Genome-wide gene expression profiling of ischemia-reperfusion injury in rat kidney, intestine and skeletal muscle implicate a common involvement of MAPK signaling pathway
- Authors:
- Published online on: January 21, 2015 https://doi.org/10.3892/mmr.2015.3235
- Pages: 3786-3793
Abstract
Introduction
Ischemia-reperfusion (I/R) injury was first described in 1968 (1). The influence of I/R on various tissues has been widely discussed, since various organs may be affected during traumatic, reconstructive and transplant surgeries (2,3). I/R injury consists of two consecutive components, which comprise ischemia, a breakdown of blood perfusion and reperfusion, where the nutrient blood supply is restored. Ischemia leads to a lack of oxygen within cells of the affected organs, resulting in the conversion of the cellular metabolism to an anaerobic state. This results in lactate accumulation, depletion of cellular adenosine triphosphate, increased production of reactive oxygen species (ROS) and dysfunction of membrane transport systems (4,5). Recent clinical and experimental studies have demonstrated that paradoxically, the major damage of I/R injury occurs during the reperfusion period (6,7). Reperfusion initiates complex reactions which lead to the induction of leukocyte accumulation, micro-vascular barrier dysfunction, edema formation, and the release of inflammatory cytokines and complement activation (8,9). The parenchymal damage of I/R injury occurs due to leukocytes being carried to the affected area and the release of inflammatory factors in response to the tissue damage caused by ischemia. The reperfusion reintroduces oxygen that can cause damage to cellular proteins, DNA and the plasma membrane, and results in an increase in release of free radicals, which initiates apoptosis. Leukocytes may additionally act on the capillaries, causing obstruction and leading to increased ischemia (4,5,10).
Numerous organs may be clinically involved in I/R injury. The intestine, kidney and skeletal muscle are the three most affected, in their function, by I/R insult. Studies investigating treatment options for I/R injury are limited in animal studies and are rarely conducted in the clinical setting due to the limited understanding of the molecular mechanisms of I/R injury (2). The mechanisms of I/R injury are challenging to study since numerous interacting bioactivities are presented at different time-points. During the shock status, the individual organs suffer from I/R insult, respectively, and receive the toxin from the other organs during reperfusion. Simplification of the method for mechanical study of I/R is important. Tracing the biological changes during I/R at the genomic level is one method that can be employed. There currently are limited reports that have used this approach, and only few pro-inflammatory genes have been identified following I/R insult. These genes include upregulated S100A4, complement C4, ADAM2, HO-1, UCP-2 and TMSB4X, and downregulated GLUL, CYP2A6 and CYP2d9 in a renal model; upregulated MRP2 and PGP in an intestinal model, and upregulated IGF-1 and p27Kp1 in a skeletal muscle model (11,12). These studies have been limited to individual or small groups of genes, which restrict the exploration of the entire mechanism. There have been no studies, to the best of our knowledge, comparing the genomic changes between different organs under the same I/R insult. In the present study, a kidney, intestine and skeletal muscle model of I/R was used to investigate the genomic changes using a DNA microarray approach, with the aim to identify target genes involved in I/R injury.
Materials and methods
Animals and experimental groups
A total number of 45 male inbred Lewis rats aged 8–12 weeks with a body weight of 270–330 g were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and used for I/R experiments. All experiments were approved by the Chang Gung Memorial Animal Research and Ethic Committee (Tao-Yuan, Taiwan). During surgery, all animals were placed under a heat lamp to prevent a decrease in body temperature, and during ischemia and reperfusion the exposed organs were covered with normal saline wet gauzes to maintain normal moisture levels. General anesthesia was induced by an intraperitoneal injection of urethane (15 mg/kg). Rats were assigned to three different study groups, and ischemia and reperfusion injury was studied individually in the kidneys, intestine, and skeletal muscle. Animals in the first group (group I, n=5) were sham operated and served as controls. Animals in the second group (group II, n=5) were subjected to 60 min of vascular occlusion. Animals in the third group (group III, n=5) were subjected to 60 min of ischemia followed by 60 min of reperfusion. According to the literature, 60 min of ischemia and reperfusion were considered appropriate to study early changes in gene expression following I/R injury (2–5,7,23–25).
Establishment of kidney I/R injury
Briefly, the abdomen was opened through a midline incision, and the pedicles of both kidneys were located and freed from surrounding tissue. The left renal artery and vein was clamped with a single microvascular clamp and ischemia was macroscopically verified by a change in color of the kidney to pale blue. For reperfusion studies, the renal clamp was removed and reestablishment of blood flow was again monitored macroscopically.
Establishment of intestinal I/R injury
To study the effects of experimental ischemia on gene expression within intestinal tissue, rats were laparotomized through a midline incision. Briefly, the superior mesenteric artery (SMA) and the supplied intestine were identified and the superior mesenteric vessels were freed from the surrounding tissue. The SMA and superior mesenteric vein (SMV) were occluded with a single vascular clamp for 60 min and ischemia was verified macroscopically by observing the color change of the intestinal segment to a dark pale color. For reperfusion, the clamp of the superior mesenteric vessels was removed and biopsies were taken after 60 min.
Establishment of skeletal muscle I/R injury
The rat hind-limb vascular occlusion model was used to study the impact of ischemia and reperfusion in the skeletal muscle. Briefly, an incision in the inner side of the hind leg, from the inguinal ligament to the tendon calcaneus insertion, was made. Other than the femoral vessels, all of the muscles, tendons, nerves and vessels were dissected and the femur head was dislocated from the acetabulum. Next, the femoral artery and vein were clamped with a single vascular clamp. For reperfusion, the clamp occluding the femoral vessel was removed to regain of the blood supply to the distal limb was monitored macroscopically.
Organ tissue collection and RNA preparation
At the endpoint of the study, organs subjected to ischemia and reperfusion were harvested under terminal anesthesia. The organs were carefully removed, gently rolled on cotton swabs and irrigated with normal saline to remove the adjacent tissue and excess blood. The organs were then blotted dry, weighed and shock-frozen in liquid nitrogen for storage and subsequent RNA extraction.
The tissue was homogenized and total RNA isolated using TRIzol™ reagent (Gibco-BRL, Carlsbad, CA, USA) according to the manufacturer’s instructions. Subsequently, two phenol/chloroform extractions were performed, followed by a DNAse digestion. Total RNA from the organs of individual rats of each experimental group was pooled and poly A+ RNA (mRNA) isolated with oligo (dT) cellulose columns (Gibco-BRL). Both total RNA and poly A+ RNA concentrations were determined spectrophotometrically at A260 and all samples were checked by formaldehyde gel electrophoresis.
Microarray experiment
The samples were prepared for microarray analysis according to the Nimblegen gene expression analysis protocol (Roche Diagnostics, Manheim, Germany). Double-stranded (ds) cDNA from 10 μg of total RNA was synthesized using the SuperScriptTM Double-Stranded cDNA Synthesis kit (Invitrogen Life Technologies, Carlsbad, CA, USA). The cDNA was treated with RNase and the total RNA was purified using phenol/chloroform/isoamyl alcohol (25:24:1 v/v) and precipitated by adding 16 μl of 7.5 M ammonium acetate, 7 μl glycogen (5 mg/ml stock solution), 326 μl ice-cold absolute ethanol. The resulting pellet was washed with 500 μl ethanol (80%) and dissolved in 20 μl water. Gel electrophoresis was used to verify successful dscDNA synthesis, which was confirmed by the presence of a smeared band of 500–2,000 bp. The reactions were labeled with Cy3–9mer primers using a Nimblegen One-Cola DNA Labeling kit, followed by precipitation using NaCl and isopropanol. The precipitate was resuspended in 25 μl distilled water.
Microarray hybridization and data analysis
Microarray hybridization was combined with 4 μg cDNA from each of the samples. A NimbleGen Hybridization kit (NimbleGen Systems; Roche) was used for the hybridization reaction according to the manufacturer’s instructions. The hybridization reaction was performed in a MAUI Hybridization system (BioMicro® Systems, Inc., Salt Lake City, UT, USA). Following hybridization, the array was washed and dried according to the NimbleGen Washing kit (NimbleGen Systems; Roche) protocol. The array image was acquired using an Axon GenePix 4000B (Axon Instruments, Inc., Union City, CA, USA) laser scanner at a 5-μm resolution and the intensity data were extracted using the NimbleScan software (NimbleGen Systems; Roche). The data was further examined using NexuExp software (BioDiscovery, El Segundo, CA, USA). Gene expression changes that were greater or less than two-fold as compared with the control group, and with a P<0.01, were considered to indicate a statistically significant difference in the expressed genes between the samples.
Quantitative polymerase chain reaction (qPCR)
SYBR® Green qPCR primers were designed using Beacon Designer software version 2 (PREMIER Biosoft International, Palo Alto, CA, USA) with the following sequences: forward, 5′-AGTCGTGGGAAGAGGGAACT-3′, and reverse, 5′-CCCTGGAAGTTGTTCATGCT-3′ for adrenomedullin (Adm); forward, 5′-ACAGAGCATGACCCTGAACC-3′, and reverse, 5′-CCGTTGCTGGACTGGATTAT-3′ for Jun; forward, 5′-CAAGACAAAAGCGTGGTTGA-3′, and reverse, 5′-TCTTCCTGAGTCCCTCCTGA-3′ for Junb; forward, 5′-AATGGAGGTGATGGCAGACA-3′, and reverse, 5′-GAGCAACCCACAGAGTACCT-3′ for c-FBJ osteosarcoma (c-Fos); forward, 5′-GGGTCACTGGTGTTTGAGGA-3′, and reverse, 5′-CCTCGGCTTTTGTGATGGAC-3′ for activating transcription factor 3 (Atf3) and forward, 5′-CTCAGCCAATTGTCCCAACC-3′, and reverse, 5′-AGGTAAGCAAGGCAGATGGT-3′ for dual specificity phosphatase 1 (Dusp1) genes,. SYBR Green reactions were performed using the SYBR Green Supermix (BioRad, Hercules, CA, USA). The qPCR reactions were then performed using the BioRad iCycler iQ Real-Time Detection system (BioRad). The cycling conditions were as follows; 3 min at 95°C, 15 sec at 95°C and 45 sec at 55°C for 45 cycles. The relative expression levels of Adm, Jun, Junb, c-fos, Atf3 and Dusp1 were analyzed using the iCycle iQ system software and presented as a ratio to the expression of the housekeeping gene, tubulin. Each sample was replicated twice from three independent sets of RNA preparations.
Statistical analysis
All values are expressed the mean + standard deviation. The results of the gene expression levels across the different groups were analyzed by analysis of variance with post-hoc comparison using Kruskal-Wallis test. A P<0.05 was considered to indicate a statistically significant difference. The statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA).
Results
Gene expression profiling in I/R models
The microarray compared the expression profile of >21486 genes, using the Nexus Expression™ analysis software (BioDiscovery). Each organ had a different number of genes that were differentially expressed during the I/R condition (Table I). As compared with the sham operation group, in the intestinal model, there were 76 genes upregulated and 429 genes downregulated in the ischemia-only group (group II) and 172 genes upregulated and 416 genes downregulated in the I/R group (group III). In the renal model, there were 903 genes upregulated and 1351 genes downregulated in the ischemia only group and 467 genes upregulated and 437 genes downregulated in the I/R group. In the skeletal muscle model, there were 2658 genes upregulated and 1972 genes downregulated in the ischemia only group and 3932 genes upregulated and 4203 genes down-regulated in I/R group (Table I).
Comparisons of the gene expression profiling in different organ models
The details of the up- and downregulated genes were markedly different between the organs. Fig. 1 shows the Venn diagram of the genes that were differentially up- and downregulated in the Is and I/R groups in all three models. As for the common up- or downregulated genes, one and 13 gene probe sets were up- and downregulated in the Is group, respectively; 20 gene probe sets were upregulated in the I/R group and no genes were downregulated in the I/R group. The details of the common up and downregulated genes are shown in Table II.
Validation of target gene expression in the I/R injury model using qPCR
In order to confirm the validity of the microarray findings with regard to the genes up- or downregulated in common in all three organ models, the same RNA samples of the three organs used for the microarrays were subjected to qPCR. Primers were selected for six representative genes associated with the MAPK pathway, including Atf3 (GenBank: NM_012912, BC078903), Jun (GenBank: BC078738), Jun b (GenBank: NM_021836), c-Fos (GenBank: NM_022197), Dusp1 (GenBank: NM_053769) and Adm (GenBank: NM_012715). The results of the qPCR expression are shown in Fig. 2. The majority of the qPCR results confirmed the upregulated gene expression. Selected gene expression status in three different organs was additionally examined by qPCR. The expression levels of each gene in each organ detected by qPCR and microarray experiments were comparable.
Discussion
The detailed mechanisms of I/R injury in individual organs have not been fully elucidated due to the molecular complexity of the condition. The present study used a single organ model and gene expression profiling method to identify specific molecules that may be important in I/R injury at an early ischemia and reperfusion time-point. After 1 h ischemia, there was only one commonly upregulated gene (Sox18; NM_001024781) and 13 downregulated genes. Overexpression of Sox18 in blood vascular endothelial cells was previously reported to induce angiogenesis and lymphangiogenesis, which is associated with the ischemic response of organs. Sox18 therefore has the potential be an organ-ischemic marker (13). Of the 13 common downregulated genes, Gata4 (NM_144730) is a downstream gene of the MAPK pathway and its downregulation may represent the inactive status of the extracellular signal-regulated protein kinase (ERK) 1/2 pathway, which corresponded to the inactivity of nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) and activator protein (AP)-1 at this time-point (14). Ischemic insult also induces apoptosis and angiogenesis in order to respond to the hypoxic status, thus the downregulation of the adversely effected genes, including Casp9 (AF517560), Adamts1 (NM_024400) and Ceacam3 (NM_012702) are expected (15–17).
After 1 h reperfusion, additional biological activities were present, in which the interacting functions increased the biological complexity. There were 20 commonly upregulated gene probe sets in the I/R group. The majority of genes were not significantly upregulated during the initial 1 h of ischemia. Among these genes, several were involved in the MAPK and NFκB pathways. These two pathways may serve as the common pathways between the three organs at this time-point and modulate the biochemical response towards I/R injury (18,19).
Six genes were identified that were involved in the MAPK signaling pathway. Four of these were associated with the heterodimeric protein AP-1, Jun, Atf3, Jun b, and Fos. AP-1 is one of the end targets of the MAPK signaling pathway, and is considered to mediate I/R-induced gene expression since numerous subunit genes are known to mediate either proliferation, differentiation, or apoptosis (Jun family predominant) by altering the expression levels of cytokines, neurotransmitters, and other intercellular signaling molecules (20,21). AP-1 is additionally known to function in the process of T-cell activation, which is a key process in transplant immunology (22). In addition, AP-1 activates numerous downstream genes which are implicated in organ damage (23,24). AP-1 consists of three major subfamilies, including Jun, Fos, and Atf (25). In the early phase following I/R stress, the high expression levels of Jun and Atf activate the JNK and P38 pathways, promoting apoptosis. The high expression levels of Jun and Fos activate the ERK1/2 pathway to promote cellular proliferation (26). The data from the present study showed that there was a higher expression of Jun, Junb and Fos, but no significant difference in the expression of Atf3. This expression pattern was compatible with the previously described theories of apoptosis (27). Atf3, however, was found to be a common gene with higher expression (26). Atf3 is a stress-inducible gene that encodes a member of the ATF/cyclic adenosine monophosphate response element binding protein family of transcription factors (28). Atf3 mRNA was observed to increase in expression within 2 h following exposure of cells to stress signals, and therefore, Aft3 is a suitable candidate for further analysis in I/R injury.
The MAPK pathway may additionally be mediated during I/R injury by higher expression levels of Dusp1 and Adm, which downregulate the MAPK pathway. Dusp1 is an oxidative stress-inducible gene that acts as a negative regulator of the JNK and p38 pathways (29). Adm selectively inhibits the JNK pathway, therefore the two genes may act in opposition to AP-1 (30). The adjustment of their expression may facilitate a reduction in I/R injury.
The NFκB pathway is another important pathway that responds to I/R injury at this time-point. Ischemic insult activates NFκB-inducing kinase, which degrades IκB kinase and releases NFκB. NFκB then translocates to the nucleus to induce bioactivities including promotion of transcription and activation of adhesion molecules, cytokines and maturing of B cells (31). According to the presented database, the upregulation of Rhob (NM_022542) may repress NFκB signaling by inhibiting dissociation and subsequent degradation of IκB, therefore further diminishing the downstream inflammatory response. Two genes were additionally identified to modulate B-cells. Btg2 (NM_017259), the p53-transcriptional target, is an anti-proliferative B-cell translocation gene. Over-expression of Btg2 has a protective role, inducing B-cell depletion, which can further reduce the inflammatory response. Conversely, Nfkbiz (GenBank: XM_221537) activates B-cell proliferation and differentiation to enhance the inflammatory response (32). The present study additionally identified prostaglandin-endoperoxide synthase 2 (Cox2; GenBank: L25925, NM_017232) to be upregulated in the three organ models. Cox2 is an enzyme that catalyzes the initial step of the synthesis of inflammatory prostaglandins from arachidonic acid. The upregulation of Cox2 can activate the NFκB pathway and perform additional downstream bioactivities (33).
The cytokines and adhesion molecules triggered by different signaling pathways function to initiate the inflammatory response towards I/R insult. According to the presented database, only interleukin 1β (IL-1β; NM_031512) was identified to be upregulated in all three organ models. However, Hbegf (GenBank: NM_012945) and Socs3 (GenBank: NM_053565) were two genes identified that act as a negative controller, eliciting protective effects against cytokine and adhesion molecules, and diminishing the inflammatory response.
Other genes were identified in the present study that have not been previously associated with I/R injury, however may be functional in the I/R response. These genes included Verge (GenBank: NM_001003403) and Tfpi2 (GenBank: NM_173141), which were noted to be associated with angiogenesis and capillary endothelial and microcirculation dysfunction, as well as Plk2 (GenBank: NM_031821, BC070878), Klf6 (NM_031642) and Nr4a1 (NM_024388), which are involved in the G1 phase of the cell cycle and can promote cellular proliferation and prevent apoptosis (34). The schematic diagram in Fig. 3 illustrates the proposed complex mechanisms of I/R conditions.
In the present study, the uniquely affected genes in the three organ models in both ischemia and reperfusion status were identified and compared. Among these genes, several were identified to be associated with the MAPK and NFκB signaling pathways. The present study focused on only two time-points following I/R insult; therefore, the kinetic changes of the specific genes require further investigation. This study provided fundamental information to the understanding of the key biomechanical changes during I/R injury.
Acknowledgements
The authors would like to thank the Chang Gung Memorial Hospital for financial support (nos. CMRPG470041, CMRPG4B0021, CMRPG4A0101 and CMRPG4A0102), and the Chang Gung Memorial Hospital Urology Laboratory.
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