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.

Targeting STUB1-tissue factor axis normalizes hyperthrombotic uremic phenotype without increasing bleeding risk.

Chronic kidney disease (CKD/uremia) remains vexing because it increases the risk of atherothrombosis and is also associated with bleeding complications on standard antithrombotic/antiplatelet therapies. Although the associations of indolic uremic solutes and vascular wall proteins [such as tissue factor (TF) and aryl hydrocarbon receptor (AHR)] are being defined, the specific mechanisms that drive the thrombotic and bleeding risks are not fully understood. We now present an indolic solute-specific animal model, which focuses on solute-protein interactions and shows that indolic solutes mediate the hyperthrombotic phenotype across all CKD stages in an AHR- and TF-dependent manner. We further demonstrate that AHR regulates TF through STIP1 homology and U-box-containing protein 1 (STUB1). As a ubiquitin ligase, STUB1 dynamically interacts with and degrades TF through ubiquitination in the uremic milieu. TF regulation by STUB1 is supported in humans by an inverse relationship of STUB1 and TF expression and reduced STUB1-TF interaction in uremic vessels. Genetic or pharmacological manipulation of STUB1 in vascular smooth muscle cells inhibited thrombosis in flow loops. STUB1 perturbations reverted the uremic hyperthrombotic phenotype without prolonging the bleeding time, in contrast to heparin, the standard-of-care antithrombotic in CKD patients. Our work refines the thrombosis axis (STUB1 is a mediator of indolic solute-AHR-TF axis) and expands the understanding of the interconnected relationships driving the fragile thrombotic state in CKD. It also establishes a means of minimizing the uremic hyperthrombotic phenotype without altering the hemostatic balance, a long-sought-after combination in CKD patients.

Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial.

It has been reported that vitamin D regulates the immune system. However, whether vitamin D repletion modulates inflammatory responses in lymphocytes from dialysis patients is unclear. In the clinical trial, thirty-two (32) dialysis patients with 25 vitamin D ≤ 20ng/mL were randomized to receive either supplementation of cholecalciferol 100,000 UI/week/3 months (16 patients) or placebo (16 patients). In the in vitro study, B and T lymphocytes from 12 healthy volunteers (HV) were incubated with or without uremic serum in the presence or absence of 25 or 1,25 vitamin D. We evaluated the intracellular expression of IL-6, IFN-γ TLR7, TLR9, VDR, CYP27b1 and CYP24a1 by flow cytometry. We observed a reduction in the expression of TLR7, TLR9, INF-γ and CYP24a1 and an increase in VDR and CYP27b1 expression in patients which were supplemented with cholecalciferol, whereas no differences were found in the placebo group. Uremic serum increased the intracellular expression of IL-6, IFN-γ, TLR7, TLR9, VDR, CYP27b1 and CYP24a1. Treatment with 25 or 1,25 vitamin D decreased IL-6 and TLR9. CYP24a1 silencing plus treatment with 25 and/or 1,25 vitamin D had an additional reduction effect on IL-6, IFN-γ, TLR7 and TLR9 expression. This is the first study showing that cholecalciferol repletion has an anti-inflammatory effect and improves vitamin D intracellular regulatory enzymes on lymphocytes from dialysis patients.

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.

Differential Expression of Lipoprotein-Associated Phospholipase A2 in Monocyte Subsets: Impact of Uremia and Atherosclerosis.

BACKGROUND: Monocytic products, such as lipoprotein-associated phospholipase A2 (Lp-PLA2), may participate in the development of atherosclerosis. Heterogeneity of monocytes is widely acknowledged. Classical, intermediate, and non-classical subsets can be discerned. Recently, an inflammatory, pro-atherogenic monocyte population could be identified in hemodialysis patients. In this study, we investigated the expression of Lp-PLA2 on leucocytes and different monocyte subpopulations and their possible role in uremia, inflammation, and atherosclerosis. METHODS: Chronic kidney disease stage 5-D (CKD5-D; n = 57), healthy control subjects with hs-C-reactive protein (CRP) levels ≤1 mg/L (CO-N, n = 22) and a control group with inflammatory activation (CRP levels >1 mg/L, CO-I, n = 29) were enrolled in this cross-sectional observation. The CKD5-D cohort was dichotomized into patients with (A+) and without subclinical atherosclerosis (A-) by carotid artery ultrasound measurement. Lp-PLA2 activity was determined in plasma samples, Lp-PLA2 mRNA expression analysis in leucocytes, and sorted monocyte subsets. Effects of Lp-PLA2 overexpression were studied in classical vs. intermediate and non-classical subsets. RESULTS: The classical monocytes expressed the highest Lp-PLA2 mRNA levels as compared to other subpopulations. CKD5-D patients revealed significantly higher Lp-PLA2 transcripts, as well as higher Lp-PLA2 plasma activity as compared to healthy and "inflammatory" controls. In vitro data confirmed that uremia significantly contributes to Lp-PLA2 mRNA upregulation. Non-classical monocytes of A+ patients revealed significant higher Lp-PLA2 mRNA compared to A-. CONCLUSION: Uremic environment but not inflammation per se increases plasma Lp-PLA2 activity and upregulates monocytic Lp-PLA2 mRNA expression. The highest Lp-PLA2 levels were found in the classical and not in the inflammatory subsets. Atherosclerosis also contributes to a subset-specific increase in Lp-PLA2 mRNA expression.

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.

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.

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.

Uremic toxins and lipases in haemodialysis: a process of repeated metabolic starvation.

Severe kidney disease results in retention of uremic toxins that inhibit key enzymes for lipid breakdown such as lipoprotein lipase (LPL) and hepatic lipase (HL). For patients in haemodialysis (HD) and peritoneal dialysis (PD) the LPL activity is only about half of that of age and gender matched controls. Angiopoietin, like protein 3 and 4, accumulate in the uremic patients. These factors, therefore, can be considered as uremic toxins. In animal experiments it has been shown that these factors inhibit the LPL activity. To avoid clotting of the dialysis circuit during HD, anticoagulation such as heparin or low molecular weight heparin are added to the patient. Such administration will cause a prompt release of the LPL and HL from its binding sites at the endothelial surface. The liver rapidly degrades the release plasma compound of LPL and HL. This results in a lack of enzyme to degrade triglycerides during the later part of the HD and for another 3-4 h. PD patients have a similar baseline level of lipases but are not exposed to the negative effect of anticoagulation.

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.

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.

Erythrocyte glutathione transferase activity: a possible early biomarker for blood toxicity in uremic diabetic patients.

Erythrocyte glutathione transferase (e-GST) displays increased activity in patients with renal damage and positive correlation with homocysteine (Hcy) in patients under maintenance hemodialysis. Here, we determined e-GST, Hcy, and erythrocyte catalase (e-CAT) in 328 patients affected by type 2 diabetes mellitus (T2DM), 61 diabetic non-nephropathic patients and 267 affected by diabetes and by chronic kidney disease (CKD) under conservative therapy subdivided into four stages according to K-DOQI lines. e-GST activity was significantly higher in all T2DM patients compared to the control group (7.90 ± 0.26 vs. 5.6 ± 0.4 U/g(Hb)), and we observed an enhanced activity in all subgroups of CKD diabetic patients. No significant correlation or increase has been found for e-CAT in all patients tested. Mean Hcy in diabetic patients is higher than that in healthy subjects (33.42 ± 1.23 vs. 13.6 ± 0.8 µM), and Hcy increases in relation to the CKD stage. As expected, a significant correlation was found between e-GST and Hcy levels. These findings suggest that e-GST hyperactivity is not caused directly by diabetes but by its consequent renal damage. e-GST, as well as Hcy, may represent an early biomarker of renal failure.

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.

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.

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.

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.

Conformational changes of blood ACE in chronic uremia.

BACKGROUND: The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. HYPOTHESIS: Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. METHODOLOGY/PRINCIPAL FINDINGS: The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The "short" ACE inhibitor enalaprilat (tripeptide analog) and "long" inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. CONCLUSIONS/SIGNIFICANCE: The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon anti-hypertensive therapy.

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.

[Appropriate pharmacotherapy in patients with chronic kidney disease - new approach - ].

The kidney is the most important organ for the excretion of drugs. It was previously thought that appropriate dosages of these drugs could be easily estimated by evaluating the kidney function of patients and the excretion rate of the drug. However, the pharmacokinetic characteristics of patients with kidney disease are as follows: 1) Active metabolites with a higher polarity can accumulate, which can induce unpredictable adverse effects. For example, over sedation with morphine or the development of the fatal toxic syndrome in the case of allopulinol are due to the accumulation of active metabolites derived from these drugs. 2) Although the renal excretion rate of acetoaminophen is only less than 5%, the accumulation of its glucuronide conjugate during multiple dosing in patients with kidney failure may induce high serum acetoaminophen trough levels via the entero-hepatic circulation. 3) Although the renal excretion rate of the drugs are negligible, a remarkable increase in the serum levels of certain drugs were observed in patients with end stage kidney disease, suggesting a significant reduction in non-renal clearance probably by the accumulation of uremic toxins. For drugs that are likely to be administered to patients with kidney disease, even including drugs that are not excreted by the kidney, a full pharmacokinetic study should be conducted in patients and the results carefully assessed. Information on dosing adjustments for impaired kidney function based on estimated glomerular filtration rates should then be clearly stated in the package insert of the drugs.

The relationship between glutathione peroxidase and bioimpedance parameters in nondiabetic hemodialysis patients.

There is growing evidence from experimental and clinical studies that oxidative stress is involved in the pathogenesis of malnutrition. This cross-sectional study aimed to investigate the relationship between glutathione peroxidase (GPx) levels as a marker of antioxidant status and the nutritional status assessed by bioimpedance analysis (BIA). Ninety-seven nondiabetic stable outpatient uremic adults undergoing chronic hemodialysis (HD) were recruited for this study. Impedance measurements were performed using a multifrequency bioelectrical impedance analyzer after dialysis. GPx levels correlated with intracellular water (ICW) (r = 0.341, P = 0.011), ICW/total body weight (r = 0.320, P = 0.017), lean body mass (r = 0.300, P = 0.026) and total body cell mass (r = 0.339, P = 0.011). When patients were divided into two groups according to mean GPx levels (83.9 U/gr hemoglobin), the patients with higher GPx (GPx > 83.9 U/gr hemoglobin) had higher albumin (P = 0.038), lean body mass (P = 0.026), ICW (P = 0.011), and total body cell mass (P = 0.011) compared with those with lower GPx (GPx ≤ 83.9 U/gr hemoglobin). Furthermore, in the patients with higher GPx, body fat; extracellular water/total body water; illness marker and body fat mass index were lower than other group. In conclusion, our results reveal correlation indicating a relationship between antioxidant status (as measured by GPx) and nutritional status as assessed by BIA in nondiabetic HD patients.