Up-regulation of HDACs, a harbinger of uraemic endothelial dysfunction, is prevented by defibrotide.

Endothelial dysfunction is an earlier contributor to the development of atherosclerosis in chronic kidney disease (CKD), in which the role of epigenetic triggers cannot be ruled out. Endothelial protective strategies, such as defibrotide (DF), may be useful in this scenario. We evaluated changes induced by CKD on endothelial cell proteome and explored the effect of DF and the mechanisms involved. Human umbilical cord vein endothelial cells were exposed to sera from healthy donors (n = 20) and patients with end-stage renal disease on haemodialysis (n = 20). Differential protein expression was investigated by using a proteomic approach, Western blot and immunofluorescence. HDAC1 and HDAC2 overexpression was detected. Increased HDAC1 expression occurred at both cytoplasm and nucleus. These effects were dose-dependently inhibited by DF. Both the HDACs inhibitor trichostatin A and DF prevented the up-regulation of the endothelial dysfunction markers induced by the uraemic milieu: intercellular adhesion molecule-1, surface Toll-like receptor-4, von Willebrand Factor and reactive oxygen species. Moreover, DF down-regulated HDACs expression through the PI3/AKT signalling pathway. HDACs appear as key modulators of the CKD-induced endothelial dysfunction as specific blockade by trichostatin A or by DF prevents endothelial dysfunction responses to the CKD insult. Moreover, DF exerts its endothelial protective effect by inhibiting HDAC up-regulation likely through PI3K/AKT.

Role of Small Interfering RNA Silencing Protein Kinase C-α Gene on the Occurrence of Ultrafiltration Failure in Peritoneal Dialysis Rats.

This study aims to explore the effect of PKC-α gene silencing on the occurrence of ultrafiltration failure (UFF) in peritoneal dialysis (PD) rats. Forty-eight male SD rats were collected to establish 5/6 renal resection uremic and uremic PD rats models. Rats were assigned into control, sham operation, uremia, PD-2 W (peritoneal dialysis for 2 weeks), PD-4 W (peritoneal dialysis for 4 weeks), negative control (NC) (peritoneal dialysis for 4 weeks, and injected 0.1 mg/kg blank plasmid into abdominal cavity) and PKC-α siRNA (peritoneal dialysis for 4 weeks, and injected 0.1 mg/kg PKC-α siRNA into abdominal cavity) groups. CD34 staining was performed to determine microvessel density (MVD) for peritoneal tissues. The mRNA and protein expression of PKC-α in peritoneal tissue were detected by qRT-PCR and Western blot. Compared with the control group, MVD, the mRNA and protein expression of PKC-α were significantly increased in rats of the uremia, PD-2 W, PD-4 W, NC, and PKC-α siRNA groups. Compared with the uremia group, MVD, the mRNA and protein expression of PKC-α were increased, the changes observed in the PD-4 W and NC groups were better obvious than in the PD-2 W group. In comparison with the PD-4 W and NC groups, MVD, the mRNA and protein expression of PKC-α in rats were decreased in the PKC-α siRNA group. PKC-α gene has a high expression in uremic PD rats, and PKC-α gene silencing is able to increase UF while decrease MVD and glucose transport in peritoneal tissues thus reversing UFF in PD rats. J. Cell. Biochem. 118: 4607-4616, 2017. © 2017 Wiley Periodicals, Inc.

Cilostazol inhibits uremic toxin-induced vascular smooth muscle cell dysfunction: role of Axl signaling.

Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, and vascular smooth muscle cell (VSMC) dysfunction plays a pivotal role in uremic atherosclerosis. Axl signaling is involved in vascular injury and is highly expressed in VSMCs. Recent reports have shown that cilostazol, a phosphodiesterase type 3 inhibitor (PDE3), can regulate various stages of the atherosclerotic process. However, the role of cilostazol in uremic vasculopathy remains unclear. This study aimed to identify the effect of cilostazol in VSMCs in the experimental CKD and to investigate whether the regulatory mechanism occurs through Axl signaling. We investigated the effect of P-cresol and cilostazol on Axl signaling in A7r5 rat VSMCs and the rat and human CKD models. From the in vivo CKD rats and patients, aortic tissue exhibited significantly decreased Axl expression after cilostazol treatment. P-cresol increased Axl, proliferating of cell nuclear antigen (PCNA), focal adhesion kinase (FAK), and matrix metalloproteinase-2 (MMP-2) expressions, decreased caspase-3 expression, and was accompanied by increased cell viability and migration. Cilostazol significantly reversed P-cresol-induced Axl, downstream gene expressions, and cell functions. Along with the increased Axl expression, P-cresol activated PLCγ, Akt, and ERK phosphorylation and cilostazol significantly suppressed the effect of P-cresol. Axl knockdown significantly reversed the expressions of P-cresol-induced Axl-related gene expression and cell functions. Cilostazol with Axl knockdown have additive changes in downstream gene expression and cell functions in P-cresol culture. Both in vitro and in vivo experimental CKD models elucidate a new signal transduction of cilostazol-mediated protection against uremic toxin-related VSMCs dysfunction and highlight the involvement of the Axl signaling and downstream pathways.

Elevated hepatic 11ß-hydroxysteroid dehydrogenase type 1 induces insulin resistance in uremia.

Insulin resistance and associated metabolic sequelae are common in chronic kidney disease (CKD) and are positively and independently associated with increased cardiovascular mortality. However, the pathogenesis has yet to be fully elucidated. 11ß-Hydroxysteroid dehydrogenase type 1 (11ßHSD1) catalyzes intracellular regeneration of active glucocorticoids, promoting insulin resistance in liver and other metabolic tissues. Using two experimental rat models of CKD (subtotal nephrectomy and adenine diet) which show early insulin resistance, we found that 11ßHSD1 mRNA and protein increase in hepatic and adipose tissue, together with increased hepatic 11ßHSD1 activity. This was associated with intrahepatic but not circulating glucocorticoid excess, and increased hepatic gluconeogenesis and lipogenesis. Oral administration of the 11ßHSD inhibitor carbenoxolone to uremic rats for 2 wk improved glucose tolerance and insulin sensitivity, improved insulin signaling, and reduced hepatic expression of gluconeogenic and lipogenic genes. Furthermore, 11ßHSD1(-/-) mice and rats treated with a specific 11ßHSD1 inhibitor (UE2316) were protected from metabolic disturbances despite similar renal dysfunction following adenine experimental uremia. Therefore, we demonstrate that elevated hepatic 11ßHSD1 is an important contributor to early insulin resistance and dyslipidemia in uremia. Specific 11ßHSD1 inhibitors potentially represent a novel therapeutic approach for management of insulin resistance in patients with CKD.

Loss of miR-125b contributes to upregulation of CYP24 in uraemic rats.

AIM: Abnormal upregulation of CYP24 contributes to vitamin D insufficiency and resistance to vitamin D therapy in chronic kidney disease (CKD), because human CYP24 is a key enzyme involved in the inactivation of 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3; calcitriol) and 1,25(OH)2D3. There are multiple mechanisms regulating CYP24 in a variety of types of tissues and diseases. An increasing body of evidence suggests that microRNA-125b (miR-125b) plays an important role in post-transcriptional regulation of CYP24 mRNA. METHODS: We sought to test hypothesis that abnormal elevation of CYP24 in CKD is a consequence of loss of miR-125b in CKD in a uraemia rat model. RESULTS: We found that expression of miR-125b was significantly inhibited in uraemic rats coupled with increased CYP24 at both protein and mRNA levels compared with normal controls. In NRK-52 kidney cells, we further found that miR-125b antagomirs increased CYP24 but miR-125b mimics decreased CYP24, and luciferase assay confirmed that CYP24 is a direct target of miR-125b. Vitamin D status exerted no significant effects on expression of both miR-125b and CYP24 in uraemic rats. CONCLUSION: These results suggest that modulation of miR-125b may be used for treatment of Vitamin D insufficiency in CKD.

Uremic toxins inhibit renal metabolic capacity through interference with glucuronidation and mitochondrial respiration.

During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2µM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13µM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.

Modulation of NADPH oxidase activity by known uraemic retention solutes.

BACKGROUND: Uraemia and cardiovascular disease appear to be associated with an increased oxidative burden. One of the key players in the genesis of reactive oxygen species (ROS) is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Based on initial experiments demonstrating a decreased inhibitory effect on NADPH oxidase activity in the presence of plasma from patients with CKD-5D after dialysis compared with before dialysis, we investigated the effect of 48 known and commercially available uraemic retention solutes on the enzymatic activity of NADPH oxidase. METHODS: Mononuclear leucocytes isolated from buffy coats of healthy volunteers were isolated, lysed and incubated with NADH in the presence of plasma from healthy controls and patients with CKD-5D. Furthermore, the leucocytes were lysed and incubated in the presence of uraemic retention solute of interest and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase. The effect on enzymatic activity of NADPH oxidase was quantified within an incubation time of 120 min. RESULTS: Thirty-nine of the 48 uraemic retention solutes tested had a significant decreasing effect on NADPH oxidase activity. Oxalate has been characterized as the strongest inhibitor of NADPH oxidase (90% of DPI inhibition). Surprisingly, none of the uraemic retention solutes we investigated was found to increase NADPH oxidase activity. Furthermore, plasma from patients with CKD-5D before dialysis caused significantly higher inhibitory effect on NADPH oxidase activity compared with plasma from healthy subjects. However, this effect was significantly decreased in plasma from patients with CKD-5D after dialysis. CONCLUSIONS: The results of this study show that uraemic retention solutes modulated the activity of the NADPH oxidase. The results of this study might be the basis for the development of inhibitors applicable as drug in the situation of increased oxidative stress.

Cyclooxygenase-2 and vascular endothelial growth factor expressions are involved in ultrafiltration failure.

BACKGROUND: Long-term peritoneal dialysis (PD) is associated with ultrafiltration failure (UFF). The aim of the study was to investigate changes in cyclooxygenase-2 (COX-2), vascular endothelial growth factor A (VEGF-A), and vascular endothelial growth factor C (VEGF-C) expressions in a rat model of UFF induced by PD solution. METHODS: Sprague-Dawley rats were divided into six groups (n = 8/group): normal untreated control group, sham operation group, uremic group (nephrectomy without PD), uremic 2-wk PD group (PD solution for 2 wk), uremic 4-wk PD group (PD solution for 4 wk), and uremic 4-wk PD + celecoxib group (PD solution plus COX-2 inhibitor celecoxib 20 mg/kg for 4 wk). Peritoneal function was determined by peritoneal equilibration test. Peritoneal morphology was determined by hematoxylin and eosin and Masson staining. Microvessel and lymphatic microvessel formation was determined by immunohistochemistry. COX-2, VEGF-A, and VEGF-C expressions were determined by real-time polymerase chain reaction and immunohistochemistry. RESULTS: Uremic rat model was successfully established. PD-induced peritoneal morphologic changes associated with UFF, characterized by inflammation, edema, and collagen accumulation. PD solution increased the density of microvessels marked by CD31 (microvessel density) and lymphatic microvessels marked by LYVE-1 (lymphatic vessel density) in peritoneum. COX-2, VEGF-A, and VEGF-C expression levels in the uremic 4-wk PD group were higher than those in the uremic group (all P < 0.05). All these changes were partially reversed by celecoxib. VEGF-A and VEGF-C protein expressions were positively correlated with microvessel density and lymphatic vessel density formation. CONCLUSIONS: COX-2 could increase VEGF-A and VEGF-C expressions in peritoneal tissue, resulting in increased formation of peritoneal microvessels and lymphatic microvessels, playing pivotal roles in the development of UFF.

Enhanced expression of glucose transporter-1 in vascular smooth muscle cells via the Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) pathway in experimental renal failure.

BACKGROUND: Chronic renal failure (CRF) is associated with increased cardiovascular mortality, and medial vascular smooth muscle cell (VSMC) hypertrophy, proliferation, and calcification play a pivotal role in uremic vasculopathy. Glucose transporter-1 (GLUT1) facilitates the transport of glucose into VSMCs, and GLUT1 overexpression associated with high glucose influx leads to a stimulation of VSMC proliferation. However, the role of GLUT1 in uremic vasculopathy remains unclear. This study aimed to identify changes in the expression of GLUT1 in VSMCs in the setting of experimental uremia and investigate whether Akt/tuberous sclerosis complex subunit 2 (TSC2)/mammalian target of rapamycin (mTOR)/ribosomal S6 protein kinase (S6K) signaling, which plays a crucial role in VSMC proliferation and glucose metabolism, is involved in the regulation of GLUT1 expression. METHODS: In vivo experimental CRF was induced in Wistar rats by 5/6 nephrectomy, and the GLUT1 expression in aortic tissue was determined by the reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemical staining. Indoxyl sulfate (IS) is a uremic retention solute proven with pro-proliferative effect on rat VSMCs, and we further studied the expression of GLUT1 in rat A7r5 rat embryonic aortic cells stimulated by IS in the presence or absence of phloretin, a GLUT1 inhibitor, to explore the pathogenic role of GLUT1 in uremic vasculopathy. The contribution of Akt/TSC2/mTOR/S6K signaling in modifying the GLUT1 expression was also assessed. RESULTS: Eight weeks after 5/6 nephrectomy, aortic tissue obtained from CRF rats exhibited increased wall thickness and VSMC hypertrophy, hyperplasia, and degeneration. Compared with the sham-operated control group, the messenger (m)RNA and protein abundance of GLUT1 were both markedly increased in CRF rats. In vitro, IS induced a significant increase in expression of GLUT1 protein as well as pro-proliferative cyclin D1 and p21 mRNA and a modest increase in expression of antiapoptotic p53 mRNA in A7r5 cells, whereas inhibition of GLUT1 mediated glucose influx reduced the pro-proliferative and antiapoptotic effects of IS. In addition to increased GLUT1 expression, IS significantly suppressed Akt and TSC2 phosphorylation after 6-hour and 12-hour treatment, but increased S6K phosphorylation after 3-hour treatment. Inactivation of mTOR downstream signaling by rapamycin treatment inhibited S6K phosphorylation and abolished the stimulatory effect of IS on GLUT1 expression. CONCLUSIONS: In vivo and in vitro experimental CRF displayed prominent GLUT1 upregulation in VSMCs. The uremic toxin IS stimulated proliferation of VSMCs possibly through induction of GLUT1 expression. The Akt/TSC/mTOR/S6K signaling pathway may be one of the mechanisms underlying the upregulation of GLUT1 expression in uremic VSMCs.

Upregulation of a disintegrin and metalloproteinase with thrombospondin motifs-7 by miR-29 repression mediates vascular smooth muscle calcification.

OBJECTIVE: Vascular calcification significantly increases cardiovascular morbidity and mortality. We recently reported that the deficiency of cartilage oligomeric matrix protein (COMP) leads to vascular mineralization. We characterized the COMP-degrading metalloproteinase, a disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7). Here, we tested whether ADAMTS-7 facilitates vascular calcification. METHODS AND RESULTS: ADAMTS-7 expression was markedly upregulated in calcifying rat vascular smooth muscle cells (VSMCs) in vitro, calcified arteries of rats with chronic renal failure in vivo, and radial arteries of uraemic patients. Silencing of ADAMTS-7 markedly reduced COMP degradation and ameliorated VSMC calcification, whereas ectopic expression of ADAMTS-7 greatly enhanced COMP degradation and exacerbated mineralization. The transcriptional activity of ADAMTS-7 promoter was not altered by high phosphate. We used bioinformatics and quantitative polymerase chain reaction analysis to demonstrate that high-phosphate upregulated ADAMTS-7 mRNA and protein via miR-29a/b repression, which directly targeted the 3' untranslated region of ADAMTS-7 in VSMCs. MicroRNA (MiR)-29a/b mimic markedly inhibited but miR-29a/b inhibitor greatly enhanced high-phosphate-induced ADAMTS-7 expression, COMP degradation, and subsequent VSMC calcification. ADAMTS-7 silencing significantly diminished miR-29a/b repression-exaggerated VSMC calcification. CONCLUSIONS: Our data reveal a novel mechanism by which ADAMTS-7 upregulation by miR-29a/b repression mediates vascular calcification, which may shed light on preventing cardiovascular morbidity and mortality.

Expression of both matrix metalloproteinase-2 and its tissue inhibitor-2 in tunica media of radial artery in uremic patients.

OBJECTIVE: To investigate the expression and clinical significance of both matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor (tissue inhibitor of metalloproteinase-2 (TIMP-2)) in tunica media of radial artery in uremic patients. METHODS: The modified radial arteries from 80 uremic patients during internal arteriovenous fistula surgery were selected and used as experimental specimens. The calcification of tunica media was observed by alizarin red staining, and the expression status of MMP-2, TIMP-2, osteoprotegerin (OPG), and osteopontin (OPN) in tunica media of radial arteries of these patients was detected by immunohistochemical method. The semiquantitative analysis and comparison were conducted based on the calcification grading and the expression of each test protein in tunica media of radial artery. RESULTS: Of the 80 cases of radial artery specimens, 37 cases were presented with various degrees of calcification of tunica media, and the calcification rate was 46.25%; the expression of MMP-2, TIMP-2, OPG, and OPN could be detected in the calcificated tunica media of the radial artery and was positively correlated with the degree of vascular calcification (p < 0.05). CONCLUSION: The incidence of vascular calcification in uremic patients was high. The occurrence of calcification in tunica media of the radial artery was correlated with the expression of MMP-2 and TIMP-2.

Elastin degradation and vascular smooth muscle cell phenotype change precede cell loss and arterial medial calcification in a uremic mouse model of chronic kidney disease.

Arterial medial calcification (AMC), a hallmark of vascular disease in uremic patients, is highly correlated with serum phosphate levels and cardiovascular mortality. To determine the mechanisms of AMC, mice were made uremic by partial right-side renal ablation (week 0), followed by left-side nephrectomy at week 2. At 3 weeks, mice were switched to a high-phosphate diet, and various parameters of disease progression were examined over time. Serum phosphate, calcium, and fibroblast growth factor 23 (FGF-23) were up-regulated as early as week 4. Whereas serum phosphate and calcium levels declined to normal by 10 weeks, FGF-23 levels remained elevated through 16 weeks, consistent with an increased phosphate load. Elastin turnover and vascular smooth muscle cell (VSMC) phenotype change were early events, detected by week 4 and before AMC. Both AMC and VSMC loss were significantly elevated by week 8. Matrix metalloprotease 2 (MMP-2) and cathepsin S were present at baseline and were significantly elevated at weeks 8 and 12. In contrast, MMP-9 was not up-regulated until week 12. These findings over time suggest that VSMC phenotype change and VSMC loss (early phosphate-dependent events) may be necessary and sufficient to promote AMC in uremic mice fed a high-phosphate diet, whereas elastin degradation might be necessary but is not sufficient to induce AMC (because elastin degradation occurred also in uremic mice on a normal-phosphate diet, but they did not develop AMC).

Uremia suppresses immune signal-induced CYP27B1 expression in human monocytes.

BACKGROUND: Local production of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) regulated by the CYP27B1 enzyme in monocytes contributes to the immunomodulatory effects of vitamin D. Uremia suppresses renal CYP27B1, but its impact on monocytic CYP27B1 is incompletely understood. The present study aimed to elucidate this issue and to define the pathogenic role of p-cresyl sulfate (PCS), indoxyl sulfate (IndS), and fibroblast growth factor 23 (FGF23). METHODS: Resting or immune (interferon-γ + lipopolysaccharide)-stimulated THP1 cells and monocytes, isolated from healthy donors, were cultured in the presence of either healthy serum, uremic serum, PCS, IndS or FGF23. RNA expression levels for CYP27B1 and cytokines were quantified by RT-PCR and enzymatic CYP27B1 activity was measured 24 h after incubation. RESULTS: Culturing THP1 cells or human monocytes in the presence of uremic serum led to higher inflammatory cytokine and CYP27B1 expression. Immune signal-induced CYP27B1 expression and activity, conversely, was impaired in the presence of uremic serum. Similar effects were observed in the presence of FGF23, although significance was reached in immune-stimulated cells only. PCS and IndS failed to show any effect. CONCLUSIONS: Monocytic baseline CYP27B1 expression is increased in uremia, probably reflecting the microinflammatory state. Immune signal-induced CYP27B1 expression, conversely, is impaired in uremic conditions. Elevated FGF23 levels, but not PCS and IndS, may account, at least partly, for the dysregulation of monocytic CYP27B1 in uremia and, as such, may contribute to the high cardiovascular and infectious burden in chronic kidney disease.

Preventive effect of a proteasome inhibitor on the formation of accelerated atherosclerosis in rabbits with uremia.

Inflammation plays a central role in the pathogenesis of atherosclerosis. This study investigated whether the proteasome inhibitor has the same preventive effect on the formation of accelerated atherosclerosis in rabbits with uremia compared with a NF-kappaB inhibitor. New Zealand white rabbits were subjected to five-sixths nephrectomy (chronic renal failure [CRF]) or to a sham operation. Rats in each group were randomly assigned into three subgroups (n = 24 in each group) and treated with repeated intramuscular injections of proteasome inhibitor MG132 or NF-kappaB inhibitor PDTC for a specified period. Compared with sham rabbits, CRF rabbits displayed typical atherosclerotic changes (endothelial cell damage, intimal thickens, and appearance of foam cells). CRF rabbits had significantly higher levels of proteasome activity, NF-kappaB mRNA, protein, and DNA binding activity as well as tumor necrosis factor-a and proliferative cell nuclear antigen protein expression in aortic wall cells. CRF rabbits also showed lower levels of IkappaBalpha. Compared with CRF rabbits, CRF rabbits treatment with proteasome inhibitor MG132 showed restoration of IkappaBalpha mRNA and protein expression and decreased NF-kappaB DNA binding activity and tumor necrosis factor-a expression. Treatment with either proteasome inhibitor MG132 or NF-kappaB inhibitor PDTC could reverse these pathologic changes in the aortic wall cells of CRF rabbits. A comparison between the inhibitory effects of the two treatments revealed no statistical difference. These results suggest that ubiquitin-proteasome activation play a pivotal role in the pathogenesis of uremia-accelerated atherosclerosis. The ubiquitin-proteasome signaling pathway in aortic cells may therefore be an important target for preventing uremia-accelerated atherosclerosis.

Resistin inhibits essential functions of polymorphonuclear leukocytes.

The serum levels of resistin, a 12-kDa protein primarily expressed in inflammatory cells in humans, are increased in patients with chronic kidney disease and in those with diabetes mellitus. Both groups of patients have an increased risk of infections mainly as a result of disturbed polymorphonuclear leukocyte (PMNL) functions. Therefore, we investigated the influence of resistin on human PMNLs. Serum resistin concentrations were determined with a sandwich enzyme immunoassay. Using PMNLs from healthy subjects, chemotaxis was tested by the under-agarose method. Flow cytometric assays to measure oxidative burst and phagocytosis were conducted in whole blood. The uptake of deoxyglucose was determined as measure of the PMNL activation state. The activity of intracellular kinases was assessed by Western blotting and by in vitro kinase assays. Resistin inhibited PMNL chemotaxis and decreased the oxidative burst stimulated by Escherichia coli and by PMA, but did not influence PMNL phagocytosis of opsonized E. coli and PMNL glucose uptake. The inhibition of PMNLs by resistin was observed at concentrations found in serum samples of uremic patients, but not in concentrations measured in healthy subjects. Experiments with specific signal transduction inhibitors and measurements of intracellular kinases suggest that PI3K is a major target of resistin. In conclusion, resistin interferes with the chemotactic movement and the stimulation of the oxidative burst of PMNL, and therefore may contribute to the disturbed immune response in patients with increased resistin serum levels such as uremic and diabetic subjects.

The effect of uremia and hemodialysis on caspase-1 and caspase-3 activity in neutrophils.

BACKGROUND: Neutrophils in uremia maintain chronic inflammation, which contributes to malnutrition and immunity disorders. The influence of hemodialysis on the life span of neutrophils is unclear, although many authors postulate its apoptotic effect. Erythropoietin (EPO) is an antiapoptotic factor for various cells. We investigated factors possibly interfering in apoptosis of neutrophils in hemodialysis (HD) patients, such as HD session, parathormone (PTH), dose of EPO, inflammation and nutrition. SUBJECTS AND METHODS: Twenty HD patients and ten healthy controls were donors of neutrophils. We evaluated caspase-1 and caspase-3 activities, related to apoptosis of neutrophils, before and after a 4-hour HD session with cuprophane or polysulphone dialyzer. We measured serum concentrations of C-reactive protein, cholesterol, albumin, prealbumin and transferrin. Time of dialysis treatment, PTH level and dose of EPO were also evaluated. RESULTS: We observed a significant increase in caspase-1 activity and decrease in caspase-3 activity in neutrophils after HD with cuprophane, but not with polysulphone. Caspase-1 activity correlated positively with EPO dose and negatively with PTH level. Caspase-3 correlated negatively with the time of dialysis treatment. No correlation between caspase activity and markers of inflammation or nutrition was noticed. CONCLUSIONS: The results suggest that HD with bioincompatible cuprophane may prolong the life span of neutrophils due to the intense inflammatory reaction and high activity of caspase-1. Time of dialysis treatment, PTH level and EPO dose potentially influence the life span of neutrophils. PTH seems to have a proapoptotic effect of neutrophils, while EPO is an antiapoptotic factor.

Hydrogen sulfide, the third gaseous signaling molecule with cardiovascular properties, is decreased in hemodialysis patients.

Hydrogen sulfide, H(2)S, is the third endogenous gas with cardiovascular properties, after nitric oxide and carbon monoxide. H(2)S is a potent vasorelaxant, and its deficiency is implicated in the pathogenesis of hypertension and atherosclerosis. Cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfurtransferase catalyze H(2)S formation. Chronic kidney disease is characterized by high prevalence of hyperhomocysteinemia, hypertension, and high cardiovascular mortality, especially in hemodialysis patients. H(2)S levels are decreased in hemodialysis patients through transcriptional deregulation of genes encoding for the H(2)S-producing enzymes. Potential implications relate to the pathogenesis of the manifestations of the uremic syndrome, such as hypertension and atherosclerosis.

Increased indoleamine 2,3-dioxygenase (IDO) activity and elevated serum levels of tryptophan catabolites in patients with chronic kidney disease: a possible link between chronic inflammation and uraemic symptoms.

BACKGROUND: Tryptophan (Trp) is catabolized by indoleamine 2,3-dioxygenase (IDO). Changes in Trp metabolism and IDO activity in chronic kidney disease (CKD) have not been widely studied, and the impact of haemodialysis is uncertain. Here we investigate Trp catabolism, IDO activity and the role of inflammation in moderate to very severe CKD and haemodialysis. METHODS: Eighty individuals were included in a prospective blinded endpoint analysis. Using tandem mass spectrometry, serum levels of Trp, kynurenine (Kyn), kynurenic-acid (Kyna), quinolinic-acid (Quin), 5-hydroxytryptophan (OH-Trp), serotonin (5-HT), estimated IDO activity and inflammatory markers were assessed in 40 CKD patients (age 57 +/- 14 years, 21 male, creatinine 4.5 +/- 2.7, n = 17 receiving haemodialysis), and in 40 healthy controls (age 34 +/- 9 years, 26 male). RESULTS: Trp levels were unchanged in CKD (P = 0.78 versus controls). Serum levels of Kyn, Kyna and Quin increased with CKD severity (stages 4, 5 versus controls all P < or = 0.01). IDO activity was significantly induced in CKD and correlated with disease severity (stages 3-5 versus controls, all P < or = 0.01) and inflammatory markers [high-sensitivity C-reactive protein (hsCRP), soluble TNF-receptor-1 (sTNFR-I); both P < or = 0.03]. IDO products (Kyn, Kyna, Quin) correlated also with hsCRP and sTNFR-I (all P < or = 0.04). Haemodialysis did not influence IDO activity (P = 0.26) and incompletely removed Kyn, Kyna, Quin, OH-Trp and 5-HT by 22, 26, 50, 44 and 34%, respectively. In multiple regression, IDO activity correlated with hsCRP and sTNFR-I (both P < or = 0.03) independent of serum creatinine, age and body weight. CONCLUSIONS: IDO activity and serum levels of tryptophan catabolites of the kynurenine pathway increase with CKD severity. In CKD, induction of IDO may primarily be a consequence of chronic inflammation.

Lipoprotein lipase disturbances induced by uremia and hemodialysis.

Factors such as malnutrition, physical inactivity, uremic toxins, and inflammation are known to influence the activity of lipoprotein lipase (LPL), an important enzyme in metabolism of blood lipids. In patients with chronic kidney disease these factors are common and may result in a decreased LPL activity. This is particularly so in patients on hemodialysis. Further, during each dialysis treatment, the use of heparin (or low molecular weight heparin) induces a release of LPL from its normal binding sites at the plasma membrane of endothelial cells. This results in an increased degradation of the enzyme and a relative lack of LPL activity for up to 10 hours from the start of the dialysis. Thus, the use of conventional anticoagulation for hemodialysis, in addition to the consequences of the uremic state, may cause a severe functional deficiency of LPL. This in turn may have deleterious effects on energy metabolism and may contribute to the increased risk for cardiovascular disease in this vulnerable group of patients.

Inhibition of brain creatine kinase activity after renal ischemia is attenuated by N-acetylcysteine and deferoxamine administration.

Encephalopathy may accompany acute or chronic renal failure, and the mechanisms responsible for neurological complications in patients with renal failure are poorly known. Considering that creatine kinase (CK) is important for brain energy homeostasis and is inhibited by free radicals, and that oxidative stress is probably involved in the pathogenesis of uremic encephalopathy, we measured CK activity (hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex) in brain if rats submitted to renal ischemia and the effect of administration of antioxidants (N-acetylcysteine, NAC and deferoxamine, DFX) on this enzyme. We verified that CK activity was not altered in cerebellum and striatum of rats. CK activity was inhibited in prefrontal cortex and hippocampus of rats 12h after renal ischemia. The treatment with antioxidants prevented such effect. Cerebral cortex was also affected, but in this area CK activity was inhibited 6 and 12h after renal ischemia. Moreover, only NAC or NAC plus DFX were able to prevent the inhibition on the enzyme. Although it is difficult to extrapolate our findings to the human condition, the inhibition of brain CK activity after renal failure may be associated to neuronal loss and may be involved in the pathogenesis of uremic encephalopathy.