Abstract
Background: Acute kidney injury (AKI), a common pathological process following hemorrhagic shock, can lead to an internal milieu disorder, which is an important factor of multiple organ failure (MOF). It has been shown that the mesenteric lymph return plays a deleterious effect on MOF induced by hemorrhagic shock. In this study, we investigated the effects of mesenteric lymph duct ligation (MLDL) on gene expression profiles of renal tissue following hemorrhagic shock with fluid resuscitation. Methods: After establishment of hemorrhagic shock model and fluid resuscitation in rats of shock and shock ligation groups, the MLDL was performed in shock ligation group, and only threading under the mesenteric lymph duct in the shock group. Then, the fixed position renal tissue was taken out for homogenate in two groups at 3 h after resuscitation, the total mRNA was extracted, reversely transcribed into cDNAs and marked with Cy3 and Cy5. The cDNAs were subjected for microarray scanning with 12,028 cDNA probes; the differentially expressed genes between two groups were analyzed. Results: In the 5812 valid dates of rat genomes transcription, there were 34 known differentially expressed genes between the two groups, of which 11 genes were up-regulated whereas 23 genes were down-regulated by MLDL. These different expressed genes encoding protein function were mainly involved in signal transduction, transcription regulation, metabolism, transport, cell growth, cell cycle, cell adhesion, cell movement, cellular component, and biological process. Conclusions: The mechanism of MLDL alleviating the AKI aftershock might be associated with up- or down-regulation of the above gene expressions.
Introduction
Acute kidney injury (AKI) is a common pathological process induced by trauma, hemorrhage, infection, and intoxication.Citation1,Citation2 During the hemorrhagic shock, fluid resuscitation based on the theory of microcirculation disturbance can significantly reduce the deteriorative effects of AKI induced directly by ischemia after shock.Citation3 However, severe hypotension and ischemia reperfusion injury still caused the AKI or acute renal failure (ARF),Citation4,Citation5 which further results in the internal milieu disorder. It is one of the important factors of hemorrhagic shock inducing multiple organ dysfunction syndrome (MODS) or multiple organ failure (MOF).Citation6,Citation7 Therefore, it is essential to elucidate the possible mechanisms and develop efficient therapeutic measures of AKI induced by hemorrhagic shock.
It has been shown that mesenteric lymph plays an important role in multiple organ injury following hemorrhagic shock.Citation8 Our previous studies demonstrated that the mesenteric lymph duct ligation (MLDL) alleviated the AKI induced by two hits of hemorrhage and lipopolysaccharideCitation9,Citation10 or severe hemorrhagic shock.Citation2 These beneficial effects of the MLDL were associated with its attenuation of the free radical injury, inflammatory reaction, and apoptosis. However, the precise mechanisms remain to be clarified.
In the past several years, DNA microarray has attracted tremendous interests among biologists, which promises to monitor the whole genome on a single chip, and is widely used in gene-expression analysis, genotyping of individuals, analysis of point mutations, and single nucleotide polymorphisms as well as other genomic or transcriptomic variations. Therefore, in the present study, we used the methods of DNA microarrays to investigate the effects of MLDL on gene expression profiles of renal tissue following hemorrhagic shock with fluid resuscitation, thereby reveal the possible mechanisms underlying mesenteric lymph induction of AKI after shock.
Methods
Animals
Twenty specific pathogen free (SPF) Wistar rats, 200 g to 220 g, were purchased from the Chinese Academy of Medical Sciences Animal Breeding Center (Beijing, China), and were maintained at an animal facility under barrier-sustained conditions with 12-h light/dark cycle at a standard conditions (temperature: 23 ± 2 °C, relative humidity: 40%--80%), and free access to standard laboratory food and water. Before the experimentation, the rats were fasted for 12 h, but were allowed free access to water. Surgical procedure in this study was reviewed and approved by the Hebei North University Animal Care Committee and conformed to National Institutes of Health guidelines. All efforts were made to minimize suffering of animals.
Hemorrhagic shock model
After rats were anesthetized with pentobarbital sodium (1%, 50 mg/kg), under sterile conditions, the right jugular vein was separated from the surrounding tissues and cannulated using a microcatheter for heparin sodium (1 mL/kg, 700 U/kg) injection to prevent blood coagulation systematically and fluid resuscitation; then, the left carotid artery was also separated, while a minimally heparinized polyethylene catheter attached a t-branch pipe was introduced into the artery to continuously monitor mean arterial pressure by RM-6240B biological signal collecting and processing system (Chengdu Instrument Factory, Chengdu, China) and hemorrhage for establishment of hemorrhagic shock model. After that, a 3-cm midline laparotomy was performed to isolate the mesenteric lymph duct from the surrounding connective tissues by blunt dissection.
After operation, all rats were allowed to stabilize for 10 min. Then, hemorrhage (one-fifth of whole blood volume, which is one-thirteenth of avoirdupois) was performed from the left carotid artery using an automatic withdrawal-infusion machine (ZCZ-50, Zhejiang University Medical Ltd., Hangzhou, China) slowly and uniformly, the whole course was completed within 3 min. The MAP was maintained at 40 ± 2 mmHg for 90 min by withdrawing or returning shed blood as required for the establishment of hemorrhagic shock model.Citation2 Subsequently, the released blood and Ringer’s solution (the total amount was the whole blood volume) was injected through the right jugular vein over 20 min at a speed of 50 mL/h using an infusion pump. After resuscitation, rats were randomly divided equally into: hemorrhagic shock group (shock group) and hemorrhagic shock plus MLDL group (shock ligation group); the mesenteric lymph duct was ligated in the shock ligation group, whereas only cotton below mesenteric lymph duct was threaded in the shock group.
Collection of renal tissue
At 3 h after resuscitation, the left kidney was obtained from each rat under deeply anesthetic conditions. Subsequently, the kidney was split by a longitudinal midline incision, half of them, including medulla and cortex, was cut into the minced tissues of 1–1.5 mm3. These minced tissues about 0.5 g, were immigrated into EP tube with 1 mL RNAlater RNA Stabilization Reagent (Qiagen, Valencia, CA), and was frozen at −80 °C for further assays. Similarly, another part of the renal tissue was frozen at −80 °C for other experiments.
Total RNA Extraction
The prepared renal homogenate (300 μL) was obtained from each tube. RNA was extracted using an RNA extraction kit (Epicentre, Madison, WI) to investigate the process, from cell lysis, nucleic acid precipitation, DNA cleavage to RNA precipitation. About 1 μL of RNA sample was prepared, and the RNA content was examined by identifying A260 and A280 values by using an ND-1000 photometer (NanoDrop, Wilmington, DL).
RNA reverse transcription
RNA (50 μg) was obtained, and 1.5 μL of oligo (dT) primer (0.5 μg/μL) was added. Water (22.5 μL) was added to the solution, which was then incubated for 10 min at 70 °C and then at −20 °C for 5 min. We then added 1 μL of RNA enzyme inhibitor (40 Μ/μL), 8 μL of 5× first strand buffer, 4 μL of 0.1 M d,l-Dithiothreitol (DTT), 1.6 μL of 12.5 mM dNTPs, 2 μL of 4 mM aa-dUTP, and 1 μL of SuperScript III reverse transcriptase (200 U/μL). The solution was mixed thoroughly and processed with transient centrifugation. The reaction lasted for 1.5 h at 50 °C, and 1 μL of reverse transcriptase was then supplemented. The solution was thoroughly mixed and processed with centrifugation. This reaction lasted for 1.5 h at 50 °C and then switched to 75 °C for 10 min. The solution was incubated at 4 °C. At the end of the reaction, 10 μL of 0.5 M EDTA mixed with the solution. About 2 μL of 5 M NaOH was added into the solution, which was then incubated at 65 °C for 15 min. After the completion of RNA lysis, 2 μL of 5 M HCl was added to attain an equilibrium reaction system.
Purification and fluorochrome labeling of amino-marked cDNA
The RNA of renal tissues obtained from five rats in each group was taken as the template (two biological replicates). Following the instructions of the kit, the obtained cDNA was purified with QIA quick PCR Purification Kit (Qiagen, Dusseldorf, Germany), and the purification products were transferred to a 0.2 mL PCR reaction tube. After content analysis, the products were dried in an SPD1010 vacuum drier (Thermo, Waltham, MA) at 50 °C and then preserved at −20 °C for further use.
For each tube of cDNA purification products, 4.5 μL of 0.1 M sodium carbonate (pH 9.3) and 4.5 μL of Cy3 or Cy5 fluorescence storage solution (Amersham, Piscataway, NJ) were added. The solution was oscillated, and the amino-labeled cDNA was re-suspended. In the Cy3 and Cy5 reaction systems, 200 μL of PB was added, and the solution was thoroughly mixed. The solution was transferred into a QIA quick column. The solution was centrifuged for 1 min at 13,000 r/min. The solution was supplied with 750 μL of the buffer. The solution was again centrifuged for 1 min at 13,000 r/min. The solution was then centrifuged for another minute at 13,000 r/min. About 40 μL of water (pH 7.5–8.0) was added in each column, and the solution was kept in the shade at 37 °C for 10 min. The solution was centrifuged for 1 min at 13,000 r/min. Fluorochrome-labeled cDNA was recycled. After content analysis, the solution was dried in a vacuum drier.
Hybridization, poaching, and scanning of microarray
In this research, rat genome cDNA microarray (covering 12,028 kinds of genes, including 11,257 unigenes) was purchased from Shanghai GeeDom Biochip Company (Shanghai, China). The microarray was placed flat in the UVC500 crosslinker (Hoefer Pharmacia Biotech Inc., San Francisco, CA). After two crosslinks at a total energy mode of 60 mJ, the microarray was washed with deionized water for 2 min and dried using an automatic hand dryer. Each of the two cDNA probes from the two groups was randomly selected for hybridization with one microarray (for two technical replicates). A total of four microarrays were used. About 50 μL of the hybridization solution was added in the target molecule labeled with dry fluorochrome. The solution was oscillated at a low velocity. The solution was denatured at 95 °C for 5 min, oscillated at a low velocity, and then centrifuged. The solution was then denatured at 98 °C for 5 min and then centrifuged at 12,000 r/min for 5 min. The sample point was added on the microarray, covered with cover glass, and placed into the hybridization chamber. The chamber was then placed in the hybridization oven (MODEL 1000, Robbins Scientific, Sunnyvale, CA) and oscillated at a low velocity. The hybridization was continued at 42 °C for 16 to 18 h.
After hybridization, the microarray was removed from the oven. The cover glass was gently shaken off. The microarray was placed on the shaft and washed with rinsing solutions I, II, and III for 2, 2, and 1 min, respectively. The microarray was then dried. The microarray was scanned at dual channels of 635 and 532 nm by using a GenePix 4100A laser confocal microscopy scanner (Axon Instruments, Foster City, CA). After reading the fluorescence signals, the images were saved. GenePix Pro 4.1 was used to process the fluorescence signals. First, the sample signals and background noises were identified, and the fluorescence signals after the deduction of background noise from the sample points were calculated. Thus, the median of correction signals (mid-value) in the sample point was obtained. During the process, hybridization points with abnormal signals (such as impurity contamination and fusion of hybridization points) were manually deleted. Data in the two-channel fluorescence signal that are smaller than twice the background value were removed. Next, the global normalization method was applied for the normalization of the bi-color fluorescence data. The ratio of expression differences of each hybridization point of gene was calculated. The ratio of the medians of the two groups of signals from each sample point was calculated and converted into log with blank 2. The mean value was then calculated. For each gene, at least four data points were obtained. Genes with fewer than two data points were not included in the downstream gene analysis.
Data processing
Excel and Significance Analysis of Microarrays (SAM) were used for the analysis of differential expression gene. The gene would be identified as a differential expression gene if the following conditions are reached: the signal ratio of Cy5 showed a difference of at least twice that of three Cy3 experiments; at least two experiments of one group of samples showed differential expression with the same direction of changes; meanwhile, p was set at ≤0.05.
The function of differential expression genes was divided into: transport, transcription regulation, signal transduction, stress, metabolism, cell growth, cellular component and biological process, cell movement, cell differentiation, cell cycle, cell adhesion, and apoptosis, according to the standard of classification from http://geneontology.org
Results
Quantitation of total RNA and cDNA
The ratios of A260nm/A280nm of total RNA in four samples were over than 1.80, the contents of total RNA were over than 0.8 µg/µL; these results indicated that the RNA had on obvious degradation and it was suitable for the experiment of RNA reverse transcription. Meanwhile, the contents of DNA were over 0.1 µg/µL, which indicated that it was suitable for the next experiment of hybridization.
Hybridization results of microarray
The scanning image of the microarray was clear with comparatively fewer background noises. The signal intensity at the hybridization points showed significant difference. After overlapping the scanning results of Cy3 and Cy5, the overlapped fluorescence signals on one point were presented. Cy3 with a stronger signal intensity showed green fluorescence, whereas Cy5 with stronger signal intensity showed red fluorescence. Close fluorescence intensity values showed yellow fluorescence (see and ).
Figure 1. The 2-dye scanning results of expression pattern cDNA microarray in renal tissue of hemorrhagic shock rats with fluid resuscitation. A: Cy3; B: Cy5.
Figure 2. Built up of the microarray scanning results in renal tissue of hemorrhagic shock rats with fluid resuscitation.
Analysis of differentially expressed genes
As shown in , after the analysis of differentially expressed genes, the 5812 valid dates of rat genomes transcription were acquired. Among them, there were 56 differentially expressed genes between shock and shock ligation groups. Meanwhile, MLDL caused 25 genes up-regulated and 31 genes down-regulated in renal tissue of hemorrhagic shock.
Figure 3. Scatter diagram of differentially expressed genes induced by mesenteric lymph duct ligation in renal tissue of hemorrhagic shock rats with fluid resuscitation. The spots-up dashed line represented the up-regulated genes; in contrast, the spots-down dashed line represented the down-regulated genes.
There were 11 known genes in 25 genes up-regulated by MLDL, including Vsnl1, P2ry1, Abcd2, Slc13a5, Hcn4, Flt1, Mif, USP7, Ipo5, Sypl1, and Chordc1 (). These proteins encoded by these up-regulated genes were involved in signal transduction, metabolism, transport, cell growth, cell movement, cellular component, and biological process.
Table 1. The genes that up-regulated in renal tissue by mesenteric lymph duct ligation in hemorrhagic shock rats with fluid resuscitation.
Meanwhile, there were 23 known genes in 31 genes down-regulated by MLDL, such as Cuta, Gabarap, Klf6, Polr2l, Polr2e, Endog, Umod, Fah, Ppp1ca, Atp5d, Tst, Gss, Gstm1, Gstp1, Tmem150a, Timm13, Folr1, Ccnd1, Rnasek, Cdh2, Klk1c7, Dnase1, and Wasf1 (). The proteins encoded by these down-regulated genes were involved in signal transduction, transcription regulation, metabolism, transport, cell growth, cell cycle, cell adhesion, and cellular component and biological process.
Table 2. The genes that down-regulated in renal tissue by mesenteric lymph duct ligation in hemorrhagic shock rats with fluid resuscitation.
Discussion
In the present study, the major novel finding was that 34 known genes of renal tissue were differentially expressed after MLDL following hemorrhagic shock with resuscitation using the methods of DNA microarrays. The results implicate that these up- or down-regulated genes may be involved in the pathogenesis of AKI and in alleviated effects of MLDL after hemorrhagic shock.
First, we found that the MLDL significantly enhanced the expressions of Vsnl1, P2ry1, which are associated with signal transduction. Vsnl1 encoding protein calcium binding protein, Visinin-like protein 1, has been shown to regulate natriuretic peptide receptor B in the heart, as target of heart failure.Citation11 P2ry1 encoding protein, P2Y1 purinergic receptor (P2Y1R), mediates extracellular regulated protein kinases 1/2 phosphorylationCitation12 and cytokine/chemokine response.Citation13 These results implied that the mechanism of MLDL lessening AKI is related to regulating the inflammation response through enhancement of the Vsnl1 and P2ry1 expressions. Recent studies,Citation14–16 have shown that the encoded proteins by Cuta, Gabarap, and Klf6 were involved in signal transduction. In the present study, we found that MLDL decreased the expressions of these genes, suggesting that the down-regulated expression of these genes may also involve AKI following hemorrhagic shock. In addition, we also found that the MLDL reduced the expressions of Polr2l, Polr2e, and Endog, which are involved in transcriptional regulation. Polr2l and Polr2e are the polypeptide L and E of RNA polymerase II, and their functions might be the same as that of RNA polymerase II. Previous studies have reported that Endog was involved in the mitochondrial damage and caspases-independent DNA fragmentation,Citation17 endogenous oxidative stresses,Citation18 and ischemia,Citation19 etc. However, these studies were focused on the mediated effects of these genes in the brain damage; the role of the genes on AKI needs further investigation.
Second, the present results showed that there were differentially expressed genes associated with metabolism, such as energy metabolism and free radical elimination. Abcd2 gene, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D, as a homodimer allowing the entry of CoA-esters of very-long chain fatty acids into the peroxisome;Citation20 Atp5d encoding protein maintains structural stability of F1 moiety of ATP synthase;Citation21 as a result, they are involved in the production of ATP. Tst, mitochondrial matrix enzyme, is related to function and structure of mitochondrion.Citation22 It has been shown that trauma and shock can cause the mitochondrion to wreak havoc in many ways.Citation23 In the present study, MLDL increased the Abcd2 expression and decreased the Atp5d expression. It is contributive for the energy metabolism of the kidney after hemorrhagic shock, but the detailed mechanism is unclear. Moreover, the MLDL down-regulated the expressions of Gss, Gstm1, and Gstp1, which play significant role in the synthesis of glutathione and neutralizing harmful metabolites. It should be pointed out that the down-regulation of these genes was related to MLDL reducing the return of harmful substances through mesenteric lymph, lessening the production of free radical. Hence, there was a beneficial effect for alleviating the kidney injury subjected to hemorrhagic shock. In addition, Hart et al.Citation24 reported that mutations of the Umod gene were responsible for the clinical changes of interstitial renal disease, polyuria, and hyperuricemia in medullary cystic kidney disease 2 and familial juvenile hyperuricemic nephropathy. Aponte et al.Citation25 reported that a point mutation of Fah gene was an important factor for disrupting tyrosine catabolism that resulted in hereditary tyrosinemia of type 1. Ppp1ca is a catalytic subunit, alpha isozyme of protein phosphatase 1 (PP1), encoding protein PP1, which regulates gephyrin cluster size by dephosphorylation of gephyrin- or cytoskeleton-associated proteins.Citation26 But to the best of our knowledge, the role of the Umod, Fah, and Ppp1ca in the pathogenesis of kidney injury is unknown.
Third, the present study revealed that MLDL significantly increased the expressions of Slc13a5 and Hcn4, and decreased the expressions of Tmem150a and Timm13, respectively. These differentially expressed genes are associated with the function of transport. The Slc13a5 gene codes protein NaCT, Na+-coupled transporter for citrate, in liver cells, astrocytes, and neurons, first reported by Gopal et al.Citation27, Yodoya et al.Citation28 and Hardel et al.Citation29 reported that Hcn4, a cardiac pacemaker channel in cardiomyocytes, was crucial for not only maintaining a homeostatic surface expression but also supplying channels for rapid adaptation of their surface expression in response to extracellular stimuli. Its function was related to cellular excitability. Tmem150a, also known as Tm6p1, encodes a membrane-integrated protein with six transmembrane domains, and have an important function during fasting-induced catabolism.Citation30 Timm13, encodes small zinc finger protein, is involved in mitochondrial carrier import. However, the mechanism of MLDL alleviating kidney injury through regulating these genes expressions needs further investigation.
Fourth, we found that the MLDL induced the higher expression of Flt1 and lower expression of Folr1, involved in cell growth. At the same time, MLDL down-regulated the expression of Ccnd1 related to cell cycle. Flt1 is tyrosine kinase receptor for vascular endothelial growth factor, and plays a key role in cell proliferation, cell survival,Citation31 and vascular inflammation and neointima formation;Citation32 The Folr1 gene codes protein folate binding protein-1 (Folbp1), which has been shown to play a vital role in embryonic development;Citation33 Ccnd1 encoding protein is a regulatory subunit of CDK4 or CDK6 to regulate the G1/S transition of the cell cycle.Citation34 Moreover, we found that the MLDL decreased the expression of Cdh2, which codes protein Cdh2, a cell–cell adhesion molecule; N-cadherin was found predominantly at cell–cell contact sites of mesangial cells where actin filaments concentrated in the rat kidney.Citation35 Recent studies have shown that increased expression of Mif, which codes protein Mif, a macrophage migration inhibitory factor, was involved in the pathogenesis of several inflammatory diseases,Citation36 and was released by necrotic renal cortical tubular cells during Escherichia coli infection.Citation37 Together, our results, in conjunction with aforementioned studies from others, indicate that the mechanism of MLDL alleviating kidney injury is related to regulating the expressions of Cdh2 and Mif, inducing the inflammatory cells adhesion, aggregation, sequestration in tissue, thereby attenuating inflammatory response.
Finally, MLDL increased the expression of USP7 and decreased the expressions of Rnasek, Klk1c7, and Dnase1, all of which are involved in the cellular component and biological process. USP7, also known as Hausp, is a deubiquitinating enzyme for the tumor suppressor protein p53Citation38; Rnasek codes RNasek, is a subtype of ribonuclease involved in the hydrolization of RNA; Dnase1 codes DNase I, has been implicated in the induction of DNA fragmentation and cell death, alternative splicing in 5′-UTR in the kidney may provide a prompt DNA-independent regulation of DNase I activity when DNA is damaged during ischemic injuryCitation39; Kal7 plays an essential role in processing of bioactive peptides, and is beneficial in acute ischemic renal disease.Citation40 Combined with these results, our findings suggest that the MLDL on the regulation of USP7, Rnasek, Klk1c7, and Dnase1 may contribute to its beneficial role in alleviating kidney injury following hemorrhagic shock; however, the precise mechanism needs further study. In addition, the present results showed that there were four differentially expressed genes in renal tissue, such as Ipo5, Sypl1, Chordc1, and Wasf1, but their function were unknown, and further identification is needed.
In summary, the current study demonstrated the differentially expressed genes induced by MLDL in renal tissue in hemorrhagic shock rats with fluid resuscitation. Because these differentially expressed genes encoding proteins are mainly involved in signal transduction, transcription regulation, metabolism, transport, cell growth, cell cycle, cell adhesion, cell movement, cellular component, and biological process, our findings provided novel evidence and indicated that the beneficial role of MLDL alleviating the AKI after hemorrhagic shock might be associated with up- or down-regulation of the above gene expressions. Certainly, the changes of encode proteins by these identified genes should be investigated in the future, by which the mechanism of MLDL alleviating the AKI after hemorrhagic shock will be further elucidated.
Declaration of interest
No benefits in any form have been received or will be received from a commercial association related directly or indirectly to the subject of this article. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
This study was supported by the National Natural Science Foundation of China (30370561); the Key Scientific and Technological Project of Hebei Province (11276103D-84), and the Foundation of Hundred Innovative Talents in Universities of Hebei Province (CPRC047 and CPRCII026).
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