Exploration of the mechanism of NORAD activation of TGF-ß1/Smad3 through miR-136-5p and promotion of tacrolimus-induced renal fibrosis.

BACKGROUND: Tacrolimus is a potent immunosuppressant, but has various side effects, with nephrotoxicity being the most common. Renal fibrosis is an important process of tacrolimus nephrotoxicity. Therefore, it is important to identify the factors that contribute to renal fibrosis after tacrolimus nephrotoxicity, and control its development. METHODS: The present study aims to determine whether tacrolimus may speed up the course of renal fibrosis by upregulating noncoding RNA activated by DNA damage (NORAD) to compete with miR-136-5p, and activating the TGF-ß1/Smad3 pathway. Furthermore, in vivo rat models and in vitro cell models were established. Then, the expression levels of NORAD and miR-136-5p were determined by RT-qPCR, while the expression of the TGF-ß1/Smad3 pathway was determined by western blot and RT-qPCR. In order to investigate the interaction between NORAD and miR-136-5p, as well as miR-136-5p and SYK, two luciferase reporters were employed. The renal fibrosis of mice was observed using Masson and PAS staining. The expression of inflammatory factors IL-1, IL-6, MCP-1 and TNF-α was detected by ELISA. RESULTS: In the in vitro experiments, NORAD was upregulated, while miR-136-5p was downregulated after tacrolimus induction. The expression of the TGF-ß1/Smad3 pathway correspondingly changed after the induction by tacrolimus. In the in vivo experiments, the expression of NORAD and miR-136-5p, and the trend for renal fibrosis were consistent with the results in the in vitro experiments. Furthermore, the inflammatory factors correspondingly changed with the severity of renal fibrosis. Moreover, the expression trend of the TGF-ß1/Smad3 pathway in tacrolimus-induced rats was consistent with that in the in vitro experiments. CONCLUSION: Through in vitro and in vivo experiments, the present study was able to successfully prove that tacrolimus upregulates NORAD to compete with miR-136-5p, resulting in a decrease in miR-136-5p expression, which in turn activates the TGF-ß1/smad3 pathway, and finally induces the aggravation of renal fibrosis.

Over-expressed microRNA-181a reduces glomerular sclerosis and renal tubular epithelial injury in rats with chronic kidney disease via down-regulation of the TLR/NF-κB pathway by binding to CRY1.

BACKGROUND: MicroRNAs (miRNAs) contribute to the progression of chronic kidney disease (CKD) by regulating renal homeostasis. This study explored the effects of miR-181a on CKD through the Toll-like receptor (TLR)/nuclear factor-kappa B (NF-κB) pathway by binding to CRY1. METHODS: Seventy male rats were selected and assigned into specific groups: miR-181a mimic, miR-181a inhibitor, and siRNA against CRY1, with each group undergoing different treatments to investigate many different outcomes. First, 24-h urinary protein was measured. ELISA was used to determine the serum levels of SOD, ROS, MDA, IL-1ß, IL-6, and TNF-α. Biochemical tests for renal function were performed to measure albumin, uric acid, and urea in urine and urea nitrogen and creatinine in serum. The glomerulosclerosis index (GSI) and renal tubular epithelial (RTE) cell apoptosis were detected using PASM staining and TUNEL staining, respectively. Finally, RT-qPCR and western blot were done to determine miR-181a, CRY1, TLR2, TLR4, and NF-κB expression. RESULTS: CRY1 is the target gene of miR-181a, according to a target prediction program and luciferase assay. Rats diagnosed with CKD presented increases in 24-h urinary protein; GSI; RTE cell apoptosis rate; serum ROS, MDA, IL-1ß, IL-6, and TNF-α; and CRY1, TLR2, TLR4, and NF-κB expression, as well as decreases in SOD level and miR-181a expression. Following transfection with either the miR-181a mimic or si-CRY1, 24-h urinary protein, renal damage, GSI, and cell apoptosis rate were all decreased. In addition, the overexpression of miR-181a or inhibition of CRY1 alleviated the degree of kidney injury through suppression of the TLR/NF-κB pathway. CONCLUSION: miR-181a alleviates both GS and RTE injury in CKD via the down-regulation of the CRY1 gene and the TLR/NF-κB pathway.

CD38 Drives Progress of Osteoarthritis by Affecting Cartilage Homeostasis.

OBJECTIVE: To observe expression of CD38, a key modulator of nicotinamide dinucleotide (NAD+) metabolism in mice with knee osteoarthritis, and protective effect of CD38 inhibition during the osteoarthritis (OA) development. METHOD: The destabilization of the medial meniscus (DMM) model was performed in mice to mimic the process of OA. Immunofluorescence of CD38 was performed to evaluate its response during the OA process. Limb bud-derived mesenchymal cells were isolated for micromass culture. 100 nM or 1 µM CD38 inhibitor (78c) treatment for 14 days and CD38 sgRNA infection were then used to explore the effects of chondrogenic differentiation via Alcian blue staining. The expressions of chondrogenic markers were detected using RT-PCR and Western blot. To explore the protective effect of CD38 inhibitor on cartilage degradation during OA in vivo, a CD38 inhibitor was injected into the knee joint after DMM operations. Micro-CT analysis and Safranin O-fast green staining were used to evaluate subchondral bone micro-architecture changes and cartilage degeneration. RESULTS: Compared to the control group, the CD38 expression in superficial cartilage was obviously increased in DMM group (P < 0.05). During the normal chondrogenic differentiation, the extracellular matrix formed and expression of Sox9, Col2, aggrecan increased apparently while CD38 expression decreased, which could be reversed with ablation of CD38 in limb bud-derived mesenchymal cells. Consistent with findings in vitro, CD38 blockage via CD38 inhibitor injection protected against osteosclerosis in medial subchondral bone and cartilage degeneration in DMM-induced experimental mice. Compared to the Sham group, DMM mice showed significantly increased values of BV and BV/TV in subchondral bone (P < 0.05) and Mankin score, which could be rescued by 78c treatment (P < 0.05). Also the CD38 inhibitor contributed to homeostasis of anabolism and catabolism by upregulating Sox9, Col2, aggrecan and downregulating Runx2, Col10 and Mmp13. CONCLUSION: This study primarily implicates CD38 as an important regulator of chondrogenic differentiation. Inhibition of CD38 demonstrated protection against cartilage degeneration, which suggests that CD38 could be a potential therapeutic target for OA.

Donor BMSC-derived small extracellular vesicles relieve acute rejection post-renal allograft through transmitting Loc108349490 to dendritic cells.

Bone marrow-derived mesenchymal stem cell (BMSC)-derived small extracellular vesicles (sEVs) are potent candidates for the suppression of acute rejection post-renal allograft and have been reported to halt dendritic cells (DCs) maturation. However, whether BMSC-derived sEVs mitigate acute rejection post-renal allograft by targeting DCs is still unclear. In this study, donor BMSC-derived sEVs (sEVs) relieved the inflammatory response and suppressed mature DCs (mDCs) location in kidney grafts, and increased regulatory T (Treg) cell population in the spleens of the rats that underwent kidney allograft. In lipopolysaccharide (LPS)-stimulated immature DCs (imDCs), sEVs suppressed the maturation and migration of DCs and inactivated toll-like receptor 4 (TLR4) signaling. Compared with LPS-treated imDCs, imDCs treated with LPS+sEVs promoted CD4+ T cells differentiated toward Treg cells. Subsequently, we found that Loc108349490, a long non-coding RNA (lncRNA) abundant in sEVs, mediated the inhibitory effect of sEVs on DC maturation and migration by promoting TLR4 ubiquitination. In rats that underwent an allograft, Loc108349490 deficiency weakened the therapeutic effect of sEVs on acute rejection. The present study firstly found that sEVs alleviated acute rejection post-renal allograft by transferring lncRNA to DCs and screened out the functional lncRNA loaded in sEVs was Loc108349490.

Incorporation of Gene-Environment Interaction Terms Improved the Predictive Accuracy of Tacrolimus Stable Dose Algorithms in Chinese Adult Renal Transplant Recipients.

The narrow therapeutic window of tacrolimus necessitates daily monitoring and predictive algorithms based on genetic and nongenetic factors. In this study, we constructed predictive algorithms for tacrolimus stable dose in a retrospective cohort of 1045 Chinese renal transplant recipients. All patients were genotyped for CYP3A4 20230T>C (rs2242480), CYP3A4 T>C (rs4646437), CYP3A5*3 6898A>G (rs776746), ABCB1 129T>C (rs3213619); ABCB1 c.1236C>T (rs01128503), ABCB1 c.2677G>T/A (rs2032582) and ABCB1 c.3435C>T (rs1045642) polymorphisms, and the effects of gene-gene and gene-environment interactions on the predictive accuracy of algorithm were evaluated. In wild-type CYP3A4 rs2242480 (TT) carriers, patients who took calcium channel blockers had lower tacrolimus stable doses than those without the concomitant medications (P < 1 × 10-4 ). In contrast, there was no significant difference in mutant type patients. Similarly, the tacrolimus stable doses in wild-type CYP3A5 rs776746 carriers who had hypertension were higher than those without hypertension (P = 4.10 × 10-3 ). More importantly, dose-predictive algorithms with interaction terms showed higher accuracy and better performance than those without interaction terms. Our finding suggested that wild-type CYP3A4 rs2242480 (TT) carriers should be more cautious to take tacrolimus when they are coadministrated with calcium channel blockers, and CYP3A5 rs776746 (AA) carriers may need higher tacrolimus dosage when they are in combination with hypertension.

Corrigendum: Application of Machine-Learning Models to Predict Tacrolimus Stable Dose in Renal Transplant Recipients.

This corrects the article DOI: 10.1038/srep42192.

Application of Machine-Learning Models to Predict Tacrolimus Stable Dose in Renal Transplant Recipients.

Tacrolimus has a narrow therapeutic window and considerable variability in clinical use. Our goal was to compare the performance of multiple linear regression (MLR) and eight machine learning techniques in pharmacogenetic algorithm-based prediction of tacrolimus stable dose (TSD) in a large Chinese cohort. A total of 1,045 renal transplant patients were recruited, 80% of which were randomly selected as the "derivation cohort" to develop dose-prediction algorithm, while the remaining 20% constituted the "validation cohort" to test the final selected algorithm. MLR, artificial neural network (ANN), regression tree (RT), multivariate adaptive regression splines (MARS), boosted regression tree (BRT), support vector regression (SVR), random forest regression (RFR), lasso regression (LAR) and Bayesian additive regression trees (BART) were applied and their performances were compared in this work. Among all the machine learning models, RT performed best in both derivation [0.71 (0.67-0.76)] and validation cohorts [0.73 (0.63-0.82)]. In addition, the ideal rate of RT was 4% higher than that of MLR. To our knowledge, this is the first study to use machine learning models to predict TSD, which will further facilitate personalized medicine in tacrolimus administration in the future.

Kidney transplantation from pediatric donors in a single Chinese center.

To report clinical outcomes of kidney transplantation from pediatric brain and cardiac death donors (DBCD) in a single Chinese center and to investigate its feasibility to expand organ donor pool. 18 recipients, transplanted between August 2011 and October 2013 in the First Affiliated Hospital of Zhengzhou University, receive a single graft from DBCD donors age ranged from 1.5 to 13 years old. Renal function expressed as serum creatinine, blood urea nitrogen as well as eGFR values at 1, 2 weeks as well as 1-, 3-, 6-, and 12-months post-transplantation was evaluated. Graft size was also monitored at the same time by ultrasonography. In addition, delayed graft function, acute rejection, surgical complication as well as patient and graft survival were also assessed. The primary causes of DBCD donors included six cases of severe brain trauma and three cases of cerebral hemorrhage. The mean age of DBCD donors was (7.2 ± 3.4) years (range 1.5-13). The mean weight of DBCD donors was (29.8 ± 15.3) kilogram (range 13-67). The mean height of DBCD donors was (118.3 ± 27.8) centimeter (range 70-173). ECMO was applied to DBCD donors to avoid warm ischemia time and the applicating time was (79.8 ± 44.5) (range 32-180) minutes.There were seven males and 11 females recipients. Among which, 16 recipients were pediatrics and two recipients were adults. The mean age of the recipients was (14.6 ± 9.7) years (range 4-47). The mean weight of recipients was (31.9 ± 12.4) kilogram (range 11-54). The mean height of recipients was (138.0 ± 23.7) centimeter (range 84-172). Renal function recovered to normal within the first-week post-operation except one recipient which occurred acute rejection. Two cases of renal artery stenosis were found 2-week and 3-month post-transplantation, respectively. They subsequently underwent ballon angioplasty and followed up for 8 and 12 months, respectively, and no recurrence was found. One recipient developed ureteral leak. Five weeks later, the ureter leak healed after adequate drainage and prolongation of indwelling catheter. Graft size significantly and continuously increased during the first year, especially in the first 3-month post-transplantation. All the 18 recipients are alive at the last follow-up. Among which, 16 recipients are followed up for 12 months and 1-year recipient/graft survival rate is 100 %. The use of single kidney graft from pediatric DBCD could yield good short-term results.