Diabetes and Hypertension Differentially Affect Renal Catecholamines and Renal Reactive Oxygen Species

Patients with diabetic hypertensive nephropathy have accelerated disease progression. Diabetes and hypertension have both been associated with changes in renal catecholamines and reactive oxygen species. With a specific focus on renal catecholamines and oxidative stress we examined a combined model of hypertension and diabetes using normotensive BPN/3J and hypertensive BPH/2J Schlager mice. Induction of diabetes (5 × 55 mg/kg streptozotocin i.p.) did not change the hypertensive status of BPH/2J mice (telemetric 24 h avg. MAP, non-diabetic 131 ± 2 vs. diabetic 129 ± 1 mmHg, n.s at 9 weeks of study). Diabetes-associated albuminuria was higher in BPH/2J vs. diabetic BPN/3J (1205 + 196/-169 versus 496 + 67/-59 μg/24 h, p = 0.008). HPLC measurement of renal cortical norepinephrine and dopamine showed significantly greater levels in hypertensive mice whilst diabetes was associated with significantly lower catecholamine levels. Diabetic BPH/2J also had greater renal catecholamine levels than diabetic BPN/3J (diabetic: norepinephrine BPN/3J 40 ± 4, BPH/2J 91 ± 5, p = 0.010; dopamine: BPN/3J 2 ± 1; BPH/2J 3 ± 1 ng/mg total protein, p < 0.001 after 10 weeks of study). Diabetic BPH/2J showed greater cortical tubular immunostaining for monoamine oxidase A and cortical mitochondrial hydrogen peroxide formation was greater in both diabetic and non-diabetic BPH/2J. While cytosolic catalase activity was greater in non-diabetic BPH/2J it was significantly lower in diabetic BPH/2J (cytosolic: BPH/2J 127 ± 12 vs. 63 ± 6 nmol/min/ml, p < 0.001). We conclude that greater levels of renal norepinephrine and dopamine associated with hypertension, together with diabetes-associated compromised anti-oxidant systems, contribute to increased renal oxidative stress in diabetes and hypertension. Elevations in renal cortical catecholamines and reactive oxygen species have important therapeutic implications for hypertensive diabetic patients

miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

OBJECTIVE: Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2. RESEARCH DESIGN AND METHODS: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy. RESULTS: Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β-dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3' untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. CONCLUSIONS: miR-200a and miR-141 significantly impact on the development and progression of TGF-β-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes

Endothelial or vascular smooth muscle cell-specific expression of human NOX5 exacerbates renal inflammation, fibrosis and albuminuria in the Akita mouse

Aims/hypothesis: Excessive production of reactive oxygen species (ROS) plays a detrimental role in the progression of diabetic kidney disease (DKD). Renal oxidative stress activates proinflammatory cytokines, chemokines and profibrotic factors in DKD. Increased expression of the prooxidant enzyme NADPH oxidase (NOX) 5 in kidneys of diabetic individuals has been hypothesised to correlate with renal injury and progression of DKD. Since the gene encoding NOX5 is not expressed in the mouse genome, we examined the effect of inducible human NOX5 expression in renal cells, selectively in either endothelial cells or vascular smooth muscle cells (VSMCs)/mesangial cells in a model of insulin-deficient diabetes, the Akita mouse. Methods: Renal structural injury, including glomerulosclerosis, mesangial expansion and extracellular matrix protein accumulation, as well as renal inflammation, ROS formation and albuminuria, were examined in the NOX5 transgenic Akita mouse model of DKD. Results: Expression of NOX5 in either endothelial cells or VSMCs/mesangial cells in diabetic Akita mice was associated with increased renal inflammation (monocyte chemoattractant protein-1, NF-κB and toll-like receptor-4) and glomerulosclerosis, as well as upregulation of protein kinase C-α and increased expression of extracellular matrix genes (encoding collagen III, fibronectin and α-smooth muscle actin) and proteins (collagen IV), most likely mediated via enhanced renal ROS production. The effect of VSMC/mesangial cell-specific NOX5 expression resulted in more pronounced renal fibrosis in comparison with endothelial cell-specific NOX5 expression in diabetic mice. In addition, albuminuria was significantly increased in diabetic VEcad+NOX5+ mice (1192 ± 194 μg/24 h) when compared with diabetic VEcad+NOX5− mice (770 ± 98 μg/24 h). Furthermore, the regulatory components of NOX5 activation, including heat shock protein 90 and transient receptor potential cation channel subfamily C member 6, were upregulated only in the presence of both NOX5 and diabetes. Conclusions/interpretation: The findings from this study highlight the importance of NOX5 in promoting diabetes-related renal injury and provide the rationale for the development of a selective NOX5 inhibitor for the prevention and/or treatment of DKD

NADPH oxidase, NOX1, mediates vascular injury in ischemic retinopathy

<b>Aims:</b> Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity. <b>Results:</b> Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831. <b>Innovation:</b> Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity. <b>Conclusions:</b> Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathie

CXCR4 Antagonism attenuates the development of diabetic cardiac fibrosis

Heart failure (HF) is an increasingly recognized complication of diabetes. Cardiac fibrosis is an important causative mechanism of HF associated with diabetes. Recent data indicate that inflammation may be particularly important in the pathogenesis of cardiovascular fibrosis. We sought to determine the mechanism by which cardiac fibrosis develops and to specifically investigate the role of the CXCR4 axis in this process. Animals with type I diabetes (streptozotocin treated mice) or type II diabetes (Israeli Sand-rats) and controls were randomized to treatment with a CXCR4 antagonist, candesartan or vehicle control. Additional groups of mice also underwent bone marrow transplantation (GFP+ donor marrow) to investigate the potential role of bone marrow derived cell mobilization in the pathogenesis of cardiac fibrosis. Both type I and II models of diabetes were accompanied by the development of significant cardiac fibrosis. CXCR4 antagonism markedly reduced cardiac fibrosis in both models of diabetes, similar in magnitude to that seen with candesartan. In contrast to candesartan, the anti-fibrotic actions of CXCR4 antagonism occurred in a blood pressure independent manner. Whilst the induction of diabetes did not increase the overall myocardial burden of GFP+ cells, it was accompanied by an increase in GFP+ cells expressing the fibroblast marker alpha-smooth muscle actin and this was attenuated by CXCR4 antagonism. CXCR4 antagonism was also accompanied by increased levels of circulating regulatory T cells. Taken together the current data indicate that pharmacological inhibition of CXCR4 significantly reduces diabetes induced cardiac fibrosis, providing a potentially important therapeutic approach

Evaluation of the zucker diabetic fatty (ZDF) rat as a model for human disease based on urinary peptidomic profiles

Representative animal models for diabetes-associated vascular complications are extremely relevant in assessing potential therapeutic drugs. While several rodent models for type 2 diabetes (T2D) are available, their relevance in recapitulating renal and cardiovascular features of diabetes in man is not entirely clear. Here we evaluate at the molecular level the similarity between Zucker diabetic fatty (ZDF) rats, as a model of T2D-associated vascular complications, and human disease by urinary proteome analysis. Urine analysis of ZDF rats at early and late stages of disease compared to age- matched LEAN rats identified 180 peptides as potentially associated with diabetes complications. Overlaps with human chronic kidney disease (CKD) and cardiovascular disease (CVD) biomarkers were observed, corresponding to proteins marking kidney damage (eg albumin, alpha-1 antitrypsin) or related to disease development (collagen). Concordance in regulation of these peptides in rats versus humans was more pronounced in the CVD compared to the CKD panels. In addition, disease-associated predicted protease activities in ZDF rats showed higher similarities to the predicted activities in human CVD. Based on urinary peptidomic analysis, the ZDF rat model displays similarity to human CVD but might not be the most appropriate model to display human CKD on a molecular level

Urinary MicroRNA Profiling in the Nephropathy of Type 1 Diabetes

Background: Patients with Type 1 Diabetes (T1D) are particularly vulnerable to development of Diabetic nephropathy (DN) leading to End Stage Renal Disease. Hence a better understanding of the factors affecting kidney disease progression in T1D is urgently needed. In recent years microRNAs have emerged as important post-transcriptional regulators of gene expression in many different health conditions. We hypothesized that urinary microRNA profile of patients will differ in the different stages of diabetic renal disease. Methods and Findings: We studied urine microRNA profiles with qPCR in 40 T1D with >20 year follow up 10 who never developed renal disease (N) matched against 10 patients who went on to develop overt nephropathy (DN), 10 patients with intermittent microalbuminuria (IMA) matched against 10 patients with persistent (PMA) microalbuminuria. A Bayesian procedure was used to normalize and convert raw signals to expression ratios. We applied formal statistical techniques to translate fold changes to profiles of microRNA targets which were then used to make inferences about biological pathways in the Gene Ontology and REACTOME structured vocabularies. A total of 27 microRNAs were found to be present at significantly different levels in different stages of untreated nephropathy. These microRNAs mapped to overlapping pathways pertaining to growth factor signaling and renal fibrosis known to be targeted in diabetic kidney disease. Conclusions: Urinary microRNA profiles differ across the different stages of diabetic nephropathy. Previous work using experimental, clinical chemistry or biopsy samples has demonstrated differential expression of many of these microRNAs in a variety of chronic renal conditions and diabetes. Combining expression ratios of microRNAs with formal inferences about their predicted mRNA targets and associated biological pathways may yield useful markers for early diagnosis and risk stratification of DN in T1D by inferring the alteration of renal molecular processes. © 2013 Argyropoulos et al

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

Local Gene Silencing of Monocyte Chemoattractant Protein-1 Prevents Vulnerable Plaque Disruption in Apolipoprotein E-Knockout Mice

Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine (CCL2), has been demonstrated to play important roles in atherosclerosis and becoming an important therapeutic target for atherosclerosis. The present study was undertaken to test the hypothesis that local RNAi of MCP-1 by site-specific delivery of adenovirus-mediated small hairpin RNA (shRNA) may enhance plaque stability and prevent plaque disruption in ApoE−/− mice. We designed an adenovirus-mediated shRNA against mouse MCP-1 (rAd5-MCP-1-shRNA). Male apolipoprotein E-knockout (ApoE−/−) mice (n = 120) were fed a high-fat diet and vulnerable plaques were induced by perivascular placement of constrictive collars around the carotid artery, intraperitoneal injection of lipopolysaccharide and stress stimulation. Mice were randomly divided into RNA interference (Ad-MCP-1i) group receiving local treatment of rAd5-MCP-1-shRNA suspension, Ad-EGFP group receiving treatment of rAd5-mediated negative shRNA and mock group receiving treatment of saline. Two weeks after treatment, plaque disruption rates were significantly lower in the Ad-MCP-1i group than in the Ad-EGFP group (13.3% vs. 60.0%, P = 0.01), and local MCP-1 expression was significantly inhibited in the Ad-MCP-1i group confirmed by immunostaining, qRT-PCR and western blot (P<0.001). Compared with the Ad-EGFP group, carotid plaques in the Ad-MCP-1i group showed increased levels of collagen and smooth muscle cells, and decreased levels of lipid and macrophages. The expression of inflammatory cytokines and activities of matrix metalloproteinases (MMPs) were lower in the Ad-MCP-1i group than in the Ad-EGFP group. In conclusion, site-specific delivery of adenoviral-mediated shRNA targeting mouse MCP-1 downregulated MCP-1 expression, turned a vulnerable plaque into a more stable plaque phenotype and prevented plaque disruption. A marked suppression of the local inflammatory cytokine expression may be the central mechanism involved