2016, Volume 32, Number 3, Page(s) 158-163
Expression of Neutrophil Gelatinase-Associated Lipocalin and Kidney Injury Molecule-1 in Wilms Tumor
Senem ERSAVAŞ1, Gülden DİNİZ1, Hülya TOSUN YILDIRIM2, Yetkin KOCA1, Dudu SOLAKOĞLU KAHRAMAN1, Duygu AYAZ1, Bengü DEMİRAĞ3
1Department of Pathology, Tepecik Education and Research Hospital, İzmir, Turkey
2Department of Pathology, Dr. Behcet Uz Children's Hospital, İzmir, Turkey
3Department of Oncology, Dr. Behcet Uz Children's Hospital, İzmir, Turkey
Keywords: Wilms Tumor, NGAL protein, KIM-1 protein
Neutrophil gelatinase-associated lipocalin (NGAL) and Kidney injury molecule-1 (KIM-1) play important roles in both immunity
and cell proliferation. It was reported previously that they are overexpressed in various human cancers. The present study was undertaken to
examine the expressions of NGAL and KIM-1 in Wilms Tumors.
Material and Method: Tissue samples of 50 Wilms Tumors were evaluated and underwent immunhistochemical staining for NGAL and KIM-1
protein expressions. The correlations between them, and some clinical prognostic factors such as tumor weight, stage and histological features
were also evaluated.
Results: Twenty-three (46%) of the cases were male while 27 (54%) were female. The mean age was found to be 3.26±2 years. The average tumor
size was 9.16 ± 2.9 cm in diameter and the average weight of the kidney was 478±312 gr. Thirteen (26%) cases were stage I, 18 (36%) cases were
stage II, 7 (14%) cases were stage III, and 6 (12%) cases were stage IV. Thirty-nine cases were alive (78%), while 11 cases (22%) were deceased.
Mean overall survival time was 68.2±39.5 (2-148) months. NGAL expression was negative in all tumors except the neutrophils within the tumors.
KIM-1 expression was positive in 37 tumors (74%), while it was absent in 13 tumors (26%). Using Mann-Whitney U Analysis, KIM-1 expression
was found to be associated with the stage of the tumor (p=0.027).
Conclusion: The preliminary data indicates that KIM-1 expression may be associated with stage in Wilms Tumor. However, further studies are
needed to validate these pilot observations and to clarify the functional and mechanistic significance of this relevance.
Wilms Tumor (WT) is the most common malignant renal
tumor of children, accounting for approximately 14% of
. Although survival rates in WT have
been improved in the past decades due to a multidisciplinary
therapeutic approach, a certain population of the patients
continue to experience poor survival and increased rates
. Mutations and abnormal expressions of the
6 WT genes basically contribute to tumorigenesis of WT
but other genes also participate in its development. Recent
studies have revealed that several genetic abnormalities are
associated with a worse prognosis in WT, even in those
with localized stage and favorable histology3,4
Neutrophil gelatinase-associated lipocalin (NGAL), a
member of the lipocalin superfamily, was first isolated
as a 25 kDA glycoprotein covalently bound to matrix
metalloproteinase 9 (MMP9) in human neutrophils5.
Although initially in neutrophils, it was later found to be expressed in most epithelial cells and to participate in the
diverse processes of growth, development, differentiation
and tumorigenesis of many tissues6,7.
Kidney injury molecule-1 (KIM-1) was first reported as
a sensitive and specific biomarker in detecting injury of
the proximal tubules in 1998 by Ichimura8. KIM-1 is a
type 1 membrane protein that contains a novel six-cysteine
immunoglobulin-like domain and a mucin domain.
Structurally, KIM-1 is a member of the immunoglobulin
gene superfamily most reminiscent of mucosal addressin
cell adhesion molecule 1 (MAdCAM-1). Human KIM-1
is also homologous to the monkey hepatitis A virus cell
receptor 1 (HAVcr-1)9. KIM-1 is expressed at a low level
in the normal kidney but is increased dramatically in the
Hitherto, many parameters have been suggested as relevant
markers for assessing the proliferative activity and tumor
cell dynamics of WT11-13. However, the presence of NGAL and KIM-1 expressions in WT has not been
investigated widely10. The aim of this study was to explore
the importance of these two markers in Wilms tumor and
also to investigate the correlations between them, and some
clinical prognostic factors such as tumor weight, stage and
WT resection specimens of 50 cases diagnosed and treated
in Dr. Behçet Uz Children’s Education and Research
Hospital between 1999 and 2014 were included in this study.
The study was approved by the Local Ethics Committee
of Tepecik Education and Research Hospital. The staging
system developed by the National Wilms Tumor Study
Group (NWTS) was used to describe the extent of spread
of these tumors14,15
For immunohistochemistry (IHC), hematoxylin and eosin
(H&E) staining was used to select appropriate paraffin
blocks and to identify the viable tumor areas. IHC was
performed by the streptavidin biotin peroxidase method
(Invitrogen, Camarillo, 85-9043). Serial 5-μm sections
were obtained and these slides were baked over-night
at 60oC, dewaxed in xylene, and hydrated with distilled
water through decreasing concentrations of alcohol. All
slides were treated with heat-induced epitope retrieval in
the microwave (in 10mM/L citrate buffer, pH 6.0, for 20
minutes, followed by cooling at room temperature for 20
minutes) and blocked for endogenous peroxidase and
biotin. An affinity purified monoclonal mouse antibodies
against NGAL (Novus Biologicals, Littleton, USA, NDP1-
90331) and KIM-1 (Bioss, Philadelphia, USA, HAVCR1)
were used at a dilution of 1: 300. Renal tissue with acute tubular necrosis was used as positive control for KIM-1
and splenic tissue for NGAL (Figure 1). The evaluation was
blinded to any of the clinical features and staining patterns
were classified according to the severity of staining. For
KIM-1, cytoplasmic staining similar to the proximal
tubules in control tissues was considered as positive (Figure
2). Focal staining occupying less than 5 % of the field or
diffuse weak staining were considered as negative. In
previous studies, it was reported that NGAL showed both
cytoplasmic and membranous expression in most tissues.
Contrary to the other studies, there was no expression of
NGAL in tumor cells. We counted the neutrophils that
infiltrated the tumors. If there were up to five cells in every
high power fields, we evaluated this as negative for NGAL.
Spearman Correlation analysis, Mann-Whitney U test,
Chi square test and Kaplan-Meier survival analyses were
performed for statistical analysis with SPSS 15.0. P values
less than 0.05 was considered to be statistically significant.
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|Figure 1: Cytoplasmic NGAL expression in red pulp of spleen
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|Figure 2: Cytoplasmic KIM-1 expression in proximal tubules of
post-ischemic kidney (KIM-1; x200).
Surgery, chemotherapy and radiotherapy were the treatment
modalities that were applied alone or in combination to the
total of 50 patients according to their individual features.
We used the NWTS protocol with surgery approach first
for patients with unilateral tumor, but pre-operative
chemotherapy was added and the combination of drugs
was changed for patients with bilateral tumors. In addition,
unfavorable histology required radiation therapy, even
in some localized diseases. Therefore we classified the
histology of all tumors as favorable or unfavorable. Thirtynine
(78%) cases had triphasic tumors, while 11 (22%) were
biphasic and the blastemal component was predominant. These latter 11 cases were evaluated as showing unfavorable
histology. In the whole series, 11 patients died at followup;
3 of these died because of bilateral tumor, and 4 from
conditions apparently unrelated to WT such as pneumonia,
sepsis, hepatic insufficiency and veno-occlusive disease.
Twenty-three (46%) of the cases were male while 27 (54%)
were female. The mean age was found to be 3.26±2 years
(ranging from 5 months to 8 years). The tumor was rightsided
in 25 (50%) cases, left-sided in 19 (38%) cases and
6 (12%) cases had bilateral tumors (stage V). The average
tumor size was 9.16 ± 2.9 cm in diameter and the average
weight of the kidney was 478±312 gr15. Thirteen (26%)
cases were stage I, 18 (36%) cases were stage II, 7 (14%)
cases were stage III, 6 (12%) cases were stage IV. Thirty-nine cases were alive (78%), while 11 cases (22%) were deceased.
Mean overall survival time was 68.2±39.5 (3-148) months.
The frequency of KIM-1 expression varied between
different components in the same tumor. KIM-1 was
negative in 13 (26%) cases. Expression was limited in the
epithelial component in 19 (38%) cases (Figure 3), while
it was limited in the blastemal in 7 (14%) cases (Figure
4) and in mesenchymal areas in 3 (6%) cases. In 8 cases
(16%), diffuse KIM-1 expressions were determined (Figure
5). NGAL expressions were determined in only NGALpositive
inflammatory cells within the WTs (Figure 6). In
most tumors, less than 5 NGAL-positive neutrophils per
high-power field were determined. Therefore NGAL was
considered as negative in all WTs.
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|Figure 3: Cytoplasmic KIM-1 expression in epithelial component
in WT (KIM-1; x200).
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|Figure 4: Cytoplasmic KIM-1 expression in blastemal component
in WT (KIM-1; x100).
Click Here to Zoom
|Figure 6: Note the NGAL-positive inflammatory cell within a
WT (NGAL; x200)
Most prognostic parameters such as kidney weight
(p=0.127), tumor diameter (p=0,271), patient age
(p=0.340) and therapy response (p=0.407) were not found
to be associated with KIM-1 expression using the Mann-
Whitney U and Chi-square Analyses. The overall survival
was 61.5±11.7 months in patients with KIM-1 positive
tumors while it was 70.8±6.3 months in KIM-1 negative
tumors. There were no relationship between the KIM-1
expression and the survival (Log Rank, p=0.932) by Kaplan-
Meier Survival Analysis (Figure 7).
Click Here to Zoom
|Figure 7: No statistical significant association with tumoral KIM-
1 expression and survival (Log Rank, p=0.932).
KIM-1 expression was positive in all stage I tumors and
most stage II and III tumors. In contrast, KIM-1 was
determined as negative in most stage IV tumors (Figure 8).
While 67.5% of KIM-1 positive tumors were in the earlystage,
46.2% of KIM-1 negative tumors were in the early
stage, excluding the bilateral tumors. The Chi square test
revealed a relationship between KIM-1 expression and
stage that was statistically significant (p= 0.027).
Click Here to Zoom
|Figure 8: Significant association with tumoral KIM-1 expression
and stage (p=0.027).
NGAL was initially defined as a useful bacteriostatic agent,
and was found to be over-expressed in many types of cancers
including breast, pancreatic and ovarian cancers5,6
reported effects of NGAL in tumors are contradictory. For
example, it was shown to have protumoral effects in breast16,
and esophageal cancers18
. In contrast,
some studies showed that NGAL demonstrates antitumor
and antimetastatic effect in anaplastic thyroid carcinoma
Recently Wang et al.22
reported that both NGAL gene
and NGAL expression in tumor tissue was down-regulated in head and neck squamous cell carcinoma (HNSCC) and
this down-regulation may correlate with tumorigenesis
in HNSCC. It was also reported that NGAL could form
a complex with MMP-9 to prevent its degradation and
increase MMP-9 activity. Moreover, NGAL is bound
to siderophores and participates in iron metabolism in
mammalians. Thus, iron homeostasis was speculated to
be involved with NGAL in promoting cancer. Based on
these studies, NGAL was speculated to be a new kind of
metastasis biomarker. However, the detailed mechanism
has not been totally understood yet16-22
. In the present
study, we evaluated the NGAL expression in WT. However,
we did not determine NGAL expression in tumor cells or
renal tissue. Therefore, we think that NGAL may not have
any role in tumorigenesis in WTs.
Clinical investigations have revealed that the prognosis of
WT correlates with stage and favorable histology, which
is characterized by the presence of all three histological
elements and the absence of diffuse anaplasia12,14,15.
These three histological components of WT have different
proliferation potentials and different responses to therapy.
Hitherto, many studies have revealed these differences.
In most reports, the lowest proliferative capacity was
determined in the mesenchymal component and this
component generally survived after chemotherapy14,15.
In the present study, KIM-1 expression was determined in
the early stage tumors. In addition, we determined KIM-
1 expressions confined to the epithelial and blastemal
components in the most cases. Our results have two
important implications. Firstly, the relationship between
the KIM-1 expression and stage suggests that KIM-1 may be used an important indicator of localized disease.
Secondly, KIM-1 expression is potentially relevant in WT
differentiation. However, further research is required to
define how KIM-1 expression status can be used to clinical
advantage in WT.
As in several body fluids, the urine is a rich reservoir of
various substances and extracellular vesicles, directly
originating from cells facing the urinary lumen. These
substances are secreted by all types of cells under both
physiological and pathological conditions. Some of them
are accepted as markers of glomerular and tubular damage,
as well as of renal regeneration. In addition, some substances
appear to be involved in the cell-to-cell communication
along the nephron and to emerge as potential amplifying
or limiting factors in renal damage. Substances secreted
from injured cells may favor the demonstration of fibrosis
or disease progression. KIM-1 is one of these substances
in the urine and it has been identified as representing an
incredible source of information for diagnostic purposes23. Several studies revealed that KIM-1 is expressed in
both proliferating and dedifferentiated epithelial cells in
regenerating proximal tubules. In addition, it is an epithelial
cell adhesion molecule up-regulated in the cells, which are
dedifferentiated and undergoing replication. KIM-1 may
play an important role in the restoration of the morphological
integrity and function to the post-ischemic kidney. KIM-1
is a sensitive and specific biomarker in detecting injury of
proximal tubules in humans and other animals10,23,24.
Recent studies indicate that KIM-1 may play an important
role in the tumorigenesis of renal cell carcinomas10,24.
Kidney development is a complex process regulated by
transcription factors, proto-oncogenes, and several growth
factors that act as signaling molecules and their receptors.
WT can be considered as a failure of this transition11. In
this study, we determined KIM-1 expression in most WTs.
This finding indicates that urinary KIM-1 may also be a
marker in WTs. KIM-1, a marker of tubular damage, may
possibly be useful to gain information about tissue damage,
regeneration and even tumorigenesis in WTs.
In summary, the preliminary data indicates that KIM-
1 is frequently expressed in WT and this expression is
negatively associated with stage in WTs. However, further
studies are needed to validate these pilot observations and
to clarify the functional and physiopathologic significance
of this relevance. Contrary to the other studies, we did
not determine any association with NGAL protein and
tumorigenesis in WT.
CONFLICT OF INTEREST
The authors declared no conflict of interest
1) An Q, Wang Y, An R, Li Y, Yao T, Zhai B, Sun X. Association
of E2F3 expression with clinicopathological features of Wilms’
tumors. J Pediatr Surg. 2013;48:2187-93.
2) Honeyman JN, Rich BS, McEvoy MP, Knowles MA, Heller
G, Riachy E, Kobos R, Shukla N, Wolden SL, Steinherz PG, La
Quaglia MP. Factors associated with relapse and survival in Wilms
tumor: A multivariate analysis. J Pediatr Surg. 2012;47:1228-33.
3) Scott RH, Murray A, Baskcomb L, Turnbull C, Loveday C, Al-
Saadi R, Williams R, Breatnach F, Gerrard M, Hale J, Kohler
J, Lapunzina P, Levitt GA, Picton S, Pizer B, Ronghe MD,
Traunecker H, Williams D, Kelsey A, Vujanic GM, Sebire NJ,
Grundy P, Stiller CA, Pritchard-Jones K, Douglas J, Rahman N.
Stratification of Wilms tumor by genetic and epigenetic analysis.
4) Diniz G, Aktas S, Cubuk C, Ortac R, Vergin C, Olgun N. Tissue
expression of MLH1, PMS2, MSH2, and MSH6 proteins and
prognostic value of microsatellite instability in Wilms tumor:
Experience of 45 cases. Pediatr Hematol Oncol. 2013;30:273-84.
5) K jeldsen L, Johnsen AH, Sengeløv H, Borregaard N. Isolation
and primary structure of NGAL, a novel protein associated with
human neutrophil gelatinase. J Biol Chem. 1993; 268:10425-32.
6) Bratt T. Lipocalins and cancer. Biochim Biophys Acta.
7) Bolignano D, Donato V, Lacquaniti A, Fazio MR, Bono C,
Coppolino G, Buemi M. Neutrophil gelatinase-associated
lipocalin (NGAL) in human neoplasias: A new protein enters the
scene. Cancer Lett. 2010;288:10-6.
8) Ichimura T, Bonventre JV, Bailly V, Wei H, Hession CA,
Cate RL, Sanicola M. Kidney injury molecule-1 (KIM-1), a
putative epithelial cell adhesion molecule containing a novel
immunoglobulin domain, is up-regulated in renal cells after
injury. J Biol Chem. 1998;273:4135-42.
9) Z hang PL, Mashni JW, Sabbisetti VS, Schworer CM, Wilson GD,
Wolforth SC, Kernen KM, Seifman BD, Amin MB, Geddes TJ,
Lin F, Bonventre JV, Hafron JM. Urine kidney injury molecule-1:
A potential non-invasive biomarker for patients with renal cell
carcinoma. Int Urol Nephrol. 2014; 46:379-88.
10) Sinha V, Vence LM, Salahudeen AK. Urinary tubular proteinbased
biomarkers in the rodent model of cisplatin nephrotoxicity:
A comparative analysis of serum creatinine, renal histology, and
urinary KIM-1, NGAL, and NAG in the initiation, maintenance,
and recovery phases of acute kidney injury. J Investig Med.
11) Diniz G, Aktas S, Turedi A, Temir G, Ortac R, Vergin C.
Telomerase reverse transcriptase catalytic subunit expression and
proliferation index in Wilms tumor. Tumour Biol. 2011;32:761-7.
12) Horster MF, Braun GS, Huber SM. Embryonic renal epithelia:
Induction, nephrogenesis, and cell differentiation. Physiol Rev.
13) Vicent S, Chen R, Sayles LC, Lin C, Walker RG, Gillespie AK,
Subramanian A, Hinkle G, Yang X, Saif S, Root DE, Huff V, Hahn
WC, Sweet-Cordero EA. Wilms tumor 1 (WT1) regulates KRASdriven
oncogenesis and senescence in mouse and human models.
J Clin Invest. 2010;120:3940-52.
14) Lanzkowsky P. Renal tumors. In: Manuel of pediatric hematology
and oncology. 5th ed. London: Elsevier; 2011. 695-714.
15) Tosun Yildirim H, Diniz G, Oymak Y, Demirağ B, Aktaş S, Ortaç
R, Solakoğlu Kahraman D, Sayhan S, Hoşgör M. Caveolin-1
expression is associated with tumor size and therapy response in
Wilms tumor. Turkiye Klinikleri J Med Sci. 2015;35:31-5.
16) Li SH, Hawthorne VS, Neal CL, Sanghera S, Xu J, Yang J, Guo H,
Steeg PS, Yu D. Upregulation of neutrophil gelatinase-associated
lipocalin by ErbB2 through nuclear factor-kappaB activation.
Cancer Res. 2009;69:9163-8.
17) K ubben FJ, Sier CF, Hawinkels LJ, Tschesche H, van Duijn W,
Zuidwijk K, van der Reijden JJ, Hanemaaijer R, Griffioen G,
Lamers CB, Verspaget HW. Clinical evidence for a protective role
of lipocalin-2 against MMP-9 autodegradation and the impact for
gastric cancer. Eur J Cancer. 2007;43:1869-76.
18) Z hang H, Xu L, Xiao D, Xie J, Zeng H, Wang Z, Zhang X, Niu Y,
Shen Z, Shen J, Wu X, Li E. Upregulation of neutrophil gelatinaseassociated
lipocalin in oesophageal squamous cell carcinoma:
Significant correlation with cell differentiation and tumour
invasion. J Clin Pathol. 2007;60:555-61.
19) Volpe V, Raia Z, Sanguigno L, Somma D, Mastrovito P, Moscato F,
Mellone S, Leonardi A, Pacifico F. NGAL controls the metastatic
potential of anaplastic thyroid carcinoma cells. J Clin Endocrinol
20) Tung MC, Hsieh SC, Yang SF, Cheng CW, Tsai RT, Wang SC,
Huang MH, Hsieh YH. Knockdown of lipocalin-2 suppresses
the growth and invasion of prostate cancer cells. Prostate.
21) Nuntagowat C, Leelawat K, Tohtong R. NGAL knockdown by
siRNA in human cholangiocarcinoma cells suppressed invasion
by reducing NGAL/MMP-9 complex formation. Clin Exp
22) Wang L, Chen C, Li F, Hua Q, Chen S, Xiao B, Dai M, Li M, Zheng
A, Yu D, Hu Z, Tao Z. Down-regulation of neutrophil gelatinaseassociated
lipocalin in head and neck squamous cell carcinoma
correlated with tumorigenesis, not with metastasis. Int J Clin Exp
23) R anghino A, Dimuccio V, Papadimitriou E, Bussolati B.
Extracellular vesicles in the urine: Markers and mediators of
tissue damage and regeneration. Clin Kidney J. 2015;8:23-30.
24) Cuadros T, Trilla E, Sarró E, Vilà MR, Vilardell J, de Torres I,
Salcedo M, López-Hellin J, Sánchez A, Ramón y Cajal S, Itarte E,
Morote J, Meseguer A. HAVCR/KIM-1 activates the IL-6/STAT-3
pathway in clear cell renal cell carcinoma and determines tumor
progression and patient outcome. Cancer Res. 2014;74:1416-28.