*Purpose: B6 recipients spontaneously accept MHC-disparate renal allografts from allogeneic DBA/2 x B6 (H-2^d x H-2^b) F1 donors via mechanisms that are in part regulatory T cell (Treg)-dependent. We hypothesized that kidney allograft production of erythropoietin (EPO), which we previously showed inhibits effector T cells while promoting Treg function, contributes to the observed spontaneous acceptance. A direct link between kidney produced EPO and allograft acceptance has not been previously tested.

*Methods: We generated a unique B6 mouse with a doxycycline(DOX)-inducible shRNA specific for EPO (shEPO+) and crossed it to a DBA/2 mouse to produce a shEPO+ x DBA/2 F1 (shEPO+ F1) animal. After verifying DOX-dependent EPO downregulation, we transplanted nephrectomized, B6 recipients with kidneys from shEPO+ F1 or control shEPO- F1 (control F1) donors and treated the recipients post-transplant with a DOX-containing diet.

*Results: By day 14 post-transplant, the recipients of shEPO+ F1 allografts had lower hematocrits (32% vs 40%, p<0.05) and higher serum creatinine levels (0.47 vs 0.2 mg/dl, p = 0.05). Allograft expression of EPO mRNA was reduced 3-fold (qPCR) in the shEPO+ F1 allografts vs controls (confirming EPO knockdown, p<0.05), and allograft histology of the shEPO+ F1 renal allografts revealed extensive mononuclear interstitial infiltration (>50% of parenchyma), consistent with acute cellular rejection, greater than in the control F1 renal allografts. Additional qPCR analyses of kidney graft mRNA showed ~3-fold increase in granzyme B (p<0.05) and a trend towards increased interferon-gamma expression (p=0.073) in the shEPO+ F1 vs control F1 grafts. To next test whether EPO contributes to peripheral self-tolerance of the native kidneys in non-transplanted mice, we examined kidneys from shEPO+ and shEPO- control mice treated with DOX for 4 months (hematocrit levels and serum EPO were significantly lower in the shEPO+ mice). Remarkably, histology of the DOX-treated shEPO+ kidneys showed diffuse perivascular and interstitial mononuclear cell infiltrates, not observed in the DOX-treated shEPO- controls.

*Conclusions: Together, our data demonstrate for the first time that kidney allograft-derived EPO is protolerogenic and that endogenous EPO contributes to peripheral tolerance of native kidneys, organs regularly exposed to a wide array of commensal microbes to which potent immune responses could be injurious.

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