Empagliflozin and Rapid Kidney Function Decline Incidence in Type 2 Diabetes: An Exploratory Analysis From the EMPA-REG OUTCOME Trial.

Rationale & Objective: Kidney function progressively declines in most patients with type 2 diabetes (T2DM). Many develop progressive chronic kidney disease (CKD), but some experience a more rapid decline, with a greater risk of kidney failure and cardiovascular disease. In EMPA-REG OUTCOME, empagliflozin was associated with slower kidney disease progression. This post hoc analysis evaluated the effect of empagliflozin (pooled doses) on the prevalence of a "rapid decliner" phenotype, defined by an annual estimated glomerular filtration rate (eGFR) decline of >3 mL/min/1.73 m2. Study Design: This was an exploratory analysis of EMPA-REG OUTCOME, a large randomized, double-blind, placebo-controlled trial in adults with T2DM, established cardiovascular disease and an eGFR of ≥30 mL/min/1.73 m2. Setting & Participants: Analysis was undertaken on 6,967 participants (99.2%) in whom serial eGFR data was available. Interventions: Patients were randomized (1:1:1) to empagliflozin 10 mg, 25 mg, or placebo in addition to standard of care. Outcomes: Annual change in eGFR over the maintenance phase of treatment (week 4 to last value on treatment) was calculated using linear regression models. Logistic regression analysis was used to investigate differences in rapid decline between the treatment groups. Results: Over the study period, a rapid decliner phenotype was observed in 188 (9.5%) participants receiving placebo and 134 (3.4%) receiving empagliflozin. After adjusting for other risk factors, this equated to a two-third reduction in odds (OR, 0.32; 95% CI, 0.25-0.40; P < 0.001) among participants receiving empagliflozin versus placebo. A comparable risk reduction was observed using a threshold of eGFR decline of >5 mL/min/1.73 m2/y (empagliflozin vs placebo, 43 [1.1%] vs 44 [2.2%] participants; OR, 0.47; 95% CI, 0.31-0.72; P < 0.001). Limitations: This is a post hoc analysis of a trial undertaken in participants with T2DM and CVD. Generalization of findings to other settings remains to be established. Conclusions: Patients receiving empagliflozin were significantly less likely to experience a rapid decline in eGFR over a median of 2.6 years of exposure to the study drug. Funding: The Boehringer Ingelheim and Eli Lilly and Company Diabetes Alliance. Trial Registration: clinicaltrials.gov ID: NCT01131676.

In most people with type 2 diabetes, their kidney function starts to decline over time. However, in some people, this can happen more rapidly, which can increase their risk of kidney or cardiovascular disease. A major study, EMPA-REG OUTCOME, has shown that empagliflozin, which helps to control blood sugar in people with type 2 diabetes, also reduced the risk of cardiovascular disease events and slowed the progression of kidney disease, when compared with people in the study who received placebo. In this new research from the same major study empagliflozin, compared with a placebo, was shown to reduce the risk of people having a rapid decline in their kidney function over the 3 years of the study.

Transient Intermittent Hyperglycemia Accelerates Atherosclerosis by Promoting Myelopoiesis.

RATIONALE: Treatment efficacy for diabetes mellitus is largely determined by assessment of HbA1c (glycated hemoglobin A1c) levels, which poorly reflects direct glucose variation. People with prediabetes and diabetes mellitus spend >50% of their time outside the optimal glucose range. These glucose variations, termed transient intermittent hyperglycemia (TIH), appear to be an independent risk factor for cardiovascular disease, but the pathological basis for this association is unclear. OBJECTIVE: To determine whether TIH per se promotes myelopoiesis to produce more monocytes and consequently adversely affects atherosclerosis. METHODS AND RESULTS: To create a mouse model of TIH, we administered 4 bolus doses of glucose at 2-hour intervals intraperitoneally once to WT (wild type) or once weekly to atherosclerotic prone mice. TIH accelerated atherogenesis without an increase in plasma cholesterol, seen in traditional models of diabetes mellitus. TIH promoted myelopoiesis in the bone marrow, resulting in increased circulating monocytes, particularly the inflammatory Ly6-Chi subset, and neutrophils. Hematopoietic-restricted deletion of S100a9, S100a8, or its cognate receptor Rage prevented monocytosis. Mechanistically, glucose uptake via GLUT (glucose transporter)-1 and enhanced glycolysis in neutrophils promoted the production of S100A8/A9. Myeloid-restricted deletion of Slc2a1 (GLUT-1) or pharmacological inhibition of S100A8/A9 reduced TIH-induced myelopoiesis and atherosclerosis. CONCLUSIONS: Together, these data provide a mechanism as to how TIH, prevalent in people with impaired glucose metabolism, contributes to cardiovascular disease. These findings provide a rationale for continual glucose control in these patients and may also suggest that strategies aimed at targeting the S100A8/A9-RAGE (receptor for advanced glycation end products) axis could represent a viable approach to protect the vulnerable blood vessels in diabetes mellitus. Graphic Abstract: A graphic abstract is available for this article.

Relationship Between Plasma 8-OH-Deoxyguanosine and Cardiovascular Disease and Survival in Type 2 Diabetes Mellitus: Results From the ADVANCE Trial.

BACKGROUND: 8-Oxo-2'-deoxyguanosine (8-oxo-2'-dG) is a biomarker of oxidative DNA damage that is associated with cardiovascular disease and premature mortality in the general population. Although oxidative stress has a proven role in cardiovascular complications in diabetes mellitus, evidence for a relationship between plasma 8-oxo-2'-dG and major cardiovascular outcomes in diabetes mellitus is weak. METHODS AND RESULTS: A case-cohort study was performed in 3766 participants with prevalent diabetes mellitus in the ADVANCE (Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation) trial (ClinicalTrials.gov number NCT00145925). The hazard ratios for mortality and major acute cardiovascular events were derived using Cox regression models. During a median of 5 years of follow-up, 695 (18.4%) participants in this enriched cohort died (including 354 deaths from cardiovascular disease). Individuals with higher levels of 8-oxo-2'-dG were more likely to die. After adjusting for cardiovascular disease risk factors, the hazard ratio for a 1-SD increase in plasma 8-oxo-2'-dG was 1.10 (95% confidence interval, 1.01-1.20; P=0.03). This was driven by an independent association between plasma 8-oxo-2'-dG and cardiovascular death (hazard ratio, 1.23; 95% confidence interval, 1.10-1.37 [P<0.001]). By contrast, no association was seen between 8-oxo-2'-dG and noncardiovascular disease death (of which cancer was the major single cause). 8-Oxo-2'-dG was also not significantly associated with either nonfatal myocardial infarction or nonfatal stroke. CONCLUSIONS: In adults with type 2 diabetes mellitus, increased levels of 8-oxo-2'-dG are independently associated with all-cause mortality and cardiovascular mortality in adults with longstanding type 2 diabetes mellitus who participated in the ADVANCE trial, consistent with the role of oxidative damage in the development and progression of cardiovascular decompensation in diabetes mellitus. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00145925.

Resveratrol Inhibits Growth of Experimental Abdominal Aortic Aneurysm Associated With Upregulation of Angiotensin-Converting Enzyme 2.

OBJECTIVE: Recent evidence suggests an important role for angiotensin-converting enzyme 2 (ACE2) in limiting abdominal aortic aneurysm (AAA). This study examined the effect of ACE2 deficiency on AAA development and the efficacy of resveratrol to upregulate ACE2 in experimental AAA. APPROACH AND RESULTS: Ace2 deletion in apolipoprotein-deficient mice (ApoE-/-Ace2-/y ) resulted in increased aortic diameter and spontaneous aneurysm of the suprarenal aorta associated with increased expression of inflammation and proteolytic enzyme markers. In humans, serum ACE2 activity was negatively associated with AAA diagnosis. ACE2 expression was lower in infrarenal biopsies of patients with AAA than organ donors. AAA was more severe in ApoE-/-Ace2-/y mice compared with controls in 2 experimental models. Resveratrol (0.05/100-g chow) inhibited growth of pre-established AAAs in ApoE-/- mice fed high-fat chow and infused with angiotensin II continuously for 56 days. Reduced suprarenal aorta dilatation in mice receiving resveratrol was associated with elevated serum ACE2 and increased suprarenal aorta tissue levels of ACE2 and sirtuin 1 activity. In addition, the relative phosphorylation of Akt and ERK (extracellular signal-regulated kinase) 1/2 within suprarenal aorta tissue and gene expression for nuclear factor of kappa light polypeptide gene enhancer in B cells 1, angiotensin type-1 receptor, and metallopeptidase 2 and 9 were significantly reduced. Upregulation of ACE2 in human aortic smooth muscle cells by resveratrol in vitro was sirtuin 1-dependent. CONCLUSIONS: This study provides experimental evidence of an important role for ACE2 in limiting AAA development and growth. Resveratrol upregulated ACE2 and inhibited AAA growth in a mouse model.

AT2R agonist, compound 21, is reno-protective against type 1 diabetic nephropathy.

The hemodynamic and nonhemodynamic effects of angiotensin II on diabetic complications are considered to be primarily mediated by the angiotensin II type 1 receptor subtype. However, its biological and functional effect mediated through the angiotensin II type 2 receptor subtype is still unclear. Activation of the angiotensin II type 2 receptors has been postulated to oppose angiotensin II type 1 receptor-mediated actions and thus attenuate fibrosis. This study aimed to elucidate the reno-protective role of the novel selective angiotensin II type 2 receptor agonist, Compound 21, in an experimental model of type 1 diabetic nephropathy. Compound 21 treatment significantly attenuated diabetes mellitus-induced elevated levels of cystatin C, albuminuria, mesangial expansion, and glomerulosclerosis in diabetic mice. Moreover, Compound 21 markedly inhibited the expression of various proteins implicated in oxidative stress, inflammation, and fibrosis, in association with decreased extracellular matrix production. These findings demonstrate that monotherapy of Compound 21 is protective against the progression of experimental diabetic nephropathy by inhibiting renal oxidative stress, inflammation, and fibrosis.

Added value of soluble tumor necrosis factor-α receptor 1 as a biomarker of ESRD risk in patients with type 1 diabetes.

OBJECTIVE: Recent studies have suggested that circulating levels of the tumor necrosis factor-α receptor 1 (sTNFαR1) may be a useful predictor for the risk of end-stage renal disease (ESRD) in patients with diabetes. However, its potential utility as a biomarker has not been formally quantified. RESEARCH DESIGN AND METHODS: Circulating levels of sTNFαR1 were assessed in 429 patients with type 1 diabetes and overt nephropathy from the Finnish Diabetic Nephropathy (FinnDiane) cohort study. Predictors of incident ESRD over a median of 9.4 years of follow-up were determined by Cox regression and Fine-Gray competing risk analyses. The added value of sTNFαR1 was estimated via time-dependent receiver operating characteristic curves, net reclassification index (NRI), and integrated discrimination improvement (IDI) for survival data. RESULTS: A total of 130 individuals developed ESRD (28%; ESRD incidence rate of 3.4% per year). In cause-specific modeling, after adjusting for baseline renal status, predictors of increased incidence of ESRD in patients with overt nephropathy were an elevated HbA1c, shorter duration of diabetes, and circulating levels of sTNFαR1. Notably, sTNFαR1 outperformed estimated glomerular filtration rate in terms of R(2). Circulating levels of the sTNFαR1 also remained associated with ESRD after adjusting for the competing risk of death. A prediction model including sTNFαR1 (as a -0.5 fractional polynomial) was superior to a model without it, as demonstrated by better global fit, an increment of R(2), the C index, and area under the curve. Estimates of IDI and NRI(>0) were 0.22 (95% CI 0.16-0.28; P < 0.0001) and 0.98 (0.78-1.23; P < 0.0001), respectively. The median increment in the risk score after including sTNFαR1 in the prediction model was 0.18 (0.12-0.30; P < 0.0001). CONCLUSIONS: Circulating levels of sTNFαR1 are independently associated with the cumulative incidence of ESRD. This association is both significant and biologically plausible and appears to provide added value as a biomarker, based on the absolute values of NRI and IDI.

Role of bone-marrow- and non-bone-marrow-derived receptor for advanced glycation end-products (RAGE) in a mouse model of diabetes-associated atherosclerosis.

RAGE (receptor for advanced glycation end-products) is expressed on multiple cell types implicated in the progression of atherosclerosis and plays a role in DAA (diabetes-associated atherosclerosis). The aim of the present study was to determine the relative role of either BM (bone marrow)- or non-BM-derived RAGE in the pathogenesis of STZ (streptozotocin)-induced DAA. Male ApoE (apolipoprotein E)-null (ApoE-/-:RAGE+/+) and ApoE:RAGE-null (ApoE-/-:RAGE-/-) mice at 7 weeks of age were rendered diabetic with STZ. At 8 weeks of age, ApoE-/- and ApoE-/-:RAGE-/- control and diabetic mice received BM from either RAGE-null or RAGE-bearing mice, generating various chimaeras. After 10 and 20 weeks of diabetes, mice were killed and gene expression and atherosclerotic lesion formation were evaluated respectively. Deletion of RAGE in either the BM cells or non-BM cells both resulted in a significant attenuation in DAA, which was associated with reduced VCAM-1 (vascular cell adhesion molecule-1) expression and translated into reduced adhesion in vitro. In conclusion, the results of the present study highlight the importance of both BM- and non-BM-derived RAGE in attenuating the development of DAA.

Coeliac disease, gluten-free diet and the development and progression of albuminuria in children with type 1 diabetes.

OBJECTIVES: Although a diagnosis of coeliac disease (CD) may be confronting to children with type 1 diabetes and their families, we hypothesize that children with CD have lower urinary albumin excretion, a marker of renal dysfunction. RESEARCH DESIGN: Twenty-four children with type 1 diabetes and biopsy-proven CD, on a gluten-free diet for at least 1 yr, were recruited from a single paediatric diabetes clinic alongside 55 children with type 1 diabetes but without CD matched for age, gender, duration of diabetes, and glycaemic control. RESULTS: Despite comparable diabetes exposure, glycaemic control and nutritional status, children with type 1 diabetes and CD had a lower urinary albumin creatinine ratio than in diabetic subjects without CD (0.9 ± 0.3 mg/mmol vs. 1.6 ± 0.3 mg/mmol; p = 0.01). Participants with CD also showed slower progression in albuminuria over 5-yr of follow-up while a small but significant increase was observed in the children with diabetes alone (1.6 ± 0.3 mg/mmol; follow-up 2.4 ± 0.5 mg/mmol; p = 0.02). CONCLUSIONS: As urinary albumin excretion is continuously associated with the risk of kidney disease, it is possible to speculate that CD or its management confers a degree of renoprotection. Larger studies are required to test this hypothesis.

Osteoprotegerin is an independent predictor of vascular events in Finnish adults with type 1 diabetes.

OBJECTIVE: Osteoprotegerin (OPG) is involved in the process of vascular calcification. We investigated whether OPG is associated with the development and progression of diabetes complications in adults with type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: Serum OPG was measured in 1,939 adults with T1D participating in the Finnish Diabetic Nephropathy (FinnDiane) Study. Patients with end-stage renal disease (dialysis or transplantation) at baseline were excluded from analysis. Data on cardiovascular (CV) events and mortality during follow-up were verified from hospital discharge registries (ICD codes) and the Finnish National Death Registry, respectively. The follow-up time was 10.4 ± 2.0 (mean ± SD) years. RESULTS: Only patients with macroalbuminuria and/or renal impairment had elevated OPG concentrations, when compared with participants without overt kidney disease. Patients with retinopathy or CV disease also had higher OPG concentrations, but this was attributable to their higher frequency of chronic kidney disease. OPG predicted an incident CV event (hazard ratio 1.21 [95% CI 1.01-1.45]; P = 0.035) and peripheral vascular disease/amputation events (1.46 [1.13-1.88]; P = 0.004) during follow-up. CONCLUSIONS: We showed that serum OPG is an independent predictor of CV complications. OPG may be directly involved in extraosseous calcification, resulting in stiffening of the arteries and subsequent vascular insufficiency in patients with T1D.

Activation of the Renin-Angiotensin system mediates the effects of dietary salt intake on atherogenesis in the apolipoprotein E knockout mouse.

Dietary salt intake is a major determinant of the activation state of renin-angiotensin-aldosterone system. Given the important role of the renin-angiotensin-aldosterone system in plaque accumulation, we investigated its role in the development of atherogenesis associated with sodium intake in apolipoprotein E knockout mice. Six-weeks of a low-salt diet (containing 0.03% sodium) resulted in a 4-fold increase in plaque accumulation in apolipoprotein E knockout mice when compared with mice receiving normal chow (containing 0.30% sodium). This was associated with activation of the renin-angiotensin-aldosterone system, increased vascular expression of adhesion molecules and inflammatory cytokines, and increased adhesion of labeled leukocytes across the whole aorta on a dynamic flow assay. These changes were blocked with the angiotensin-converting enzyme inhibitor perindopril (2 mg/kg per day). A high-salt diet (containing 3% sodium) attenuated vascular inflammation and atherogenesis, associated with suppression of the renin-angiotensin-aldosterone system, although systolic blood pressure levels were modestly increased (5 ± 1 mmHg). Constitutive activation of the renin-angiotensin-aldosterone system in angiotensin-converting enzyme 2 apolipoprotein E knockout mice was also associated with increased atherosclerosis and vascular adhesion, and this was attenuated by a high-salt diet associated with suppression of the renin-angiotensin-aldosterone system. By contrast, a low-salt diet failed to further activate the renin-angiotensin-aldosterone system or to increase atherosclerosis in angiotensin-converting enzyme 2 apolipoprotein E knockout mice. Together, these data validate a relationship between salt-mediated renin-angiotensin-aldosterone system activation and atherogenesis, which may partly explain the inconclusive or paradoxical findings of recent observational studies, despite clear effects on blood pressure.

TNF-related apoptosis-inducing ligand significantly attenuates metabolic abnormalities in high-fat-fed mice reducing adiposity and systemic inflammation.

TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] has recently been shown to ameliorate the natural history of DM (diabetes mellitus). It has not been determined yet whether systemic TRAIL delivery would prevent the metabolic abnormalities due to an HFD [HF (high-fat) diet]. For this purpose, 27 male C57bl6 mice aged 8 weeks were randomly fed on a standard diet, HFD or HFD+TRAIL for 12 weeks. TRAIL was delivered weekly by intraperitoneal injection. Body composition was evaluated; indirect calorimetry studies, GTT (glucose tolerance test) and ITT (insulin tolerance test) were performed. Pro-inflammatory cytokines, together with adipose tissue gene expression and apoptosis, were measured. TRAIL treatment reduced significantly the increased adiposity associated with an HFD. Moreover, it reduced significantly hyperglycaemia and hyperinsulinaemia during a GTT and it improved significantly the peripheral response to insulin. TRAIL reversed the changes in substrate utilization induced by the HFD and ameliorated skeletal muscle non-esterified fatty acids oxidation rate. This was associated with a significant reduction of pro-inflammatory cytokines together with a modulation of adipose tissue gene expression and apoptosis. These findings shed light on the possible anti-adipogenic and anti-inflammatory effects of TRAIL and open new therapeutic possibilities against obesity, systemic inflammation and T2DM (Type 2 DM).

High nephron endowment protects against salt-induced hypertension.

While low nephron number is associated with increased risk of developing cardiovascular and renal disease, the functional consequences of a high nephron number are unknown. We tested the hypothesis that a high nephron number provides protection against hypertensive and renal insults. Mean arterial pressure (MAP) and renal function were characterized in male wild-type (WT) and transforming growth factor-ß2 heterozygous (Tgfb2(+/-)) mice under basal conditions and following a chronic high-salt diet. Kidneys were collected for unbiased stereological analysis. Baseline MAP and renal function were indistinguishable between genotypes. The chronic high-salt diet (5% NaCl for 4 wk followed by 8% NaCl for 4 wk) led to similar step-wise increases in urine volume, Na(+) excretion, and albuminuria in the genotypes. The 5% NaCl diet induced modest and similar increases in MAP (3.5 ± 1.6 and 3.4 ± 0.8 mmHg in WT and Tgfb2(+/-), respectively). After the step up to the 8% NaCl diet, MAP increased further in WT (+15.9 ± 5.1 mmHg), but not Tgfb2(+/-) (-0.1 ± 1.0 mmHg), mice. Nephron number was 30% greater in Tgfb2(+/-) than WT mice and was not affected by the chronic high-salt diet. Mean glomerular volume was lower in Tgfb2(+/-) than WT mice, and the chronic high-salt diet induced significant glomerular hypertrophy. In a separate cohort of mice, an acute, 7-day, 8% NaCl diet induced similar rises in MAP in the genotypes. This is the first study to examine the physiological characteristics of a model of high nephron number, and the findings are consistent with this phenotype providing protection against chronic, but not acute, hypertensive insults.

The arterial depressor response to chronic low-dose angiotensin II infusion in female rats is estrogen dependent.

The complex role of the renin-angiotensin-system (RAS) in arterial pressure regulation has been well documented. Recently, we demonstrated that chronic low-dose angiotensin II (ANG II) infusion decreases arterial pressure in female rats via an AT(2)R-mediated mechanism. Estrogen can differentially regulate components of the RAS and is known to influence arterial pressure regulation. We hypothesized that AT(2)R-mediated depressor effects evident in females were estrogen dependent and thus would be abolished by ovariectomy and restored by estrogen replacement. Female Sprague-Dawley rats underwent ovariectomy or sham surgery and were treated with 17ß-estradiol or placebo. Mean arterial pressure (MAP) was measured via telemetry in response to a 2-wk infusion of ANG II (50 ng·kg(-1)·min(-1) sc) or saline. MAP significantly decreased in females treated with ANG II (-10 ± 2 mmHg), a response that was abolished by ovariectomy (+4 ± 2 mmHg) and restored with estrogen replacement (-6 ± 2 mmHg). Cardiac and renal gene expression of components of the RAS was differentially regulated by estrogen, such that overall, estrogen shifted the balance of the RAS toward the vasodilatory axis. In conclusion, estrogen-dependent mechanisms offset the vasopressor actions of ANG II by enhancing RAS vasodilator pathways in females. This highlights the potential for these vasodilator pathways as therapeutic targets, particularly in women.

Osteoprotegerin promotes vascular fibrosis via a TGF-ß1 autocrine loop.

BACKGROUND: This study was designed to evaluate the potential role of osteoprotegerin (OPG) in arterial fibrosis. METHODS: Aortic samples were analyzed after in vivo treatment of ApoE(-/-) mice with recombinant human OPG. Mouse vascular smooth muscle cells (VSMC) were exposed in vitro to recombinant OPG and analyzed for markers of inflammation and fibrosis, such as fibronectin, collagen I, III, IV and transforming growth factor-ß1 (TGF-ß1). Conversely, the potential modulation of endogenous OPG expression and release by VSMC was analyzed in response to different pro-atherosclerotic cytokines, TGF-ß1, platelet derived growth factor (PDGF) and angiogensin II (Ang II). RESULTS: In vivo treatment with human OPG induced signs of fibrosis and up-regulated the arterial expression of TGF-ß1. Consistently, in vitro treatment of VSMC with human OPG induced the expression of fibronectin, collagen type I, III, IV, metalloprotein-2 (MMP-2) and MMP-9, as well as of TGF-ß1. On the other hand, exposure to recombinant TGF-ß1 promoted the expression/release of endogenous OPG and mediated the increase of OPG release induced by PDGF and Ang II in VSMC. CONCLUSIONS: Taken together, these data support a pathogenic role for OPG in the development and progression of atherosclerotic lesions and suggest the existence of a vicious circle between TGF-ß1 and OPG.

miR-200a Prevents renal fibrogenesis through repression of TGF-ß2 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-ß1 and TGF-ß2. RESEARCH DESIGN AND METHODS: Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-ß1 and TGF-ß2 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-ß signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy. RESULTS: Both TGF-ß1 and TGF-ß2 induced EMT and fibrogenesis in NRK52E cells. TGF-ß1 and TGF-ß2 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-ß-dependent EMT. miR-200a also downregulated the expression of TGF-ß2, via direct interaction with the 3' untranslated region of TGF-ß2. 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-ß-dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-ß 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.

Genetic Ace2 deficiency accentuates vascular inflammation and atherosclerosis in the ApoE knockout mouse.

RATIONALE: Angiotensin-converting enzyme (ACE)2 opposes the actions of angiotensin (Ang) II by degrading it to Ang 1-7. OBJECTIVE: Given the important role of Ang II/Ang 1-7 in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis. METHODS AND RESULTS: C57Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C57Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-α. Similarly, selective inhibition of ACE2 with MLN-4760 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (10(-7) mol/L). CONCLUSIONS: Genetic Ace2 deficiency is associated with upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli. In atherosclerosis-prone ApoE KO mice, these changes potentially contribute to increased plaque accumulation. These findings emphasize the potential utility of ACE2 repletion as a strategy to reduce atherosclerosis.

The molecular mediators of type 2 epithelial to mesenchymal transition (EMT) and their role in renal pathophysiology.

Common to all forms of chronic kidney disease is the progressive scarring of the tubulo-interstitial space, associated with the acquisition and accumulation of activated myofibroblasts. Many of these myofibroblasts are generated when tubular epithelial cells progressively lose their epithelial characteristics (cell-cell contact, microvilli, tight-junction proteins, apical-basal polarity) and acquire features of a mesenchymal lineage, including stress fibres, filopodia and augmented matrix synthesis. This process, known as epithelial to mesenchymal transition (EMT), plays an important role in progressive kidney disease. For EMT to occur in tubular cells, the transcriptional activation (and derepression) of genes required to sustain mesenchymal-type structures and functions (e.g. vimentin, alpha-smooth muscle actin) must occur alongside repression (or deactivation) of genes that act to maintain the epithelial phenotype (e.g. E-cadherin, bone morphogenic protein 7). Several factors have been suggested as potential initiators of EMT. With a few key exceptions, these triggers require the induction of transforming growth factor beta (TGF-beta) and downstream mediators, including SMADs, CTGF, ILK and SNAI1. Activation of TGF-beta receptors is also able to stimulate a range of additional pathways (so-called non-SMAD activation), including RhoA, mitogen-activated protein kinase and phosphoinositide 3-kinase signalling cascades, that also contribute to EMT and renal fibrogenesis. This review examines in detail the molecular mediators of EMT in tubular cells and its potential role as a long-lasting mediator of metabolic stress.

Disparate effects on renal and oxidative parameters following RAGE deletion, AGE accumulation inhibition, or dietary AGE control in experimental diabetic nephropathy.

Advanced glycation end products (AGEs) and the receptor for AGEs (RAGE) generate ROS, and therefore this study evaluated the effects of RAGE deletion, decreasing AGE accumulation, or lowering dietary AGE content on oxidative parameters in diabetic nephropathy (DN). Control and diabetic male wild-type and RAGE-deficient (RAGE-/-) mice were fed high- or low-AGE diets, with two groups given the inhibitor of AGE accumulation, alagebrium chloride, and followed for 24 wk. Diabetic RAGE-/- mice were protected against albuminuria, hyperfiltration, glomerulosclerosis, decreased renal mitochondrial ATP production, and excess generation of both mitochondrial and cytosolic superoxide. Whereas glomerulosclerosis, tubulointerstitial expansion, and hyperfiltration were improved in diabetic mice treated with alagebrium, there was no effect on urinary albumin excretion. Both diabetic RAGE-/- and alagebrium-treated mice had an attenuation of renal RAGE expression and decreased renal and urinary AGE (carboxymethyllysine) levels. Low-AGE diets did not confer renoprotection, lower the AGE burden or renal RAGE expression, or improve cytosolic or mitochondrial superoxide generation. Renal uncoupling protein-2 gene expression and mitochondrial membrane potential were attenuated by all therapeutic interventions in diabetic mice. In the present study, diverse approaches to block the AGE-RAGE axis had disparate effects on DN, which has potential clinical implications for the way this axis should be targeted in humans.

The management of diabetes in indigenous Australians from primary care.

BACKGROUND: Indigenous Australians have high rates of diabetes and its complications. This study examines ethnic differences in the management of patients with type 2 diabetes in Australian primary care. METHODS: Diabetes management and outcomes in Indigenous patients enrolled in the NEFRON study (n = 144) was systematically compared with that in non-Indigenous patients presenting consecutively to the same practitioner (n = 449), and the NEFRON cohort as a whole (n = 3893). RESULTS: Indigenous Australians with diabetes had high rates of micro- and macrovascular disease. 60% of Indigenous patients had an abnormal albumin to creatinine ratio compared to 33% of non-Indigenous patients (p < 0.01). When compared to non-Indigenous patients, Indigenous patients were more likely to have established macrovascular disease ((adjusted Odds ratio 2.7). This excess in complications was associated with poor glycemic control, with an HbA1c >or= 8.0%, observed in 55% of all Indigenous patients, despite the similar frequency use of oral antidiabetic agents and insulin. Smoking was also more common in Indigenous patients (38%vs 10%, p < 0.01). However, the achievement of LDL and blood pressure targets was the same or better in Indigenous patients. CONCLUSION: Although seeing the same doctors and receiving the same medications, glycaemic and smoking cessation targets remain unfulfilled in Indigenous patients. This cross-sectional study confirms Aboriginal ethnicity as a powerful risk factor for microvascular and macrovascular disease, which practitioners should use to identify candidates for intensive multifactorial intervention.