Impact of Steroids on the Inflammatory Response after Ischemic Acute Kidney Injury in Rats.

Inflammation plays a crucial role in acute kidney injury (AKI). The current study was designed to analyze the influence of prednisolone treatment on the inflammatory reaction during the first 96 h after AKI induction in a rat model. AKI was induced by unilateral clipping of the renal vessels. The treatment group received prednisolone 5 mg/kg s.c. daily. Infiltration rates of macrophages, leukocytes, and T-cells (24, 96 h) as well as plasma concentrations of the inflammatory markers intercellular adhesion molecule, interleukin-1 beta (IL-1ß), IL-18, IL-6, and tumor necrosis factor-alpha (0, 6, 24, 96 h) were determined by fluorescence-activated cell sorting (FACS) analysis only. Ninety-six hours after AKI induction, the prednisolone group demonstrated significantly lower creatinine concentrations compared to the control group (P < 0.05). Twenty-four hours after induction of AKI, a significantly higher rate of infiltrating leukocytes was detectable with FACS analysis in the control group (P < 0.01) with a corresponding significantly higher rate of macrophages after 96 h (P < 0.01). IL-6 and IL-1ß demonstrated a peak after 6 h with a significantly higher release in the control group (IL-6: P < 0.01; IL-1ß: P < 0.05). In contrast to the control group, the prednisolone group demonstrated no further incline of IL-18 after 24 h. The results demonstrate the importance of stretching the observation period in an ischemia-reperfusion-induced AKI setting beyond the first 24 h. Despite the demonstrated protective effects of a continuous prednisolone application, it seems that this single anti-inflammatory agent will not be able to completely suppress the inflammatory response after an ischemia-reperfusion-induced AKI.

Dopamine treatment of brain-dead Fisher rats improves renal histology but not early renal function in Lewis recipients after prolonged static cold storage.

BACKGROUND: Brain death (BD) and cold preservation are major risk factors for an unfavorable transplantation outcome. Although donor dopamine treatment in brain-dead rats improves renal function and histology in allogeneic recipients, it remains to be assessed if this also holds true for the combinations of BD and prolonged static cold preservation. METHODS: BD was induced in F344 donor rats, which were subsequently treated with NaCl 1 mL/h (BD, n = 11), NaCl/hydroxy ethyl starch (BD-norm, n = 10), or 10 µg/min/kg dopamine (BD-dopa, n = 10). Renal grafts were harvested 4 h after BD and transplanted into bilateral nephrectomized Lewis recipients 6 h after cold preservation in University of Wisconsin solution. Renal function was evaluated by use of serum creatinine and urea concentrations at days 0, 1, 3, 5, and 10. Ten days after transplantation, recipients were killed and the renal allografts were processed for light microscopy and immune histology. RESULTS: Serum urea concentrations at days 5 and 10 were significantly lower in recipients that received a renal graft from dopamine-treated rats; for serum creatinine, only a trend was observed at day 10. Immune histology revealed a lower degree of ED1-positive cells in the donor dopamine-treated group. Under light microscopy, Banff classification revealed significantly less intimal arteritis in these grafts (P < .05). CONCLUSIONS: Although donor dopamine treatment clearly improves renal histology in this model, the beneficial effect on early renal function was marginal. It remains to be assessed if donor dopamine treatment has a beneficial effect on renal function in long-term follow-up.

The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: a comparison of primary human hepatocytes and OATP-transfected HEK293 cells.

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and -LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

Modulation of brain dead induced inflammation by vagus nerve stimulation.

Because the vagus nerve is implicated in control of inflammation, we investigated if brain death (BD) causes impairment of the parasympathetic nervous system, thereby contributing to inflammation. BD was induced in rats. Anaesthetised ventilated rats (NBD) served as control. Heart rate variability (HRV) was assessed by ECG. The vagus nerve was electrically stimulated (BD + STIM) during BD. Intestine, kidney, heart and liver were recovered after 6 hours. Affymetrix chip-analysis was performed on intestinal RNA. Quantitative PCR was performed on all organs. Serum was collected to assess TNFalpha concentrations. Renal transplantations were performed to address the influence of vagus nerve stimulation on graft outcome. HRV was significantly lower in BD animals. Vagus nerve stimulation inhibited the increase in serum TNFalpha concentrations and resulted in down-regulation of a multiplicity of pro-inflammatory genes in intestinal tissue. In renal tissue vagal stimulation significantly decreased the expression of E-selectin, IL1beta and ITGA6. Renal function was significantly better in recipients that received a graft from a BD + STIM donor. Our study demonstrates impairment of the parasympathetic nervous system during BD and inhibition of serum TNFalpha through vagal stimulation. Vagus nerve stimulation variably affected gene expression in donor organs and improved renal function in recipients.

Toxin mixture in cyanobacterial blooms--a critical comparison of reality with current procedures employed in human health risk assessment.

Cyanobacteria are the oldest life forms on earth known to produce a broad spectrum of secondary metabolites. The functions/advantages of most of these secondary metabolites (peptides and alkaloids) are unknown, however, some of them have adverse effects in humans and wildlife, especially when ingested, inhaled or upon dermal exposure. Surprisingly, some of these cyanobacteria are ingested voluntarily. Indeed, for centuries mankind has used cyanobacteria as a protein source, primarily Spirulina species. However, recently also Aphanizomenon flos-aquae are used for the production of so called blue green algae supplements (BGAS), supposedly efficacious for treatment of various diseases and afflictions. Unfortunately, traces of neurotoxins and protein phosphatases (inhibiting compounds) have been detected in BGAS, making these health supplements a good example for human exposure to a mixture of cyanobacterial toxins in a complex matrix. The discussion of this and other possible exposure scenarios, e.g. drinking water, contact during recreational activity, or consumption of contaminated food, can provide insight into the question of whether or not our current risk assessment schemes for cyanobacterial blooms and the toxins contained therein suffice for protection of human health.

High mobility group box 1 and adenosine are both released by endothelial cells during hypothermic preservation.

Hypothermic preservation of solid allografts causes profound damage of vascular endothelial cells. This, in turn, might activate innate immunity. In the present study we employed an in vitro model to study to what extent supernatants of damaged endothelial cells are able to activate innate immunity and to study the nature of these signals. The expression of high mobility group box 1 (HMGB1) and adhesion molecules on human umbilical vein endothelial cell was studied by immunofluorescence, fluorescence activated cell sorter and Western blotting. Cytokine production was performed by enzyme-linked immunosorbent assay. HMGB1 expression was lost completely in endothelial cells after hypothermic preservation. This was associated with cell damage as it occurred only in untreated endothelial cell but not in cells rendered resistant to hypothermia-mediated damage by dopamine treatment. Only supernatants from hypothermia susceptible cells up-regulated the expression of interleukin (IL)-8 and adhesion molecules in cultured endothelial cells in an HMGB1-dependent manner. In whole blood assays, both supernatants of hypothermia susceptible and resistant cells inhibited tumour necrosis factor (TNF)-alpha production concomitantly with an increased IL-10 secretion. The activity of the supernatants was already found after 6 h of hypothermic preservation, and paralleled the decrease in intracellular adenosine triphosphate (ATP) levels. Modulation of TNF-alpha and IL-10 production by these supernatants was abrogated completely by prior treatment with adenosine deaminase and was similar to the response of an A2R agonist. Our study demonstrates that both HMGB1 and adenosine are released during hypothermic preservation. While release of HMGB1 is caused by cell damage, release of adenosine seems to be related to ATP hydrolysis, occurring in both susceptible and resistant cells.

Anaesthesia of small rodents during magnetic resonance imaging.

The use of experimental animals for magnetic resonance studies requires anaesthesia to provide immobility and acquire signals with minimal stress and maximal reproducibility. However, the conduct of anaesthesia within a magnetic resonance imaging (MRI) suite implicates many problems, because most of the anaesthetic and monitoring equipment contains ferromagnetic substances. To decrease disturbances during anaesthesia and make data interpretation more accurate, it is mandatory that investigators become familiar with methods and physiologic effects of anaesthesia under these special conditions. This article is intended to give an overview of anaesthetic medication, administration routes and practical instructions for anaesthesia in small rodents during MRI.

Analytical and functional characterization of microcystins [Asp3]MC-RR and [Asp3,Dhb7]MC-RR: consequences for risk assessment?

The microcystin (MC) producing P. rubescens occurs in pre-alpine lakes and may impact fishery success, bathing, and raw water quality. P. rubescens extracts, characterized via LC-MS, contained the two MC-RR variants [Asp3]MC-RR and [Asp3,Dhb7]MC-RR. The protein-phosphatase-inhibition assay (cPPIA with phosphatases 1 and 2A) in its capability to quantify [Asp3]MC-RR, [Asp3,Dhb7]MC-RR, and MC-RR was compared to HPLC-DAD and anti-Adda-ELISA. The IC50 values (PP1 and PP2A) determined for MC-LR, MC-RR, and [Asp3]MC-RR were in the same range (1.9-3.8 and 0.45-0.75 nM). A 50-fold higher concentration of [Asp3,Dhb7]MC-RR (29.8 nM) was necessary to inhibit the PP2A by 50%. The PP1-IC50 of [Asp3,Dhb7]MC-RR was 22-fold higher (56.4 nM) than those of the other MCs, suggesting that specific structural characteristics are responsible for its weaker PPI capacity. Western blots demonstrated that [Asp3,Dhb7]MC-RR does not covalently bind to PP1. [Asp3,Dhb7]MC-RR has comparable in vivo LD50 values to MC-RR, despite a far lower PP-inhibiting capacity, suggesting that toxicodynamic and toxicokinetic characteristics of [Asp3,Dhb7]MC-RR are responsible for its high in vivo toxicity. The data demonstrate that cPPIA analysis of [Asp3,Dhb7]MC-RR-containing samples prevent reliable MC determination and lead to underestimation of potential toxicity.

Effect of pre-treatment with catecholamines on cold preservation and ischemia/reperfusion-injury in rats.

Treatment of organ donors with catecholamines reduces acute rejection episodes and improves long-term graft survival after renal transplantation. The aim of this study was to investigate the effect of catecholamine pre-treatment on ischemia/reperfusion (I/R)- and cold preservation injury in rat kidneys. I/R-injury was induced by clamping the left kidney vessels for 60 min along with a contralateral nephrectomy. Cold preservation injury was induced by storage of the kidneys for 24 h at +4 degrees Celsius in University of Wisconsin solution, followed by syngeneic transplantation. Rats were pre-treated with either dopamine (DA), dobutamine (DB), or norepinephrine (2, 5, and 10 microg/kg/min, each group) intravenously via an osmotic minipump for 24 h before I/R- and cold preservation injury. Pre-treatment with DA (2 or 5 microg/kg/min) and DB (5 microg/kg/min) improved recovery of renal function after I/R-injury and dose dependently reduced mononuclear and major histocompatibility complex class II-positive cells infiltrating the kidney after I/R-injury. One day after I/R-injury, upregulation of transforming growth factor (TGF)-beta 1 and 2 and phosphorylation of p42/p44 mitogen-activated protein kinases was observed in kidneys of animals treated with DA or DB. DA (5 microg/kg/min) and DB (5 microg/kg/min) pre-treatment reduced endothelial cell damage after 24 h of cold preservation. Only DA pre-treatment improved renal function and reduced renal inflammation after 24 h of cold preservation and syngeneic transplantation. Our results demonstrate a protective effect of pre-treatment with catecholamines on renal inflammation and function after I/R- or cold preservation injury. This could help to explain the potent organoprotective effects of catecholamine pre-treatment observed in human kidney transplantation.