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Background and Aims Chronic kidney disease (CKD) is connected with accumulation of uremic toxins that leads to dysfunction of many systems including skeletal system. Indoxyl sulfate (IS) is one of the most potent protein-bound uremic toxin. However, its impact on bone and mineral disorders remains unclear. Sirtuins (SIRT) are members of the class III type nicotinamide adenine dinucleotide-dependent histone deacetylases. Their roles are commonly linked to aging, metabolism, and antioxidant defense. However, SIRT1, SIRT3 and SIRT7 maintain also bone formation. In turn, reduction of SIRT2 has a protective role due to reduced activation of osteoclasts-mediated bone resorption. Thus, the aim of this work was to evaluate IS impact on bone metabolism and bone properties, and SIRT genes expression. Method Male Wistar rats weighting 180 – 210 g were divided into 2 groups: experimental receiving IS in the dose of 200 mg/kg of b.w. for four weeks (200IS), and control receiving pure water (CON). After four weeks the femurs and tibiae were collected, and geometrical parameters (CI – cortical index, CSA – cortical sectional area, CSMI – cross sectional moment of inertia, MRWT – mean relative wall thickness) were measured. The IS bone (cortical and trabecular), plasma and urine levels were determined by high-performance liquid chromatography (HPLC). The concentrations of Ca, Mg, Zn, Fe, Mn, and Cu in bone tissue were determined by the flame atomic absorption spectrometry. Bone turnover markers (alkaline phosphatase – ALP, and tartrate-resistant acid phosphatase - TRAP) were determined in homogenates from trabecular and cortical left femurs. The expression of ALP, TRAP, SIRT1, SIRT2, SIRT3 and SIRT7 gene was assessed by QRT-PCR in right femurs. Results Plasma, urine, cortical and trabecular bone IS concentrations were increased in 200IS compared to CON. Geometrical properties of femur (CI, CSA, CSMI and MRWT) were significantly reduced in 200IS. There were no differences in relative femur weight and length between studied groups. The activity of ALP and TRAP in bone tissue were significantly lower in 200IS compared to CON. In turn, the expression of TRAP gene was increased in 200IS. There were no difference in concentrations of Ca, Mg, Fe, Mn, and Cu in bone tissue. Only Zn concentration in cortical bone was reduced in 200IS. The expressions of SIRT3 and SIRT7 were decreased in 200IS, whereas SIRT2 was increased in 200IS. Chronic exposure to IS did not affect expression of SIRT1. Conclusion Obtained results suggest that IS affects bone properties and bone metabolism, and has impact on expression of SIRT2, SIRT3, and SIRT7 genes. Our findings may help to better understand mechanisms leading to dysfunction of skeletal system during CKD. This work was supported by Polish National Science Centre (Grant No. 2017/27/N/NZ4/00033).

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.

Background and Aims Abnormalities in bone metabolism represent the most complex disorder accompanying a chronic kidney disease (CKD) development. Serotonin – one of the tryptophan metabolites, has received intensive attention due to its potential role in bone metabolism. The synthesis of peripheral serotonin is initiated by the tryptophan hydroxylase-1 (TPH-1) in the gut. LP533401 is a small-molecule inhibitor of TPH-1. Previously, we found that inhibition of TPH-1 by LP533401 may improve bone mineralization in rats with experimentally induced CKD (Pawlak et al, Bone, 2018). The aim of this study was to establish whether the inhibition of serotonin synthesis by LP533401 may affect kynurenine pathway activity in bone tissue of nephrectomized rats, and to determine the potential consequence of this process in relation to osteoblastogenesis. Method Nephrectomized rats were randomized into: untreated (CKD), treated with vehicle (VEH), treated with LP533401 at a dose of 30 (LP 30) and 100 mg/kg (LP 100) daily for 8 weeks. The shame-operated animals served as controls (CON). Tryptophan (TRP) and kynurenine (KYN) concentrations in trabecular and cortical homogenates of femoral bone were determined using high-performance liquid chromatography (HPLC). The expression of tryptophan 2,3-dioxygenase (TDO) as well as genes associated with osteoblastogenesis: activating transcription factor 4 (ATF4), forkhead box protein O1 (FOXO1), Cyclin D1, osteocalcin (Bglap) and sclerostin (Sost) were assessed using the QRT-PCR method. Results We demonstrated a presence of TDO-dependent, paracrine kynurenic system in bone homogenates of rats with CKD. The significant decrease in TDO mRNA level was observed in the bone of all nephrectomized groups in comparison with CON (all p In LP 100 group, the TDO expression was still lower than in CON, but was significantly higher than in VEH and LP 30-treated groups (both p Conclusion This study for the first time demonstrated the presence of a TDO-dependent, paracrine kynurenic system in the bone of rats with CKD. The modulation of this system during LP533401 treatment was closely associated with the expression of genes participating in osteoblastogenesis, particularly with osteoblast maturation markers. These results represent the next step in studying the role of tryptophan metabolites in renal osteodystrophy.

Background and Aims Chronic kidney disease - mineral bone disorder (CKD–MBD) is one of the major clinical complications in patients with chronic kidney disease (CKD). Bone remodeling has been suggested to play a fundamental role in the maintenance of skeletal integrity via a balance between the bone formation/resorption process. The parathyroid hormone (PTH) is a key hormone controlling bone metabolism. PTH is known to affect bone with a net catabolic and anabolic effect but the mechanisms responsible for these differing effects are poorly understood. Previously, we demonstrated the distinct effect of mild degree of CKD on trabecular and cortical bone strength in rapidly growing rats (Pawlak et al. PlosOne, 2016). The aim of the present study was to evaluate the effect of endogenous PTH, its receptor PTH1R and activating transcription factor 4 (ATF4) – the major regulators of the anabolic PTH response in osteoblasts on bone remodeling and growth of young rats with experimental CKD. Method Four-week old Wistar male rats were divided into 2 groups: with CKD induced by surgical 5/6 subtotal nephrectomy, and sham-operated (CON). After one (CON-1; CKD-1) and three months (CON-3; CKD-3) of the surgery the femurs were collected and their lengths were measured. The activity of alkaline phosphatase (ALP), a bone formation marker, and tartrate-resistant acid phosphatase (TRACP5b) reflecting bone resorption were determined in homogenates from trabecular and cortical left femurs. The expression of PTH1R and ATF4 gene was determined by QRT-PCR in right femurs. Serum PTH was analyzed using ELISA kit. Computations were performed using Statistica ver.10 computer software. Results Serum PTH was increased in CKD-3 compared to CKD-1, moreover the slight increase in PTH levels was noted in CKD-3 compared to appropriate controls (both p Conclusion The endogenous PTH, through PTH1R/ATF4 axis, inhibited trabecular bone remodeling. In contrary, PTH/PTH1R/ATF4 system intensified bone resorption in cortical bone region. This opposite effect of PTH on bone remodeling was associated with the intensification of growth process in the long bones of young rats with CKD.

Background and Purpose Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase involved in the activation of signaling 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 anemia, which is treated with an erythropoietin supplement. The goal of the present study was to assess the effects of combination therapy with erythropoietin beta (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, cell viability, expression of EpoR, BTK, Akt at mRNA, and protein levels were assessed. Nude mice were inoculated with adenocarcinoma cells and treated with Epo and LFM-A13. Key Results Simultaneous use of Epo and LFM-A13 exerts a mostly synergistic inhibitory effect on colon cancer cell growth. The featured therapeutic scheme resulted in effective cell killing, accompanied by attenuation of the BTK signaling pathways. Epo+LFM-A13 also prevented the normal process of microtubule assembly during mitosis via the down-regulation of PLK1 expression. The combined administration of Epo and LFM-A13 significantly reduced the growth rate of tumor cells, while it showed high-profile safety with no induced nephrotoxicity, hepatotoxicity, or changes in the hematological parameters. Conclusion and Implications Our findings demonstrate that Epo significantly enhances the antitumor activity of LFM-A13, indicating the potential use of a combination of Epo and LFM-A13 as an effective therapeutic approach for patients with colorectal cancer.

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.

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 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.

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.

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.

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 β (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.

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.

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.

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.

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.

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.

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.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.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 p0.001), and with SOX markers: Cu/Zn SOD (both p0.0001) and MDA levels (p0.05, and p0.0001; respectively) in the whole ESRD group. The positive relationship were between Cu/Zn SOD and KYN (p0.010), 3-HKYN and QA levels (both p0.0001), whereas MDA was correlated with 3-HKYN and QA concentrations (both p0.05). 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.Our study demonstrated that kynurenine metabolites and increased oxidative status are independently and significantly associated with endothelial dysfunction in ESRD patients.

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

Although there are some in vitro evidence that angiotensin II (Ang II) may promote thrombosis, there is still no data concerning effect of Ang II on arterial thrombus formation. In the present study we have investigated the influence of Ang II on electrically induced arterial thrombosis in a common carotid artery of renovascular hypertensive rats. Furthermore, we examined if Ang II effect is mediated via AT1 receptor. We measured some coagulation and fibrinolytic parameters at the same time. Since platelets play crucial role in the initiation of arterial thrombosis their contribution in the mode of Ang II action was also determined. Intravenous infusion of Ang II caused significant increase in arterial thrombus weight, which was reversed by losartan, selective AT1 receptor antagonist. The prothrombotic effect of Ang II was accompanied by increase in haemostatic and decrease in fibrinolytic potential of rat plasma. While number of data has clearly demonstrated that Ang II can augment human platelets aggregation, at least in rats, platelets were not involved in the mechanism of Ang II action. Our study shows that Ang II via AT1 receptor accelerates arterial thrombosis in renovascular hypertensive rat, therefore may be considered as a risk factor of myocardial infarction or stroke.

The aim of this study was to determine whether AT 1 -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 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 A 2 mimetic U46619 and this action was preserved in L-NAME-pretreated animals. Our results demonstrate the ability of AT 1 -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 A 2 /prostaglandin H 2 receptor, could be of potential clinical relevance, particularly in conditions in which synthesis of endogenous nitric oxide is impaired.

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.