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.

Bone marrow mesenchymal stem cells repress renal transplant immune rejection by facilitating the APRIL phosphorylation to induce regulation B cell production.

Bone marrow mesenchymal stem cells (BMSCs) exert pivotal roles in suppressing immune rejection in organ transplantation. However, the function of BMSCs on immune rejection in renal transplantation remains unclear. This study aimed to evaluate the effect and underlying mechanism of BMSCs on immune rejection in renal transplantation. Following the establishment of the renal allograft mouse model, the isolated primary BMSCs were injected intravenously into the recipient mice. Enzyme-linked immunosorbent assay, flow cytometry, hematoxylin-eosin staining, and Western blot assays were conducted to investigate BMSCs' function in vivo and in vitro. Mechanistically, the underlying mechanism of BMSCs on immune rejection in renal transplantation was investigated in in vivo and in vitro models. Functionally, BMSCs alleviated the immune rejection in renal transplantation mice and facilitated B cell activation and the production of IL-10+ regulatory B cells (Bregs). Furthermore, the results of mechanism studies revealed that BMSCs induced the production of IL-10+ Bregs by facilitating a proliferation-inducing ligand (APRIL) phosphorylation to enhance immunosuppression and repressed renal transplant rejection by promoting APRIL phosphorylation to induce IL-10+ Bregs. BMSCs prevent renal transplant rejection by facilitating APRIL phosphorylation to induce IL-10+ Bregs.

Immature dendritic cells derived exosomes promotes immune tolerance by regulating T cell differentiation in renal transplantation.

OBJECTIVE: To investigate the mechanism of immature dendritic cells-derived exosomes (imDECs) in the regulation of T cell differentiation and immune tolerance in renal allograft model mice. RESULTS: imDECs significantly improved the percent of survival, relieved inflammatory response, and reduced CD4+T cell infiltration. In addition, imDECs reduced the rejection associated cytokines in allograft mice, and increased the percentage of Foxp3+CD4+T cells in spleen and kidney tissues. imDECs suppressed the IL17+CD4+T cells and promoted the Foxp3+CD4+T cells under Th17 polarization condition. Moreover, miR-682 was found to be highly expressed in imDECs which suppressed the IL17+CD4+T cells and promoted the Foxp3+CD4+T cells. Luciferase reporter assay showed ROCK2 was a target of miR-682, and ROCK mRNA level was negative correlated with miR-682 mRNA level. CONCLUSION: miR-682 was highly expressed in imDECs, and imDECs-secreted miR-682 promoted Treg cell differentiation by negatively regulating ROCK2 to promote immune tolerance in renal allograft model mice. METHODS: Renal allograft model mice were established, and imDECs or mature dendritic cells-derived exosomes (mDECs) were injected into model mice. Rejection associated cytokines IFN-γ, IL-2, IL-17 levels in plasma were detected by ELISA. IL-17A, Foxp3, miR-682, ROCK2, p-STAT3, p-STAT5 expressions were measured by qRT-PCR or western blot.

Bortezomib ameliorates acute allograft rejection after renal transplant by inhibiting Tfh cell proliferation and differentiation via miR-15b/IRF4 axis.

OBJECTIVE: The present study aimed to investigate the functional role of bortezomib in the development of acute allograft rejection (AR) after renal transplant. METHODS: The mouse model of AR was established by allograft kidney transplant followed by the treatment of bortezomib. The serum cytokines, renal function, and the percentage of T follicular helper (Tfh) cells in CD4+ T cells were measured. The effect of miR-15b and interferon-regulatory factor 4 (IRF4) on Tfh cell proliferation and differentiation was assessed by cell transfection technology and CCK-8 assay. The interaction between miR-15b and IRF4 was assessed by luciferase reporter assay. RESULTS: Bortezomib relieved acute AR after renal transplant by suppressing Tfh cell proliferation and differentiation. Meanwhile, bortezomib treatment markedly increased miR-15b expression in AR renal tissues. The upregulation of miR-15b inhibited Tfh cell proliferation and differentiation by reducing IRF4. In addition, bortezomib ameliorated AR by suppressing Tfh cell proliferation and differentiation through miR-15b/IRF4 axis in vitro and in vivo. CONCLUSION: Our findings indicated the mechanism underlying the bortezomib in treating acute AR after renal transplant, and suggested the critical role of miR-15b in Tfh cell proliferation and differentiation, which provided a therapeutic target in attenuating acute AR.