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Am J Physiol Renal Physiol 295: F1554-F1562, 2008. First published September 24, 2008; doi:10.1152/ajprenal.90250.2008
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Lipocalin-2-induced renal regeneration depends on cytokines

Eugenia Vinuesa,2 Anna Sola,1,2,* Michaela Jung,2 Vicente Alfaro,3 and Georgina Hotter1,2,*

1El Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Networking Center on Bioengineering, Biomaterials, and Nanomedicine, 2Department of Experimental Pathology, Instituto de Investigactiones Biomédicas de Barcelona-Consejo Superior de Investigaciones Científicas-Institut de Investigacions Biomèdiques August Pi i Sunyer, and 3Department of Physiology (Biology), University of Barcelona, Barcelona, Spain

Submitted 15 April 2008 ; accepted in final form 16 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 Lcn2-mediated 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 min of renal ischemia with a reperfusion period of 24 h (early reperfusion, expected time for maximum inflammation) and 96 h (late reperfusion, expected time for maximum regeneration). Different experimental groups underwent I/R, I/R with iv anti-mouse Lcn2 monoclonal antibody injected during the early/inflammatory or late/recovery phase, and I/R with proinflammatory cytokine cocktail administration (recombinant mouse IL-1β, TNF-{alpha}, and IFN-{gamma}). Compared with control nonischemic 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. Proinflammatory 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.

Lcn2; mice; kidney; tubular cells; ischemia-reperfusion; cell regeneration

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 h 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 upregulated 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 vs. reparative phases of I/R might both be mediated by different Lcn2 effects.

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 date, 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 I/R.

The present study was designed to investigate whether the injury vs. 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.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mouse Model of Renal I/R 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 nontraumatic vascular clamp for 30 min, 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 h. After 96 h 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 the kidneys were harvested. One-half of each kidney was snap-frozen 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) (see Table 1).


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  		Table 1. Experimental groups in the study

 
Control (C). In the control group, animals were subjected to the identical maneuvers as the I/R group, except that the renal pedicles were not clamped.

Ischemia/early reperfusion (I/early reperfusion). Animals underwent 30 min of bilateral ischemia and were killed after 24 h of reperfusion.

Ischemia/late reperfusion (I/late reperfusion). Animals underwent 30 min of bilateral ischemia and were killed after 96 h 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 iv bolus) in the inflammatory phase (24 h).

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 iv bolus) in the regenerative phase (72 h). 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 iv bolus) in the regenerative phase (72 h).

Proinflammatory cytokine cocktail administration (I/late reperfusion+ cytokines). Animals underwent the same procedure as the I/R group but also received an infusion of a proinflammatory cytokine cocktail (200 µl ip) 1 day before death.

Lcn2 Antibody and Rat IgG Administration

Mice were injected intravenously via the tail vein with 150 µl of PBS containing 75 µg of monoclonal anti-mouse Lcn2 antibody (R&D Systems) or rat IgG (R&D Systems), respectively.

Cytokine Administration

To modulate the inflammatory response, mice were treated intraperitoneally with a proinflammatory cytokine cocktail containing 200 µl of PBS with recombinant mouse IL-1β (100 ng), TNF-{alpha} (2 µg), and IFN-{gamma} (750 ng). All cytokines were 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

BUN was determined in plasma using a Sigma diagnostic kit, and the results are expressed as milligrams per deciliter. 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 (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 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 h 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/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 reentry of cells into the cell cycle and the onset of cell proliferation (7), whereas PCNA labels proliferating nuclei.

Quantitative Real-Time RT-PCR

Total tissue and cell RNAs 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 using an iScript cDNA Synthesis Kit (Bio-Rad) in a final volume of 40 µl.

Stathmin, PCNA, Ki-67, Lcn2, Lcn2R, megalin, TNF-{alpha}, MCP-1, and IL-10 mRNA expression normalized to the housekeeping gene GADPH were measured by quantitative (q) real-time RT-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.


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  		Table 2. Sequences, annealing temperature, and expected fragment size of primers utilized in real-time qRT-PCR

 
In Vitro Test to Study Antibody's Effect as Blocker of Lcn2 Activity

Mouse RAW 264.7 macrophages (European Collection of Cell Culture) were cultured in DMEM 1:1 F-12 nutrient supplement with high glucose, 15 mM HEPES, and stable L-glutamine, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (vol/vol) 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 six-well plate at a density of 1 x 106 cells/well. Cells were incubated with LPS (15 µg/ml, Sigma) for 24 h to stimulate Lcn2 production (24, 37) before antibody treatment. Cells were washed with PBS and fresh culture medium containing either 75 µg of a monoclonal antibody against mouse Lcn2 or rat IgG to LPS-stimulated macrophages and incubated for 24 h.

Lcn2 ELISA

Supernatants from the in vitro text cultures were collected and clarified by centrifugation. Fifty microliters of each sample were applied to an ELISA as previously described (19). Briefly, 96-well-plates were previously covered with catch anti-mouse lipocalin-2/NGAL monoclonal antibody (R&D Systems) and blocked for 1 h. After sample incubation, biotinylated anti-mouse lipocalin-2/NGAL antibody (R&D Systems) was added. Afterward, horseradish peroxidase-conjugated avidin (Invitrogen) was incubated for 1 h. 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 means ± SE. The means of different groups were compared using one-way ANOVA. The Student-Newman-Keuls test was performed to evaluate significant differences between groups, which were assumed to exist when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lcn2 Influences Tubule Cell Proliferation after Ischemic Injury

Representative kidney sections stained with antibodies to stathmin and PCNA are shown in Fig. 1. Nonischemic control kidneys had a minimal presence of proliferating cells (2.8 ± 0.60 for stathmin and 1.5 ± 0.45 for PCNA) (Fig. 1A); eight fields were counted per sample and averaged. Kidneys subjected to I/R showed a high number 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 (Fig. 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 h) had a 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) (Fig. 1C). In contrast, animals that were treated with anti-Lcn2 antibody during late reperfusion (72 h, 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) (Fig. 1D).


Figure 1
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  		Fig. 1. Representative immunofluorescence pictures of stathmin (green) and proliferating-cell nuclear antigen (PCNA; red) staining of kidney samples. A: control. B: ischemia (I)/late reperfusion (30 min of ischemia followed by 96 h of reperfusion). C: I/early reperfusion+anti-mLcn2 [ischemia-reperfusion (I/R) with Lcn2 antibody administration in early reperfusion, 24 h]. D: I/late reperfusion+anti-mLcn2 (I/R with Lcn2 antibody administration in late reperfusion, 72 h). Images are viewed under x400 magnification.

 
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 (Fig. 2).


Figure 2
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  		Fig. 2. Blood urea nitrogen (BUN; A) and plasma creatinine (B) at different time points of anti-mouse lipocalin-2 (Lcn2) antibody administration. C, control; I/early reperfusion, 30 min of ischemia followed by 24 h of reperfusion; I/late reperfusion, 30 min of ischemia followed by 96 h of reperfusion; I/early reperfusion+anti-mLcn2, I/R with Lcn2 antibody administration at 24 h postreperfusion; I/late reperfusion+anti-mLcn2, I/R with Lcn2 antibody administration at 72 h; I/late reperfusion+cytokines, I/R with proinflammatory cytokine cocktail administration at 72 h; I/late reperfusion+rat IgG, I/R with rat IgG administration at 72 h. Values are means ± SE (n = 5/group). *P < 0.05 vs. control. +P < 0.05 vs. I/late reperfusion.

 
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 (Fig. 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 periods. These results provide evidence of a dual role for Lcn2 as an inducer or inhibitor of renal cell proliferation.


Figure 3
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  		Fig. 3. Patterns of normalized stathmin (A), PCNA (B), and Ki-67 (C) mRNA expression assessed by quantitative (q) RT-PCR at different time points of anti-mouse Lcn2 antibody administration. C, control; I/early reperfusion, 30 min of ischemia followed by 24 h of reperfusion; I/late reperfusion, 30 min of ischemia followed by 96 h of reperfusion; I/early reperfusion+anti-mLcn2, I/R with Lcn2 antibody administration at 24 h postreperfusion; I/late reperfusion+anti-mLcn2, I/R with Lcn2 antibody administration at 72 h; I/late reperfusion+cytokines, I/R with proinflammatory cytokine cocktail administration at 72 h; I/late reperfusion+rat IgG, I/R with rat IgG administration at 72 h. Values are means ± SE (n = 5/group). *P < 0.05 vs. control. +P < 0.05 vs. I/late reperfusion.

 
Effect of Proinflammatory 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 (Fig. 4). The significant increase in the expression of the three proliferation markers observed in the I/R group was reversed when the proinflammatory cytokine cocktail was administered.


Figure 4
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  		Fig. 4. Effect of cytokines on patterns of normalized stathmin (A), 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 h of reperfusion; I/late reperfusion, animals subjected to 30 min of bilateral ischemia and killed after 96 h of reperfusion; I/late reperfusion+cytokines, I/R with cytokine administration (recombinant mouse 100 ng IL-1β, 2 µg TNF-{alpha}, and 750 ng INF-{gamma} ). mRNA expression normalized to GAPDH is shown as means ± SE (n = 5 each group). *P < 0.05 vs. control. +P < 0.05 vs. I/late reperfusion.

 
Cytokine Expression

We analyzed cytokine expression by quantitative RT-PCR (Fig. 5). MCP-1 expression was increased in the I/R group compared with controls, although, as expected, the highest expression was observed when the proinflammatory cytokine cocktail was administered (Fig. 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 compared with controls, but addition of the proinflammatory cytokine cocktail did not significantly change IL-10 expression (Fig. 5B).


Figure 5
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  		Fig. 5. Cytokine profiles. qRT-PCR from representative renal injury cytokines is shown. A: MCP-1 expression. B: IL-10 expression. C, control; I/early reperfusion, animals subjected to 30 min of bilateral ischemia and killed after 24 h of reperfusion; I/late reperfusion, animals subjected to 30 min of bilateral ischemia and killed after 96 h of reperfusion; I/late reperfusion+cytokines, I/R with cytokine administration. Values are means ± SE (n = 5/group). *P < 0.05 vs. control. +P < 0.05 vs. I/late reperfusion.

 
Lcn2, Lcn2R and Megalin Expression

Lcn2 and Lcn2-receptor mRNA expression was analyzed by RT-PCR in renal tissue (Fig. 6). Lcn2 expression was increased in kidneys in the I/R groups with respect to controls. But significantly higher levels were observed during early reperfusion periods (24 h) with respect to the late reperfusion and regenerative phase (96 h). Cytokine administration induced significant increases in Lcn2 levels (Fig. 6A).


Figure 6
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  		Fig. 6. Patterns of normalized stathmin Lcn2 (A), Lcn2 receptor (Lcn2R; B), and megalin (C) mRNA expression in renal tissue assessed by quantitative real-time RT-PCR at 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 h of reperfusion; I/late reperfusion, animals subjected to 30 min of bilateral ischemia and killed after 96 h of reperfusion; I/late reperfusion+cytokines, I/R with cytokine cocktail administration (100 ng IL-1β, 2 µg TNF-{alpha}, and 750 ng IFN-{gamma} ). mRNA expression normalized to GAPDH is shown as means ± SE (n = 5/group). *P < 0.05 vs. control. +P < 0.05 vs. I/late reperfusion.

 
By contrast, Lcn2R and megalin expression profiles showed the opposite behavior to that observed for Lcn2 expression. Lcn2R and megalin mRNA decreased in the early/inflammatory phase of ischemia (24 h), increased during the late/reparative phase (96 h), and decreased with cytokine administration (Fig. 6, B and C).

In Vitro Test to Study Antibody's Effect as Blocker of Lcn2 Activity

As shown in Fig. 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-{alpha} mRNA, and Lcn2. Addition of the anti-mLcn2 antibody decreased Lcn2 mRNA expression and Lcn2 protein, and increased TNF-{alpha} mRNA, while administration of rat IgG had no effect, indicating the ability of the anti-mLcn2 antibody to block various effects classically attributed to Lcn2.


Figure 7
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  		Fig. 7. Biological effect of anti-mLcn2 antibody. Normalized Lcn2 (A) and TNF-{alpha} (B) mRNA expression assessed by qRT-PCR, and Lcn2 (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.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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 postischemic reperfusion. However, Lcn2 blockade with a 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 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 vs. 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 phases 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 upregulated in response to inflammation in neoplastic tissues and in inflammatory bowel diseases, and its expression also increases in epithelial cells after stimulation with proinflammatory 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-{alpha}, IL-1β, and IFN-{gamma}) reduces renal regenerative effects in a period in which regeneration is modulated by Lcn2. In addition, administration of proinflammatory cytokines increased the expression of proinflammatory cytokines such as MCP-1 (Fig. 5A), known for its properties in the pathogenesis of renal I/R (13). Overall, these findings indicate that the induction of a proinflammatory 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 h or in the cytokine-administered groups) (Fig. 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. However, 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 was reduced (see Lcn2R and megalin expression in 24 h, 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 receptor 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 proinflammatory milieu appears to minimize the regenerative effects of Lcn2. In later phases of reperfusion, transition from a proinflammatory to an anti-inflammatory environment results in an increased upregulation of Lcn2, which contributes to repair.

In summary, we report that renal regeneration after I/R is influenced by Lcn2 and cytokines. This Lcn2-mediated cell regeneration is dependent on the inflammatory cytokines of the milieu, as the presence of a 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.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by FIS 05/0219 (awarded to G. Hotter), FIS 06/0173 (awarded to G. Hotter), European Project PL 036813 (awarded to G. Hotter), and FIS 05/0156 (awarded to A. Sola). E. Vinuesa is supported by the Institut de Investigacions Biomèdiques August Pi i Sunye.


   ACKNOWLEDGMENTS
 
The authors thank Angeles Muñoz for excellent technical support and Martín Cullell-Young for English assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Hotter, Dept. of Experimental Pathology, IIBB-CSIC-IDIBAPS, Rosselló, 161, 7aplanta, 08036 Barcelona, Spain (e-mail: ghcbam{at}iibb.csic.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. Sola and G. Hotter contributed equally to this study. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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