Tomasz Domaniewski

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.

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 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.

Erythropoietin accelerates tumor growth through increase of erythropoietin receptor (EpoR) as well as by the stimulation of angiogenesis in DLD-1 and Ht-29 xenografts.

Anemia is a relatively common symptom coexisting with colorectal carcinoma. Besides having a positive impact on hematological parameters, erythropoietin (Epo) has the serious adverse effect of promoting the neoplastic process. The role of Epo in colon cancer has not been clearly shown. The aim of this study was to assess the effects of Epo therapy on colorectal carcinoma cells both in in vitro and in animal models. Human colon adenocarcinoma cells DLD-1 and Ht-29 were cultured in medium with Epo beta in normoxia. Cell proliferation was measured with an automated cell counter. Expression of erythropoietin receptor (EpoR) mRNA, Akt mRNA, and their proteins were assessed by RT-PCR and confocal microscopy, respectively. Nude mice were inoculated with adenocarcinoma cells and treated with a therapeutic dose of Epo. Expression of EpoR, VEGF, Flt-1 and CD31 was evaluated in xenograft tumors. We identified that Epo through EpoR activates Akt, which promotes colon cancer cell growth and proliferation. Epo, and high levels of phosphorylated EpoR, directly accelerates tumor growth through its proliferative and proangiogenic effects. This study demonstrated that Epo had enhanced carcinogenesis through increase of EpoR and Flt-1 expression, and thereby contributed to tumor development. These results suggest that both EpoR-positive and EpoR-negative cancer cells could be regulated by exogenous Epo. However, an increased response to erythropoietin was observed in the EpoR-positive cells. Thus, erythropoietin increases the risk of tumor progression in colon cancer and should not be used to treat anemia in this type of cancer.

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.

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.

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.

ERYTHROPOIETIN INCREASES Epo AND EpoR EXPRESSION IN DLD-1 CELLS

Introduction. Supplementation of recombinant human erythropoietin (rHuEpo) is one of the methods for the treatment of anemia for patients with colon cancer. However, the results of in vitro studies investigating the influence of rHuEpo on cancer cells are contradictory. Aim. The aim of the present study was an assessment of the effect of rHuEpo on proliferation, as well as Epo and EpoR protein expressions in normoxia and hypoxia conditions on human colon adenocarcinoma cells (DLD-1). Materials and methods. The cells were cultured in medium with rHuEpo in con centrations of 1 and 3 IU without (normoxia) or with (hypoxia) cadmium chloride for 48 hours. Cell viability was counted using a haematocytometer and trypan blue 0.4% (w/v) dye. Expression of Epo and EpoR protein was assessed by western blot. Results and Discussion. We observed a decrease in the number of colon cancer cells in hypoxia. Addition of rHuEpo did not modify cell numbers in normoxia and hypoxia. We found a significant increase of EpoR expression in all cells growing in medium with cobalt chloride in comparison with respective normoxic cells. We also noted that rHuEpo in concentration of 3 IU significantly increased expression of Epo and EpoR protein in colon cancer cells in normoxia and hypoxia conditions. Conclusions. We concluded that Epo and EpoR are constitutively expressed in DLD-1 cells. In hypoxia as well as in the presence of rHuEpo the increase of Epo and EpoR protein was found. However, the expression of Epo and EpoR protein in these cells does not seem essential to their growth.

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.