PP2Ac upregulates PI3K-Akt signaling and induces hepatocyte apoptosis in liver donor after brain death.

Multiple research groups have demonstrated that the outcome of patients receiving liver grafts from brain death donors (DBD) is poorer when compared with patients receiving grafts from living donors. This might be due to an increased hepatocyte apoptosis induced after brain death (BD). In this work, we found that the activity of PP2A-Akt pathway is significantly increased in clinical donor ex vivo hepatocytes after BD by iTRAQ protein quantification analysis. The same results were confirmed in animal models. A time-dependent promotion of apoptosis was also found in DBD rabbit liver, as demonstrated by the increased levels of cleaved Caspase 3 and the decreased of Bcl-2. To further investigate the roles of PP2A and Akt in regulating apoptosis of hepatocytes after BD, we cultivated human liver cell line L02 with serum deprivation and hypoxia, to simulate the ischemic and hypoxic conditions of hepatocytes in DBD. Increased apoptosis and decreased viability were observed during the time in this model. Meanwhile PP2A activity and Akt activity were respectively increased and decreased. Notably, the proportion of Akt phosphorylation at Ser473 decreased, while other known targets of PP2A (p38, JNK and ERK) were not affected in terms of protein levels or phosphorylation. These results suggested that PP2A is involved in apoptotic induction of hepatocytes after brain death by specific suppression of Akt. This discovery was further confirmed with pharmaceutical and genetic methods. Our work implied potential targets for reducing liver cell apoptosis and improving organ donor quality after BD.

Outcome Improvement for Hypothermic Machine Perfusion Versus Cold Storage for Kidneys From Cardiac Death Donors.

Organ shortage has led to an increased use of kidneys from cardiac death donors (DCDs), but controversies about the methods of organ preservation still exist. This study aims to compare the effect of machine perfusion (MP) and cold storage (CS) in protecting kidneys harvested from DCDs. 141 kidney pairs from DCDs between July 2010 and July 2015 were included in this randomized controlled study. One kidney from each donor was randomly assigned to MP and the contralateral kidney was assigned to CS. Delayed graft function (DGF) rate, resistance index of renal arteries, early renal function, and survival rates were used to estimate the effect of preservation. The results showed that MP decreased the rate of DGF from 33.3 to 22.0% (P = 0.033). Ultrasound of the kidneys within 48 h after transplantation showed that the resistance index of renal main artery (0.673 ± 0.063 vs. 0.793 ± 0.124, P < 0.001), sub segmental artery (0.66 ± 0.062 vs. 0.764 ± 0.077, P < 0.001) and interlobular artery (0.648 ± 0.056 vs. 0.745 ± 0.111, P = 0.023) were all significantly lower in the MP group than those in the CS group. Furthermore, compared to the CS group, in the first 7 days following transplantation, the median urine volume was significantly higher (4080 mL vs. 3000 mL, P = 0.047) in kidneys sustained using MP and the median serum creatinine was remarkably lower (180 µmol/L vs. 390 µmol/L, P = 0.024). More importantly, MP group had higher 1- and 3-year graft survival rates (98% vs. 93%, P = 0.026; 93% vs. 82%, P = 0.036, respectively). Hypothermic MP improved the outcomes of DCD kidney transplantation.

Mechanisms of Hypothermic Machine Perfusion to Decrease Donation After Cardiac Death Graft Inflammation: Through the Pathway of Upregulating Expression of KLF2 and Inhibiting TGF-ß Signaling.

Hypothermic machine perfusion (HMP) has been known as an efficient way to improve kidney graft function, but the underlying mechanisms remain unclear. Here, we adopt a rabbit reperfusion mode to investigate the upstream mechanisms of end-ischemic HMP of kidneys from donors after cardiac death (DCD), with static cold storage (CS) as a control. Eighteen New Zealand healthy male rabbits (12 weeks old, with a weight of 3.0 ± 0.2 kg) were randomly divided into three groups: HMP group, CS group, and Normal group (n = 6). The left kidney of rabbits underwent warm ischemia for 25 min through clamping the left renal pedicle and then reperfusion for 1 h. Then the left kidneys were preserved by CS or HMP (4°C for 4 h) ex vivo respectively, after they were autotransplanted and rabbits were submitted to a right nephrectomy. Twenty-four hours after reperfusion, all left renal specimens were collected. Finally, the expression of Krüppel-like factor 2 (KLF2), transforming growth factor-ß (TGF-ß) and SMAD4 protein in renal cortical tissue were detected by immunoblotting, and the TGF-ß and SMAD4 expressions were further confirmed by immunohistochemistry analysis. We found that expression of KLF2 in HMP group was significantly higher than CS group (P = 0.011), while expression of TGF-ß and SMAD4 in HMP group were significantly lower than CS group (P = 0.002, P = 0.01, respectively); Compared with normal group, the expression of TGF-ß and SMAD4 in HMP and CS group significantly increased (P<0.05). Compared with CS group, TGF-ß and SMAD4 protein were equally down-regulated in glomerular and the tubular epithelial cells in HMP group confirmed by immunohistochemistry. In conclusion, HMP may decrease DCD kidneys inflammation through the pathway of upregulating expression of KLF2 and inhibiting TGF-ß signaling after transplantation.

Aldehyde Dehydrogenase 2 Protects the Kidney from Ischemia-Reperfusion Injury by Suppressing the IκBα/NF-κB/IL-17C Pathway.

Objective: Ischemia-reperfusion injury (IRI) is an important cause of delayed functional recovery after transplantation. This study is aimed at investigating the molecular mechanism of ALDH2 in a kidney ischemia-reperfusion model based on RNA-seq. Methods: We performed kidney ischemia-reperfusion in ALDH2-/- and WT mice and evaluated kidney function and morphology using SCr, HE staining, TUNEL staining, and TEM. We used RNA-seq to compare mRNA expression in ALDH2-/- and WT mice after IR, and then, we verified the related molecular pathways by PCR and western blotting. In addition, activators and inhibitors of ALDH2 were used to alter the activity of ALDH2. Finally, we established a model of hypoxia and reoxygenation in HK-2 cells and clarified the role of ALDH2 in IR by interfering with ALDH2 and using an NF-κB inhibitor. Results: After kidney ischemia-reperfusion, the SCr value increased significantly, kidney tubular epithelial cells were damaged, and the apoptosis rate increased. In the microstructure, mitochondria were swollen and deformed, and ALDH2 deficiency aggravated these changes. The NF-κB pathway and IL-17 pathway were significantly enriched in ALDH2-/- mice compared with WT mice according to KEGG enrichment analysis of the RNA-seq data. The PCR results showed that the mRNA expression levels of IκBα and IL-17B, C, D, E, and F were significantly higher than those in the WT-IR group. Western blot verification results showed that ALHD2 knockdown resulted in increased phosphorylation of IκBα, increased phosphorylation of NF-κB, and increased expression of IL-17C. When we used ALDH2 agonists, the number of lesions and the expression levels of the corresponding proteins were reduced. Knockdown of ALDH2 in HK-2 cells resulted in a higher proportion of apoptotic cells after hypoxia and reoxygenation, but inhibiting the phosphorylation of NF-κB prevented the increase in apoptosis and reduced the protein expression level of IL-17C. Conclusion: ALDH2 deficiency can lead to the aggravation of kidney ischemia-reperfusion injury. RNA-seq analysis and validation by PCR and western blotting revealed that this effect may be due to the promotion of IκBα/NF-κB p65 phosphorylation during ischemia-reperfusion caused by ALDH2 deficiency, which then leads to an increase in inflammatory factors, including IL-17C. Thus, cell death is promoted, and kidney IRI is eventually aggravated. We link ALDH2 deficiency with inflammation, revealing a new idea for ALDH2-related research.

Dual-network polyacrylamide/carboxymethyl chitosan-grafted-polyaniline conductive hydrogels for wearable strain sensors.

Conductive, wearable, and flexible hydrogel-based sensors are considered as promising applications in human motion detection and physiological signal monitoring. However, it is still a problem to integrate multiple functions into one material for the next-generation smart devices. Herein, we fabricated an ionic/electronic dual conductive hydrogel by combining the chemically crosslinked polyacrylamide (PAM) and the physically crosslinked carboxymethyl chitosan-grafted-polyaniline (CMCS-g-PANI)/Ag+ network. The double-network hydrogel displays a high stretchability, repeatable adhesiveness, antibacterial activities, and biocompatibility. It also has high sensitivity and stable electrical performance for wearable strain sensors. Furthermore, we assembled a self-powered strain sensor based on the conversion of chemical energy to electrical energy. It can be used for human motion detection even without external power supply. This work provides an avenue for the development of multifunctional hydrogels with outstanding mechanical and electronic performances for application in wearable electronic devices.

Single-Cell Transcriptome Analysis of Chronic Antibody-Mediated Rejection After Renal Transplantation.

Renal transplantation is currently the most effective treatment for end-stage renal disease. However, chronic antibody-mediated rejection (cABMR) remains a serious obstacle for the long-term survival of patients with renal transplantation and a problem to be solved. At present, the role and mechanism underlying immune factors such as T- and B- cell subsets in cABMR after renal transplantation remain unclear. In this study, single-cell RNA sequencing (scRNA-seq) of peripheral blood monocytes (PBMCs) from cABMR and control subjects was performed to define the transcriptomic landscape at single-cell resolution. A comprehensive scRNA-seq analysis was performed. The results indicated that most cell types in the cABMR patients exhibited an intense interferon response and release of proinflammatory cytokines. In addition, we found that the expression of MT-ND6, CXCL8, NFKBIA, NFKBIZ, and other genes were up-regulated in T- and B-cells and these genes were associated with pro-inflammatory response and immune regulation. Western blot and qRT-PCR experiments also confirmed the up-regulated expression of these genes in cABMR. GO and KEGG enrichment analyses indicated that the overexpressed genes in T- and B-cells were mainly enriched in inflammatory pathways, including the TNF, IL-17, and Toll-like receptor signaling pathways. Additionally, MAPK and NF-κB signaling pathways were also involved in the occurrence and development of cABMR. This is consistent with the experimental results of Western blot. Trajectory analysis assembled the T-cell subsets into three differentiation paths with distinctive phenotypic and functional prog rams. CD8 effector T cells and γδ T cells showed three different differentiation trajectories, while CD8_MAI T cells and naive T cells primarily had two differentiation trajectories. Cell-cell interaction analysis revealed strong T/B cells and neutrophils activation in cABMR. Thus, the study offers new insight into pathogenesis and may have implications for the identification of novel therapeutic targets for cABMR.

Endoplasmic reticulum stress induces liver cells apoptosis after brain death by suppressing the phosphorylation of protein phosphatase 2A.

The present study aimed to investigate whether brain death (BD) induces the activation of endoplasmic reticulum stress (ERS) and protein phosphatase 2A (PP2A), and reveal the possible association with BD­induced liver cell apoptosis. A total of 30 healthy adult male Sprague­Dawley rats were randomized into three groups: Sham­operated group (S), BD group and 4­phenylbutyric acid group (BD + 4­PBA), with 10 rats in each group. All rats were anesthetized. The model of BD was established by inflating a balloon catheter that was placed into the extradural space after anesthesia. 4­PBA was administered via an intraperitoneal injection when the BD model was established. Anesthesia of the S group of rats was maintained for 6 h. Liver tissues were harvested after 6 h of BD. HE staining was used to evaluate the damage of liver. Terminal deoxynucleotidyl transferase­mediated 2'­deoxyuridine 5'­triphosphate nick­end labeling staining was used to observe the apoptosis of liver cells. Activation of ERS and PP2A was examined by western blotting and immunohistochemical staining. We reported that the apoptosis of liver cells after BD was significantly promoted than in the S group. Activation of ERS and PP2A was induced in the BD group when compared with S group. Phosphorylation of PP2A was suppressed in BD group. Application of 4­PBA decreased the activation of ERS and apoptosis rate compared with the BD group. In addition, activation of PP2A in the BD + 4­PBA group was decreased due to the reduction of PP2A phosphorylation compared with the BD group, but the levels were higher than in the S group. (P<0.05). In summary, our results indicated that BD induced ERS, then activated PP2A by suppressing the phosphorylation of PP2A, resulting in the apoptosis of liver cells.

MMP7 damages the integrity of the renal tubule epithelium by activating MMP2/9 during ischemia-reperfusion injury.

Renal ischemia-reperfusion (IR) injury is a common issue in urological surgery, and the renal tubules, particularly the proximal tubules, are extremely vulnerable to IR injury. In this work, we detected the differently expressed genes (DEGs) between normal rabbit kidneys and IR kidneys by RNA-sequencing, then identified that matrix metalloproteinase-7 (MMP7) played an important role in the progress of IR injury. Indeed, A time-dependent promotion of renal injury was detected in rabbit model, as demonstrated by the increased levels of MMP2/7/9, and the decreased of tight junction protein-1 (TJP1). Furtherly, similar results were confirmed in human renal proximal tubule epithelial (HK-2) cells model. Notably, downregulation of MMP7 affected the activity of MMP2/9 by suppressing expression of cleaved-MMP2/9 not the pro-MMP2/9 protein, which directly alleviated the degradation of TJP1 in HK-2 model. On the contrary, MMP7 had not been affected by inhibiting MMP2/9. In addition, coimmunoprecipitation assay showed that knockdown MMP7 restrained the interaction between MMP2/9 and TJP1. Collectively, this study suggested that MMP7 could serve as early biomarkers for renal tubular injury, and revealed that MMP7 could destroy the integrity of tubular epithelium through degrading TJP1 by activating MMP2/9.

Aldehyde dehydrogenase 2 regulates autophagy via the Akt-mTOR pathway to mitigate renal ischemia-reperfusion injury in hypothermic machine perfusion.

AIMS: Ischemia-reperfusion injury (IRI) is harmful to patients following kidney transplantation. Hypothermic machine perfusion (HMP) can be adopted to preserve grafts and reduce consequential injury. We hypothesized that aldehyde dehydrogenase 2 (ALDH2) partly mitigates kidney IRI via regulating excessive autophagy in HMP. MATERIALS AND METHODS: The rabbits were assigned to 5 groups: Normal, HMP, HMP + Alda-1, HMP + CYA and cold storage (CS). After the rabbit autologous kidney transplantation, renal pathology and function were evaluated by histological analysis, glomerular related proteins (desmin, nephrin), tubular injury factors (NGAL, Ki67), serum creatinine (Cr) and blood urea nitrogen (BUN). Oxidative stress molecular Malondialdehyde (MDA) and superoxide dismutase (SOD2) expression, as well as inflammatory cytokines (TNF-α, IL-6, IL-10) were assessed by immunohistochemistry. The expression of LC3, p62, ALDH2, p-Akt, mTOR, PTEN, p-PTEN, and 4-HNE were measured by immunohistochemistry, RT-PCR, Western blot analysis or ELISA. KEY FINDINGS: HMP was more effective than CS for kidney preservation, with p- ALDH2 expressed in greater quantities in HMP. The results of kidney pathology and function in HMP + Alda-1 were the best. The MDA & SOD2 and the Vyacheslav score were improved in HMP + CYA. ALDH2 reduced 4-HNE-induced oxidative stress, inflammatory infiltration, the expression of LC3, p62 and inhibited autophagy accompanied by activation of p-Akt and mTOR via p-PTEN/PTEN. SIGNIFICANCE: Akt-mTOR autophagy pathway is a novel target for ALDH2 to reduce renal IRI partly by inhibition of 4-HNE in HMP, then protecting the donated kidney received after cardiac death (DCD).

Ischemic Postconditioning Alleviates Intestinal Ischemia-Reperfusion Injury by Enhancing Autophagy and Suppressing Oxidative Stress through the Akt/GSK-3ß/Nrf2 Pathway in Mice.

AIMS: Ischemic postconditioning (IPO) has a strong protective effect against intestinal ischemia-reperfusion (IIR) injury that is partly related to autophagy. However, the precise mechanisms involved are unknown. METHODS: C57BL/6J mice were subjected to unilateral IIR with or without IPO. After 45 min ischemia and 120 min reperfusion, intestinal tissues and blood were collected for examination. HE staining and Chiu's score were used to evaluate pathologic injury. We test markers of intestinal barrier function and oxidative stress. Finally, we used WB to detect the expression of key proteins of autophagy and the Akt/GSK-3ß/Nrf2 pathway. RESULTS: IPO significantly attenuated IIR injury. Expression levels of LC3 II/I, Beclin-1, and p62 were altered during IIR, indicating that IPO enhanced autophagy. IPO also activated Akt, inhibited GSK-3ß/Nrf2 pathway. CONCLUSION: Our study indicates that IPO can ameliorate IIR injury by evoking autophagy, activating Akt, inactivating GSK-3ß, and activating Nrf2. These findings may provide novel insights for the alleviation of IIR injury.ß/Nrf2 pathway.