Rictor/mTORC2 signalling contributes to renal vascular endothelial-to-mesenchymal transition and renal allograft interstitial fibrosis by regulating BNIP3-mediated mitophagy.

BACKGROUND: Renal allograft interstitial fibrosis/tubular atrophy (IF/TA) constitutes the principal histopathological characteristic of chronic allograft dysfunction (CAD) in kidney-transplanted patients. While renal vascular endothelial-mesenchymal transition (EndMT) has been verified as an important contributing factor to IF/TA in CAD patients, its underlying mechanisms remain obscure. Through single-cell transcriptomic analysis, we identified Rictor as a potential pivotal mediator for EndMT. This investigation sought to elucidate the role of Rictor/mTORC2 signalling in the pathogenesis of renal allograft interstitial fibrosis and the associated mechanisms. METHODS: The influence of the Rictor/mTOR2 pathway on renal vascular EndMT and renal allograft fibrosis was investigated by cell experiments and Rictor depletion in renal allogeneic transplantation mice models. Subsequently, a series of assays were conducted to explore the underlying mechanisms of the enhanced mitophagy and the ameliorated EndMT resulting from Rictor knockout. RESULTS: Our findings revealed a significant activation of the Rictor/mTORC2 signalling in CAD patients and allogeneic kidney transplanted mice. The suppression of Rictor/mTORC2 signalling alleviated TNFα-induced EndMT in HUVECs. Moreover, Rictor knockout in endothelial cells remarkably ameliorated renal vascular EndMT and allograft interstitial fibrosis in allogeneic kidney transplanted mice. Mechanistically, Rictor knockout resulted in an augmented BNIP3-mediated mitophagy in endothelial cells. Furthermore, Rictor/mTORC2 facilitated the MARCH5-mediated degradation of BNIP3 at the K130 site through K48-linked ubiquitination, thereby regulating mitophagy activity. Subsequent experiments also demonstrated that BNIP3 knockdown nearly reversed the enhanced mitophagy and mitigated EndMT and allograft interstitial fibrosis induced by Rictor knockout. CONCLUSIONS: Consequently, our study underscores Rictor/mTORC2 signalling as a critical mediator of renal vascular EndMT and allograft interstitial fibrosis progression, exerting its impact through regulating BNIP3-mediated mitophagy. This insight unveils a potential therapeutic target for mitigating renal allograft interstitial fibrosis.

Association of UGT1A Gene Polymorphisms with BKV Infection in Renal Transplantation Recipients.

BACKGROUND: BK virus (BKV) infection is an opportunistic infectious complication and constitutes a risk factor for premature graft failure in kidney transplantation. Our research aimed to identify associations and assess the impact of single-nucleotide polymorphisms (SNPs) on metabolism-related genes in patients who have undergone kidney transplantation with BKV infection.

Material/Methods: The DNA samples of 200 eligible kidney transplant recipients from our center, meeting the inclusion criteria, have been collected and extracted. Next-generation sequencing was used to genotype SNPs on metabolism-associated genes (CYP3A4/5/7, UGT1A4/7/8/9, UGT2B7). A general linear model (GLM) was used to identify and eliminate confounding factors that may influence the outcome events. Multiple inheritance models and haplotype analyses were utilized to identify variation loci associated with infection caused by BKV and ascertain haplotypes, respectively.

Results: A total of 141 SNPs located on metabolism-related genes were identified. After Hardy-Weinberg equilibrium (HWE) and minor allele frequency (MAF) analysis, 21 tagger SNPs were selected for further association analysis. Based on GLM results, no confounding factor was significant in predicting the incidence of BK polyomavirus-associated infection. Then, multiple inheritance model analyses revealed that the risk of BKV infection was significantly associated with rs3732218 and rs4556969. Finally, we detect significant associations between haplotype T-A-C of block 2 (rs4556969, rs3732218, rs12468274) and infection caused by BKV (P = 0.0004).

Conclusions: We found that genetic variants in the UGT1A gene confer BKV infection susceptibility after kidney transplantation.

Enhanced humification via lignocellulosic pretreatment in remediation of agricultural solid waste.

Stover and manure are the main solid waste in agricultural industry. The generation of stover and manure could lead to serious environmental pollution if not handled properly. Composting is the potential greener solution to remediate and reduce agricultural solid waste, through which stover and manure could be remediated and converted into organic fertilizer, but the long composting period and low efficiency of humic substance production are the key constraints in such remediation approach. In this study, we explore the effect of lignocellulose selective removal on composting by performing chemical pretreatment on agricultural waste followed by utilization of biochar to assist in the remediation by co-composting treatment and reveal the impacts of different lignocellulose component on organic fertilizer production. Aiming to discover the key factors that influence humification during composting process and improve the composting quality as well as comprehensive utilization of agricultural solid waste. The results demonstrated that the removal of selective lignin or hemicellulose led to the shift of abundances lignocellulose-degrading bacteria, which in turn accelerated the degradation of lignocellulose by almost 51.2%. The process also facilitated the remediation of organic waste via humification and increased the humic acid level and HA/FA ratio in just 22 days. The richness of media relies on their lignocellulose content, which is negatively correlated with total nitrogen content, humic acid (HA) content, germination index (GI), and pH, but positively correlated with fulvic acid (FA) and total organic carbon (TOC). The work provides a potential cost effective and efficient framework for agricultural solid waste remediation and reduction.

Left ventricular remodeling and its association with mineral and bone disorder in kidney transplant recipients.

BACKGROUND: The assessment of left ventricular (LV) remodeling and its association with mineral and bone disorder (MBD) in kidney transplant recipients (KTRs) have not been systematically studied. We aimed to evaluate LV remodeling changes one year after kidney transplantation (KT) and identify their influencing factors. METHODS: Ninety-five KTRs (68 males; ages 40.2 ± 10.8 years) were followed before and one year after KT. Traditional risk factors and bone metabolism indicators were assessed. Left ventricular mass index (LVMI), left ventricular ejection fraction (LVEF) and left ventricular diastolic dysfunction (LVDD) were measured using two-dimensional transthoracic echocardiography. The relationship between MBD and LV remodeling and the factors influencing LV remodeling were analyzed. RESULTS: One year after KT, MBD was partially improved, mainly characterized by hypercalcemia, hypophosphatemia, hyperparathyroidism, 25-(OH) vitamin D deficiency, elevated bone turnover markers, and bone loss. LVMI, the prevalence of left ventricular hypertrophy (LVH), and the prevalence of LVDD decreased, while LVEF increased. LVH was positively associated with postoperative intact parathyroid hormone (iPTH) and iPTH nonnormalization. △LVMI was positively associated with preoperative type-I collagen N-terminal peptide and postoperative iPTH. LVEF was negatively associated with postoperative phosphorous. △LVEF was negatively associated with postoperative iPTH. LVDD was positively associated with postoperative lumbar spine osteoporosis. Preoperative LVMI was negatively associated with △LVMI and positively associated with △LVEF. Advanced age, increased BMI, diabetes, longer dialysis time, lower albumin level, and higher total cholesterol and low-density lipoprotein levels were associated with LV remodeling. CONCLUSIONS: LV remodeling partially improved after KT, showing a close relationship with MBD.

A retrospective study of mineral and bone disorder in kidney transplant recipients: Single-center experience.

BACKGROUND: The status of mineral and bone disorder (MBD) after kidney transplantation is not fully understood, and the assessment of abnormal mineral and bone metabolism in kidney transplant recipients (KTRs) has not been standardized. MATERIALS AND METHODS: We performed a retrospective analysis of 292 KTRs in our center. The levels of biochemical markers of bone metabolism and bone mineral density (BMD) were assessed. We evaluated the influencing factors of BMD using linear regression analysis. And correlation test was used for the correlation analysis between bone metabolism indicators and other indicators. RESULTS: Postoperative MBD mainly manifested as hypercalcemia (8.9%), hypophosphatemia (27.1%), low levels of 25-hydroxyvitamin D(25(OH)vitD) (67.0%), hyperparathyroidism (50.6%), and high levels of bone turnover markers (BTMs). The prevalence of osteopenia/osteoporosis in the femoral neck (FN) and lumbar spine (LS) was 20.1%/2.8% and 26.1%/3.6%, respectively. Multivariate analysis indicated that FN BMD was positively associated with body mass index (BMI) and negatively associated with acute rejection history (p < 0.05); while LS BMD was positively associated with BMI, and negatively associated with intact parathyroid hormone (iPTH) (p < 0.05). Biochemical markers of bone metabolism were affected by age, sex, preoperative dialysis mode and time, postoperative time, transplanted kidney function, and iPTH levels. LS BMD was negatively correlated with iPTH and BTMs (p < 0.05). CONCLUSION: MBD persisted after kidney transplantation. Decreased bone mass was associated with persistent hyperparathyroidism, acute rejection history, low BMI, advanced age, and menopause. Dynamic monitoring of bone metabolism index and BMD helps to assess MBD after kidney transplantation.

Construction of predictive model of interstitial fibrosis and tubular atrophy after kidney transplantation with machine learning algorithms.

Background: Interstitial fibrosis and tubular atrophy (IFTA) are the histopathological manifestations of chronic kidney disease (CKD) and one of the causes of long-term renal loss in transplanted kidneys. Necroptosis as a type of programmed death plays an important role in the development of IFTA, and in the late functional decline and even loss of grafts. In this study, 13 machine learning algorithms were used to construct IFTA diagnostic models based on necroptosis-related genes. Methods: We screened all 162 "kidney transplant"-related cohorts in the GEO database and obtained five data sets (training sets: GSE98320 and GSE76882, validation sets: GSE22459 and GSE53605, and survival set: GSE21374). The training set was constructed after removing batch effects of GSE98320 and GSE76882 by using the SVA package. The differentially expressed gene (DEG) analysis was used to identify necroptosis-related DEGs. A total of 13 machine learning algorithms-LASSO, Ridge, Enet, Stepglm, SVM, glmboost, LDA, plsRglm, random forest, GBM, XGBoost, Naive Bayes, and ANNs-were used to construct 114 IFTA diagnostic models, and the optimal models were screened by the AUC values. Post-transplantation patients were then grouped using consensus clustering, and the different subgroups were further explored using PCA, Kaplan-Meier (KM) survival analysis, functional enrichment analysis, CIBERSOFT, and single-sample Gene Set Enrichment Analysis. Results: A total of 55 necroptosis-related DEGs were identified by taking the intersection of the DEGs and necroptosis-related gene sets. Stepglm[both]+RF is the optimal model with an average AUC of 0.822. A total of four molecular subgroups of renal transplantation patients were obtained by clustering, and significant upregulation of fibrosis-related pathways and upregulation of immune response-related pathways were found in the C4 group, which had poor prognosis. Conclusion: Based on the combination of the 13 machine learning algorithms, we developed 114 IFTA classification models. Furthermore, we tested the top model using two independent data sets from GEO.

Vascular calcification progression and its association with mineral and bone disorder in kidney transplant recipients.

BACKGROUND: The assessment and prevention of vascular calcification (VC) in kidney transplant recipients (KTRs) have not been systematically studied. We aimed to evaluate VC change one year after kidney transplantation (KT) and identify their influencing factors. METHODS: 95 KTRs (68 males; ages 40.2 ± 10.8 years) were followed one year after KT. Changes in bone mineral density (BMD) and bone metabolism biomarkers were assessed. Coronary artery calcification (CAC) and thoracic aortic calcification (TAC) were measured using 192-slice third-generation dual-source CT. The relationship between bone metabolism indicators and VC and the factors influencing VC were analyzed. RESULTS: Postoperative estimated glomerular filtration rate was 79.96 ± 24.18 mL/min*1.73 m2. One year after KT, serum phosphorus, intact parathyroid hormone (iPTH), osteocalcin, type I collagen N-terminal peptide (NTx), type I collagen C-terminal peptide, and BMD decreased, 25-hydroxyvitamin D remained low, and VC increased. Post-CAC and TAC were negatively correlated with pre-femoral neck BMD, and TAC was positively correlated with post-calcium. CAC and TAC change were positively correlated with post-calcium and 25-hydroxyvitamin D. Increased CAC was positively associated with hemodialysis and pre-femoral neck osteopenia. CAC change was positively associated with prediabetes, post-calcium, and pre-CAC and negatively associated with preoperative and postoperative femoral neck BMD, and NTx change. Increased TAC was positively associated with age, prediabetes, preoperative parathyroid hyperplasia/nodule, post-calcium, and post-femoral neck osteopenia. TAC change was positively associated with age, diabetes, pre-triglyceride, pre-TAC, dialysis time, post-calcium and post-iPTH, and negatively associated with post-femoral neck BMD. CONCLUSIONS: Mineral and bone disorders persisted, and VC progressed after KT, showing a close relationship.

Association between fibrosis-related gene polymorphism and long-term allograft outcome in renal transplant recipients.

BACKGROUND: Renal allograft fibrosis is one of characteristic causes of long-term renal function loss. The purpose of our study is to investigate the association between fibrosis-related genes single nucleotide polymorphism (SNPs) and kidney function in 5 years after kidney transplantation. METHODS: A total of 143 recipients were eligible for screening with 5-year follow-up information and SNP sequencing information from blood samples were included in this study. Minor Allele Frequency (MAF) and Hardy-Weinberg Equilibrium (HWE) analysis was conducted to identify tagger single-nucleotide polymorphisms (SNPs) and haplotypes. SNPs associated with the fifth year chronic kidney disease (CKD) staging were screened by SPSS and the "SNPassoc" package in RStudio and used for subsequent prediction model construction. RESULTS: A total of 275 renal transplant-related SNPs identified after target sequencing analysis. 64 Tagger SNPs were selected, and two SNPs (rs13969 and rs243849) were statistically significant for stage of CKD in 5 years. Finally, a model based on Gender, Age, rs1396, and rs243849 was constructed by multivariate linear regression analysis. Additionally, this model has a good performance in predicting uremia five years after kidney transplantation. CONCLUSION: Two SNPs (rs13969 and rs243849) were identified to be significantly associated with long-term renal allograft function. Based on this, a prediction model for long-term allograft function was established containing Gender, Age, rs1396, and rs243849. However, an independent cohort should be enrolled to validate the predicting performance.

Efficacy of iguratimod on mineral and bone disorders after kidney transplantation: a preliminary study.

BACKGROUND: Iguratimod has been shown to promote bone formation and inhibit bone resorption in rheumatoid arthritis patients. We aimed to explore its effect on bone metabolism and vascular calcification (VC) in kidney transplant recipients (KTRs). METHODS: A post hoc analysis was conducted among the subjects in our previous randomized clinical trial (NCT02839941). Forty-three KTRs completing bone metabolism 52 weeks after enrollment were selected for this analysis, among whom 27 patients received VC examinations. In the iguratimod group, iguratimod (25 mg twice daily) was added adjuvant to the traditional triple regimen. At the 52-week follow-up, the following parameters were assessed: serum calcium, phosphorus, 25-hydroxyvitamin D, intact parathyroid hormone (iPTH), bone alkaline phosphatase (BALP), osteocalcin, type I collagen N-terminal peptide (NTx), type I collagen C-terminal peptide (CTx), bone mineral density (BMD) of the femoral neck and lumbar spine, coronary artery calcification (CAC) and thoracic aortic calcification (TAC). Bone metabolic and VC indices were compared between the two groups using the independent samples t test and Wilcoxon nonparametric test. RESULTS: At 52 weeks after enrollment, the iguratimod group had lower osteocalcin (p = 0.010), BALP (p = 0.015), NTx (p = 0.007), CTx (p = 0.012), CAC (p = 0.080) and TAC scores (p = 0.036) than the control group. There was no significant difference in serum calcium, phosphorus, 25-hydroxyvitamin D, iPTH and BMD between the groups. Iguratimod could reduce bone turnover markers (BTMs) at both high and low iPTH levels. The adverse effect of iguratimod was mild and tolerable. CONCLUSION: Iguratimod is safe, can reduce BTMs and may could attenuate VC in the first year after KT.

Mineral and bone disorder after kidney transplantation: a single-center cohort study.

BACKGROUND: The assessment and prevention of mineral and bone disorder (MBD) in kidney transplant recipients (KTRs) have not been standardized. This study aimed to evaluate MBD one year after kidney transplantation (KT) and identify the influencing factors of MBD. METHODS: A total of 95 KTRs in our center were enrolled. The changes in bone mineral density (BMD) and bone metabolism biochemical markers, including serum calcium (Ca), phosphorus(P), 25-hydroxyvitamin D(25(OH)vitD), intact parathyroid hormone (iPTH), bone alkaline phosphatase, osteocalcin (OC), type I collagen N-terminal peptide and type I collagen C-terminal peptide (CTx), over one year after KT were assessed. The possible influencing factors of BMD were analyzed. The relationships between bone metabolism biochemical markers were evaluated. The indicators between groups with or without iPTH normalization were also compared. RESULTS: MBD after KT was manifested as an increased prevalence of hypophosphatemia and bone loss, persistent 25(OH)vitD deficiency, and partially decreased PTH and bone turnover markers (BTMs). Femoral neck BMD was positively correlated with body mass index (BMI) and postoperative 25(OH)vitD, and negatively correlated with postoperative PTH. Lumbar spine BMD was positively correlated with BMI and preoperative TG, and negatively correlated with preoperative OC and CTx. BMD loss was positively associated with glucocorticoid accumulation. Preoperative and postoperative iPTH was negatively correlated with postoperative serum P and 25(OH)vitD, and positively correlated with postoperative Ca and BTMs. The recipients without iPTH normalization, who accounted for 41.0% of all KTRs, presented with higher Ca, lower P, higher BTMs, advanced age, and a higher prevalence of preoperative parathyroid hyperplasia. CONCLUSIONS: MBD persisted after KT, showing a close relationship with hyperparathyroidism, high bone turnover, and glucocorticoid accumulation.

Involvement of angiogenesis in cancer-associated acinar-to-ductal metaplasia lesion of pancreatic cancer invasive front.

PURPOSE: This study aimed to demonstrate the involvement of angiogenesis in cancer-associated acinar-to-ductal metaplasia (CA-ADM) lesion of invasive front pancreatic ductal adenocarcinoma (PDAC) and investigate the possible mechanism. METHODS: Tissue samples from 128 patients with PDAC and 36 LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre mice were analyzed. Immunohistochemical assay was performed using HE, anti-CK19 and anti-amylase to confirm the presence of CA-ADM lesions, using anti-CD34 and anti-CD31 to measure microvessel density (MVD), and using anti-CD68, anti-CD163, anti-iNOS, or anti-MMP9 to evaluate the immune microenvironment. We performed multiplex immunohistochemical assay to detect the co-expression of MMP9 and CD68 on macrophage. We examined clinical outcomes and other clinicopathological factors to determine the significance of high-level MVD of CA-ADM on survival and liver metastasis. We performed tube formation assay to evaluate the effect of macrophage on angiogenic capacity in vitro. RESULTS: Angiogenesis was significantly abundant in CA-ADM lesions compared with that in PDAC lesions in human and mouse tissues. High-level MVD in CA-ADM lesions was an independent predictor of poor prognosis (P = 0.0047) and the recurrence of liver metastasis (P = 0.0027). More CD68-positive and CD163-positive macrophages were detected in CA-ADM lesions than in PDAC. The percentage of CD68-positive macrophages was positively correlated with MVD in CA-ADM lesions. Multiplex-immunostaining revealed that MMP9 was expressed in CD68-positive macrophages of CA-ADM lesions. In CA-ADM lesions, the percentage of macrophages was positively correlated with MMP9 expression, which positively correlated with microvessel density. CONCLUSION: CA-ADM related angiogenesis is a promising predictive marker for poor prognosis of PDAC and may provide an attractive therapeutic target for PDAC.

Causal effects of human serum metabolites on occurrence and progress indicators of chronic kidney disease: a two-sample Mendelian randomization study.

Background: Chronic kidney disease (CKD) is often accompanied by alterations in the metabolic profile of the body, yet the causative role of these metabolic changes in the onset of CKD remains a subject of ongoing debate. This study investigates the causative links between metabolites and CKD by leveraging the results of genomewide association study (GWAS) from 486 blood metabolites, employing bulk two-sample Mendelian randomization (MR) analyses. Building on the metabolites that exhibit a causal relationship with CKD, we delve deeper using enrichment analysis to identify the metabolic pathways that may contribute to the development and progression of CKD. Methods: In conducting the Mendelian randomization analysis, we treated the GWAS data for 486 metabolic traits as exposure variables while using GWAS data for estimated glomerular filtration rate based on serum creatinine (eGFRcrea), microalbuminuria, and the urinary albumin-to-creatinine ratio (UACR) sourced from the CKDGen consortium as the outcome variables. Inverse-variance weighting (IVW) analysis was used to identify metabolites with a causal relationship to outcome. Using Bonferroni correction, metabolites with more robust causal relationships are screened. Additionally, the IVW-positive results were supplemented with the weighted median, MR-Egger, weighted mode, and simple mode. Furthermore, we performed sensitivity analyses using the Cochran Q test, MR-Egger intercept test, MR-PRESSO, and leave-one-out (LOO) test. Pathway enrichment analysis was conducted using two databases, KEGG and SMPDB, for eligible metabolites. Results: During the batch Mendelian randomization (MR) analyses, upon completion of the inverse-variance weighted (IVW) approach, sensitivity analysis, and directional consistency checks, 78 metabolites were found to meet the criteria. The following four metabolites satisfy Bonferroni correction: mannose, N-acetylornithine, glycine, and bilirubin (Z, Z), and mannose is causally related to all outcomes of CKD. By pathway enrichment analysis, we identified eight metabolic pathways that contribute to CKD occurrence and progression. Conclusion: Based on the present analysis, mannose met Bonferroni correction and had causal associations with CKD, eGFRcrea, microalbuminuria, and UACR. As a potential target for CKD diagnosis and treatment, mannose is believed to play an important role in the occurrence and development of CKD.

Iguratimod Attenuates Macrophage Polarization and Antibody-Mediated Rejection After Renal Transplant by Regulating KLF4.

Background: This study aimed to explore the effect and mechanism of iguratimod (IGT) on M1 macrophage polarization and antibody-mediated rejection (ABMR) after renal transplant. Methods: Bioinformatics analysis was performed using three public databases derived from the GEO database. Sprague-Dawley (SD) rats were pre-sensitized with donors of Wistar rats in skin transplantation and a rat renal transplant ABMR model was established from the donors to skin pre-sensitized recipients. Subsequently, IGT was treated on the ABMR model. Routine staining and immunofluorescence (IF) staining were performed to observe the pathological changes in each group and flow cytometry was performed to detect the changes of DSA titers in peripheral blood. In addition, bone-marrow-derived macrophage (BMDM) was extracted and interfered with IGT to explore the effect of IGT in vivo. PCR, IF staining, and Western blot were used to detect the expression of related genes and proteins. Results: Bioinformatics analysis revealed that several immune cells were significantly infiltrated in the ABMR allograft, while M1 macrophage was noticed with the most significance. Results of IF staining and PCR proved the findings of the bioinformatics analysis. Based on this, IGT was observed to significantly attenuate the degree of peritubular capillary vasculitis and arteriolitis in the rat renal transplant ABMR model, whereas it decreases the expression of C4d and reduces the titer of DSA. Results in vitro suggested that M1 macrophage-related transcripts and proteins were significantly reduced by the treatment of IGT in a dose- and time-dependent manner. Furthermore, IGT intervention could remarkably decrease the expression of KLF4. Conclusion: Polarization of M1 macrophages may aggravate ABMR after renal transplant by promoting DSA-mediated endothelial cell injury, and IGT may attenuate the pathogenesis of ABMR by targeting KLF4.

Efficacy and Safety of Iguratimod Supplement to the Standard Immunosuppressive Regimen in Highly Mismatched Renal Transplant Recipients: A Pilot Study.

Iguratimod (IGU) can mitigate the symptoms of rheumatoid arthritis through its anti-inflammatory effects. The objective of this study was to investigate the clinical efficacy and safety of IGU in highly HLA-mismatched renal transplant recipients, in combination with standard immunosuppressive regimen. This pilot study was designed as an open-label, blank-control, randomized clinical trial on patients recruited from a single transplant center in China. Patients who met the inclusion criteria were randomized to the IGU (n=27) and blank control (n=27) groups. IGU was administrated with the conventional triple immunosuppressive protocol for 52 weeks after kidney transplantation. The incidence of biopsy-proven acute rejection rate was 14.8% (4/27) in the IGU group and 29.6% (8/27) in the control group, P = 0.19. The clinical rejection rate was also substantially reduced in the IGU group (3.7% vs. 18.5%, P = 0.08). De novo donor-specific antibody also showed a decline trend in the IGU group after 52 weeks. The graft function and incidence of adverse events were similar between the two groups. In addition, IGU intervention significantly decreased the number of NK cells throughout the follow-up. In conclusion, our study has shown the possibility that IGU could reduce the allograft rejection rate and de novo DSA with appreciable safety in combination with conventional immunosuppressants. Formal clinical trials were warranted based on current findings.

Single Nucleotide Polymorphisms of IL-33 Gene Correlated with Renal Allograft Fibrosis in Kidney Transplant Recipients.

BACKGROUND: Nowadays, renal allograft survival is confined by the development of allograft fibrosis. Previous studies have reported interleukin-33 (IL-33) upregulated significantly in patients with chronic renal allograft dysfunction, and it could induce renal tubular epithelial to mesenchymal transition (EMT), which eventually contributed to renal allograft fibrosis. Our study intended to detect the underlying association between single nucleotide polymorphisms (SNPs) of IL-33 gene and renal allograft fibrosis in kidney transplant recipients. METHODS: We collected blood samples from 200 renal transplant recipients for the identification of SNPs and transplanted kidney tissue samples for identifying differentially expressed genes (DEGs). Intersection of SNP-related genes and DEGs was conducted for further analysis. Relationships between these SNPs and renal allograft fibrosis were evaluated by the inheritance models. Immunohistochemical (IHC) staining and western blotting (WB) were used to detect the expression of IL-33 and the markers of EMT in human kidney tissues obtained from control and chronic renal allograft dysfunction (CAD) patients. In vitro, we detected the progressions of EMT-related markers and the levels of MAPK signaling pathway mediators after transfecting IL-33 mutant plasmids in HK2 cells. RESULTS: Three intersected genes including IL-33 genes were significantly expressed. IL-33 expression was validated in kidney tissues by IHC and WB. Thirty-nine IL-33-related SNPs were identified in targeted sequencing, in which 26 tagger SNPs were found by linkage disequilibrium analysis for further analysis. General linear models indicated sirolimus administration significantly influenced renal allograft fibrosis (P < 0.05), adjustment of which was conducted in the following analysis. By multiple inheritance model analyses, SNP rs10975519 of IL-33 gene was found closely related to renal allograft fibrosis (P < 0.005). Furthermore, HK2 cells transfected with mutated plasmid of rs10975519 showed stronger mobility and migration ability. Moreover, IL-33 mutant plasmids could promote the IL-33-induced EMT through the sustained activation of p38 MAPK signaling pathway in HK2 cells. CONCLUSION: In our study, rs10975519 on the IL-33 gene was found to be statistically associated with the development of renal allograft fibrosis in kidney transplant recipients. This process may be related to the IL-33-induced EMT and sustained activation of p38 MAPK signaling pathway.

Impaired ATG16L-Dependent Autophagy Promotes Renal Interstitial Fibrosis in Chronic Renal Graft Dysfunction Through Inducing EndMT by NF-κB Signal Pathway.

Chronic renal graft dysfunction (CAD) is caused by multiple factors, including glomerular sclerosis, inflammation, interstitial fibrosis and tubular atrophy (IF/TA). However, the most prominent elements of CAD are IF/TA. Our studies have confirmed that endothelial-mesenchymal transition (EndMT) is an important source to allograft IF/TA. The characteristic of EndMT is the loss of endothelial marker and the acquisition of mesenchymal or fibroblastic phenotypes. Autophagy is an intracellular degradation pathway that is regulated by autophagy-related proteins and plays a vital role in many fibrotic conditions. However, whether or not autophagy contributes to fibrosis of renal allograft and how such mechanism occurs still remains unclear. Autophagy related 16 like gene (ATG16L) is a critical autophagy-related gene (ARG) necessary for autophagosome formation. Here, we first analyzed kidney transplant patient tissues from Gene Expression Omnibus (GEO) datasets and 60 transplant patients from our center. Recipients with stable kidney function were defined as non-CAD group and all patients in CAD group were histopathologically diagnosed with CAD. Results showed that ATG16L, as one significant differential ARG, was less expressed in CAD group compared to the non-CAD group. Furthermore, we found there were less autophagosomes and autolysosomes in transplanted kidneys of CAD patients, and downregulation of autophagy is a poor prognostic factor. In vitro, we found out that the knockdown of ATG16L enhanced the process of EndMT in human renal glomerular endothelial cells (HRGECs). In vivo, the changes of EndMT and autophagic flux were then detected in rat renal transplant models of CAD. We demonstrated the occurrence of EndMT, and indicated that abundance of ATG16L was accompanied by the dynamic autophagic flux change along different stages of kidney transplantation. Mechanistically, knockdown of ATG16L, specifically in endothelial cells, reduced of NF-κB degradation and excreted inflammatory cytokines (IL-1ß, IL-6 and TNF-α), which could facilitate EndMT. In conclusion, ATG16L-dependent autophagic flux causing by transplant showed progressive loss increase over time. Inflammatory cytokines from this process promoted EndMT, thereby leading to progression of CAD. ATG16L served as a negative regulator of EndMT and development of renal graft fibrosis, and autophagy can be explored as a potential therapeutic target for chronic renal graft dysfunction.

Combined Immunotherapy With Belatacept and BTLA Overexpression Attenuates Acute Rejection Following Kidney Transplantation.

Background: Costimulatory blockade provides new therapeutic opportunities for ensuring the long-term survival of kidney grafts. The adoption of the novel immunosuppressant Belatacept has been limited, partly due to concerns regarding higher rates and grades of acute rejection in clinical trials. In this study, we hypothesized that a combined therapy, Belatacept combined with BTLA overexpression, may effectively attenuate acute rejection after kidney transplantation. Materials and Methods: The rat kidney transplantation model was used to investigate graft rejection in single and combined therapy. Graft function was analyzed by detecting serum creatinine. Pathological staining was used to observe histological changes in grafts. The expression of T cells was observed by immunohistochemistry and flow cytometry. In vitro, we constructed an antigen-stimulated immune response by mixed lymphocyte culture, treated with or without Belatacept and BTLA-overexpression adenovirus, to observe the proliferation of receptor cells and the expression of cytokines. In addition, western blot and qRT-PCR analyses were performed to evaluate the expression of CTLA-4 and BTLA at various time points during the immune response. Results: In rat models, combined therapy reduced the serum creatinine levels and prolonged graft survival compared to single therapy and control groups. Mixed acute rejection was shown in the allogeneic group and inhibited by combination treatment. Belatacept reduced the production of DSA and the deposition of C4d in grafts. Belatacept combined with BTLA overexpression downregulated the secretion of IL-2 and IFN-γ, as well as increasing IL-4 and IL-10 expression. We also found that Belatacept combined with BTLA overexpression inhibited the proliferation of spleen lymphocytes. The duration of the elevated expression levels of CTLA-4 and BTLA differentially affected the immune response. Conclusion: Belatacept combined with BTLA overexpression attenuated acute rejection after kidney transplantation and prolonged kidney graft survival, which suggests a new approach for the optimization of early immunosuppression after kidney transplantation.

Association Between a TLR2 Gene Polymorphism (rs3804099) and Proteinuria in Kidney Transplantation Recipients.

Background: The occurrence of proteinuria is one of the evaluation indicators of transplanted kidney damage and becomes an independent risk factor for poor prognosis after kidney transplantation. Our research sought to understand these potential associations and detect the underlying impact of single-nucleotide polymorphisms (SNPs) on proteinuria in kidney transplant recipients. Materials and Methods: There were 200 recipients enrolled in this study, from which blood samples were extracted for SNP mutation-related gene detection. RNA sequencing was performed in kidney tissues after kidney transplantation, and the significantly differentially expressed genes (DEGs) were analyzed between the control group and the proteinuria group. Then, the intersection of genes with SNP mutations and DEGs was conducted to obtain the target genes. Multiple genetic models were used to investigate the relationship between SNPs and proteinuria. In addition, the effect of SNP mutation in the target gene was further validated in human renal podocytes. Results: According to the sequencing results, 26 significant SNP mutated genes and 532 DEGs were found associated with proteinuria after kidney transplantation. The intersection of SNP mutated genes and DEGs showed that the Toll-like receptor 2 (TLR2) gene was significantly increased in the transplanted renal tissues of patients with proteinuria after kidney transplantation, which was consistent with the results of immunohistochemical staining. Further inheritance model results confirmed that mutations at rs3804099 of the TLR2 gene had significant influence on the occurrence of proteinuria after kidney transplantation. In the in vitro validation, we found that, after the mutation of rs3804099 on the TLR2 gene, the protein expressions of podocalyxin and nephrin in podocytes were significantly decreased, while the protein expressions of desmin and apoptosis markers were significantly increased. The results of flow cytometry also showed that the mutation of rs3804099 on the TLR2 gene significantly increased the apoptotic rate of podocytes. Conclusion: Our study suggested that the mutation of rs3804099 on the TLR2 gene was significantly related to the generation of proteinuria after kidney transplantation. Our data provide insights into the prediction of proteinuria and may imply potential individualized therapy for patients after kidney transplantation.

Everolimus Alleviates Renal Allograft Interstitial Fibrosis by Inhibiting Epithelial-to-Mesenchymal Transition Not Only via Inducing Autophagy but Also via Stabilizing IκB-α.

Chronic allograft dysfunction (CAD) is the major cause of late graft loss in long-term renal transplantation. In our previous study, we found that epithelial-mesenchymal transition (EMT) is a significant event in the progression of renal allograft tubulointerstitial fibrosis, and impaired autophagic flux plays a critical role in renal allograft fibrosis. Everolimus (EVR) has been reported to be widely used to prevent the progression of organ fibrosis and graft rejection. However, the pharmacological mechanism of EVR in kidney transplantation remains to be determined. We used CAD rat model and the human kidney 2 (HK2) cell line treated with tumor necrosis factor-α (TNF-α) and EVR to examine the role of EVR on TNF-α-induced EMT and transplanted renal interstitial fibrosis. Here, we found that EVR could attenuate the progression of EMT and renal allograft interstitial fibrosis, and also activate autophagy in vivo. To explore the mechanism behind it, we detected the relationship among EVR, autophagy level, and TNF-α-induced EMT in HK2 cells. Our results showed that autophagy was upregulated upon mTOR pathway inhibition by EVR, which could significantly reduce expression of TNF-α-induced EMT. However, the inhibition of EVR on TNF-α-induced EMT was partly reversed following the addition of autophagy inhibitor chloroquine. In addition, we found that TNF-α activated EMT through protein kinase B (Akt) as well as nuclear factor kappa B (NF-κB) pathway according to the RNA sequencing, and EVR's effect on the EMT was only associated with IκB-α stabilization instead of the Akt pathway. Together, our findings suggest that EVR may retard impaired autophagic flux and block NF-κB pathway activation, and thereby prevent progression of TNF-α-induced EMT and renal allograft interstitial fibrosis.

Bortezomib limits renal allograft interstitial fibrosis by inhibiting NF-κB/TNF-α/Akt/mTOR/P70S6K/Smurf2 pathway via IκBα protein stabilization.

Chronic allograft dysfunction is a major cause of late graft failure after kidney transplantation. One of the histological changes is interstitial fibrosis, which is associated with epithelial-mesenchymal transition. Bortezomib has been reported to prevent the progression of fibrosis in organs. We used rat renal transplantation model and human kidney 2 cell line treated with tumor necrosis factor-α (TNF-α) to examine their response to bortezomib. To explore the mechanism behind it, we assessed the previously studied TNF-α/protein kinase B (Akt)/Smad ubiquitin regulatory factor 2 (Smurf2) signaling and performed RNA sequencing. Our results suggested that bortezomib could attenuate the TNF-α-induced epithelial-mesenchymal transition and renal allograft interstitial fibrosis in vitro and in vivo. In addition to blocking Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase/Smurf2 signaling, bortezomib's effect on the epithelial-mesenchymal transition was associated with inhibition of nuclear factor kappa B (NF-κB) pathway by stabilizing inhibitor of NF-κB. The study highlighted the therapeutic potential of bortezomib on renal allograft interstitial fibrosis. Such an effect may result from inhibition of NF-κB/TNF-α/Akt/mTOR/p70S6 kinase/Smurf2 signaling via stabilizing protein of inhibitor of NF-κB.