Min Gu

Background: Iguratimod (IGT) is a small-molecule anti-inflammatory agent that effectively attenuates the progression of antibody-mediated rejection (ABMR). However, the underlying mechanism of action of IGT remains poorly understood. Thus, this study aimed to investigate the mechanism through which IGT regulates macrophage polarization and ABMR. Methods: A mouse model of ABMR was established by performing kidney transplantation after skin graft-induced sensitization. Experimental interventions included neurogenic locus notch homolog protein 4 (NOTCH4) knockout mice and oral administration of IGT. RNA sequencing, quantitative polymerase chain reaction, Western blotting (WB), flow cytometry, and immunofluorescence (IF) staining were performed to investigate M1 macrophage polarization and the Jagged1–NOTCH4 signaling pathway. Results: This study found that IGT significantly reduced M1 macrophage infiltration into renal allografts in a mouse model of ABMR. Transcriptomic sequencing and subsequent biological experiments indicated that IGT may regulate macrophage polarization through the Jagged1–NOTCH4 pathway. Conclusion: M1 macrophage polarization may contribute to the initiation and progression of ABMR following kidney transplantation. IGT attenuates M1 macrophage polarization by inhibiting Jagged1–NOTCH4–Krüppel-like Factor 4 (KLF4) signaling.

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.

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 ( ), 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 ( ). 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.

To the Editor:

Purpose: To investigate a modified laparoscopic retroperitoneal lymph node dissection technique using an extraperitoneal approach and to evaluate its feasibility. Patients and Methods: A group of consecutive patients from a single institution underwent extraperitoneal laparoscopic retroperitoneal lymph node dissection (EL-RLND) at weeks after orchiectomy for primary testicular nonseminomatous germ cell tumors (NSGCT). All patients were placed in the supine position and tilted up 15 degrees on the affected side. Four trocars were introduced. The templates of lymph node dissection conformed to those of Weissbach and Boedefeld. Results: EL-RLND was successfully performed in 15 patients (left, 6 patients; right, 9 patients). The mean total operative time for the entire series was 164±49 minutes. The mean blood loss was 118±74 mL. One intraoperative complication was injury of the vena cava, which occurred in one patient. Ureteral obstruction occurred in another patient. The mean postoperative intestinal function recovery time was 2 days. The mean postoperative hospitalization was 5.5±1.1 days. Normal antegrade ejaculation was preserved in all patients. Pathologic studies revealed positive lymph nodes in three (20%) patients. No recurrence or distant metastasis occurred during 2 to 32 months of follow-up. Conclusions: EL-RLND is a safe and feasible procedure using an improved extraperitoneal approach that provides minimal invasion and rapid recovery of patients.