Free heme and hemopexin in acute kidney injury after cardiopulmonary bypass and transient renal ischemia.

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

Cell-Free Hemoglobin in Acute Kidney Injury after Lung Transplantation and Experimental Renal Ischemia/Reperfusion.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

Ischemia Reperfusion Injury Triggers CXCL13 Release and B-Cell Recruitment After Allogenic Kidney Transplantation.

Ischemia reperfusion injury (IRI) is linked with inflammation in kidney transplantation (ktx). The chemokine CXCL13, also known as B lymphocyte chemoattractant, mediates recruitment of B cells within follicles of lymphoid tissues and has recently been identified as a biomarker for acute kidney allograft rejection. The goal of this study was to explore whether IRI contributes to the up-regulation of CXCL13 levels in ktx. It is demonstrated that systemic levels of CXCL13 were increased in mouse models of uni- and bilateral renal IRI, which correlated with the duration of IRI. Moreover, in unilateral renal IRI CXCL13 expression in ischemic kidneys was up-regulated. Immunohistochemical studies revealed infiltration of CD22+ B-cells and, single-cell RNA sequencing analysis a higher number of cells expressing the CXCL13 receptor CXCR5, in ischemic kidneys 7 days post IRI, respectively. The potential relevance of these findings was also evaluated in a mouse model of ktx. Increased levels of serum CXCL13 correlated with the lengths of cold ischemia times and were further enhanced in allogenic compared to isogenic kidney transplants. Taken together, these findings indicate that IRI is associated with increased systemic levels of CXCL13 in renal IRI and ktx.

Chemokine CXCL13 as a New Systemic Biomarker for B-Cell Involvement in Acute T Cell-Mediated Kidney Allograft Rejection.

The presence of B-cell clusters in allogenic T cell-mediated rejection (TCMR) of kidney allografts is linked to more severe disease entities. In this study we characterized B-cell infiltrates in patients with TCMR and examined the role of serum CXCL-13 in these patients and experimentally. CXCL-13 serum levels were analyzed in 73 kidney allograft recipients at the time of allograft biopsy. In addition, four patients were evaluated for CXCL13 levels during the first week after transplantation. ELISA was done to measure CXCL-13 serum levels. For further mechanistic understanding, a translational allogenic kidney transplant (ktx) mouse model for TCMR was studied in BalbC recipients of fully mismatched transplants with C57BL/6 donor kidneys. CXCL-13 serum levels were measured longitudinally, CD20 and CD3 composition and CXCL13 mRNA in tissue were examined by flow cytometry and kidneys were examined by histology and immunohistochemistry. We found significantly higher serum levels of the B-cell chemoattractant CXCL13 in patients with TCMR compared to controls and patients with borderline TCMR. Moreover, in patients with acute rejection within the first week after ktx, a >5-fold CXCL13 increase was measured and correlated with B-cell infiltrates in the biopsies. In line with the clinical findings, TCMR in mice correlated with increased systemic serum-CXCL13 levels. Moreover, renal allografts had significantly higher CXCL13 mRNA expression than isogenic controls and showed interstitial CD20+ B-cell clusters and CD3+ cell infiltrates accumulating in the vicinity of renal vessels. CXCL13 blood levels correlate with B-cell involvement in TCMR and might help to identify patients at risk of a more severe clinical course of rejection.