Alistair J. Ingram

Low-Intensity Warfarin Is Ineffective for the Prevention of PTFE Graft Failure in Patients on Hemodialysis: A Randomized Controlled Trial

Polytetrafluoroethylene (PTFE) dialysis grafts in patients with end-stage renal disease (ESRD) are prone to thrombotic failure. The objective of this multicenter, randomized, double-blind, placebo-controlled clinical trial was to determine if warfarin reduces the risk of failure of PTFE dialysis grafts. Patients with ESRD and newly placed PTFE grafts were studied at community and academic dialysis centers in Southwestern Ontario. Patients were allocated to receive warfarin or matching placebo, with the warfarin administered to achieve a target INR of 1.4 to 1.9. Time to graft failure was the main outcome measure. A total of 107 patients (56 allocated to warfarin) were randomized. The time-to-event analysis revealed no significant difference in the likelihood of graft survival between the two groups (odds ratio, 1.76 in favor of placebo; 95% confidence interval, 0.72 to 4.34). Six major bleeds occurred in five patients allocated to warfarin compared with none in the patients who received placebo (P = 0.03). In conclusion, low-dose warfarin was associated with an excess of clinically important major bleeding in patients with ESRD enrolled in this study. Furthermore, low-intensity, monitored-dose warfarin does not appear to prolong PTFE graft survival.

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 SM22&agr; or a SM22&agr; 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 &agr;-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.

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.

RhoA/Rho-kinase Contribute to the Pathogenesis of Diabetic Renal Disease

OBJECTIVE—Accumulation of glomerular matrix proteins is central to the pathogenesis of diabetic nephropathy, with resident mesangial cells (MCs) known to upregulate matrix protein synthesis in response to high glucose. Because activation of the GTPase RhoA has been implicated in matrix upregulation, we studied its role in induction of the matrix protein fibronectin in diabetic MCs and in vivo in diabetic nephropathy. RESEARCH DESIGN AND METHODS—Glucose (30 mmol/l)-induced RhoA/Rho-kinase, AP-1 activation, and fibronectin upregulation were assessed by immunoblotting, luciferase, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, real-time PCR, Northern blots, and immunofluorescence. Streptozotocin-induced diabetic rats were treated with the ρ-kinase inhibitor fasudil, which was compared with enalapril, and functional and pathologic parameters were assessed. RESULTS—Glucose led to RhoA and downstream Rho-kinase activation. Mannitol was without effect. Activity of the transcription factor AP-1, increased in diabetic MCs and kidneys, is important in the profibrotic effects of glucose, and this was dependent on Rho-kinase signaling. Upregulation of fibronectin by glucose, shown to be mediated by activator protein-1 (AP-1), was prevented by Rho-kinase inhibition. RhoA siRNA and dominant-negative RhoA also markedly attenuated fibronectin upregulation by high glucose. Applicability of these findings were tested in vivo. Fasudil prevented glomerular fibronectin upregulation, glomerular sclerosis, and proteinuria in diabetic rats, with effectiveness similar to enalapril. CONCLUSIONS—High glucose activates RhoA/Rho-kinase in MCs, leading to downstream AP-1 activation and fibronectin induction. Inhibition of this pathway in vivo prevents the pathologic changes of diabetic nephropathy, supporting a potential role for inhibitors of RhoA/Rho in the treatment of diabetic renal disease.

Prolonged sulfonylurea-induced hypoglycemia in diabetic patients with end-stage renal disease.

Renal impairment is a recognized risk factor for prolonged hypoglycemia, but predisposing characteristics in patients with advanced renal impairment have not been studied. We observed prolonged hypoglycemia in a number of patients with end-stage renal disease (ESRD) and conducted a case-control study at two Canadian centers to identify such risk factors. Through hospital, pharmacy, and dialysis program records, we retrospectively identified 7 case patients and 31 controls with ESRD and type 2 diabetes using oral hypoglycemic monotherapy. Control patients had no history of hospital admission for prolonged hypoglycemia. All case patients and 28 controls were receiving glyburide (glibenclamide in Europe); the remainder were treated with tolbutamide. Duration of intravenous treatment for hypoglycemia ranged from 28 to 256 hours, with 83 g to 2 kg of glucose administered per episode. Preceding treatment with glyburide varied from 2 days to 13 years. Univariate analyses showed a recent decline in oral intake (odds ratio [OR], 81; 95% confidence interval [CI], 3.6 to 1,840), previous hypoglycemic episodes (OR, 15; 95% CI, 0.77 to 297), longer duration of diabetes (22 versus 12 years; P = 0.008), and a history of cerebrovascular disease (OR, 7. 0; 95% CI, 1.0 to 47) to be associated with prolonged hypoglycemia. No association between prolonged hypoglycemia and age, sex, beta blockers, angiotensin-converting enzyme inhibitors, oral hypoglycemic dose, or duration of treatment was identified. This study describes the potentially devastating effect of sulfonylurea-based oral hypoglycemic therapy in ESRD. Patients at greatest risk appear to be those with reduced intake, previous hypoglycemic episodes, and longer duration of diabetes. We describe the mechanisms for observed hypoglycemia and suggest that alternative drugs may be considered in this patient group.

PKC-β1 Mediates Glucose-Induced Akt Activation and TGF-β1 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.

Nitric oxide inhibits stretch-induced MAPK activation in mesangial cells through RhoA inactivation.

Glomerular capillary hypertension is an important determinant of glomerulosclerosis in rats with subtotal renal ablation. Dietary supplementation with L-arginine increases renal nitric oxide (NO) production and limits glomerular injury in this model, and early benefits are seen without altered glomerular capillary pressure. In an in vitro model of hemodynamically mediated signaling, the authors have reported that subjecting MC to cyclic stretch/relaxation activates the mitogen-activated protein kinase p42/44 (Erk) cascade and that NO and cyclic GMP abrogate stretch-induced Erk activation by inducing actin cytoskeletal disassembly. The actin cytoskeleton is regulated by the Rho family of GTPases, including RhoA; therefore, the authors examined the role of RhoA in stretch-induced Erk activation and as an NO target. In primary rat MC subjected to cyclic mechanical strain, RhoA activity was maximally increased (2.4-fold) after 1 min of stretch, and Erk activation temporally followed. The Rho-kinase inhibitor Y-27632 attenuated Erk activation in a dose-dependent manner and prevented stretch-induced actin stress fiber formation. The NO donors S-nitroso-N-acetylpenicillamine and cGMP both inhibited stretch-induced RhoA and Erk activation and stress fiber formation. Infection of MC with the RhoA mutant RhoA-Ala188, which is resistant to NO-dependent phosphorylation, abrogated the effects of NO and cGMP on stretch-induced Erk activation and stress fiber formation. The authors conclude that the early activation of RhoA is essential for stretch-induced actin stress fiber formation and Erk activation in MC, events which are prevented by NO and cGMP through their action on RhoA. Inhibition of RhoA may thus be a new approach to the prevention of hemodynamically mediated glomerular injury.

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.

Regulation of the Tumor Suppressor PTEN through Exosomes: A Diagnostic Potential for Prostate Cancer

PTEN is a potent tumor-suppressor protein. Aggressive and metastatic prostate cancer (PC) is associated with a reduction or loss of PTEN expression. PTEN reduction often occurs without gene mutations, and its downregulation is not fully understood. Herein, we show that PTEN is incorporated in the cargo of exosomes derived from cancer cells. PTEN is not detected in exosomes derived from normal, noncancerous cells. We found that PTEN can be transferred to other cells through exosomes. In cells that have a reduction or complete loss of PTEN expression, the transferred PTEN is competent to confer tumor-suppression activity to acceptor cells. In PC patients, we show that PTEN is incorporated in the cargo of exosomes that circulate in their blood. Interestingly, normal subjects have no PTEN expression in their blood exosomes. Further, we found that the prostate-specific antigen (PSA) is incorporated in PC patients’ and normal subjects’ blood exosomes. These data suggest that exosomal PTEN can compensate for PTEN loss in PTEN deficient cells, and may have diagnostic value for prostate cancer.

Shank-interacting protein–like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells

Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein-like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell-derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.