Purpose: Distinct macrophage M∅ subsets are recruited into the kidney allografts during different phases of transplant rejection and the number of graft-infiltrating M∅ (GIMs) correlates to the severity of kidney injury. However, the molecular mechanisms underlying their pathogenic roles in kidney transplant injury are largely unknown. We have previously demonstrated that IRE1a-XBP1 mediated endoplasmic reticulum (ER) stress response regulates M∅ function during inflammation. In this study, we aimed to investigate the role of Xbp1, a key transcription factor regulating cellular ER stress response in GIMs in kidney transplant ischemia-reperfusion injury (IRI) and allograft rejection.
Methods: Kidneys harvested from WT BALB/c donors were stored in cold UW solution for 3 hrs and then transplanted into syngeneic BALB/c (syn-Tx), or allogeneic   B6 WT (allo-Tx) or M∅-specific Xbp1 knockout (mXbp-1 KO) recipients. The kidney graft, spleen, and serum were collected post-transplant to analyze kidney graft function and changes in the expression of genes associated with ER stress, inflammation, and tissue regeneration. Single cell-RNA sequencing (scRNA-seq) of the allografts was performed at 2 days post-transplant. Additionally, to investigate M∅-RTEC interactions, we utilized an in vitro IRI model of primary renal tubular epithelial cells (RTECs). 
Results: Both syn-Txs and allo-Txs displayed significantly increased Cr and BUN at 24 hr post-Tx, indicative of IRI mediated impairment of renal function. Expression of ER stress sensors (GPR78, IRE-1a) was significantly upregulated by 24 hr post-Tx in transplanted kidneys. Interestingly, their downstream TFs (XBP-1u and its activated form, XBP1s) were upregulated within 3 hr after transplant, indicating that the IRE-1a/XBP-1 ER stress pathway was activated in IRI. Compared with the WT allo-Txs, Xbp1 deletion from GIM improved early renal graft function at POD1 and POD3, which was coincided with decreased expression of kidney injury biomarkers, e.g. Kim-1 and NGAL and increased expression of genes associated with cell differentiation and proliferation (e.g. Klf4 and cMyc), Furthermore, increased expression of MCM2 in RTECs of mXbp-1 ko allograft, suggesting an enhanced RTEC proliferation. Moreover, we found that soluble factors derived from mXbp-1 KO M∅ enhanced the growth of TECs and protected them from cell death in the in vitro IRI model of RTEC. Data from scRNA-seq revealed distinct changes in transcriptome landscapes and GIM phenotypes comparing allografts in WT vs KO recipients 2 days post-transplantation. Intercellular communication network analysis showed that TWEAK-Fn14 dependent crosstalk between GIMs and TECs was diminished in the mXbp-1 ko allo-Txs. 
Conclusion: Activation of ER stress pathways in GIM is linked to IRI following kidney transplantation; abrogating Xbp1 in GIMs improves renal function and tubular regeneration, as well as mitigates renal injury via regulating TWEAK-Fn14 signaling pathway.