http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, 2014; 36(2): 271–277 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2013.844623
LABORATORY STUDY
Effect of mesenteric lymph duct ligation on gene expression profiles of renal tissue in hemorrhagic shock rats with fluid resuscitation Zi-Gang Zhao1,2, Chun-Yu Niu2, Jing-Fu Qiu2, Xiao-Dong Chen3, and Ji-Cheng Li1 1
Institute of Cell Biology, Zhejiang University, Hangzhou, Zhejiang, PR China, 2Institute of Microcirculation, Hebei North University, Zhangjiakou, Hebei, PR China, and 3Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT, Australia Abstract
Keywords
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 downregulated 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 downregulation of the above gene expressions.
Acute kidney injury, DNA microarrays, hemorrhagic shock, ligation, mesenteric lymph duct
Acute kidney injury (AKI) is a common pathological process induced by trauma, hemorrhage, infection, and intoxication.1,2 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.3 However, severe hypotension and ischemia reperfusion injury still caused the AKI or acute renal failure (ARF),4,5 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).6,7 Therefore, it is essential to elucidate the possible mechanisms and develop
Address correspondence to Ji-Cheng Li, Institute of Cell Biology, Zhejiang University, Yuhangtang Road 388, Hangzhou, 310058 Zhejiang, PR China. Tel: 86-571-88208088; Fax: 86-571-88208088; E-mail: [email protected] Chun-Yu Niu, Institute of Microcirculation, Hebei North University, Diamond South Road 11, Zhangjiakou, 075000 Hebei, PR China. Tel: 86-313-4029168; Fax: 86-313-4029168; E-mail: [email protected]
Received 28 July 2013 Revised 25 August 2013 Accepted 30 August 2013 Published online 18 October 2013
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.8 Our previous studies demonstrated that the mesenteric lymph duct ligation (MLDL) alleviated the AKI induced by two hits of hemorrhage and lipopolysaccharide9,10 or severe hemorrhagic shock.2 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.
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Introduction
History
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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.2 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
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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 mL) 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 mL 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 mg) was obtained, and 1.5 mL of oligo (dT) primer (0.5 mg/mL) was added. Water (22.5 mL) 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 mL of RNA enzyme inhibitor (40 L/mL), 8 mL of 5 first strand buffer, 4 mL of 0.1 M D,L-Dithiothreitol (DTT), 1.6 mL of 12.5 mM dNTPs, 2 mL of 4 mM aa-dUTP, and 1 mL of SuperScript III reverse transcriptase (200 U/mL). The solution was mixed thoroughly and processed with transient centrifugation. The reaction lasted for 1.5 h at 50 C, and 1 mL 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 mL of 0.5 M EDTA mixed with the solution. About 2 mL 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 mL of 5 M HCl was added to attain an equilibrium reaction system. Purification and fluorochrome labeling of aminomarked 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 mL of 0.1 M sodium carbonate (pH 9.3) and 4.5 mL 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 mL 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 mL 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 mL 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.
MLDL and renal gene expression profiles in HS
DOI: 10.3109/0886022X.2013.844623
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 mL 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
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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 mg/mL; 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 mg/mL, 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 Figures 1 and 2). Analysis of differentially expressed genes As shown in Figure 3, 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 upregulated and 31 genes down-regulated in renal tissue of hemorrhagic shock. 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 (Table 1). These proteins encoded by these up-regulated genes were involved in signal transduction, metabolism, transport, cell growth, cell movement, cellular component, and biological process. Meanwhile, there were 23 known genes in 31 genes downregulated 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 (Table 2). 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.
Discussion In the present study, the major novel finding was that 34 known genes of renal tissue were differentially expressed after
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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.
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.
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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.11 P2ry1 encoding protein, P2Y1 purinergic receptor (P2Y1R), mediates extracellular regulated protein kinases 1/2 phosphorylation12 and cytokine/chemokine response.13 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,14–16 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,17 endogenous oxidative stresses,18 and ischemia,19 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;20 Atp5d encoding protein maintains structural stability of F1 moiety of ATP synthase;21 as a result, they are involved in the production of ATP. Tst, mitochondrial matrix enzyme, is related to function and structure of mitochondrion.22 It has been shown that trauma and shock can cause the mitochondrion to wreak havoc in many ways.23 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.24 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.25 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
MLDL and renal gene expression profiles in HS
DOI: 10.3109/0886022X.2013.844623
Significant: 59 Median # false significant: 0.68582
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Delta 0.81257 SAM Plot 7 6 5 4
Observed
3 2 1 0 −1.5
−1
−0.5
0
0.5
1
−1 −2 −3 −4 Expected
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. Table 1. The genes that up-regulated in renal tissue by mesenteric lymph duct ligation in hemorrhagic shock rats with fluid resuscitation. Gene name
Gene ID
Cy5/Cy3
Function category
Visinin-like 1 (Vsnl1) Purinergic receptor P2Y, G-protein coupled, 1 (P2ry1) ATP-binding cassette, subfamily D (ALD), member 2 (Abcd2) Solute carrier family 13 (sodium-dependent citrate transporter), member 5 (Slc13a5) Hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (Hcn4) FMS-related tyrosine kinase 1 (Flt1) Macrophage migration inhibitory factor (Mif) Ubiquitin specific peptidase 7 (herpes virus-associated) (USP7)
24877 25265 84356 266998 59266 54251 81683 360471
2.1 2.1 2.2 10.3 2.0 2.4 3.4 2.2
Importin 5 (Ipo5) Synaptophysin-like 1 (Sypl1) Cysteine and histidine-rich domain (CHORD)-containing 1 (Chordc1)
306182 366595 315447
2.3 2.3 2.0
Signal transduction Signal transduction Metabolism Transport Transport Cell growth Cell movement Cellular component and biological process Unknown Unknown Unknown
size by dephosphorylation of gephyrin- or cytoskeletonassociated proteins.26 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, Naþ-coupled transporter for citrate, in liver cells, astrocytes, and neurons, first reported by Gopal et al.27, Yodoya et al.28 and Hardel et al.29 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.30 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,31 and vascular inflammation and neointima formation;32 The Folr1 gene codes protein folate binding protein-1 (Folbp1), which has been shown to play a vital role in embryonic development;33 Ccnd1 encoding protein is a regulatory subunit of CDK4 or CDK6 to regulate the G1/S transition of the cell cycle.34 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.35 Recent studies have shown
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Table 2. The genes that down-regulated in renal tissue by mesenteric lymph duct ligation in hemorrhagic shock rats with fluid resuscitation. Gene name
Gene ID Cy3/Cy5
CutA divalent cation tolerance homolog (E. coli) (Cuta) GABA(A) receptor-associated protein (Gabarap) Kruppel-like factor 6 (Klf6) Polymerase (RNA) II (DNA directed) polypeptide L (Polr2l) Polymerase (RNA) II (DNA directed) polypeptide E (Polr2e) Endonuclease G (Endog) Uromodulin (Umod) Fumarylacetoacetate hydrolase (Fah) Protein phosphatase 1, catalytic subunit, Alpha isozyme (Ppp1ca) ATP synthase, Hþ transporting, mitochondrial F1 complex, delta subunit (Atp5d) Thiosulfate sulfurtransferase (Tst) Glutathione synthetase (Gss) Glutathione S-transferase mu 1 (Gstm1) Glutathione S-transferase pi 1 (Gstp1) Transmembrane protein 150A (Tmem150a) Translocase of inner mitochondrial membrane 13 homolog (yeast) (Timm13) Folate receptor 1 (adult) (Folr1) Cyclin D1 (Ccnd1) Cadherin 2 (Cdh2) Ribonuclease, RNase K (Rnasek) Kallikrein 1-related peptidase C7 (Klk1c7) Deoxyribonuclease 1 (Dnase1) WAS protein family, member 1 (Wasf1)
294288 58974 58954 502374 690966 362100 25128 29383 24668 245965 25274 25458 24423 24426 245966 252928 171049 58919 83501 287453 24523 25633 294568
that increased expression of Mif, which codes protein Mif, a macrophage migration inhibitory factor, was involved in the pathogenesis of several inflammatory diseases,36 and was released by necrotic renal cortical tubular cells during Escherichia coli infection.37 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 p5338; 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 50 -UTR in the kidney may provide a prompt DNA-independent regulation of DNase I activity when DNA is damaged during ischemic injury39; Kal7 plays an essential role in processing of bioactive peptides, and is beneficial in acute ischemic renal disease.40 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
2.4 2.5 2.3 1.9 2.5 2.1 2.3 2.3 2.1 3.4 2.9 2.1 2.2 3.5 2.6 3.6 2.4 2.4 2.4 2.2 2.0 2.9 2.4
Function category Signal transduction Signal transduction Signal transduction Transcription regulation Transcription regulation Transcription regulation Metabolism Metabolism Metabolism Metabolism Metabolism Metabolism Metabolism Metabolism Transport Transport Cell growth Cell cycle Cell adhesion Cellular component and biological process Cellular component and biological process Cellular component and biological process Unknown
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 downregulation 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).
References 1. Wei Q, Dong Z. Regulation and pathological role of bid in ischemic acute kidney injury. Ren Fail. 2007;29(8):935–940. 2. Niu CY, Zhao ZG, Ye YL, et al. Mesenteric lymph duct ligation against renal injury in rats after hemorrhagic shock. Ren Fail. 2010; 32(5):584–591. 3. Anderson RJ. Plumbing the depths of blood pressure: hypotensive hemorrhage and acute kidney injury. Crit Care Med. 2011; 39(9):2196–2197. 4. Mayeur N, Minville V, Jaafar A, et al. Morphologic and functional renal impact of acute kidney injury after prolonged hemorrhagic shock in mice. Crit Care Med. 2011;39(9):2131–2138. 5. Wang Y, Yan J, Xi L, et al. Protective effect of crocetin on hemorrhagic shock-induced acute renal failure in rats. Shock. 2012; 38(1):63–67.
DOI: 10.3109/0886022X.2013.844623
6. Mizock BA. The multiple organ dysfunction syndrome. Dis Mon. 2009;55(8):476–526. 7. Wohlauer MV, Sauaia A, Moore EE, et al. Acute kidney injury and post trauma multiple organ failure: the canary in the coal mine. J Trauma Acute Care Surg. 2012;72(2):373–380. 8. Deitch EA. Gut lymph and lymphatics: a source of factors leading to organ injury and dysfunction. Ann N Y Acad Sci. 2010; 1207(Suppl 1):E103–E111. 9. Niu CY, Li JC, Zhao ZG, et al. Effect of intestinal lymphatic circulation blockage in two-hit rats. World J Gastroenterol. 2006; 12(36):5805–5812. 10. Zhao ZG, Niu CY, Zhang J, et al. Effects of mesenteric lymph duct ligation on apoptosis of renal tubule epithelial cells in rats after two-hits. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2007; 19(12):724–726. 11. Buttgereit J, Qadri F, Monti J, et al. Visinin-like protein 1 regulates natriuretic peptide receptor B in the heart. Regul Pept. 2010;161(1– 3):51–57. 12. Chen J, Wang L, Zhang Y, et al. P2Y1 purinoceptor inhibition reduces extracellular signal-regulated protein kinase 1/2 phosphorylation in spinal cord and dorsal root ganglia: implications for cancer-induced bone pain. Acta Biochim Biophys Sin (Shanghai). 2012;44(4):367–372. 13. Kuboyama K, Harada H, Tozaki-Saitoh H, et al. Astrocytic P2Y(1) receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia. J Cereb Blood Flow Metab. 2011;31(9):1930–1941. 14. Arnesano F, Banci L, Benvenuti M, et al. The evolutionarily conserved trimeric structure of CutA1 proteins suggests a role in signal transduction. J Biol Chem. 2003;278(46):45999–46006. 15. Alam J, Deharo D, Redding KM, et al. C-terminal processing of GABARAP is not required for trafficking of the angiotensin II type 1A receptor. Regul Pept. 2010;159(1–3):78–86. 16. Qi W, Chen X, Holian J, et al. Transcription factors Kru¨ppel-like factor 6 and peroxisome proliferator-activated receptor-{gamma} mediate high glucose-induced thioredoxin-interacting protein. Am J Pathol. 2009;175(5):1858–1867. 17. Zhang J, Ye J, Altafaj A, et al. EndoG links Bnip3-induced mitochondrial damage and caspase-independent DNA fragmentation in ischemic cardiomyocytes. PLoS One. 2011;6(3):e17998. 18. Ishihara Y, Shimamoto N. Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress. J Biol Chem. 2006;281(10):6726–6733. 19. Nielsen M, Zimmer J, Diemer NH. Endonuclease G expression in thalamic reticular nucleus after global cerebral ischemia. Exp Brain Res. 2008;190(1):81–89. 20. Genin EC, Geillon F, Gondcaille C, et al. Substrate specificity overlap and interaction between adrenoleukodystrophy protein (ALDP/ABCD1) and adrenoleukodystrophy-related protein (ALDRP/ABCD2). J Biol Chem. 2011;286(10):8075–8084. 21. Ko YH, Hullihen J, Hong S, et al. Mitochondrial F(0)F(1) ATP synthase. Subunit regions on the F1 motor shielded by F(0), functional significance, and evidence for an involvement of the unique F(0) subunit F(6). J Biol Chem. 2000; 275(42):32931–32939. 22. Gerhard DS, Wagner L, Feingold EA, et al. The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome Res. 2004;14(10B):2121–2127. 23. Hubbard WJ, Bland KI, Chaudry IH. The role of the mitochondrion in trauma and shock. Shock. 2004;22(5):395–402.
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24. Hart TC, Gorry MC, Hart PS, et al. Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy. J Med Genet. 2002; 39(12):882–892. 25. Aponte JL, Sega GA, Hauser LJ, et al. Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc Natl Acad Sci USA. 2001;98(2):641–645. 26. Bausen M, Weltzien F, Betz H, et al. Regulation of postsynaptic gephyrin cluster size by protein phosphatase 1. Mol Cell Neurosci. 2010;44(3):201–209. 27. Gopal E, Miyauchi S, Martin PM, et al. Expression and functional features of NaCT, a sodium-coupled citrate transporter, in human and rat livers and cell lines. Am J Physiol Gastrointest Liver Physiol. 2007;292(1):G402–G408. 28. Yodoya E, Wada M, Shimada A, et al. Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons. J Neurochem. 2006;97(1):162–173. 29. Hardel N, Harmel N, Zolles G, et al. Recycling endosomes supply cardiac pacemaker channels for regulated surface expression. Cardiovasc Res. 2008;79(1):52–60. 30. Zhang J, D’Ercole AJ, Underwood LE. Identification of a new gene (rat TM6P1) encoding a fasting-inducible, integral membrane protein with six transmembrane domains. Biochim Biophys Acta. 2000;1492(1):280–284. 31. Herse F, Fain JN, Janke J, et al. Adipose tissue-derived soluble FMS-like tyrosine kinase 1 is an obesity-relevant endogenous paracrine adipokine. Hypertension. 2011;58(1):37–42. 32. Li XD, Chen J, Ruan CC, et al. Vascular endothelial growth factorinduced osteopontin expression mediates vascular inflammation and neointima formation via Flt-1 in adventitial fibroblasts. Arterioscler Thromb Vasc Biol. 2012;32(9):2250–2258. 33. Spiegelstein O, Cabrera RM, Bozinov D, et al. Folate-regulated changes in gene expression in the anterior neural tube of folate binding protein-1 (Folbp1)-deficient murine embryos. Neurochem Res. 2004;29(6):1105–1112. 34. Modi PK, Komaravelli N, Singh N, et al. Interplay between MEKERK signaling, cyclin D1, and cyclin-dependent kinase 5 regulates cell cycle reentry and apoptosis of neurons. Mol Biol Cell. 2012; 23(18):3722–3730. 35. Nameta M, Yaoita E, Kato N, et al. Mesangial cells connected by the N-cadherin-catenin system in the rat kidney. Nephron Exp Nephrol. 2009;112(4):e92–e98. 36. Liu A, Fang H, Dirsch O, et al. Early release of macrophage migration inhibitory factor after liver ischemia and reperfusion injury in rats. Cytokine. 2012;57(1):150–157. 37. Hong MY, Tseng CC, Chuang CC, et al. Uropathogenic Escherichia coli causes cortical tubular necrotic cell death and the release of macrophage migration inhibitory factor. Cytokine. 2013;61(3):945–952. 38. Lee HJ, Kim MS, Kim YK, et al. HAUSP, a deubiquitinating enzyme for p53, is polyubiquitinated, polyneddylated, and dimerized. FEBS Lett. 2005;579(21):4867–4872. 39. Basnakian AG, Singh AB, Shah SV. Identification and expression of deoxyribonuclease (DNase) I alternative transcripts in the rat. Gene. 2002;289(1–2):87–96. 40. Chiang WC, Chien CT, Lin WW, et al. Early activation of bradykinin B2 receptor aggravates reactive oxygen species generation and renal damage in ischemia/reperfusion injury. Free Radic Biol Med. 2006;41(8):1304–1314.
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LABORATORY STUDY
Effect of mesenteric lymph duct ligation on gene expression profiles of renal tissue in hemorrhagic shock rats with fluid resuscitation Zi-Gang Zhao1,2, Chun-Yu Niu2, Jing-Fu Qiu2, Xiao-Dong Chen3, and Ji-Cheng Li1 1
Institute of Cell Biology, Zhejiang University, Hangzhou, Zhejiang, PR China, 2Institute of Microcirculation, Hebei North University, Zhangjiakou, Hebei, PR China, and 3Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, ACT, Australia Abstract
Keywords
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 downregulated 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 downregulation of the above gene expressions.
Acute kidney injury, DNA microarrays, hemorrhagic shock, ligation, mesenteric lymph duct
Acute kidney injury (AKI) is a common pathological process induced by trauma, hemorrhage, infection, and intoxication.1,2 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.3 However, severe hypotension and ischemia reperfusion injury still caused the AKI or acute renal failure (ARF),4,5 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).6,7 Therefore, it is essential to elucidate the possible mechanisms and develop
Address correspondence to Ji-Cheng Li, Institute of Cell Biology, Zhejiang University, Yuhangtang Road 388, Hangzhou, 310058 Zhejiang, PR China. Tel: 86-571-88208088; Fax: 86-571-88208088; E-mail: [email protected] Chun-Yu Niu, Institute of Microcirculation, Hebei North University, Diamond South Road 11, Zhangjiakou, 075000 Hebei, PR China. Tel: 86-313-4029168; Fax: 86-313-4029168; E-mail: [email protected]
Received 28 July 2013 Revised 25 August 2013 Accepted 30 August 2013 Published online 18 October 2013
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.8 Our previous studies demonstrated that the mesenteric lymph duct ligation (MLDL) alleviated the AKI induced by two hits of hemorrhage and lipopolysaccharide9,10 or severe hemorrhagic shock.2 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.
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History
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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.2 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
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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 mL) 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 mL 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 mg) was obtained, and 1.5 mL of oligo (dT) primer (0.5 mg/mL) was added. Water (22.5 mL) 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 mL of RNA enzyme inhibitor (40 L/mL), 8 mL of 5 first strand buffer, 4 mL of 0.1 M D,L-Dithiothreitol (DTT), 1.6 mL of 12.5 mM dNTPs, 2 mL of 4 mM aa-dUTP, and 1 mL of SuperScript III reverse transcriptase (200 U/mL). The solution was mixed thoroughly and processed with transient centrifugation. The reaction lasted for 1.5 h at 50 C, and 1 mL 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 mL of 0.5 M EDTA mixed with the solution. About 2 mL 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 mL of 5 M HCl was added to attain an equilibrium reaction system. Purification and fluorochrome labeling of aminomarked 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 mL of 0.1 M sodium carbonate (pH 9.3) and 4.5 mL 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 mL 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 mL 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 mL 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.
MLDL and renal gene expression profiles in HS
DOI: 10.3109/0886022X.2013.844623
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 mL 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
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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 mg/mL; 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 mg/mL, 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 Figures 1 and 2). Analysis of differentially expressed genes As shown in Figure 3, 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 upregulated and 31 genes down-regulated in renal tissue of hemorrhagic shock. 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 (Table 1). These proteins encoded by these up-regulated genes were involved in signal transduction, metabolism, transport, cell growth, cell movement, cellular component, and biological process. Meanwhile, there were 23 known genes in 31 genes downregulated 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 (Table 2). 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.
Discussion In the present study, the major novel finding was that 34 known genes of renal tissue were differentially expressed after
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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.
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.
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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.11 P2ry1 encoding protein, P2Y1 purinergic receptor (P2Y1R), mediates extracellular regulated protein kinases 1/2 phosphorylation12 and cytokine/chemokine response.13 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,14–16 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,17 endogenous oxidative stresses,18 and ischemia,19 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;20 Atp5d encoding protein maintains structural stability of F1 moiety of ATP synthase;21 as a result, they are involved in the production of ATP. Tst, mitochondrial matrix enzyme, is related to function and structure of mitochondrion.22 It has been shown that trauma and shock can cause the mitochondrion to wreak havoc in many ways.23 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.24 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.25 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
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DOI: 10.3109/0886022X.2013.844623
Significant: 59 Median # false significant: 0.68582
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Delta 0.81257 SAM Plot 7 6 5 4
Observed
3 2 1 0 −1.5
−1
−0.5
0
0.5
1
−1 −2 −3 −4 Expected
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. Table 1. The genes that up-regulated in renal tissue by mesenteric lymph duct ligation in hemorrhagic shock rats with fluid resuscitation. Gene name
Gene ID
Cy5/Cy3
Function category
Visinin-like 1 (Vsnl1) Purinergic receptor P2Y, G-protein coupled, 1 (P2ry1) ATP-binding cassette, subfamily D (ALD), member 2 (Abcd2) Solute carrier family 13 (sodium-dependent citrate transporter), member 5 (Slc13a5) Hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (Hcn4) FMS-related tyrosine kinase 1 (Flt1) Macrophage migration inhibitory factor (Mif) Ubiquitin specific peptidase 7 (herpes virus-associated) (USP7)
24877 25265 84356 266998 59266 54251 81683 360471
2.1 2.1 2.2 10.3 2.0 2.4 3.4 2.2
Importin 5 (Ipo5) Synaptophysin-like 1 (Sypl1) Cysteine and histidine-rich domain (CHORD)-containing 1 (Chordc1)
306182 366595 315447
2.3 2.3 2.0
Signal transduction Signal transduction Metabolism Transport Transport Cell growth Cell movement Cellular component and biological process Unknown Unknown Unknown
size by dephosphorylation of gephyrin- or cytoskeletonassociated proteins.26 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, Naþ-coupled transporter for citrate, in liver cells, astrocytes, and neurons, first reported by Gopal et al.27, Yodoya et al.28 and Hardel et al.29 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.30 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,31 and vascular inflammation and neointima formation;32 The Folr1 gene codes protein folate binding protein-1 (Folbp1), which has been shown to play a vital role in embryonic development;33 Ccnd1 encoding protein is a regulatory subunit of CDK4 or CDK6 to regulate the G1/S transition of the cell cycle.34 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.35 Recent studies have shown
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Table 2. The genes that down-regulated in renal tissue by mesenteric lymph duct ligation in hemorrhagic shock rats with fluid resuscitation. Gene name
Gene ID Cy3/Cy5
CutA divalent cation tolerance homolog (E. coli) (Cuta) GABA(A) receptor-associated protein (Gabarap) Kruppel-like factor 6 (Klf6) Polymerase (RNA) II (DNA directed) polypeptide L (Polr2l) Polymerase (RNA) II (DNA directed) polypeptide E (Polr2e) Endonuclease G (Endog) Uromodulin (Umod) Fumarylacetoacetate hydrolase (Fah) Protein phosphatase 1, catalytic subunit, Alpha isozyme (Ppp1ca) ATP synthase, Hþ transporting, mitochondrial F1 complex, delta subunit (Atp5d) Thiosulfate sulfurtransferase (Tst) Glutathione synthetase (Gss) Glutathione S-transferase mu 1 (Gstm1) Glutathione S-transferase pi 1 (Gstp1) Transmembrane protein 150A (Tmem150a) Translocase of inner mitochondrial membrane 13 homolog (yeast) (Timm13) Folate receptor 1 (adult) (Folr1) Cyclin D1 (Ccnd1) Cadherin 2 (Cdh2) Ribonuclease, RNase K (Rnasek) Kallikrein 1-related peptidase C7 (Klk1c7) Deoxyribonuclease 1 (Dnase1) WAS protein family, member 1 (Wasf1)
294288 58974 58954 502374 690966 362100 25128 29383 24668 245965 25274 25458 24423 24426 245966 252928 171049 58919 83501 287453 24523 25633 294568
that increased expression of Mif, which codes protein Mif, a macrophage migration inhibitory factor, was involved in the pathogenesis of several inflammatory diseases,36 and was released by necrotic renal cortical tubular cells during Escherichia coli infection.37 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 p5338; 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 50 -UTR in the kidney may provide a prompt DNA-independent regulation of DNase I activity when DNA is damaged during ischemic injury39; Kal7 plays an essential role in processing of bioactive peptides, and is beneficial in acute ischemic renal disease.40 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
2.4 2.5 2.3 1.9 2.5 2.1 2.3 2.3 2.1 3.4 2.9 2.1 2.2 3.5 2.6 3.6 2.4 2.4 2.4 2.2 2.0 2.9 2.4
Function category Signal transduction Signal transduction Signal transduction Transcription regulation Transcription regulation Transcription regulation Metabolism Metabolism Metabolism Metabolism Metabolism Metabolism Metabolism Metabolism Transport Transport Cell growth Cell cycle Cell adhesion Cellular component and biological process Cellular component and biological process Cellular component and biological process Unknown
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 downregulation 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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DOI: 10.3109/0886022X.2013.844623
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