Dose-dependent exposure to indoxyl sulfate alters AHR signaling, sirtuins gene expression, oxidative DNA damage, and bone mineral status in rats.

Indoxyl sulfate (IS), an agonist of aryl hydrocarbon receptors (AhR), can accumulate in patients with chronic kidney disease, but its direct effect on bone is not clear. The present study investigated the effect of chronic exposure to low (100 mg/kg b.w.; 100 IS) and high (200 mg/kg b.w.; 200 IS) dose of IS on bone AhR pathway, sirtuins (SIRTs) expression, oxidative DNA damage and bone mineral status in Wistar rats. The accumulation of IS was observed only in trabecular bone tissue in both doses. The differences were observed in the bone parameters, depending on the applied IS dose. The exposure to 100 IS increased AhR repressor (AhRR)-CYP1A2 gene expression, which was associated with SIRT-1, SIRT-3 and SIRT-7 expression. At the low dose group, the oxidative DNA damage marker was unchanged in the bone samples, and it was inversely related to the abovementioned SIRTs expression. In contrast, the exposure to 200 IS reduced the expression of AhRR, CYP1A, SIRT-3 and SIRT-7 genes compared to 100 IS. The level of oxidative DNA damage was higher in trabecular bone in 200 IS group. Femoral bone mineral density was decreased, and inverse relations were noticed between the level of trabecular oxidative DNA damage and parameters of bone mineral status. In conclusion, IS modulates AhR-depending signaling affecting SIRTs expression, oxidative DNA damage and bone mineral status in a dose dependent manner.

Serum PTH, PTH1R/ATF4 pathway, and the sRANKL/OPG system in bone as a new link between bone growth, cross-sectional geometry, and strength in young rats with experimental chronic kidney disease.

The progression of chronic kidney disease (CKD) in children is associated with deregulated parathyroid hormone (PTH), growth retardation, and low bone accrual. PTH can cause both catabolic and anabolic impact on bone, and the activating transcription factor 4 (ATF4), a downstream target gene of PTH, is related to its anabolic effect. Osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) are PTH-dependent cytokines, which may play an important role in the regulation of bone remodeling. This study aimed to evaluate the impact of endogenous PTH and the bone RANKL/OPG system on bone growth, cross-sectional geometry and strength utilizing young, nephrectomized rats. The parameters of cross-sectional geometry were significantly elevated in rats with CKD during the three-month experimental period compared with the controls, and they were strongly associated with serum PTH levels and the expression of parathyroid hormone 1 receptor (PTH1R)/ATF4 genes in bone. Low bone soluble RANKL (sRANKL) levels and sRANKL/OPG ratios were also positively correlated with cross-sectional bone geometry and femoral length. Moreover, the analyzed geometric parameters were strongly related to the biomechanical properties of femoral diaphysis. In summary, the mild increase in endogenous PTH, its anabolic PTH1R/ATF4 axis and PTH-dependent alterations in the bone RANKL/OPG system may be one of the possible mechanisms responsible for the favorable impact on bone growth, cross-sectional geometry and strength in young rats with experimental CKD.

Modulation of the Paracrine Kynurenic System in Bone as a New Regulator of Osteoblastogenesis and Bone Mineral Status in an Animal Model of Chronic Kidney Disease Treated with LP533401.

An increase in the peripheral synthesis of serotonin and kynurenine, observed during the chronic kidney disease (CKD) course, is negatively associated with bone health. Serotonin and kynurenine are connected by the common precursor, tryptophan. LP533401 is an inhibitor of peripheral serotonin synthesis. This study aimed to establish if the inhibition of serotonin synthesis by LP533401 may affect the kynurenine pathway activity in bone tissue and its potential consequence with regard to osteogenesis and bone mineral status. Nephrectomized rats were treated with LP533401 at a dose of 30 and 100 mg/kg daily for eight weeks. Tryptophan and kynurenine concentrations were determined, and tryptophan 2,3-dioxygenase (TDO) expression was assessed. We discovered the presence of a TDO-dependent, paracrine kynurenic system in the bone of rats with CKD. Its modulation during LP533401 treatment was associated with impaired bone mineral status. Changes in TDO expression affecting the kynurenine pathway activity were related to the imbalance between peripheral serotonin and 25-hydroxyvitamin D. There were also close associations between the expression of genes participating in osteoblastogenesis and activation of the kynurenine pathway in the bones of LP53301-treated rats. Our results represent the next step in studying the role of tryptophan metabolites in renal osteodystrophy.

Inhibition of peripheral serotonin synthesis by LP533401 and disturbances in calciotropic hormones attenuated excessive osteoblastogenesis with simultaneous improvement of bone mineral status in 5/6 nephrectomized rats.

Chronic kidney disease (CKD) is a pathological condition associated with renal osteodystrophy for which there are limited treatment options. Gut-derived serotonin (GDS) is one of the key signaling factors controlling the osteoblast proliferation. Previously, we shown that inhibition of GDS synthesis by LP533401 improved bone mineral status of rats with 5/6 nephrectomy-induced CKD model. Here, we investigated whether the use of LP533401 can modify GDS-dependent molecular pathway involved in osteoblast formation and bone mineralization in CKD rats. The 8-weeks of pharmacological manipulation after a complete CKD development reduced GDS and lead to the advantage of endogenous vitamin D [25(OH)D] over serotonin and parathyroid hormone (PTH) in rats treated with LP533401. The imbalance between GDS - 25(OH)D - PTH resulted in the intensified expression of cAMP- responsive element-binding protein (Creb), whereas the expression of myelocytomatosis oncogene (c-Myc) was simultaneously reduced. This lead to disruption of Foxo1- activating transcription factor 4 (Atf4) complex, and decrease in the expression of the major osteogenic markers. The weakening of excessive osteoblastogenesis was associated with better bone mineral status in all rats with CKD, and especially in LP533401-treated animals. In conclusion, the inhibition of GDS synthesis resulted in the mitigation of osteoblastogenesis observed in CKD, which translated into improvement of bone mineral status. This study provides key mechanistic insights into how modification of GDS-dependent molecular pathway affects bone mineral status in CKD and lays the groundwork for translating the role of functional serotonin signaling in the origin of impaired bone mineral status in patients with CKD.

The use of LP533401 as a therapeutic option for renal osteodystrophy affects, renal calcium handling, vitamin D metabolism, and bone health in uremic rats.

BACKGROUND: Klotho is a key regulator of phosphate and Ca2+-transport in the kidney. Recently, we showed that treatment with LP533401 improved bone health in rats with chronic kidney disease (CKD) via the normalization of serum phosphate resulting from the reduced renal expression of phosphate cotransporters, including Klotho. METHODS: We evaluated the effect of LP533401 therapy on Klotho-expression-dependent Ca2+-transporters, renal calcium handling, and the potential consequences for the bone of uremic rats. RESULTS: Treatment with LP533401 and its vehicle resulted in the inhibition of transient receptor potential vanilloid receptor subtypes 5 and 6 (TRPV5, TRPV6) and calbindin (CaBP-28k, CaBP-9k) expression. The compensatory acceleration in renal expression of Na+/Ca2+-exchanger, 25-hydroxyvitamin d-1α-hydroxylase (CYP27B1), the intensification of vitamin D metabolism, and disruption of sophisticated balance between 1,25-dihydroxyvitamin D-serotonin was observed, especially in rats treated with LP533401. The imbalance between 1,25-dihydroxyvitamin D-serotonin levels led to intensified bone remodeling and improvement in bone geometry, mineral status, and strength in animals treated with LP533401. CONCLUSION: The modulation of circulating serotonin and its relation to other regulators of calcium handling can play an important role in calcium homeostasis and bone integrity in CKD rats treated with LP533401.

Indoxyl Sulfate Promotes Arterial Thrombosis in Rat Model via Increased Levels of Complex TF/VII, PAI-1, Platelet Activation as Well as Decreased Contents of SIRT1 and SIRT3.

Patients suffering from chronic kidney disease (CKD) are at a 20-fold higher risk of dying due to cardiovascular diseases (CVDs), primarily thrombosis following vascular injury. CKD is connected with retention of uremic toxins, especially indoxyl sulfate (IS), which are currently considered as a non-classical CKD-specific risk factor for CVDs. The present study aimed to examine the effect of chronic exposure to IS on the hemostatic system and arterial thrombosis in a model without greater interferences from the uremic milieu consisting of additional uremic toxins. Forty-eight male Wistar Crl:WI (cmdb) rats were divided into three groups: one control group and two experimental groups, which were exposed to 100 or 200 mg/kg of b.w./day of IS in drinking water for a period of 28 days. The control group received water without IS. At the end of the experiment, the induction of arterial thrombosis was performed. We investigated the impact of IS on thrombosis incidence, kinetics and strength of clot formation, platelet activity, aortic contents of sirtuin (SIRT) 1 and sirtuin 3 (SIRT3), hemostatic system, cardiorespiratory parameters, biochemistry of plasma and urine as well as histology of the thrombus, kidney, and liver. Obtained data revealed that chronic exposure to IS promotes arterial thrombosis via increased levels of complex tissue factor/factor VII, plasminogen activator inhibitor-1 (PAI-1), platelet activation, as well as decreased aortic levels of SIRT1 and SIRT3. Therefore, we hypothesize that IS enhances primary hemostasis leading to augmented formation of platelet plug with increased amounts of fibrin and affects secondary hemostasis through the influence on plasma coagulation and fibrinolysis factors, which results in the increased kinetics and strength of clot formation. The findings described may contribute to a better understanding of the mechanisms leading to increased thrombotic events in patients with CKD with elevated levels of IS.

LP533401 restores bone health in 5/6 nephrectomized rats by a decrease of gut-derived serotonin and regulation of serum phosphate through the inhibition of phosphate co-transporters expression in the kidneys.

LP533401 is an orally bioavailable small molecule that inhibits tryptophan hydroxylase-1, an enzyme responsible for the synthesis of gut-derived serotonin (GDS). Recently, we showed that increased GDS in rats with chronic kidney disease (CKD) affected bone strength and metabolism. We tested the hypothesis that treatment with LP533401 could reverse CKD-induced bone loss in uremia. Sixteen weeks after 5/6 nephrectomy, rats were randomized into untreated (CKD), treated with vehicle (VEH) and LP533401 at a dose of 30 or 100 mg/kg daily for 8 weeks. Treatment with LP533401 decreased serotonin turnover and restored bone mineral status, microarchitecture, and strength in CKD rats to the values observed in the controls. In parallel with the reduction of serotonin, serum phosphate levels also decreased, particularly in the LP533401, 100 mg/kg group. The mechanism underlying this phenomenon resulted from decreased expression of the renal VDR/FGF1R/Klotho/Npt2a/Npt2c axis, leading to elevated phosphate excretion in the kidneys. The elevated urinary phosphate excretion resulted in improved bone mineral status and strength in LP533401-treated rats. Unexpectedly, the standard VEH used in this model was able to reduce renal VDR/FGF1R/Klotho/Npt2a expression, leading to a compensatory increase in Npt2c mRNA levels, secondary disturbances in phosphate-regulated hormones and partial improvement in the mineral status of the trabecular bone. The decrease of serotonin synthesis together with the simultaneous reduction of renal Npt2a and Npt2c expression in rats treated with LP533401, 100 mg/kg led to an increase in 1,25(OH)2D3 levels; this mechanism seems to be particularly beneficial in relation to the mineral status of cortical bone.

RANKL/OPG system regulation by endogenous PTH and PTH1R/ATF4 axis in bone: Implications for bone accrual and strength in growing rats with mild uremia.

Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), and parathyroid hormone (PTH) play a central role in the regulation of bone turnover in chronic kidney disease (CKD), but their influence on bone mineral density (BMD) and strength remains unclear, particularly in children. We studied the clinical significance of OPG and RANKL in relation to PTH, femur weight, BMD, and bone biomechanical properties in growing rats after one month (CKD-1) and three months (CKD-3) of surgically-induced mild CKD. Gene expression of parathyroid hormone 1 receptor (PTH1R) and activating transcription factor 4 (ATF4), major regulators of anabolic PTH response in bone, was also determined. Serum PTH and bone PTH1R/ATF4 expression was elevated in CKD-3 compared with other groups, and it positively correlated with femur weight, BMD, and the biomechanical properties of the femoral diaphysis reflecting cortical bone strength. In contrast, bone RANKL/OPG ratios were decreased in CKD-3 rats compared with other groups, and they were inversely correlated with PTH and the other abovementioned bone parameters. However, the PTH-PTH1R-ATF4 axis exerted an unfavorable effect on the biomechanical properties of the femoral neck. In conclusion, this study showed for the first time an inverse association between serum PTH and the bone RANKL/OPG system in growing rats with mild CKD. A decrease in the RANKL/OPG ratio, associated with PTH-dependent activation of the anabolic PTH1R/ATF4 pathway, seems to be responsible for the unexpected, beneficial effect of PTH on cortical bone accrual and strength. Simultaneously, impaired biomechanical properties of the femoral neck were observed, making this bone site more susceptible to fractures.

Simultaneous use of erythropoietin and LFM-A13 as a new therapeutic approach for colorectal cancer.

BACKGROUND AND PURPOSE: Bruton's tyrosine kinase (Btk) is a non-receptor tyrosine kinase involved in the activation of signalling pathways responsible for cell maturation and viability. Btk has previously been reported to be overexpressed in colon cancers. This kind of cancer is often accompanied by anaemia, which is treated with an erythropoietin supplement. The goal of the present study was to assess the effects of combination therapy with erythropoietin ß (Epo) and LFM-A13 (Btk inhibitor) on colon cancer in in vitro and in vivo models. EXPERIMENTAL APPROACH: DLD-1 and HT-29 human colon adenocarcinoma cells were cultured with Epo and LFM-A13. Cell number and viability, and mRNA and protein levels of Epo receptors, Btk and Akt were assessed. Nude mice were inoculated with adenocarcinoma cells and treated with Epo and LFM-A13. KEY RESULTS: The combination of Epo and LFM-A13 mostly exerted a synergistic inhibitory effect on colon cancer cell growth. The therapeutic scheme used effectively killed the cancer cells and attenuated the Btk signalling pathways. Epo + LFM-A13 also prevented the normal process of microtubule assembly during mitosis by down-regulating the expression of Polo-like kinase 1. The combination of Epo and LFM-A13 significantly reduced the growth rate of tumour cells, while it showed high safety profile, inducing no nephrotoxicity, hepatotoxicity or changes in the haematological parameters. CONCLUSION AND IMPLICATIONS: Epo significantly enhances the antitumour activity of LFM-A13, indicating that a combination of Epo and LFM-A13 has potential as an effective therapeutic approach for patients with colorectal cancer.

Elevated Levels of Peripheral Kynurenine Decrease Bone Strength in Rats with Chronic Kidney Disease.

The diagnosis and treatment of bone disorders in patients with chronic kidney disease (CKD) represent a clinical challenge. CKD leads to mineral and bone complications starting early in the course of renal failure. Recently, we have observed the positive relationship between intensified central kynurenine turnover and bone strength in rats with subtotal 5/6 nephrectomy (5/6 Nx)-induced CKD. The aim of the present study was to determine the association between peripheral kynurenine pathway metabolites and bone strength in rats with 5/6 Nx-induced CKD. The animals were sacrificed 1 and 3 months after 5/6 Nx or sham operation. Nephrectomized rats presented higher concentrations of serum creatinine, urea nitrogen, and parathyroid hormone both 1 and 3 months after nephrectomy. These animals revealed higher concentrations of kynurenine and 3-hydroxykynurenine in the serum and higher gene expression of aryl hydrocarbon receptor (AhR) as a physiological receptor for kynurenine and AhR-dependent cytochrome in the bone tissue. Furthermore, nephrectomy significantly increased the number of osteoclasts in the bone without affecting their resorptive activity measured in serum. These changes were particularly evident in rats 1 month after 5/6 Nx. The main bone biomechanical parameters of the tibia were unchanged between nephrectomized and sham-operated rats but were significantly increased in older compared to younger animals. A similar trend was observed for geometrical parameters measured with calipers, bone mineral density based on Archimedes' method and image of bone microarchitecture obtained from micro-computed tomography analyses of tibial cortical bone. In nephrectomized animals, peripheral kynurenine levels correlated negatively with the main parameters of bone biomechanics, bone geometry, and bone mineral density values. In conclusion, our data suggest that CKD-induced elevated levels of peripheral kynurenine cause pathological changes in bone structure via AhR pathway. This finding opens new opportunities for the treatment/prevention of osteoporosis in CKD.

The impact of peripheral serotonin on leptin-brain serotonin axis, bone metabolism and strength in growing rats with experimental chronic kidney disease.

Chronic kidney disease (CKD) results in decreased bone strength. Serotonin (5-HT) is one of the critical regulators of bone health, fulfilling distinct functions depending on its synthesis site: brain-derived serotonin (BDS) favors osteoblast proliferation, whereas gut-derived serotonin (GDS) inhibits it. We assessed the role of BDS and peripheral leptin in the regulation of bone metabolism and strength in young rats with 5/6 nephrectomy. BDS synthesis was accelerated during CKD progression. Decreased peripheral leptin in CKD rats was inversely related to BDS content in the hypothalamus, brainstem and frontal cortex. Serotonin in these brain regions affected bone strength and metabolism in the studied animals. The direct effect of circulating leptin on bone was not shown in uremia. At the molecular level, there was an inverse association between elevated GDS and the expression of cAMP responsive element-binding protein (Creb) gene in bone of CKD animals. In contrast, increased expression of activating transcription factor 4 (Atf4) was shown, which was associated with GDS-dependent transcription factor 1 (Foxo1), clock gene - Cry-1, cell cycle genes: c-Myc, cyclins, and osteoblast differentiation genes. These results identified a previously unknown molecular pathway, by which elevated GDS can shift in Foxo1 target genes from Creb to Atf4-dependent response, disrupting the leptin-BDS - dependent gene pathway in the bone of uremic rats. Thus, in the condition of CKD the effect of BDS and GDS on bone metabolism and strength can't be distinguished.

A link between central kynurenine metabolism and bone strength in rats with chronic kidney disease.

BACKGROUND: Disturbances in mineral and bone metabolism represent one of the most complex complications of chronic kidney disease (CKD). Serotonin, a monoamine synthesized from tryptophan, may play a potential role in bone metabolism. Brain-derived serotonin exerts a positive effect on the bone structure by limiting bone resorption and enhancing bone formation. Tryptophan is the precursor not only to the serotonin but also and primarily to kynurenine metabolites. The ultimate aim of the present study was to determine the association between central kynurenine metabolism and biomechanical as well as geometrical properties of bone in the experimental model of the early stage of CKD. METHODS: Thirty-three Wistar rats were randomly divided into two groups (sham-operated and subtotal nephrectomized animals). Three months after surgery, serum samples were obtained for the determination of biochemical parameters, bone turnover biomarkers, and kynurenine pathway metabolites; tibias were collected for bone biomechanical, bone geometrical, and bone mass density analysis; brains were removed and divided into five regions for the determination of kynurenine pathway metabolites. RESULTS: Subtotal nephrectomized rats presented higher serum concentrations of creatinine, urea nitrogen, and parathyroid hormone, and developed hypocalcemia. Several biomechanical and geometrical parameters were significantly elevated in rats with experimentally induced CKD. Subtotal nephrectomized rats presented significantly higher kynurenine concentrations and kynurenine/tryptophan ratio and significantly lower tryptophan levels in all studied parts of the brain. Kynurenine in the frontal cortex and tryptophan in the hypothalamus and striatum correlated positively with the main parameters of bone biomechanics and bone geometry. DISCUSSION: In addition to the complex mineral, hormone, and metabolite changes, intensified central kynurenine turnover may play an important role in the development of bone changes in the course of CKD.

Erythropoietin Enhances the Cytotoxic Effect of Hydrogen Peroxide on Colon Cancer Cells.

BACKGROUND: Cancer patients treated with alkylating agents and radiotherapy are exposed to high level of reactive oxygen species (ROS) in tissues. ROS can involve superoxide free radicals, peroxynitrite, singlet oxygen, nitric oxide and hydrogen peroxide. It is well documented that increased exposure to oxygen through a high metabolic rate could lead to a shortened life span. Ionizing radiation, use of drugs and the development of cancer can lead to cancer-induced anemia. Recombinant human erythropoietin (Epo) supplementation is one of the methods for treating anemia. Erythropoietin through an increase in the number of erythrocytes, improves oxygenation tissue. The aim of this work was to study the effect of Epo on colon adenocarcinoma cells (DLD-1) given alone or in combination with hydrogen peroxide (H2O2). Cell proliferation and number were measured. METHODS: Expression of EpoR, Bcl-2 and Akt1 protein was assessed by RT-PCR, Western blot, and confocal microscopy. RESULTS: The results show that the coadministration of Epo and H2O2 indicates antitumor action, which occurs via a dose-dependent inhibition of DLD-1 cell growth and proliferation. Moreover, the coadministration of Epo and H2O2 resulted in a decrease of cell numbers, as well as Bcl-2 expression. The incubation of DLD-1 cells with those agents led to a decrease in EpoR and phosphorylated EpoR expression and an increase in Akt1 and phosphorylated Akt expression. The addition of Epo to H2O2 intensified the cytotoxic effect of the latter. CONCLUSION: These preclinical results suggest that Epo during chemotherapy or radiotherapy may possess potential benefits in colon cancer patients.

The Association between Elevated Levels of Peripheral Serotonin and Its Metabolite - 5-Hydroxyindoleacetic Acid and Bone Strength and Metabolism in Growing Rats with Mild Experimental Chronic Kidney Disease.

Chronic kidney disease (CKD) is associated with disturbances in bone strength and metabolism. The alterations of the serotonergic system are also observed in CKD. We used the 5/6 nephrectomy model of CKD to assess the impact of peripheral serotonin and its metabolite- 5-hydroxyindoleacetic acid on bone biomechanical properties and metabolism in growing rats. The animals were sacrificed one and three months after nephrectomy. Biomechanical properties were determined on two different bone types: the cortical bone of the femoral diaphysis using three-point bending test and the mixed cortico-trabecular bone by the bending test of the femoral neck. Biomechanical tests revealed preserved cortical bone strength, whereas work to fracture (W) and yield load (Fy) of mixed cortico-trabecular bone were significantly lower in CKD compared to controls. Serum activity of alkaline phosphatase (ALP), a bone formation marker, and tartrate-resistant acid phosphatase (TRACP 5b) reflecting bone resorption, were similar in CKD and controls. ALP was associated with lower femoral stiffness and strength, and higher displacements and W. TRACP 5b was inversely associated with cortical Fu and W. The elevated peripheral serotonergic system in CKD was: inversely associated with stiffness but positively related to the displacements and W; inversely associated with cortical Fy but positively correlated with this parameter in cortico-trabecular bone; inversely associated with ALP in controls but positively correlated with this biomarker in CKD animals. In conclusion, this study demonstrates the distinct effect of mild degree of CKD on bone strength in rapidly growing rats. The impaired renal function affects the peripheral serotonin metabolism, which in turn may influence the strength and metabolism of bones in these rats. This relationship seems to be beneficial on the biomechanical properties of the cortico-trabecular bone, whereas the cortical bone strength can be potentially reduced.

Serum metabolic fingerprinting after exposure of rats to quinolinic acid.

Quinolinic acid (QUIN), one of the end metabolites in the kynurenine pathway, plays an important role in the pathogenesis of several diseases. Serum QUIN concentration rises in patients with renal dysfunction, liver cirrhosis, and many other inflammatory diseases. In the present study, osmotic minipumps containing QUIN (0.3 and 1mg/day) were implanted intraperitoneally into rats for 28days. Then, the physiological and toxicological variables were evaluated and LC-QTOF-MS serum metabolic fingerprinting was performed. QUIN significantly decreased the serum concentrations of several amino acids (phenylalanine, valine, tyrosine, and tryptophan), pantothenic acid, branched chain C4 acylcarnitine, total cholesterol, and glucose; increased the serum concentrations of amides (pentadecanoic amide, palmitic amide, oleamide, and stearamide), polyamines (spermine and spermidine), sphingosine, and deoxy-prostaglandin; caused alterations in phospholipids. This is the first report of comprehensive metabolites analysis after chronic intraperitoneal administration of QUIN. Further studies could develop new therapeutics for patients with disorders accompanied by increased serum level of QUIN.

Effect of erythropoietin, 5-fluorouracil and SN-38 on the growth of DLD-1 cells.

Supplementation of recombinant human erythropoietin (rHuEpo) is one of the methods for the treatment of anemia. The influence of rHuEpo on proliferation or clonogenic growth of cancer cells is not clear and some of the published results are conflicting. The aim of this work was to study the effect of rHuEpo on colon cancer cells when given alone or in combination with cytostatics. Human colon adenocarcinoma cells (DLD-1) were cultured in medium with rHuEpo, 5-fluorouracil (5-FU) and an active metabolite of irinotecan (SN-38). Cell viability was determined using a hematocytometer and 0.4% (w/v) trypan blue dye. Cell proliferation was measured by the MTT assay. Expression of EpoR, Bax, Bcl-2 and Akt1 protein was assessed by Western blot. The results of this study indicate a dose-dependent inhibitory effect of rHuEpo on DLD-1 cell growth and proliferation. Moreover, the combined treatment of rHuEpo and cytotoxic agents such as 5-FU and SN-38 increases the antitumor action, which is indicated by decreases in proliferation in the MTT test, cell numbers and DNA synthesis. We found a significant increase in EpoR, Bcl-2 and Akt1 protein expression in all cells grown in medium containing 3 IU of rHuEpo. We observed that EpoR is constitutively expressed in DLD-1 cells. Our results indicate that rHuEpo acts via EpoR to directly inhibit DLD-1 cell growth and indirectly modulate the cytostatics effects of 5-FU and SN-38.

Kynurenines and oxidative status are independently associated with thrombomodulin and von Willebrand factor levels in patients with end-stage renal disease.

INTRODUCTION: Increased oxidative stress (SOX) is one of the most potent inductors of endothelial dysfunction in end-stage renal disease (ESRD) patients. Kynurenines are the metabolites of tryptophan (TRP) degradation in mammals. However, the role of kynurenines in the function of the endothelium is still not recognized. MATERIALS AND METHODS: We determined the plasma concentrations of TRP, kynurenine (KYN), 3-hydroxykynurenine (3-HKYN), quinolinic acid (QA); markers of SOX: Cu/Zn superoxide dismutase (Cu/Zn SOD), malondialdehyde (MDA); and endothelial dysfunction markers: thrombomodulin (TM) and von Willebrand factor (vWF) levels in 148 ESRD patients and healthy controls. RESULTS: TM, vWF, KYN, 3-HKYN and QA levels were significantly elevated in ESRD patients compared to controls. TRP concentrations in uremics were significantly lower than in healthy people. Both dialyzed groups showed a significant increase Cu/Zn SOD and MDA levels compared to controls. TM and vWF were positively associated with kynurenine pathway metabolites: KYN, 3-HKYN, QA (all p<0.001), and with SOX markers: Cu/Zn SOD (both p<0.0001) and MDA levels (p<0.05, and p<0.0001; respectively) in the whole ESRD group. The positive relationship were between Cu/Zn SOD and KYN (p<0.010), 3-HKYN and QA levels (both p<0.0001), whereas MDA was correlated with 3-HKYN and QA concentrations (both p<0.05). Multiple stepwise regression analysis showed that KYN metabolites and oxidative status were the independent variables significantly associated with increased both TM and vWF levels in uremic patients. CONCLUSIONS: Our study demonstrated that kynurenine metabolites and increased oxidative status are independently and significantly associated with endothelial dysfunction in ESRD patients.

The kynurenines are associated with oxidative stress, inflammation and the prevalence of cardiovascular disease in patients with end-stage renal disease.

Increased oxidative stress (SOX), inflammation and prevalence of cardiovascular disease (CVD) have been reported in end-stage renal disease (ESRD), but their associations with kynurenine (KYN) pathway activation remain unknown. We determined the plasma concentrations of tryptophan (TRP), KYN, 3-hydroxykynurenine (3-HKYN); two distinct SOX markers: Cu/Zn superoxide dismutase (Cu/Zn SOD) and total peroxide; and high sensitivity C-reactive protein (hs CRP) as a indicator of inflammation in 146 ESRD patients and healthy controls. Analysis of TRP degradation through the KYN pathway demonstrated that in uremia the concentrations of this aminoacid were decreased by 40-60% in comparison with controls. In contrast, the plasma levels of KYN and 3-HKYN in ESRD patients were increased by 32-96% and 184-306%, respectively. These changes were accompanied by significant increase in the kyn/trp ratios by 140-240%, and 3-hkyn/kyn ratios by 40-154% in uremics compared to controls. ESRD patients showed a significant increase in Cu/Zn SOD, total peroxide and hs CRP levels between controls and all patients group. KYN and 3-HKYN were positively associated with inflammation and SOX markers in uremics. Logistic regression analysis showed that age, gender, presence of DM (all p<0.001), elevated hs CRP (p<0.01) and 3-HKYN levels (p<0.05) were independently associated with the presence of CVD in this population. These results suggest a relationship between KYN pathway activation and increased SOX, inflammation and CVD prevalence in ESRD patients.

Nitric oxide-dependent antiplatelet action of AT1-receptor antagonists in a pulmonary thromboembolism in mice.

The aim of this study was to determine whether AT1-receptor antagonists could inhibit platelet activation-dependent pulmonary thromboembolism in mice and to investigate the involvement of nitric oxide in this action. Losartan, its active metabolite EXP3174, and valsartan given intraperitoneally 1 hour before the thrombotic challenge (in doses of 3, 10, or 30 mg/kg) protected mice from death or hind-limb paralysis in response to intravenous injection of a mixture of collagen and epinephrine; losartan was effective in all doses used, whereas EXP3174 and valsartan reduced mortality only in the two higher doses. The protective action of EXP3174 and valsartan was abolished when nitric oxide synthase was inhibited with l-NAME, whereas that of losartan was only partially reduced. Moreover, only losartan protected mice from death caused by intravenous injection of the thromboxane A2 mimetic U46619 and this action was preserved in l-NAME-pretreated animals. Our results demonstrate the ability of AT1-receptor antagonists to inhibit platelet activation in vivo in a nitric oxide-dependent mechanism. Stronger antiplatelet activity of losartan, most likely due to its blockade of thromboxane A2/prostaglandin H2 receptor, could be of potential clinical relevance, particularly in conditions in which synthesis of endogenous nitric oxide is impaired.