S. Shukla1,2, W. Yuen2, X. Zheng1,2, Z. Zhang1,2 1Feinberg School Of Medicine – Northwestern University, Department Of Surgery, Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University, Comprehensive Transplant Center, Microsurgery Core, Chicago, IL, USA
Introduction: Macrophages are crucial immune cells involved in ischemic reperfusion Injury (IRI), tissue healing, inflammatory response, and infection response. Previous studies have demonstrated that severity of allograft rejection and kidney graft survival longevity is linked to macrophage infiltration. Unfolded protein response is triggered when the endoplasmic reticulum (ER) is overrun by unfolded proteins, directly influenced by macrophage activity. The XBP1 gene encodes an essential transcription factor that controls gene expression related to protein folding and ER stress management. While the XBP1 pathway has been linked to the development of multiple diseases, including renal disease, and has been found to influence inflammatory responses in macrophages, its role in macrophages during kidney transplantation has not been studied. Hence, the aim of this study is to explore the function of the macrophage-derived Xbp-1 ER stress pathway in mitigating IRI after kidney transplantation.
Methods: To investigate the impact of macrophage-derived Xbp-1 ER stress pathway in transplant ischemia-reperfusion injury (IRI), we utilized conditional Knockout( KO) mice that lack Xbp-1specifically in macrophages as recipients and transplanted with MHC-mismatched allogeneic kidney transplants. The post-transplant kidney allograft function was compared with the wildtype (WT) recipients. We employed RNA sequencing to determine the impact of XBP-1 on transcriptome landscapes and putative pathways that are associated with kidney transplant IRI. For validation, we employed a protocol to KO the XBP1 gene in macrophages using SiRNA and mimicked the IRI. Proliferation dye imaging was utilized to assess cell proliferation and the resulting RtPCR data were analyzed using T-tests.
Results: The Xbp1 KO group exhibited decreased kidney injury biomarkers. Furthermore, the graft function was superior in the Xbp1 KO group at 24 hours and 3 days post-transplantation. HE staining of WT and Xbp1 mice revealed more severe kidney injury in WT mice. MCM2 staining, a kidney regeneration marker, showed that epithelial cells treated with supernatant from Xbp1 KO macrophages experienced better survival, evidenced by the results of Live/Dead and Ki67 staining. RNA sequencing was performed on macrophages from KO and WT mice, in which 54,432 genes were studied. 333 genes exhibited changes following stimulation, with 159 genes upregulated and 174 genes downregulated exhibiting crucial functions such as protein processing in the ER and cell regulation. Our preliminary data on the SiRNA XBP1-KO confirmed that both XBP1S and XBP1U gene expression was significantly lower in the SiRNA XBP1 KO model (p< 0.005).
Conclusion: Recipient-derived macrophage XBP1 pathway could be a viable therapeutic target for mitigating IRI in grafts. Our research indicates that macrophages can promote renal tubular epithelial cell proliferation, and SiRNA-induced XBP1 gene KO may bolster this process, potentially improving graft survival.