Inhibition of Circ-Snrk ameliorates apoptosis and inflammation in acute kidney injury by regulating the MAPK pathway.

BACKGROUND: Circular RNA (circRNA) is involved in the process of acute kidney injury (AKI), but only a few circRNAs have been reported. In the study, we investigated a new circRNA and its association with AKI. METHODS: An AKI model was established in Sprague-Dawley rats, followed by serum creatinine and urea nitrogen tests measured by a biochemical analyzer. The pathological changes and apoptosis in the renal tissue were detected by Hematoxylin and Eosin, and TUNEL staining. Then, circRNA expression in AKI was determined by quantitative real-time-PCR (qRT-PCR). NRK-52E cells were induced with hypoxia/reoxygenation (H/R) as in vitro models and the circ-Snrk level was tested by qRT-PCR. The effects of circ-Snrk in H/R-induced NRK-52E cells were assessed by flow cytometry, western blot, and enzyme-linked immunosorbent assay. Finally, RNA sequencing and western blot analysis were used to validate the mRNA profile and pathways involved in circ-Snrk knockdown in H/R-induced NRK-52E. RESULTS: A reliable AKI rat model and H/R cell model were established. qRT-PCR demonstrated that circ-Snrk level was upregulated in AKI left kidney tissue and NRK-52E cells with H/R treatment. Circ-Snrk knockdown inhibited apoptosis of NRK-52E cells and secretion of inflammatory factors (IL-6 and TNF-α). RNA sequencing showed that the mRNA profile changed after inhibition of circ-Snrk and differential expression of mRNA mainly enriched various signaling pathways, including MAPK signaling pathway. Furthermore, western blot indicated that circ-Snrk knockdown could inhibit the activation of p-JNK and p-38 transcription factors. CONCLUSIONS: Circ-Snrk is involved in AKI development and associated with the MAPK signaling pathway in AKI.

METTL3 contributes to renal ischemia-reperfusion injury by regulating Foxd1 methylation.

To investigate the mechanism of renal ischemia-reperfusion injury (IRI) via regulation of N6-methyl-adenosine (m6A) and relevant genes, IRI was induced in Sprague-Dawley rats, and urine and serum creatinine levels and tissue structure changes were observed. m6A and methyltransferase-like 3 (METTL3) protein levels were assessed via dot-blot and Western blot analyses, respectively. The hypoxia/reoxygenation (H/R) cell model was constructed using NRK-52E cells, and METTL3 protein levels were assessed. METTL3 was inhibited to observe its impact on NRK-52E cell apoptosis and m6A expression in H/R processes. Methylated RNA immunoprecipitation (MeRIP) sequencing was conducted followed by MeRIP-quantitative RT-PCR and quantitative RT-PCR validation. Our results indicated that urine and serum creatinine levels increased and that renal injury and cell apoptosis were both observed in the IRI model. In additon, m6A expression increased in the IRI model, and METTL3 protein levels significantly increased in the IRI and H/R models. When METTL3 was inhibited, m6A levels were accordingly decreased and cell apoptosis was suppressed in the H/R in vitro model. Based on MeRIP sequencing, transcription factor activating enhancer binding protein 2α (tfap2a), cytochrome P-450 1B1 (cyp1b1), and forkhead box D1 (foxd1) were significantly differentially expressed, as was m6A, which is involved in the negative regulation of cell proliferation and kidney development. We confirmed that foxd1 mRNA and its methylation levels contributed to IRI and H/R.

Allogeneic mesenchymal stem cells as induction therapy are safe and feasible in renal allografts: pilot results of a multicenter randomized controlled trial.

BACKGROUND: Kidneys from deceased donors are being used to meet the growing need for grafts. However, delayed graft function (DGF) and acute rejection incidences are high, leading to adverse effects on graft outcomes. Optimal induction intervention should include both renal structure injury repair and immune response suppression. Mesenchymal stem cells (MSCs) with potent anti-inflammatory, regenerative, and immune-modulatory properties are considered a candidate to prevent DGF and acute rejection in renal transplantation. Thus, this prospective multicenter paired study aimed to assess the clinical value of allogeneic MSCs as induction therapy to prevent both DGF and acute rejection in deceased donor renal transplantation. METHODS: Forty-two renal allograft recipients were recruited and divided into trial and control groups. The trial group (21 cases) received 2 × 106/kg human umbilical-cord-derived MSCs (UC-MSCs) via the peripheral vein before renal transplantation, and 5 × 106 cells via the renal artery during the surgical procedure. All recipients received standard induction therapy. Incidences of DGF and biopsy-proven acute rejection were recorded postoperatively and severe postoperative complications were assessed. Graft and recipient survivals were also evaluated. RESULTS: Treatment with UC-MSCs achieved comparable graft and recipient survivals with non-MSC treatment (P = 0.97 and 0.15, respectively). No increase in postoperative complications, including DGF and acute rejection, were observed (incidence of DGF: 9.5% in the MSC group versus 33.3% in the non-MSC group, P = 0.13; Incidence of acute rejection: 14.3% versus 4.8%, P = 0.61). Equal postoperative estimated glomerular filtration rates were found between the two groups (P = 0.88). All patients tolerated the MSCs infusion without adverse clinical effects. Additionally, a multiprobe fluorescence in situ hybridization assay revealed that UC-MSCs administered via the renal artery were absent from the recipient's biopsy sample. CONCLUSIONS: Umbilical-cord-derived MSCs can be used as clinically feasible and safe induction therapy. Adequate timing and frequency of UC-MSCs administration may have a significant effect on graft and recipient outcomes. Trial registration NCT02490020 . Registered on June 29 2015.

Donation after brain death followed by circulatory death, a novel donation pattern, confers comparable renal allograft outcomes with donation after brain death.

BACKGROUND: Organ donation after brain death (DBD) is the standard strategy for organ transplantation; however, the concept of brain death is not universally accepted due to cultural beliefs and barriers amongst billions of people worldwide. Hence, a novel donation pattern has been established in China which outlines the concept of donation after brain death followed by circulatory death (DBCD). Differently from any current donation classification, this new concept is formulated based on combination of recognizing brain death and circulatory death. Should approval be gained for this definition and approach, DBCD will pave a novel donation option for billions of people who cannot accept DBD due to their cultural beliefs. METHODS: A multi-center, cohort study was conducted from February 2012 to December 2015. 523 kidney transplant recipients from four kidney transplant institutions were enrolled into the study, of which, 383 received kidneys from DBCD, and 140 from DBD. Graft and recipient survivals following transplantation were retrospectively analyzed. Postoperative complications including delayed graft function,, and acute rejection, were also analyzed for both groups. RESULTS: DBCD could achieve comparable graft and recipient survivals in comparison with DBD (Log-rank P = 0.32 and 0.86,respectively). One-year graft and recipient survivals were equal between DBCD and DBD groups (97.4% versus 97.9%, P = 0.10;98.4% versus 98.6%, P = 1.0, respectively). Furthermore, DBCD did not increase incidences of postoperative complications compared with DBD, including delayed graft function (19.3% versus 22.1%, P = 0.46) and acute rejection (9.1% versus 8.6%, P = 1.0). Additionally, antithymocyte globulin as induction therapy and shorter warm ischemia time decreased incidence of delayed graft function in DBCD group (16.8% on antithymocyte globulin versus 27.2% on basiliximab, P = 0.03; 16.7% on ≤18 min versus 26.7% on > 18 min group, P = 0.03). CONCLUSIONS: Kidney donation through DBCD achieves equally successful outcomes as DBD, and could provide a feasible path to graft availability for billions of people who face barriers to organ donation from DBD.

Ultrasound Assessment of the Gastrocnemius Muscle as a Potential Tool for Identifying Sarcopenia in Patients with Type 2 Diabetes.

Objective: This study aims to examine the clinical significance of ultrasound evaluation of the gastrocnemius muscle (GM) in identifying sarcopenia in patients with type 2 diabetes (T2D). Methods: One hundred and fifty-three patients with T2D were included in this study. We measured the appendicular skeletal muscle mass index (ASMI), handgrip strength, and 6-meter walking speed. The US-derived muscle thickness (MT), cross-sectional area (CSA), and shear wave ultrasound elastography (SWE) of GM were also measured. We assessed the correlations between clinical indicators and US features. The model for screening sarcopenia was established using stepwise logistic regression. Stepwise linear regression was used to identify a set of variables that jointly estimated ASMI. The model's ability to identify sarcopenia and low muscle mass was assessed by receiver operating characteristic (ROC) curve analysis. Results: The prevalence of sarcopenia in this study was 24.2%. The CSA, MT and SWE values of the patients with sarcopenia were lower than those of patients without sarcopenia (all p < 0.05). ASMI was positively correlated with CSA (r = 0.56, p < 0.001) and MT (r = 0.39, p < 0.001). Handgrip strength was positively correlated with CSA (r = 0.45, p < 0.001), MT (r = 0.25, p < 0.001), and SWE (r = 0.26, p = 0.002). A diagnostic model for sarcopenia was established with a sensitivity of 81.1%, specificity of 75.0%, and an area under the curve (AUC) of 0.800. The estimated ASMI equation was developed and found to have a positive correlation with actual ASMI (r = 0.70, p < 0.001). It was also effective in diagnosing low muscle mass, with an AUC of 0.787 for males and 0.783 for females. Conclusion: Ultrasonographic assessment of the gastrocnemius muscle was found to be a useful and convenient method for detecting sarcopenia in patients with T2D.

A randomized controlled trial to evaluate efficacy and safety of early conversion to a low-dose calcineurin inhibitor combined with sirolimus in renal transplant patients.

BACKGROUND: The calcineurin inhibitor (CNI)-based immune maintenance regimen that is commonly used after renal transplantation has greatly improved early graft survival after transplantation; however, the long-term prognosis of grafts has not been significantly improved. The nephrotoxicity of CNI drugs is one of the main risk factors for the poor long-term prognosis of grafts. Sirolimus (SRL) has been employed as an immunosuppressant in clinical practice for over 20 years and has been found to have no nephrotoxic effects on grafts. Presently, the regimen and timing of SRL application after renal transplantation vary, and clinical data are scarce. Multicenter prospective randomized controlled studies are particularly rare. This study aims to investigate the effects of early conversion to a low-dose CNI combined with SRL on the long-term prognosis of renal transplantation. METHODS: Patients who receive four weeks of a standard regimen with CNI + mycophenolic acid (MPA) + glucocorticoid after renal transplantation in multiple transplant centers across China will be included in this study. At week 5, after the operation, patients in the experimental group will receive an additional administration of SRL, a reduction in the CNI drug doses, withdrawal of MPA medication, and maintenance of glucocorticoids. In addition, patients in the control group will receive the maintained standard of care. The patients' vital signs, routine blood tests, routine urine tests, blood biochemistry, serum creatinine, BK virus (BKV)/ cytomegalovirus (CMV), and trough concentrations of CNI drugs and SRL at the baseline and weeks 12, 24, 36, 48, 72, and 104 after conversion will be recorded. Patient survival, graft survival, and estimated glomerular filtration rate will be calculated, and concomitant medications and adverse events will also be recorded. CONCLUSION: The study data will be utilized to evaluate the efficacy and safety of early conversion to low-dose CNIs combined with SRL in renal transplant patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800017277.

Mycoparasitism of endophytic fungi isolated from reed on soilborne phytopathogenic fungi and production of cell wall-degrading enzymes in vitro.

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of beta-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of beta-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.

Anastomotic leakage after intrathoracic versus cervical oesophagogastric anastomosis for oesophageal carcinoma in Chinese population: a retrospective cohort study.

OBJECTIVE: To investigate the characteristics and predictors for anastomotic leakage after oesophagectomy for oesophageal carcinoma from the perspective of anastomotic level. DESIGN: Retrospective cohort study. SETTINGS: A single tertiary medical centre in China. PARTICIPANTS: From January 2010 to December 2016, all patients with oesophageal cancer of the distal oesophagus or gastro-oesophageal junction undergoing elective oesophagectomy with a curative intent for oesophageal carcinoma with intrathoracic oesophagogastric anastomosis (IOA) versus cervical oesophagogastric anastomosis (COA) were included. We investigated anastomotic level and perioperative confounding factors as potential risk factors for postoperative leakage by univariate and multivariate logistic regression. PRIMARY OUTCOME MEASURES: The primary outcome was the odds of anastomotic leakage by different confounding factors. Secondary outcome was the association of IOA versus COA with other postoperative outcomes. RESULTS: Of 458 patients included, 126 underwent cervical anastomosis and 332 underwent intrathoracic anastomosis. Anastomotic leakage developed in 55 patients (12.0%), with no statistical differences between COA and IOA (16.6% vs 10.2%; p=0.058). Multivariable analysis identified active diabetes mellitus (OR 2.001, p=0.047), surgical procedure (open: reference; minimally invasive: OR 1.770, p=0.049) and anastomotic method (semimechanical: reference; stapled: OR 1.821; handsewn: OR 2.271, p=0.048) rather than anastomotic level (IOA: reference; COA: OR 1.622, p=0.110) were independent predictors of leakage. CONCLUSIONS: Surgical and anastomotic techniques rather than the level of anastomotic site were independent predictors of postoperative anastomotic leakage in patients undergoing oesophageal cancer surgery.

Allogeneic mesenchymal stem cell as induction therapy to prevent both delayed graft function and acute rejection in deceased donor renal transplantation: study protocol for a randomized controlled trial.

BACKGROUND: Using kidneys from deceased donors is an available strategy to meet the growing need of grafts. However, higher incidences of delayed graft function (DGF) and acute rejection exert adverse effects on graft outcomes. Since ischemia-reperfusion injury (IRI) and ongoing process of immune response to grafts are the major causes of DGF and acute rejection, the optimal induction intervention should possess capacities of both repairing renal structure injury and suppressing immune response simultaneously. Mesenchymal stem cells (MSCs) with potent anti-inflammatory, regenerative and immune-modulatory properties are considered as a candidate to prevent both DGF and acute rejection in renal transplantation. Previous studies just focused on the safety of autologous MSCs on living-related donor renal transplants, and lack of concomitant controls and the sufficient sample size and source of MSCs. Here, we propose a prospective multicenter controlled study to assess the clinical value of allogeneic MSCs in preventing both DGF and acute rejection simultaneously as induction therapy in deceased-donor renal transplantation. METHODS/DESIGN: Renal allograft recipients (n = 100) will be recruited and divided into trial and control groups, and 50 patients in the trial group will be administered with a dose of 2 × 106 per kilogram human umbilical-cord-derived MSCs (UC-MSCs) via peripheral vein injection preoperatively, and a dose of 5 × 106 cells via renal arterial injection during surgery, with standard induction therapy. Incidences of postoperative DGF and biopsy-proved acute rejection (BPAR) will be recorded and analyzed. Additionally, other clinical parameters such as baseline demographics, graft and recipient survival and other severe postoperative complications, including complicated urinary tract infection, severe pneumonia, and severe bleeding, will be also assessed. DISCUSSION: This study will clarify the clinical value of UC-MSCs in preventing DGF and acute rejection simultaneously in deceased-donor renal transplantation, and provide evidence as to whether allogeneic MSCs can be used as clinically feasible and safe induction therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02490020 . Registered on 29 June 2015.

Inhibition of xenogeneic response in porcine endothelium using RNA interference.

BACKGROUND: Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells. METHODS: alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively. RESULTS: RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed. CONCLUSIONS: Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.

[The role RNA interference of alpha1, 3GT plays in resistance to complement mediated cytotoxicity of porcine endothelial cells].

OBJECTIVE: To evaluate whether RNA interference can protect porcine endothelial cells from complement mediated cytotoxicity. METHODS: Immortalized porcine aortic endothelial cells of the line PED were cultured and transfected with alpha1,3-galactosyltransferase (alpha1, 3-GT) specific siRNAs. Cells transfected with mismatch SiRNA was used as negative controls. Forty-eight hours later the cells were collected. The expression of alpha1, 3-GT mRNA was examined by RT-PCR. The expression of alpha-Gal was examined by flow cytometry. PED cells ere labeled with (51)Cr and mixed with normal human serum (NHS). The release of (51)Cr was measured by gamma-ray counter. Heat inactivated NHS (HINHS) was used as control. RESULTS: Two isoforms (isoform 1 and isoform 2) were amplified from the PED cells. The expression of alpha1, 3-GT in the PED cells transfected with SiRNA-1 was lower by 70% in comparison with the mock group (69% for the isoform 1 and 72% for the isoform 2, both P < 0.05). However, the expression of alpha1, 3-GT in the PED cells transfected with SiRNA21 was not different from those in the mock group and mismatch group (both P > 0.05). Flow cytometry showed that the average fluorescence intensity of the PED cells transfected with SiRNA-1 was 52, 9, significantly lower than that of the mismatch group and mock group (493.9 and 5-5.7 respectively, both P < 0.0). Fluorescence microscopy observed the "silence effect" of alphaGal after SiRNA-1 transfection. Added with 20% and 40% NHS, the cell dissolution rate of the SiRNA transfection group was lower than that of the mock group by 70% and 60% respectively. CONCLUSIONS: alpha1, 3GT gene silencing actually occurs following transfection of SiRNA-1. Porcine endothelial cells can be the targets of RNAi.