Superoxide potently induces ceramide formation in glomerular endothelial cells.

Recent evidence suggests that the sphingolipid-derived second messenger ceramide and oxidative stress are intimately involved in apoptosis induction. Here we report that exposure of microcapillary glomerular endothelial cells to superoxide-generating substances, including hypoxanthine/xanthine oxidase and the redox cyclers DMNQ and menadione results in a dose-dependent and delayed increase in the lipid signaling molecule ceramide. Long-term incubation of endothelial cells for 2-30 h with either DMNQ or hypoxanthine/xanthine oxidase leads to a continuous increase in ceramide levels. In contrast, short-term stimulation for 1 min up to 1 h had no effect on ceramide formation. The DMNQ-induced delayed ceramide formation is dose-dependently inhibited by reduced glutathione, whereas oxidized glutathione was without effect. Furthermore, N-acetylcysteine completely blocks DMNQ-induced ceramide formation. All superoxide-generating substances were found to dose-dependently trigger endothelial cell apoptosis. In addition, glutathione and N-acetylcysteine also prevented superoxide-induced apoptosis and implied that ceramide represents an important mediator of superoxide-triggered cell responses like apoptosis.

Suppression of apoptosis by glucocorticoids in glomerular endothelial cells: effects on proapoptotic pathways.

Tumour necrosis factor-alpha (TNF-alpha)- and lipopolysaccharide (LPS)-induced apoptosis of bovine glomerular endothelial cells is now recognized as an important part in the pathogenesis of glomerulonephritis characterized by early mitochondrial cytochrome c release, mitochondrial permeability transition, Bak protein upregulation, Bcl-X(L) protein downregulation and caspase-3 activation. Co-treatment of cells with 10 nM dexamethasone and TNF-alpha or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. The action of glucocorticoids could be documented in that they prevented all apoptotic markers such as DNA laddering, DNA fragmentation measured by the diphenylamine assay as well as morphological alterations. To mechanistically elucidate the action of glucocorticoids we evaluated whether glucocorticoids elicit a time-dependent effect. For dexamethasone, to maximally inhibit DNA fragmentation a preincubation period was not required. Even if dexamethasone was supplemented 6 h following TNF-alpha or LPS we observed a maximal inhibitory effect. Concerning its influence on TNF-alpha and LPS signal transduction, we found that dexamethasone only partially prevented cytochrome-c-release as a first sign of apoptotic cell death but efficiently blocked mitochondrial permeability transition. Moreover, TNF-alpha- and LPS-induced Bak upregulation, Bcl-X(L)-downregulation, and the activation of caspase-3-like proteases, measured fluorometrically using DEVD-AMC and PARP cleavage, were efficiently blocked by dexamethasone. We postulate that glucocorticoids exert their inhibitory action upstream of the terminal death pathways but downstream of primary receptor mediated signals by blocking pro-apoptotic signals pre- and/or post cytochrome c release and mitochondrial signalling.

Suppression and recovery of adrenal response after short-term, high-dose glucocorticoid treatment.

BACKGROUND: Suppression of the adrenal response is an unpredictable consequence of glucocorticoid treatment. To investigate the kinetics of the adrenal response after short-term, high-dose glucocorticoid treatment, we measured the adrenal response to the low-dose (1 microg) corticotropin stimulation test. METHODS: We studied 75 patients who received the equivalent of at least 25 mg prednisone daily for between 5 days and 30 days. After discontinuation of glucocorticoid treatment, 1 microg corticotropin was administered intravenously, and stimulated plasma cortisol concentrations were measured 30 min later. In patients with a suppressed response to 1 microg corticotropin, the test was repeated until stimulated plasma cortisol concentrations reached the normal range. FINDINGS: The adrenal response to 1 microg corticotropin was suppressed in 34 patients and normal in 41. Subsequent low-dose corticotropin tests showed a steady recovery of the adrenal response within 14 days. In two patients, the adrenal response remained suppressed for several months. There was no correlation between plasma cortisol concentrations and the duration or dose of glucocorticoid treatment. INTERPRETATION: Suppression of the adrenal response is common after short-term, high-dose glucocorticoid treatment. The low-dose corticotropin test is a sensitive and simple test to assess the adrenal response after such treatment.

[Spontaneous bacterial peritonitis with Streptococcus constellatus in an HIV positive patient]. / Spontane bakterielle Peritonitis mit Streptococcus constellatus bei einem HIV-positiven Patienten.

Liver diseases are an important cause of high morbidity and mortality in HIV-infected patients, and liver cirrhosis is the commonest cause of ascites in this population. We describe the case of a 38-year-old HIV-positive male (CDC stage B3, CD4 cell count 199/mm3) with a history of hepatitis C-associated liver cirrhosis. Following pneumonia he developed spontaneous bacterial peritonitis due to Streptococcus constellatus. Clinically noticeable was the gradually worsening course with few symptoms, despite the initially high ascitic fluid leucocyte count of over 11,000/microliter, but a favourable response to betalactam antibiotics.

Glucocorticoids potently block tumour necrosis factor-alpha- and lipopolysaccharide-induced apoptotic cell death in bovine glomerular endothelial cells upstream of caspase 3 activation.

1. Endothelial cell damage in glomeruli and kidney arterioles appears to play a pivotal role in glomerular inflammatory diseases. Glomerular endothelial cells, a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration, die by apoptosis in response to tumour necrosis factor-alpha (TNF-alpha), TNF-alpha/basic fibroblast growth factor (bFGF), TNF-alpha/cycloheximide, and bacterial lipopolysaccharide (LPS). Apoptotic cell death is characterized by extensive DNA cleavage, DNA ladder formation, and characteristic morphological alterations. 2. In search for apoptosis-preventing signals, we identified glucocorticoids as potent death preventing factors. Co-treatment of cells with 10 nM dexamethasone and TNF-alpha, TNF-alpha/bFGF, TNF-alpha/cycloheximide, or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. 3. Similarly to dexamethasone (TNF-alpha- and LPS-induced apoptosis are prevented with IC50 values of 0.8 and 0.9 nM, respectively), other synthetic and natural forms of glucocorticoids, such as fluocinolone, prednisolone, hydrocortisone, and corticosterone potently inhibited cell death with IC50 values of 0.2, 6, 50 and 1000 nM, for TNF-alpha and 0.7, 8, 100 and 500 nM for LPS, respectively. 4. Apart from glucocorticoids, mineralocorticoids such as aldosterone also blocked TNF-alpha/LPS-induced apoptosis (IC50 approximately 500 nM for TNF-alpha and approximately 500 nM for LPS), whereas sex hormones, i. e. beta-estradiol and testosterone remained without effect. 5. The protective effect of glucocorticoids (and mineralocorticoids) required glucocorticoid receptor binding as it could be antagonized by the glucocorticoid receptor antagonist RU-486. Concerning TNF-alpha and LPS signal transduction, we found that dexamethasone efficiently prevented TNF-alpha- and LPS-induced activation of caspase-3-like proteases. Therefore, we postulate inhibitory mechanisms upstream of terminal death pathways.

[Interdisciplinary discussion about euthanasia--viewpoint of the clinical physicians]. / Sterbehilfe--interdisziplinär betrachtet--aus der Sicht der Spitalärztin.

In western cities more than 80% of deaths occur in the hospital. Thus, we should be familiar with the professional care for dying patients. However, reports of euthanasia in the Netherlands and interviews of patients in other countries demonstrate that medical care for patients with end stage diseases frequently is insufficient. The need for palliative care, which encloses medical, psychological, social and spiritual aspects of the dying becomes apparent. The physical symptoms (e.g. pain) are only one aspect of the suffering of the terminally ill. Following the WHO guidelines for use of analgesic drugs pain control is achieved in the majority of patients. Palliative care may individually tailor the treatment and care to achieve symptom control. Legalization of euthanasia will diminish the interest in practicing palliative care and may also limit the enthusiasm in research in this field as seen in the Netherlands. Data analysis report significant increase of physician-assisted suicide and euthanasia in the Netherlands within five years time from 1990 to 1995 (total: 3.7% to 4.7%, euthanasia: 1.7% to 2.4%). In addition, each year about 1000 patients were not competent at the time euthanasia was performed (euthanasia without request)! Furthermore, a patients illness did not have to be in end stage when he required euthanasia. This information should rise concern about future developments! The public enthusiasm for legalization of euthanasia in Switzerland may reflect the fear of dying and the belief that physicians and other medical professionals are not equipped to adequately care for the dying. Indeed, professional competence of palliative medicine to treat the symptoms of terminally ill patients particularly with cancer has frequently been insufficient. The majority of Swiss dying with assisted suicide (Switzerland belongs to the few countries where assisted suicide is not illegal) in 1996 and more than 80% in the Netherlands dying by euthanasia had cancer. For the terminally ill euthanasia and assisted suicide may seem the only solution. Enhancing education in palliative medicine is a necessary first step to improve the care for the dying patients.

Revival of tetracyclines--in the treatment of visceral leishmaniasis?

A 37-year-old immigrant from Kosovo who had been in Switzerland for 2 years developed fever, cough, weight loss and malaise. Serology (complement binding reaction) was positive for leptospirosis. The symptoms resolved very rapidly under vibramycin 2 x 100 mg/day for 3 weeks. However, a flare-up occurred after cessation of medication. Reexposure to tetracyclines improved the symptoms though they did not subside completely. Bone marrow analysis demonstrated intracellular leishmania (amastigotes). Analysis of frozen serum preserved since the first hospitalisation and samples from the second admission were positive for leishmania (indirect fluorescence antibody test) and confirmed the diagnosis of visceral leishmaniasis. Reevaluation of the serology for leptospirosis was negative using the specific microagglutination method. Treatment with antimony for 28 days resolved all symptoms. The parasites of visceral leishmaniasis grow intracellularly and eradication may be impossible in patients with an impaired cellular immune response. Flare-ups thus recur in 60-100% of patients with organ transplants or AIDS, despite regular treatment. Our finding raises the question whether relapses are suppressed in immunocompromised patients by tetracyclines, drugs known to be well tolerated even under long-term exposure. Randomised studies are required in this setting.

Tumor necrosis factor-alpha and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells.

BACKGROUND: The glomerular endothelial cell is a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration. During gram-negative sepsis, glomerulonephritis, and acute renal failure, bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) may cause severe cell damage. Our aim was to study and compare the direct effects of TNF-alpha and LPS on the induction of apoptosis in bovine glomerular endothelial cells. METHODS: Primary bovine glomerular endothelial cells were stimulated with TNF-alpha or LPS, and apoptotic cell death was investigated by DNA fragmentation analysis, morphological studies, measurement of cytochrome c efflux and mitochondrial permeability transition, Bak, Bad, Bax, Bcl-2, Bcl-xL protein expression, and caspase-3-like protease activity. RESULTS: TNF-alpha, as well as LPS, elicited apoptotic cell death both time and concentration dependently. Along with DNA ladder formation, we detected the formation of 50 kbp high molecular weight DNA fragments, nuclear condensation, and mitochondrial permeability transition. Concerning all parameters, LPS signaling proved to be more rapid than TNF-alpha. Mechanistically, TNF-alpha-induced cell death was preceded by an efflux of mitochondrial cytochrome c into the cytosol and, subsequently, by a marked increase in the proapoptotic protein Bak and a decrease in the anti-apoptotic Bcl-xL protein content. Comparable but more pronounced effects were seen with LPS. Later, caspase-3-like protease activity was first detectable after 10 hours and was continuously increased up to 24 hours in both TNF-alpha- and LPS-stimulated cells. Correspondingly, we detected an extended cleavage of the nuclear enzyme poly(ADP-ribose) polymerase. Caspase inhibitors Z-Asp-CH2-DCB and Z-VAD-fmk blocked both TNF-alpha- and LPS-induced apoptosis in a comparable manner. Only Z-Asp-CH2-DCB was able to block apoptotic cell death completely. CONCLUSION: Both bacterial LPS and TNF-alpha potently induced apoptotic cell death in glomerular endothelial cells. Direct endotoxin-induced apoptosis may therefore be relevant in the progression of acute renal failure, which is a frequent complication of gram-negative sepsis.

[Rhabdomyolysis and cholestatic hepatitis under treatment with simvastatin and chlorzoxazone]. / Rhabdomyolyse und cholestatische Hepatitis unter der Behandlung mit Simvastatin und Chlorzoxazon.

Acute rhabdomyolysis under treatment with HMG-CoA reductase inhibitors ("statins") is a group-specific if rare side effect. Muscle toxicity of statins can be potentiated by medication influencing their metabolism. Here drug interactions on the level of the microsomal cytochrome P450 enzymes play an important role. We report the first case of marked rhabdomyolysis with cholestatic hepatitis in a 73-year-old woman treated with simvastatin and chlorzoxazone. Withdrawal of the causal medication and conservative therapy with volume substitution and forced diuresis was followed by almost complete resolution of the symptoms with normalisation of the blood tests. Possible mechanisms involved in the drug interactions are discussed. Thorough knowledge of the enzyme systems involved in drug metabolism helps to predict possible adverse drug interactions and prevent toxic effects.

Nitric oxide stimulates chronic ceramide formation in glomerular endothelial cells.

Exposure of glomerular endothelial cells for 24 h to compounds releasing NO, including spermine-NO, MAHMA-NO, and S-nitroso-glutathione, results in a dose-dependent and delayed (after 24 h) increase in the lipid signaling molecule ceramide. This NO-induced stimulation occurs in a cGMP-independent fashion since the membrane-permeant cGMP analogue dibutyryl cGMP has no effect on chronic ceramide production. Short-term incubation of endothelial cells for 20 min reveals that NO and dibutyryl cGMP fail to stimulate an acute ceramide increase, whereas TNF-alpha, a well-known activator of sphingomyelinases, is able to acutely increase ceramide formation. Interestingly, N-oleoylethanolamine, an acidic ceramidase inhibitor, potentiates NO-induced chronic ceramide production, indicating that ceramide generation rather than ceramide metabolism is modulated by NO. Furthermore, NO-induced delayed ceramide formation is partially inhibited by the thiol-specific inhibitor iodoacetamide and the radical scavenger alpha-tocopherol, suggesting a regulatory role of thiol-containing enzymes and the involvement of a redox-sensitive mechanism. In addition, NO causes an increased DNA fragmentation in glomerular endothelial cells which is further enhanced by N-oleoylethanolamine and can be mimicked by exogenous ceramide. In summary, these results imply that ceramide represents an important mediator of NO-triggered chronic cell responses like apoptosis. Inhibition of ceramide synthesis may provide a new therapeutic approach to the treatment of pathological conditions involving increased NO formation.

[Neurosarcoidosis]. / Die Neurosarkoidose.

Signs and symptoms of neurosarcoidosis are variable and depend on location and size of granulomas. Clinical studies suggest a rate of 5% and autopsy results a rate of more than 25% of central nervous system (CNS) involvement in sarcoidosis. Statistical analysis of 57,789 patients admitted to the Department of Medicine in Lucerne over an 11-year period revealed 51 patients (0.9/1000) with the diagnosis of sarcoidosis. Six of these (12%) had sarcoidosis affecting the CNS. Neurosarcoidosis presented as: leptomeningeal granulomas, cranial nerve palsy, hypothalamic-pituitary syndrome, diabetes insipidus, pareses, paresthesia, pyramidal signs, dementia, urine retention, and asymptomatic granulomas. Neurosarcoidosis has predilections for the base of the brain, cranial nerves (facial nerve palsy is the most common) and meninges, but any part of the CNS may be affected. Therefore, the diagnosis of neurosarcoidosis may be extremely difficult, especially when it occurs as an isolated finding. Positive findings in transbronchial biopsy and lavage may demonstrate asymptomatic pulmonary involvement in as many as 50% of patients with neurosarcoidosis. Angiotensin-converting enzyme levels may be raised in the blood or cerebrospinal fluid in some 50% of cases. Kveim test has a low sensitivity in neurosarcoidosis and thus is of little use. Gallium uptake may demonstrate extracranial granuloma available for biopsy. All these tests, and also computed tomography and magnetic resonance imaging, may be helpful. However, when in selected cases with isolated CNS disease standard investigations are not conclusive, meningeal or cerebral biopsy may be required in order to exclude other causes such as other granulomatous disorders, tumor metastasis, lymphoma, vasculitis, Sjögren syndrome, infection, neurologic disease such as multiple sclerosis, or systemic diseases such as Whipple's disease. CNS involvement in the acute phase of the disease has a favorable prognosis, while chronic courses respond less well to therapy. Treatment is initiated most frequently with corticosteroids (0.5-1 mg/kg body weight/day or pulses of 1 g/day of methylprednisolone in severe cases). Improvement is seen within 1-2 months. Side effects of corticosteroids, aggressive disease or frequent recurrences may require other immunosuppressive drugs (methotrexate, azathioprine, chlorambucil, cyclosporine A). Cerebral irradiation may be successful in some cases when other treatments fail.

Feedback regulation of extracellular ATP-stimulated phosphoinositide hydrolysis by protein kinase C-alpha in bovine glomerular endothelial cells.

In glomerular endothelial cells, extracellular ATP stimulates a phospholipase C with subsequent hydrolysis of polyphosphoinositides and an increase in cytosolic free Ca2+ concentration ([Ca2+]i). Short-term (30 min) pretreatment of endothelial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C (PKC), decreases the ATP-stimulated phosphoinositide degradation and Ca2+ mobilization. However, this inhibition was lost after incubating the cells for four hours with TPA. Longer-term pretreatment (10 to 48 hr) even potentiated ATP-induced phosphoinositide breakdown and Ca2+ mobilization. In addition, pretreating the cells for 30 minutes with the specific PKC inhibitor Ro 31-8220 dose-dependently increased ATP-stimulated phosphoinositide hydrolysis, thus clearly indicating a regulatory role for PKC in the inositol lipid signaling pathway in glomerular endothelial cells. By using specific antibodies recognizing the different PKC isoenzymes, it is observed that glomerular endothelial cells express five isoenzymes: PKC-alpha, -delta, -epsilon, -zeta and -theta. No PKC-beta, -gamma, -eta and -mu isoenzymes were detected. On exposure to TPA, a complete depletion of PKC-alpha is observed within four hours. In contrast, PKC-epsilon was more resistant to phorbol ester, and even after 48 hours of TPA treatment, only 60% of PKC-epsilon was down-regulated. PKC-theta decreased very slowly from the cytosol (47% left after 24 hr of phorbol ester treatment) and translocated to the Triton X100-insoluble fraction. Moreover, PKC-delta and PKC-zeta were not significantly affected by 48 hours of phorbol ester incubation. Thus, only PKC-alpha is depleted with a kinetic that corresponds to the loss of feedback inhibition of ATP-stimulated phosphoinositide turnover. In the next step, [Ca2+]i changes were measured in single cells loaded with Fura-2 after microinjection of neutralizing PKC isoenzyme-specific antibodies. Injection of antibodies specific for PKC-alpha potently increased Ca2+ mobilization in response to ATP stimulation when compared to cells injected with buffer only or antibodies specific for PKC-epsilon. These results provide evidence that PKC-alpha mediates feedback inhibition of ATP-stimulated phosphoinositide hydrolysis in glomerular endothelial cells.

Agonist-induced activation of a non-selective ion current in glomerular endothelial cells.

The control of intracellular calcium activity ([Ca2+]i) and membrane voltage (Vm) play an important role in regulating functions of glomerular endothelial cells (GEC). We investigated the effect of extracellular ATP on the intracellular [Ca2+]i, Vm and ion conductances in GEC. ATP (100 mumol/liter) induced a rapid increase of [Ca2+]i in GEC from 20 +/- 6 to 442 +/- 84 nmol/liter, which was followed by a sustained Ca2+ plateau of 112 +/- 29 nmol/liter. In a bath solution with a low extracellular Ca2+ concentration the ATP-induced [Ca2+]i peak was still present, but the [Ca2+]i plateau was completely prevented. In 186 experiments with the patch clamp technique the addition of ATP (1 to 100 mumol/liter) to GEC induced a transient small hyperpolarization, which was followed by a depolarization. During the ATP-induced depolarization an increase of the whole cell conductance was found. The Ca2+ ionophore A23187 (10 mumol/liter) mimicked the effect of ATP on Vm. Reduction of the extracellular Ca2+ to 1 mumol/liter itself depolarized GEC reversibly from -88 +/- 2 to -60 +/- 12 mV and increased the ATP-induced depolarization to -18 +/- 3 mV. In the absence of Na+ in the bathing solution (replacement by NMDG+) ATP induced only an attenuated depolarization and no inward current was activated. Flufenamate (100 mumol/liter), a blocker of non-selective ion channels inhibited the ATP-induced depolarization of Vm significantly by 58 +/- 13%, whereas nicardipine (10 mumol/liter) or amiloride (10 mumol/liter) had no effect. Our data indicate that the resting Vm of GEC cells is almost completely dominated by K+ conductances and that ATP activates a Ca2+ dependent non-selective ion conductance in GEC.

Nitric oxide donors induce apoptosis in glomerular mesangial cells, epithelial cells and endothelial cells.

Renal mesangial cells exposed to inflammatory cytokines produce high concentrations of nitric oxide (NO) which may exert cytotoxic actions. We report here that glomerular mesangial cells, endothelial cells and epithelial cells in culture are themselves targets for NO and undergo apoptotic cell death upon exposure to high concentrations of NO. NO generated from different NO-releasing compounds as well as NO-saturated solution induce apoptosis in all three cell types as demonstrated by internucleosomal DNA fragmentation, an enrichment of cytosolic DNA/histone complexes, an increasing number of cellular 3'-OH-fragmented DNA ends and typical nuclear chromatin condensation. Induction of apoptosis was found to be dependent on protein synthesis and is preceded by expression of the tumour suppressor gene product p53 in mesangial cells. Induction of inducible NO synthase in mesangial cells by interleukin-1 beta leads to excessive formation of NO by the cells as measured by nitrite production. However, there was no evidence for apoptotic changes in mesangial cells triggered by endogenously produced NO. Co-cultures of glomerular endothelial or epithelial cells with interleukin-1 beta-activated mesangial cells expressing inducible NO synthase do not show apoptotic alterations in endothelial or epithelial cells. Moreover, preincubation of mesangial cells with interleukin-1 beta protects the cells from apoptosis induced by subsequent addition of exogenous NO thus suggesting that interleukin-1 beta not only triggers the expression of inducible NO synthase and massive NO formation but simultaneously stimulates a protecting principle in the cells. In summary, these results suggest that exogenous NO can induce apoptosis in all three types of intrinsic glomerular cells. However, whether endogenously produced NO can fulfil this function critically depends on a balance between a yet to be defined protective mechanism and inducible NO synthase expression in mesangial cells in response to interleukin-1 beta and eventually other inflammatory cytokines.

Extracellular ATP regulates glomerular endothelial cell function.

1. Glomerular endothelial cells form the inner part of the filtration barrier and are involved in pathophysiological processes in the glomerulum. New techniques for culturing glomerular endothelial cells have been established recently. The effect of extracellular ATP on membrane voltage and intracellular calcium activity was examined in bovine glomerular endothelial cells (GEC) in culture. 2. Membrane voltage was measured with the patch clamp technique in the fast whole cell configuration. GEC possess a stable membrane voltage of -88 mV. ATP induced a small transient hyperpolarization, which was followed by a depolarization. The ATP-induced depolarization was significantly inhibited by flufenamate, a blocker of non-selective ion channels. 3. The intracellular calcium activity [Ca2+]i was measured in single cells with the fura-2 technique. ATP stimulated an increase of [Ca2+]i. The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free solution, whereas the plateau was inhibited. 4. The order of potency of different purine nucleotides in stimulating [Ca2+]i and inositol formation was UTP = ATP > ATP-gamma-S > 2-methylthio ATP > [alpha,beta-CH2]ATP. 5. The data indicate that ATP regulates membrane voltage and [Ca2+]i in glomerular endothelial cells by a P2y2 receptor.