.014-0.378, p=0.002 by multiple logistic regres-
sion analysis) in patients with the S/S genotype compared to
the risk in patients with the M/M, L/M, or L/L genotype.
Courtney et al. (25) reported that HO-1 (GT)n promoter
polymorphism was not related with the progression to end
stage renal disease in patients with IgA nephropathy. In
their study, the dependent variable compared between the
genotypic groups was the mean age of entry into the renal
replacement therapy program. The mean age of entry into
this program might be a surrogate marker for renal survival
in genetic renal diseases like autosomal dominant polycystic
kidney disease but is not usually accepted as a primary out-
come in IgA nephropathy. Although the data at the time of
renal biopsy might not be the data at the disease onset
point, we should consider the clinical status at the time of
renal biopsy and adjust the differences of clinical status at
diagnosis between patient groups to estimate the risk of
disease progression, statistically.
Although the glomerular injury in IgA nephropathy is
usually provoked by IgA-induced mesangial cell activation
and complement activation leading to pro-inflammatory and
pro-fibrotic phenotype transformation in mesangial cells (26),
increased oxidative stress was reported to play some roles in
the development and progression of IgA nephropathy (27,
28). Chen et al. (28) observed that the renal infiltration of
polymorphonuclear leukocytes, which has a high potential
for the production of reactive oxygen species, increased in
patients with IgA nephropathy. Advanced oxidation protein
products increased in progressed IgA nephropathy compared
with in stable IgA nephropathy and was an independent risk
factor to renal outcome of IgA nephropathy (27).
The HO-1 antioxidant effect is mediated by the removal
of pro-oxidant heme, generation of biliverdin and bilirubin,
coinduction of ferritin, reduction of hydroxyl radical produc-
tion, and suppression of the pro-oxidant MCP-1 (reviewed in
1). Renal up-regulation of HO-1 attenuates inducible nitric
oxide synthase expression and proteinuria in experimental
glomerulonephritis (16). For consideration of the pathogenic
mechanism of IgA nephropathy and the roles of the HO-1
gene, the relationship between the renal impairment of IgA
nephropathy at diagnosis and the inducibility of the HO-1
gene promoter has logical relevance.
The relationship between HO-1 promoter length poly-
morphism and renal impairment was evident only in males,
partially because of the gender-difference of HO-1 inducibil-
ity to various stresses. The induction of HO-1 expression
and activity was more enhanced in females than in males
after trauma and hemorrhagic shock (35) and myocardial
ischemia (36). Actually, the S/S genotype was related to a
lower rate of renal impairment in females older than 50 yr,
which is older than the mean age of menopause in Korean
women of 47 yr (37). The HO-1 inducibility was not relat-
edto the renal impairment of patients with more severe clin-
ical findings such as hypertension and high proteinuria, sug-
gesting that the influence of the HO-1 gene might be over-
whelmed by the clinical risk factors.
This study had several limitations. The devices for mea-
suring serum creatinine were different among institutions.
Jaffe’s colorimetric method was used to measure the level of
serum creatinine in all of the participating institutions, but
the device models differed. We did not standardize the results
by calibrating the serum creatinine value of each institution
to the result of one standard device. The mortality data were
from the national mortality database and we could not assess
whether or not the primary cause of mortality was correct.
In conclusion, the short allele of HO-1 gene promoter
length polymorphism was related to the lower rate of renal
impairment in IgA nephropathy at diagnosis, which is an
important risk factor for the mortality of IgA nephropathy.
APPENDIX
Members of The PREMIER group: Institutions that par-
ticipated in the study (Investigators).
Cheju National University Hospital (Eun Hee Jang), Chon-
buk National University Medical School (Won Kim), Chon-
nam National University Medical School (Nam Ho Kim,
Woo Kyun Bae), Chungbuk National University College of
Medicine (Hye Young Kim), Chungnam National Univer-
sity College of Medicine (Young-Tai Shin, Kang Wook Lee,
Ki-Ryang Na), Daegu Catholic University Medical Center
(Ki Sung Ahn), Dankook University Hospital (Jong Tae Cho,
Eun Kyeong Lee), Dong-A University College of Medicine

Page 7

S36 H.J. Chin, H.J. Cho, T.W. Lee, et al.
(Ki Hyun Kim, WonSuk An, Seong Eun Kim), Ewha Wom-
ans University School of Medicine (Choi Gyu Bog, Seung-
Jung Kim), Gachon University of Medicine and Science
(Woo Kyung Chung, Hyun Hee Lee, Jaeseok Yang, Sejoong
Kim), Gyeongsang National University Hospital (Se-Ho
Chang), Hallym University College of Medicine (Jung Woo
Noh, Young Ki Lee, Seong Gyun Kim , Jieun Oh, Young
Rim Song), Inha University College of Medicine (Moon Jae
Kim, Seoung Woo Lee), Inje University College of Medicine
(Yeong Hoon Kim, Won Do Park), Keimyung university
school of medicine (Hyun Chul Kim, Sung Bae Park), Kon-
kuk University School of Medicine (Kyo-Soon Kim), Korea
University Anam Hospital (Won Yong Cho, Hyoung Kyu
Kim, Sang-Kyung Jo), Korea University Ansan Hospital
(Cha Dae Ryong, Kang Young Sun), Korea University Col-
lege of Medicine Guro Hospital (Young-Joo Kwon), Kyung-
pook National University School of Medicine (Yong-Lim
Kim, Sun-Hee Park, Chan-Duck Kim), Pochon CHA Uni-
versity College of Medicine (Dong Ho Yang), Pusan Nation-
al University School of Medicine (Ihm Soo Kwak, Soo Bong
Lee, Dong Won Lee, Sang Heon Song, Eun Young Seoung),
Seoul Medical Center (Su-Jin Yoon), Seoul National Univer-
sity Bundang Hospital (Dong-Wan Chae, Ki Young Na, Ho
Jun Chin), Seoul National University College of Medicine
Boramae Medical Center (Chun Soo Lim, Yoon Kyu Oh),
Seoul National University Hospital (Kook Hwan Oh, Kwon
Wook Joo, Yon-Su Kim, Curie Ahn, Jin Suk Han, Suhngg-
won Kim), Seoul National University Hosptial Clinical Insti-
tute (Hyung Jin Yoon), Sungkyunkwan University School
of Medicine (Kyu-Beck Lee), Sungkyunkwan University
School of Medicine Samsung Medical Center (Yoon Goo
Kim, Jung Eun Lee), Ulsan University College of Medicine,
Asan Medical Center (Sang Koo Lee), Yeungnam Universi-
ty College of Medicine (Jun-Young Do, Jong-Won Park,
Kyung-Woo Yoon), ordered by alphabet.
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