Researches Of Genomics And Proteomics On Acute Kidney Injury Induced By Mesenteric Lymph Based On Hemorrhagic Shock In Rats

A variety of pathogenic factors, including trauma, hemorrhagie, body fluid loss, burns, surgical accidents, traffic accidents, and natural disasters such as earthquakes, can induce the reduction of effective circulating blood volume, lead to uncontrolled or controlled hemorrhagic shock. The internal milieu disorder induced by kidney injury following hemorrhagic shock is one of the key links of hemorrhagic shock leading to multiple organ injuries and/or multiple organ dysfunction syndrome (MODS). It has been shown that mesenteric lymph re turn plays an important role in multiple organ injuries based on hemorrhagic shock. Our previous studies demonstrated that the decreased mesenteric lymph return with the methods of mesenteric lymph duct ligation and mesenteric lymph drainage could alleviate the hemorrhagic shock induced-acute kidney injury (AKI). And its mechanism were related to reducing the produce and release of free radical, nitric oxide, and pro-inflammatory mediators, enhancing the activity of membrance ATPase, lessening the apoptosis, decreasing the bacteria/endotoxin translocation (BET) through the intestinal lymphatic pathway. However, the precise mechanisms of mesenteric lymph return aggravating AKI or mesenteric lymph drainage ameliorating AKI base on hemorrhagic shock remain to be clarified.Objective: In this study, based on the research basis of the mesenteric lymph return is an important mechanism of AKI after hemorrhagic shock, we investigated the effects of mesenteric lymph duct ligation on gene expression profile and mesenteric lymph drainage on protein expression profile of renal tissue following hemorrhagic shock with fluid resuscitation, using the techniques of genomics and proteomics. Furthermore, we searched the differentially expressed genes or proteins in renal tissue induced by post-hemorrhagic shock mesenteric lymph; thereby revealed the possible mechanisms underlying mesenteric lymph induction of AKI after hemorrhagic shock., and provided the experimental and theoretical basises for the prevention and treatment of severe shock and it induced-AKI, targeted mesenteric lymph.Methods:1) Effect of mesenteric lymph duct ligation on gene expression profiles of renal tissue in hemorrhagic shock ratsTwenty male Wistar rats were randomly divided into shock group and shock+ligation group. After general anesthesia, under sterile conditions, the left carotid artery and right jugular vein were isolated by minimal dissection and aseptically cannulated respectively with a catheter for mean arterial pressure (MAP) monitoring, hemorrhage and fluid resuscitation. After that, a3-cm midline laparotomy was performed to isolate the mesenteric lymph duct from the surrounding connective tissues and superior mesenteric artery by blunt dissection for mesenteric lymph duct ligation. After operation, all rats were allowed to stabilize for10min. 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 slowly and uniformly, the whole course was completed within3min. The MAP was maintained at40mmHg for90minutes by withdrawing or returning shed blood as required for the establishment of hemorrhagic shock model. Subsequently, the released blood and Ringer’s solution (the total amount was the whole blood volume) was injected through the right jugular vein over20min at a speed of50mL/h using an infusion pump. After resuscitation, the mesenteric lymph duct (MLD) was ligated in the shock+ligation group, whereas only cotton below MLD was threaded in the shock group. At3h after resuscitation, the left kidney was obtained from each rat under deeply anesthetic conditions for homogenate. Then, the total mRNA was extracted, reversely transcribed into cDNAs and marked with Cy3and Cy5. The cDNAs were subjected for microarray scanning with12028cDNA probes; the differentially expressed genes between two groups were analyzed. Subsequently, the hemorrhagic shock model was extablished for the total mRNA extraction in renal tissue, and then, the part of differentially expressed genes was validated using the method of real-time fluorescent quantitative PCR (qRT-PCR).2) Effect of post-hemorrhagic shock mesenteric lymph (PHSML) drainage on renal tissue proteome in hemorrhagic shock ratsTwenty-seven healthy and specific pathogen-free male Wistar rats were randomly divided into the sham, shock, and shock+drainage groups. After rats were anesthetized with pentobarbital sodium, the right femoral vein and artery were isolated and cannulated with a minimally heparinized polyethylene catheter for continuous monitoring of the MAP and fluid resuscitation. The left femoral artery was then isolated, cannulated, and attached in-line to an automatic withdrawal-infusion machine for hemorrhage. Afterwards, the mesenteric lymph duct was isolated for PHSML or sham drainage. After a30min stabilization period, rats in the shock and shock+drainage groups were subjected to hemorrhage slowly and uniformly to decrease MAP to40mmHg within10min. MAP was maintained at40+-2mmHg for60min by withdrawing or returning shed blood, as required for the preparation of hemorrhagic shock model. The rats were then resuscitated with the shed blood plus an equal volume of Ringer’s solution within30min. After fluid resuscitation, the mesenteric lymphatic duct was cannulated using a homemade bending needle for lymph drainage, which lasted for180min for rats in the shock+drainage group. Subsequently, under deep anesthesia with pentobarbital, the kidneys were collected from each group of rats in a fixed position and were frozen at-80℃for the proteome analysis using two-dimensional fluorescence difference gel electrophoresis. As follows:preparation of renal homogenate, elimination of high-abundant protein and confirmation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF) was performed using an IEF apparatus, proteins were then separated by12%SDS-PAGE using an Ettan DAL Tsix instrument, silver staining, scanning and data analysis. Subsequently, prepared IPG strips were de-stained with Coomassie Brilliant Blue. Differential proteins with intensities that either increased or decreased by1.5-fold or greater were selected for trypsin digestion and analyzed by matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry and tandem TOF/TOF mass spectrometry. Furthermore, ELISA was used to validate the identified partial proteins.Results:1) Effect of mesenteric lymph duct ligation on gene expression profiles of renal tissue in hemorrhagic shock ratsIn this part of study, the5,812valid dates of rat genomes transcription were acquired after the analysis of differentially expressed genes. Among them, there were56differentially expressed genes between shock and shock+ligation groups. Meanwhile, MLD ligation (MLDL) caused25genes up-regulated and31genes down-regulated in renal tissue of hemorrhagic shock.There were11known genes in25genes up-regulated by MLDL, including visinin-like1(Vsnl1), purinergic receptor P2Y, G-protein coupled,1(P2ryl), ATP-binding cassette, subfamily D (ALD), member2(Abcd2), solute carrier family13(sodium-dependent citrate transporter), member5(Slc13a5), hyperpolarization activated cyclic nucleotide-gated potassium channel4(Hcn4), FMS-related tyrosine kinase1(Fltl), macrophage migration inhibitory factor (Mif), ubiquitin specific peptidase7(USP7), importin5(Ipo5), synaptophysin-like1(Sypll), cysteine and histidine-rich domain (CHORD)-containing1(Chordcl). 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 were23known genes in31genes down-regulated by MLDL, such as cutA divalent cation tolerance homolog (Cuta), GABA(A) receptor-associated protein (Gabarap), kruppel-like factor6(Klf6), polymerase (RNA) Ⅱ (DNA directed) polypeptide L (Polr21), polymerase (RNA) Ⅱ (DNA directed) polypeptide E (Polr2e), endonuclease G (Endog), uromodulin (Umod), fumarylacetoacetate hydrolase (Fah), protein phosphatase1, catalytic subunit, alpha isozyme (Ppplca), ATP synthase, H+transporting, mitochondrial F1complex, delta subunit (Atp5d), thiosulfate sulfurtransferase (Tst), glutathione synthetase (Gss), glutathione S-transferase mu1(Gstm1), glutathione S-transferase pi1(Gstp1), transmembrane protein150A (Tmem150a), translocase of inner mitochondrial membrane13homolog (yeast)(Timml3), folate receptor1(Folrl), cyclin D1(Ccndl), ribonuclease, RNase K (Rnasek),cadherin2(Cdh2), kallikrein1-related peptidase C7(Klklc7), deoxyribonuclease1(Dnasel), WAS protein family, member1(Wasfl). 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.Results qRT-PCR analysis showed that the expressions of Cdh2, Gss, Tst mRNA in renal tissue in shock+ligation group were decreased than that of the shock group; meanwhile, the expression of Mif mRNA in shock+ligation group was increased compared to the shock group. The results of qRT-PCR were consistent with DNA microarray.2) Effect of post-hemorrhagic shock mesenteric lymph (PHSML) drainage on renal tissue proteome in hemorrhagic shock ratsIn this study, five2D-DIGE gels were established for subsequent proteomic analysis. A total of about2,000spots were detected in various Differential In-gel Analysis (DIA) workspaces in all gels using DeCyder6.5software. In the biological variation analysis module, five spots were found to be differentially expressed based on the following criteria:an average increased or decreased in ratio of1.5-fold or greater and a student t-test P<0.05between the hemorrhagic shocked and sham rats. Twelve spots were found to be upregulated or downregulated in the rats in the shock+drainage group compared with the sham group. Three spots were found to be upregulated or downregulated in the rats of the shock+drainage group compared with the shock group. After visual review,14protein spots were selected for MALDI-TOF/TOF MS analysis. Several proteins were successfully identified in nine spots. These differentially expressed proteins were classified into several categories, such as cell proliferation, energy metabolism, cell motility and cytoskeleton. In addition, the other five protein spots failed to be identified because their Mascot scores were lower than59.Compared with the sham group, heterogeneous nuclear ribonucleoprotein C (hnRNPC) and serine-threonine kinase receptor-associated protein (Strap) were decreased in the renal tissue of the shock group; Meanwhile, trifunctional enzyme subunit alpha, mitochondrial (Hadha) and solute carrier family25, member13(Slc25a13), ATP synthase subunit beta, mitochondrial (Atp5b), multiple variants of actin, such as actin, alpha skeletal muscle (Actal), actin, gamma-enteric smooth muscle (Actg2),33kDa protein (Actgl) and actin, gamma1propeptide-like (LOC684969), and40S ribosomal protein S3(Rps3), as well as hnRNPC and Strap, were downregulated in the renal tissue of the hemorrhagic shock+drainage group. Furthermore, multiple variants of actin, including actin, alpha cardiac muscle1(Actcl); actin, aortic smooth muscle (Acta2); Actg2; and actin, cytoplasmic1(Actb), and Atp5b were decreased in the shock+drainage group compared with the shock group.Results ELISA analysis showed that there were no significant differences were found between the shock and sham groups in terms of Atp5b and Actg2levels in renal tissue, and the Atp5b and Actg2levels in the shock+drainage group were significantly decreased compared with that of the sham and shock groups.Conclusions:1) The major novel finding was that34known genes of renal tissue were differentially expressed after MLDL following hemorrhagic shock with resuscitation using the methods of DNA microarrays. 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. The results implicate 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.2) The current results showed several differentially expressed proteins in renal tissue after hemorrhagic shock using the methods of DIGE and MS/MS identification. PHSML drainage decreased the expression of several proteins. These differentially expressed proteins were classified into several categories, such as cell proliferation, energy metabolism, cell motility and cytoskeleton. The results imply that these differentially expressed proteins may be involved in the pathogenesis of AKI and in mitigating the effects of PHSML drainage after hemorrhagic shock.