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Einleitung und Fragestellung / Introduction and Background

Despite advances in immunosuppressive regimens having significantly increased short-term graft outcome, overall long-term graft survival has not dramatically changed. Frequently used immunosuppressive drugs e.g. cyclosporine A (CsA) or tacrolimus (Tac) primarily target T cells, whereas their influence on other immune subsets such as NK cells might be limited. As NK cells have recently been recognized as key players in chronic allograft failure comprehensive studies are required to address whether NK cells can escape conventional immunosuppressants and play a role in recurrence of allograft rejection.

Methodik / Methods

We characterized the effects of CsA on murine NK cells and further assessed its influence on NK cells during transplantation using a murine model of allogenic KTX (Balb/C to C57Bl/6). Immunophenotyping of NK cells and other graft infiltrating lymphocytes was performed by flow cytometry. Allograft function was assessed by measuring serum creatinine and urea levels. Histology of the HE stained tissue sections were performed following Banff criteria. Expression of cytokines was performed by RT-qPCR.

Ergebnisse und Schlussfolgerungen / Results and Conclusions

NK cells isolated from CsA treated C57BL/6 mice (10 mg/kg) revealed normal function regarding degranulation and IFNγ production, whereas CD8⁺ T cells were functionally impaired. In vivo, application of CsA to C57BL/6 recipients of fully allogeneic Balb/C kidneys resulted in a significant reduction of creatinine levels at day 7. Flow cytometric analysis revealed a CsA mediated reduction of intragraft CD4⁺ and CD8⁺ T cells by half, whereas intragraft NK cell frequencies significantly increased and remained unaffected within the spleen or liver. Importantly, the additional depletion of NK cells resulted in a further improvement of kidney function, associated with reduced intragraft and splenic IFNγ expression. Taken together, CsA insufficiently targets murine NK cells and their depletion combined with CsA synergistically improves graft function in an acute transplantation setting.

Einleitung und Fragestellung / Introduction and Background

Natural Killer (NK) cells play a crucial role during infections and inflammatory processes. NK cells represent an enriched population within the intrahepatic lymphocytes (IHLs) by constituting up to 40% of this population. In animal models, specific hepatic NK cells have shown to confer hapten- or virus-specific responses. While data obtained in mice have defined a subset of liver-resident NK (lr-NK) cells, human lr-NKs remain poorly characterized.

The main objective of this study was to characterize lr-NK cells using IHLs and compare their phenotype and function to conventional peripheral blood NK cells from 15 matched donors.

Methodik / Methods

Matched liver tissues and blood samples were obtained from patients undergoing a liver transplantation, signed written consent according to the ethical guidelines of the Universitäts-Klinikum Hamburg. Liver samples were processed through hashing and filtering protocols, PBMCs were obtained by Ficoll-gradient purification. Recovered cells were immediately stained, fixed and measured with an LRS Fortessa.

Ergebnisse und Schlussfolgerungen / Results and Conclusions

Using Principal Component Analysis as an untargeted approach, we observed that NK cells derived from IHLs possessed a distinct phenotypic signature when compared to conventional NK (cNK) cells. CD56^bright NK cells were more frequent in IHLs compared to peripheral blood (p<0,001). NK cells from IHLs exhibited a less activated and a more immature phenotype, with lower expression of CD57 (p<0,001), DNAM-1 (p=0,002) and higher expression of CD34 (p=0,002). CD49a was exclusively expressed on IHL NK cells (p<0,001), as previously reported for mice lr-NK cells. These human CD49a⁺ NK cells showed higher expression levels of CD25 (p=0,002), CD34 (p=0,002) and CXCR3 (p=0,002).

Taken together, intrahepatic NK cells differ phenotypically from blood NK cells derived from matched sample individuals, and include liver-specific CD49a⁺CD25⁺ and CD49a⁺CD34⁺ NK cell populations, suggesting different functional features including self-renewal and persistence.

Einleitung und Fragestellung / Introduction and Background

Renal transplantation is the best available renal replacement therapy for the treatment of patients with end stage renal disease although the necessary immunosuppressive therapy can result in severe side effects, mostly caused by systemic effects of the drugs.

Aim of this study was the evaluation of a modified version of the immunosuppressive drug prednisolone in a rat model of allogeneic kidney transplantation. Coupled to a polypeptide the compound is selectively taken up by the kidney where the prednisolone is released, avoiding systemic effects.

Methodik / Methods

Six groups of animals were used (N=5-6): syngeneically (Lewis Brown Norway F1 to Lewis Brown Norway F1) and allogeneically (Lewis Brown Norway F1 to Lewis) transplanted rats without immunosuppressive therapy, as well as allogeneically transplanted animals receiving either systemically acting or kidney-specific prednisolone at two different concentrations (4mg/kg/12h or 16mg/kg/12h, i.p). Treatment efficiency was evaluated at day 4 post surgery by ¹⁸F-FDG positron emission tomography and histological analysis. Blood glucose levels were measured to assess possible systemic effects.

Ergebnisse und Schlussfolgerungen / Results and Conclusions

Compared with allogeneically transplanted animals receiving no immunosuppressive drugs, high-dose treatment with systemically acting prednisolone significantly reduced renal ¹⁸F-FDG accumulation and histological signs of rejection, low dose treatment had no effect. Animals treated with kidney-specific prednisolone showed significant amelioration of graft rejection already at low dose treatment, high dose treatment resulted in further improvement. Elevated blood glucose levels were only observed after administration of systemically acting prednisolone.

We conclude that kidney-specific prednisolone can effectively prevent graft rejection and partially even outperform systemically acting prednisolone. Because we did not find systemic effects on blood glucose further evaluations seem to be promising

Einleitung und Fragestellung / Introduction and Background

Acute kidney injury (AKI) is a frequent complication after solid organ transplantation. Especially, lung, heart and liver transplantation are associated with substantial blood loss and the need of packed red blood cell (pRBC) transfusions. Although pRBC are beneficial and in many cases lifesaving, blood products also have adverse effects.  Release of toxic extracellular hemoglobin (hb) and heme has been shown to contribute to acute organ (especially lung) injury. In this study, the effect of free hb / heme on ischemia-induced subclinical AKI was investigated in a mouse model.

Methodik / Methods

Renal ischemia reperfusion injury (IRI) was induced by bilateral 15 min renal pedicle clamping in mice. Mice after sham surgery served as controls. Afterwards, free hb, free heme or vehicle was infused. Clinical chemistry for renal function parameters and functional magnetic resonance imaging (fMRI) was done to quantify creatinine elevation, renal perfusion impairment and tissue edema. qPCR for cytokine expression, histology and immunohistochemistry for acute kidney injury and inflammation were done.

Ergebnisse und Schlussfolgerungen / Results and Conclusions

Free hb/heme infusion resulted in marked aggravation of AKI with elevated s-creatinine and BUN whereas vehicle treatment did not cause a relevant impairment in renal function. By fMRI significant decrease of renal perfusion was measured due to heme injection after IRI but not after sham surgery.  Inflammation and acute tubular injury were more prominent in mice after free hb/heme infusion than in those given vehicle. Tissue levels of pro-inflammatory cytokineswere significantly higher in free hb/heme-treated mice than in vehicle treated animals.

Taken togehter, transfusion of aged pRBC is pro-inflammatory and aggravates AKI in an experimental mouse model of mild renal IRI.