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.

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.

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.

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.

Atypical Polycystic Kidney Disease as defined by Imaging.

Using age- and height-adjusted total kidney volume, the Mayo Clinic Imaging Classification provides a validated approach to assess the risk of chronic kidney disease (CKD) progression in autosomal dominant polycystic kidney disease (ADPKD), but requires excluding patients with atypical imaging patterns, whose clinical characteristics have been poorly defined. We report an analysis of the prevalence, clinical and genetic characteristics of patients with atypical polycystic kidney disease by imaging. Patients from the extended Toronto Genetic Epidemiology Study of Polycystic Kidney Disease recruited between 2016 and 2018 completed a standardized clinical questionnaire, kidney function assessment, genetic testing, and kidney imaging by magnetic resonance or computed tomography. We compared the prevalence, clinical features, genetics, and renal prognosis of atypical versus typical polycystic kidney disease by imaging. Forty-six of the 523 (8.8%) patients displayed atypical polycystic kidney disease by imaging; they were older (55 vs. 43 years; P < 0.001), and less likely to have a family history of ADPKD (26.1% vs. 74.6%; P < 0.001), a detectable PKD1 or PKD2 mutation (9.2% vs. 80.4%; P < 0.001), or progression to CKD stage 3 or stage 5 (P < 0.001). Patients with atypical polycystic kidney disease by imaging represent a distinct prognostic group with a low likelihood of progression to CKD.

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.

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.

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.

Systems biology of autosomal dominant polycystic kidney disease (ADPKD): computational identification of gene expression pathways and integrated regulatory networks.

To elucidate the molecular pathways that modulate renal cyst growth in ADPKD, we performed global gene profiling on cysts of different size (<1 ml, n = 5; 10-20 ml, n = 5; >50 ml, n = 3) and minimally cystic tissue (MCT, n = 5) from five PKD1 human polycystic kidneys using Affymetrix HG-U133 Plus 2.0 arrays. We used gene set enrichment analysis to identify overrepresented signaling pathways and key transcription factors (TFs) between cysts and MCT. We found down-regulation of kidney epithelial restricted genes (e.g. nephron segment-specific markers and cilia-associated cystic genes such as HNF1B, PKHD1, IFT88 and CYS1) in the renal cysts. On the other hand, PKD1 cysts displayed a rich profile of gene sets associated with renal development, mitogen-mediated proliferation, cell cycle progression, epithelial-mesenchymal transition, hypoxia, aging and immune/inflammatory responses. Notably, our data suggest that up-regulation of Wnt/beta-catenin, pleiotropic growth factor/receptor tyrosine kinase (e.g. IGF/IGF1R, FGF/FGFR, EGF/EGFR, VEGF/VEGFR), G-protein-coupled receptor (e.g. PTGER2) signaling was associated with renal cystic growth. By integrating these pathways with a number of dysregulated networks of TFs (e.g. SRF, MYC, E2F1, CREB1, LEF1, TCF7, HNF1B/ HNF1A and HNF4A), our data suggest that epithelial dedifferentiation accompanied by aberrant activation and cross-talk of specific signaling pathways may be required for PKD1 cyst growth and disease progression. Pharmacological modulation of some of these signaling pathways may provide a potential therapeutic strategy for ADPKD.

Minimal Change Disease After First Dose of Pfizer-BioNTech COVID-19 Vaccine: A Case Report and Review of Minimal Change Disease Related to COVID-19 Vaccine.

RATIONALE: While severe complications are generally uncommon with novel coronavirus disease 2019 (COVID-19) vaccine, there has been a steady increase in the number of patients presenting with nephrotic syndrome and acute kidney injury after the administration of COVID-19 vaccine. Physicians should be made aware of minimal change disease as a potential complication associated with COVID-19 vaccine. PRESENTING CONCERNS: A 60-year-old male without significant past medical history presented with new onset of nephrotic syndrome approximately 10 days after his first dose of Pfizer-BioNTech COVID-19 vaccine. Laboratory findings showed hypoalbuminemia (20 g/L), elevated urine albumin/creatinine ratio (668 mg/mmol), and elevated creatinine of 116 µmol/L from a baseline of 79 µmol/L. DIAGNOSIS: A diagnostic kidney biopsy was performed 6 weeks after the onset of the edema and approximately 8 weeks after his first dose of Pfizer-BioNTech COVID-19 vaccine. The kidney biopsy findings were consistent with minimal change disease with focal acute tubular injury. INTERVENTIONS: The patient was treated conservatively with ramipril 10 mg and furosemide 80 mg daily 5 weeks after the onset of swelling. Prednisone 1 mg/kg was initiated immediately when the kidney biopsy result became available (approximately 6 weeks after the onset of edema). OUTCOMES: The patient remitted with rapid weight loss starting 2 weeks post prednisone initiation. NOVEL FINDINGS: De novo minimal change disease with acute tubular injury is a kidney manifestation following the administration of Pfizer-BioNTech COVID-19 vaccine. Minimal change disease is potentially a rare complication of Pfizer-BioNTech COVID-19 vaccine.

PKC-beta1 mediates glucose-induced Akt activation and TGF-beta1 upregulation in mesangial cells.

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. The serine/threonine kinase Akt mediates glucose-induced upregulation of collagen I in mesangial cells through transactivation of the EGF receptor (EGFR). In addition, in renal tubular cells, glucose-induced secretion of TGF-beta requires phosphoinositide-3-OH kinase, suggesting a possible role for Akt in the modulation of TGF-beta expression, but the mechanisms of Akt activation and its involvement in TGF-beta regulation are unknown. Here, in primary mesangial cells, high glucose induced AktS473 phosphorylation, which correlates with its activation, in a protein kinase C beta (PKC-beta)-dependent manner. Glucose led to PKC-beta1 membrane translocation and association with Akt, and PKC-beta1 immunoprecipitated from glucose-treated cells phosphorylated recombinant Akt on S473. PKC is known to mediate glucose-induced TGF-beta1 upregulation through the transcription factor AP-1; here, inhibitors of phosphoinositide-3-OH kinase, PKC-beta and Akt, and dominant-negative Akt all prevented glucose-induced activation of AP-1 and upregulation of TGF-beta1. Finally, pharmacologic and dominant negative inhibition of EGFR blocked glucose-induced activation of PKC-beta1, phosphorylation of AktS473, activation of AP-1, and upregulation of TGF-beta1. In vivo, the PKC-beta inhibitor ruboxistaurin prevented Akt activation in the renal cortex of diabetic rats. In conclusion, PKC-beta1 is an Akt S473 kinase in glucose-treated mesangial cells, and TGF-beta1 transcriptional upregulation requires EGFR/PKC-beta1/Akt signaling. New therapeutic approaches for diabetic nephropathy may result from targeting components of this pathway, particularly the initial EGFR transactivation.

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.

Caveolin-1 phosphorylation is required for stretch-induced EGFR and Akt activation in mesangial cells.

Increased glomerular hydrostatic pressure is an important determinant of glomerulosclerosis and can be modeled in vitro by exposure of mesangial cells (MC) to cyclic mechanical strain. We have recently shown that Akt mediates the stretch-induced production of type I collagen, an important contributor to sclerosis, in MC. Here we studied the upstream mediators of Akt activation. Primary rat MC were exposed to 1 Hz cyclic strain for 10 min, previously shown to induce maximal Akt activation. Neither the integrin inhibitor GRDGSP nor cytoskeletal disruptors had any effect on stretch-induced Akt activation. Akt activation was, however, mediated by transactivation of the epidermal growth factor receptor (EGFR), and this required receptor kinase activity since Akt activation did not occur in cells expressing kinase-dead EGFR (K721A). Src was further shown to be upstream of the EGFR, with its inhibitor SU6656 preventing both EGFR and Akt activation. The membrane microdomains caveolae were found to be required for this signaling to occur. Chemical disruption of caveolae with cyclodextrin or filipin prevented Akt activation, and both EGFR and Akt activation were lost in caveolin-1 (cav-1) knockout MC. The latter was rescued with reexpression of cav-1. Further, Src-mediated phosphorylation of cav-1 on Y14 was required for stretch-induced EGFR and Akt activation, since these were abrogated in MC expressing the nonphosphorylatable cav-1 Y14A mutant. Thus, mechanical strain-induced activation of Akt in MC is independent of integrin activation and the actin cytoskeleton, but depends upon EGFR transactivation. EGFR transactivation requires intact caveolae and the Src-mediated phosphorylation of cav-1 on Y14. These studies define a novel function for cav-1 and caveolae in EGFR transactivation leading to Akt activation by mechanical stress.

p14ARF inhibits the growth of p53 deficient cells in a cell-specific manner.

While p14(ARF) suppression of tumorigenesis in a p53-dependent manner is well studied, the mechanism by which p14(ARF) inhibits tumorigenesis independently of p53 remains elusive. A variety of factors have been reported to play a role in this latter process. We report here that p14(ARF) displays different effects on the anchorage-dependent and -independent growth of p53-null/Mdm2 wild type cells. p14(ARF) blocks both the anchorage-dependent and-independent (soft agar) proliferation of 293T and p53(-/-) HCT116, but not p53-null H1299 lung carcinoma cells. While p14(ARF) had no effect on the anchorage-dependent proliferation of p53(-/-) MEFs and Ras12V-transformed p53(-/-) MEFs, it inhibited the growth of Ras12V-transformed p53(-/-) MEFs in soft agar. Furthermore, ectopic expression of p14(ARF) did not lead to degradation of the E2F1 protein and did not result in the reduction of E2F1 activity detected by two E2F1 responsible promoters, Apaf1 and p14(ARF) promoter, in 293T, p53(-/-) HCT116, and H1299 cells. This is consistent with our observations that p14(ARF) did not result in G1 arrest, but induced apoptosis via Bax up-regulation. Taken together, our data demonstrate that the response of p53-null cells to ARF is cell type dependent and involves factors other than Mdm2 and E2F1.

Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

Identification of a novel Wee1 isoform.

We have identified a novel isoform of Wee1 kinase (Wee1i), which uses Met215 of Wee1 as its initiation codon. RT-PCR, Western blot, and in situ hybridization verified wee1i expression in mammalian cells, rat brain, and rat thymus. Recombinant and partially purified Wee1i from rat thymus displayed kinase activity comparable to or higher than Wee1. The N-terminal 214 residues of Wee1 facilitate its ubiquitin-dependent degradation to trigger mitotic entry. Since Wee1i, lack of these 214 residues, it may evade this degradation and thus provide constitutive Wee1-like kinase activity to inhibit mitotic cell proliferation. Thus, Wee1i may play an important role in differentiation and in tumor suppression.

Stretch-induced Raf-1 activation in mesangial cells requires actin cytoskeletal integrity.

Glomerular capillary hypertension is a determinant of glomerulosclerosis and is modelled in vitro by exposure of mesangial cells to cyclic mechanical strain. In response to strain, Erk is activated and mediates extracellular matrix accumulation and mesangial cell proliferation. Erk activation is dependent on an intact cytoskeleton. Since Raf-1 lies upstream of Erk in response to numerous stimuli, and since its activation is dependent on membrane recruitment, we postulated that the cytoskeleton was essential for Raf-1 membrane recruitment and Erk activation. Primary rat mesangial cells (passages 8-20) were stretched at 1 Hz and 27 kPa. Raf-1 was both phosphorylated on serine-338 (S338) and activated within 2 min of strain. The Raf-1 inhibitor, GW5074, dose-dependently blocked strain-induced Erk activation and Raf-1 phosphorylation. Although phosphatidylinositol-3-kinase (PI3-K) may mediate Raf-1 activation, PI3-K inhibition with wortmannin or LY294002 had no effect on stretch-induced Raf-1 activation. Cytoskeletal disruption with cytochalasin D and the Rho-kinase inhibitor, Y-27632, however, blocked both Raf-1 phosphorylation and activation. Furthermore, membrane localization of Raf-1 was increased by strain and prevented by cytoskeletal disruption. Thus, strain leads to rapid membrane localization, S338 phosphorylation, and activation of Raf-1. These events are independent of PI3-K, but require Rho-kinase activation and an intact actin cytoskeleton.

ATM activity contributes to the tumor-suppressing functions of p14ARF.

P14/p19ARF (ARF) plays a major role in the activation of p53 by oncogenic signals. The biochemical basis of this has not been fully elucidated. We report here that forced expression of p14ARF enhances phosphorylation of p53 serine 15 (p53S15) in NIH3T3, IMR90 and MCF7 cells. Ectopic expression of the oncogenes c-myc, E2F1 and E1A, all of which activate p53 at least partially via ARF, lead to p53S15 phosphorylation in IMR90 cells. In addition, ectopic expression of p53 also results in p53S15 phosphorylation, suggesting that this is a common event in the ARF-p53 tumor suppression system. Furthermore, p53-, p14ARF-, c-myc- and E2F1-, but not E1A-, induced p53S15 phosphorylation was substantially reduced in AT fibroblasts (GM05823). Downregulation of ATM in MCF7 cells using RNA interference (RNAi) technology significantly attenuated p14ARF- and p53-induced phosphorylation of p53S15. Ectopically expressed ARF in NIH3T3 cells induced ATM nuclear foci and activated ATM kinase. Functionally, ectopic expression of p14ARF and c-myc inhibited the proliferation of IMR90 but not ATM null GM05823 cells, and p14ARF-induced inhibition of MCF7 cell proliferation was significantly attenuated by downregulation of ATM by RNAi. Taken together, these data show a functional role for ATM in ARF-mediated tumor suppression.

Flux through the hexosamine pathway is a determinant of nuclear factor kappaB- dependent promoter activation.

The hexosamine pathway may mediate some of the toxic effects of glucose. We hypothesized that flux through this pathway might regulate the activity of nuclear factor kappaB (NF-kappaB)-dependent genes in mesangial cells (MCs). In MCs, RT-PCR revealed that high glucose (30 mmol/l) and glucosamine (1 mmol/l) increased mRNA levels for vascular cell adhesion molecule 1 (VCAM-1) and increased the activity of an NF-kappaB enhancer by 1.5- and 2-fold, respectively. Overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for flux through the hexosamine pathway, led to a 2.2-fold increase in NF-kappaB enhancer activity; the combination of GFAT overexpression and high glucose increased activity 2.8-fold, and these increases were prevented by 40 micromol/l O-diazoacetyl-L-serine (azaserine) or 6-diazo-5-oxonorleucine. High glucose, glucosamine, and GFAT overexpression increased binding of MC nuclear proteins to NF-kappaB consensus sequences. Immunoblotting revealed that the p65 subunit of NF-kappaB was O-glycosylated in MC cultured in physiologic glucose and that significant enhancement occurred with high glucose and glucosamine. Both glucose and glucosamine dose-dependently increased human VCAM-1 promoter activity. In addition, GFAT overexpression activated the VCAM-1 promoter (2.25-fold), with further augmentation by high glucose and abrogation by inhibitors of GFAT, NF-kappaB, and O-glycosylation. Inactivation of the two NF-kappaB sites in the VCAM-1 promoter abolished its response to high glucose, glucosamine, and GFAT overexpression. These results suggest that increased flux through the hexosamine pathway leads to NF-kappaB-dependent promoter activation in MCs.

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.