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.

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.

Acute kidney injury (AKI) frequently complicates major surgery and can be associated with hypertension and progress to chronic kidney disease, but reports on blood pressure normalization in AKI are conflicting. In the present study, we investigated the effects of an angiotensin-converting enzyme inhibitor, enalapril, and a soluble epoxide hydrolase inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), on renal inflammation, fibrosis, and glomerulosclerosis in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. Male CD1 mice underwent unilateral IRI for 35 min. Blood pressure was measured by tail cuff, and mesangial matrix expansion was quantified on methenamine silver-stained sections. Renal perfusion was assessed by functional MRI in vehicle- and TPPU-treated mice. Immunohistochemistry was performed to study the severity of AKI and inflammation. Leukocyte subsets were analyzed by flow cytometry, and proinflammatory cytokines were analyzed by quantitative PCR. Plasma and tissue levels of TPPU and lipid mediators were analyzed by liquid chromatography mass spectrometry. IRI resulted in a blood pressure increase of 20 mmHg in the vehicle-treated group. TPPU and enalapril normalized blood pressure and reduced mesangial matrix expansion. However, inflammation and progressive renal fibrosis were severe in all groups. TPPU further reduced renal perfusion on days 1 and 14. In conclusion, early antihypertensive treatment worsened renal outcome after AKI by further reducing renal perfusion despite reduced glomerulosclerosis.

Nephrotoxic drugs can cause acute kidney injury (AKI) and analgesic nephropathy. Diclofenac is potentially nephrotoxic and frequently prescribed for pain control. In this study, we investigated the effects of single and repetitive oral doses of diclofenac in the setting of pre-existing subclinical AKI on the further course of AKI and on long-term renal consequences. Unilateral renal ischemia–reperfusion injury (IRI) for 15 min was performed in male CD1 mice to induce subclinical AKI. Immediately after surgery, single oral doses (100 mg or 200 mg) of diclofenac were administered. In a separate experimental series, repetitive treatment with 100 mg diclofenac over three days was performed after IRI and sham surgery. Renal morphology and pro-fibrotic markers were investigated 24 h and two weeks after the single dose and three days after the repetitive dose of diclofenac treatment using histology, immunofluorescence, and qPCR. Renal function was studied in a bilateral renal IRI model. A single oral dose of 200 mg, but not 100 mg, of diclofenac after IRI aggravated acute tubular injury after 24 h and caused interstitial fibrosis and tubular atrophy two weeks later. Repetitive treatment with 100 mg diclofenac over three days aggravated renal injury and caused upregulation of the pro-fibrotic marker fibronectin in the setting of subclinical AKI, but not in sham control kidneys. In conclusion, diclofenac aggravated renal injury in pre-existing subclinical AKI in a dose and time-dependent manner and already a single dose can cause progression to chronic kidney disease (CKD) in this model.

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.

We hypothesized that multiparametric MRI is able to non-invasively assess, characterize and monitor renal allograft pathology in a translational mouse model of chronic allograft rejection. Chronic rejection was induced by allogenic kidney transplantation (ktx) of BALB/c-kidneys into C57BL/6-mice (n = 23). Animals after isogenic ktx (n = 18) and non-transplanted healthy animals (n = 22) served as controls. MRI sequences (7T) were acquired 3 and 6 weeks after ktx and quantitative T1, T2 and apparent diffusion coefficient (ADC) maps were calculated. In addition, in a subset of animals, histological changes after ktx were evaluated. Chronic rejection was associated with a significant prolongation of T1 time compared to isogenic ktx 3 (1965 ± 53 vs. 1457 ± 52 ms, p < 0.001) and 6 weeks after surgery (1899 ± 79 vs. 1393 ± 51 ms, p < 0.001). While mean T2 times and ADC were not significantly different between allogenic and isogenic kidney grafts, histogram-based analysis of ADC revealed significantly increased tissue heterogeneity in allografts at both time points (standard derivation/entropy/interquartile range, p < 0.05). Correspondingly, histological analysis showed severe inflammation, graft fibrosis and tissue heterogeneity in allogenic but not in isogenic kidney grafts. In conclusion, renal diffusion weighted imaging and mapping of T2 and T1 relaxation times enable detection of chronic renal allograft rejection in mice. The combined quantitative assessment of mean values and histograms provides non-invasive information of chronic changes in renal grafts and allows longitudinal monitoring.