TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

OBJECTIVE: Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. METHODS AND RESULTS: Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. CONCLUSIONS: Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

Intrafamilial Variability of ADPKD.

INTRODUCTION: Discordance in kidney disease severity between affected relatives is a recognized feature of autosomal dominant polycystic kidney disease (ADPKD). Here, we report a systematic study of a large cohort of families to define the prevalence and clinical features of intrafamilial discordance in ADPKD. METHODS: The extended Toronto Genetic Epidemiology Study of Polycystic Kidney Disease (eTGESP) cohort includes 1390 patients from 612 unrelated families with ADPKD ascertained in a regional polycystic kidney disease center. All probands underwent comprehensive PKD1 and PKD2 mutation screening. Total kidney volume by magnetic resonance imaging (MRI) was available in 500 study patients. RESULTS: Based on (i) rate of estimated glomerular filtration rate (eGFR) decline, (ii) age at onset of end-stage renal disease (ESRD), and (iii) Mayo Clinic Imaging Classification (MCIC), 20% of patients were classified as having mild disease, and 33% as having severe disease. Intrafamilial ADPKD discordance with at least 1 mild and 1 severe case was observed in 43 of 371 (12%) families, at a similar frequency regardless of the responsible gene (PKD1/PKD2/no mutation detected) or mutation type (protein-truncating versus nontruncating). Intrafamilial discordance was more common in larger families and was present in 30% of families with more than 5 affected members. The heritability of age at onset of ESRD was similar between different mutation types. CONCLUSION: Extreme kidney disease discordance is present in at least 12% of families with ADPKD, regardless of the underlying mutated gene or mutation class. Delineating genetic and environmental modifiers underlying the observed intrafamilial ADPKD variability will provide novel insights into the mechanisms of progression in ADPKD.

Coronary artery calcification, cardiovascular events, and death: a prospective cohort study of incident patients on hemodialysis.

BACKGROUND: Coronary calcification in patients with end-stage renal disease (ESRD) is associated with an increased risk of cardiovascular outcomes and death from all causes. Previous evidence has been limited by short follow-up periods and inclusion of a heterogeneous cluster of events in the primary analyses. OBJECTIVE: To describe coronary calcification in patients incident to ESRD, and to identify whether calcification predicts vascular events or death. DESIGN: Prospective substudy of an inception cohort. SETTING: Tertiary care haemodialysis centre in Ontario (St Joseph's Healthcare Hamilton). PARTICIPANTS: Patients starting haemodialysis who were new to ESRD. MEASUREMENTS: At baseline, clinical characterization and spiral computed tomography (CT) to score coronary calcification by the Agatston-Janowitz 130 scoring method. A primary outcome composite of adjudicated stroke, myocardial infarction, or death. METHODS: We followed patients prospectively to identify the relationship between cardiac calcification and subsequent stroke, myocardial infarction, or death, using Cox regression. RESULTS: We recruited 248 patients in 3 centres to our main study, which required only biochemical markers. Of these 164 were at St Joseph's healthcare, and eligible to participate in the substudy; of these, 51 completed CT scanning (31 %). Median follow up was 26 months (Q1, Q3: 14, 34). The primary outcome occurred in 16 patients; 11 in the group above the median and 5 in the group below (p = 0.086). There were 26 primary outcomes in 16 patients; 20 (77 %) events in the group above the coronary calcification median and 6 (23 %) in the group below (p = 0.006). There were 10 deaths; 8 in the group above the median compared with 2 in the group below (p = 0.04). The hazard ratios for coronary calcification above, compared with below the median, for the primary outcome composite were 2.5 (95 % CI 0.87, 7.3; p = 0.09) and 1.7 (95 % CI 0.55, 5.4; p = 0.4), unadjusted and adjusted for age, respectively. For death, the hazard ratios were 4.6 (95 % CI 0.98, 21.96; p = 0.054) and 2.4 (95 % CI 0.45, 12.97; p = 0.3) respectively. LIMITATIONS: We were limited by a small sample size and a small number of events. CONCLUSIONS: Respondent burden is high for additional testing around the initiation of dialysis. High coronary calcification in patients new to ESRD has a tendency to predict cardiovascular outcomes and death, though effects are attenuated when adjusted for age.

CONTEXTE: La calcification de l'artère coronaire chez les patients atteints d'insuffisance rénale terminale (IRT) est associée à un risque accru de troubles cardiovasculaires et de mortalité, toutes causes confondues. Les données précédemment recueillies se limitaient à un suivi de courte durée, de même qu'à l'inclusion de séries d'accidents non liés lors de l'analyse préliminaire. OBJECTIFS: Décrire la calcification de l'artère coronaire chez les patients atteints d'IRT et déterminer si la calcification de l'artère coronaire peut prédire des accidents vasculaires et la mort. TYPE D'ÉTUDE: Sous-étude prospective de cohorte selon le mode d'installation. CADRE: Une unité de soins tertiaires en dialyse, en Ontario (St Joseph's Healthcare Hamilton). PARTICIPANTS: Des patients qui sont nouvellement atteints d'IRT et qui entament une hémodialyse. MESURES: En début de traitement, une caractérisation clinique et une tomodensitométrie (TDM) hélicoïdale qui permettent de mesurer la calcification de l'artère coronaire sur 130, selon l'échelle d'Agatston-Janowitz. L'indicateur principal des résultats comprend l'AVC, l'infarctus du myocarde ou la mort. MÉTHODES: Nous avons suivi les patients de manière prospective, afin de cibler la relation entre la calcification de l'artère coronaire et l'AVC, l'infarctus du myocarde ou la mort subséquente, en utilisant la régression de Cox. RÉSULTATS: Nous avons recruté 248 patients dans trois unités, dans le cadre de l'étude principale, qui ne requérait que des biomarqueurs chimiques. De ces patients, 164 étaient de St Joseph's Healthcare, et étaient admissibles à la sous-étude; 51 avaient effectué une tomographie par ordinateur (31 %). Le suivi médian s'étendait sur 26 mois (Q1, Q3: 14, 34). L'indicateur principal a été observé chez 16 patients; 11 dans le groupe se trouvant au-dessus de la médiane, et 5 dans le groupe inférieur (p?=?0,086). On a observé 26 indicateurs principaux chez 16 patients; 20 (77 %) accidents dans le groupe se trouvant au-dessus de la médiane en ce qui a trait à la calcification et 6 (23 %) dans le groupe inférieur (p?=?0,006). Il y a eu 10 décès; 8 dans le groupe se trouvant au-dessus de la médiane et 2 dans le groupe inférieur (p?=?0,04). Les taux de risque de calcification de l'artère coronaire se trouvant au-dessus et sous la médiane, pour les indicateurs principaux, étaient respectivement de 2,5 (95 % IC 0,98; 21,96; p?=?0,054) et 2,4 (95 % IC 0,45, 12,97; p?=?0,3). LIMITES DE L'ÉTUDE: Nous avons été limités par la taille restreinte de l'échantillon, de même que par le petit nombre d'accidents. CONCLUSION: Le fardeau du répondant repose sur des examens supplémentaires au moment de commencer la dialyse. Un fort taux de calcification de l'artère coronaire chez les patients nouvellement atteints d'IRT tend à prédire des accidents cardiovasculaires et la mort, bien que les effets soient atténués après révision en fonction de l'âge.

4-Phenylbutyrate inhibits tunicamycin-induced acute kidney injury via CHOP/GADD153 repression.

Different forms of acute kidney injury (AKI) have been associated with endoplasmic reticulum (ER) stress; these include AKI caused by acetaminophen, antibiotics, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic known to induce ER stress and is a commonly used inducer of AKI. 4-phenylbutyrate (4-PBA) is an FDA approved substance used in children who suffer from urea cycle disorders. 4-PBA acts as an ER stress inhibitor by aiding in protein folding at the molecular level and preventing misfolded protein aggregation. The main objective of this study was to determine if 4-PBA could protect from AKI induced by ER stress, as typified by the TM-model, and what mechanism(s) of 4-PBA's action were responsible for protection. C57BL/6 mice were treated with saline, TM or TM plus 4-PBA. 4-PBA partially protected the anatomic segment most susceptible to damage, the outer medullary stripe, from TM-induced AKI. In vitro work showed that 4-PBA protected human proximal tubular cells from apoptosis and TM-induced CHOP expression, an ER stress inducible proapoptotic gene. Further, immunofluorescent staining in the animal model found similar protection by 4-PBA from CHOP nuclear translocation in the tubular epithelium of the medulla. This was accompanied by a reduction in apoptosis and GRP78 expression. CHOP(-/-) mice were protected from TM-induced AKI. The protective effects of 4-PBA extended to the ultrastructural integrity of proximal tubule cells in the outer medulla. When taken together, these results indicate that 4-PBA acts as an ER stress inhibitor, to partially protect the kidney from TM-induced AKI through the repression of ER stress-induced CHOP expression.

Colchicine attenuates renal injury in a model of hypertensive chronic kidney disease.

Hypertension is a risk factor for chronic kidney disease, particularly when associated with impaired renal autoregulation and thereby increased intraglomerular pressure (Pgc). Elevated Pgc can be modeled in vitro by exposing glomerular mesangial cells to mechanical strain. We previously showed that RhoA mediates strain-induced matrix production. Here, we show that RhoA activation is dependent on an intact microtubule network. Upregulation of the profibrotic cytokine connective tissue growth factor (CTGF) by mechanical strain is dependent on RhoA activation and inhibited by microtubule disruption. We tested the effects of the microtubule depolymerizing agent colchicine in 5/6 nephrectomized rats, a model of chronic kidney disease driven by elevated Pgc. Colchicine inhibited glomerular RhoA activation and attenuated both glomerular sclerosis and interstitial fibrosis without affecting systemic blood pressure. Upregulation of the matrix proteins collagen I and fibronectin, as well as CTGF, was attenuated by colchicine. Activity of the profibrotic cytokine TGF-ß, as assessed by Smad3 phosphorylation, was also inhibited by colchicine. Microtubule disruption significantly decreased renal infiltration of lymphocytes and macrophages. Our studies thus indicate that colchicine modifies hypertensive renal fibrosis. Its protective effects are likely mediated by inhibition of RhoA signaling and renal infiltration of inflammatory cells. Already well-established in clinical practice for other indications, prevention of hypertension-associated renal fibrosis may represent a new potential use for colchicine.

Stroke prevention in atrial fibrillation patients with chronic kidney disease.

Chronic kidney disease (CKD) is prevalent in elderly patients with atrial fibrillation and is an independent risk factor for stroke. Warfarin anticoagulation is efficacious for stroke prevention in atrial fibrillation patients with moderate CKD (stage III, estimated glomerular filtration rate 30-59 mL/min), but recent observational studies have challenged its value for patients with end-stage renal disease requiring dialysis. The novel oral anticoagulants (i.e., dabigatran, apixaban, rivaroxaban) all undergo renal metabolism to varying degrees, and hence dosing, efficacy, and safety require special consideration in CKD patients. In randomized trials to date involving 11,169 patients with moderate CKD, the novel oral anticoagulants performed well, with similar efficacy and safety profiles as for non-CKD patients. For atrial fibrillation patients with stage III CKD, the available data are strongest for dabigatran 150 mg twice daily as superior to warfarin for stroke prevention and for apixaban as superior to warfarin regarding reduced major hemorrhage. Renal function should be monitored at least annually in patients receiving a novel oral anticoagulant, and more often in elderly patients and those with underlying CKD or comorbidities who are at special risk for dehydration and deterioration of renal function. Much remains to be learned about the optimal use of the novel oral anticoagulants in CKD patients; additional studies about optimal dosing of the novel oral anticoagulants and frequency of monitoring renal function in CKD patients with atrial fibrillation are needed. Anticoagulation options for hemodialysis patients require testing in randomized trials.

TGFß receptor I transactivation mediates stretch-induced Pak1 activation and CTGF upregulation in mesangial cells.

Increased intraglomerular pressure is an important pathogenic determinant of kidney fibrosis in the progression of chronic kidney disease, and can be modeled by exposing glomerular mesangial cells (MC) to mechanical stretch. MC produce extracellular matrix and profibrotic cytokines, including connective tissue growth factor (CTGF) when stretched. We show that p21-activated kinase 1 (Pak1) is activated by stretch in MC in culture and in vivo in a process marked by elevated intraglomerular pressures. Its activation is essential for CTGF upregulation. Rac1 is an upstream regulator of Pak1 activation. Stretch induces transactivation of the type I transforming growth factor ß1 receptor (TßRI) independently of ligand binding. TßRI transactivation is required not only for Rac1/Pak1 activation, but also for activation of the canonical TGFß signaling intermediate Smad3. We show that Smad3 activation is an essential requirement for CTGF upregulation in MC under mechanical stress. Pak1 regulates Smad3 C-terminal phosphorylation and transcriptional activation. However, a second signaling pathway, that of RhoA/Rho-kinase and downstream Erk activation, is also required for stretch-induced CTGF upregulation in MC. Importantly, this is also regulated by Pak1. Thus, Pak1 serves as a novel central mediator in the stretch-induced upregulation of CTGF in MC.

Propagation of human prostate cancer stem-like cells occurs through EGFR-mediated ERK activation.

Prostate cancer stem-like cells (PCSCs) are being intensely investigated largely owing to their contributions towards prostate tumorigenesis, however, our understanding of PCSC biology, including their critical pathways, remains incompletely understood. While epidermal growth factor (EGF) is widely used in maintaining PCSC cells in vitro, the importance of EGF-dependent signaling and its downstream pathways in PCSC self-renewal are not well characterized. By investigating DU145 sphere cells, a population of prostate cancer cells with stem-like properties, we report here that epidermal growth factor receptor (EGFR) signaling plays a critical role in the propagation of DU145 PCSCs. Activation of EGFR signaling via addition of EGF and ectopic expression of a constitutively-active EGFR mutant (EGFRvIII) increased sphere formation. Conversely, inhibition of EGFR signaling by using EGFR inhibitors (AG1478 and PD168393) and knockdown of EGFR significantly inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signaling, activation of the MEK-ERK pathway contributed to EGFR-facilitated PCSC propagation. Modulation of EGFR signaling affected extracellular signal-related kinase (ERK) activation. Inhibition of ERK activation through multiple approaches, including treatment with the MEK inhibitor U0126, ectopic expression of dominant-negative MEK1(K97M), and knockdown of either ERK1 or ERK2 resulted in a robust reduction in PCSC propagation. Collectively, the present study provides evidence that EGFR signaling promotes PCSC self-renewal, in part, by activating the MEK-ERK pathway.

BRCA1 is a novel target to improve endothelial dysfunction and retard atherosclerosis.

OBJECTIVE: BRCA1, a tumor suppressor gene implicated in breast and ovarian cancers, exerts multiple effects on DNA repair and affords resistance against cellular stress responses. We hypothesized that BRCA1 limits endothelial cell apoptosis and dysfunction, and via this mechanism attenuates atherosclerosis. METHODS: Loss and gain of function were achieved in cultured endothelial cells by silencing and overexpressing BRCA1, respectively. In vivo loss and gain of function were performed by generating endothelial cell-specific knockout (EC-BRCA1(-/-)) mice and administering a BRCA1 adenovirus. Well-established cell and animal models of angiogenesis and atherosclerosis were used. RESULTS: BRCA1 is basally expressed in endothelial cells. BRCA1 overexpression protected and BRCA1 silencing exaggerated inflammation- and doxorubicin-induced endothelial cell apoptosis. Key indices of endothelial function were modulated in a manner consistent with an effect of BRCA1 to limit endothelial cell apoptosis and improve endothelial function. BRCA1 overexpression strongly attenuated the production of reactive oxygen species and upregulated endothelial nitric oxide synthase, phosphorylated endothelial nitric oxide synthase, phosphorylated Akt, and vascular endothelial growth factor-a expression. BRCA1 overexpression also improved capillary density and promoted blood flow restoration in mice subjected to hind-limb ischemia. BRCA1-overexpressing ApoE(-/-) mice fed a Western diet developed significantly less aortic plaque lesions, exhibited reduced macrophage infiltration, and generated less reactive oxygen species. Lung sections and aortic segments from EC-BRCA1(-/-) mice demonstrated greater inflammation-associated apoptosis and impaired endothelial function, respectively. BRCA1 expression was attenuated in the plaque region of human atherosclerotic carotid artery samples compared with the adjacent plaque-free area. CONCLUSIONS: These data collectively highlight a previously unrecognized role of BRCA1 as a gatekeeper of inflammation-induced endothelial cell function and a target to limit atherosclerosis. Translational studies evaluating endothelial function and atherosclerosis in individuals with BRCA1 mutations are suggested.

MicroRNA-145 targeted therapy reduces atherosclerosis.

BACKGROUND: MicroRNA are essential posttranscriptional modulators of gene expression implicated in various chronic diseases. Because microRNA-145 is highly expressed in vascular smooth muscle cells (VSMC) and regulates VSMC fate and plasticity, we hypothesized that it may be a novel regulator of atherosclerosis and plaque stability. METHODS AND RESULTS: Apolipoprotein E knockout mice (ApoE(-/-)) mice were treated with either a microRNA-145 lentivirus under the control of the smooth muscle cell (SMC)-specific promoter SM22α or a SM22α control lentivirus before commencing the Western diet for 12 weeks. The SMC-targeted microRNA-145 treatment markedly reduced plaque size in aortic sinuses, ascending aortas, and brachiocephalic arteries. It also significantly increased fibrous cap area, reduced necrotic core area, and increased plaque collagen content. Cellular plaque composition analyses revealed significantly less macrophages in ApoE(-/-) mice treated with the SMC-specific microRNA-145. These mice also demonstrated marked increases in calponin levels and α-smooth muscle actin-positive SMC areas in their atherosclerotic lesions. Furthermore, lentiviral delivery of microRNA-145 resulted in reduced KLF4 and elevated myocardin expression in aortas from ApoE(-/-) mice, consistent with an effect of microRNA-145 to promote a contractile phenotype in VSMC. CONCLUSIONS: VSMC-specific overexpression of microRNA-145 is a novel in vivo therapeutic target to limit atherosclerotic plaque morphology and cellular composition, shifting the balance toward plaque stability vs plaque rupture.

Anticoagulants in atrial fibrillation patients with chronic kidney disease.

Atrial fibrillation is an important cause of preventable, disabling stroke and is particularly frequent in patients with chronic kidney disease (CKD). Stage 3 CKD is an independent risk factor for stroke in patients with atrial fibrillation. Warfarin anticoagulation is efficacious for stroke prevention in atrial fibrillation patients with stage 3 CKD, but recent observational studies have challenged its value for patients with end-stage renal disease and atrial fibrillation. Novel oral anticoagulants such as dabigatran, apixaban and rivaroxaban are at least as efficacious as warfarin with reduced risks of intracranial haemorrhage. However, all these agents undergo renal clearance to varying degrees, and hence dosing, efficacy, and safety require special consideration in patients with CKD. Overall, the novel oral anticoagulants have performed well in randomized trials of patients with stage 3 CKD, with similar efficacy and safety profiles as for patients without CKD, albeit requiring dosing modifications. The required period of discontinuation of novel oral anticoagulants before elective surgery is longer for patients with CKD owing to their reduced renal clearance. Although much remains to be learned about the optimal use of these new agents in patients with CKD, they are attractive anticoagulation options that are likely to replace warfarin in coming years.

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death.

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca(2+)-independent phospholipase A(2) (iPLA(2)ß), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.

SREBP-1 activation by glucose mediates TGF-ß upregulation in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent studies showed that overexpression of the transcription factor sterol-responsive element-binding protein (SREBP)-1 induces pathology reminiscent of diabetic nephropathy, and SREBP-1 upregulation was observed in diabetic kidneys. We thus studied whether SREBP-1 is activated by high glucose (HG) and mediates its profibrogenic responses. In primary rat mesangial cells, HG activated SREBP-1 by 30 min, seen by the appearance of its cleaved nuclear form (nSREBP-1), EMSA, and by activation of an SREBP-1 response element (SRE)-driven green fluorescent protein construct. Activation was dose dependent and not induced by an osmotic control. Site 1 protease was required, since its inhibition by AEBSF prevented SREBP-1 activation. SCAP, the ER-associated chaperone for SREBP-1, was also necessary since its inhibitor fatostatin also blocked SREBP-1 activation. Signaling through the EGFR/phosphatidylinositol 3-kinase (PI3K) pathway, which we previously showed mediates HG-induced TGF-ß1 upregulation, and through RhoA, were upstream of SREBP-1 activation (Wu D, Peng F, Zhang B, Ingram AJ, Gao B, Krepinsky JC. Diabetologia 50: 2008-2018, 2007; Wu D, Peng F, Zhang B, Ingram AJ, Kelly DJ, Gilbert RE, Gao B, Krepinsky JC. J Am Soc Nephrol 20: 554-566, 2009). Fatostatin and AEBSF prevented HG-induced TGF-ß1 upregulation by Northern blot analysis, and HG-induced TGF-ß1 promoter activation was inhibited by both fatostatin and dominant negative SREBP-1a. Chromatin immunoprecipitation analysis confirmed that HG led to SREBP-1 binding to the TGF-ß1 promoter in a region containing a putative SREBP-1 binding site (SRE). Thus HG-induced SREBP-1 activation requires EGFR/PI3K/RhoA signaling and SCAP-mediated transport to the Golgi for its proteolytic cleavage. Activated SREBP-1 binds to the TGF-ß promoter, resulting in TGF-ß1 upregulation in response to HG. SREBP-1 thus provides a potential novel therapeutic target for the treatment of diabetic nephropathy.

Anticoagulants in patients with atrial fibrillation and end-stage renal disease.

Atrial fibrillation (AF) is a common cardiac arrhythmia and is associated with an increased risk for thromboembolic stroke. Anticoagulant therapy has been shown to reduce the risk for ischemic stroke in patients with AF; however, these studies have excluded patients with end-stage renal disease (ESRD). This review examines the relationships between ESRD, AF, and the use of anticoagulants to prevent ischemic stroke. Medline and Embase were used to identify relevant articles. Identified review articles and their references were searched. The prevalence of AF in patients with ESRD is higher than that in the general population; ESRD appears to be an independent risk factor for AF. The presence of AF in patients with ESRD increases the risk for stroke, although this effect is less pronounced when compared with the general population. The presence of ESRD confers an increased risk for bleeding; warfarin appears to enhance this risk. Observational data suggest that warfarin increases the rate of hemorrhagic stroke in patients with ESRD, but are unclear on its utility in reducing ischemic stroke. In addition to increasing the risk for bleeding, warfarin may also promote vascular calcification in this population. Currently, there are no oral anticoagulants other than warfarin that are approved for use in patients with ESRD. Recent guidelines suggest that warfarin only be used for secondary prevention in patients with ESRD and AF. Randomized controlled trials are needed to clarify the role of warfarin or other anticoagulants in preventing stroke in patients with ESRD and AF.

α-Mannosidase 2C1 attenuates PTEN function in prostate cancer cells.

PTEN dephosphorylates the 3-position phosphate of phosphatidylinositol 3,4,5 triphosphate (PIP(3)), thereby inhibiting AKT activation. Although attenuation of PTEN function has a major role in tumourigenesis, the underlying mechanisms remain unclear. Here we show that α-mannosidase 2C1 (MAN2C1) inhibits PTEN function in prostate cancer (PC) cells and is associated with a reduction in PTEN function in primary PC. MAN2C1 activates AKT and promotes the formation of PTEN-positive DU145 cell-derived xenograft tumours by imparing endogenous PTEN function. In 659 PC patients who were examined, ~60% of tumours were PTEN positive with elevated AKT activation. Of these, 80% display MAN2C1 overexpression that co-localizes with PTEN. Increases in MAN2C1 were detected only in PTEN-positive prostatic intraepithelial neoplasia and carcinomas, and showed a significant association with PC recurrence only in patients with PTEN-positive PCs. Mechanistically, MAN2C1 binds PTEN thereby inhibiting its PIP(3) phosphatase activity. These findings show that MAN2C1 function as a PTEN-negative regulator in PC cells.

Low-intensity adjusted-dose warfarin for the prevention of hemodialysis catheter failure: a randomized, controlled trial.

BACKGROUND AND OBJECTIVES: To determine whether warfarin prolongs the time to first mechanical-catheter failure. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This was a multicenter parallel-group randomized controlled trial with blinding of participants, trial staff, clinical staff, outcome assessors, and data analysts. Randomization was in a 1:1 ratio in blocks of four and was concealed by use of fax to a central pharmacy. Hemodialysis patients with newly-placed catheters received low-intensity monitored-dose warfarin, target international normalized ratio (INR) 1.5 to 1.9, or placebo, adjusted according to schedule of sham INR results. The primary outcome was time to first mechanical-catheter failure (inability to establish a circuit or blood flow less than 200 ml/min). RESULTS: We randomized 174 patients: 87 to warfarin and 87 to placebo. Warfarin was associated with a hazard ratio (HR) of 0.90 (P=0.60; 95% confidence interval [CI], 0.57, 1.38) for time to first mechanical-catheter failure. Secondary analyses were: time to first guidewire exchange or catheter removal for mechanical failure (HR 0.78; 95% CI, 0.37, 1.6); time to catheter removal for mechanical failure (HR 0.67; 95% CI, 0.19, 2.37); and time to catheter removal for any cause (HR 0.89; 95% CI, 0.42, 1.81). Major bleeding occurred in 10 participants assigned to warfarin and seven on placebo (relative risk, 1.43; 95% CI, 0.57, 3.58; P=0.61). CONCLUSIONS: We found no evidence for efficacy of low-intensity, monitored-dose warfarin in preventing mechanical-catheter failure.

Induction of the unfolded protein response after monocyte to macrophage differentiation augments cell survival in early atherosclerotic lesions.

Endoplasmic reticulum (ER) stress causes macrophage cell death within advanced atherosclerotic lesions, thereby contributing to necrotic core formation and increasing the risk of atherothrombotic disease. However, unlike in advanced lesions, the appearance of dead/apoptotic macrophages in early lesions is less prominent. Given that activation of the unfolded protein response (UPR) is detected in early lesion-resident macrophages and can enhance cell survival against ER stress, we investigated whether UPR activation occurs after monocyte to macrophage differentiation and confers a cytoprotective advantage to the macrophage. Human peripheral blood monocytes were treated with monocyte colony-stimulating factor to induce macrophage differentiation, as assessed by changes in ultrastructure and scavenger receptor expression. UPR markers, including GRP78, GRP94, and spliced XBP-1, were induced after macrophage differentiation and occurred after a significant increase in de novo protein synthesis. UPR activation after differentiation reduced macrophage cell death by ER stress-inducing agents. Further, GRP78 overexpression in macrophages was sufficient to reduce ER stress-induced cell death. Consistent with these in vitro findings, UPR activation was observed in viable lesion-resident macrophages from human carotid arteries and from the aortas of apoE(-/-) mice. However, no evidence of apoptosis was observed in early lesion-resident macrophages from the aortas of apoE(-/-) mice. Thus, our findings that UPR activation occurs during macrophage differentiation and is cytoprotective against ER stress-inducing agents suggest an important cellular mechanism for macrophage survival within early atherosclerotic lesions.

Mechanical strain-induced RhoA activation requires NADPH oxidase-mediated ROS generation in caveolae.

Increased intraglomerular pressure leads to kidney fibrosis, and can be modeled by exposing glomerular mesangial cells (MC) to mechanical strain. We previously showed that RhoA mediates strain-induced matrix production. Here we investigate whether reactive oxygen species (ROS) are required for RhoA activation. Maximal RhoA activation (1 min) was inhibited by ROS scavenge or NADPH oxidase inhibition. Strain activated NADPH oxidase, with Rac1, p47(phox), and p67(phox) membrane translocation, and Rac1 activation, observed within 30 sec. Epidermal growth factor receptor (EGFR) inhibition blocked RhoA and Rac1 activation, p67(phox) membrane translocation, and ROS generation. However, EGFR activation was unaffected by ROS inhibitors, placing it upstream of ROS generation. We previously showed, using chemical disruption, that caveolae mediate strain-induced EGFR and RhoA activation. In MC from caveolin-1 knockout mice, which lack caveolae, RhoA and Rac1 activation, p67(phox) membrane translocation, and ROS generation were absent. These were rescued by caveolin-1 re-expression. ROS generation, Rac1 activation, and p67(phox) membrane translocation were also prevented by Src inhibition. They were absent in MC stably infected with caveolin-1 Y14A, a mutant resistant to Src phosphorylation. In MC, caveolae are thus important mediators of strain-induced ROS generation through NADPH oxidase, mediating a signaling cascade which results in RhoA activation.

Mechanical stretch-induced RhoA activation is mediated by the RhoGEF Vav2 in mesangial cells.

Increased intraglomerular pressure is an important hemodynamic determinant of glomerulosclerosis, and can be modelled in vitro by exposing mesangial cells (MC) to cyclic mechanical stretch. We have previously shown that the GTPase RhoA mediates stretch-induced fibronectin production. Here we investigate the role of the RhoGEF Vav2 in the activation of RhoA by stretch. Primary rat MC were exposed to 1 Hz cyclic stretch, previously shown to induce maximal RhoA activation at 1 min. Total Vav2 tyrosine phosphorylation and specific phosphorylation on Y172, required for activation, were increased by 1 min of stretch. Overexpression of dominant-negative Vav2 Y172/159F in COS-1 cells or downregulation of Vav2 by siRNA in MC prevented stretch-induced RhoA activation. Vav2 is known to be activated in response to growth factors, and we have previously shown the epidermal growth factor receptor (EGFR) to be transactivated by stretch in MC. Both Vav2 Y172 phosphorylation and RhoA activation were blocked by the EGFR inhibitor AG1478 and prevented in MC overexpressing kinase inactive EGFR. Stretch led to physical association between the EGFR and Vav2, and this was dependent on EGFR activation. EGFR Y992 phosphorylation, required for growth factor-induced Vav2 phosphorylation, was also induced by stretch. Activation of both Src and PI3K were necessary upstream mediators of stretch-induced Vav2 Y172 phosphorylation and RhoA activation. In summary, stretch-induced RhoA activation is dependent on transactivation of the EGFR and activation of the RhoGEF Vav2. Src and PI3K are both required upstream of Vav2 and RhoA activation.