Characterizing hub biomarkers for post-transplant renal fibrosis and unveiling their immunological functions through RNA sequencing and advanced machine learning techniques.

BACKGROUND: Kidney transplantation stands out as the most effective renal replacement therapy for patients grappling with end-stage renal disease. However, post-transplant renal fibrosis is a prevalent and irreversible consequence, imposing a substantial clinical burden. Unfortunately, the clinical landscape remains devoid of reliable biological markers for diagnosing post-transplant renal interstitial fibrosis. METHODS: We obtained transcriptome and single-cell sequencing datasets of patients with renal fibrosis from NCBI Gene Expression Omnibus (GEO). Subsequently, we employed Weighted Gene Co-Expression Network Analysis (WGCNA) to identify potential genes by integrating core modules and differential genes. Functional enrichment analysis was conducted to unveil the involvement of potential pathways. To identify key biomarkers for renal fibrosis, we utilized logistic analysis, a LASSO-based tenfold cross-validation approach, and gene topological analysis within Cytoscape. Furthermore, histological staining, Western blotting (WB), and quantitative PCR (qPCR) experiments were performed in a murine model of renal fibrosis to verify the identified hub genes. Moreover, molecular docking and molecular dynamics simulations were conducted to explore possible effective drugs. RESULTS: Through WGCNA, the intersection of core modules and differential genes yielded a compendium of 92 potential genes. Logistic analysis, LASSO-based tenfold cross-validation, and gene topological analysis within Cytoscape identified four core genes (CD3G, CORO1A, FCGR2A, and GZMH) associated with renal fibrosis. The expression of these core genes was confirmed through single-cell data analysis and validated using various machine learning methods. Wet experiments also verified the upregulation of these core genes in the murine model of renal fibrosis. A positive correlation was observed between the core genes and immune cells, suggesting their potential role in bolstering immune system activity. Moreover, four potentially effective small molecules (ZINC000003830276-Tessalon, ZINC000003944422-Norvir, ZINC000008214629-Nonoxynol-9, and ZINC000085537014-Cobicistat) were identified through molecular docking and molecular dynamics simulations. CONCLUSION: Four potential hub biomarkers most associated with post-transplant renal fibrosis, as well as four potentially effective small molecules, were identified, providing valuable insights for studying the molecular mechanisms underlying post-transplant renal fibrosis and exploring new targets.

Periosteum-derived mesenchymal stem cell alleviates renal fibrosis through mTOR-mediated Treg differentiation.

BACKGROUND: Mesenchymal stem cells (MSCs) are the hotspots of cellular therapy due to their low immunogenicity, potent immunoregulation, and unique renoprotection. The present study aimed to investigate the effects of periosteum-derived MSCs (PMSCs) in ischemia-reperfusion (IR)-mediated renal fibrosis. METHODS: Using cell proliferation assay, flow cytometry, immunofluorescence, and histologic analysis, the differences in cell characteristics, immunoregulation, and renoprotection of PMSCs were compared to the bone marrow-derived MSCs (BMSCs), the most frequently studied stem cells in cellular therapy. In addition, the mechanism of PMSC renoprotection was investigated by 5' end of the RNA transcript sequencing (SMART-seq) and mTOR knockout mice. RESULTS: The proliferation and differentiation capabilities of PMSCs were stronger than those of BMSCs. Compared with BMSCs, the PMSCs exerted a better effect on alleviating renal fibrosis. Meanwhile, the PMSCs more effectively promote Treg differentiation. Treg exhaustion experiment indicated that Tregs exerted an important effect on inhibiting renal inflammation and acted as a critical mediator in PMSC renoprotection. Additionally, SMART-seq results implied that the PMSCs promoted Treg differentiation, possibly via the mTOR pathway. In vivo and in vitro experiments showed that PMSC inhibited mTOR phosphorylation of Treg. After mTOR knockout, the PMSCs failed to promote Treg differentiation. CONCLUSIONS: Compared with BMSCs, the PMSCs exerted stronger immunoregulation and renoprotection that was mainly attributed to PMSC promotion for Treg differentiation by inhibiting the mTOR pathway.

Integration of transcriptomics and metabolomics reveals pathways involved in MDSC supernatant attenuation of TGF-ß1-induced myofibroblastic differentiation of mesenchymal stem cells.

Overexposure to transforming growth factor b1 (TGF-ß1) induces myofibroblastic differentiation of mesenchymal stem cells (MSCs), which could be attenuated by myeloid-derived suppressor cell (MDSC) supernatant. However, the promyofibroblastic effects of TGF-ß1 and the antimyofibroblastic effects of MDSC supernatant in MSCs have not been fully elucidated. To further clarify the latent mechanism and identify underlying therapeutic targets, we used an integrative strategy combining transcriptomics and metabolomics. Bone marrow MSCs were collected 24 h following TGF-ß1 and MDSC supernatant treatment for RNA sequencing and untargeted metabolomic analysis. The integrated data were then analyzed to identify significant gene-metabolite correlations. Differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) were assessed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for exploring the mechanisms of myofibroblastic differentiation of MSCs. The integration of transcriptomic and metabolomic data highlighted significantly coordinated changes in glycolysis/gluconeogenesis and purine metabolism following TGF-ß1 and MDSC supernatant treatment. By combining transcriptomic and metabolomic analyses, this study showed that glycolysis/gluconeogenesis and purine metabolism were essential for the myofibroblastic differentiation of MSCs and may serve as promising targets for mechanistic research and clinical practice in the treatment of fibrosis by MDSC supernatant.

Estimation of Mycophenolic Acid Exposure in Chinese Renal Transplant Patients by a Joint Deep Learning Model.

BACKGROUND: To predict mycophenolic acid (MPA) exposure in renal transplant recipients using a deep learning model based on a convolutional neural network with bilateral long short-term memory and attention methods. METHODS: A total of 172 Chinese renal transplant patients were enrolled in this study. The patients were divided into a training group (n = 138, Ruijin Hospital) and a validation group (n = 34, Zhongshan Hospital). Fourteen days after renal transplantation, rich blood samples were collected 0-12 hours after MPA administration. The plasma concentration of total MPA was measured using an enzyme-multiplied immunoassay technique. A limited sampling strategy based on a convolutional neural network-long short-term memory with attention (CALS) model for the prediction of the area under the concentration curve (AUC) of MPA was established. The established model was verified using the data from the validation group. The model performance was compared with that obtained from multiple linear regression (MLR) and maximum a posteriori (MAP) methods. RESULTS: The MPA AUC 0-12 of the training and validation groups was 54.28 ± 18.42 and 41.25 ± 14.53 µg·ml -1 ·h, respectively. MPA plasma concentration after 2 (C 2 ), 6 (C 6 ), and 8 (C 8 ) hours of administration was the most significant factor for MPA AUC 0-12 . The predictive performance of AUC 0-12 estimated using the CALS model of the validation group was better than the MLR and MAP methods in previous studies (r 2 = 0.71, mean prediction error = 4.79, and mean absolute prediction error = 14.60). CONCLUSIONS: The CALS model established in this study was reliable for predicting MPA AUC 0-12 in Chinese renal transplant patients administered mycophenolate mofetil and enteric-coated mycophenolic acid sodium and may have good generalization ability for application in other data sets.

Transcriptional profile changes after treatment of ischemia reperfusion injury-induced kidney fibrosis with 18ß-glycyrrhetinic acid.

INTRODUCTION: Chronic kidney disease (CKD) is characterized by renal fibrosis without effective therapy. 18ß-Glycyrrhetinic acid (GA) is reported to have detoxification and anti-inflammatory functions and promotes tissue repair. However, the role of GA in CKD remains unclear. In this study, we investigated whether GA has a potential therapeutic effect in kidney fibrosis. METHODS: A renal fibrosis mouse model was established by ischemia/reperfusion (I/R) injury via clamping unilateral left renal pedicle for 45 min; then, the mice were treated with vehicle or GA. Kidney tissues and blood samples were extracted 14 days after reperfusion and renal function, histopathological staining, quantitative PCR, and western blotting were performed. RNA-seq was performed to explore the changes in the transcriptional profile after GA treatment. RESULTS: Renal function, pathological and molecular analysis displayed that fibrosis was successfully induced in the I/R model. In the GA treatment group, the severity of fibrosis gradually reduced with the best effect seen at a concentration of 25 mg kg -1. A total of 970 differentially expressed genes were identified. Pathway enrichment showed that reduced activation and migration of inflammatory cells and decreased chemokine interaction in significant pathways. Protein-protein interaction networks were constructed and 15 hub genes were selected by degree rank, including chemokines, such as C3, Ccl6, Ccr2, Ptafr, Timp1, and Pf4. CONCLUSIONS: GA may alleviate renal fibrosis by inhibiting the inflammatory response. GA is a promising therapy that may perhaps be used in treating renal fibrosis and CKD.

Carbamazepine-induced immune thrombocytopenia confirmed by modified MASPAT test.

A 76-year-old man suffering post-herpetic neuralgia developed severe thrombocytopenia 15 days after the administration of carbamazepine. Carbamazepine-dependent platelet antibodies were proved to be present in the patient's serum by a modified Monoclonal Antibody Solid-phase Platelet Antibody Test (MASPAT), and the diagnosis of carbamazepine-induced immune thrombocytopenia was confirmed. For the patient, carbamazepine should be advised to be avoided permanently. The present report advocated the application of a modified MASPAT test for the detection of carbamazepine-dependent platelet antibodies.

Association between preoperative lipid profiles and new-onset diabetes after transplantation in Chinese kidney transplant recipients: A retrospective cohort study.

BACKGROUND: This study investigated the association between the preoperative lipid profiles and new-onset diabetes after transplantation (NODAT) in Chinese kidney transplant recipients (KTRs). METHODS: In this study, of 1140 KTRs registered between January 1993 and March 2018 in Zhongshan Hospital, Fudan University, 449 were enrolled. Clinical data, obtained through a chart review of the patient records in the medical record system, were evaluated, and NODAT was diagnosed based on the American Diabetes Association guidelines. Multivariate Cox regression analysis was conducted to determine whether the preoperative lipid profiles in KTRs were independently associated with NODAT incidence. The preoperative lipid profiles were analyzed as continuous variables and grouped into tertiles. Smooth curve fitting was used to confirm the linear associations. RESULTS: During a median follow-up of 28.03 (interquartile range 12.00-84.23) months, 104 of the 449 (23.16%) participants developed NODAT. The multivariate model analysis, adjusted for all potential covariates, showed that increased values of the following parameters were associated with NODAT (hazard ratio, 95% confidence interval): preoperative total cholesterol (TC; 1.25, 1.09-1.58, p = 0.0495), low-density lipoprotein cholesterol (LDL-C; 1.33, 1.02-1.75, p = 0.0352), non-high-density lipoprotein cholesterol (non-HDL-C; 1.41, 1.09-1.82, p = 0.0084), TC/HDL-C (1.28, 1.06-1.54, p = 0.0109), and non-HDL-C/HDL-C (1.26, 1.05-1.52, p = 0.0138). However, the association between the preoperative triglyceride, HDL-C, or TG/HDL-C and NODAT was not significant. CONCLUSIONS: Preoperative TC, LDL-C, non-HDL-C, TC/HDL-C, and non-HDL-C/HDL-C were independent risk factors for NODAT.

High-mobility group box 1 protein antagonizes the immunosuppressive capacity and therapeutic effect of mesenchymal stem cells in acute kidney injury.

BACKGROUND: Kidney ischemia reperfusion injury (IRI) is a common cause of acute kidney injury and an unavoidable consequence of kidney transplantation and still lacks specific therapeutics. Recently, mesenchymal stem cell (MSC) has been emerging as a promising cell-based therapy for IRI in the context of transplantation. MSC negatively regulates the secretion of pro-inflammatory as well as the activation of immune cells during IRI through its unique immunosuppressive property. METHODS: We employed mice kidney IRI model and MSC cell line to monitor the IRI related checkpoints. siRNAs were utilized to knock down the potential key factors for mechanistic analysis. Statistical analysis was performed by using one-way ANOVA with Tukey's post hoc procedure by SPSS. RESULTS: The expression of high-mobility group box 1 protein (HMGB1) is increased in the acute phase as well as the recovery stage of IRI. Importantly, the HMGB1 upregulation is correlated with the injury severity. HMGB1 diminishes the MSC induced immunosuppressive capacity in the presence of pro-inflammatory cytokines in vitro. Toll like receptor 4 (TLR4)-mediated inducible nitric oxide synthase (iNOS) inhibition contributes to the negative effect of HMGB1 on MSCs. HMGB1-TLR4 signaling inhibition augments the therapeutic efficacy of MSCs in mice renal IRI model. CONCLUSIONS: These findings demonstrate that HMGB1 plays a crucial role in shaping the immunoregulatory property of MSCs within the microenvironments, providing novel insights into the crosstalk between MSCs and microenvironment components, suggesting HMGB1 signals as a promising target to improve MSC-based therapy.

Complement Inhibitor CRIg/FH Ameliorates Renal Ischemia Reperfusion Injury via Activation of PI3K/AKT Signaling.

Complement activation is involved in the pathogenesis of ischemia reperfusion injury (IRI), which is an inevitable process during kidney transplantation. Therefore, complement-targeted therapeutics hold great potential in protecting the allografts from IRI. We observed universal deposition of C3d and membrane attack complex in human renal allografts with delayed graft function or biopsy-proved rejection, which confirmed the involvement of complement in IRI. Using FB-, C3-, C4-, C5-, C5aR1-, C5aR2-, and C6-deficient mice, we found that all components, except C5aR2 deficiency, significantly alleviated renal IRI to varying degrees. These gene deficiencies reduced local (deposition of C3d and membrane attack complex) and systemic (serum levels of C3a and C5a) complement activation, attenuated pathological damage, suppressed apoptosis, and restored the levels of multiple local cytokines (e.g., reduced IL-1ß, IL-9, and IL-12p40 and increased IL-4, IL-5, IL-10, and IL-13) in various gene-deficient mice, which resulted in the eventual recovery of renal function. In addition, we demonstrated that CRIg/FH, which is a targeted complement inhibitor for the classical and primarily alternative pathways, exerted a robust renoprotective effect that was comparable to gene deficiency using similar mechanisms. Further, we revealed that PI3K/AKT activation, predominantly in glomeruli that was remarkably inhibited by IRI, played an essential role in the CRIg/FH renoprotective effect. The specific PI3K antagonist duvelisib almost completely abrogated AKT phosphorylation, thus abolishing the renoprotective role of CRIg/FH. Our findings suggested that complement activation at multiple stages induced renal IRI, and CRIg/FH and/or PI3K/AKT agonists may hold the potential in ameliorating renal IRI.

Discrepant mRNA and Protein Expression in Immune Cells.

With the development of single-cell mRNA sequencing (scRNA-seq), researchers have attempted to identify new methods for performing in-depth studies of immune cells. However, the discrepancies between the mRNA levels and the levels of surface proteins have confused many researchers. Here, we report a significant and interesting phenomenon in which the mRNA and protein expression levels were mismatched in immune cells. We concluded that scRNA-seq should be combined with other sequencing methods in single-cell studies (e.g., CITE-seq). The simultaneous assessment of both mRNA and protein expression will enhance the precision and credibility of the results.

Single-cell Sequencing in the Field of Stem Cells.

Variation and heterogeneity between cells are the basic characteristics of stem cells. Traditional sequencing analysis methods often cover up this difference. Single-cell sequencing technology refers to the technology of high-throughput sequencing analysis of genomes at the single-cell level. It can effectively analyze cell heterogeneity and identify a small number of cell populations. With the continuous progress of cell sorting, nucleic acid extraction and other technologies, single-cell sequencing technology has also made great progress. Encouraging new discoveries have been made in stem cell research, including pluripotent stem cells, tissue-specific stem cells and cancer stem cells. In this review, we discuss the latest progress and future prospects of single-cell sequencing technology in the field of stem cells.

Prediction of renal allograft chronic rejection using a model based on contrast-enhanced ultrasonography.

OBJECTIVE: To evaluate the application of contrast-enhanced ultrasonography (CEUS) for the diagnosis of renal allograft chronic rejection (CR). METHODS: A total of 104 patients who were suspected to have AR or CR were enrolled in this study (derivation group, n = 66; validation group, n = 38). Before biopsy, all patients received an ultrasound examination. RESULTS: In the CR group, rising time (RT) and time to peak (TTP) of medulla (RTm and TTPm, respectively) were significantly longer compared to those in the AR group. The kidney volume was significantly decreased in the CR group but was increased in the AR group. In the derivation group, age, change in kidney volume, and TTPm were identified as independent predictors by multivariate analysis. Based on the multivariate analysis results and area under receiver operating characteristic (ROC) curves (AUROCs) of individual markers, we constructed a new index as follows: P = -5.424 + 0.074 × age -9.818 × kidney volume change + 0.115 × TTPm; New Index = eP /(1 + eP ). The new index discriminates CR from AR and had better AUROCs than any other parameters. CONCLUSION: In conclusion, the new index provides a new diagnosis model for CR.

Resveratrol Alleviates Inflammatory Responses and Oxidative Stress in Rat Kidney Ischemia-Reperfusion Injury and H2O2-Induced NRK-52E Cells via the Nrf2/TLR4/NF-κB Pathway.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major causes of postoperative renal allograft dysfunction, which is mainly the result of proinflammatory reactions including inflammatory responses, oxidative stress, and metabolic disorders. Resveratrol (RSV) plays an important role in protecting various organs in IRI because it reduces oxidative stress, lessens the inflammatory response, and exerts anti-apoptotic effects. The aim of this study was to demonstrate the renoprotective effect of RSV in inhibiting inflammatory responses, reducing oxidative stress, and decreasing cell apoptosis in vivo and in vitro. METHODS: RSV was administered before renal ischemia and H2O2 induction. Serum and kidneys were harvested 24 h after reperfusion and NRK-52E cells were collected 4 h after H2O2 stimulation. Serum creatinine and blood urea nitrogen were used to assess renal function. Hematoxylin and eosin staining was performed to assess histological injury. Quantitative real-time PCR and enzyme-linked immunosorbent assay were used to assess proinflammatory cytokine expression. Oxidative stress-related proteins, such as Nrf2 and TLR4, were evaluated by western blot. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay was used to detect apoptotic cells in tissues, and western blot was used to evaluate the expression of caspase-3, -8, and -9 in this study. RESULTS: RSV inhibited inflammatory responses and improved renal function after renal IRI. Additionally, RSV decreased oxidative stress and reduced cell apoptosis by upregulating Nrf2 expression, downregulating the TLR4/NF-κB signaling pathway, and by decreasing caspase-3 activity and caspase cascades. CONCLUSION: Our study demonstrated the mechanisms underlying RSV renoprotection. We found that RSV exerts its greatest effects by blocking inflammatory responses, lowering oxidative stress, and reducing apoptosis via the Nrf2/TLR4/NF-κB pathway.

Transplantation of Telocytes Attenuates Unilateral Ureter Obstruction-Induced Renal Fibrosis in Rats.

BACKGROUND/AIMS: Previous studies imply that telocytes may have a protective effect on fibrosis in various organs, including the liver, colon, and heart. The effect of telocytes on renal fibrosis remains unknown. Herein, this study was designed to investigate the effect of telocytes on renal fibrosis and the potential mechanisms involved. METHODS: In a unilateral ureteral obstruction (UUO)-induced renal fibrosis model, telocytes were injected via the tail vein every other day for 10 days. The degree of renal damage and fibrosis was determined using histological assessment. The expression of collagen I, fibronectin, epithelial-mesenchymal transition markers, and Smad2/3 phosphorylation was examined by western blot analyses. Real-time PCR and enzyme-linked immunosorbent assay were performed in vivo to detect the levels of transforming growth factor (TGF)-ß1 and various growth factors. RESULTS: Telocytes attenuated renal fibrosis, as evidenced by reduced interstitial collagen accumulation, decreased expression of fibronectin and collagen I, upregulation of E-cadherin, and downregulation of α-smooth muscle actin. Furthermore, telocytes decreased serum TGF-ß1 levels, suppressed Smad2/3 phosphorylation, and increased the expression of hepatocyte growth factor (HGF) in rat kidney tissue following UUO. Blockage of HGF counteracted the protective effect of telocytes on UUO-treated kidneys. Through the detection of HGF mRNA levels in vitro, we found that telocytes had no effect on HGF expression compared with renal fibroblasts. CONCLUSION: Telocytes attenuated UUO-induced renal fibrosis in rats, likely through enhancing the expression of HGF in an indirect manner.

GC/MS-based urine metabolomics analysis of renal allograft recipients with acute rejection.

BACKGROUND: Acute renal allograft rejection is a common complication after renal transplantation that often leads to chronic rejection and ultimate graft loss. While renal allograft biopsy remains the gold standard for diagnosis of acute rejection, the possibility of biopsy-associated complications cannot be overlooked. The development of noninvasive methods for accurate detection of acute renal allograft rejection is thus of significant clinical importance. METHODS: Gas chromatography-mass spectrometry (GC/MS) was employed for analysis of urine metabolites in 15 renal allograft recipients with acute rejection and 15 stable renal transplant recipients. Partial least squares (PLS) regression and leave-one-out analyses were performed to ascertain whether the metabolites identified could be exploited to distinguish acute rejection from stable groups as well as their sensitivity and specificity. RESULTS: Overall, 14 metabolites were significantly altered in the acute rejection group (11 and 3 metabolites displayed higher and lower levels, respectively) relative to the stable transplant group. Data from PLS and leave-one-out analyses revealed that the differential metabolites identified not only distinguished acute rejection from stable transplant recipients but also showed high sensitivity and specificity for diagnosis of renal allograft recipients with acute rejection. CONCLUSION: Urine metabolites identified with GC/MS can effectively distinguish acute rejection from stable transplant recipients, supporting the potential utility of metabolome analysis in non-invasive diagnosis of acute rejection.

A novel cytoprotective peptide protects mesenchymal stem cells against mitochondrial dysfunction and apoptosis induced by starvation via Nrf2/Sirt3/FoxO3a pathway.

BACKGROUND: Mesenchymal stem cell (MSC) has been widely explored in the past decade as a cell-based treatment for various diseases. However, poor survival of adaptively transferred MSCs limits their clinical therapeutic potentials, which is largely ascribed to the nutrient starvation. In this study, we determined whether a novel kidney protective peptide CHBP could protect MSCs against starvation and invested the underlying mechanisms. METHODS: MSCs were subjected to serum deprivation and CHBP of graded concentrations was administered. Cell viability and apoptosis were detected by CCK-8, Annexin V/PI assay and Hoechst staining. ROS generation, mitochondrial membrane potential indicated by JC-1 and mitochondrial mass were measured by flow cytometry. The location of cytochrome c within cells was observed under fluorescence microscopy. Expressions of Nrf2, Sirt3, and FoxO3a were analyzed by western blot. In addition, preconditioning MSCs with CHBP was applied to test the possible protection against starvation. Finally, the effect of CHBP on the differentiation and self-renewal capacity of MSCs was also examined. RESULTS: CHBP improved cell viability and suppressed apoptosis in a dose dependent manner. Starvation resulted in the mitochondrial dysfunction and treatment of CHBP could alleviate mitochondrial stress by diminishing oxidative injury of ROS, restoring mitochondrial membrane potential and maintaining mitochondrial membrane integrity. Importantly, Nrf2/Sirt3/FoxO3a pathway was activated by CHBP and Sirt3 knockdown partially abolished the protection of CHBP. Moreover, MSCs pretreated with CHBP were more resistant to starvation. Under normal condition, CHBP exerted little effects on the differential and self-renewal capacity of MSCs. CONCLUSIONS: The present study demonstrated the efficient protection of CHBP upon MSCs against starvation-induced mitochondrial dysfunction and apoptosis and indicated possible involvement of Nrf2/Sirt3/FoxO3a pathway in the protective effect.

Estimation of Mycophenolic Acid Area Under the Curve With Limited-Sampling Strategy in Chinese Renal Transplant Recipients Receiving Enteric-Coated Mycophenolate Sodium.

BACKGROUND: The enteric-coated mycophenolate sodium (EC-MPS), whose active constituent is mycophenolic acid (MPA), has been widely clinically used for organ transplant recipients. However, its absorption is delayed due to its special designed dosage form, which results in difficulty to monitor the exposure of the MPA in patients receiving the EC-MPS. This study was aimed at developing a relatively practical and precise model with limited sampling strategy to estimate the 12-hour area under the concentration-time curve (AUC0-12 h) of MPA for Chinese renal transplant recipients receiving EC-MPS. METHODS: A total of 36 Chinese renal transplant recipients receiving the EC-MPS and tacrolimus were recruited in this study. The time point was 2 weeks after the transplantation for all the patients. The MPA concentrations were measured with enzyme-multiplied immunoassay technique for 11 blood specimens collected predose and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and 12 hours after the morning dose of EC-MPS. The measured AUC was calculated with these 11 points of MPA concentrations with the linear trapezoidal rule. Limited sampling strategy was used to develop models for estimated AUC in the model group (n = 18). The bias and precision of different models were evaluated in the validation group (n = 18). RESULTS: C4 showed the strongest correlation with the measured AUC. The best 3 time point equation was 6.629 + 8.029 × C0 + 0.592 × C3 + 1.786 × C4 (R = 0.910; P < 0.001), whereas the best 4 time point equation was 3.132 + 5.337 × C0 + 0.735 × C3 + 1.783 × C4 + 3.065 × C8 (R = 0.959; P < 0.001). When evaluated in the validation group, the 4 time point model had a much better performance than the 3 time point model: for the 4 time point model: R = 0.873, bias = 0.505 [95% confidence interval (CI), -10.159 to 11.170], precision = 13.370 (95% CI, 5.186-21.555), and 77.8% of estimated AUCs was within 85%-115% of the measured AUCs; for the 3 time point model: R = 0.573, bias = 6.196 (95% CI, -10.627 to 23.018), precision = 21.286 (95% CI, 8.079-34.492), and 50.0% of estimated AUCs was within 85%-115% of the measured AUCs. CONCLUSIONS: It demanded at least 4 time points to develop a relatively reliable model to estimate the exposure of MPA in renal transplant recipients receiving the EC-MPS. The long time span needed restricted its application, especially for the outpatients, but it could be a useful tool to guide the personalized prescription for the inpatients.

Inhibition of histone methyltransferase EZH2 ameliorates early acute renal allograft rejection in rats.

BACKGROUND: Although histone methyltransferases EZH2 has been proved to have significant regulatory effect on the immune rejection after hematopoietic stem cell transplantation, its role in solid-organ transplantation remains uncovered. In this study, we investigate whether histone methylation regulation can impact renal allograft rejection in rat models. RESULTS: Allogeneic rat renal transplantation model (Wistar to Lewis) was established, and the recipients were administrated with EZH2 inhibitor DZNep after transplantation. Renal allografts and peripheral blood were collected on day 5 after transplantation for histological examination and mechanism investigation. We found that inhibition of EZH2 by DZNep after transplantation significantly ameliorated acute rejection (AR), with decreased histological injury and reduced inflammatory infiltration in renal allografts. Attenuation of AR was due to the prohibited activation of alloreactive T cells, the subsequent impaired production of inflammatory cytokines, and also the elevated apoptosis of alloreactive T cells in both renal allografts and periphery. However, inhibition of EZH2 did not increase the regulatory T cells during the AR. CONCLUSIONS: Disruption of EZH2 by DZNep suppressed the immune responses of alloreactive T cells and ameliorated AR of renal allografts. This suggests a therapeutic potential of targeting histone methyltransferases EZH2 in treating allograft rejection after solid organ transplantation.

Endothelin Receptor Down-Regulation Mediated Ligand Regulation Mechanisms Protect Against Cellular Hypoxia Injury in Rat Vascular Endothelial Cells.

OBJECTIVE: Investigation of the effect of endothelin receptor A (ETaR)-targeting small interfering RNA (siRNA) on rat vascular endothelial cellular hypoxia injury, as well as its underlying mechanism. METHODS: An in vitro rat vascular smooth muscle cells - endothelial cells co-culture model was established and transfected with ETaR siRNA before hypoxia treatment. Cell culture supernatant, cellular protein and RNA were collected and examined at 0.5hrs, 1hrs, 2hrs, 4hrs, 8hrs, 16hrs, 24hrs and 48hrs of hypoxia with 1% oxygen. The time point at which the best silencing effect was achieved was chosen, eNOS inhibitor L-NAME was added, and post hypoxia cell culture supernatant, cellular protein and RNA was collected for further examination. RESULTS: After hypoxic treatment, endothelial-1 (ET-1) and ETaR expression levels gradually increased as oxygen deprivation extended. ET-1 and ETaR expression levels were significantly lower in the ETaR siRNA group compared with the Hypoxia group (P<0.001). Such difference peaked at 4hrs of hypoxia. ELISA examination of cell culture supernatant revealed that the amount of ET-1 and TGF-ßin the ETaR siRNA group were significantly lower compared to the Hypoxia group at all times, while the amount of NO and eNOS was higher. After 4 hrs of hypoxia, Smad2, Smad3, HIF-1, TNF-α, IFN-γ, IL-6, MCP-1, NF-κb, ET-1 and ANG II mRNA expression in endothelial cells and ETaR mRNA expression in A-10 cells of the ETaR siRNA group were lower than those of the Hypoxia siRNA group, while such results were much higher in the L-NAME group. Western Blot results showed lower expression of ETaR in the ETaR siRNA group compared with the hypoxia and negative siRNA groups, as well as significantly higher ETaR expression in the L-NAME group compared with the ETaR siRNA group. PI3K and p-AKT expression levels were mildly elevated after mild oxygen deprivation, and ETaR siRNA was able to enhance such elevation induced by hypoxia. In the L-NAME group, PI3K and p-AKT expression was much higher than the ETaR siRNA group. PKG and sGC expression levels significantly descended after mild oxygen deprivation. While such levels were higher in the ETaR siRNA group, compared with the hypoxia and negative siRNA groups, the L-NAME group had lower levels of PKG and sGC compared with the ETaR siRNA group. CONCLUSION: ETaR siRNA is capable of down-regulating the expression of inflammatory and transcription factors among endothelial cells treated with hypoxia. Down-regulation of ET-1 is triggered by altered nucleus transcription factor activity through the sGC/PKG signal pathway, and results in enhanced eNOS activity through the PI3K/Akt signal pathway. We suspect this to be the mechanism of the protective effect of ETaR siRNA.

A novel proteolysis-resistant cyclic helix B peptide ameliorates kidney ischemia reperfusion injury.

Helix B surface peptide (HBSP), derived from erythropoietin, displays powerful tissue protection during kidney ischemia reperfusion (IR) injury without erythropoietic side effects. We employed cyclization strategy for the first time, and synthesized thioether-cyclized helix B peptide (CHBP) to improve metabolic stability and renoprotective effect. LC-MS/MS analysis was adopted to examine the stability of CHBP in vitro and in vivo. The renoprotective effect of CHBP in terms of renal function, apoptosis, inflammation, extracellular matrix deposition, and histological injury was also detected in vivo and in vitro. Antibody array and western blot were performed to analyze the signal pathway of involvement by CHBP in the IR model and renal tubular epithelial cells. In this study, thioether-cyclized peptide was significantly stable in vivo and in vitro. One dose of 8nmol/kg CHBP administered intraperitoneally at the onset of reperfusion improved renal protection compared with three doses of 8nmol/kg linear HBSP in a 48h murine IR model. In a one-week model, the one dose CHBP-treated group exhibited remarkably improved renal function over the IR group, and attenuated kidney injury, including reduced inflammation and apoptosis. Interestingly, we found that the phosphorylation of autophagy protein mTORC1 was dramatically reduced upon CHBP treatment. We also demonstrated that CHBP induced autophagy via inhibition of mTORC1 and activation of mTORC2, leading to renoprotective effects on IR. Our results indicate that the novel metabolically stable CHBP is a promising therapeutic medicine for kidney IR injury treatment.