NSD2 modulates Drp1-mediated mitochondrial fission in chronic renal allograft interstitial fibrosis by methylating STAT1.

Renal interstitial fibrosis/tubular atrophy (IF/TA) is a prominent pathological feature of chronic allograft dysfunction (CAD). Our previous study has demonstrated that epithelial-mesenchymal transition (EMT) plays a significant role in shaping the development of IF/TA. Nuclear SET domain (NSD2), a histone methyltransferase catalyzing methylation at lysine 36 of histone 3, is crucially involved in the development and progression of solid tumors. But its role in the development of renal allograft interstitial fibrosis has yet to be elucidated. Here, we characterize NSD2 as a crucial mediator in the mouse renal transplantation model in vivo and a model of tumor necrosis factor-α (TNF-α) stimulated-human renal tubular epithelial cells (HK-2) in vitro. Functionally, NSD2 knockdown inhibits EMT, dynamin-related protein 1 (Drp1)-mediated mitochondrial fission in mice. Conversely, NSD2 overexpression exacerbates fibrosis-associated phenotypes and mitochondrial fission in tubular cells. Mechanistically, tubular NSD2 aggravated the Drp-1 mediated mitochondrial fission via STAT1/ERK/PI3K/Akt signaling pathway in TNF-α-induced epithelial cell models. Momentously, mass spectrometry (MS) Analysis and site-directed mutagenesis assays revealed that NSD2 interacted with and induced Mono-methylation of STAT1 on K173, leading to its phosphorylation, IMB1-dependent nuclear translocation and subsequent influence on TNF-α-induced EMT and mitochondrial fission in NSD2-dependent manner. Collectively, these findings shed light on the mechanisms and suggest that targeting NSD2 could be a promising therapeutic approach to enhance tubular cell survival and alleviate interstitial fibrosis in renal allografts during CAD.

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.

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.

Macrophage polarization induces endothelium-to-myofibroblast transition in chronic allograft dysfunction.

Our research explores the role of M1 macrophage polarization in endothelium-to-myofibroblast transition (EndMT) and chronic allograft dysfunction (CAD). GSE21374 transcriptome sequencing data were obtained. Transplanted nephrectomy specimens from CAD patients were collected and studied to explore the infiltration of M1 and M2 macrophages using immunofluorescence, PCR, and Western blotting (WB). A co-culture model of M1 macrophages, polarized from mouse bone marrow-derived macrophages (BMDM) or Raw264.7, and aortic endothelial cells was established, and EndMT was tested using PCR and WB. RNA-sequencing was performed on the macrophages from the mouse BMDM. The TNF-α secreted from the polarized M1 macrophages was verified using ELISA. Based on the GEO public database, it was observed that macrophages were significantly infiltrated in CAD allograft tissues, with CD68(+) iNOS(+) M1 macrophages significantly infiltrating the glomeruli of allograft tissues, and CD68(+)CD206(+) M2 macrophages notably infiltrating the allograft interstitial area. The mRNA expression of the M1 macrophage marker inducible nitric oxide synthase (iNOS) was significantly increased (p < 0.05) and M1 macrophages were found to significantly promote the EndMT process in vitro. RNA-Sequencing analysis revealed that TNF signaling could be involved in the EndMT induced by M1 macrophages, and in vitro studies confirmed that TNF-α in the supernatant was significantly higher. The renal allograft tissues of CAD patients were found to be significantly infiltrated by M1 macrophages and could promote the progression of CAD by secreting the cytokine TNF-α to induce EndMT in endothelial cells.

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.

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.

Iguratimod reduces panel reactive antibody in high mismatched renal transplant recipients: One single-center experience.

OBJECTIVE: To evaluate the clinical efficacy and safety of iguratimod (IGU) for reducing panel reactive antibody (PRA) in high-mismatched renal transplant recipients. METHODS: Eligible recipients positive for PRAs who received or did not receive IGU treatment were enrolled. We retrospectively reviewed, collected, and analyzed statistically the clinical data of the recipients. RESULTS: A total of 80 recipients were included for further analysis. After IGU was administered for 9 months, no significant difference was found in the change rates of donor specific antibodies between two groups. Meanwhile, the reduction in the PRAs in the IGU group was greater than that in the non-IGU group in anti-human leukocyte antigen (HLA) class I and class II, anti-HLA class I, anti-HLA class II, anti-HLA A, and anti-HLA DR antibodies. However, no differences were found in the anti-HLA B, anti-HLA Cw, anti-HLA DP, and anti-HLA DQ antibodies between the two groups. No serious adverse events were reported, and the incidence of adverse events was comparable between the two groups. CONCLUSION: PRA levels in high-mismatched renal transplant recipients were significantly reduced after the administration of IGU. The high safety of IGU was also determined.

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.

Acute pancreatitis due to tacrolimus in kidney transplant and review of the literature.

WHAT IS KNOWN AND OBJECTIVE: The purpose of this case report is to increase the awareness of tacrolimus-induced acute pancreatitis in renal transplantation patients. CASE SUMMARY: We present a case of tacrolimus-induced acute pancreatitis with positive rechallenge. The 24-year-old male patient underwent kidney transplant and received immunosuppressive therapy with tacrolimus. On day 10 post-transplant, he presented with abdominal pain. A laboratory analysis showed elevated serum amylase and serum lipase levels. An abdominal computed tomography scan showed large-volume ascites and pelvic cavity effusion. These findings led to a diagnosis of acute pancreatitis. After tacrolimus was temporarily stopped and altered with cyclosporine, his symptoms decreased and he was restarted with tacrolimus. On day 61, laboratory tests again revealed significant elevations of serum amylase and serum lipase. A computed tomography scan of the abdomen showed increased pancreatic tail fluid collections. We excluded other possible causes and concluded that tacrolimus was the definite inducer of pancreatitis. The patient was switched from tacrolimus to cyclosporine again. Serum amylase and serum lipase were gradually decreased to normal, and he was discharged home with no relapse. WHAT IS NEW AND CONCLUSION: With the consideration of the wide use of tacrolimus, it is important that healthcare providers are aware of tacrolimus-induced acute pancreatitis. Future studies are needed to confirm and quantify the risk of tacrolimus-induced acute pancreatitis.

Bortezomib attenuates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF-α-Smurf1-Akt-mTOR-P70S6K pathway.

Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial-mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK-2) treated with tumour necrosis factor-alpha (TNF-α) in various doses or at various time points by Western Blotting or qRT-PCR. We knockdown or overexpressed Smurf1 in HK-2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF-α significantly promoted the development of EMT in a time-dependent and dose-dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF-α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF-α. Bortezomib can attenuate the Sumrf1-mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.

Impact of single nucleotide polymorphisms on P450 oxidoreductase and peroxisome proliferator-activated receptor alpha on tacrolimus pharmacokinetics in renal transplant recipients.

The P450 oxidoreductase (POR) and peroxisome proliferator-activated receptor alpha (PPARA) genes are associated with the activity of cytochrome P450 enzymes in vivo. We aimed to investigate the impact of single nucleotide polymorphisms (SNPs) in the POR and PPARA genes on the pharmacokinetics of tacrolimus (TAC) in renal transplant recipients. A total of 220 recipients were assessed and 105 recipients were included for final quantitative analysis. Blood samples were collected and DNA was extracted. Targeting sequencing based on next-generation sequencing was applied to detect the SNPs in the POR and PPARA genes. In addition, a systematic review and meta-analysis was performed to comprehensively evaluate the influence of POR and PPARA mutations on the TAC concentrations. A total of 81 SNPs were obtained. Three SNPs (POR*28, Chr7:75619677 and Chr7:75614288) were found to be significantly associated with the TAC pharmacokinetics at 3 months, 6 months, and more than 12 months. No significant association was observed in the combined effect analysis of CYP3A4*1G and CYP3A5*3 with three significant SNPs in the POR gene. Age, post-transplant duration, and the use of sirolimus were identified as the most important factors that influenced the TAC concentrations. A meta-analysis of four studies results and our cohort indicated that compared with recipients carrying the CT or TT genotypes, recipients carrying the CC genotypes of POR*28 showed significantly higher TAC concentrations. Our study suggested the positive influence of mutations in the POR gene on TAC exposure at 3 months after kidney transplantation.

A Single-Nucleotide Polymorphism (rs1131243) of the Transforming Growth Factor Beta Signaling Pathway Contributes to Risk of Acute Rejection in Chinese Renal Transplant Recipients.

BACKGROUND Acute rejection (AR) is a common complication of kidney transplantation. The transforming growth factor beta (TGF-ß) signaling pathway has been observed to be involved in several cellular functions. Our study aimed to investigate the correlations between single-nucleotide polymorphisms (SNPs) in TGF-ß-related genes and the risk of AR in renal transplant recipients. MATERIAL AND METHODS This retrospective, single-center study included 200 Chinese renal transplant recipients. All exons, exon/intron boundaries, and flanking regions of the TGF-ß signaling pathway were detected by targeting sequencing (TS) based on next-generation sequencing technology. Tagger SNPs and haplotypes were identified after adjustment. A general linear model (GLM) was used to explore the confounding effect of clinical variables. Five adjusted inheritance models were utilized to investigate the influence of SNPs on AR, and Banff score was applied to evaluate the effect of related SNPs on pathological changes. RESULTS A total of 188 SNPs on TGF-ß genes were detected. Analysis of adjustment led to identification of 31 tagger SNPs and 10 haplotype blocks. After the analysis of a general linear model and 5 sirolimus-adjusted multiple inheritance models, 1 of the SNPs - rs1131243 on the TGF-ßR3 gene - was observed to be significantly associated with the occurrence of AR. Based on Banff score, no significant association was observed between SNPs and pathological changes. CONCLUSIONS In this study, we observed that the SNP rs1131243 on the TGF-ßR3 gene was significantly associated with the occurrence of AR in Chinese renal transplant recipients.

Polymorphisms of nucleotide factor of activated T cells cytoplasmic 2 and 4 and the risk of acute rejection following kidney transplantation.

BACKGROUND: Acute rejection (AR) is a common complication of kidney transplantation. Nuclear factors of activated T cells (NFATs) are transcription factors involved in the activation of T lymphocytes, but their association with AR is unclear. METHODS: This retrospective, case-control study included 200 renal transplant recipients who were divided into the AR group (n = 69) and stable group (n = 131). Their blood samples were collected, and DNA was extracted from the whole blood. High-throughput next-generation sequencing was used to identify single nucleotide polymorphisms (SNPs) of the NFATC2 and NFATC4 genes. The correlation of these SNPs with AR was determined by logistic analysis. RESULTS: Seventy-one SNPs of the NFATC2 and NFATC4 genes were identified by the sequencing and Hardy-Weinberg equilibrium analyses. After adjusting for age, gender and immunosuppressive protocols, 27 SNPs were correlated with AR, of which the SNP rs2426295 of the NFATC2 gene showed a significant correlation with AR in the HET model (AA vs. AC: OR = 0.43, 95% CI = 0.19-0.98, P = 0.045), but no significant NFATC4 SNPs were identified. CONCLUSIONS: Our study shows that the rs2426295 variant of the NFATC2 gene is significantly associated with the occurrence of AR following kidney transplantation. And patients with AA genotypes in rs2426295 are inclined to suffer from AR pathogenesis.

Role of B and T Lymphocyte Attenuator in Renal Transplant Recipients with Biopsy-Proven Acute Rejection.

BACKGROUND Acute rejection is a common predisposing cause of allograft dysfunction in kidney transplantation. Recently, the B and T lymphocyte attenuator (BTLA)/herpes virus entry mediator (HVEM)/lymphotoxin (LIGHT)/CD160 pathway was found to be potentially involved in the regulation of T cell activation. This could mean that this pathway is involved in graft rejection in kidney transplantation; the present study aimed to explore this possibility. MATERIAL AND METHODS The expression of BTLA, HVEM, LIGHT and CD160 on peripheral CD4+, CD8+ and CD19+ lymphocytes were analyzed by flow cytometry in recipients with biopsy-proven acute rejection (BPAR) or stable allograft function, as well as in healthy volunteers. Moreover, we performed HE staining and immunohistochemical staining to assess the expression of BTLA and HVEM in kidney samples from recipients with BPAR and patients who underwent the surgery of radical nephrectomy. RESULTS We observed the significantly lower expression of BTLA on CD4+ T cells in recipients from the BPAR group than in recipients from the stable group. The expression of BTLA on CD8+ T cells among recipients both from the BPAR and stable group was statistically increased than that in the healthy volunteers. A significant difference in the expression of CD160 in the stable group was found when compared with the BPAR group or control group. Moreover, there was no significance in the expression of HVEM, LIGHT or CD160 on other subtypes of T cells between the 3 groups or in the expression of BTLA on CD4+ T cells between the BPAR and control group. CONCLUSIONS The findings indicate that the BTLA/HVEM pathway does be involved in pathogenesis of acute rejection following kidney transplantation, as well as the induction of transplant tolerance. This pathway may therefore be a useful target for therapy against acute rejection after kidney transplantation.

Role of endothelial-to-mesenchymal transition induced by TGF-ß1 in transplant kidney interstitial fibrosis.

Chronic allograft dysfunction (CAD) induced by kidney interstitial fibrosis is the main cause of allograft failure in kidney transplantation. Endothelial-to-mesenchymal transition (EndMT) may play an important role in kidney fibrosis. We, therefore, undertook this study to characterize the functions and potential mechanism of EndMT in transplant kidney interstitial fibrosis. Proteins and mRNAs associated with EndMT were examined in human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor-beta1 (TGF-ß1) at different doses or at different intervals with western blotting, qRT-PCR and ELISA assays. Cell motility and migration were evaluated with motility and migration assays. The mechanism of EndMT induced by TGF-ß1 was determined by western blotting analysis of factors involved in various canonical and non-canonical pathways. In addition, human kidney tissues from control and CAD group were also examined for these proteins by HE, Masson's trichrome, immunohistochemical, indirect immunofluorescence double staining and western blotting assays. TGF-ß1 significantly promoted the development of EndMT in a time-dependent and dose-dependent manner and promoted the motility and migration ability of HUVECs. The TGF-ß/Smad and Akt/mTOR/p70S6K signalling pathways were found to be associated with the pathogenesis of EndMT induced by TGF-ß1, which was also proven in vivo by the analysis of specimens from the control and CAD groups. EndMT may promote transplant kidney interstitial fibrosis by targetting the TGF-ß/Smad and Akt/mTOR/p70S6K signalling pathways, and hence, result in the development of CAD in kidney transplant recipients.

MiR-122 promotes renal cancer cell proliferation by targeting Sprouty2.

MicroRNAs are short non-coding RNAs, which have been implicated in several biological processes. Aberrant expression of the microRNA miR-122 has frequently been reported in malignant cancers. However, the mechanism underlying the effects of miR-122 in renal cell carcinoma remains unknown. The aim of this study was to determine the biological function of miR-122 in renal cell carcinoma and to identify a novel molecular target regulated by miR-122. We measured the expression levels of Sprouty2 in six renal cell carcinoma tissue samples and adjacent non-tumor tissues by western blot analysis. We then used reverse transcription polymerase chain reaction to measure miR-122 levels in 40 primary renal cell carcinoma and adjacent non-malignant tissue samples. The effects of miR-122 down-regulation or Sprouty2 knockdown were evaluated via Cell Counting Kit-8 assay, flow cytometry, and western blot analysis. The relationship between miR-122 and Sprouty2 was determined using dual-luciferase reporter assays. Sprouty2 was down-regulated in renal cell carcinoma tissue samples compared with adjacent normal tissue. In contrast, miR-122 was up-regulated in primary renal cell carcinoma tissue samples compared with adjacent normal tissue samples. Down-regulation of miR-122 substantially weakened the proliferative ability of renal cell carcinoma cell lines in vitro. In contrast, Sprouty2 knockdown promoted the in vitro proliferation of renal cell carcinoma cell lines. The spry2 gene could therefore be a direct target of miR-122. In conclusion, miR-122 could act as a tumor promoter and potentially target Sprouty2. MiR-122 promotes renal cell carcinoma cell proliferation, migration, and invasion and could be a molecular target in novel therapies for renal cell carcinoma.

Proton Nuclear Magnetic Resonance (¹H-NMR)-Based Metabolomic Evaluation of Human Renal Allografts from Donations After Circulatory Death.

BACKGROUND Delayed graft function (DGF) is a common complication that impairs allograft function after kidney transplantation. However, the mechanism of DGF remains unclear. Nuclear magnetic resonance (NMR)-based analysis has been widely used in recent times to assess changes in metabolite levels. MATERIAL AND METHODS Samples of perfusate from allografts donated after circulatory death were collected prior to transplantation, during static cold storage. ¹H-NMR-based metabolomics combined with the statistical methods, orthogonal partial least-squares discriminant analysis (OPLS-DA), and principle-component analysis (PCA), were employed to test different levels of metabolites between the allografts that exhibited DGF and those that exhibited immediate graft function (IGF). RESULTS The study population consisted of 36 subjects, 11 with DGF and 25 with IGF. Of the 37 detected and identified metabolites, a-glucose and citrate were significantly elevated in the perfusate of DGF allografts, and taurine and betaine were significantly decreased. CONCLUSIONS ¹H-NMR analysis of DGF and IGF perfusates revealed some significant differences in their metabolite profiles, which may help explain the mechanisms of kidney ischemia-reperfusion injury and DGF.

Application of Ultrasound Elastography for Chronic Allograft Dysfunction in Kidney Transplantation.

Interstitial fibrosis is the main characteristic of chronic allograft dysfunction, which remains the key factor affecting long-term allograft survival after kidney transplantation. Ultrasound elastography (UE), including real-time elastography, transient elastography, and acoustic radiation force impulse, has been applied widely in breast, thyroid, and liver diseases, especially in the assessment of liver fibrosis. Recently, numerous studies have reported the efficacy of UE methods in evaluating renal allograft fibrosis. This review aims to investigate the clinical applications, limitations, and future roles of UE in current clinical practice in light of changing management paradigms. In current clinical practice, UE methods, especially transient elastographic measurement, appear to be useful for ruling out fibrosis but do not have sufficient accuracy to distinguish between various stages of allograft fibrosis. Moreover, there remain considerable issues to be solved for the application of UE in kidney transplantation. Thus, UE methods cannot replace the crucial role of renal allograft biopsy in the diagnosis and evaluation of allograft fibrosis in kidney transplantation. Perhaps UE methods could be of more importance in the long-term observation and evaluation of allograft fibrosis during follow-up.

Advanced glycation end products accelerate arteriosclerosis after renal transplantation through the AGE/RAGE/ILK pathway.

BACKGROUND: The effects of advanced glycation end products (AGEs) on arteriosclerosis (AS) after kidney transplantation and the molecular mechanisms involved remain unclear. METHODS: Samples were collected from 30 healthy volunteers and 30 renal transplant recipients (RTRs) to determine the levels of AGEs and to observe both histological changes and α-smooth muscle actin (α-SMA) and osteopontin (OPN) expression. Furthermore, we analyzed α-SMA, OPN and integrin-linked kinase (ILK) in rat vascular smooth muscle cells (VSMCs) that were treated with AGEs and in ILK plasmid transfected rat VSMCs treated with AGEs. Finally, we measured the expression of ILK and the receptor for advanced glycation end (RAGE) products in rat VSMCs treated with AGEs and an anti-RAGE antibody. RESULTS: Significant differences in the histological changes, serum AGEs, and expression of α-SMA and OPN in arterial walls were noted between healthy volunteers and RTRs. Significant OPN and ILK overexpression and reduced α-SMA expression were detected in a time-dependent manner in rat VSMCs after treatment with AGEs. Similar outcomes were observed regarding the overexpression of ILK, and these results could be prevented via RAGE inhibition. CONCLUSIONS: AGEs may play a critical role in the formation and progression of AS after renal transplantation by inducing VSMCs-to-osteoblast trans-differentiation through the AGE/RAGE/ILK pathway.