A Rat Model of Orthotopic Kidney Transplantation Based on Nonanastomotic Technique.

BACKGROUND: Renal transplantation is an effective treatment for end-stage renal disease, which involves pathophysiologic processes such as ischemia-reperfusion injury and immune rejection. The degree of ischemia-reperfusion injury is closely related to the functional state of the transplanted kidney. At present, the allogeneic kidney transplantation model has been widely used in related research. The traditional kidney transplantation model has the disadvantages of complicated vascular anastomosis, difficulty in ureteral reconstruction. The aim of this study was to establish a rat autologous orthotopic kidney transplantation model based on non-anastomotic technique. METHODS: Inbred Wistar rats weighing 260 to 280 g were selected. The rats were anesthetized by intraperitoneal injections of 40 mg/kg body weight pentobarbital sodium. We exposed and freed the left kidney after laparotomy and separated the left renal artery and left renal vein, abdominal aorta, and posterior vena cava. A purse-string suture with a diameter of 1 to 2 mm was made through the tunica media of the abdominal aorta. A puncture was made through the center of the purse-string suture. The in-dwelling needle was placed in the renal artery along the blood flow direction, and was infused with constant flow of 4°C heparinized lactated ringer's solution until the kidney became pale yellow. The renal vein was ligated and the renal artery was clamped. The in-dwelling needle was removed, purse-string suture was ligated, and the kidney was stored in a self-made autologous kidney transplant cold storage bag for 4 hours. We then opened the vein and artery, removed the cold storage bag, and rewarmed with 37°C normal saline. The abdomen was then closed layer by layer. RESULTS: Fifty-two orthotopic renal transplantations were performed, which included pre-experimental (40 operations) and experimental stages (12 operations). The success rates of the 2 stages were 75% and 91.7%, respectively. The main causes of failure were intraoperative hemorrhagic shock and postoperative infection. The operation time of orthotopic renal transplantation was 360 ± 30 minutes, including 30 ± 10 minutes for dissociation and management of kidney and blood vessels, 1 ± 0.5 minutes for warm ischemia and 240 ± 10 minutes for cold storage. Rats were sacrificed at 1 day and 7 day respectively. The rats were in good condition after operation. They could eat and drink freely. At 24 hours and 1 week after transplantation, the kidney's blood supply was good, the intestine was light or showed no adhesions, and the abdominal cavity had no ascites or peculiar smell. Hematoxylin & eosin (H&E) staining showed that there were no obvious pathologic changes in the sham group. The orthotopic kidney transplantation 1-day group showed pathologic changes of ischemia-reperfusion, such as swelling, necrosis, shedding, and cast formation of renal tubular cells. The orthotopic kidney transplantation 7-day group recovered well, with mild dilation of the renal capsule and mild dilatation of the renal tubules. CONCLUSION: The new model of autologous kidney transplantation is simple to use, does not require vascular anastomosis and ureteral reconstruction, and has a high success rate.

Role of HIF-1α in Cold Ischemia Injury of Rat Donor Heart Via the miR-21/PDCD4 Pathway.

BACKGROUND: Hypoxia-inducible factor 1 alpha (HIF-1α) is a transcription factor that plays a major role under hypoxia conditions. Cold storage during heart transplantation causes the donor heart long-term hypoxia. There is some evidence indicating a conceivable HIF-1α/microRNA-21 (miR-21)/phosphatase and tensin homolog (PTEN)/programmed cell death 4 (PDCD4) pathway. We assessed the hypothesis that HIF-1α has a positive effect during donor heart cold storage by making the miR-21 upregulate to reduce the expression of PDCD4. METHODS: We established the rat heart cold storage model and stratified it into 6-hour groups from 0 to 24 hours. Western blot and quantitative reverse transcription polymerase chain reaction were performed to detect the expression of HIF-1α, miR-21, PDCD4, and PTEN. RESULTS: After cold storage the expression of HIF-1α increased from 0 to 6 hours and then gradually decreased, but the expression level was relatively higher compared with the control group. The miR-21 was upregulated from 0 to 12 hours then downregulated. The messenger RNA expression of PDCD4 was upregulated gradually, but the protein expression was significantly downregulated at 12th hour then continued to upregulate. Interestingly, the expression level of miR-21 was highest in the 12th hour, which indicated miR-21 could inhibit the PDCD4. We subsequently detected the messenger RNA of PTEN, which can inhibit HIF-1α and be inhibited by miR-21. The expression of PTEN was also significantly downregulated at the 12th hour. CONCLUSION: In conclusion, there is possible interaction between HIF-1α and miR-21, and the conceivable HIF-1α/miR-21/PTEN/PDCD4 pathway plays a protective role in cold storage of the heart.

Sialyltransferase7A promotes angiotensin II-induced cardiomyocyte hypertrophy via HIF-1α-TAK1 signalling pathway.

AIMS: Sialylation is up-regulated during the development of cardiac hypertrophy. Sialyltransferase7A (Siat7A) mRNA is consistently over-expressed in the hypertrophic left ventricle of hypertensive rats independently of genetic background. The aims of this study were: (i) to detect the Siat7A protein levels and its roles in the pathological cardiomyocyte hypertrophy; (ii) to elucidate the effect of sialylation mediated by Siat7A on the transforming-growth-factor-ß-activated kinase (TAK1) expression and activity in cardiomyocyte hypertrophy; and (iii) to clarify hypoxia-inducible factor 1 (HIF-1) expression was regulated by Siat7A and transactivated TAK1 expression in cardiomyocyte hypertrophy. METHODS AND RESULTS: Siat7A protein level was increased in hypertrophic cardiomyocytes of human and rats subjected to chronic infusion of angiotensin II (ANG II). Delivery of adeno-associated viral (AAV9) bearing shRNA against rat Siat7A into the left ventricular wall inhibited ventricular hypertrophy. Cardiac-specific Siat7A overexpression via intravenous injection of an AAV9 vector encoding Siat7A under the cardiac troponin T (cTNT) promoter aggravated cardiac hypertrophy in ANG II-treated rats. In vitro, Siat7A knockdown inhibited the induction of Sialyl-Tn (sTn) antigen and cardiomyocyte hypertrophy stimulated by ANG II. Mechanistically, ANG II induced the activation of TAK1-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in parallel to up-regulation of Siat7A in hypertrophic cardiomyocytes. Siat7A knockdown inhibited activation of TAK1-NF-κB pathway. Interestingly, HIF-1α expression was increased in cardiomyocytes stimulated by ANG II but decreased after Siat7A knockdown. HIF-1α knockdown efficiently decreased TAK1 expression. ChIP and luciferase assays showed that HIF-1α transactivated the TAK1 promoter region (nt -1285 to -1274 bp) in the cardiomyocytes following ANG II stimulus. CONCLUSION: Siat7A was up-regulated in hypertrophic myocardium and promoted cardiomyocyte hypertrophy via activation of the HIF-1α-TAK1-NF-κB pathway.

Changes to Liver Structure and Energy Metabolism During Cold Storage of Transplanted Liver in Mice.

OBJECTIVES: In this study, we aimed to investigate the pathologic and ultrastructural changes in transplanted mouse livers after different durations of cold storage by testing indicators of liver function and energy metabolism. We aimed to describe the effects of cold storage on liver function and the mechanisms of cold storage damage. MATERIALS AND METHODS: We randomly placed 8-weekold male C57BL/6 mice into the following 4 groups to establish a cold-preserved mouse model of liver transplant: a normal control group and 3 cold storage groups, in which livers were stored for 4, 12, and 24 hours. Hepatic morphology, ultrastructural changes, and glycogenolysis were observed by hematoxylin and eosin staining, periodic acid-Schiff staining, and transmission electron microscopy. After different durations of cold storage, livers were reperfused with 4°C University of Wisconsin solution to obtain perfusion fluid, and alanine and aspartate aminotransferase levels were measured. Glycogen synthase, hypoxia-inducible factor-1α, Krüppel-like factor 2, and endothelial nitric oxide synthase mRNA expression levels in liver tissues were detected by real-time polymerase chain reaction, and aquaporin 8 protein expression levels in liver tissues were detected by Western blot. RESULTS: Hematoxylin and eosin staining and electron microscopy ofliver showed signs ofinjury after 12 hours of cold storage, which included mainly cytoplasmic edema characterized by loose liver cell arrangement, increased hepatic sinus fissure, mitochondrial swelling, and nuclear pyknosis. Periodic acid-Schiff staining showed that glycogen content was significantly reduced, with glycogen synthase levels also reduced. Alanine aminotransferase and aspartate aminotransferase levels gradually increasedwith cold storage. Glycogen synthase, Krüppel-like factor 2, endothelial nitric oxide synthase, and aquaporin 8 expression levels also gradually increased in liver tissue. These levels gradually decreased, but hypoxia-inducible factor-1α increased. CONCLUSIONS: Mouse livers showed progressive damage to structure and function during cold storage, with mitochondrial damage perhaps showing the earliest damage.

Influence of gut microbiota and intestinal barrier on enterogenic infection after liver transplantation.

Liver transplantation is currently a standard therapy for patients with end-stage liver diseases and hepatocellular carcinoma. Given that liver transplantation has undergone a thriving development in these decades, the survival rates after liver transplantation have markedly improved as a result of the critical advancement in surgical techniques, immunosuppressive therapies, and post-operative care. However, infection remains a fatal complication after liver transplantation surgery. In particular, enterogenic infection represents a major complication in liver transplant recipients. This article gives an overview of infection cases after liver transplantation and focuses on the discussion of enterogenic infection in terms of its pathophysiology, risk factor, outcome, and treatment.

Protective effect of dehydroandrographolide on obstructive cholestasis in bile duct-ligated mice.

BACKGROUND: Dehydroandrographolide (DA) is the main contributor to the therapeutic properties of the medicinal plant Andrographis paniculata (AP). However, it is unknown whether DA has a hepatoprotective effect on obstructive cholestasis in mice and humans. METHODS: We administered DA to mice for 5 days prior to bile duct ligation (BDL) and for the 7 days. Liver function markers, liver histology and necrosis, compensatory responses of hepatocytes, liver fibrosis and the expression of hepatic fibrogenesis markers were evaluated in BDL mice and/or human LX-2 cells. RESULTS: Mice treated with DA demonstrated lower levels of serum alanine transarninase (ALT), milder liver damage, liver necrosis and fibrosis formation than in vehicle control with carboxymethylcellulose (CMC) mice after BDL. DA treatment also enhanced the Mrp3 expression of hepatocytes but not Mrp4 following BDL. Further, DA treatment in BDL mice significantly reduced liver mRNA and/or protein expression of Tgf-ß, Col1a1, α-Sma and Mmp2. This result was also supported by hydroxyproline analysis. The molecular mechanisms of DA treatment were also assessed in human hepatic stellate cell line (LX-2 cell). DA treatment significantly inhibited Tgf-ß-induced Col1a1, Mmp2 and α-Sma expression in human LX-2 cells. These data suggested that DA treatment reduced liver damage through development of a hepatic adaptive response and inhibition of the activation of HSCs, which led to a reduction in liver fibrosis formation in BDL mice. CONCLUSIONS: DA treatment protected against liver damage and fibrosis following BDL and might be an effective therapy for extrahepatic cholestasis due to bile duct obstruction.

Sialyltransferase7A, a Klf4-responsive gene, promotes cardiomyocyte apoptosis during myocardial infarction.

Myocardial infarction (MI) is one major cause of heart failure through its induction of cardiomyocyte death. However, the molecular mechanisms associated with MI-induced cardiomyocyte apoptosis in the context of sialylation of heart are not yet understood. In this study, we found that sialyltransferase7A (Siat7A), one of the members of sialyltransferase family, was significantly increased in the ischemic myocardium, as well as in the human cardiomyocyte cell line AC16 under hypoxic condition. The Sialyl-Tn antigen (Neu5Acα2-6GalNAc-O-Ser/Thr) synthesized by Siat7A also increased in the AC16 cardiomyocytes following hypoxic stimulus. Increased Siat7A promoted cardiomyocyte apoptosis. The knockdown of Siat7A expression reduced cardiomyocyte apoptosis in both of vivo and vitro. Furthermore, the decreased extracellular signal-regulated kinase ERK1 and ERK2 (ERK1/2) activity was involved in the Siat7A-induced cardiomyocyte apoptosis. Notably, we showed that Krüppel-like factor 4 (Klf4), one of the transcription factors, specifically bound to the Siat7A promoter by ChIP assays. Deletion and mutagenesis analysis identified that Klf4 could transactivate the Siat7A promoter region (nt -655 to -636 bp). The upregulated Siat7A expression, which was paralleled by the increased Klf4 in the ischemic myocardium, contributed to cardiomyocyte apoptosis. Our study suggests Siat7A could be a valuable target for developing treatments for MI patients.