Articles in PresS. Am J Physiol Renal Physiol (September 24, 2008). doi:10.1152/ajprenal.90250.2008
Lipocalin-2-induced renal regeneration depends on cytokines
Eugenia Vinuesa 2, Anna Sola1,2*, Michaela Jung 2, Vicente Alfaro 3,
Georgina Hotter 1,2*
* These authors directed this study and contributed equally.
1 CIBER-BBN, Networking Center on Bioengineering, Biomaterials and
Nanomedicine, Barcelona, Spain.
2 Department of Experimental Pathology, IIBB-CSIC-IDIBAPS, Rosselló, 161,
7ª planta, 08036 Barcelona, Spain.
3 Department of Physiology (Biology), University of Barcelona, Avinguda
Diagonal 645, 08028 Barcelona, Spain.
Running head: Lipocalin-2 regeneration and cytokines
Author for correspondence:
Georgina Hotter
Dept. of Experimental Pathology,
IIBB-CSIC-IDIBAPS, Rosselló, 161, 7ª planta.
08036 Barcelona, Spain
Phone: 34 93 3638334
Fax:
+34.93. 3638301
e-mail: [email protected]
1
Copyright © 2008 by the American Physiological Society.
ABSTRACT
This study investigated whether the renal regeneration occurring in the recovery
phase of kidney ischemia/reperfusion (I/R) is mediated by endogenously
generated lipocalin-2 (Lcn2). A second objective was to examine whether Lcn2mediated cell effects could be regulated by the inflammatory cytokines in the
environment through their action on Lcn2 receptors (Lcn2R and megalin).
Male Swiss mice were subjected to 30 minutes of renal ischemia with a
reperfusion period of 24 hours (early reperfusion, expected time for maximum
inflammation) and 96 hours (late reperfusion, expected time for maximum
regeneration). Different experimental groups underwent I/R, I/R with i.v. antimouse Lcn2 monoclonal antibody injected during the early/inflammatory or
late/recovery
phase,
and
I/R
with
pro-inflammatory
cytokine
cocktail
administration (recombinant mouse IL-1β, TNF-α and IFN-γ). Compared to
control non-ischemic mice, the expression of three proliferation markers
(stathmin, PCNA and Ki-67, analyzed by quantitative RT-PCR) increased
significantly in the I/R treated animals. Blockade of Lcn2 by addition of anti-Lcn2
antibody significantly decreased the expression of these three proliferation
markers when administered in the late/reparative phase, but had the opposite
effect when administered in the early/inflammatory phase. Pro-inflammatory
cytokine cocktail administration reduced the proliferative effects of Lcn2, and
repressed Lcn2R and megalin expression. In conclusion, endogenously
generated Lcn2 induces renal cell regeneration depending on the inflammatory
cytokines in kidney I/R.
Key words:
Lcn2; mice; kidney; tubular cells; ischemia/reperfusion; cell
regeneration.
2
INTRODUCTION
Acute or chronic low grade ischemia is the main cause of renal failure
(10). The initial phase of ischemia/reperfusion (I/R) (i.e., the first 24 hours after
reperfusion) involves the onset and progression of tissue injury, and is
characterized by loss of cell polarity, increased cell swelling, loss of adherence
to the extracellular matrix and cell death (32). The injury phase is followed by
the recovery phase, which at the cellular level is manifested by an increase in
dedifferentiated and mitotic cells in the damaged areas (16). The presence of
these dedifferentiated, mitotic cells indicates an ongoing regenerative process
after kidney I/R injury. Nevertheless, cells involved in the regenerative process
of renal tissue include a smaller number of cells derived from the bone marrow
(i.e., stem cells) (14).
The identification of interventions that might enhance the recovery phase
is of considerable interest, because improved knowledge of the mediators of
this cell regeneration may be critical for developing new innovative and effective
therapies. Previous studies have shown that neutrophil gelatinase-associated
lipocalin (Lcn2/NGAL), a novel early urinary biomarker for ischemic renal injury,
is up-regulated in tubular epithelial cells undergoing proliferation in the postischemic kidney (21). Renoprotective effects have been found for Lcn2
administered in vivo to I/R injured mice (22). Lcn2 plays a regulatory role in
epithelial morphogenesis in the mouse by promoting the organization of cells
into tubular structures (15). As Lcn2 is massively upregulated after renal tubular
injury and may participate in limiting kidney damage (30), we hypothesized that
the injury versus reparative phases of I/R might both be mediated by different
Lcn2 effects.
3
In renal tubular cells, Lcn2 may be induced by the local release of
cytokines such as HGF from renal epithelial cells or infiltrated inflammatory cells
(6; 11; 15). This is also postulated in other pathologies, in which Lcn2 induction
is the result of interactions between inflammatory cells or cytokines and the
epithelial lining (1; 3; 18; 23; 35). Although the effects of cytokines on Lcn2
induction have been previously assessed, no studies so far have investigated
the effect of cytokines on the modification of Lcn2 function.
Megalin, a multifunctional scavenger receptor highly expressed in kidney
epithelial cells, binds Lcn2 with high affinity (2) and functions as a receptor for
Lcn2 (17). Devireddy et al. (9) isolated a specific cell-surface receptor of Lcn2
(a highly conserved protein named brain type organic cation transporter
(BOCT)) Lcn2R, from murine cells, and later Richardson et al (27) presented
evidence that some oncoproteins that increase expression of apo-NGAL (the
human homolog of Lcn2) could repress the expression of its receptor (NGALR).
To data, no studies have attempted to identify the effect of cytokines on Lcn2R
expression.
Bearing in mind that renal regeneration is dependent on inflammatory
cytokines (8; 13; 33) and that Lcn2, being an inducer of renal regeneration,
could be modulated by cytokines, we decided to investigate the involvement of
both
factors
in
the
regenerative
process
associated
with
renal
ischemia/reperfusion.
The present study was designed to investigate whether the injury versus
reparative phases of I/R might both be mediated by different Lcn2 effects and
whether Lcn2-mediated cell effects might be regulated by the cytokine
environment through its action on Lcn2 receptors.
4
MATERIALS AND METHODS
Mouse Model of Renal Ischemia-Reperfusion Injury
All animal procedures were approved by the Institutional Animal Care and Use
Committee (IACUC) and followed the European Union guidelines for the
handling and care of laboratory animals. Male Swiss mice (Charles River)
weighing 25-30 g were housed with 12:12-h light:dark cycle and were allowed
free access to food and water. The animals were anesthetized with isoflurane
and body temperature was maintained at 37°C. Renal pedicles were occluded
with a non-traumatic vascular clamp for 30 minutes, while the kidney was kept
warm and moist. Thereafter, clamps were removed, return of kidney blood was
observed and the incision was sutured. The reperfusion period was selected
based on a previous study (33) in which the maximum plateau for regeneration
was observed at 96 hours. After 96 hours of reperfusion, the animals were reanesthetized, the abdominal cavity was opened and blood was obtained via
puncture of the inferior vena cava to measure blood urea nitrogen (BUN). Mice
were killed and kidneys were harvested. One half of each kidney was snapfrozen in liquid nitrogen and stored at -80°C until further processing. A sample
was fixed in formalin, paraffin-embedded and sectioned (4 µm).
Experimental groups
The following experimental groups were studied (n=5 each):
● Control (C). In the control group, animals were subjected to the identical
manoeuvres as the I/R group, except that the renal pedicles were not clamped.
5
● Ischemia / early reperfusion (I/early Reperfusion): animals underwent 30
minutes of bilateral ischemia and were killed after 24 hours of reperfusion.
● Ischemia / late reperfusion (I/late Reperfusion): animals underwent 30
minutes of bilateral ischemia and were sacrified after 96 hours of reperfusion.
● Ischemia / early reperfusion + anti-mLcn2 administration (I/early
reperfusion+anti-mLcn2): animals underwent the same procedure as the I/R
group, but were treated with monoclonal anti-mouse Lcn2 antibody (75 µg i.v.
bolus) in the inflammatory phase (24 hours).
● Ischemia / late reperfusion + anti-mLcn2 administration (I/late
reperfusion+anti-mLcn2): animals underwent the same procedure as the I/R
group, but were treated with monoclonal anti-mouse Lcn2 antibody (75 µg i.v.
bolus) in the regenerative phase (72 hours). Administration of anti-Lcn2
antibody has previously been found effective in reversing Lcn2-associated
effects (26).
● Rat IgG administration (I/late Reperfusion + rat IgG): as a control group,
animals underwent the same procedure as the I/R group, but were treated with
(75 µg i.v. bolus) in the regenerative phase (72 hours).
● Pro-inflammatory cytokine cocktail administration (I/late Reperfusion +
Cytokines): animals underwent the same procedure as the I/R group but also
received an infusion of a pro-inflammatory cytokine cocktail (200 µl i.p.) one day
before sacrifice.
6
Lcn2 antibody and rat IgG administration
Mice were injected i.v. via tail vein with 150 µl of phosphate-buffered saline
(PBS) containing 75 µg of monoclonal anti-mouse Lcn2 antibody (R&D
Systems) or rat IgG (R&D) respectively.
Cytokine Administration
To modulate the inflammatory response, mice were treated i.p. with a proinflammatory cytokine cocktail containing 200 µl of PBS with recombinant
mouse IL-1β (100 ng), TNF-α (2 µg) and IFN-γ (750 ng) (all cytokines provided
by Invitrogen). These or similar doses of mouse/rat cytokines have previously
been found to exert biological effects in murine inflammation models (5; 20; 29;
34).
Plasma urea and creatinine
Blood urea (BUN) was determined in plasma using a Sigma diagnostic kit, and
the results are expressed as mg/dl. Creatinine concentrations was determined
by a standardized colorimetric assay using alkaline picrate with a Advia 2400
automated analyzer (Siemens Medical Solutions Diagnostics) (4; 31), at the
Clinical Hospital of Barcelona, Spain.
Proliferation Assays
Kidneys were fixed in 4% paraformaldehyde, embedded in paraffin and cut into
sections of 4 µm. Immunofluorescent staining of stathmin and proliferating cell
nuclear antigen (PCNA) were prepared, as previously described (33; 36).
Briefly, paraformaldehyde-fixed paraffin-embedded sections were washed in
7
PBS and blocked. Tissue sections were incubated with rabbit anti-stathmin
polyclonal antibody (Calbiochem), which labels dedifferentiated, mitotically
active epithelial cells, and with mouse anti-PCNA monoclonal antibody (Santa
Cruz Biotechnology), which labels cells in the G1, S, and G2 phases of the cell
cycle, followed by incubation with secondary antibodies (rabbit anti-goat IgG
conjugated with Alexa Fluor 488 for anti-stathmin antibody and goat anti-mouse
IgG conjugated with Alexa Fluor 568 for anti-PCNA antibody (Molecular Probes)
for 2 hours at room temperature. Sections were mounted with mowiol
(Calbiochem) and viewed using a Leica TCS NT laser microscope (Leica
Microsystems). Slides were examined in a blinded manner, and proliferation
was quantified by counting the number of stathmin and PCNA-positive cells per
100 cells counted in an average of five high-power fields (x40) in each section.
Stathmin is a ubiquitously expressed cytosolic phosphoprotein (25; 28) that has
been identified as a marker of cell proliferation in the recovery phase of acute
ischemic renal failure (36). Increased expression of stathmin is associated with
the re-entry of cells into the cell cycle and the onset of cell proliferation (7),
whereas PCNA labels proliferating nuclei.
Quantitative Real Time Reverse Transcriptase-Polymerase Chain Reaction
(qRT-PCR)
Total tissue and cells RNA were extracted from homogenized tissue with TRIzol
Reagent (Invitrogen) according to the manufacturer’s instructions, and RNA
concentrations were calculated from A260 determinations. One microgram of
RNA was reverse transcribed by iScript cDNA Synthesis Kit (Bio-Rad) in a final
volume of 40 µl.
8
Stathmin, PCNA, Ki-67, Lcn2, Lcn2R, megalin, TNF-α, MCP-1 and IL-10 mRNA
expression normalized to the housekeeping gene GADPH were measured by
qRT-PCR, using the appropriate primers (Table 2), performed in a Bio-Rad
iCycler iQ Real-Time PCR Detection System. Amplifications were carried out in
a 20-µl reaction volume, using IQ SYBR Green Supermix (Bio-Rad), according
to the manufacturer's instructions.
In vitro test to study the antibody effect as blocker of Lcn2 activity
Mouse RAW 264.7 macrophages (European Collection of Cell Culture,) were
cultured in Dulbecco’s modified Eagle’s Medium (DMEM) 1:1 F-12 nutrient
supplement with high glucose, 15mM Hepes and stable L-glutamine,
supplemented with 100 units/ml penicillin, 100µg/ml streptomycin, and 10%
(v/v) fetal bovine serum (Invitrogen). Cells were kept in a humidified atmosphere
of 5% CO2 in air at 37°C. RAW 264.7 macrophages were transferred at 70-80%
confluence by cell scraping. For the experiments, macrophages were
transferred to a 6-well plate at a density of 1x106 cells per well. Cells were
incubated with LPS (15µg/ml) (Sigma) for 24h to stimulate Lcn2 production (24;
37) before antibody treatment. Cells were washed with PBS and fresh culture
medium containing either 75 µg of monoclonal antibody against mouse Lcn2 or
rat IgG to LPS-stimulated macrophages and incubated for 24h.
Lcn2 ELISA
Supernatants from the in vitro text cultures were collected and clarified by
centrifugation. 50 µl of each sample were applied to an ELISA as previously
described (19). Briefly, 96-well-plate previously covered with catch Anti-mouse
9
Lipocalin-2/NGAL monoclonal antibody (R&D) and blocked for 1 hour. After
sample incubation, biotinylated anti-mouse Lipocalin-2/NGAL antibody (R&D)
was added. Afterwards, HRP-conjugated avidin (Invitrogen) was incubated for 1
hour. Finally, color reagent (OPD tablets, Dako) was added and absorbance
was read at 492 nm in a plate reader. All steps were performed at room
temperature.
Statistical Analyses
Data were recorded as mean ± standard error of mean (SEM). The means of
different groups were compared using one-way ANOVA. The Student-NewmanKeuls test was performed in order to evaluate significant differences between
groups, which were assumed to exist when p<0.05.
RESULTS
Lcn2 Influences Tubule Cell Proliferation after Ischemic Injury
Representative kidney sections stained with antibodies to stathmin and PCNA
are shown in Figure 1. Non-ischemic control kidneys had a minimal presence of
proliferating cells (2.8 ± 0.60 for stathmin and 1.5 ± 0.45 for PCNA) (Figure 1A);
8 fields counted per sample and averaged). Kidneys subjected to I/R showed a
high amount of cells expressing stathmin and PCNA (57 ± 1.67 for stathmin and
26.50 ± 1.09 for PCNA) when samples were obtained during late reperfusion
(Figure 1B); this number was less when samples were obtained during early
reperfusion (14.3 ± 0.82 for stathmin and 9.1 ± 1.21 for PCNA). Animals that
were treated with anti-Lcn2 antibody during early reperfusion (24 hours) had a
10
significant increase in the number of stathmin- and PCNA-positive proximal
tubule epithelial cells (66.2 ± 2.05 for stathmin and 36.8 ± 1.95 for PCNA)
(Figure 1C). In contrast, animals that were treated with anti-Lcn2 antibody
during late reperfusion (72 hours, regenerative phase) showed a marked
decrease in proliferating proximal tubule cells with respect to the I/R group (12 ±
0.81 for stathmin and 7 ± 0.63 for PCNA) (Figure 1D).
Kidney injury, evaluated as BUN and plasma creatinine, presented higher injury
development during early reperfusion than during late reperfusion periods. The
different treatments did not modify the increased injury markers (Figure 2).
Three markers of proliferation (stathmin, PCNA and Ki-67) were analyzed by
quantitative RT-PCR to determine the involvement of Lcn2 in the recovery of
renal tissue after I/R injury (Figure 3). The increase in the three proliferation
markers after I/late Reperfusion was dramatically reversed after blockage of
Lcn2 at late reperfusion stages. By contrast, anti-Lcn2 antibody administration
during early reperfusion significantly increased the expression of the three
proliferation markers. Administration of rat IgG during late reperfusion did not
modify the increases observed in the three proliferation markers in the late
reperfusion. These results provide evidence of a dual role for Lcn2 as an
inducer or inhibitor of renal cell proliferation.
Effect of pro-inflammatory cytokines on regeneration
Three markers of proliferation (stathmin, PCNA and Ki-67) were analyzed by
quantitative RT-PCR to determine the involvement of cytokines in the recovery
phase of renal tissue after I/R injury (Figure 4). The significant increase in the
11
expression of the three proliferation markers observed in the I/R group was
reversed when the pro-inflammatory cytokine cocktail was administered.
Cytokine Expression
We analyzed cytokine expression by quantitative RT-PCR (Figure 5). MCP-1
expression was increased in the I/R group compared to controls although, as
expected, the highest expression was observed when the pro-inflammatory
cytokine cocktail was administered (Figure 5A) and in the inflammatory phase
(I/early reperfusion). However, the anti-inflammatory cytokine IL-10 showed
different results. I/R showed increased IL-10 expression when compared to
controls, but addition of the pro-inflammatory cytokine cocktail did not
significantly change IL-10 expression (Figure 5B).
Lcn2, Lcn2R and megalin expression
Lcn2 and Lcn2-receptor mRNA expression was analyzed by RT-PCR in renal
tissue (Figure 6). Lcn2 expression was increased in kidney in the I/R groups
with respect to controls. But significantly higher levels were observed during
early reperfusion periods (24 hours) with respect to the late reperfusion and
regenerative phase (96 hours). Cytokine administration induced significant
increases in Lcn2 levels (Figure 6A).
By contrast, Lcn2R and megalin expression profile showed the opposite
behaviour to that observed for Lcn2 expression. Lcn2R and megalin mRNA
decreased in the early/inflammatory phase of ischemia (24 hours), increased
during the late/reparative phase (96 hours) and decreased with cytokine
administration (Figures 6B and 6C).
12
In vitro test to study the antibody effect as blocker of Lcn2 activity
As shown in figure 7, under normal cell culture conditions, murine macrophages
(RAW 264.7) responded directly to the addition of LPS by an increase in the
expression of Lcn2 mRNA, TNF-α mRNA and Lcn2 protein. Addition of antimLcn2 antibody decreased Lcn2 mRNA expression and Lcn2, and increased
TNF-α mRNA, while administration of rat IgG had no effect, indicating the ability
of anti-mLcn2 antibody to block various effects classically attributed to Lcn2.
DISCUSSION
In this study we found that renal regeneration after I/R is mediated by
endogenously generated Lcn2. The regenerative function of Lcn2 is influenced
by the kidney inflammatory status and the expression of Lcn2 receptors.
Lcn2 is an acute phase protein involved in multiple apoptotic events and
in proliferation. In the kidney, it is known that Lcn2 is produced at sites of injury
and that may modulate renal repair and, therefore, kidney integrity (15; 22). In
our study, we found that Lcn2 expression increased significantly in the
early/inflammatory and also in the late/reparative phases of post-ischemic
reperfusion. However, Lcn2 blockade with specific anti-Lcn2 antibody generates
different effects according to the time of administration; results showed that
Lcn2 blockade was able to reduce the three different markers of regeneration
(stathmin, PCNA and Ki-67) when administered in the regenerative phase, but
had the opposite effect when applied in the inflammatory phase, in which
antibody administration promotes regeneration. This finding indicates a dual
13
role of the endogenously generated Lcn2, which may be either pro- or antireparative depending on the phase of reperfusion.
Previous studies have shown that cell regeneration versus injury during
the renal I/R process are directly related to inflammatory activity. Maximum cell
regeneration occurred during phases in which an anti-inflammatory environment
(represented by maximum levels of IL-10) (33) prevailed. Our results indicate
that during the early/inflammatory phase of reperfusion, Lcn2 does not induce
proliferation, but that during the anti-inflammatory and reparative phase it acts
as an inducer of proliferation, suggesting that an anti-inflammatory environment
may be a requisite for Lcn2 to induce renal cell regeneration. In fact, we have
detected that during the late/reparative phase, increases in the proliferative
markers are concomitant with significant increases in the anti-inflammatory
cytokine IL-10.
The effect of inflammatory cytokines on Lcn2 expression has been
studied previously. Lcn2 is up-regulated in response to inflammation in
neoplastic tissues and in inflammatory bowel diseases and its expression also
increases in epithelial cells after stimulation with pro-inflammatory cytokines
(12; 23). However, the effect of inflammatory cytokines on counteracting the
reparative role of Lcn2 has not been studied to date.
The results of the present study confirm that the addition of the proinflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ) reduces renal
regenerative effects in a period in which regeneration is modulated by Lcn2. In
addition, administration of pro-inflammatory cytokines increased the expression
of pro-inflammatory cytokines such as MCP-1 (Figure 5A), known for its
properties in the pathogenesis of renal I/R (13). Overall, these findings indicate
14
that the induction of pro-inflammatory environment is able to modify the
regenerative profile induced by Lcn2.
As Lcn2 levels were higher in the groups in which the regeneration was
decreased (see Lcn2 levels at 24 hours or in the cytokine administered groups,
figure 6A), the observed effects on regeneration cannot have been due to
increased Lcn2 production. Devireddy and colleagues (9) presented evidence
indicating that expression of apo-NGAL (the human homolog of Lcn2) and
repression of its receptor (NGALR) may be observed in other pathologies. But
to our knowledge no data are reported concerning the effect of cytokines on
Lcn2R expression.
In adult kidneys, megalin appears to be the predominant receptor for
Lcn2 (2; 17). We therefore tested the expression of megalin mRNA in the
different experimental conditions. Our results indicate that receptor expression
was repressed in the groups in which Lcn2 was overexpressed, and
regeneration reduced (see Lcn2R and megalin expression in 24 hours, or in the
cytokine administered group). This suggested that the effect of cytokines on the
regenerative action of Lcn2 could be attributed to their effect on receptors
expression, not to an effect on Lcn2 expression or Lcn2 synthesis by itself.
Thus, the inflammatory cytokines exert an effect on Lcn2 receptors.
The Lcn2-induced regenerative process could be described as follows:
inflammation due to kidney I/R appears to increase endogenous Lcn2
production in renal tissue, already in the first phases of reperfusion after the
ischemic insult. This early pro-inflammatory milieu appears to minimize the
regenerative effects of Lcn2. In later phases of reperfusion, transition from a
15
pro-inflammatory to an anti-inflammatory environment results in an increased
up-regulation of Lcn2, which contributes to repair.
In summary, we report that renal regeneration after ischemia reperfusion
is influenced by Lcn2 and cytokines. This Lcn2-mediated cell regeneration is
dependent on the inflammatory cytokines of the milieu, as the presence of proinflammatory cytokine cocktail resulted in decreased Lcn2-induced cell
regeneration. The effects of cytokines on Lcn2 function may be mediated by
their action on the expression of the Lcn2 receptors.
16
ACKNOWLEDGMENTS
The authors want to thank Angeles Muñoz for her excellent technical support and
Martín Cullell-Young for English edition.
DISCLOSURE
The authors confirm that there is no conflict of interest to be disclosed
FUNDING SOURCES:
This study was supported by FIS 05/0219 (awarded to GH), FIS 06/0173
(awarded to GH), European Project PL 036813 (awarded to GH), and FIS
05/0156 (awarded to AS). Eugenia Vinuesa is supported by IDIBAPS.
17
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PC and Brown G. Stathmin expression is a feature of proliferating cells of
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29. Sarraf P, Frederich RC, Turner EM, Ma G, Jaskowiak NT, Rivet DJ, III,
Flier JS, Lowell BB, Fraker DL and Alexander HR. Multiple cytokines
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31. Schwartz GJ, Furth S, Cole SR, Warady B and Munoz A. Glomerular
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macrophages. Mol Endocrinol 22: 1416-1426, 2008.
24
FIGURE LEGENDS
Figure 1. Representative immunofluorescence pictures of stathmin
(green) and proliferating cell nuclear antigen (PCNA) (red) staining of
kidney samples.
(A) Control; (B) I/late Reperfusion (30 minutes of ischemia followed by 96
hours of reperfusion); I/early Reperfusion + anti-mLcn2 (I/R with Lcn2
antibody administration in early reperfusion 24 hours); (C) I/late Reperfusion +
anti-mLcn2 (I/R with Lcn2 antibody administration in late reperfusion 72 hours).
Images are viewed under x400 magnification.
Figure 2. BUN (A) and plasma creatinine (B) in different time points of antimouse Lcn2 antibody administration C: Control; I/early Reperfusion: 30
minutes of ischemia followed by 24 hours of reperfusion; I/late Reperfusion: 30
minutes of ischemia followed by 96 hours of reperfusion; I/early Reperfusion +
anti-mLcn2: I/R with Lcn2 antibody administration at 24 hours post-reperfusion;
I/late Reperfusion + anti-mLcn2: I/R with Lcn2 antibody administration at 72
hours.
I/late Reperfusion + Cytokines: I/R with pro-inflammatory cytokine
cocktail administration at 72 hours. I/late Reperfusion + rat IgG: I/R with rat
IgG administration at 72 hours. Data shown are mean ± SEM (n=5 each group).
*p<0.05 vs. C; + p<0.05 vs. I/late Reperfusion.
Figure 3. Patterns of normalized stathmin (A), proliferating cell nuclear
antigen (PCNA) (B) and Ki-67 (C) mRNA expression assessed by qRT-PCR
in different time points of anti-mouse Lcn2 antibody administration.
25
C: Control; I/early Reperfusion: 30 minutes of ischemia followed by 24 hours
of reperfusion; I/late Reperfusion: 30 minutes of ischemia followed by 96 hours
of reperfusion; I/early Reperfusion + anti-mLcn2: I/R with Lcn2 antibody
administration at 24 hours post reperfusion; I/late Reperfusion + anti-mLcn2:
I/R with Lcn2 antibody administration at 72 hours. I/late Reperfusion +
Cytokines: I/R with pro-inflammatory cytokine cocktail administration at 72
hours. I/late Reperfusion + rat IgG: I/R with rat IgG administration at 72 hours.
Data shown are mean ± SEM (n=5 each group). *p<0.05 vs. C; + p<0.05 vs.
I/late Reperfusion.
Figure 4. Effect of cytokines on patterns of normalized stathmin (A),
proliferating cell nuclear antigen (PCNA) (B) and Ki-67 (C) mRNA
expression assessed by real-time RT-PCR.
C: control; I/early Reperfusion: animals subjected to 30 min of bilateral
ischemia and killed after 24 hours of reperfusion; I/late Reperfusion: animals
subjected to 30 min of bilateral ischemia and killed after 96 hours of reperfusion;
I/late Reperfusion + Cytokines: I/R with cytokine administration (recombinant
mouse 100 ng IL-1β, 2 µg TNF-α and 750 ng INF-γ). mRNA expression
normalized to GAPDH is shown as mean ± SEM (n=5 each group). * p<0.05 vs.
C; + p<0.05 vs. I/late Reperfusion.
Figure 5. Cytokine profiles. Quantitative RT-PCR from representative renal
injury cytokines. A, monocyte chemotactic protein 1 (MCP-1) expression; B,
interleukin 10 (IL-10) expression.
26
C: control; I/early Reperfusion: animals subjected to 30 min of bilateral
ischemia and killed after 24 hours of reperfusion; I/late Reperfusion: animals
subjected to 30 min of bilateral ischemia and killed after 96 hours of reperfusion;
I/late Reperfusion + Cytokines : I/R with cytokine administration
Data shown are mean ± SEM (n=5 each group). * p<0.05 vs. C; + p<0.05 vs.
I/late Reperfusion.
Figure 6. Patterns of normalized stathmin Lipocalin-2 (A), Lipocalin-2
Receptor (B), Megalin (C) mRNA expression in renal tissue assessed by
quantitative real-time RT-PCR in different time points of anti-mouse Lcn2
antibody administration.
C: control; I/early Reperfusion: animals subjected to 30 min of bilateral
ischemia and killed after 24 hours of reperfusion; I/late Reperfusion: animals
subjected to 30 min of bilateral ischemia and killed after 96 hours of reperfusion;
I/late Reperfusion + Cytokines: I/R with cytokine cocktail administration
(100ng IL-1β, 2 µg TNF-α and 750 ng IFN-γ). mRNA expression normalized to
GAPDH is shown as mean ± SEM (n=5 each group). * p<0.05 vs. C; + p<0.05
vs. I/late Reperfusion.
Figure 7. Biological effect of anti-mLcn2 antibody. Normalized Lipocalin-2
(A) and TNF-α (B) mRNA expression assessed by qRT-PCR, and Lipocalin-2
(C) assessed by ELISA in LPS-stimulated RAW 264.7 cell culture, under the
action of anti-mouse Lcn2 antibody and rat IgG. * p<0.05 vs. resting RAW
264.7; + p<0.05 vs. LPS-stimulated RAW 264.7.
27
A
B
C
D
Figure 1
A
*+
50
Blood Urea Nitrogen
[mg/dl]
40
*
30
*
20
*
*
*
10
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es
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Lc
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Plasma Creatinine
[mg/dl]
0.4
*
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*
*+
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0.2
0.1
0.0
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I/
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Figure 2
Stathmin mRNA expression
normalized to GAPDH
A
*+
7
6
*
5
*
4
*
3
*+
2
1
0
2
2
n
n
cn
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tR
a
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te
arl
I/la
I/e
8
C
B
*+
PCNA mRNA expression
normalized to GAPDH
7
6
5
*
*
4
*
3
+
2
1
0
n2
n2
ion
ion
IgG
Lc
Lc
us
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-m
-m
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i
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erf
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+
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Re
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a
l
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I/
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I/la
I/e
C
*+
10
KI-67 mRNA expression
normalized to GAPDH
C
9
8
*
*
7
6
5
4
*+
3
*
2
1
0
n2
n2
ion
ion
IgG
Lc
Lc
us
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rat
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Figure
I/la
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3
A
6
Stathmin mRNA expression
normalized to GAPDH
5
*
4
*+
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3
2
1
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s
n
n
ine
sio
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rfu
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tok
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PCNA mRNA expression
normalized to GAPDH
C
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*
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R
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KI-67 mRNA expression
normalized to GAPDH
6
5
4
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Figure 4
A
200
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MCP-1 mRNA expression
normalized to GAPDH
175
150
*+
125
100
*
75
50
25
0
C
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arl
I/e
Re
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*
30
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IL-10 mRNA expression
normalized to GAPDH
25
20
*+
15
10
5
0
C
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I/e
Re
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tok
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Figure 5
A
30
Lipocalin-2 mRNA expression
normalized to GAPDH
25
*+
*+
20
*
15
10
5
0
s
n
n
ine
sio
sio
rfu
rfu
to k
e
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ep e
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B
4.0
*
Lcn2R mRNA expression
normalized to GAPDH
3.5
+
3.0
2.5
*+
2.0
1.5
1.0
0.5
0.0
s
n
n
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C
4.5
Megalin mRNA expression
normalized to GAPDH
4.0
3.5
3.0
+
2.5
*
2.0
+
1.5
1.0
0.5
0.0
C
r
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si
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a
Figure
I/l
on
6
A
20.0
*
22.5
*
15.0
12.5
*+
10.0
20.0
TNF-α mRNA expression
normalized to GAPDH
Lcn2 mRNA expression
normalized to GAPDH
17.5
*+
25.0
B
7.5
5.0
*
17.5
15.0
*
12.5
10.0
7.5
5.0
2.5
2.5
0.0
0.0
Raw 264.7
+
+
+
+
Raw 264.7
+
+
+
+
LPS
-
+
+
+
LPS
-
+
+
+
Anti-mLcn2
-
-
-
+
Anti-mLcn2
-
-
-
+
Rat IgG
-
-
+
-
Rat IgG
-
-
+
-
20
C
*
18
Lcn2 [ng / 10 6 cells]
16
*
14
12
10
5
4
3
2
1
*+
0
Raw 264.7
+
+
+
+
LPS
-
+
+
+
Anti-mLcn2
-
-
-
+
Rat IgG
-
-
+
-
Figure 7
Table 1. Experimetal groups performed in the study.
Reperfusion Period (hours)
*(administration point) /
+ (sacrifice point)
24 (EARLY)
72
*
Anti-Lcn2
administration
96 (LATE)
+
*
+
Rat IgG
administration
*
+
Cytokine cocktail
administration
*
+
Group
I/early Reperfusion
+ anti-mLcn2
I/late Reperfusion
+ anti-mLcn2
I/late Reperfusion
+ Rat IgG
I/late Reperfusion
+ Cytokines
Table 2. Sequences, annealing temperature and expected fragment size of primers utilized in real-time qRT-PCR.
Gene
(accession number)
Source
GAPDH (NM_008084)
Invitrogen
STATHMIN (NM_019641)
PCNA (NM_011045)
KI-67 (NM_001081117)
LIPOCALIN-2 (NM_008491)
TNF-α (NM_013693)
Primer sequences
Forward
5’-TGA-AGC-AGG-CAT-CTG-AGG-G-3’
Reverse
5’-CGA-AGG-TGG-AAG-AGT-GGG-AG-3’
Forward
5’-CTT-GCG-AGA-GAA-GGA-CAA-GC-3
Reverse
5-CGG-TCC-TAC-ATC-GGC-TTC-TA-3’
Forward
5’-AAT-GGG-GTG-AAG-TTT-TCT-GC-3’
Reverse
5’-CAG-TGG-AGT-GGC-TTT-TGT-GA-3’
Forward
5’-CAG-TAC-TCG-GAA-TGC-AGC-AA -3’
Reverse
5’-CAG-TCT-TCA-GGG-GCT-CTG-TC-3’
Forward
5’-CAA-GTG-GCC-GAC-ACT-GAC-TA-3’
Reverse
5’-GGT-GGG-AAC-AGA-GAA-AAC-GA-3’
Forward
5'-AAC-TCC-CAG-AAA-AGC-AAG-CA-3'
Reverse
5’-CGA-GCA-GGA-ATG-AGA-AGA-GG-3’
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Annealing
(ºC)
Amplicon length
(bp)
49
102
49
200
49
173
49
170
49
192
50
212
MEGALIN (NM_001081088)
Qiagen
QuantiTect Primer Assay for SybGreen detection
49
84
IL-10 (NM_010548)
Qiagen
QuantiTect Primer Assay for SybGreen detection
50
103
MCP-1 (NM_011333)
Qiagen
QuantiTect Primer Assay for SybGreen detection
55
118