Orellanine specifically targets renal clear cell carcinoma.

Renal cell carcinoma (RCC), arising from the proximal tubule in the kidney, accounts for approximately 85% of kidney cancers and causes over 140,000 annual deaths worldwide. In the last decade, several new therapies have been identified for treatment of metastatic RCC. Although these therapies increase survival time compared to standard care, none of them has curative properties. The nephrotoxin orellanine specifically targets proximal tubular epithelial cells, leaving other organs unaffected. We therefore hypothesized that the selective toxicity of orellanine extends to clear cell RCC (ccRCC) cells since they emanate from proximal tubular cells. Orellanine would thus target both primary and metastatic ccRCC in vitro and in vivo. We found that orellanine induces dose-dependent cell death in proximal tubular cells and in all ccRCC cells tested, both primary and cell lines, with no toxicity detected in control cells. The toxic action of orellanine involve decreased protein synthesis, disrupted cell metabolism and induction of apoptosis. In nude rats carrying human ccRCC xenografts, brief orellanine treatment eliminated more than 90% of viable tumor mass compared to control rats. This identifies orellanine as a potential treatment concept for ccRCC patients on dialysis, due to its unique selective toxicity towards ccRCC.

The glomerular endothelial cell coat is essential for glomerular filtration.

The endothelial cell surface layer (ESL) is believed to contribute to the glomerular barrier, and the nature of its molecular structure is still largely unknown. The ESL consists of the membrane-bound glycocalyx and the loosely attached endothelial cell coat (ECC). A brief injection of hypertonic sodium chloride into the left renal artery was used to displace, elute, and collect non-covalently bound components of the renal ESL in rats. This procedure increased the fractional clearance of albumin 12-fold without detectable morphological changes as assessed by electron microscopy compared with the control group injected with isotonic saline. Mathematical modeling suggested a reduced glomerular charge density. Mass spectrometry of the renal eluate identified 17 non-covalently bound proteins normally present in the ECC. One of these proteins, orosomucoid, has previously been shown to be important for capillary permselectivity. Another protein, lumican, is expressed by glomerular endothelial cells and likely contributes to maintaining an intact barrier. Thus, the absence of one or more of these proteins causes proteinuria and illustrates the importance of the ECC in glomerular permselectivity.

Impaired glomerular and tubular antioxidative defense mechanisms in nephrotic syndrome.

The molecular mechanisms behind acquired nephrotic syndrome (NS) are still largely unknown. One possible explanation for the development of proteinuria is oxidative damage to the glomerular cells. Our hypothesis was that the oxidative defense is weakened in NS, and we focused on measurements of the oxidative-antioxidative status in the glomerular and tubular parts of the nephron. Gene expression was analyzed in renal biopsies from patients with NS. In addition, to compare the acute and chronic phases of the disease, we studied puromycin-treated rats. In the biopsy material, the expression of enzymes involved in the antioxidative defense was higher in the tubulointerstitial compartment than in the glomerular cells. Real-time PCR analysis revealed a decreased glomerular expression in nephrotic kidneys for the antioxidant enzymes catalase and glutathione peroxidase-3, and -4. The tubular gene expression was downregulated for catalase, glutathione peroxidase-3, and thioredoxin reductase-1 and -2. The altered gene expression was accompanied by increased lipid peroxidation in urine. In rats, serum concentrations of ascorbyl-free radicals, measured with electron spin resonance, were elevated in the acute phase of the disease, suggesting increased oxidative stress in the circulation. In addition, we saw an increase in the plasma antioxidant capacity combined with a decreased oxidation of proteins in sera from nephrotic rats, but not from humans. In conclusion, there is a marked downregulation of several antioxidative enzymes in nephrotic kidneys, especially in glomerular structures. Our data suggest that oxidative damage to glomerular cells may contribute significantly to the course and prognosis of nephrotic syndrome.

The fungal nephrotoxin orellanine simultaneously increases oxidative stress and down-regulates cellular defenses.

Confusion of various nephrotoxic Cortinarius species with edible mushrooms occurs every year throughout Europe and North America. The toxin, orellanine (OR), accumulates selectively in renal tubular epithelium with ensuing renal failure after several days as the only clinical manifestation. This study was performed to clarify the mechanisms behind the kidney damage. Sprague-Dawley rats, 100 g bw, received various doses of purified OR ip (0-5 mg/kg bw). One week later, renal function (GFR) was determined (51Cr-EDTA), ascorbyl radicals in venous blood were analyzed using electron spin resonance, and oxidative protein damage was evaluated immunohistochemically. One OR-treated group (3.5 mg/kg) simultaneously received superoxide dismutase (SOD) targeted to tubular epithelium (HC-SOD; 10 mg/kg ip daily for 5 days). RT-PCR was used for analysis of mRNA expression of genes related to oxidative stress. OR caused a dose-dependent decrease in GFR, paralleled by increased levels of ascorbyl radicals and oxidative protein damage. Antioxidant treatment with HC-SOD decreased renal function even more and also increased tissue damage and mortality. Renal mRNA levels for key components in the antioxidative defense were strongly decreased, whereas those for several cytokines were increased. The data strongly suggest that OR nephrotoxicity in vivo is mediated by oxidative stress, including a virtual shutdown of important antioxidative enzymes. We interpret the unexpected effect of HC-SOD in terms of unbalanced SOD and catalase levels in the presence of OR, leading to massive generation of *OH and cell death.

Transplantation of the uterus in the sheep: oxidative stress and reperfusion injury after short-time cold storage.

OBJECTIVE: To study the effects of cold ischemia and reperfusion after transplantation of the sheep uterus and to compare the preservation solution Perfadex (Vitrolife, Mölndal, Sweden) with Ringer's acetate. DESIGN: Experimental animal study. SETTING: University hospital. ANIMAL(S): Adult, female sheep. INTERVENTION(S): One uterine horn with the common uterine cavity and cervix of sexually mature ewes was auto-transplanted after 1 hour of cold ischemic storage in either Perfadex (n = 5) or Ringer's acetate (n = 5). During 3 hours of reperfusion, uterine venous blood was collected and analyzed for several parameters that were indicative of oxidative stress and reperfusion injury. A biopsy was taken for histological analysis at the end of the experiment. MAIN OUTCOME MEASURE(S): Lipid peroxidation and ascorbyl radicals in uterine venous blood during reperfusion. Light microscopy and quantification of neutrophils in tissue after 3 hours of reperfusion. RESULT(S): A decline in pH and a rise in lactate and pCO(2)-pO(2), as well as an elevation of antioxidant capacity, lipid peroxidation, and intensity of ascorbyl radical electron spin resonance signal, was found that was more prominent after storage in Ringer's acetate. The histological analysis revealed mild inflammation in both study groups. CONCLUSION(S): Short-time cold ischemic storage of the sheep uterus does not induce any severe reperfusion damage, but the use of the protective buffer Perfadex decreases oxidative stress and inflammation when compared with a more simple solution.

Neurochemical markers of brain damage in cerebrospinal fluid during induction treatment of acute lymphoblastic leukemia in children.

BACKGROUND: Central nervous system (CNS) irradiation has been replaced by systemic high-dose methotrexate (MTX) and intrathecal MTX in acute lymphoblastic leukemia treatment due to the risk of late effects. However, treatment without CNS irradiation might also cause brain damage. PROCEDURE: Cerebrospinal fluid (CSF) was analyzed in 121 patients in an attempt to detect CNS injury. Seventy-three samples were analyzed for neuron-specific enolase (NSE), 108 for glial fibrillary acidic protein (GFAp), 110 for neurofilament protein light chain (NFp), and 70 for ascorbyl radical (AsR). Samples were taken at day 0, 8, 15, and 29 during induction treatment, including intrathecal MTX. Levels at days 8, 15, and 29 were compared with the levels before treatment. RESULTS: NSE levels were 9.0 (+/-3.5) microg/L (mean (+/-SD)) at day 0, 15.0 (+/-5.3) at day 8 (P < 0.001), 13.6 (+/-4.7) at day 15 (P < 0.001) and 11.1 (+/-4.3) at day 29 (P < 0.001). GFAp were 177 (+/-98) ng/L at day 0, 206 (+/-101) at day 8 (P < 0.001), 200 (+/-106) at day 15 (n.s.) and 228 (+/-137) at day 29 (P < 0.001). NFp were below the detection limit 125 ng/L at day 0 in all 110 CSF samples analyzed, and increased significantly above the detection limit in 6/77 samples at day 8, in 11/84 at day 15 and in 22/91 at day 29. The AsR content did not change significantly. CONCLUSIONS: Levels of NSE, GFAp, and NFp increased in CSF, which can be interpreted as early signs of brain damage. AsR levels do not show any convincing signs of oxidative stress.

Non-protein-bound iron in brain interstitium of newborn pigs after hypoxia.

Oxidative damage is implied in perinatal hypoxic-ischemic brain injury, most importantly in white matter. Nonprotein-bound iron (NPBI) catalyzes the formation of toxic hydroxyl radicals. We measured the extracellular level of NPBI through microdialysis in the cortex, striatum, and periventricular white matter before, during and after severe hypoxia in newborn pigs. NPBI was analyzed by a new spectrophotometric method in which ferrous iron is chelated by bathophenanthroline. NPBI was present in all brain areas under baseline conditions and increased in white matter from 0.97 (0.69) to 2.75 (1.85) micromol/l (not corrected for recovery rate) during early reoxygenation. NPBI may contribute to oxidative injury after perinatal hypoxic insults.

A new method for monitoring nitric oxide production using Teflon membrane microdialysis.

The measurement of nitric oxide (NO) by electron spin resonance (ESR) is complicated by potentially toxic spin-trapping agents, which may affect the NO-producing cells per se and/or cause artifacts and systemic side effects. These problems can be addressed by preventing direct interaction between the agent and the biological system. In the present study, we utilized Teflon as a barrier between the spin trap and the living cell, since the material is permeable to gas only. Our aim was to investigate if NO could diffuse across the membrane in sufficient amounts to be trapped and quantified by ESR. We used standard microdialysis equipment and specially designed dialysis probes, or tubing, with Teflon membranes. Sodium nitroprusside was used as a NO donor and Fe-N-dithiocarboxysarcosine (Fe(DTCS)2) as a spin trap. NO readily diffuses through Teflon and could be quantified in concentrations considerably below 50 nM in a reproducible and accurate manner. In cell cultures of activated murine macrophages, NO synthesis from iNOS could be monitored and we noted a huge increase in NO concentration by superoxide dismutase. We conclude that spin trapping of NO by Fe(DTCS)2 across Teflon membranes is an attractive approach for quantifying and monitoring nitric oxide production without interfering with cell viability.

White matter injury following prolonged free radical formation in the 0.65 gestation fetal sheep brain.

Free radicals seem to be involved in the development of cerebral white matter damage after asphyxia in the premature infant. The immature brain may be at increased risk of free radical mediated injury, as particularly the preterm infant has a relative deficiency in brain antioxidants systems, such as superoxide dismutase and glutathione peroxidase. In vitro studies show that immature oligodendrocytes express an intrinsic vulnerability to reactive oxygen species and free radical scavengers are able to protect immature oligodendrocytes from injury. The aim of this study was to examine the formation of ascorbyl radicals as a marker of oxidative stress in the preterm brain in association with cerebral white matter injury after intrauterine asphyxia. Fetal sheep at 0.65 gestation were chronically instrumented with vascular catheters and an occluder cuff around the umbilical cord. A microdialysis probe was placed in the periventricular white matter. Fetal asphyxia was induced by occlusion of the umbilical cord for 25 min (n = 10). Microdialysis samples were collected for 72 h and analyzed for ascorbyl radicals using electron spin resonance. Five instrumented fetuses served as controls. Three days after the insult, fetal brains were examined for morphologic injury. Umbilical cord occlusion resulted in prolonged and marked increase in ascorbyl radical production in the brain in connection with white matter injury, with activation of microglia cells in periventricular white matter and axonal injury. These data suggest that reperfusion injury following asphyxia in the immature brain is associated with marked free radical production.

A simple and rapid method for the determination of "free" iron in biological fluids.

We present a convenient method for determining "free" or non-protein-bound iron in biological fluids. The new method is based on the bathophenantroline method for determination of total serum iron, and comprises binding of iron by a chromogenic chelator (bathophenantroline-disulphonate, BPS), which is specific for ferrous iron. The ferrous complex of BPS absorbs strongly at 535 nm, and the detection limit is less than 1 microM in a sample size of 50 microliters. The chelator does not liberate iron from either haemoglobin or transferrin. Interference from copper or zinc in concentrations up to 50 microM does not significantly disturb measurements. The main problem when measuring in blood plasma, the high and fluctuating background in the region around 535 nm, has been overcome through filtering techniques. Data from measurements of ferrous iron in microdialysate, cerebrospinal fluid, and blood plasma in different animal models and clinical conditions are presented as illustrative examples of the usefulness of the method. The method allows the determination of ferric, as well as ferrous, iron in the same sample.