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.

Podocytes are terminally differentiated cells of the kidney filtration barrier. They are subjected to physiological filtration pressure and considerable mechanical strain, which can be further increased in various kidney diseases. When injury causes cytoskeletal reorganization and morphological alterations of these cells, the filtration barrier may become compromised and allow proteins to leak into the urine (a condition called proteinuria). Using time-resolved proteomics, we showed that podocyte injury stimulated the activity of the transcriptional coactivator YAP and the expression of YAP target genes in a rat model of glomerular disease before the development of proteinuria. Although the activities of YAP and its ortholog TAZ are activated by mechanical stress in most cell types, injury reduced YAP and TAZ activity in cultured human and mouse podocyte cell lines grown on stiff substrates. Culturing these cells on soft matrix or inhibiting stress fiber formation recapitulated the damage-induced YAP up-regulation observed in vivo, indicating a mechanotransduction-dependent mechanism of YAP activation in podocytes. YAP overexpression in cultured podocytes increased the abundance of extracellular matrix-related proteins that can contribute to fibrosis. YAP activity was increased in mouse models of diabetic nephropathy, and the YAP target was highly expressed in renal biopsies from glomerular disease patients. Although overexpression of human YAP in mice induced mild proteinuria, pharmacological inhibition of the interaction between YAP and its partner TEAD in rats ameliorated glomerular disease and reduced damage-induced mechanosignaling in the glomeruli. Thus, perturbation of YAP-dependent mechanosignaling is a potential therapeutic target for treating some glomerular diseases.

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.

Abstract. 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 M 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.Diabetic nephropathy (DN) is characterized by mesangial matrix expansion, thickening of the glomerular basement membrane (GBM), and a concomitant loss of heparan sulfate proteoglycans (HSPG) (1-4). HSPG consist of a core protein to which one or more HS glycosaminoglycan (GAG) side chains are attached. To date, three HSPG core proteins-perlecan, agrin, and collagen XVIII (5-7)-have been identified in the GBM.The permselectivity of the GBM is partly due to the high negative charge of HSPG and their interaction with other matrix components (8 -10). Most studies on the decrease of glomerular HSPG in patients with DN have suggested that a selective dysregulation in sulfation of HSPG is underlying the observed reduction in HSPG expression (11-13). However, we recently provided in vivo and in vitro evidence that under hyperglycemic conditions, the expression of the core protein of agrin in the GBM of these patients and in cultured podocytes is also affected (14).In patients with DN, an initial increase in the GFR is followed by a linear decrease in GFR over time (15). It is believed that in these patients, autoregulation of renal blood flow is impaired and, consequently, systemic pressure is transferred to the glomerular capillary loops, resulting in hyperfiltration (15,16). Several clinical studies have demonstrated that antihypertensive drugs...

SummaryInterindividual 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), Eselectin)], 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 δ δ δ ). Type I responders also displayed a low response towards tumour necrosis factor (TNF)-α α α α . In general, maximal activation of nuclear factor (NF)-κ κ κ κ B 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 α α α stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients.