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.

Transactivation of RAGE mediates angiotensin-induced inflammation and atherogenesis.

Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through pathways independent of classical Gq signaling that regulate vascular homeostasis. Here, we report that the AT1 receptor preformed a heteromeric complex with the receptor for advanced glycation endproducts (RAGE). Activation of the AT1 receptor by angiotensin II (Ang II) triggered transactivation of the cytosolic tail of RAGE and NF-κB-driven proinflammatory gene expression independently of the liberation of RAGE ligands or the ligand-binding ectodomain of RAGE. The importance of this transactivation pathway was demonstrated by our finding that adverse proinflammatory signaling events induced by AT1 receptor activation were attenuated when RAGE was deleted or transactivation of its cytosolic tail was inhibited. At the same time, classical homeostatic Gq signaling pathways were unaffected by RAGE deletion or inhibition. These data position RAGE transactivation by the AT1 receptor as a target for vasculoprotective interventions. As proof of concept, we showed that treatment with the mutant RAGE peptide S391A-RAGE362-404 was able to inhibit transactivation of RAGE and attenuate Ang II-dependent inflammation and atherogenesis. Furthermore, treatment with WT RAGE362-404 restored Ang II-dependent atherogenesis in Ager/Apoe-KO mice, without restoring ligand-mediated signaling via RAGE, suggesting that the major effector of RAGE activation was its transactivation.

Lipoxins Protect Against Inflammation in Diabetes-Associated Atherosclerosis.

Increasing evidence points to the fact that defects in the resolution of inflammatory pathways predisposes individuals to the development of chronic inflammatory diseases, including diabetic complications such as accelerated atherosclerosis. The resolution of inflammation is dynamically regulated by the production of endogenous modulators of inflammation, including lipoxin A4 (LXA4). Here, we explored the therapeutic potential of LXA4 and a synthetic LX analog (Benzo-LXA4) to modulate diabetic complications in the streptozotocin-induced diabetic ApoE-/- mouse and in human carotid plaque tissue ex vivo. The development of diabetes-induced aortic plaques and inflammatory responses of aortic tissue, including the expression of vcam-1, mcp-1, il-6, and il-1ß, was significantly attenuated by both LXA4 and Benzo-LXA4 in diabetic ApoE-/- mice. Importantly, in mice with established atherosclerosis, treatment with LXs for a 6-week period, initiated 10 weeks after diabetes onset, led to a significant reduction in aortic arch plaque development (19.22 ± 2.01% [diabetic]; 12.67 ± 1.68% [diabetic + LXA4]; 13.19 ± 1.97% [diabetic + Benzo-LXA4]). Secretome profiling of human carotid plaque explants treated with LXs indicated changes to proinflammatory cytokine release, including tumor necrosis factor-α and interleukin-1ß. LXs also inhibited platelet-derived growth factor-stimulated vascular smooth muscle cell proliferation and transmigration and endothelial cell inflammation. These data suggest that LXs may have therapeutic potential in the context of diabetes-associated vascular complications.

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.

Lipoxins Regulate the Early Growth Response-1 Network and Reverse Diabetic Kidney Disease.

Background The failure of spontaneous resolution underlies chronic inflammatory conditions, including microvascular complications of diabetes such as diabetic kidney disease. The identification of endogenously generated molecules that promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. Lipoxins (LXs) are lipid mediators that promote the resolution of inflammation.Methods We investigated the potential of LXA4 and a synthetic LX analog (Benzo-LXA4) as therapeutics in a murine model of diabetic kidney disease, ApoE-/- mice treated with streptozotocin.Results Intraperitoneal injection of LXs attenuated the development of diabetes-induced albuminuria, mesangial expansion, and collagen deposition. Notably, LXs administered 10 weeks after disease onset also attenuated established kidney disease, with evidence of preserved kidney function. Kidney transcriptome profiling defined a diabetic signature (725 genes; false discovery rate P≤0.05). Comparison of this murine gene signature with that of human diabetic kidney disease identified shared renal proinflammatory/profibrotic signals (TNF-α, IL-1ß, NF-κB). In diabetic mice, we identified 20 and 51 transcripts regulated by LXA4 and Benzo-LXA4, respectively, and pathway analysis identified established (TGF-ß1, PDGF, TNF-α, NF-κB) and novel (early growth response-1 [EGR-1]) networks activated in diabetes and regulated by LXs. In cultured human renal epithelial cells, treatment with LXs attenuated TNF-α-driven Egr-1 activation, and Egr-1 depletion prevented cellular responses to TGF-ß1 and TNF-αConclusions These data demonstrate that LXs can reverse established diabetic complications and support a therapeutic paradigm to promote the resolution of inflammation.

ACE2 deficiency shifts energy metabolism towards glucose utilization.

BACKGROUND: This study aimed at investigating the effects of genetic angiotensin-converting enzyme (ACE) 2 deficiency on glucose homeostasis in the pancreas and skeletal muscle and their reversibility following ACE inhibition. PROCEDURES: ACE2-knockout and C57bl6J mice were placed on a standard diet (SD) or a high-fat diet (HFD) for 12 weeks. An additional group of ACE2-knockout mice was fed a SD and treated with the ACE inhibitor, perindopril (2 mg kg(-1)day(-1)). Glucose and insulin tolerance tests, indirect calorimetry measurements and EchoMRI were performed. Non-esterfied 'free' fatty acid oxidation rate in skeletal muscle was calculated by measuring the palmitate oxidation rate. ß-cell mass was determined by immunostaining. Insulin, collectrin, glucose transporter protein, and peroxisome proliferator-activated receptor-γ expression were analysed by RT-PCR. Markers of mithocondrial biogenesis/content were also evaluated. MAIN FINDINGS: ACE2-knockout mice showed a ß-cell defect associated with low insulin and collectrin levels and reduced compensatory hypertrophy in response to a HFD, which were not reversed by perindopril. On the other hand, ACE2 deficiency shifted energy metabolism towards glucose utilization, as it increased the respiratory exchange ratio, reduced palmitate oxidation and PCG-1α expression in the skeletal muscle, where it up-regulated glucose transport proteins. Treatment of ACE2-knockout mice with perindopril reversed the skeletal muscle changes, suggesting that these were dependent on Angiotensin II (Ang II). PRINCIPAL CONCLUSIONS: ACE2-knockout mice display a ß-cell defect, which does not seem to be dependent on Ang II but may reflect the collectrin-like action of ACE2. This defect seemed to be compensated by the fact that ACE2-knockout mice shifted their energy consumption towards glucose utilisation via Ang II.

Osteoprotegerin increases in metabolic syndrome and promotes adipose tissue proinflammatory changes.

BACKGROUND: Inflammation is believed to link obesity to insulin resistance, as in the setting of metabolic syndrome (MetS). Osteoprotegerin (OPG) is a soluble protein that seems to exert proatherogenic and prodiabetogenic effects. This study aims at determining OPG levels in MetS and whether OPG might contribute to MetS development and progression. METHODOLOGY/PRINCIPAL FINDINGS: Circulating OPG was measured in 46 patients with MetS and 63 controls, and was found significantly elevated in those with MetS. In addition, circulating and tissue OPG was significantly increased in high-fat diet (HFD) fed C57BL6 mice, which is one of the animal models for the study of MetS. To evaluate the consequences of OPG elevation, we delivered this protein to C57BL6 mice, finding that it promoted systemic and adipose tissue proinflammatory changes in association with metabolic abnormalities. CONCLUSIONS/SIGNIFICANCE: These data suggest that OPG may trigger adipose tissue proinflammatory changes in MetS/HFD-induced obesity.

Alagebrium reduces glomerular fibrogenesis and inflammation beyond preventing RAGE activation in diabetic apolipoprotein E knockout mice.

Advanced glycation end products (AGEs) are important mediators of diabetic nephropathy that act through the receptor for AGEs (RAGE), as well as other mechanisms, to promote renal inflammation and glomerulosclerosis. The relative contribution of RAGE-dependent and RAGE-independent signaling pathways has not been previously studied in vivo. In this study, diabetic RAGE apoE double-knockout (KO) mice with streptozotocin-induced diabetes were treated with the AGE inhibitor, alagebrium (1 mg/kg/day), or the ACE inhibitor, quinapril (30 mg/kg/day), for 20 weeks, and renal parameters were assessed. RAGE deletion attenuated mesangial expansion, glomerular matrix accumulation, and renal oxidative stress associated with 20 weeks of diabetes. By contrast, inflammation and AGE accumulation associated with diabetes was not prevented. However, treatment with alagebrium in diabetic RAGE apoE KO mice reduced renal AGE levels and further reduced glomerular matrix accumulation. In addition, even in the absence of RAGE expression, alagebrium attenuated cortical inflammation, as denoted by the reduced expression of monocyte chemoattractant protein-1, intracellular adhesion molecule-1, and the macrophage marker cluster of differentiation molecule 11b. These novel findings confirm the presence of important RAGE-independent as well as RAGE-dependent signaling pathways that may be activated in the kidney by AGEs. This has important implications for the design of optimal therapeutic strategies for the prevention of diabetic nephropathy.

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).

Angiotensin-converting enzyme 2 regulates renal atrial natriuretic peptide through angiotensin-(1-7).

Deficiency of ACE2 (angiotensin-converting enzyme 2), which degrades Ang (angiotensin) II, promotes the development of glomerular lesions. However, the mechanisms explaining why the reduction in ACE2 is associated with the development of glomerular lesions have still to be fully clarified. We hypothesized that ACE2 may regulate the renoprotective actions of ANP (atrial natriuretic peptide). The aim of the present study was to investigate the effect of ACE2 deficiency on the renal production of ANP. We evaluated molecular and structural abnormalities, as well as the expression of ANP in the kidneys of ACE2-deficient mice and C57BL/6 mice. We also exposed renal tubular cells to AngII and Ang-(1-7) in the presence and absence of inhibitors and agonists of RAS (renin-angiotensin system) signalling. ACE2 deficiency resulted in increased oxidative stress, as well as pro-inflammatory and profibrotic changes. This was associated with a down-regulation of the gene and protein expression on the renal production of ANP. Consistent with a role for the ACE2 pathway in modulating ANP, exposing cells to either Ang-(1-7) or ACE2 or the Mas receptor agonist up-regulated ANP gene expression. This work demonstrates that ACE2 regulates renal ANP via the generation of Ang-(1-7). This is a new mechanism whereby ACE2 counterbalances the renal effects of AngII and which explains why targeting ACE2 may be a promising strategy against kidney diseases, including diabetic nephropathy.

High-salt diet increases glomerular ACE/ACE2 ratio leading to oxidative stress and kidney damage.

BACKGROUND: Angiotensin II (AngII) contributes to salt-driven kidney damage. In this study, we aimed at investigating whether and how the renal damage associated with a high-salt diet could result from changes in the ratio between angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2). METHODS: Forty-eight rats randomly allocated to three different dietary contents of salt were studied for 4 weeks after undergoing a left uninephrectomy. We focussed on kidney functional, structural and molecular changes. At the same time, we studied kidney molecular changes in 20 weeks old Ace2-knockout mice (Ace2KO), with and without ACE inhibition. RESULTS: A high salt content diet significantly increased the glomerular ACE/ACE2 ratio. This was associated with increased oxidative stress. To assess whether these events were related, we measured renal oxidative stress in Ace2KO, and found that the absence of ACE2 promoted oxidative stress, which could be prevented by ACE inhibition. CONCLUSION: One of the mechanisms by which a high-salt diet leads to renal damage seems to be the modulation of the ACE/ACE2 ratio which in turn is critical for the cause of oxidative stress, through AngII.

Hyperglycemia induces a dynamic cooperativity of histone methylase and demethylase enzymes associated with gene-activating epigenetic marks that coexist on the lysine tail.

OBJECTIVE: Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as "hyperglycemic memory." We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS: Models of transient hyperglycemia were used to link NFkappaB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFkappaB-p65 chromatin. RESULTS: The sustained upregulation of the NFkappaB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS: These studies indicate that the active transcriptional state of the NFkappaB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

Cardiac inflammation associated with a Western diet is mediated via activation of RAGE by AGEs.

A diet high in fat induces cardiac hypertrophy, inflammation, and oxidative stress. Although such actions have largely been ascribed to fat deposition, the accumulation of advanced glycation end products (AGEs) and subsequent activation of the receptor for AGEs (RAGE) may also represent important mediators of cardiac injury following exposure to a Western diet. In this study, male C57BL6J and RAGE knockout mice were placed on either a standard diet (7% fat) or a Western "fast-food" diet (21% fat). Animals receiving a high-fat diet were further randomized to receive the AGE inhibitor alagebrium chloride (1 mg.kg(-1).day(-1)) and followed for 16 wk. A Western diet was associated with cardiac hypertrophy, inflammation, mitochondrial-dependent superoxide production, and cardiac AGE accumulation in wild-type mice. Although RAGE-KO mice fed a Western diet also became obese and accumulated intramyocardial lipid, cardiomyocyte hypertrophy, inflammation, and oxidative stress were attenuated compared with wild-type mice. Similarly, mice of both strains receiving alagebrium chloride had reduced levels of inflammation and oxidative stress, in association with a reduction in cardiac AGEs and RAGE. This study suggests that AGEs represent important mediators of cardiac injury associated with a Western fast-food diet. These data point to the potential utility of AGE-reducing strategies in the prevention and management of cardiac disease.

Advances in the renin-angiotensin-aldosterone system: relevance to diabetic nephropathy.

Hypertension is now recognized as a key contributory factor to the development and progression of kidney disease in both type 1 and type 2 diabetes. The renin angiotensin system (RAS) and its effector molecule angiotensin II, in particular, have a range of hemodynamic and nonhemodynamic effects that contribute not only to the development of hypertension, but also to renal disease. As a result, therapeutic inhibition of the RAS with angiotensin-converting enzyme inhibitors and/or selective angiotensin II type 1 receptor blockers has been proposed as a key strategy for reducing kidney damage beyond the expected effects one would observe with blood pressure reduction per se. Although the relationship between the RAS and the progression of diabetic renal disease has been known for many decades, recent advances have revealed a more complex paradigm with the discovery of a number of new components. Thus, further understanding of these new components of the renin angiotensin aldosterone system (RAAS), such as the angiotensin type 2 receptor subtype, angiotensin converting enzyme 2, and the recently cloned renin receptor, is likely to have therapeutic implications for disorders such as diabetic nephropathy, where interruption of the RAAS is widely used.

Increased atherosclerosis following treatment with a dual PPAR agonist in the ApoE knockout mouse.

OBJECTIVE: Recent reports have suggested that dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonists are associated with adverse cardiovascular events. This study aimed to investigate the actions of the non-thiazolidinedione PPARalpha/gamma agonist, compound 3q, on plaque development in the apolipoprotein E knockout (apoE KO) mouse, a recognised model of accelerated plaque development. METHODS: Six-week-old male apoE KO mice were randomised to receive the dual PPARalpha/gamma agonist, compound 3q (3 mg/kg/day), the PPARgamma agonist, rosiglitazone (20 mg/kg/day), the PPARalpha agonist, gemfibrozil (100 mg/kg/day) by gavage or no treatment for 20 weeks (n=12/group). RESULTS: Gemfibrozil and rosiglitazone significantly reduced lesion area. However, compound 3q was associated with a three-fold increase in total plaque area (versus control p<0.001). This was associated with an upregulation of markers of plaque instability including vascular cell adhesion molecule-1 (3.5-fold, p<0.001), P-selectin (3.4-fold, p<0.001) monocyte chemoattractant protein-1 (3.4-fold; p<0.001) as well as the scavenger receptor, CD36 (2-fold, p<0.01). These disparate effects were observed with the dual PPAR agonist despite lowering LDL cholesterol and improving insulin sensitivity to a similar extent to PPARalpha and gamma agonists used individually. CONCLUSION: The finding of increased atherogenesis following a dual PPARalpha/gamma agonist is consistent with recent clinical findings. These data provide an important framework for further exploring the potential utility and safety of combinatorial approaches.

Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: implications for diabetic renal disease.

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

Interactions between renin angiotensin system and advanced glycation in the kidney.

Although hemodynamic and metabolic factors are individually implicated in the development of diabetic nephropathy, their interaction has not been defined clearly. In this study, the effects of angiotensin II (Ang II) and advanced glycation end products (AGE) both individually on each other are explored and compared. In the first study arm, Sprague-Dawley rats received a continuous infusion of AGE-modified rat serum albumin (RSA) or unmodified RSA for 4 wk with or without the angiotensin receptor type 1 antagonist valsartan. In the second arm, animals received a continuous infusion of Ang II (58.3 ng/kg per min) with or without the AGE inhibitor pyridoxamine. Components of the intrarenal renin-angiotensin system were measured using real time reverse transcription-PCR, immunohistochemistry, and standard angiotensin-converting enzyme (ACE) activity assays. Renal and serum AGE were quantified by immunohistochemistry, ELISA, and AGE-fluorescence. After an infusion of AGE-RSA, renal expression of angiotensinogen, ACE, renin, and angiotensin receptor type 1 were increased significantly (all P < 0.01), and ACE activity was elevated. This was associated with tubular and glomerular hypertrophy and AGE accumulation, which could be antagonized by valsartan. However, valsartan had no effect on increased filtration fraction associated with an AGE-RSA infusion. At the same time, an infusion of Ang II increased the serum and renal accumulation of AGE and advanced oxidation protein products and induced renal hypertrophy and salt retention that could be antagonized by pyridoxamine. However, pyridoxamine had no effect on renal vasoconstriction manifested by reduced renal blood flow. AGE and Ang II have overlapping activities in the kidney. The beneficial effects of blockade of either pathway underline the importance of this interaction in diabetic renal disease and the aging kidney.

Why blockade of the renin-angiotensin system reduces the incidence of new-onset diabetes.

Recent trials have suggested that inhibitors of the renin-angiotensin system (RAS), such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs), may reduce the incidence of new-onset diabetes in patients with or without hypertension and at high risk of developing diabetes. In this review, we critically evaluate the evidence from recent clinical trials for such a potential preventive effect of ACE inhibitors and ARBs, including a meta-analysis of these recent trials. The reduced incidence of diabetes in patients at high risk of developing diabetes by ACE inhibitors or ARBs has been explained by haemodynamic effects, such as improved delivery of insulin and glucose to the peripheral skeletal muscle, and non-haemodynamic effects, including direct effects on glucose transport and insulin signalling pathways, all of which decrease insulin resistance. There is now evidence that the pancreas may contain an in situ active RAS, which appears to be upregulated in an animal model of type 2 diabetes. Thus, ACE inhibitors and ARBs may act by attenuating the deleterious effect of angiotensin II on vasoconstriction, fibrosis, inflammation, apoptosis and beta-cell death in the pancreas, thereby protecting a critical beta-cell mass essential for insulin production. New evidence is presented that ACE inhibitors and ARBs may delay or prevent the development of insulin resistance and diabetes, for which novel mechanisms are suggested. The actions of agents that interrupt the RAS on insulin resistance, obesity and diabetes warrant further investigation in other animal models. Prospective clinical studies with the primary endpoint of the prevention of diabetes are now indicated to (i) further explore whether the inhibitors of the RAS are superior compared to other antihypertensive agents such as calcium channel blockers (CCBs) and (ii) to evaluate the potential beneficial effects of combination antihypertensive regimens on the development of diabetes.

Myocardial infarction increases ACE2 expression in rat and humans.

AIMS: Angiotensin converting enzyme (ACE) 2 catalyses the cleavage of angiotensin (Ang) I to Ang 1-9 and of Ang II to Ang 1-7. ACE2 deficiency impairs cardiac contractility and upregulates hypoxia-induced genes, suggesting a link with myocardial ischaemia. We studied the expression of ACE2 after myocardial infarction (MI) in the rat as well as in human failing hearts. METHODS AND RESULTS: Rats were killed at days 1, 3, and 28 after MI, or treated for 4 weeks with the ACE inhibitor ramipril (1 mg/kg). Cardiac gene and protein expression of ACE and ACE2 were assessed by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry/activity assays/in vitro autoradiography, respectively. Both ACE (P = 0.022) and ACE2 (P = 0.015) mRNA increased in the border/infarct area compared with the viable area at day 3 after MI. By day 28, increases in ACE (P = 0.005) and ACE2 (P = 0.006) mRNA were also seen in the viable myocardium of MI rats compared with myocardium of control rats. ACE2 protein localized to macrophages, vascular endothelium, smooth muscle, and myocytes. Ramipril attenuated cardiac hypertrophy and inhibited cardiac ACE. In contrast, ramipril had no effect on cardiac ACE2 mRNA, which remained elevated in all areas of the MI rat heart. Immunoreactivity of both ACE and ACE2 increased in failing human hearts. CONCLUSION: The increase in ACE2 after MI suggests that it plays an important role in the negative modulation of the renin angiotensin system in the generation and degradation of angiotensin peptides after cardiac injury.

The role of advanced glycation in reduced organic cation transport associated with experimental diabetes.

Tubular dysfunction is an important early manifestation of diabetic nephropathy. Reduced renal expression of organic cation transporters (OCTs) potentially contributes to impaired cation clearance in diabetes. This study examines the role of advanced glycation end-products (AGEs) in mediating these changes. Experimental diabetes was induced with streptozotocin (55 mg/kg). Rats were randomly treated with the AGE inhibitor aminoguanidine for 32 weeks. In a second protocol, diabetic rats were followed with and without low-dose insulin therapy (2 U/day) for 4 weeks. Expression of OCTs was determined by real-time RT-PCR (reverse transcription-polymerase chain reaction) and Western blotting. As a marker of cation transport, the fractional clearance of endogenous N-methylnicotinamide (NMN) was determined by high-performance liquid chromatography. Both short- and long-term diabetes was associated with reduced gene and protein expression of the three renal OCT isotypes. This was associated with a reduction in the fractional clearance of NMN compared with control animals by over 50%. These changes correlated with the accumulation of renal and plasma AGEs. Treatment with the AGE inhibitor aminoguanidine restored the expression of OCT-2 and OCT-3 in diabetic animals and normalized renal NMN clearance. NMN clearance was also improved in diabetic animals receiving low-dose insulin, correlating with a reduction in AGEs and improvement in effective renal plasma flow. These studies demonstrate an early impairment of expression of OCTs and cation clearance associated with diabetes. These changes correlate with the accumulation of AGEs and may be partly attenuated by an AGE inhibitor, implicating a role for AGEs in organic cation transport.