Hypoxia-inducible factor-1α-dependent induction of miR122 enhances hepatic ischemia tolerance

Hepatic ischemia and reperfusion (IR) injury contributes to the morbidity and mortality associated with liver transplantation. microRNAs (miRNAs) constitute a family of noncoding RNAs that regulate gene expression at the posttranslational level through the repression of specific target genes. Here, we hypothesized that miRNAs could be targeted to enhance hepatic ischemia tolerance. A miRNA screen in a murine model of hepatic IR injury pointed us toward the liver-specific miRNA miR122. Subsequent studies in mice with hepatocyte-specific deletion of miR122 (miR122loxP/loxP Alb-Cre+ mice) during hepatic ischemia and reperfusion revealed exacerbated liver injury. Transcriptional studies implicated hypoxia-inducible factor-1α (HIF1α) in the induction of miR122 and identified the oxygen-sensing prolyl hydroxylase domain 1 (PHD1) as a miR122 target. Further studies indicated that HIF1α-dependent induction of miR122 participated in a feed-forward pathway for liver protection via the enhancement of hepatic HIF responses through PHD1 repression. Moreover, pharmacologic studies utilizing nanoparticle-mediated miR122 overexpression demonstrated attenuated liver injury. Finally, proof-of-principle studies in patients undergoing orthotopic liver transplantation showed elevated miR122 levels in conjunction with the repression of PHD1 in post-ischemic liver biopsies. Taken together, the present findings provide molecular insight into the functional role of miR122 in enhancing hepatic ischemia tolerance and suggest the potential utility of pharmacologic interventions targeting miR122 to dampen hepatic injury during liver transplantation

Biobanking for glomerular diseases: a study design and protocol for KOrea Renal biobank NEtwoRk System TOward NExt-generation analysis (KORNERSTONE)

Abstract Backgrounds Glomerular diseases, a set of debilitating and complex disease entities, are related to mortality and morbidity. To gain insight into pathophysiology and novel treatment targets of glomerular disease, various types of biospecimens linked to deep clinical phenotyping including clinical information, digital pathology, and well-defined outcomes are required. We provide the rationale and design of the KOrea Renal biobank NEtwoRk System TOward Next-generation analysis (KORNERSTONE). Methods The KORNERSTONE, which has been initiated by Korea Centres for Disease Control and Prevention, is designed as a multi-centre, prospective cohort study and biobank for glomerular diseases. Clinical data, questionnaires will be collected at the time of kidney biopsy and subsequently every 1 year after kidney biopsy. All of the clinical data will be extracted from the electrical health record and automatically uploaded to the web-based database. High-quality digital pathologies are obtained and connected in the database. Various types of biospecimens are collected at baseline and during follow-up: serum, urine, buffy coat, stool, glomerular complementary DNA (cDNA), tubulointerstitial cDNA. All data and biospecimens are processed and stored in a standardised manner. The primary outcomes are mortality and end-stage renal disease. The secondary outcomes will be deterioration renal function, remission of proteinuria, cardiovascular events and quality of life. Discussion Ethical approval has been obtained from the institutional review board of each participating centre and ethics oversight committee. The KORNERSTONE is designed to deliver pioneer insights into glomerular diseases. The study design allows comprehensive, integrated and high-quality data collection on baseline laboratory findings, clinical outcomes including administrative data and digital pathologic images. This may provide various biospecimens and information to many researchers, establish the rationale for future more individualised treatment strategies for glomerular diseases. Trial registration NCT03929887

Remote Ischemic Preconditioning and Diazoxide Protect from Hepatic Ischemic Reperfusion Injury by Inhibiting HMGB1-Induced TLR4/MyD88/NF-κB Signaling

Remote ischemic preconditioning (RIPC) is known to have a protective effect against hepatic ischemia-reperfusion (IR) injury in animal models. However, the underlying mechanism of action is not clearly understood. This study examined the effectiveness of RIPC in a mouse model of hepatic IR and aimed to clarify the mechanism and relationship of the ATP-sensitive potassium channel (KATP) and HMGB1-induced TLR4/MyD88/NF-κB signaling. C57BL/6 male mice were separated into six groups: (i) sham-operated control, (ii) IR, (iii) RIPC+IR, (iv) RIPC+IR+glyburide (KATP blocker), (v) RIPC+IR+diazoxide (KATP opener), and (vi) RIPC+IR+diazoxide+glyburide groups. Histological changes, including hepatic ischemia injury, were assessed. The levels of circulating liver enzymes and inflammatory cytokines were measured. Levels of apoptotic proteins, proinflammatory factors (TLR4, HMGB1, MyD88, and NF-κB), and IκBα were measured by Western blot and mRNA levels of proinflammatory cytokine factors were determined by RT-PCR. RIPC significantly decreased hepatic ischemic injury, inflammatory cytokine levels, and liver enzymes compared to the corresponding values observed in the IR mouse model. The KATP opener diazoxide + RIPC significantly reduced hepatic IR injury demonstrating an additive effect on protection against hepatic IR injury. The protective effect appeared to be related to the opening of KATP, which inhibited HMGB1-induced TRL4/MyD88/NF-kB signaling

A new method for purification of functional recombinant GST-cyclophilin A protein from E. coli

374-378The expression of glutathione-S-transferase (GST) fusion protein is extensively utilized in the study of protein-protein interactions. In the commonly used purification method, the overexpressed GST fusion protein is bound to the glutathione (GSH)-coupled resins via affinity chromatography, and then eluted by an excessive quantity of reduced GSH. However, this technique has certain limitations, such as low product purity, retention of GSH in the sample, as well as relatively high cost. To overcome these limitations, in this study, elution buffer containing 2% formic acid was utilized rather than GSH to elute the GST-fusion protein, and thereafter the acidic samples were neutralized using collecting buffer. By using this method, highly purified GST-cyclophilin A (CypA) fusion protein was obtained, without affecting the structural and functional characteristics such as PPIase and chaperone activities. Moreover, the procedure is also cost-effective, due to the low cost of formic acid as compared with GSH