The serum- and glucocorticoid-inducible kinase 1 (SGK1) is a serine threonine protein kinase activated through the phosphatidylinositol 3-kinase (PI3-kinase) pathway and counteracting apoptosis. Protein expression and activation of SGK1 are increased in various models of cell stress. The present study explored the role of SGK1 in renal hypoxia/ischemia induced apoptosis. HEK 293 cells were exposed in vitro to hypoxia/reoxygenation (H/R), which increased SGK1 transcript levels, SGK1 protein abundance and SGK1 phosphorylation. H/R injury further enhanced the percentage of apoptotic cells, an effect significantly blunted by prior SGK1 overexpression. In vivo renal ischemia/reperfusion (I/R) injury increased SGK1 transcript levels and SGK1 protein abundance. I/R enhanced apoptosis, an effect significantly more pronounced in gene targeted mice lacking SGK1. In conclusion, SGK1 is up-regulated and counteracts apoptosis following H/R in vitro and ischemia in vivo.

Ischaemia followed by reperfusion (I/R) can induce inflammation and injury and is a risk factor for delayed graft function and rejection of transplanted kidneys. Inflammation is regulated by NF-kappa B transcription factors which induce pro-inflammatory molecules in endothelial cells (EC). We examined whether A20, a negative regulator of NF-kappa B, can protect kidneys from I/R injury. To mimic the fluctuations in endothelial oxygenation that occur during I/R we exposed cultured human umbilical vein EC (HUVEC) to hypoxia (1% O(2) for 4 h) followed by re-oxygenation (21% O(2) for 1 h-24 h). We observed transient expression of pro-inflammatory molecules (E-selectin, VCAM-1 and IL-8) and sustained expression of A20 in HUVEC exposed to hypoxia/re-oxygenation. The effect of A20 on endothelial responses to hypoxia/re-oxygenation was assessed. We observed that pre-treatment of HUVEC with an adenovirus containing A20 (Ad-A20) suppressed activation of NF-kappa B and induction of pro-inflammatory molecules by hypoxia/re-oxygenation, whereas a control adenovirus had little or no effect. Thus the induction of A20 may form a negative feedback loop in pro-inflammatory signalling in cells exposed to hypoxia/re-oxygenation. To validate our cell culture experiments we examined the role of A20 in renal responses to I/R. We observed that A20 was induced in rat kidneys exposed to I/R. Moreover, pre-treatment of animals with Ad-A20 significantly reduced acute tubular necrosis, renal expression of VCAM-1 and NF-kappa B activation in response to I/R, whereas pre-treatment with control adenovirus did not. Our observations suggest that A20 maintains physiological homeostasis in kidneys exposed to I/R by protecting them from inflammation and injury.

Interleukin (IL)-1 is a major contributor to inflammation and apoptosis during ischemia/reperfusion (I/R) injury. Its deleterious effects are primarily mediated by the activation of nuclear factor-kappaB (NF-kappaB). Receptor-binding and signaling of IL-1 can be blocked by the IL-1 receptor antagonist (IL-1ra). The aim of our study was to characterize effects and mechanisms of IL-1ra administration on inflammation, apoptosis, and infiltration in renal I/R injury. Renal ischemia was induced in Lewis rats by clamping of the left renal artery for 45 min. Kidneys were removed for histological and molecular analysis 24 h or 5 days after reperfusion. IL-1ra ameliorated I/R induced renal injury and inflammation. Furthermore, the number of apoptotic tubular cells was lower in IL-1ra-treated animals 24 h after ischemia, which was paralleled by a Bax/Bcl-2 mRNA ratio towards anti-apoptotic effects. IL-1ra reduced the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA at 24 h and 5 days and that of intracellular adhesion molecule-1 (ICAM-1) expression at 24 h in the ischemic reperfused kidneys. Our results indicate that IL-1ra treatment ameliorates renal I/R injury and this protective effect might be mediated by reduced induction of NF-kappaB mediated MCP-1, ICAM-1, and a decreased ratio between Bax and Bcl-2 mRNA expression.

Objective. In this study, we used combined treatment with cyclosporine (CsA)/everolimus (EVR) or CsA/FIY720 to affect ongoing chronic allograft nephropathy (CAN) compared with monotherapy with EVR or FTY720. Background. CAN is an important cause of renal allograft loss. Immunosuppressive therapy is based on calcineurin inhibitors, which are associated with nephrotoxicity and decreasing graft function. Thus, alternative treatment regimens, including new immuno-suppressants, such as EVR or FTY720, are of interest. We asked whether the combination of CsA with EVR or FTY720 ameliorated the development of CAN more effectively than monotherapy with EVR and/or FTY720 during the later stages of CAN. Methods. Kidneys from Fisher rats were orthotopically transplanted into Lewis rats. Animals received CsA (1.5 mg/kg/d) for the first 10 days after transplantation. Animals were assigned to 6 groups: EVR (0.5 mg/kg/d), FTY720 (0.5 mg/kg/d), CsA (1.5 mg/kg/d), CsA+EVR (1.5 + 0.5 mg/kg/d), CsA+FTY720 (1.5 + 0.5 mg/kg/d), and vehicle (VEH). Treatment started at week 20. The observation period ended after 28 weeks. Results. Treatment with EVR and FTY720 reduced proteinuria and glomerulosclerosis, suppressed lymphocyte and macrophage infiltration, and resulted in a greater number of apoptotic tubular and interstitial cells compared with the combined treatment groups and controls. Conclusion. Although EVR and FTY720 monotherapy delayed the progression of CAN, their combination with CsA had no beneficial effect.

Background. Activated T cells play a key role in allograft rejection. T cell activation requires signaling via the T cell receptor as well as costimulatory signals. Inducible costimulatory molecule (ICOS), with its ligand B7RP-1, is a recently discovered costimulatory molecule of the CD28 family. The role of this signaling pathway during the early phases of kidney allograft rejection is not clear so far.Methods. Kidneys were orthotopically transplanted from BALB/c to C57BL/6 mice. Animals were assigned to five experimental groups: blocking anti-ICOS monoclonal antibody, ICOS fusion protein, anti-B7RP1 monoclonal antibody, B7RP-1 fusion protein, and control immunoglobulin G.Results. Survival was significantly reduced in animals treated with ICOS monoclonal antibody (mAb) and B7RP-1 Fc as compared with controls. These animals had also a lower number of apoptotic graft infiltrating T cells, whereas the expression of intracellular interferon-gamma in CD3(+) CD4(+) T cells was increased. Animals treated with ICOS Fc and B7RP-1 mAb had similar survival and numbers of apoptotic T cells as compared with controls.Conclusions. In summary, the blockade of ICOS with ICOS mAb or B7RP-1 Fc reduced the amount of apoptosis of infiltrating lymphocytes and resulted in continuous inflammatory processes with progressive tissue damage and graft failure.

Background. Chronic allograft nephropathy (CAN) still belongs to the leading causes of graft loss over the long term. The leflunomide derivative FK778 is a novel immunosuppressant with improved pharmacokinetic properties that effectively prolonged graft survival in several transplantation models. In the present study, we investigated the effects of FK778 at different phases after transplantation on the progression of CAN. Methods. Fisher 344 kidneys were orthotopically transplanted into Lewis recipients. Recipients were treated with FK778 (5 mg/kg/day) over different time periods (early: days 0–10 only, continuous: day 0 to week 24, or late: weeks 16–24 only posttransplantation). Proteinuria was measured every 4 weeks, whereas grafts were harvested at 24 weeks posttransplantation for morphological and immunohistochemical analysis as well as transforming growth factor–&bgr; and platelet derived growth factor–B chain expression. Results. Continuous treatment with FK778 ameliorated the progression of CAN, whereas late treatment reduced proteinuria and resulted in a similar grade of CAN as compared to animals with continuous treatment. In contrast, FK778 given only during the early phase after transplantation had no effect on the progression of CAN as compared to controls. Conclusion. In summary, FK778 is a potent immunosuppressive drug that can delay the progression of CAN, even when given at later stages after transplantation.