Bioengineered kidney tubules efficiently excrete uremic toxins.

The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs.

Effect of dipyridamole on myocardial reperfusion injury: A double-blind randomized controlled trial in patients undergoing elective coronary artery bypass surgery.

Dipyridamole reduces reperfusion-injury in preclinical trials and may be beneficial in patients undergoing coronary angioplasty, but its effect on patients undergoing coronary artery bypass grafting (CABG) is unknown. We hypothesized that dipyridamole limits myocardial reperfusion-injury in patients undergoing CABG. The trial design was a double-blind trial randomizing between pretreatment with dipyridamole or placebo. In all, 94 patients undergoing elective on-pump CABG were recruited between February 2010 and June 2012. The primary endpoint was plasma high-sensitive (hs-) troponin-I at 6, 12, and 24 hours after reperfusion. Secondary endpoints were the occurrence of bleeding, arrhythmias, need for inotropic support, and intensive care unit length of stay. Finally, 79 patients (33 dipyridamole) were included in the per-protocol analysis. Dipyridamole did not significantly affect postoperative hs-troponin-I (change in plasma hs-troponin I -3% [95% confidence interval -23% to 36%]; P > 0.1). Secondary endpoints did not differ between groups. Dipyridamole prior to CABG does not significantly reduce postoperative hs-troponin release.

Can sulfasalazine therapy induce or exacerbate Wegener's granulomatosis?

Sulfasalazine (SSZ) can induce serological and clinical autoimmune reactions but the occurrence of SSZ-related Wegener's granulomatosis (WG) has not been reported before. We describe two patients with rheumatoid factor (RF)-positive rheumatoid arthritis (RA) who developed biopsy-proven WG with serious organ involvement during SSZ therapy. The pathogenetic mechanism that explains the relationship between SSZ and the occurrence of a de novo anti-neutrophil cytoplasmic antibody (ANCA)-related vasculitis or a flare is discussed. We propose that WG can be a rare complication of SSZ therapy and that this, like other autoimmune adverse events of this drug, is mediated by SSZ-induced apoptosis.

Multidrug resistance protein mrp2 mediates ATP-dependent transport of classic renal organic anion p-aminohippurate.

p-Aminohippurate (PAH) is widely used as a model substrate to characterize organic anion transport in kidney proximal tubules. The carrier responsible for uptake of PAH across the basolateral membrane has been cloned and well characterized, whereas transporters mediating PAH excretion across the brush-border (apical) membrane are yet unknown. In this study we investigated whether PAH is a substrate for the apical multidrug resistance protein 2 (Mrp2). Overexpression of recombinant rabbit Mrp2 in Sf9 cells significantly increased ATP-dependent [(14)C]PAH uptake into isolated membrane vesicles compared with endogenous ATP-dependent uptake. The Michaelis-Menten constant and maximal velocity for Mrp2-mediated ATP-dependent [(14)C]PAH transport were 1.9 +/- 0.8 mM and 187 +/- 29 pmol. mg(-1). min(-1), respectively. On the basis of the inhibitory profile, the endogenous ATP-dependent PAH transporter does not appear to be an ortholog of Mrp2. Together, our results show that Mrp2 is a low-affinity ATP-dependent PAH transporter, indicating that Mrp2 might contribute to urinary PAH excretion.

Mechanisms and interaction of vinblastine and reduced glutathione transport in membrane vesicles by the rabbit multidrug resistance protein Mrp2 expressed in insect cells.

The present study examined how the multidrug resistance protein (MRP) 2, which is an ATP-dependent anionic conjugate transporter, also mediates transport of the chemotherapeutic cationic drug vinblastine (VBL). We show that ATP-dependent [(3)H]VBL (0.2 microM) uptake into membrane vesicles from Sf9 cells infected with a baculovirus encoding rabbit Mrp2 (Sf9-Mrp2) was similar to vesicles from mock-infected Sf9 cells (Sf9-mock) but could be stimulated by reduced glutathione (GSH) with a half-maximum stimulation of 1.9 +/- 0.1 mM. At 5 mM GSH, initial ATP-dependent [(3)H]VBL uptake rates were saturable with an apparent K(m) of 1.5 +/- 0.3 microM. The inhibitory effect of VBL on Mrp2-mediated ATP-dependent transport of the anionic conjugate [(3)H]leukotriene C(4) was potentiated by increasing GSH concentrations. Membrane vesicles from Sf9-Mrp2 cells exhibited a approximately 7-fold increase in initial GSH uptake rates compared with membrane vesicles from Sf9-mock cells. Uptake of [(3)H]GSH was osmotically sensitive, independent of ATP, and was trans-inhibited by GSH. The anionic conjugates estradiol-17beta-D-glucuronide and leukotriene C(4) cis-inhibited [(3)H]GSH uptake but only in the presence of ATP. Whereas ATP-dependent [(3)H]VBL uptake was stimulated by GSH, VBL did not affect [(3)H]GSH uptake. Our results show that GSH is required for Mrp2-mediated ATP-dependent VBL transport and that Mrp2 transports GSH independent of VBL.

Prevention of oxidant-induced cell death in Caco-2 colon carcinoma cells after inhibition of poly(ADP-ribose) polymerase and Ca2+ chelation: involvement of a common mechanism.

The human colon carcinoma cell line Caco-2 was exposed to the oxidative stress-inducing agents menadione (MEN), 2,3-dimethoxy-1,4-naphthoquinone, and hydrogen peroxide. All three agents caused DNA damage which was assessed by alkaline unwinding. Further, all three agents induced intensive NAD+ depletion, followed by a decrease in intracellular ATP and viability. Inhibition of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) by 3-aminobenzamide prevented the depletion of NAD+. These cells had a higher viability and ATP content. The most pronounced effect was observed with 25 microM of MEN, while at higher levels a partial preservation of NAD+ was observed with no effect on ATP or viability. The chelation of intracellular calcium by bis-(o-aminophenoxy)-ethane-N,N,N1,N1-tetraacidic acid/tetraacetoxymethyl) ester also prevented the dramatic loss of NAD+, demonstrating that Ca2+ is an activating factor in PARP-mediated cell killing.

Quinone toxicity in DT-diaphorase-efficient and -deficient colon carcinoma cell lines.

The human colon carcinoma cell lines Caco-2 and HT-29 were exposed to three structurally related naphthoquinones. Menadione (MEN), 1,4-naphthoquinone (NQ), and 2,3-dimethoxy-1,4-naphthoquinone (DIM) redoxcycle at similar rates, NQ is a stronger arylator than MEN, and DIM does not arylate thiols. The Caco-2 cell line was particularly vulnerable to NQ and MEN and displayed moderate toxic effects of DIM. The HT-29 cell line was only vulnerable to NQ and MEN after inhibition of DT-diaphorase (DTD) with dicoumarol, whereas dicoumarol did not affect the toxicity of quinones to Caco-2 cells. DTD activity in the HT-29 and Caco-2 cell lines, as estimated by the dicoumarol-sensitive reduction of 2,6-dichlorophenolindophenol, was 393.7 +/- 46.9 and 6.4 +/- 2.2 nmol NADPH x min(-1) x mg protein(-1), respectively. MEN depleted glutathione to a small extent in the HT-29 cell line, but a rapid depletion similar to Caco-2 cells was achieved when dicoumarol was added. The data demonstrated that the DTD-deficient Caco-2 cell line was more vulnerable to arylating or redoxcycling quinones than DTD-expressing cell lines. Exposure of the Caco-2 cell line to quinones produced a rapid rise in protein disulphides and oxidised glutathione. In contrast to NQ and DIM, no intracellular GSSG was observed with MEN. The relatively higher levels of ATP in MEN-exposed cells may account for the efficient extrusion of intracellular GSSG. The reductive potential of the cell as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction was only increased by MEN and not with NQ and DIM. We conclude that arylation is a major contributing factor in the toxicity of quinones. For this reason, NQ was the most toxic quinone, followed by MEN, and the pure redoxcycler DIM elicited modest toxicity in Caco-2 cells.

Effects of the peroxisome proliferator mono(2-ethylhexyl)phthalate in primary hepatocyte cultures derived from rat, guinea pig, rabbit and monkey. Relationship between interspecies differences in biotransformation and peroxisome proliferating potencies.

Primary hepatocyte cultures derived from rat, rabbit, guinea pig and monkey have been treated in vitro with metabolites of di(2-ethylhexyl)phthalate, i.e. mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (metabolite V) and mono(2-ethyl-5-oxohexyl)phthalate (metabolite VI). In rat hepatocyte cultures MEHP and metabolite VI were equally potent in inducing peroxisome proliferation, while metabolite V was much less potent. In rat hepatocytes a 50% increase in both peroxisomal palmitoyl-CoA oxidase activity and microsomal lauric acid omega-hydroxylation activity was found after treatment with 5-15 microM MEHP. In guinea pig, rabbit and monkey hepatocyte cultures, a 50% increase in peroxisomal palmitoyl-CoA oxidase activity was found after treatment with 408-485 microM MEHP. No induction of lauric acid omega-hydroxylation activity was found. These results indicate that peroxisome proliferation can be induced by MEHP in rabbit, guinea pig and monkey hepatocytes, but that these species are at least 30-fold less sensitive to peroxisome proliferation induction than rats. The proposed mechanistic inter-relationship between induction of lauric acid omega-hydroxylation activity and peroxisome proliferation is found in rat hepatocytes, but not in hepatocytes of the other three species. Treatment of guinea pig hepatocyte cultures with MEHP resulted in an increase in triglyceride concentrations in the hepatocytes. In rat and rabbit hepatocyte cultures, triglyceride concentrations were much less altered by MEHP. In monkey hepatocytes a decrease in hepatic triglyceride concentration was found after treatment with MEHP. These effects are in agreement with in vivo effects observed before. After treatment of primary hepatocyte cultures with MEHP, high concentrations of omega- and (omega-1)-hydroxylated metabolites of MEHP were found in media from rat, rabbit and guinea pig cultures. The formation of these metabolites did not decline in time. During treatment the metabolite profile in media from rat hepatocyte cultures moved towards omega-hydroxy metabolites of MEHP. In media from monkey hepatocyte cultures the lowest concentrations of hydroxylated metabolites were determined. No major species differences were found in the potency to form oxidized MEHP metabolites, and thus no unique metabolite differences were found, which could explain the species differences in sensitivity for peroxisome proliferation.

Microsomal lauric acid hydroxylase activities after treatment of rats with three classical cytochrome P450 inducers and peroxisome proliferating compounds.

In order to investigate a proposed relationship between induction of hepatic microsomal lauric acid hydroxylase activity and peroxisome proliferation in the liver, male Wistar rats were treated with peroxisome proliferating compounds, and the lauric acid hydroxylase activity, the immunochemical detectable levels of cytochrome P450 4A1 and the activities of peroxisomal enzymes were determined. In addition, the levels of cytochrome P450 4A1 and lauric acid hydroxylase activities were studied after treatment of rats with three cytochrome P450 inducers. After treatment with aroclor-1254, phenobarbital or 3-methylcholanthrene total cytochrome P450 was 1.7-2.7 times induced. However, no induction of lauric acid omega-hydroxylase activities or P450 4A1 levels were found. After treatment of rats with di(2-ethylhexyl)phthalate (DEHP) a dose-dependent induction of lauric acid omega-hydroxylase activities, levels of cytochrome P450 4A1 and peroxisomal fatty acid beta-oxidation was found. Even at a dose-level of 100 mg DEPH/kg body weight per day a significant induction of these activities was observed. The main metabolites of DEHP, mono(2-ethylhexyl)phthalate and 2-ethyl-1-hexanol, also caused an induction of levels of P450 4A1, lauric acid omega-hydroxylase activities and the activity of peroxisomal palmitoyl-CoA oxidase. 2-Ethyl-1-hexanoic acid did not influence lauric acid omega-hydroxylase activities, but did induce levels of P450 4A1 and palmitoyl-CoA oxidase activities. Three other compounds (perfluoro-octanoic acid, valproate and nafenopin) induced both lauric acid omega-hydroxylase activity and peroxisomal palmitoyl-CoA oxidase activity. The plasticizer, di(2-ethylhexyl)adipate, did not induce levels of P450 4A1, lauric acid omega-hydroxylase activities or palmitoyl-CoA oxidase activities. With the compounds tested a close association between the induction of lauric acid omega-hydroxylase activities and peroxisomal palmitoyl-CoA oxidase activity was found. These data support the theory that peroxisome proliferating compounds do induce lauric acid omega-hydroxylase activities and that there might be a mechanistic inter-relationship between peroxisome proliferation and induction of lauric acid omega-hydroxylase activities.

The use of a grommet bone liner for flexible hinge implant arthroplasty of the great toe.

Press-fit titanium grommets were developed to shield flexible hinged silicone implants used for arthroplasty of the radiocarpal, metacarpophalangeal, and metatarsophalangeal joints. Since 1985, 179 titanium circumferential grommets were used in 90 first metatarsophalangeal joints with excellent, pain-free, functional results and favorable bone response around the implant stems and at the bone-grommet interface. There were no complications due to particulate reactivity, implant fracture, or grommet fracture. The use of circumferential titanium grommets appears to be a safe and effective method to improve the long-term durability of flexible hinge implant arthroplasty of the first metatarsophalangeal joint.

Urinary cyclophosphamide assay as a method for biological monitoring of occupational exposure to cyclophosphamide.

Urine of twenty hospital workers was monitored for the excretion of the cytostatic drug cyclophosphamide using GC-MSD. The drug was found to be present above the detection limit of 0.5 microgram/24 h urine in five cases. A clear relationship between cyclophosphamide handling and the detectability of excretion existed. This method developed can be of use for biological monitoring studies directed toward the finding of exposure hazards.

Isolation, partial identification and quantitative determination of four guaiphenesin glucuronides in plasma and urine of the horse by high-performance liquid chromatography.

The isolation, partial identification and quantitative determination of four guaiphenesin glucuronides in plasma and urine of the horse is described. The identity of the glucuronides was checked by UV and fluorescence spectrophotometry, by NMR spectrometry and by mass spectrometry after permethylation. The applicability of the procedure to pharmacokinetic studies is demonstrated.

Dopamine, noradrenaline and serotonin spinal fluid metabolites in temporal lobe epileptic patients with schizophrenic symptomatology.

Cerebrospinal fluid metabolites of dopamine, norepinephrine and serotonin were studied in two groups of left temporal lobe epileptic patients, the only difference between the two groups being that one group had paranoid schizophrenic symptomatology. Homovanillic acid accumulation was decreased in this group. This finding may be a biochemical indicator of a genetic predisposition to schizophrenic symptomatology in certain epileptic patients.