Glucagon-like peptide-1 receptor signaling modifies the extent of diabetic kidney disease through dampening the receptor for advanced glycation end products-induced inflammation.

Glucagon like peptide-1 (GLP-1) is a hormone produced and released by cells of the gastrointestinal tract following meal ingestion. GLP-1 receptor agonists (GLP-1RA) exhibit kidney-protective actions through poorly understood mechanisms. Here we interrogated whether the receptor for advanced glycation end products (RAGE) plays a role in mediating the actions of GLP-1 on inflammation and diabetic kidney disease. Mice with deletion of the GLP-1 receptor displayed an abnormal kidney phenotype that was accelerated by diabetes and improved with co-deletion of RAGE in vivo. Activation of the GLP-1 receptor pathway with liraglutide, an anti-diabetic treatment, downregulated kidney RAGE, reduced the expansion of bone marrow myeloid progenitors, promoted M2-like macrophage polarization and lessened markers of kidney damage in diabetic mice. Single cell transcriptomics revealed that liraglutide induced distinct transcriptional changes in kidney endothelial, proximal tubular, podocyte and macrophage cells, which were dominated by pathways involved in nutrient transport and utilization, redox sensing and the resolution of inflammation. The kidney-protective action of liraglutide was corroborated in a non-diabetic model of chronic kidney disease, the subtotal nephrectomised rat. Thus, our findings identify a novel glucose-independent kidney-protective action of GLP-1-based therapies in diabetic kidney disease and provide a valuable resource for exploring the cell-specific kidney transcriptional response ensuing from pharmacological GLP-1R agonism.

Targeted deletion of nicotinamide adenine dinucleotide phosphate oxidase 4 from proximal tubules is dispensable for diabetic kidney disease development.

BACKGROUND: The nicotinamide adenine dinucleotide phosphate oxidase isoform 4 (Nox4) mediates reactive oxygen species (ROS) production and renal fibrosis in diabetic kidney disease (DKD) at the level of the podocyte. However, the mitochondrial localization of Nox4 and its role as a mitochondrial bioenergetic sensor has recently been reported. Whether Nox4 drives pathology in DKD within the proximal tubular compartment, which is densely packed with mitochondria, is not yet known. METHODS: We generated a proximal tubular-specific Nox4 knockout mouse model by breeding Nox4flox/flox mice with mice expressing Cre recombinase under the control of the sodium-glucose cotransporter-2 promoter. Subsets of Nox4ptKO mice and their Nox4flox/flox littermates were injected with streptozotocin (STZ) to induce diabetes. Mice were followed for 20 weeks and renal injury was assessed. RESULTS: Genetic ablation of proximal tubular Nox4 (Nox4ptKO) resulted in no change in renal function and histology. Nox4ptKO mice and Nox4flox/flox littermates injected with STZ exhibited the hallmarks of DKD, including hyperfiltration, albuminuria, renal fibrosis and glomerulosclerosis. Surprisingly, diabetes-induced renal injury was not improved in Nox4ptKO STZ mice compared with Nox4flox/flox STZ mice. Although diabetes conferred ROS overproduction and increased the mitochondrial oxygen consumption rate, proximal tubular deletion of Nox4 did not normalize oxidative stress or mitochondrial bioenergetics. CONCLUSIONS: Taken together, these results demonstrate that genetic deletion of Nox4 from the proximal tubules does not influence DKD development, indicating that Nox4 localization within this highly energetic compartment is dispensable for chronic kidney disease pathogenesis in the setting of diabetes.

Inhibitors of Advanced Glycation End Product (AGE) Formation and Accumulation.

A range of chemically different compounds are known to inhibit the formation and accumulation of advanced glycation end products (AGEs) or disrupt associated signalling pathways. There is evidence that some of these agents can provide end-organ protection in chronic diseases including diabetes. Whilst this group of therapeutics are structurally and functionally different and have a range of mechanisms of action, they ultimately reduce the deleterious actions and the tissue burden of advanced glycation end products. To date it remains unclear if this is due to the reduction in tissue AGE levels per se or the modulation of downstream signal pathways. Some of these agents either stimulate antioxidant defence or reduce the formation of reactive oxygen species (ROS), modify lipid profiles and inhibit inflammation. A number of existing treatments for glucose lowering, hypertension and hyperlipidaemia are also known to reduce AGE formation as a by-product of their action. Targeted AGE formation inhibitors or AGE cross-link breakers have been developed and have shown beneficial effects in animal models of diabetic complications as well as other chronic conditions. However, only a few of these agents have progressed to clinical development. The failure of clinical translation highlights the importance of further investigation of the advanced glycation pathway, the diverse actions of agents which interfere with AGE formation, cross-linking or AGE receptor activation and their effect on the development and progression of chronic diseases including diabetic complications. Advanced glycation end products (AGEs) are (1) proteins or lipids that become glycated as a result of exposure to sugars or (2) non-proteinaceous oxidised lipids. They are implicated in ageing and the development, or worsening, of many degenerative diseases, such as diabetes, atherosclerosis, chronic kidney and Alzheimer's disease. Several antihypertensive and antidiabetic agents and statins also indirectly lower AGEs. Direct AGE inhibitors currently investigated include pyridoxamine and epalrestat, the inhibition of the formation of reactive dicarbonyls such as methylglyoxal as an important precursor of AGEs via increased activation of the detoxifying enzyme Glo-1 and inhibitors of NOX-derived ROS to reduce the AGE/RAGE signalling.

Mapping time-course mitochondrial adaptations in the kidney in experimental diabetes.

Oxidative phosphorylation (OXPHOS) drives ATP production by mitochondria, which are dynamic organelles, constantly fusing and dividing to maintain kidney homoeostasis. In diabetic kidney disease (DKD), mitochondria appear dysfunctional, but the temporal development of diabetes-induced adaptations in mitochondrial structure and bioenergetics have not been previously documented. In the present study, we map the changes in mitochondrial dynamics and function in rat kidney mitochondria at 4, 8, 16 and 32 weeks of diabetes. Our data reveal that changes in mitochondrial bioenergetics and dynamics precede the development of albuminuria and renal histological changes. Specifically, in early diabetes (4 weeks), a decrease in ATP content and mitochondrial fragmentation within proximal tubule epithelial cells (PTECs) of diabetic kidneys were clearly apparent, but no changes in urinary albumin excretion or glomerular morphology were evident at this time. By 8 weeks of diabetes, there was increased capacity for mitochondrial permeability transition (mPT) by pore opening, which persisted over time and correlated with mitochondrial hydrogen peroxide (H2O2) generation and glomerular damage. Late in diabetes, by week 16, tubular damage was evident with increased urinary kidney injury molecule-1 (KIM-1) excretion, where an increase in the Complex I-linked oxygen consumption rate (OCR), in the context of a decrease in kidney ATP, indicated mitochondrial uncoupling. Taken together, these data show that changes in mitochondrial bioenergetics and dynamics may precede the development of the renal lesion in diabetes, and this supports the hypothesis that mitochondrial dysfunction is a primary cause of DKD.

Deletion of bone-marrow-derived receptor for AGEs (RAGE) improves renal function in an experimental mouse model of diabetes.

AIMS/HYPOTHESIS: The AGEs and the receptor for AGEs (RAGE) are known contributors to diabetic complications. RAGE also has a physiological role in innate and adaptive immunity and is expressed on immune cells. The aim of this study was to determine whether deletion of RAGE from bone-marrow-derived cells influences the pathogenesis of experimental diabetic nephropathy. METHODS: Groups (n = 8/group) of lethally irradiated 8 week old wild-type (WT) mice were reconstituted with bone marrow from WT (WT → WT) or RAGE-deficient (RG) mice (RG → WT). Diabetes was induced using multiple low doses of streptozotocin after 8 weeks of bone marrow reconstitution and mice were followed for a further 24 weeks. RESULTS: Compared with diabetic WT mice reconstituted with WT bone marrow, diabetic WT mice reconstituted with RG bone marrow had lower urinary albumin excretion and podocyte loss, more normal creatinine clearance and less tubulo-interstitial injury and fibrosis. However, glomerular collagen IV deposition, glomerulosclerosis and cortical levels of TGF-ß were not different among diabetic mouse groups. The renal tubulo-interstitium of diabetic RG → WT mice also contained fewer infiltrating CD68(+) macrophages that were activated. Diabetic RG → WT mice had lower renal cortical concentrations of CC chemokine ligand 2 (CCL2), macrophage inhibitory factor (MIF) and IL-6 than diabetic WT → WT mice. Renal cortical RAGE ligands S100 calgranulin (S100A)8/9 and AGEs, but not high mobility box protein B-1 (HMGB-1) were also decreased in diabetic RG → WT compared with diabetic WT → WT mice. In vitro, bone-marrow-derived macrophages from WT but not RG mice stimulated collagen IV production in cultured proximal tubule cells. CONCLUSIONS/INTERPRETATION: These studies suggest that RAGE expression on haemopoietically derived immune cells contributes to the functional changes seen in diabetic nephropathy by promoting macrophage infiltration and renal tubulo-interstitial damage.

Advanced glycation end products (AGEs) are cross-sectionally associated with insulin secretion in healthy subjects.

It has been postulated that chronic exposure to high levels of advanced glycation end products (AGEs), in particular from dietary sources, can impair insulin secretion. In the present study, we investigated the cross-sectional relationship between AGEs and acute insulin secretion during an intravenous glucose tolerance test (IVGTT) and following a 75 g oral glucose tolerance test (OGTT) in healthy humans. We report the cross-sectional association between circulating AGE concentrations and insulin secretory function in healthy humans (17 F: 27 M, aged 30 ± 10 years) with a wide range of BMI (24.6-31.0 kg/m(2)). Higher circulating concentrations of AGEs were related to increased first phase insulin secretion during IVGTT (r = 0.43; p < 0.05) and lower 2-h glucose concentrations during OGTT (r = -0.31; p < 0.05). In addition, fasting (r = -0.36; p < 0.05) and 2-h glucose concentrations were negatively related to circulating levels of soluble receptor for AGE (RAGE) isoforms (r = -0.39; p < 0.01). In conclusion, in healthy humans, we show a cross-sectional association between advanced glycation end products and acute insulin secretion during glucose tolerance testing.

Plasma advanced glycation end products (AGEs) and NF-κB activity are independent determinants of diastolic and pulse pressure.

BACKGROUND: High levels of circulating advanced glycation end products (AGEs) can initiate chronic low-grade activation of the immune system (CLAIS) with each of these factors independently associated with cardiovascular (CV) morbidity and mortality. Therefore, our objective was to characterize the relationship between serum AGEs, CLAIS and other risk factors for CV disease in normotensive non-diabetic individuals. METHODS: We measured body mass index (BMI), waist-to-hip ratio (WHR), blood pressure, lipid and glucose profile in 44 non-diabetic volunteers (17 female, 27 males). Carboxymethyl-lysine (CML) was measured by ELISA as a marker for circulating AGEs and NF-κB p65 activity as an inflammatory marker by DNA-binding in peripheral blood mononuclear cells lysates (PBMC). RESULTS: Plasma CML concentrations were related to diastolic blood pressure (r=-0.51, p<0.01) independently of age, sex, BMI and WHR (p<0.05). Diastolic blood pressure was also related to NF-κB activity in PBMC (r=0.47, p<0.01) before and after adjustment for age, sex, BMI and WHR (p<0.05). Plasma CML concentrations were related to the pulse pressure before (r=0.42; p<0.05) and after adjustment for age, sex, BMI and waist (p<0.05). Neither CML nor NF-κB activity were related to systolic blood pressure (both p=ns). Plasma CML concentrations were not associated with plasma lipid or glucose concentrations (all p=ns). CONCLUSIONS: Plasma AGE levels and NF-κB activity in PBMC were independent determinants of diastolic and pulse pressure in healthy normotensive individuals. This association suggests a role for AGEs in the etiology of hypertension, possibly via the initiation of CLAIS and aortic stiffening.

The role of activator protein-1 (AP-1) complex in diabetes associated atherosclerosis: Insights from single cell RNA sequencing.

Despite advances in the treatment of atherosclerotic cardiovascular disease, it remains the leading cause of death in patients with diabetes. Even when risk factors are mitigated, the disease progresses, and thus newer targets need to be identified that directly inhibit the underlying pathobiology of atherosclerosis in diabetes. A single cell sequencing approach was utilised to distinguish the proatherogenic transcriptional profile in aortic cells in diabetes using a streptozotocin induced-diabetic Apoe-/- mouse model. Human carotid endarterectomy specimens from individuals with and without diabetes were also evaluated via immunohistochemical analysis. Further mechanistic studies were performed in human aortic endothelial cells and human THP-1 derived macrophages. We then performed a preclinical study using an AP-1 inhibitor in a diabetic Apoe-/- mouse model. Single cell RNA sequencing analysis identified the AP-1 complex as a novel target in diabetes-associated atherosclerosis. AP-1 levels were elevated in carotid endarterectomy specimens from diabetic when compared to non-diabetic individuals. AP-1 was validated as a mechanosensitive transcription factor via immunofluorescence staining for regional heterogeneity of endothelial cells of the aortic region exposed to turbulent blood flow and by performing microfluidics experiments in HAECs. AP-1 inhibition with T-5224 blunted endothelial cell activation as assessed by a monocyte adhesion assay and expression of genes relevant to endothelial function. Furthermore, AP-1 inhibition attenuated foam cell formation. Critically, treatment with T-5224 attenuated atherosclerosis development in diabetic Apoe-/- mice. This study has identified the AP-1 complex as a novel target, inhibition of which treats the underlying pathobiology of atherosclerosis in diabetes.

An Engineered Nanosugar Enables Rapid and Sustained Glucose-Responsive Insulin Delivery in Diabetic Mice.

Glucose-responsive insulin-delivery platforms that are sensitive to dynamic glucose concentration fluctuations and provide both rapid and prolonged insulin release have great potential to control hyperglycemia and avoid hypoglycemia diabetes. Here, biodegradable and charge-switchable phytoglycogen nanoparticles capable of glucose-stimulated insulin release are engineered. The nanoparticles are "nanosugars" bearing glucose-sensitive phenylboronic acid groups and amine moieties that allow effective complexation with insulin (≈95% loading capacity) to form nanocomplexes. A single subcutaneous injection of nanocomplexes shows a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg dL-1 ) for up to 13 h. The morphology of the nanocomplexes is found to be key to controlling rapid and extended glucose-regulated insulin delivery in vivo. These studies reveal that the injected nanocomplexes enabled efficient insulin release in the mouse, with optimal bioavailability, pharmacokinetics, and safety profiles. These results highlight a promising strategy for the development of a glucose-responsive insulin delivery system based on a natural and biodegradable nanosugar.

Endothelial reactive oxygen-forming NADPH oxidase 5 is a possible player in diabetic aortic aneurysm but not atherosclerosis.

Atherosclerosis and its complications are major causes of cardiovascular morbidity and death. Apart from risk factors such as hypercholesterolemia and inflammation, the causal molecular mechanisms are unknown. One proposed causal mechanism involves elevated levels of reactive oxygen species (ROS). Indeed, early expression of the ROS forming NADPH oxidase type 5 (Nox5) in vascular endothelial cells correlates with atherosclerosis and aortic aneurysm. Here we test the pro-atherogenic Nox5 hypothesis using mouse models. Because Nox5 is missing from the mouse genome, a knock-in mouse model expressing human Nox5 in its physiological location of endothelial cells (eNOX5ki/ki) was tested as a possible new humanised mouse atherosclerosis model. However, whether just on a high cholesterol diet or by crossing in aortic atherosclerosis-prone ApoE-/- mice with and without induction of diabetes, Nox5 neither induced on its own nor aggravated aortic atherosclerosis. Surprisingly, however, diabetic ApoE-/- x eNOX5ki/ki mice developed aortic aneurysms more than twice as often correlating with lower vascular collagens, as assessed by trichrome staining, without changes in inflammatory gene expression, suggesting that endothelial Nox5 directly affects extracellular matrix remodelling associated with aneurysm formation in diabetes. Thus Nox5-derived reactive oxygen species are not a new independent mechanism of atherosclerosis but may enhance the frequency of abdominal aortic aneurysms in the context of diabetes. Together with similar clinical findings, our preclinical target validation opens up a first-in-class mechanism-based approach to treat or even prevent abdominal aortic aneurysms.

Targeted reduction of advanced glycation improves renal function in obesity.

Obesity is highly prevalent in Western populations and is considered a risk factor for the development of renal impairment. Interventions that reduce the tissue burden of advanced glycation end-products (AGEs) have shown promise in stemming the progression of chronic disease. Here we tested if treatments that lower tissue AGE burden in patients and mice would improve obesity-related renal dysfunction. Overweight and obese individuals (body mass index (BMI) 26-39 kg/m(2)) were recruited to a randomized, crossover clinical trial involving 2 weeks each on a low- and a high-AGE-containing diet. Renal function and an inflammatory profile (monocyte chemoattractant protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF)) were improved following the low-AGE diet. Mechanisms of advanced glycation-related renal damage were investigated in a mouse model of obesity using the AGE-lowering pharmaceutical, alagebrium, and mice in which the receptor for AGE (RAGE) was deleted. Obesity, resulting from a diet high in both fat and AGE, caused renal impairment; however, treatment of the RAGE knockout mice with alagebrium improved urinary albumin excretion, creatinine clearance, the inflammatory profile, and renal oxidative stress. Alagebrium treatment, however, resulted in decreased weight gain and improved glycemic control compared with wild-type mice on a high-fat Western diet. Thus, targeted reduction of the advanced glycation pathway improved renal function in obesity.

Advanced glycation urinary protein-bound biomarkers and severity of diabetic nephropathy in man.

BACKGROUND/AIMS: The formation of advanced glycation end products (AGEs) is accelerated in patients with diabetic nephropathy. The aim of this study was to ascertain if the urinary excretion of proteins modified by advanced glycation can be used as biomarkers for albuminuria in individuals with type 1 or type 2 diabetes. METHODS: Community-based patients with type 1 (n = 68) or type 2 diabetes (n = 216) attending a diabetes clinic of a tertiary referral hospital were classified as having normoalbuminuria (Normo, albumin excretion rate (AER) <20 µg/min), microalbuminuria (Micro, AER 20-200 µg/min) or macroalbuminuria (Macro, AER ≥200 µg/min). Serum and urine AGE-modified proteins were measured. RESULTS: In patients with both type 1 diabetes and type 2 diabetes, there was a clear association between the degree of albuminuria and urinary AGE-modified proteins (p < 0.0001). Exclusive to patients with type 1 diabetes, urinary excretion of the AGE carboxymethyllysine correlated with AER, whereas patients with type 2 diabetes and macroalbuminuria had an increase in urinary methylglyoxal, an AGE intermediate. These changes were independent of isotopic glomerular filtration rate levels. Serum concentrations of AGEs or soluble receptor for AGEs were not consistently associated with albuminuria in either type 1 or type 2 diabetes. CONCLUSIONS: Urinary excretion of proteins modified by AGEs may be useful biomarkers of albuminuria in individuals with type 1 and type 2 diabetes, warranting prospective investigation in larger diabetic cohorts.

The AGE receptor, OST48 drives podocyte foot process effacement and basement membrane expansion (alters structural composition).

AIMS: The accumulation of advanced glycation end products is implicated in the development and progression of diabetic kidney disease. No study has examined whether stimulating advanced glycation clearance via receptor manipulation is reno-protective in diabetes. Podocytes, which are early contributors to diabetic kidney disease and could be a target for reno-protection. MATERIALS AND METHODS: To examine the effects of increased podocyte oligosaccharyltransferase-48 on kidney function, glomerular sclerosis, tubulointerstitial fibrosis and proteome (PXD011434), we generated a mouse with increased oligosaccharyltransferase-48kDa subunit abundance in podocytes driven by the podocin promoter. RESULTS: Despite increased urinary clearance of advanced glycation end products, we observed a decline in renal function, significant glomerular damage including glomerulosclerosis, collagen IV deposition, glomerular basement membrane thickening and foot process effacement and tubulointerstitial fibrosis. Analysis of isolated glomeruli identified enrichment in proteins associated with collagen deposition, endoplasmic reticulum stress and oxidative stress. Ultra-resolution microscopy of podocytes revealed denudation of foot processes where there was co-localization of oligosaccharyltransferase-48kDa subunit and advanced glycation end-products. CONCLUSIONS: These studies indicate that increased podocyte expression of oligosaccharyltransferase-48 kDa subunit results in glomerular endoplasmic reticulum stress and a decline in kidney function.

Processed foods drive intestinal barrier permeability and microvascular diseases.

Intake of processed foods has increased markedly over the past decades, coinciding with increased microvascular diseases such as chronic kidney disease (CKD) and diabetes. Here, we show in rodent models that long-term consumption of a processed diet drives intestinal barrier permeability and an increased risk of CKD. Inhibition of the advanced glycation pathway, which generates Maillard reaction products within foods upon thermal processing, reversed kidney injury. Consequently, a processed diet leads to innate immune complement activation and local kidney inflammation and injury via the potent proinflammatory effector molecule complement 5a (C5a). In a mouse model of diabetes, a high resistant starch fiber diet maintained gut barrier integrity and decreased severity of kidney injury via suppression of complement. These results demonstrate mechanisms by which processed foods cause inflammation that leads to chronic disease.

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.

Circulating high-molecular-weight RAGE ligands activate pathways implicated in the development of diabetic nephropathy.

The accumulation of advanced glycation end products is thought to be a key factor in the initiation and progression of diabetic nephropathy. Here we determined whether the size of the ligands for the receptor for advanced glycation end products (RAGEs) that were present in the serum of patients with type 2 diabetes modulates their pathogenic potential. Serum was collected from control subjects and patients with type 2 diabetes with varying degrees of renal disease (normo-, micro-, or macroalbuminuria). The titers of the RAGE ligands N-carboxymethyllysine (CML), S100A, S100B, and high-mobility group box 1 (HMGB1) were measured by enzyme-linked immunosorbent assay in serum as well as in pooled size-fractionated serum. We also measured cellular binding of serum fractions to mesangial cells transfected with RAGE and examined the downstream signaling pathways. Circulating CML was increased in patients with type 2 diabetes, whereas HMGB1 was decreased. S100A8, S100BA9, and soluble RAGE were unchanged. The high-molecular-weight (over 50 kDa) serum fraction contained the greatest proportion of RAGE ligands, with all immunoreactivity and cellular binding observed only with serum fractions over 30 kDa. High-molecular-weight serum from macroalbuminuric patients showed greater RAGE binding capacity, modulation of cell-surface RAGE expression, increased phospho-protein kinase C-alpha, and p65 nuclear factor kappaB DNA-binding activity, which were competitively inhibited by soluble RAGE or CML neutralizing antibodies. These data show that ligands that activate RAGE present in the circulation of patients with type 2 diabetes and nephropathy are predominantly of high molecular weight.

RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes.

Damaged mitochondria generate an excess of superoxide, which may mediate tissue injury in diabetes. We hypothesized that in diabetic nephropathy, advanced glycation end-products (AGEs) lead to increases in cytosolic reactive oxygen species (ROS), which facilitate the production of mitochondrial superoxide. In normoglycemic conditions, exposure of primary renal cells to AGEs, transient overexpression of the receptor for AGEs (RAGE) with an adenoviral vector, and infusion of AGEs to healthy rodents each induced renal cytosolic oxidative stress, which led to mitochondrial permeability transition and deficiency of mitochondrial complex I. Because of a lack of glucose-derived NADH, which is the substrate for complex I, these changes did not lead to excess production of mitochondrial superoxide; however, when we performed these experiments in hyperglycemic conditions in vitro or in diabetic rats, we observed significant generation of mitochondrial superoxide at the level of complex I, fueled by a sustained supply of NADH. Pharmacologic inhibition of AGE-RAGE-induced mitochondrial permeability transition in vitro abrogated production of mitochondrial superoxide; we observed a similar effect in vivo after inhibiting cytosolic ROS production with apocynin or lowering AGEs with alagebrium. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in mice. Taken together, these studies suggest that AGE-RAGE-induced cytosolic ROS production facilitates mitochondrial superoxide production in hyperglycemic environments, providing further evidence of a role for the advanced glycation pathway in the development and progression of diabetic nephropathy.

Disparate Effects of Diabetes and Hyperlipidemia on Experimental Kidney Disease.

It is well established that diabetes is the major cause of chronic kidney disease worldwide. Both hyperglycemia, and more recently, advanced glycation endproducts, have been shown to play critical roles in the development of kidney disease. Moreover, the renin-angiotensin system along with growth factors and cytokines have also been shown to contribute to the onset and progression of diabetic kidney disease; however, the role of lipids in this context is poorly characterized. The current study aimed to compare the effect of 20 weeks of streptozotocin-induced diabetes or western diet feeding on kidney disease in two different mouse strains, C57BL/6 mice and hyperlipidemic apolipoprotein (apo) E knockout (KO) mice. Mice were fed a chow diet (control), a western diet (21% fat, 0.15% cholesterol) or were induced with streptozotocin-diabetes (55 mg/kg/day for 5 days) then fed a chow diet and followed for 20 weeks. The induction of diabetes was associated with a 3-fold elevation in glycated hemoglobin and an increase in kidney to body weight ratio regardless of strain (p < 0.0001). ApoE deficiency significantly increased plasma cholesterol and triglyceride levels and feeding of a western diet exacerbated these effects. Despite this, urinary albumin excretion (UAE) was elevated in diabetic mice to a similar extent in both strains (p < 0.0001) but no effect was seen with a western diet in either strain. Diabetes was also associated with extracellular matrix accumulation in both strains, and western diet feeding to a lesser extent in apoE KO mice. Consistent with this, an increase in renal mRNA expression of the fibrotic marker, fibronectin, was observed in diabetic C57BL/6 mice (p < 0.0001). In summary, these studies demonstrate disparate effects of diabetes and hyperlipidemia on kidney injury, with features of the diabetic milieu other than lipids suggested to play a more prominent role in driving renal pathology.

A new perspective on therapeutic inhibition of advanced glycation in diabetic microvascular complications: common downstream endpoints achieved through disparate therapeutic approaches?

A commonality among the chemically disparate compounds that inhibit the formation and accumulation of advanced glycation end products (AGEs) or their signalling pathways is their end organ protection in experimental models of diabetes complications. Although this group of therapeutics are structurally and functionally distinct with numerous mechanisms of action, the most important factor governing their therapeutic capability is clearly their ability to alleviate the tissue burden of advanced glycation, rather than the biochemical mechanism by which this is achieved. However, it remains to be determined if it is the reduction in tissue AGE levels per se or inhibition of downstream signal pathways which is ultimately required for end organ protection. For example, a number of these agents stimulate antioxidant defences, modify lipid profiles and inhibit low-grade inflammation. These novel actions emphasise the importance of further examination of the advanced glycation pathway and in particular the diverse action of these agents in ameliorating the development of diabetic complications such as nephropathy.

Globally elevating the AGE clearance receptor, OST48, does not protect against the development of diabetic kidney disease, despite improving insulin secretion.

The accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic kidney disease (DKD). There has been interest in investigating the potential of AGE clearance receptors, such as oligosaccharyltransferase-48 kDa subunit (OST48) to prevent the detrimental effects of excess AGE accumulation seen in the diabetic kidney. Here the objective of the study was to increase the expression of OST48 to examine if this slowed the development of DKD by facilitating the clearance of AGEs. Groups of 8-week-old heterozygous knock-in male mice (n = 9-12/group) over-expressing the gene encoding for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST+/-) and litter mate controls were randomised to either (i) no diabetes or (ii) diabetes induced via multiple low-dose streptozotocin and followed for 24 weeks. By the study end, global over expression of OST48 increased glomerular OST48. This facilitated greater renal excretion of AGEs but did not affect circulating or renal AGE concentrations. Diabetes resulted in kidney damage including lower glomerular filtration rate, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. In diabetic mice, tubulointerstitial fibrosis was further exacerbated by global increases in OST48. There was significantly insulin effectiveness, increased acute insulin secretion, fasting insulin concentrations and AUCinsulin observed during glucose tolerance testing in diabetic mice with global elevations in OST48 when compared to diabetic wild-type littermates. Overall, this study suggested that despite facilitating urinary-renal AGE clearance, there were no benefits observed on kidney functional and structural parameters in diabetes afforded by globally increasing OST48 expression. However, the improvements in insulin secretion seen in diabetic mice with global over-expression of OST48 and their dissociation from effects on kidney function warrant future investigation.