Urinary complement proteins and risk of end-stage renal disease: quantitative urinary proteomics in patients with type 2 diabetes and biopsy-proven diabetic nephropathy.

PURPOSE: To investigate the association between urinary complement proteins and renal outcome in biopsy-proven diabetic nephropathy (DN). METHODS: Untargeted proteomic and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses and targeted proteomic analysis using parallel reaction-monitoring (PRM)-mass spectrometry was performed to determine the abundance of urinary complement proteins in healthy controls, type 2 diabetes mellitus (T2DM) patients, and patients with T2DM and biopsy-proven DN. The abundance of each urinary complement protein was individually included in Cox proportional hazards models for predicting progression to end-stage renal disease (ESRD). RESULTS: Untargeted proteomic and functional analysis using the KEGG showed that differentially expressed urinary proteins were primarily associated with the complement and coagulation cascades. Subsequent urinary complement proteins quantification using PRM showed that urinary abundances of C3, C9, and complement factor H (CFAH) correlated negatively with annual estimated glomerular filtration rate (eGFR) decline, while urinary abundances of C5, decay-accelerating factor (DAF), and CD59 correlated positively with annual rate of eGFR decline. Furthermore, higher urinary abundance of CFAH and lower urinary abundance of DAF were independently associated with greater risk of progression to ESRD. Urinary abundance of CFAH and DAF had a larger area under the curve (AUC) than that of eGFR, proteinuria, or any pathological parameter. Moreover, the model that included CFAH or DAF had a larger AUC than that with only clinical or pathological parameters. CONCLUSION: Urinary abundance of complement proteins was significantly associated with ESRD in patients with T2DM and biopsy-proven DN, indicating that therapeutically targeting the complement pathway may alleviate progression of DN.

Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune-inflammatory responses in diabetic Apoe(-/-) mice.

AIMS/HYPOTHESIS: Enhanced vascular inflammation, immune cell infiltration and elevated production of reactive oxygen species (ROS) contribute significantly to pro-atherogenic responses in diabetes. We assessed the immunomodulatory role of NADPH oxidase (NOX)-derived ROS in diabetes-accelerated atherosclerosis. METHODS: Diabetes was induced in male Apoe(-/-) mice with five daily doses of streptozotocin (55 mg kg(-1) day(-1)). Atherosclerotic plaque size, markers of ROS and immune cell accumulation were assessed in addition to flow cytometric analyses of cells isolated from the adjacent mediastinal lymph nodes (meLNs). The role of NOX-derived ROS was investigated using the NOX inhibitor, GKT137831 (60 mg/kg per day; gavage) administered to diabetic and non-diabetic Apoe(-/-) mice for 10 weeks. RESULTS: Diabetes increased atherosclerotic plaque development in the aortic sinus and this correlated with increased lesional accumulation of T cells and CD11c(+) cells and altered T cell activation in the adjacent meLNs. Diabetic Apoe(-/-) mice demonstrated an elevation in vascular ROS production and expression of the proinflammatory markers monocyte chemoattractant protein 1, vascular adhesion molecule 1 and IFNγ. Blockade of NOX-derived ROS using GKT137831 prevented the diabetes-mediated increase in atherosclerotic plaque area and associated vascular T cell infiltration and also significantly reduced vascular ROS as well as markers of inflammation and plaque necrotic core area. CONCLUSIONS/INTERPRETATION: Diabetes promotes pro-inflammatory immune responses in the aortic sinus and its associated lymphoid tissue. These changes are associated with increased ROS production by NOX. Blockade of NOX-derived ROS using the NOX inhibitor GKT137831 is associated with attenuation of these changes in the immune response and reduces the diabetes-accelerated development of atherosclerotic plaques in Apoe(-/-) mice.

Urotensin II receptor antagonism confers vasoprotective effects in diabetes associated atherosclerosis: studies in humans and in a mouse model of diabetes.

AIMS/HYPOTHESIS: The small, highly conserved vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis. However, its effects in diabetes-associated atherosclerosis have not been assessed. METHODS: Endothelial cells were grown in normal- and high-glucose (5 and 25 mmol/l) media with and without UII (10⁻8 mol/l) and/or the UII receptor antagonist, SB-657510 (10⁻8 mol/l). Apoe knockout (KO) mice with or without streptozotocin-induced diabetes were treated with or without SB-657510 (30 mg kg⁻¹ day⁻¹; n = 20 per group) and followed for 20 weeks. Carotid endarterectomy specimens from diabetic and non-diabetic humans were also evaluated. RESULTS: In high (but not normal) glucose medium, UII significantly increased CCL2 (encodes macrophage chemoattractant protein 1 [MCP-1]) gene expression (human aortic endothelial cells) and increased monocyte adhesion (HUVECs). UII receptor antagonism in diabetic Apoe KO mice significantly attenuated diabetes-associated atherosclerosis and aortic staining for MCP-1, F4/80 (macrophage marker), cyclooxygenase-2, nitrotyrosine and UII. UII staining was significantly increased in carotid endarterectomies from diabetic compared with non-diabetic individuals, as was staining for MCP-1. CONCLUSIONS/INTERPRETATION: This is the first report to demonstrate that UII is increased in diabetes-associated atherosclerosis in humans and rodents. Diabetes-associated plaque development was attenuated by UII receptor antagonism in the experimental setting. Thus UII may represent a novel therapeutic target in the treatment of diabetes-associated atherosclerosis.

Fine-mapping of 5q12.1-13.3 unveils new genetic contributors to caries.

Caries is a multifactorial disease and little is still known about the host genetic factors influencing susceptibility. Our previous genome-wide linkage scan has identified the interval 5q12.1-5q13.3 as linked to low caries susceptibility in Filipino families. Here we fine-mapped this region in order to identify genetic contributors to caries susceptibility. Four hundred and seventy-seven subjects from 72 pedigrees with similar cultural and behavioral habits and limited access to dental care living in the Philippines were studied. DMFT scores and genotype data of 75 single-nucleotide polymorphisms were evaluated in the Filipino families with the Family-Based Association Test. For replication purposes, a total 1,467 independent subjects from five different populations were analyzed in a case-control format. In the Filipino cohort, statistically significant and borderline associations were found between low caries experience and four genes spanning 13 million base pairs (PART1, ZSWIM6, CCNB1, and BTF3). We were able to replicate these results in some of the populations studied. We detected PART1 and BTF3 expression in whole saliva, and the expression of BTF3 was associated with caries experience. Our results suggest BTF3 may have a functional role in protecting against caries.

Association of HbA1c levels with vascular complications and death in patients with type 2 diabetes: evidence of glycaemic thresholds.

AIMS/HYPOTHESIS: There is conflicting evidence regarding appropriate glycaemic targets for patients with type 2 diabetes. Here, we investigate the relationship between HbA(1c) and the risks of vascular complications and death in such patients. METHODS: Eleven thousand one hundred and forty patients were randomised to intensive or standard glucose control in the Action in Diabetes and Vascular disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. Glycaemic exposure was assessed as the mean of HbA(1c) measurements during follow-up and prior to the first event. Adjusted risks for each HbA(1c) decile were estimated using Cox models. Possible differences in the association between HbA(1c) and risks at different levels of HbA(1c) were explored using linear spline models. RESULTS: There was a non-linear relationship between mean HbA(1c) during follow-up and the risks of macrovascular events, microvascular events and death. Within the range of HbA(1c) studied (5.5-10.5%), there was evidence of 'thresholds', such that below HbA(1c) levels of 7.0% for macrovascular events and death, and 6.5% for microvascular events, there was no significant change in risks (all p > 0.8). Above these thresholds, the risks increased significantly: every 1% higher HbA(1c) level was associated with a 38% higher risk of a macrovascular event, a 40% higher risk of a microvascular event and a 38% higher risk of death (all p < 0.0001). CONCLUSIONS/INTERPRETATION: In patients with type 2 diabetes, HbA(1c) levels were associated with lower risks of macrovascular events and death down to a threshold of 7.0% and microvascular events down to a threshold of 6.5%. There was no evidence of lower risks below these levels but neither was there clear evidence of harm.

Soluble receptor for AGE (RAGE) is a novel independent predictor of all-cause and cardiovascular mortality in type 1 diabetes.

AIMS/HYPOTHESIS: Activation of the receptor for AGE (RAGE) is implicated in the development and progression of vascular complications of diabetes. In this study, we explore factors and mortality outcomes associated with soluble RAGE (sRAGE) in a multicentre nationwide cohort of Finnish adults with type 1 diabetes. METHODS: Baseline sRAGE concentrations were estimated in 3,100 adults with type 1 diabetes. Clinical and biological variables independently associated with sRAGE were identified using multivariate regression analysis. Independent predictors of mortality were determined using Cox and Fine-Gray proportional-hazards models. RESULTS: The main independent determinants of sRAGE concentrations were estimated glomerular filtration rate, albuminuria, body mass index, age, duration of diabetes, HbA(1c) and insulin dose (all p < 0.05). During a median of 9.1 years of follow-up there were 202 deaths (7.4 per 1,000 patient years). sRAGE was independently associated with all-cause (Cox model: HR 1.03) and cardiovascular mortality (Fine-Gray competing risks model: HR 1.06) such that patients with the highest sRAGE concentrations had the greatest risk of mortality, after adjusting for age, sex, macrovascular disease, HDL-cholesterol, HbA(1c), triacylglycerol, high-sensitivity C-reactive protein (hsCRP) and the presence and severity of chronic kidney disease. Although polymorphisms in the gene coding for RAGE were significantly associated with sRAGE concentrations, none were associated with mortality outcomes. CONCLUSIONS/INTERPRETATION: Increased concentrations of sRAGE are associated with increased all-cause and cardiovascular mortality in type 1 diabetes, potentially reflecting the activation and production of RAGE in the context of accelerated vascular disease. These novel findings highlight the importance of the RAGE activation in the prevention and management of diabetic complications.

Increased serum potassium affects renal outcomes: a post hoc analysis of the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) trial.

AIMS/HYPOTHESIS: To assess the effect of an angiotensin receptor blocker (ARB) on serum potassium and the effect of a serum potassium change on renal outcomes in patients with type 2 diabetes and nephropathy. METHODS: We performed a post hoc analysis in patients with type 2 diabetes participating in the Reduction of Endpoints in NIDDM with the Angiotensin II Antagonist Losartan (RENAAL) study. Renal outcomes were defined as a composite of doubling of serum creatinine or end-stage renal disease. RESULTS: At month 6, 259 (38.4%) and 73 (10.8%) patients in the losartan group and 151 (22.8%) and 34 (5.1%) patients in the placebo group had serum potassium ≥5.0 mmol/l and ≥5.5 mmol/l, (p < 0.001), respectively. Losartan was an independent predictor for serum potassium ≥5.0 mmol/l at month 6 (OR 2.8; 95% CI 2.0-3.9). Serum potassium at month 6 ≥ 5.0 mmol/l was in turn associated with increased risk for renal events (HR 1.22; 95% CI 1.00-1.50), independent of other risk factors. Adjustment of the overall treatment effects for serum potassium augmented losartan's renoprotective effect from 21% (6-34%) to 35% (20-48%), suggesting that the renoprotective effects of losartan are offset by its effect on serum potassium. CONCLUSIONS/INTERPRETATION: In this study, we found that treatment with the ARB losartan is associated with a high risk of increased serum potassium levels, which is in turn associated with an increased risk of renal outcomes in patients with diabetes and nephropathy. Whether additional management of high serum potassium would further increase the renal protective properties of losartan is an important clinical question.

Delayed intervention with AGE inhibitors attenuates the progression of diabetes-accelerated atherosclerosis in diabetic apolipoprotein E knockout mice.

AIMS/HYPOTHESIS: Formation of AGEs is increased in the diabetic milieu, which contributes to accelerated atherogenesis. We studied whether delayed treatment with AGE-inhibiting compounds, alagebrium chloride and pyridoxamine dihydrochloride, affected established atherosclerosis in experimental diabetes in comparison with the angiotensin-converting enzyme inhibitor, quinapril. METHODS: Streptozotocin-induced diabetic male Apoe (-/-) mice (n = 24 per group) received, by oral gavage, from week 10 to 20 of diabetes: no treatment; alagebrium (1 mg kg(-1) day(-1)); pyridoxamine (1 g/l in drinking water); or quinapril (30 mg kg(-1) day(-1)). Atherosclerotic lesion area (en face analysis) was evaluated for all groups. RESULTS: Delayed intervention with alagebrium decreased plaque area in the diabetic Apoe (-/-) mice compared with untreated mice (total plaque area: alagebrium 10.6 ± 1.6%, untreated, 15.1 ± 1.5%, p < 0.05). This anti-atherosclerotic effect was comparable with that achieved with quinapril (quinapril 8.4 ± 1.4%, vs untreated, p < 0.05). Pyridoxamine also attenuated plaque development in diabetic mice (5.7 ± 1.2% vs untreated 11.9 ± 1.1%, p < 0.05). The anti-atherosclerotic effect conferred by alagebrium and quinapril was associated with a significant reduction in vascular oxidative stress and circulating AGEs and methylglyoxal, although preformed AGEs were not removed from the vascular wall with either delayed intervention. CONCLUSIONS/INTERPRETATION: Inhibition of AGE accumulation, using a delayed intervention with alagebrium or pyridoxamine, significantly attenuated the progression of established diabetes-associated atherosclerosis, similar to results obtained with quinapril. These findings provide further evidence that blockade of AGE-mediated pathways may present a novel therapy for the prevention of atherosclerosis in diabetes.

Receptor for advanced glycation end-products (RAGE) provides a link between genetic susceptibility and environmental factors in type 1 diabetes.

AIMS/HYPOTHESIS: This group of studies examines human genetic susceptibility conferred by the receptor for advanced glycation end-products (RAGE) in type 1 diabetes and investigates how this may interact with a western environment. METHODS: We analysed the AGER gene, using 13 tag SNPs, in 3,624 Finnish individuals from the FinnDiane study, followed by AGER associations with a high risk HLA genotype (DR3)-DQA1*05-DQB1*02/DRB1*0401-DQB1*0302 (n = 546; HLA-DR3/DR4), matched in healthy newborn infants from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study (n = 373) using allelic analysis. We also studied islets and circulating RAGE in NODLt mice. RESULTS: The rs2070600 and rs17493811 polymorphisms predicted increased risk of type 1 diabetes, whereas the rs9469089 SNP was related to decreased risk, on a high risk HLA background. Children from the DIPP study also showed a decline in circulating soluble RAGE levels, at seroconversion to positivity for type 1 diabetes-associated autoantibodies. Islet RAGE and circulating soluble RAGE levels in prediabetic NODLt mice decreased over time and were prevented by the AGE lowering therapy alagebrium chloride. Alagebrium chloride also decreased the incidence of autoimmune diabetes and restored islet RAGE levels. CONCLUSIONS/INTERPRETATION: These studies suggest that inherited AGER gene polymorphisms may confer susceptibility to environmental insults. Declining circulating levels of soluble RAGE, before the development of overt diabetes, may also be predictive of clinical disease in children with high to medium risk HLA II backgrounds and this possibility warrants further investigation in a larger cohort.

Large artery biomechanics and diastolic dysfunctionin patients with Type 2 diabetes.

AIMS: To comprehensively characterize large artery biomechanical properties and examine their relationship to cardiac function in patients with Type 2 diabetes mellitus (DM). METHODS: Fifty-five individuals with Type 2 DM were compared with 66 age- and sex-matched healthy control subjects. Arterial biomechanical properties were assessed by systemic arterial compliance (SAC; two-element Windkessel model), carotid-femoral pulse wave velocity (PWVc-f), femoral-dorsalis pedis pulse wave velocity (PWVf-d) and carotid augmentation index. Cardiac structure and function were assessed by echocardiography. RESULTS: Individuals with Type 2 DM had lower SAC and higher PWVc-f when compared with the healthy population. The PWVc-f was significantly lower than the PWVf-d in control individuals, but this difference was not evident in individuals with Type 2 DM due to higher PWVc-f. Augmentation index was similar in both groups, but the time to the first systolic inflection (time to reflection) was shorter in the individuals with Type 2 DM. The individuals with Type 2 DM had a greater prevalence of diastolic abnormalities when compared with the control group. Arterial stiffness indices, including SAC and pulse pressure, correlated with left ventricular filling pressure (defined as peak velocity during early diastolic filling divided by the velocity of movement of the mitral valve annulus in early diastole; r = -0.33 and 0.36 respectively. CONCLUSIONS: Patients with Type 2 DM on standard medication showed preferential stiffening of the large central arteries. However, carotid augmentation index was not different between the two groups and is therefore not a reliable indicator of large artery stiffening in this patient group. Diastolic dysfunction, present in a significant proportion of this population with Type 2 DM, was closely associated with arterial stiffening, suggesting a common aetiology.

Increased risk of cardiovascular disease in Type 1 diabetes: arterial exposure to remnant lipoproteins leads to enhanced deposition of cholesterol and binding to glycated extracellular matrix proteoglycans.

AIMS: To determine fasting and postprandial metabolism of apolipoprotein B48 (apoB48) remnant lipoproteins in subjects with Type 1 diabetes and the relationship to progressive cardiovascular disease, and to investigate the impact of remnant lipoprotein cholesterol accumulation associated with arterial wall biglycan using a rodent model of Type 1 diabetes. METHODS: Normolipidaemic subjects (n = 9) with long-standing Type 1 diabetes (and advanced cardiovascular disease) and seven healthy control subjects were studied. Fasting and postprandial apoB48 concentration was determined following a sequential meal challenge. A rodent model of streptozotocin-induced diabetes was used to investigate the ex vivo retention of fluorescent-conjugated remnants. Binding of remnant lipoproteins to human recombinant biglycan was assessed in vitro. RESULTS: A significantly higher concentration of fasting plasma apoB48 remnants was observed in patients with Type 1 diabetes compared with control subjects. Patients with Type 1 diabetes exhibited a greater total plasma apoB48 area under the curve (AUC) and an increased incremental AUC following a second sequential meal compared with control subjects. The arterial retention of remnants ex vivo and associated cholesterol was increased sevenfold in Type 1 diabetes rats relative to controls. Remnants were shown to bind with significant affinity to human biglycan in vitro and a further 2.3-fold increased binding capacity was observed with glycated biglycan. Remnants were shown to colocalize with both arterial biglycan and glycated matrix proteins in the Type 1 diabetes rodent model. CONCLUSION: Impaired metabolism of remnant lipoproteins associated with enhanced binding to proteoglycans appears to contribute to the arterial cholesterol deposition in Type 1 diabetes. Our findings support the hypothesis that impaired remnant metabolism may contribute to accelerated progression of atherosclerosis in the hyperglycaemic and insulin-deficient state.

Angiotensin II subtype 2 receptor blockade and deficiency attenuate the development of atherosclerosis in an apolipoprotein E-deficient mouse model of diabetes.

AIMS/HYPOTHESIS: Most of the known actions of angiotensin II have been considered primarily to be the result of angiotensin II subtype 1 receptor activation. However, recent data suggest that the angiotensin II subtype 2 receptor (AT(2)R) may modulate key processes linked to atherosclerosis. The aim of this study was to investigate the role of AT(2)R in diabetes-associated atherosclerosis using pharmacological blockade and genetic deficiency. METHODS: Aortic plaque deposition was assessed in streptozotocin-induced diabetic apolipoprotein E (Apoe) knockout (KO) and At ( 2 ) r (also known as Agtr2)/Apoe double-KO (DKO) mice. Control and diabetic Apoe-KO mice received an AT(2)R antagonist PD123319 (5 mg kg(-1) day(-1)) via osmotic minipump for 20 weeks (n = 7-8 per group). RESULTS: Diabetes was associated with a sixfold increase in plaque area (diabetic Apoe-KO: 12.7 +/- 1.4% vs control Apoe-KO: 2.3 +/- 0.4%, p < 0.001) as well as a significant increase in aortic expression of the gene At ( 2 ) r (also known as Agtr2). The increase in plaque area with diabetes was attenuated in AT(2)R antagonist-treated diabetic Apoe-KO mice (7.1 +/- 0.5%, p < 0.05) and in diabetic At ( 2 ) r/Apoe DKO mice (9.2 +/- 1.3%, p < 0.05). These benefits occurred independently of glycaemic control or BP, and were associated with downregulation of a range of pro-inflammatory cytokines, adhesion molecules, chemokines and various extracellular matrix proteins. CONCLUSIONS/INTERPRETATION: This study provides evidence for AT(2)R playing a role in the development of diabetes-associated atherosclerosis. These findings suggest a potential utility of AT(2)R blockers in the prevention and treatment of diabetic macrovascular complications.

Cell division autoantigen 1 plays a profibrotic role by modulating downstream signalling of TGF-beta in a murine diabetic model of atherosclerosis.

AIMS/HYPOTHESIS: Excess accumulation of vascular extracellular matrix (ECM) is an important pathological process in cardiovascular diseases including diabetes-associated atherosclerosis. We explored how a recently identified molecule, cell division autoantigen 1 (CDA1), influences the profibrotic TGF-beta pathway leading to vascular ECM accumulation. METHODS: Expression levels of genes encoding for CDA1, TGF-beta and connective tissue growth factor (CTGF) were examined in aorta from Apoe(-/-) mice with or without diabetes. We used retroviral and adenoviral constructs to knockdown or overexpress Tspyl2, the gene encoding CDA1, in mouse vascular smooth muscle cells (VSMCs) with or without TGF-beta treatment in order to demonstrate the role of CDA1 in TGF-beta signalling. RESULTS: In vivo studies indicated that the mRNA levels of CDA1-encoding gene Tspyl2 and protein levels of CDA1 were elevated in the aorta of diabetic Apoe(-/-) mice, accompanied by increased levels of Tgf-beta (also known as Tgfb1), Ctgf and ECM accumulation. In vitro studies in vascular cells showed that TGF-beta treatment rapidly increased CDA1 protein levels, which then amplified TGF-beta signalling leading to upregulation of ECM genes. Knockdown of CDA1-encoding gene Tspyl2 to reduce cellular CDA1 level markedly attenuated TGF-beta-stimulated MAD homologue 3 (drosophila; SMAD3) phosphorylation and transcriptional activities. CDA1 overproduction increased and Tspyl2 knockdown decreased expression of TGF-beta receptor type I, TbetarI (also known as Tgfbr1), but not TGF-beta receptor type II, TbetarII (also known as Tgfbr2), providing a mechanism for CDA1's action in modulating TGF-beta signalling. Knockdown of CDA1-encoding gene Tspyl2 also blocked the profibrotic effect of TGF-beta in VSMCs. CONCLUSIONS/INTERPRETATION: CDA1 plays an important role in vascular ECM accumulation by amplifying TGF-beta signalling. This is critical for the profibrotic effect of TGF-beta in the vasculature. CDA1 is therefore a potential target for attenuating vascular ECM accumulation caused by enhanced TGF-beta action, as seen in diabetic atherosclerosis.

The endothelin receptor antagonist avosentan ameliorates nephropathy and atherosclerosis in diabetic apolipoprotein E knockout mice.

AIMS/HYPOTHESIS: There is convincing evidence that the endothelin system contributes to diabetic nephropathy and cardiovascular disease. This study aimed to assess the effects of the non-peptidergic endothelin receptor A (ETA) antagonist avosentan in a mouse model of accelerated diabetic nephropathy and atherosclerosis in comparison with the ACE inhibitor, quinapril. METHODS: Apolipoprotein E (Apoe) knockout (KO) mice (n = 20 per group, five groups) were randomised to the following groups: non-diabetic controls and streptozotocin-induced diabetic animals gavaged daily for 20 weeks with placebo, avosentan (high dose: 30 mg/kg, or low dose: 10 mg/kg) or quinapril (given in drinking water, 30 mg/kg). RESULTS: BP was unchanged by avosentan treatment but decreased with quinapril treatment. Diabetes-associated albuminuria was significantly attenuated by high-dose avosentan after 10 and 20 weeks of treatment. Diabetic animals showed a decreased creatinine clearance, which was normalised by avosentan treatment. In diabetic mice, high-dose avosentan treatment significantly attenuated the glomerulosclerosis index, mesangial matrix accumulation, glomerular accumulation of the matrix proteins collagen IV, and renal expression of genes encoding connective tissue growth factor, vascular endothelial growth factor, transforming growth factor beta and nuclear factor kappaB (p65 subunit). Furthermore, high-dose avosentan treatment was also associated with reduced expression of the genes for ETA, ETB and angiotensin receptor 1. The renoprotective effects of avosentan were comparable or superior to those observed with quinapril. High-dose avosentan also significantly attenuated diabetes-associated aortic atherosclerosis in Apoe KO mice and reduced macrophage infiltration and aortic nitrotyrosine expression. CONCLUSIONS/INTERPRETATION: This study demonstrates that ETA blockade with avosentan may provide an alternate therapeutic strategy for the treatment of diabetic micro- and macrovascular complications.

Receptor for AGEs (RAGE) blockade may exert its renoprotective effects in patients with diabetic nephropathy via induction of the angiotensin II type 2 (AT2) receptor.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) contributes to the development and progression of diabetic nephropathy. In this study, we examined whether the protective effects of RAGE blockade are exerted via modulation of the renal angiotensin II type 2 (AT2) receptor. METHODS: Control and streptozotocin diabetic mice, wild-type or deficient in the AT2 receptor (At2 knockout [KO]) or RAGE (Rage KO), were studied for 24 weeks. Adenoviral overexpression of full-length Rage in primary rat mesangial cells was also used to determine the effects on AT2 production. RESULTS: With diabetes, Rage-deficient mice had less albuminuria, and an attenuation of hyperfiltration and glomerulosclerosis as compared with diabetic wild-type and At2 KO mice. Renal gene and protein expression of RAGE was elevated with diabetes. Diabetic Rage KO mice had a greater increase in renal AT2 receptor protein than was seen in diabetic wild-type mice. Diabetes-induced increases in renal cytosolic and mitochondrial superoxide generation were prevented in diabetic Rage KO mice, but enhanced in all At2 KO mice. Adenoviral overexpression of RAGE or AGE treatment decreased cell surface AT2 expression, in association with increasing superoxide generation; both were reversed using antioxidants N-acetylcysteine and apocynin, and soluble RAGE in primary mesangial cells. CONCLUSIONS/INTERPRETATION: RAGE appears to be a common and key modulator of AT2 receptor expression, a finding that would implicate a newly defined RAGE-AT2 axis in the development and progression of diabetic nephropathy.

Angiotensin converting enzyme 2 in the kidney.

1. Angiotensin converting enzyme 2 (ACE2) is an important homeostatic component of the renin angiotensin system (RAS). ACE2 both degrades the vasoconstrictor, angiotensin II and generates the potent vasodilator peptide, angiotensin 1-7. These actions counterbalance those of ACE. 2. ACE2 is highly expressed in the healthy kidney, particularly in the proximal tubules, where it colocalizes with ACE and angiotensin receptors. 3. Kidney disease and subtotal nephrectomy is associated with a reduction in renal ACE2 expression, possibly facilitating the damaging effects of angiotensin II in the failing kidney. Acquired or genetic ACE2 deficiency also appears to exacerbate renal damage and albuminuria in experimental models, supporting this hypothesis. 4. ACE2 also has an important role in blood pressure control. Many models of hypertension are associated with reduced ACE2 expression. Although ACE2 KO animals are normotensive, in states associated with activation of the RAS, ACE2 overexpression improves blood pressure control and reduces angiotensin responsiveness.

Enamel formation genes are associated with high caries experience in Turkish children.

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes and their interaction with Streptococcus mutans levels may contribute to caries. For the present study, we used DNA samples collected from 173 unrelated children from Istanbul: 91 children with 4 or more affected tooth surfaces and 82 caries-free children. Six single-nucleotide polymorphism markers were genotyped in selected candidate genes (ameloblastin, amelogenin, enamelin, tuftelin 1 and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Regression analysis was used for the evaluation of individual gene effects, environmental effects and gene-environment interactions. Overrepresentation of the C allele of the amelogenin marker was seen in cases with dmft scores higher than 8 (p = 0.01) when compared to controls. Also, overrepresentation of the T allele of the ameloblastin marker was seen in cases with dmfs scores higher than 10 (p = 0.05) when compared to controls. In addition, the CT genotype of the tuftelin rs3790506 marker was overrepresented in cases with dmft scores higher than 5 (p = 0.05) and dmfs scores higher than 6 (p = 0.05) compared to controls. The best-fitting model showed that dmfs is increased when the following factors are present: (1) females and both the anterior and posterior teeth are affected simultaneously, (2) when the T allele of the tuftelin rs3790506 is involved, and (3) the C allele of the amelogenin rs17878486 is involved. Our study provides support that genes involved in enamel formation modify caries susceptibility in humans.

Vascular hypertrophy in experimental diabetes. Role of advanced glycation end products.

The accelerated formation of advanced glycation end products (AGEs) and the overexpression of transforming growth factor beta (TGF-beta) have both been implicated in the pathogenesis of diabetic microvascular and macrovascular complications. Previous studies in our laboratory have demonstrated that the vascular changes in diabetes include hypertrophy of the mesenteric vasculature. To examine the role of AGEs in this process, streptozotocin-induced diabetic rats and control animals were randomized to receive aminoguanidine, an inhibitor of AGE formation, or no treatment. Animals were studied at 7 d, 3 wk, and 8 mo after induction of diabetes. When compared with control animals, diabetes was associated with an increase in mesenteric vascular weight and an increase in media wall/lumen area. By Northern analysis, TGF-beta1 gene expression was increased 100-150% (P < 0.01) and alpha1 (IV) collagen gene expression was similarly elevated to 30-110% compared to controls (P < 0.05). AGEs and extracellular matrix were present in abundance in diabetic but not in control vessels. Treatment of diabetic rats with aminoguanidine resulted in significant amelioration of the described pathological changes including overexpression of TGF-beta1 and alpha1 (IV) collagen. These data implicate the formation of AGEs in TGF-beta overexpression and tissue changes which accompany the diabetic state.

Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE).

Tubulointerstitial disease, a prominent phenomenon in diabetic nephropathy, correlates with decline in renal function. The underlying pathogenic link between chronic hyperglycemia and the development of tubulointerstitial injury has not been fully elucidated, but myofibroblast formation represents a key step in the development of tubulointerstitial fibrosis. RAGE, the receptor for advanced glycation end products (AGEs), induces the expression of TGF-beta and other cytokines that are proposed to mediate the transdifferentiation of epithelial cells to form myofibroblasts. Here we report specific binding of (125)I-AGE-BSA to cell membranes prepared from a rat proximal tubule cell line and show that the binding site was RAGE. AGE exposure induced dose-dependent epithelial-myofibroblast transdifferentiation determined by morphological changes, de novo alpha smooth-muscle actin expression, and loss of epithelial E-cadherin staining. These effects could be blocked with neutralizing Ab's to RAGE or to TGF-beta. Transdifferentiation was also apparent in the proximal tubules of diabetic rats and in a renal biopsy from a patient with type 1 diabetes. The AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711) reduced transdifferentiation in diabetic rats in association with reduced tubular AGE and TGF-beta expression. This study provides a novel mechanism to explain the development of tubulointerstitial disease in diabetic nephropathy and provides a new treatment target.

Role of the AGE crosslink breaker, alagebrium, as a renoprotective agent in diabetes.

The biochemical process of advanced glycation appears to play a central role in the development and progression of diabetic vascular complications. A number of strategies to influence this pathway have been designed, one of which involves the putative advanced glycation end-product (AGE) crosslink breaker, alagebrium which has been shown in in vitro studies to cleave preformed AGE crosslinks. This agent has been studied in various models of diabetic complications and has been shown to attenuate diabetic renal disease, cardiac dysfunction, and atherosclerosis. In addition to the ability of alagebrium to reduce tissue levels of AGEs, this drug appears to inhibit activation of certain protein kinase C isoforms. Planned clinical studies in diabetic subjects at risk of complications should assist in determining the role of alagebrium in the prevention, retardation, and reversal of diabetic micro- and macrovascular disease.