Advances in Bioscience and Biotechnology, 2011, 2, 33-39 ABB
doi:10.4236/abb.2011.21006 Published Online February 2011 (http://www.SciRP.org/journal/abb/).
Published Online February 2011 in SciRes. http://www.scirp.org/journal/ABB
The gene expression of NGAL and TLR9 in glomerulus and
tubulo-interstitium of patients with lupus nephritis
Jianxin Lu1,3, Bonnie Ching-Ha Kwan1, Fernand Mac-Moune Lai2, Paul Cheung-Lung Choi2,
Lai-Shan Tam1, Edmund Kwok-Ming Li1, Kai-Ming Chow1, Gang Wang1,4, Philip Kam-Tao Li1,
Cheuk-Chun Szeto1
1Department of Medicine & Therapeutics and 2Department of Anatomical & Cellular Pathology, Prince of Wales Hospital, The Chi-
nese University of Hong Kong, Shatin, Hong Kong, China;
3Department of Nephrology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai,
China;
4Division of Nephrology, Renmin Hospital of Wuhan University, Wuhan, China.
Received 10 December 2010; revised 15 February 2011; accepted 17 February 2010
ABSTRACT
Background: The role of neutrophil gelatinase associ-
ated lipocalin (NGAL) and Toll-like receptor 9 (TLR9)
in the pathogenesis of lupus nephritis remain elusive.
Methods: We quantified the glomerular and tubu-
lointerstitial mRNA expression of NGAL and TLR9
in 42 patients with lupus nephritis (LN group) and 10
controls. Results: As compared to controls, LN group
had higher glomerular expression of TLR9, and
higher tubulointerstitial expression of NGAL and
TLR9. Tubulointerstitial NGAL expression signifi-
cantly correlated with proteinuria (r = 0.492; p =
0.003), renal function (r = –0.386; p = 0.022) and his-
tological chronicity index (r = 0.540; p = 0.004). Pro-
teinuria had significant correlation with glomerular
(r = 0.554; p = 0.001) and tubulointerstitial (r = 0.379;
p = 0.043) TLR9 expression. Furthermore, there was
a signific ant diff er ence in t ubuloin terstiti al expr e ssion
of NGAL between treatment response groups. Con-
clusion: There is an increase in intra-renal mRNA
expression of NGAL and TLR9 in LN. Although tu-
bulointerstitial expression of NGAL does not corre-
late with systemic disease activity, it correlates with
proteinuria, renal function, and therapeutic response.
The role of NGAL in the pathogensis in LN, as well as
its application as biomarker for lupus nephritis, re-
quires further study.
Keywords: Lupus Nephritis; Immunology; Cytokine
1. INTRODUCTION
Systemic lupus erythematosus (SLE) is a multisystem
autoimmune disease characterized by aberrant cytokines
milieu. Although almost all organs in the body could be
involved, lupus nephritis (LN) remains the leading cause
of morbidity and mortality in SLE. The precise patho-
genic mechanism of SLE and LN, however, is not com-
pletely understood. Cytokine aberration is a cardinal
phenomenon of LN. It is important to realize that cyto-
kines may not only be involved in the generation of ab-
errant immune regulation [1], but also participates in the
local inflammatory processes that ultimately lead to tis-
sue destruction. Unfortunately, most studies focused on
the cytokine profile of peripheral blood in SLE patients,
and the results are often inconsistent [2,3]. Since specific
organ or tissue involvement in SLE probably involve
local cytokine aberrations that do not appear in the sys-
temic circulation, study of the immunopathogenesis
should focus at the specific sites of disease involvement.
Neutrophil gelatinase associated lipocalin (NGAL) is
a member of the lipocalin superfamily, which is first
found in the specific granules of human neutrophil [4].
NGAL is a promising biomarker for both acute kidney
injury (AKI) and chronic kidney disease (CKD) [5].
Urinary NGAL levels correspond to disease activity in
lupus nephritis more closely than standard markers [6].
Toll-like receptor 9 (TLR9) is a pattern-associated re-
ceptor functioning in innate immunity that recognizes
unmethylated CpG sequences in DNA molecules; it may
be involved in the recognition of self DNA-antigens and
the production of pathogenic auto-antibodies. Previous
study showed that a higher expression of TLR9 on pe-
ripheral blood B cells from patients with active SLE
correlated with SLEDAI [7]. Notably, TLR9 causes the
activation of NF-B [8], which is recruited to the NGAL
promoter and is essential for the transcriptional activa-

J. X. Lu et al. / Advances in Bioscience and Biotechnology 2 (2011) 33-39 34
tion of NGAL promoters [9]. We hypothesize that in-
tra-renal TLR9 and NGAL are related to the develop-
ment of LN.
2. PATIENTS AND METHODS
2.1 Patient Selection
We studied 42 consecutive SLE patients with active re-
nal disease and required kidney biopsy. All patients ful-
filled the American College of Rheumatology diagnostic
criteria of SLE [10]. The uninvolved pole of 10 kidneys
that were removed for renal cell carcinoma and had no
morphological evidence of renal disease were used as
control. The study was approved by the Clinical Re-
search Ethical Committee of the Chinese University of
Hong Kong; all patients provided informed consent.
2.2. Clinical and Histological Assessment
The disease activity of SLE was assessed clinically by
the Systemic Lupus Erythematosus Disease Activity
Index (SLEDAI) [11] on the day of kidney biopsy by an
independent physician. Baseline serum creatinine, urine
protein, complement levels (C3 and C4) and anti-double
strand (ds) DNA antibody titre were also measured.
Glomerular filtration rate (GFR) was estimated by a
standard equation [12].
Kidney biopsy specimen was evaluated according to
the International Society of Nephrology (ISN) classify-
cation of lupus nephritis [13]. The activity index (AI)
and chronicity index (CI) of each biopsy specimen were
scored by standard methods [14]. Briefly, AI is the sum
of semiquantitative manual scores (0-3 each) of the fol-
lowing parameters: Endocapillary hypercellularity; leu-
cocyte infiltration, subendothelial hyaline deposits; in-
terstitial inflammation, necrosis and cellular crescents.
Scores of the last two parameters are counted double,
making a total AI of 0 to 24.
2.3. Laser Microdissection
The method of laser microdissection has been described
in our previous studies [14,15]. Briefly, cryosections of
10 µm thickness were prepared on a cryostat (Leica Mi-
crosystems, Wetzlar, Germany) using disposable micro-
tome blades (Leica Microsystem) in RNase-free condi-
tions, and were mounted on MembraneSlide 0.17 PEN
slides (Carl Zeiss PALM Microlaser Technologies, Bern-
ried, Germany). Immediately after taking the slides out
of the cryostat, the sections were fixed in 70% ethanol
and dehydrated in 100% ethanol. Sections were air-
dried at room temperature.
Laser microdissection of the snap-frozen kidney bi-
opsy specimens was performed using the PALM Mi-
crolaser System (PALM Microlaser Technologies),
which is equipped with a pulsed high-quality laser beam,
computer-controlled microscope stage and microma-
nipulator. Under direct visual control, areas of interest in
the histological specimens were selected through the
PALM RoboSoftware (PALM Microlaser Technologies)
by moving the computer mouse, and microdissected by
cutting the contour of the selected areas with the ad-
justed laser beam. The isolated tissue was then laser-
catapulted into a microcentrifuge cap filled with gua-
nidine thiocyanate containing lysis buffer for the subse-
quent RNA isolation. Approximately 20-30 glomerulus
and 20 randomly selected tubulointerstitial area were
isolated from each specimen. The tissue lysate of
glomerulus and tubulointerstitium were kept at –80˚C
until RNA extraction was performed with the RNAque-
ous®-Micro Kit (Applied Biosystems, USA), following
manufacturer’s instruction.
2.4. Quantification of Intrarenal Gene
Expression
The method of total RNA extraction, reverse transcrip-
tion and real-time quantitative polymerase chain reaction
(RT-QPCR) was described in our previous studies
[14,16]. In the present work, we quantified the mRNA
expression of NGAL and TLR9 in glomerulus and tubu-
lointerstitium. Taqman primers and probes of each target
were purchased from Applied Biosystems (Foster City,
California, USA). The RT-QPCR was performed by ABI
Prism 7900HT Sequence Detector System (Applied Bio-
systems) following the manufacturer’s instruction. The
mRNA expression for each signal was calculated by us-
ing the Ct procedure according to manufacturer’s in-
struction, with glyceraldehyde-3-phosphate dehydroge-
nase (GAPDH) used as the housekeeping gene for nor-
malisation among samples. All primers and probes were
tested with purified DNA as the template in RT-QPCR to
ensure that they do not amplify genomic DNA. All re-
sults were analysed by Sequence Detection Software
version 1.7 (Applied Biosystems).
Gene expression of each target was calculated by us-
ing the difference-in-threshold-cycle (Ct) procedure,
according to manufacturer’s instruction. For GAPDH
and each target, the relative efficiency of amplification
over various starting template concentrations was deter-
mined. Approximately equal efficiencies for other targets
with GAPDH amplifications were verified by an abso-
lute value of <0.1 for the slope of log input complemen-
tary DNA amount versus Ct, which was obtained by
subtracting the threshold cycle (Ct) value of GAPDH
from that of the target. Therefore, it was possible to de-
tect GAPDH in the same tube with other targets. The
relative quantification of using multiplex reaction with a
comparative method was determined by the formula
2–(Ct), where the Ct was calculated by the subtrac-

J. X. Lu et al. / Advances in Bioscience and Biotechnology 2 (2011) 33-39 35
tion of Ct of the calibrator from Ct of the sample.
2.5. Clinical Follow-Up
After the renal biopsy, all patients were followed for at
least 6 months. The clinical management was decided by
individual nephrologist and not affected by the study. In
general, patients were treated with corticosteroid, to-
gether with cyclophosphamide or mycophenolate ac-
cording to published protocols. Therapeutic response to
induction immunosuppressant was assessed at 12 weeks
after treatment and classified into complete remission,
partial remission, and no response as defined previously
[17]. Briefly, complete remission was defined as a value
for urinary protein excretion that was less than 0.3 g/24
hours, with normal urinary sediment, a normal serum
albumin concentration, and values for both serum cre-
atinine and estimated GFR that were 15% or less above
the base-line values. Partial remission was defined as a
value for urinary protein excretion that was between 0.3
and 2.9 g/24 hours, with a serum albumin concentration
of at least 30 g/dL and stable renal function. Treatment
failure was defined as a value for urinary protein excre-
tion that remained at or above 3 g/24 hours or a value of
0.3 to 2.9 g/24 hours but with a serum albumin below 30
g/dL, an increase in the serum creatinine concentration ≥
50 µmol/l, or a value for estimated GFR that was more
than 15% above the base-line value, or the discontinua-
tion of treatment due to side effects.
2.6. Statistical Analysis
Statistical analysis will be performed by SPSS for Win-
dows software version 15.0 (SPSS Inc., Chicago, IL).
All the results from this series of experiments are quan-
titative. The results are presented as mean ± SD unless
otherwise specified. Since the data on gene expression
are highly skewed, they are compared between groups
by Kruskall Wallis test or Mann-Whitney U test as ap-
propriate. Correlations between continuous variables are
calculated by Spearman’s rank correlation coefficient. A
p value of less than 0.05 is considered as statistically
significantly. All probabilities are two tailed.
3. RESULT
We studied 42 SLE patients. Their baseline demographic
and clinical data are summarized in Table 1. The histo-
logical diagnoses were proliferative nephritis (class III
or IV, 9 cases), pure membranous nephritis (class V, 9
cases), class II nephritis (3 cases), and mixed prolifera-
tive and membranous nephritis (21 cases). The mean
histological Activity and Chronicity Indices were 7.1 ±
4.3 and 2.7 ± 2.2, respectively.
Table 1. Baseline demographic and clinical data.
Patient group Control group
No. of patient 40 10
Sex (M:F) 2:40 4:6
Age (years) 40.8 ± 11.4 36.4  13.7
SLEDAI score 7.6 ± 4.5 0
Serum creatinine (mol/l) 125.4 ± 115.5 82.7  14.0
Proteinuria (g/day) 2.33 ± 1.84 0.01  0.03
Glomerular filtration rate
(mL/min/1.73m2) 71.2 ± 37.2 106.6 ± 22.0
Renal histology
Interstitial fibrosis (%) 13.8 ± 17.9 1.4  2.344
Glomerosclorosis (%) 10.8 ± 15.5 2.6  5.3
SLEDAI, Systemic Lupus Erythematosus Disease Activity Index.
3.1. Gene Expression Level between Groups
The glomerular and tubulointerstitial mRNA expression
levels of NGAL and TLR9 are summarized in Figure 1.
As compared to controls, LN patients had higher glomeru-
lar expression of TLR9 (p < 0.001) but not NGAL. In
contrast, LN patients had higher tubulointerstitial ex-
pression of TLR9 (p = 0.011) and NGAL (p < 0.001) as
compared to controls. There was a modest internal cor-
relation between tubulointerstitial mRNA expression
levels of NGAL and TLR9 (r = 0.386; p = 0.047). The
glomerular and tubulointerstitial expression of NGAL
are also significantly correlated (r = 0.410; p = 0.03),
while those of TLR9 had no significant correlation (r =
0199, p = 0.388). There were no significant differences
in glomerular or tubulointerstitial expression of TLR9 or
NGAL between histological groups (details not shown).
3.2. Relation with Clinical and Histological
Parameters
We further explore the correlation between gene ex-
pression and baseline clinical and histological pa-
rameters within patients with lupus nephritis. We
found that tubulointerstitial, but not glomerular, NGAL
significantly correlated with proteinuria (r = 0.492; p
= 0.003), estimated GFR (r = –0.386; p = 0.022) and
histological CI (r = 0.54; p = 0.004) (Figure 2). In
contrast, both glomerular (r = 0.554; p = 0.001) and
tubulointerstitial (r = 0.379; p = 0.043) TLR9 expres-
sion significantly correlated with proteinuria, but not
estimated GFR or histological CI (Figure 3). The
glomerular or tubulointerstitial expressions of TLR9
or NGAL did not correlate with markers of systemic
disease activity, including SLEDAI score, serum
complement, or anti-ds DNA titre (details not shown).

J. X. Lu et al. / Advances in Bioscience and Biotechnology 2 (2011) 33-39 36
glomerular expression of NGAL
0
1
2
3
4
LN CTL
glomerular express ion of TLR9
0
2
4
6
8
LN CTL
tubulointerstitial expression of NGAL 0
5
10
15
20
LN CTL
tubulointerstitial expression of TLR9
0
10
20
30
LN CTL
p = 0.6p < 0.001
p = 0.011
p < 0.001
(A) (B)
(C) (D)
Figure 1. Comparison of glomerular expression of (a) NGAL and (b) TLR9, and tubulointersti-
tial expression of (c) NGAL and (d) TLR9, between groups. Whisker-box plot, with the boxes
indicate median, 25 and 75 percentile; whisker caps indicate 5 and 95 percentile; circles indi-
cate outliers. Data are compared by Mann-Whitney U test. (LN, lupus nephritis group; CTL,
control group)
proteinuria (g/day)
0246810
glomerular NGAL e x p r e ssio n
0
1
2
3
4
estimated GFR (ml/ mi n/1.73m2)
020406080100 120 140
0
1
2
3
4
activity index
0 2 4 6 81012141618
0
1
2
3
4
chronicity index
01234567
0
1
2
3
4
proteinuria (g/d a y)
0246810
tubulointerstitial NGAL expression
0
2
4
6
8
estimated GFR (ml/min/1.73m2)
020 4060 80100120140
0
2
4
6
8
activity ind e x
0246810121416
0
2
4
6
8
chronicity index
0246810
0
2
4
6
8
r = 0.106
p = 0.5r = -0.047
p = 0.8r = 0.181
p = 0.3r = -0.064,
p = 0.7
r = 0.492
p = 0.003
r = -0.386
p = 0.022r = 0.153 p = 0.5r = 0.540
p = 0.004
(A)
(B)
Figure 2. Correlation between (a) glomerular and (b) tubulointerstitial expression of NGAL and the degree of pro-
teinuria, estimated glomerular filtration rate (GFR), histological activity index and chronicity index. Data are com-
pared by Spearman’s correlation coefficient. (Some of the circles overlap; each circle on the figure may represent
more than one patient.)
(a) (b)
(c) (d)
(a)
(b)

J. X. Lu et al. / Advances in Bioscience and Biotechnology 2 (2011) 33-39 37
proteinuria (g/day)
0246810
glomerular TLR9 expression
0
1
2
3
4
estimated GFR (ml/min/1.73m2)
0 20406080100120140
0
1
2
3
4
activity index
024681012141618
0
1
2
3
4
chronicity index
01234567
0
1
2
3
4
proteinuria (g/day)
0246810
tubulointerstitial TLR9 expression
0
2
4
6
8
estimated GFR (ml/min/1.73m2)
0 20406080100120140
0
2
4
6
8
activity index
024681012141618
0
2
4
6
8
chronicity index
0246810
0
2
4
6
8
r = 0.554
p = 0.001r = -0.215
p = 0.254
r = -0.057
p = 0.805
r = -0.226
p = 0.342
r = 0.379
p =0.043r = -0.287
p = 0.132
r = 0.116
p = 0.637r = 0.044
p = 0.853
(A)
(B)
Figure 3. Correlation between (a) glomerular and (b) tubulointerstitial expression of TLR9 and the degree of proteinuria,
estimated glomerular filtration rate (GFR), histological activity index and chronicity index. Data are compared by Spear-
man’s correlation coefficient. (Some of the circles overlap; each circle on the figure may represent more than one patient.)
3.3. Relation with Therapeutic Responses
After 6 months follow up, 14 cases had complete remis-
sion (CR), 21 cases had partial remission (PR) cases and
7 cases had no response (NR). The expressions of the
two target genes are compared between treatment re-
sponse groups (Figure 4). There was a significant dif-
ference in tubulointerstitial expression of NGAL between
treatment response groups. Post hoc analysis showed that,
as compared to CR group, NR group had lower tubu-
lointerstitial expression of NGAL (p = 0.039). There was
no significant difference in the glomerular expression of
NGAL, or glomerular or tubulointerstitial expression of
TLR9, because treatment response groups.
4. DISCUSSION
In the present study, we found that NGAL expression
only increased in the tubulointerstitium of LN. Our re-
sult is similar to that of Brunner et al., who found sig-
nificantly greater urinary NGAL levels in individuals
with active renal disease as compared to others [18]. Our
findings also suggest that NGAL expression was abun-
dant in tubulointerstitium but not glomerulus, which is
consistent with the general belief. Furthermore, we
found that tubulointerstitial NGAL expression signifi-
cantly correlated with proteinuria, GFR and CI. The re-
sult agrees with the report of Bolignano et al., who also
showed that urinary NGAL concentrations correlated
with renal function and proteinuria [19]. In addition, we
found that the tubulointerstitial expression of NGAL is
related to treatment response. Our results concur with
the report of Hinze et al. [20], which also showed that
urinary and plasma NGAL are predictors of the course
of LN in childhood-onset SLE patients. Taken together,
the data suggest that the alteration of tubulointerstitial
NGAL in LN is functionally relevant.
The site of renal TLR9 expression has been a matter
of debate. In a recent study, Machida et al. [21] reported
injured glomerular podocytes in active LN expressed
TLR9, which disappeared during remission and was not
expressed in a normal control kidney. Furthermore,
TLR9 expression correlated with the degree of proteinu-
ria and anti-dsDNA antibody titre. On the other hand,
Benigni et al. [22] showed a robust TLR9 expression in
the proximal tubular epithelial cells in an animal lupus
model and patients with LN, and TLR9 level correlated
with proteinuria and tubular damage. Our result showed
that TLR9 expression increased in both glomerulus and
tubulointerstitium of LN, which is consistent with the
report of Papadimitraki et al. [23].
The role of TLR9 in LN is relatively well studied. The
up-regulation of TLR9 in the renal parenchyma of LN
may be a response to inflammatory cytokines or the nu-
cleosomes present within the lesion [23]. In return,
TLR9 induces macrophages and B-cells to produce cy-
tokines and chemokines, thus aggravates inflammation
and tissue damage [24-26]. Studies showed that inter
(a)
(b)

J. X. Lu et al. / Advances in Bioscience and Biotechnology 2 (2011) 33-39 38
glomerular expression of NGAL
0
1
2
3
CRPR NR
glomerular expression of TLR9
0
1
2
3
4
CRPR NR
tubulointerstitial expression of NGAL 0
5
10
15
20
25
30
CRPR NR
tubulointerstitial expression of TLR9
0
5
10
15
20
25
CRPR NR
(A) (B)
(D)
(C)
P=0.53
P=0.045 P=0.99
P=0.16
P=0.141P=0.077
P=0.039
Figure 4. Comparison of glomerular expression of (a) NGAL and (b) TLR9, and tubu lointer-
stitial expression of (c) NGAL and (d) TLR9, between treatment response groups. Data are
compared by Kruskal Wallis test. The box indicates median, 25 and 75 percentile; whisker
caps indicate 5 and 95 percentile; outliers are represented as closed circles. (Key for response
groups: CR, complete remission; PR, partial remission; NR, no response.)
nalization of SLE immune complexes into subcellular
lysosomes containing TLR9 induced a signaling cascade
that led to the activation of dendritic cells and produc-
tion of cytokines and chemokines [27], suggesting that
TLR9 may have a role in the onset and progression of
LN. In the present study, we found a modest, although
statistically significant, correlation between tubulointer-
stitial expression of NGAL and TLR9. Our result indi-
cates that tubulointerstitial NGAL expression is gov-
erned by factors other than TLR9.
Several limitations of the current study need to be ad-
dressed. First, the sample size of our study was small, so
that subgroup comparison of gene expression between
different histological classes of lupus nephritis may not
be reliable. Because of the limitated amount of patho-
logical specimen available, we did not quantify intra-
renal TLR9 or NGAL at protein level by immunohisto-
chemistry or western blotting. In addition, we could not
examine renal tissue of lupus patients without kidney
problem as the control group. Furthermore, we do not
examine the longitudinal change of intra-renal gene ex-
pression or the response to immunosuppressive therapy
by serial renal biopsy. Further experiments are necessary
to solve these limitations and confirm our findings.
In summary, we found an increase in intra-renal
mRNA expression of NGAL and TLR9 in LN. Although
tubulointerstitial expression of NGAL does not correlate
with systemic disease activity, it correlates with protein-
uria, renal function, and therapeutic response. The role
of NGAL in the pathogensis in LN, as well as its appli-
cation as biomarker for lupus nephritis, require further
study.
5. ACKNOWLEDGEMENT
This study was supported in part by the CUHK research account
6901031. All authors declare no conflict of interest. The results pre-
sented in this paper have not been published previously in whole or
part, except in abstract format.
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