Certain determinants of ischemic resistance in the Brown Norway rat strain have been proposed, but no studies to date have focused on the role of the Wnt pathway in the ischemic resistance mechanism. We performed a comparative genomic study in Brown Norway vs. Sprague-Dawley rats. Selective manipulations of the Wnt pathway in vivo and in vitro allowed us to study whether the action of the Wnt pathway on apoptosis through the regulation of osteopontin was critical to the maintenance of inherent ischemic resistance mechanisms. The results revealed a major gene upregulation of the Wnt family in Brown Norway rats after renal ischemia-reperfusion. Manipulation of the Wnt signaling cascade by selective antibodies increased mitochondrial cytochrome c release and caspase 3 activity. The antiapoptotic role of Wnt was mediated by osteopontin, a direct Wnt target gene. Osteopontin was reduced by Wnt antibody administration in vivo, and osteopontin gene silencing in vitro significantly increased mitochondrial cytochrome c release. The overexpression of Wnt pathway genes detected in Brown Norway rats is critical in the maintenance of their inherent ischemic resistance. Activation of the Wnt signaling cascade reduces mitochondrial cytochrome c release and caspase 3 activity through the action of osteopontin.
- acute renal failure
renal ischemia-reperfusion (I/R) injury is of great clinical relevance and may lead to fatal disease, including acute renal failure (ARF) (7, 17, 18). In the last few years, numerous studies employing a range of experimental models have sought to understand the processes and mechanisms underlying the development of I/R injury (2, 9, 10, 21, 22, 27, 28, 32, 33). However, little is known about the mechanisms responsible for the inherent resistance to I/R ascribed to certain animal strains, for example Brown Norway (BN) rat strains.
The lower degree of histological and functional damage in resistant BN rats compared with the Sprague-Dawley (SD) strain is due to a lower incidence of apoptosis and a better antioxidant/oxidant balance during I/R processes (25). Basile et al. (3) reported less damage to the cytoskeleton of the BN strain after renal I/R injury and identified several proteins that were pivotal to the greater resistance of BN rats to I/R-induced ARF.
Wnt proteins are cell-surface molecules that play important roles in a variety of cellular processes. They bind to “frizzled” receptors and activate them upon coupling (37). Interestingly, these molecules are expressed during ARF, and their involvement in the transcriptional regulation of different cyclins (as regulators of the restriction point of the transition of G1-to-S phase) and in cell cycle progression in renal tubular cells seems to play a critical role during the recovery and healing of the damaged kidney (35).
The Wnt pathway is further involved in cell survival via its function as an apoptosis inhibitor (6), blocking the release of cytochrome c and the subsequent caspase-9 activation by Wnt 1 signaling. Wnt proteins have also been implicated in cytoskeleton preservation via the Wnt/planar cell polarity pathway, acting on the induction of the cytoskeletal regulators JNK, Rho kinase, and Rac (13).
Nevertheless, despite the beneficial role of Wnt proteins in ARF, the possible relationship between the Wnt pathway and the inherent resistance to renal ischemic damage has not yet been explored. What is more, the possible pathway relating Wnt and apoptosis to confer ischemic resistance remains unknown.
Osteopontin (OPN) has been reported to be a Wnt target gene. This molecule is an extracellular matrix protein that binds to integrins and the CD44 family of receptors to propagate cellular signals. OPN has been related to antiapoptotic signaling in several experimental models. Soluble OPN inhibits apoptosis in adherent endothelial cells via induction of Bcl-xL (38). Studies in colon cancer and gastric cancer cells indicate that OPN treatment increases resistance to ultraviolet-induced apoptosis via integrin activation (38).
In addition, OPN may prevent apoptosis via NF-κB activation (31). NF-κB may promote cell survival by enhancing the expression of genes encoding antiapoptotic proteins, such as Bcl-xL and BFL1, which act on the mitochondria and inhibit cytochrome c release, or IAP1, IAP2, and XIAP, which block caspase activation (15, 24).
We aimed to study the mechanism responsible for ischemic resistance. We hypothesized that resistance may be due to the ability of the Wnt pathway to prevent mitochondrial cytochrome c release through the action of OPN. To test this hypothesis, we conducted a comparative genetic study of the BN and SD rat strains and carried out selective manipulations of the Wnt pathway in vivo and in vitro. We scrutinized the role of OPN by gene knockdown using small interfering (si) RNA in vitro.
The results indicate that the modifying action of the Wnt pathway on apoptosis in the course of renal I/R determines the inherent resistance of BN rats. We also show that the Wnt pathway, by regulating its target gene OPN, inhibits mitochondrial cytochrome c release and thus reduces apoptosis.
MATERIALS AND METHODS
The study was performed using male SD and inbred BN (BN/OrlCrl) rats (Ifa Credo, Barcelona, Spain) weighing 250–300 g. Animals were anesthetized using an injection of pentobarbital sodium (30 mg/kg ip) and placed in a supine position. Body temperature was maintained between 36 and 37°C. All procedures were conducted under the supervision of our institution's Research Commission and followed European Union guidelines for the handling and care of laboratory animals.
After a laparotomy and dissection of both renal pedicles, bilateral ischemia was induced by occluding the renal pedicles with nontraumatic microvascular clamps for 45 min. The immediate color change of the kidneys, signifying the stoppage of blood flow, was taken to indicate successful occlusion. During reperfusion, clamps were removed and blood flow to the kidneys was reestablished with visual verification of blood return. Animals were sutured, and reperfusion was performed for 24 h as previously reported (36).
The following experimental groups of BN and SD rats (n = 6/group for the functional and histological analyses, and n = 3 for the microarray study) were studied.
Groups 1 and 2: control BN (C BN) and SD rats (C SD), where animals were subjected to identical maneuvers as the I/R group, except that the renal pedicles were not clamped.
Group 3: I/R in BN rats (I/RBN), where BN rats were subjected to 45 min of bilateral ischemia and 24 h of reperfusion.
Group 4: I/R in SD rats (I/RSD), where SD rats were subjected to I/R.
Groups 5 and 6: I/R in BN and SD rats treated with antibody against the Wnt receptor Frizzled 5 (I/R+afz5), where animals were subjected to I/R with previous intravenous addition of the antibody (75 μg of antibody administered 15 min before I/R process, Rockland).
Groups 7 and 8: I/R in BN and SD rats treated with antibody against the Wnt protein Wnt1 (I/R+awnt1), where animals were subjected to I/R with previous intravenous addition of the antibody (75 μg of antibody administered 15 min before I/R process, Millipore, Barcelona, Spain).
Group 9: I/R in SD rats treated with the Wnt agonist 6-bromoindirubin-3′-oxime (I/RSD+BIO; Sigma, Madrid, Spain), where SD rats were subjected to I/R with the previous administration of BIO (0.1 mg/kg administered intravenously 15 min before I/R process).
A cortical segment of the left kidney was collected, immediately frozen, and stored at −80°C until use for the various determinations. Kidney tissue was also obtained and embedded in 4% formalin-fixed paraffin and cut into 3-μm sections to perform the histological analysis.
In vitro model.
The rat proximal tubular epithelial cell line NRK-52e (European Collection of Cell Culture, Porton Down, Wiltshire, UK) was cultured in DMEM-F-12 (1:1) 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) heat-inactivated fetal bovine serum (Invitrogen, Barcelona, Spain). NRK-52e cells were used at passage 10 and grown until 90% confluence. To split the cells, we used 1 mM EDTA/0.025% trypsin (Invitrogen) for 5 min in accordance with our standard laboratory protocol.
To emulate the hypoxic process, we subjected the cells to an overnight anoxic period (0.5% O2-5% CO2) followed by 6 h of normoxia (20.7% O2-5% CO2). This cell line has been previously used in anoxia procedures to mimic ischemia (23).
The following experimental groups were studied.
Group 1: control groups (C): rat renal epithelial NRK-52e cells without treatment; C+BIO, control group with the addition of 10 μM BIO; C+ OPN−, control group subjected to an OPN gene-silencing process by means of siRNA.
Group 2: anoxia group, where rat renal epithelial NRK-52e cells were subjected to the anoxia-reoxygenation (A/R) procedure.
Group 3: anoxia+BIO group (A/R+BIO), where rat renal epithelial NRK-52e cells were subjected to the A/R procedure with the addition of 10 μM BIO before anoxia induction.
Group 4: anoxia+BIO+OPN− group (A/R+BIO+OPN−), where rat renal epithelial NRK-52e cells were subjected to the anoxia procedure with the addition of 10 μM BIO and to an OPN gene-silencing process by means of siRNA 12 h before the treatment with BIO.
RNA isolation and quality control.
Total RNA was isolated from homogenized tissue and cells with TRIzol Reagent (Invitrogen) and purified by passage through RNeasy minicolumns (Qiagen, Barcelona, Spain) according to the manufacturer's protocols for RNA clean-up. The purity and quality of extracted RNA were evaluated using an RNA 6000 LabChip and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Only high-quality RNA with RNA integrity numbers (RINs) >7.5 were used in the microarray experiments and in the RT-PCR determinations.
Microarray gene expression profiling.
Gene expression profiling was performed using the Applied Biosystems Rat Genome Survey Microarray platform, which has 26,857 probes.
All RNA targets were labeled using the Applied Biosystems RT-IVT Labeling Kit, version 2.0, and then digoxigenin-labeled cRNA targets were hybridized to Applied Biosystems Rat Whole Genome Survey Microarrays using an Applied Biosystems Chemiluminescent Detection Kit. Image acquisition and analysis were performed using the Applied Biosystems Chemiluminescence Detection Kit and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following the manufacturer's protocols. Images were autogridded, and the chemiluminescent signals were quantified, corrected for background and, finally, spot and spatially normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software, version 1.1. Gene expression profiles were obtained from control and I/R groups in the two rat strains in three biological replicates, resulting in 12 hybridizations from 2 groups × 2 strains × 3 biological replicates.
Data analysis and microarray validation.
All data analyses were carried out by Integromics, Information Technologies for Life Sciences (Granada, Spain), using the Integromics Application Package for Data Analysis of AB1700 Chemiluminiscence Expression System software. Data normalization was performed using the robust nonparametric multidimensional normalization quantile method.
Increases or decreases in gene expression were evaluated using an unpaired two class t-test with the Benjamini-Hochberg correction for multiple comparisons. Results for comparison pairs are expressed as a fold-change. Statistical significance was assigned at a minimum 1.5-fold change and P < 0.01.
To validate the expression levels obtained in the microarray experiments, we used quantitative real-time RT-PCR, as described in materials and methods.
siRNA gene silencing.
The siRNA sequences used for targeted silencing of the OPN gene were validated siRNA oligonucleotides provided by bioNova (Madrid, Spain). The OPN siRNA oligonucleotides selected were siOPN (sense strand) 5′-CCAAGCAACUCCAAUGAAATT −3′ and siOPN (antisense strand) 5′-UUUCAUUGGAAGUUGCUUGGTT-3′. A siRNA targeted to no known gene (Stealth RNAi negative control, Invitrogen) was used as a negative control. Additionally, effective siRNA delivery was verified by fluorescent staining of transfected cells using the siRNA Reporter Block-it system (Invitrogen). The synthetic double-stranded siRNA oligonucleotides were delivered to NRK cells using Lipofectamine 2000 according to the manufacturer's recommended protocol (Invitrogen). OPN gene expression was measured by real-time RT-PCR at 24 h posttransfection.
Total RNA was isolated as described above. Fifty nanograms of total RNA was applied to measure gene expression levels by real-time RT-PCR (TaqMan) using validated primers provided by Applied Biosystems: SPP1 (OPN), S100-A8 (S100 calcium-binding protein A8), Fbn1 (fibrillin), Foxm1 (forkhead protein) tbb-6 (tubulin β chain), muc 1 (mucin 1), cyclin F (cyclin F), and Wnt1-inducible signaling pathway protein 2 (Wisp 2) were selected to validate the feasibility of the microarray assay.
Caspase-3 activity was determined by measuring proteolytic cleavage of the specific substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC; Biomol, Plymouth Meeting, PA). The AMC released from the samples was quantified for 1 h at 37°C by fluorospectrophotometry using 380-nm excitation and measurement of 450-nm emission.
Western blot analysis and isolation of renal mitochondria.
Mitochondria were extracted by differential centrifugations following Reinhart et al. (30) with some modifications. Equal amounts of protein from mitochondrial and cytosolic fractions (50 μg) were electrophoresed on a 15% SDS gel and transferred to nitrocellulose membranes, which were subsequently blocked with 5% nonfat dry milk in 0.06% Tween-Tris-buffered saline (TTBS) for 1 h. The membranes were incubated overnight with primary antibody (anti-cytochrome c, 1:1,000) at 4°C, washed five times with TTBS, and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG antibody (dilution 1:2,000) at room temperature, followed by enhanced chemiluminescence detection.
Confocal microscopic analyses.
Confocal images were taken with a Leica TCS NT laser microscope (Leica Microsystems, Wetzlar, Germany). To analyze the changes in the cytoskeleton and the F-actin stress fibers, cells and tissue sections were fixed in 3.7% paraformaldehyde for 15 min at 37°C and washed with PBS. The slides were then incubated with Alexa Fluor 568-phalloidin (dilution 1:40, Molecular Probes, Eugene, OR) in PBS with 1% BSA for 30 min.
Renal function test.
Blood urea nitrogen and creatinine were analyzed in plasma using an ADVIA 2400 (Siemens Medial Diagnostics) multichannel analyzer at the Hospital Clínic (Barcelona, Spain).
Data are expressed as means ± SE. Means of different groups were compared using one-way ANOVA. The Student-Newman-Keuls test was used for the evaluation of significant differences between groups. Significant differences were assumed when P < 0.05.
Whole microarray analysis of BN and SD gene expression profiles.
The comparison of the two strains revealed that 79 genes were differentially overexpressed in BN but not in SD rats after I/R (see list 1, supplementary information; all supplementary material for this article is available on the journal web site). When these genes were clustered, the most significant clusters identified by PANTHER were related to the extracellular matrix, select calcium-binding protein, receptor, signaling molecule, cell adhesion molecule, and select regulatory molecule. Among these clusters, many genes were related to the Wnt pathway and other putative injury-protective pathways. Figure 1 shows the most significant clustering of upregulated genes in BN compared with SD after I/R.
Additionally, microarray statistical analysis in BN rats identified 202 transcripts as differentially expressed, 143 transcripts as overexpressed, and 59 as downregulated at a significance of P < 0.01 in the I/R vs. the control group (see lists 2 and 3, supplementary information). This analysis also showed that 162 transcripts were differentially expressed at a significance of P < 0.01 in the I/R vs. the control group in the SD rats, 106 upregulated, and 56 downregulated (see lists 4 and 5, supplementary information).
To validate the data obtained from microarray analysis, we determined some of the most representative transcripts selected that were differentially expressed in the BN and SD strains after I/R by RT-PCR. The results were similar to those obtained from the microarray analysis (Fig. 2). The genes analyzed were SPP1 (osteopontin), Foxm1 (forkhead protein), tbb-6 (tubulin β chain), S100-A8 (S100 calcium-binding protein A8), Fbn1 (fibrillin), muc1 (mucin 1), cyclin F (cyclin F), and Wisp 2 (Wnt1-inducible signaling pathway protein 2). These genes were also upregulated after I/R in BN vs. SD rats. These transcripts were selected based on their widely described association with the cytoskeleton (1, 5, 11), cell survival (16, 29), or the Wnt pathway (19), among others.
Wnt pathway in vivo manipulation.
Wnt pathway inhibition in vivo was performed by using selective antibodies against two key elements of this pathway, namely, anti-Wnt 1 and anti-Frizzled 5 for Wnt receptor inhibition. Both antibodies were applied intravenously 15 min before the onset of I/R. RT-PCR of the osteopontin gene, a direct target of the Wnt pathway (8), showed a significant decrease in its expression after antibody administration, indicating the effectiveness of the antibodies chosen (Fig. 3).
Apoptosis induction measured by caspase-3 activity revealed a direct relationship between the administration of Wnt1 and Frizzled 5 antibodies and the increase in cleaved caspase-3. As shown in Fig. 4, administration of the antibodies to BN rats increased caspase-3 activity and achieved levels similar to those detected for the SD strain, suggesting a key antiapoptotic role of the Wnt pathway in BN rats.
Figure 5 shows the Western blot of mitochondrial cytochrome c released to the cytosol in the different groups. In the control groups of both strains, the quantity of cytochrome c detected in the cytosolic fraction was significantly lower than in the mitochondrial fraction, indicating minimal apoptosis at basal levels. The same tendency was present in the I/RBN group, confirming the protection from apoptosis induction in BN rats. By contrast, in the I/RSD group, released cytochrome c was significantly higher in the cytosolic fraction, indicating apoptosis induction by the translocation from the mitochondrial fraction to the cytosol, and corroborating previous results from caspase-3 measurements. Interestingly, when antibodies were applied to block the Wnt pathway in BN rats, the animals lost their resistance to apoptosis induced by I/R, further indicating the key role of Wnt signaling in ischemic resistance mechanisms. When the same antibodies were applied in SD rats, the previous incidence of apoptosis was maintained.
To analyze actin cytoskeleton alterations, we performed a study of cytoskeletal integrity using the fluorescent dye phalloidin, which detects F-actin fibers. The results also revealed differences between the two rat strains: cytoskeleton integrity was maintained in the BN but not in the SD strain after I/R (Fig. 6, B and C). The administration of antibodies against Frizzled 5 or Wnt 1 to BN rats before I/R reduced cytoskeleton integrity with respect to the I/R group (Fig. 6D). In SD rats, treatment with those antibodies did not significantly increase cytoskeleton disruption (Fig. 6E).
Our results showed greater histological damage in SD than in BN rats and a higher incidence of necrosis and tubular obstruction in the SD strain (Fig. 7, B and C). When the antibodies were administered to BN rats, the previous ischemic protection virtually disappeared (Fig. 7D). In SD rats, the damage after I/R plus antibodies was similar to that recorded in the I/R group (Fig. 7E).
Both plasma creatinine and blood urea nitrogen levels were higher in the SD strain than in BN after I/R, in line with the results obtained in the cytoskeletal studies described above. On the other hand, the use of antibodies to inhibit the Wnt pathway impaired renal function. SD rats were further subjected to treatment with BIO, a Wnt pathway agonist. In this case, the ischemic injury suffered by the I/R-sensitive strain, SD, was drastically reduced (Fig. 8, A and B).
Wnt pathway activation in vitro.
To further assess the action of the Wnt pathway on cytoskeleton integrity and mitochondrial cytochrome c release under hypoxic/ischemic conditions in vitro, we stimulated Wnt pathway activation by the use of BIO. BIO was added 6 h before the anoxic phase and was present throughout the whole experiment. To check the effectiveness of this compound as an activator of the Wnt pathway, we performed RT-PCR for OPN, a direct target of the Wnt pathway, which revealed the increase in OPN gene expression after BIO administration (Fig. 9). On the other hand, when the OPN gene was silenced with siRNA, the gene expression measured by RT-PCR fell significantly.
Caspase-3 activity revealed a protective effect of the Wnt pathway under anoxia in vitro. Cells treated with the Wnt inductor BIO suffered less apoptosis after the anoxia-normoxia period than untreated cells (Fig. 10). Moreover, OPN gene silencing significantly increased apoptotic activity, despite BIO application. OPN− silencing in controls also increased the basal apoptosis level, confirming the importance of OPN in the protection against cell death.
Figure 11 shows apoptosis induction measured by mitochondrial cytochrome c released into the cytosol in NRK cells subjected to anoxia. In the anoxia group, the cytochrome c release was increased compared with both the control and the anoxia+BIO group. These findings show a direct relationship between the Wnt pathway and anoxia-induced apoptosis. The fact that this resistance was not present in the anoxia+BIO+OPN− group indicates the key role of OPN in Wnt-mediated ischemic resistance.
To assess the effect of A/R and the Wnt pathway on cytoskeleton disruption, cells were stained with phalloidin. In controls and in controls with BIO administration, the F-actin stress fibers were quite well conserved (Fig. 12A). By contrast, when A/R was performed in the absence of BIO, a redistribution of the cytoskeleton was clearly revealed, with a progressive loss of the stress fibers and their accumulation in a peripheral functional ring surrounding the cells, finally resulting in total depolymerization. This tendency was also observed when OPN was silenced in both the control and the Anox+BIO+OPN− groups (Fig. 12B). Nevertheless, the induction of the Wnt pathway through BIO in the anoxia group reversed the cytoskeleton damage caused by ischemia (Fig. 12C).
This study presents a whole genome comparison, conducted by means of the Applied Biosystems Rat Genome Survey Microarray platform (used in previous studies) (34), to identify diverse genes that are differentially expressed during the I/R process in two rat strains considered as sensitive (SD) and resistant (BN) to ARF. Earlier studies by Basile et al. (3) and Nilakantan et al. (25) identified key protein families and postulated that these families contributed to the ischemic resistance of BN rats.
In this study, a microarray chip analysis revealed the overexpression of different transcripts in the I/R group compared with those in controls, with a higher number of genes upregulated in BN rats than in the SD strain. Among these overexpressed genes, Wisp 2, laminin, dynamin 3, mucin 1, diverse cycline family proteins, and osteopontin were selected and could be considered as new contributors to the resistance to ischemia in BN rats. Interestingly, all were components of the Wnt pathway. Indeed, it has previously been suggested that Wnt signaling promotes protection and regeneration during ARF (35), but there are as yet no data that indicate its role in the inherent resistance of BN rats to ischemic injury.
Moreover, in addition to the Wnt pathway, clusters related to cell structure and cell survival were highlighted among the gene groups, with the greatest number of differentially overexpressed genes in BN rats. Some of these genes have been reported elsewhere, which stresses the reliability of the array format used in our experimental set-up (4).
Those clusters have been widely associated with a renal-protective function. Fibrillin and tubulin-B6 are two cell structure-related proteins that have been considered responsible for the maintenance of kidney integrity during pathological processes and for the generation and maintenance of epithelial cell polarity, since their loss contributes to structural changes that occur after I/R (1, 5, 29). Other genes, such as FoxM1 and S100A8, were also differentially expressed in BN rats and were therefore selected for validation in this study, since they can be considered protective against ischemic or inflammatory injury. In this regard, FoxM1 and S100A reduce the overproduction of nitric oxide from activated neutrophils and macrophages. In the case of FoxM1, there are also reports that it plays a pivotal role in neutralizing the activity of proinflammatory cytokines linked to regenerative processes (12, 20). Furthermore, osteopontin (OPN), a phosphoprotein related to the Wnt pathway, was identified as one of the most affected genes in BN ischemic resistance. This protein has multiple cellular functions, the most interesting being its role as a survival factor for tubular cells in acute renal injury (11, 19), since the disruption of the OPN gene in null mice resulted in a more severe injury after I/R (26).
Surprisingly, the genes implicated in antioxidation and free radical removal were not overexpressed in our model. This result does not support results reported elsewhere (25) when protein expression as opposed to gene expression is analyzed. Nevertheless, we cannot rule out the possibility that genes related to the antioxidant process may be activated in our model, possibly during a very early phase of renal I/R. Further studies with different reperfusion times might help clarify this point.
Since the Wnt pathway exerted its effects as an apoptosis inhibitor and/or as a cytoskeleton regulator (3, 25) and since genes related to cell survival or the cytoskeleton were overexpressed in our model, we hypothesized that ischemic resistance in BN rats was mediated by the Wnt pathway, which in turn was able to preserve cytoskeleton integrity and protect against apoptosis. To our knowledge, such a direct link between the Wnt pathway and the inherent resistance to renal ischemic damage in BN rats has not been previously reported.
In this study, we detected the protective role of the Wnt pathway by its manipulation in both in vivo and in vitro models. Its role as an apoptotic protector was mediated by OPN expression, a direct Wnt target gene. OPN has antiapoptotic, chemotactic, and proliferative properties and may modulate actin cytoskeleton regulation(14). In our study, OPN was reduced as a consequence of Wnt antibody administration in vivo. OPN siRNA gene silencing in vitro significantly increased mitochondrial cytochrome c release.
Under I/R conditions, Wnt pathway inhibition provoked a significant increase in apoptosis, a strong depolymerization of the previously well-conserved cytoskeleton and damage to renal histology in BN rats. In SD rats, in contrast, the activation of this pathway reduced the ischemia-associated injury profile. This provides evidence that the mechanism for ischemic resistance present in BN rats is also present in a sensitive strain but is not upregulated sufficiently to induce a protective response. This mechanism is, then, a candidate to induce protection against renal ischemia.
Moreover, Wnt pathway induction by BIO in anoxic NRK cells protected against cellular damage, showing decreased apoptosis incidence and better cytoskeleton integrity in the cells treated with BIO. Interestingly, when cells were selectively silenced for OPN using siRNA, the positive effect of BIO on the resistance to injury induced by A/R disappeared almost completely. These findings indicate that OPN is crucial to Wnt pathway-dependent anoxic resistance mechanisms, since the possible expression of other Wnt target genes, potentially activated by the agonist BIO, did not influence injury outcome in the absence of OPN.
To summarize, the overexpression of Wnt pathway genes detected in BN rats is critical in the maintenance of their inherent ischemic resistance. The Wnt signaling cascade via OPN activation determines mitochondrial cytochrome c release and cytoskeleton integrity.
This work was supported by EU Grant LSHB-CT-2006-036813 (to G. Hotter), FISS PS09/00057 (to G. Hotter), 2009 SGR 1094 (to G. Hotter), FISS PS09/01288 (to A. Sola), and Miguel Servet CP08/00138 (to A. Sola). C. Mastora is supported by the University of Barcelona.
No conflicts of interest, financial or otherwise, are declared by the authors.
The authors thank M. Ángeles Muñoz and Maite del Hierro for excellent technical support.
- Copyright © 2010 the American Physiological Society