Paul-Thomas Brinkkoetter

Hypothermic injury: the mitochondrial calcium, ATP and ROS love-hate triangle out of balance.

Background/Aims: Catecholamines prevent hypothermic cell death which accounts for severe tissue damage and impaired allograft function after prolonged organ preservation. Here, we identified cellular processes which govern hypothermia-mediated cell death in endothelial cells and how they are influenced by dopamine. Methods: Lactate dehydrogenase assay, intracellular ATP, reactive oxygen species and reduced thio-group measurement, intracellular calcium measurement and mitochondrial calcium staining were performed in the study. Results: Intracellular ATP was almost completely depleted within 12 hrs of hypothermic preservation in untreated human umbilical vein endothelial cells (HUVEC), while dopamine pre-treatment significantly delayed ATP depletion. 4 hrs after hypothermia a redox imbalance was observed in untreated cells, which increased with the duration of hypothermia. The redox imbalance was primarily caused by depletion of SH reduction equivalents and was significantly inhibited by dopamine. In addition, hypothermia-induced Ca2+ influx and mitochondrial Ca2+ accumulation were both prevented by dopamine. The protective effect of dopamine was abrogated by ionomycin and sodium azide and partly by oligomycin and CCCP. Conclusions: Our data demonstrated that loss of intracellular ATP, generation of a redox imbalance and accumulation of intracellular Ca2+ underlie cold preservation injury. Dopamine improves the redox balance, prevents intracellular Ca2+ accumulation and delays ATP depletion.

Hypothermia-induced loss of endothelial barrier function is restored after dopamine pretreatment: role of p42/p44 activation.

Background. Donor dopamine usage is associated with improved immediate graft function after renal transplantation. Although prolonged cold preservation results in an increased vascular permeability, the present study was conducted to examine in vitro and in vivo if dopamine pretreatment influences endothelial barrier function under such conditions. Methods. To assess cold preservation injury in vitro and in vivo, cultured human umbilical vein endothelial cells (HUVEC) and Lewis donor rats were pretreated with dopamine or isotonic saline prior to cold storage. Injury was determined by lactate dehydrogenase (LDH) release, histology, and functional analysis. Results. In vitro cold storage resulted in intercellular gap formation in both untreated and dopamine pretreated HUVEC. In the latter monolayer integrity was completely restored upon rewarming and paracellular transport of fluorescein isothiocyanate-dextran was significantly reduced. In dopamine treated HUVEC, intercellular gap formation was independent of cell death and was associated with redistribution of junctional proteins and condensation of cytoskeleton proteins. In untreated HUVEC proteolysis and cell death were clearly evident after hypothermia. Closing of intercellular gaps was dependent on p42/p44 activation. Regeneration of adenosine triphosphate was only observed in dopamine pretreated cells. Only in dopamine treated Lewis renal allografts subjected to cold storage, activation of p42/p44 occurred upon rewarming. These grafts had a better renal function and displayed less inflammatory cells five days after transplantation. Conclusion. Our study demonstrates beneficial effects of dopamine treatment on cold storage induced endothelial barrier disturbances. This may contribute to the positive effects of catecholamines on immediate graft function of renal allografts in men.

Angiotensin II Type 1–Receptor Mediated Changes in Heparan Sulfate Proteoglycans in Human SV40 Transformed Podocytes

In patients with diabetic nephropathy, glomerular staining for heparan sulfate proteoglycans (HSPG) side chains and for agrin is decreased. In the present study, the influence of angiotensin II (AngII) on the production of HSPG in SV40 transformed podocytes was investigated. SV40 transformed human podocytes were cultivated with or without 1 microM AngII, and HSPG production was measured by sequential DEAE-anion exchange chromatography and HPLC-DEAE separation. Expression of agrin was studied by indirect immunofluorescence and Western blot analysis using specific mono- and polyclonal antibodies. DEAE separation of total glycosaminoglycans (GAG) revealed a significant increase of GAG in the culture supernatant and decrease in the cell and matrix layer when podocytes were cultured for 72 h in the presence of AngII. This was particularly found for HS-GAG. Qualitative analysis of HSPG, using gel filtration of HNO(2)-treated fractions, showed that AngII treatment decreased N-sulfation of HS-GAG side chains. Indirect immunofluorescence staining with anti-agrin polyclonal antibody was strongly decreased after AngII stimulation. A reduction in agrin expression in cell extracts could also be detected in Western blot analysis using an mAb. No changes in agrin mRNA were found after AngII stimulation. It is concluded from this study that AngII decreases the amount of HSPG on the cell surface and in the extracellular matrix of podocytes. Because HSPG play a fundamental role in the permselectivity of the glomerular basement membrane, these results thus may explain at least partially the antiproteinuric effects of angiotensin-converting enzyme inhibition in patients with diabetic nephropathy.

Atorvastatin interferes with activation of human CD4+ T cells via inhibition of small guanosine triphosphatase (GTPase) activity and caspase‐independent apoptosis

Although a beneficial effect of hydroxy‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors, i.e. statins, on cell‐mediated immunity has been suggested in vivo and in vitro, little is known about the molecular and biochemical events by which statins inhibit T cell proliferation. To address this question, we investigated the effects of atorvastatin (AT) on intracellular cytokine production, T cell activation markers, cell cycle progression and apoptosis in human CD4+ T cells. AT did not influence intracellular cytokine production after short‐term stimulation of whole blood with phorbol myristate acetate (PMA)/ionomycin or superantigen (SEB). In contrast, AT influenced CD45RA to RO switching dose‐dependently, as well as CD25 expression, and caused cell cycle arrest in the G1 phase after long‐term T cell stimulation. This occurred in conjunction with a reduced expression of cyclin‐dependent kinases 2 and 4 and p21wav1/cip1 and was paralleled by an increased protein expression of p27kip1. In addition to G1 arrest, increased apoptosis was observed in AT‐treated cells. In line with this, the expression of Bcl‐xl and pBad were decreased by AT. Apoptosis was independent of caspases 3 and 9 activation. The inhibitory effect of AT on T cell proliferation could be overcome by addition of mevalonic acid or geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate or squalen, suggesting reduced protein prenylation. Activation of Rho, Rac and Ras were strongly reduced in AT‐treated T cells, suggesting that impaired geranylation of these molecules might underlie the inhibitory effect of AT on T cell proliferation.

Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factors

Interindividual differences of endothelial cells in response to endotoxins might contribute to the diversity in clinical outcome among septic patients. The present study was conducted to test the hypothesis that endothelial cells (EC) with high and low proinflammatory potential exist and to dissect the molecular basis underlying this phenomenon. Thirty human umbilical vein endothelial cell (HUVEC) lines were stimulated for 24 h with lipopolysaccharide (LPS) and screened for interleukin (IL)‐8 production. Based on IL‐8 production five low and five high producers, tentatively called types I and II responders, respectively, were selected for genome‐wide gene expression profiling. From the 74 genes that were modulated by LPS in all type II responders, 33 genes were not influenced in type I responders. Among the 41 genes that were increased in both responders, 17 were expressed significantly stronger in type II responders. Apart from IL‐8, significant differences in the expression of proinflammatory related genes between types I and II responders were found for adhesion molecules [intercellular adhesion molecule (ICAM‐1), E‐selectin)], chemokines [monocyte chemoattractant protein (MCP‐1), granulocyte chemotactic protein (GCP‐2)], cytokines (IL‐6) and the transcription factor CCAAT/enhancer binding protein‐delta (C/EBP‐δ). Type I responders also displayed a low response towards tumour necrosis factor (TNF)‐α. In general, maximal activation of nuclear factor (NF)‐κB was achieved in type I responders at higher concentrations of LPS compared to type II responders. In the present study we demonstrate that LPS‐mediated gene expression differs quantitatively and qualitatively in types I and II responders. Our results suggest a pivotal role for common transcription factors as a low inflammatory response was also observed after TNF‐α stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients.

Altered CD46-mediated T cell co-stimulation in haemodialysis patients

While most of our understanding of immune dysfunction in dialysis patients involves alterations in CD28–CD80/86 signalling, nothing is known of CD46‐mediated co‐stimulation of T cells in these patients. Because C3b/C4b bind to CD46 and complement activation occurs during haemodialysis (HD), we addressed whether CD46‐mediated T cell activation is altered in HD (n = 9), peritoneal dialysis (PD) (n = 10) and predialysis patients (n = 8) compared to healthy controls (HC) (n = 8). T cell surface markers, T cell proliferation and interleukin (IL)‐10 production were studied in CD4+T cells. In addition, CD46 splice‐variants and IL‐10 promoter gene polymorphisms were studied by reverse transcription (RT) or amplification refractory mutation system‐polymerase chain reaction (ARMS‐PCR), respectively. In all uraemic patients, irrespective of the stage of renal insufficiency or dialysis modality, a significant increase in the percentage of CD25 positivity in naive CD4+T cells was found (64% ± 21%versus 23% ± 18%, P < 0·001). Lymphocytes of HD patients proliferated in greater numbers and produced more IL‐10 after co‐stimulation with anti‐CD46 than after co‐stimulation with anti‐CD28. This was also found in CD4+T cells of PD patients, albeit to a lesser extent. In contrast, with T cells of predialysis patients and of HC, co‐stimulation via CD28 was more efficient. The observed alterations in T cell proliferation and IL‐10 production were associated neither with CD46 splice variants nor with IL‐10 promoter gene polymorphisms. Lymphocytes of HD patients show an increased response on CD46 co‐stimulation. These data suggest that ongoing complement activation in HD patients may lead to alterations in acquired immunity.