Dietary resistant starch enhances immune health of the kidney in diabetes via promoting microbially-derived metabolites and dampening neutrophil recruitment.

BACKGROUND: Dietary-resistant starch is emerging as a potential therapeutic tool to limit the negative effects of diabetes on the kidneys. However, its metabolic and immunomodulatory effects have not yet been fully elucidated. METHODS: Six-week-old db/db mice were fed a diet containing 12.5% resistant starch or a control diet matched for equivalent regular starch for 10 weeks. db/m mice receiving the control diet were utilised as non-diabetic controls. Freshly collected kidneys were digested for flow cytometry analysis of immune cell populations. Kidney injury was determined by measuring albuminuria, histology, and immunohistochemistry. Portal vein plasma was collected for targeted analysis of microbially-derived metabolites. Intestinal histology and tight junction protein expression were assessed. RESULTS: Resistant starch limited the development of albuminuria in db/db mice. Diabetic db/db mice displayed a decline in portal vein plasma levels of acetate, propionate, and butyrate, which was increased with resistant starch supplementation. Diabetic db/db mice receiving resistant starch had a microbially-derived metabolite profile similar to that of non-diabetic db/m mice. The intestinal permeability markers lipopolysaccharide and lipopolysaccharide binding protein were increased in db/db mice consuming the control diet, which was not seen in db/db mice receiving resistant starch supplementation. Diabetes was associated with an increase in the kidney neutrophil population, neutrophil activation, number of C5aR1+ neutrophils, and urinary complement C5a excretion, all of which were reduced with resistant starch. These pro-inflammatory changes appear independent of fibrotic changes in the kidney. CONCLUSIONS: Resistant starch supplementation in diabetes promotes beneficial circulating microbially-derived metabolites and improves intestinal permeability, accompanied by a modulation in the inflammatory profile of the kidney including neutrophil infiltration, complement activation, and albuminuria. These findings indicate that resistant starch can regulate immune and inflammatory responses in the kidney and support the therapeutic potential of resistant starch supplementation in diabetes on kidney health.

Therapeutic Potential of Targeting Complement C5a Receptors in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) affects 30-40% of patients with diabetes and is currently the leading cause of end-stage renal disease (ESRD). The activation of the complement cascade, a highly conserved element of the innate immune system, has been implicated in the pathogenesis of diabetes and its complications. The potent anaphylatoxin C5a is a critical effector of complement-mediated inflammation. Excessive activation of the C5a-signalling axis promotes a potent inflammatory environment and is associated with mitochondrial dysfunction, inflammasome activation, and the production of reactive oxygen species. Conventional renoprotective agents used in the treatment of diabetes do not target the complement system. Mounting preclinical evidence indicates that inhibition of the complement system may prove protective in DKD by reducing inflammation and fibrosis. Targeting the C5a-receptor signaling axis is of particular interest, as inhibition at this level attenuates inflammation while preserving the critical immunological defense functions of the complement system. In this review, the important role of the C5a/C5a-receptor axis in the pathogenesis of diabetes and kidney injuries will be discussed, and an overview of the status and mechanisms of action of current complement therapeutics in development will be provided.

Valproic acid attenuates cellular senescence in diabetic kidney disease through the inhibition of complement C5a receptors.

Despite increasing knowledge about the factors involved in the progression of diabetic complications, diabetic kidney disease (DKD) continues to be a major health burden. Current therapies only slow but do not prevent the progression of DKD. Thus, there is an urgent need to develop novel therapy to halt the progression of DKD and improve disease prognosis. In our preclinical study where we administered a histone deacetylase (HDAC) inhibitor, valproic acid, to streptozotocin-induced diabetic mice, albuminuria and glomerulosclerosis were attenuated. Furthermore, we discovered that valproic acid attenuated diabetes-induced upregulation of complement C5a receptors, with a concomitant reduction in markers of cellular senescence and senescence-associated secretory phenotype. Interestingly, further examination of mice lacking the C5a receptor 1 (C5aR1) gene revealed that cellular senescence was attenuated in diabetes. Similar results were observed in diabetic mice treated with a C5aR1 inhibitor, PMX53. RNA-sequencing analyses showed that PMX53 significantly regulated genes associated with cell cycle pathways leading to cellular senescence. Collectively, these results for the first time demonstrated that complement C5a mediates cellular senescence in diabetic kidney disease. Cellular senescence has been implicated in the pathogenesis of diabetic kidney disease, thus therapies to inhibit cellular senescence such as complement inhibitors present as a novel therapeutic option to treat diabetic kidney disease.

The Complement Pathway: New Insights into Immunometabolic Signaling in Diabetic Kidney Disease.

Significance: The metabolic disorder, diabetes mellitus, results in microvascular complications, including diabetic kidney disease (DKD), which is partly believe to involve disrupted energy generation in the kidney, leading to injury that is characterized by inflammation and fibrosis. An increasing body of evidence indicates that the innate immune complement system is involved in the pathogenesis of DKD; however, the precise mechanisms remain unclear. Recent Advances: Complement, traditionally thought of as the prime line of defense against microbial intrusion, has recently been recognized to regulate immunometabolism. Studies have shown that the complement activation products, Complement C5a and C3a, which are potent pro-inflammatory mediators, can mediate an array of metabolic responses in the kidney in the diabetic setting, including altered fuel utilization, disrupted mitochondrial respiratory function, and reactive oxygen species generation. In diabetes, the lectin pathway is activated via autoreactivity toward altered self-surfaces known as danger-associated molecular patterns, or via sensing altered carbohydrate and acetylation signatures. In addition, endogenous complement inhibitors can be glycated, whereas diet-derived glycated proteins can themselves promote complement activation, worsening DKD, and lending support for environmental influences as an additional avenue for propagating complement-induced inflammation and kidney injury. Critical Issues: Recent evidence indicates that conventional renoprotective agents used in DKD do not target the complement, leaving this web of inflammatory stimuli intact. Future Directions: Future studies should focus on the development of novel pharmacological agents that target the complement pathway to alleviate inflammation, oxidative stress, and kidney fibrosis, thereby reducing the burden of microvascular diseases in diabetes. Antioxid. Redox Signal. 37, 781-801.

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.

Targeting Methylglyoxal in Diabetic Kidney Disease Using the Mitochondria-Targeted Compound MitoGamide.

Diabetic kidney disease (DKD) remains the number one cause of end-stage renal disease in the western world. In experimental diabetes, mitochondrial dysfunction in the kidney precedes the development of DKD. Reactive 1,2-dicarbonyl compounds, such as methylglyoxal, are generated from sugars both endogenously during diabetes and exogenously during food processing. Methylglyoxal is thought to impair the mitochondrial function and may contribute to the pathogenesis of DKD. Here, we sought to target methylglyoxal within the mitochondria using MitoGamide, a mitochondria-targeted dicarbonyl scavenger, in an experimental model of diabetes. Male 6-week-old heterozygous Akita mice (C57BL/6-Ins2-Akita/J) or wildtype littermates were randomized to receive MitoGamide (10 mg/kg/day) or a vehicle by oral gavage for 16 weeks. MitoGamide did not alter the blood glucose control or body composition. Akita mice exhibited hallmarks of DKD including albuminuria, hyperfiltration, glomerulosclerosis, and renal fibrosis, however, after 16 weeks of treatment, MitoGamide did not substantially improve the renal phenotype. Complex-I-linked mitochondrial respiration was increased in the kidney of Akita mice which was unaffected by MitoGamide. Exploratory studies using transcriptomics identified that MitoGamide induced changes to olfactory signaling, immune system, respiratory electron transport, and post-translational protein modification pathways. These findings indicate that targeting methylglyoxal within the mitochondria using MitoGamide is not a valid therapeutic approach for DKD and that other mitochondrial targets or processes upstream should be the focus of therapy.

Long Term High Protein Diet Feeding Alters the Microbiome and Increases Intestinal Permeability, Systemic Inflammation and Kidney Injury in Mice.

SCOPE: This study evaluates the effects of a chronic high protein diet (HPD) on kidney injury, intestinal permeability and gut microbiota perturbations in a mouse model. METHOD AND RESULTS: Mice are fed a diet containing either 20% or 52% energy from protein for 24 weeks; protein displaced an equivalent amount of wheat starch. The HPD does not alter glycemic control or body weight. The HPD induces kidney injury as evidenced by increase in albuminuria, urinary kidney injury molecule-1, blood urea nitrogen, urinary isoprostanes and renal cortical NF-κB p65 gene expression. HPD decreases intestinal occludin gene expression, increases plasma endotoxin and plasma monocyte chemoattractant protein-1, indicating intestinal leakiness and systemic inflammation. Cecal microbial analysis reveals that HPD feeding does not alter alpha diversity; however, it does alter beta diversity, indicating an altered microbial community structure with HPD feeding. Predicted metagenome pathway analysis demonstrates a reduction in branched-chain amino acid synthesis and an increase of the urea cycle with consumption of a HPD. CONCLUSION: These results demonstrate that long term HPD consumption in mice causes albuminuria, systemic inflammation, increase in gastrointestinal permeability and is associated with gut microbiome remodeling with an increase in the urea cycle pathway, which may contribute to renal injury.

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.

Exploring the role of the metabolite-sensing receptor GPR109a in diabetic nephropathy.

Alterations in gut homeostasis may contribute to the progression of diabetic nephropathy. There has been recent attention on the renoprotective effects of metabolite-sensing receptors in chronic renal injury, including the G protein-coupled receptor (GPR)109a, which ligates the short-chain fatty acid butyrate. However, the role of GPR109a in the development of diabetic nephropathy, a milieu of diminished microbiome-derived metabolites, has not yet been determined. The present study aimed to assess the effects of insufficient GPR109a signaling, via genetic deletion of GPR109a, on the development of renal injury in diabetic nephropathy. Gpr109a-/- mice or their wild-type littermates (Gpr109a+/+) were rendered diabetic with streptozotocin. Mice received a control diet or an isocaloric high-fiber diet (12.5% resistant starch) for 24 wk, and gastrointestinal permeability and renal injury were determined. Diabetes was associated with increased albuminuria, glomerulosclerosis, and inflammation. In comparison, Gpr109a-/- mice with diabetes did not show an altered renal phenotype. Resistant starch supplementation did not afford protection from renal injury in diabetic nephropathy. While diabetes was associated with alterations in intestinal morphology, intestinal permeability assessed in vivo using the FITC-dextran test was unaltered. GPR109a deletion did not worsen gastrointestinal permeability. Furthermore, 12.5% resistant starch supplementation, at physiological concentrations, had no effect on intestinal permeability or morphology. The results of this study indicate that GPR109a does not play a critical role in intestinal homeostasis in a model of type 1 diabetes or in the development of diabetic nephropathy.

Complement C5a Induces Renal Injury in Diabetic Kidney Disease by Disrupting Mitochondrial Metabolic Agility.

The sequelae of diabetes include microvascular complications such as diabetic kidney disease (DKD), which involves glucose-mediated renal injury associated with a disruption in mitochondrial metabolic agility, inflammation, and fibrosis. We explored the role of the innate immune complement component C5a, a potent mediator of inflammation, in the pathogenesis of DKD in clinical and experimental diabetes. Marked systemic elevation in C5a activity was demonstrated in patients with diabetes; conventional renoprotective agents did not therapeutically target this elevation. C5a and its receptor (C5aR1) were upregulated early in the disease process and prior to manifest kidney injury in several diverse rodent models of diabetes. Genetic deletion of C5aR1 in mice conferred protection against diabetes-induced renal injury. Transcriptomic profiling of kidney revealed diabetes-induced downregulation of pathways involved in mitochondrial fatty acid metabolism. Interrogation of the lipidomics signature revealed abnormal cardiolipin remodeling in diabetic kidneys, a cardinal sign of disrupted mitochondrial architecture and bioenergetics. In vivo delivery of an orally active inhibitor of C5aR1 (PMX53) reversed the phenotypic changes and normalized the renal mitochondrial fatty acid profile, cardiolipin remodeling, and citric acid cycle intermediates. In vitro exposure of human renal proximal tubular epithelial cells to C5a led to altered mitochondrial respiratory function and reactive oxygen species generation. These experiments provide evidence for a pivotal role of the C5a/C5aR1 axis in propagating renal injury in the development of DKD by disrupting mitochondrial agility, thereby establishing a new immunometabolic signaling pathway in DKD.

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.

Use of Readily Accessible Inflammatory Markers to Predict Diabetic Kidney Disease.

Diabetic kidney disease is a common complication of type 1 and type 2 diabetes and is the primary cause of end-stage renal disease in developed countries. Early detection of diabetic kidney disease will facilitate early intervention aimed at reducing the rate of progression to end-stage renal disease. Diabetic kidney disease has been traditionally classified based on the presence of albuminuria. More recently estimated glomerular filtration rate has also been incorporated into the staging of diabetic kidney disease. While albuminuric diabetic kidney disease is well described, the phenotype of non-albuminuric diabetic kidney disease is now widely accepted. An association between markers of inflammation and diabetic kidney disease has previously been demonstrated. Effector molecules of the innate immune system including C-reactive protein, interleukin-6, and tumor necrosis factor-α are increased in patients with diabetic kidney disease. Furthermore, renal infiltration of neutrophils, macrophages, and lymphocytes are observed in renal biopsies of patients with diabetic kidney disease. Similarly high serum neutrophil and low serum lymphocyte counts have been shown to be associated with diabetic kidney disease. The neutrophil-lymphocyte ratio is considered a robust measure of systemic inflammation and is associated with the presence of inflammatory conditions including the metabolic syndrome and insulin resistance. Cross-sectional studies have demonstrated a link between high levels of the above inflammatory biomarkers and diabetic kidney disease. Further longitudinal studies will be required to determine if these readily available inflammatory biomarkers can accurately predict the presence and prognosis of diabetic kidney disease, above and beyond albuminuria, and estimated glomerular filtration rate.

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.

Nrf2 Activation Is a Potential Therapeutic Approach to Attenuate Diabetic Retinopathy.

Purpose: Oxidative stress is a causal factor in the development of diabetic retinopathy; however, clinically relevant strategies to treat the disease by augmenting antioxidant defense mechanisms have not been fully explored. We hypothesized that boosting nuclear factor erythroid-2-related factor 2 (Nrf2) antioxidant capacity with the novel Nrf2 activator dh404, would protect the retina in diabetes including vision-threatening breakdown of the blood-retinal barrier (BRB) and associated damage to macroglial Müller cells. Methods: Sprague-Dawley rats were randomized to become diabetic or nondiabetic and administered dh404 by gavage for 10 weeks. Complementary in vitro studies were performed in cultured Müller cells exposed to hyperglycemia. Results: In diabetes, dh404 prevented vascular leakage into the retina and vitreous cavity as well as upregulation of the vascular permeability and angiogenic factors, VEGF, and angiopoietin-2, and inflammatory mediators, including TNF-α and IL-6. Müller cells, which maintain BRB integrity and become gliotic in diabetes with increased immunolabeling for glial fibrillary acidic protein, were protected by dh404. In diabetes, dh404 bolstered the antioxidant capacity of the retina with an increase in hemeoxygenase-1, nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) quinine oxidoreductase-1, and Nrf2. Further, dh404 attenuated the diabetes-induced increase in oxidative stress as measured by dihydroethidium and 8-oxo-2'-deoxyguanosine (8-OHdG) immunolabeling as well as NADPH oxidase isoform expression. Studies in Müller cells supported these findings with dh404 attenuating the hyperglycemia-induced increase in vascular permeability, angiogenic and inflammatory mediators, and oxidative stress. Conclusions: Our data demonstrate the ability of dh404 to protect the retina against diabetes-induced damage and potentially prevent vision loss.

Dietary Advanced Glycation End Products and Risk Factors for Chronic Disease: A Systematic Review of Randomised Controlled Trials.

Dietary advanced glycation end-products (AGEs) form during heating and processing of food products and are widely prevalent in the modern Western diet. Recent systematic reviews indicate that consumption of dietary AGEs may promote inflammation, oxidative stress and insulin resistance. Experimental evidence indicates that dietary AGEs may also induce renal damage, however, this outcome has not been considered in previous systematic reviews. The purpose of this review was to examine the effect of consumption of a high AGE diet on biomarkers of chronic disease, including chronic kidney disease (CKD), in human randomized controlled trials (RCTs). Six databases (SCOPUS, CINHAL, EMBASE, Medline, Biological abstracts and Web of Science) were searched for randomised controlled dietary trials that compared high AGE intake to low AGE intake in adults with and without obesity, diabetes or CKD. Twelve dietary AGE interventions were identified with a total of 293 participants. A high AGE diet increased circulating tumour necrosis factor-alpha and AGEs in all populations. A high AGE diet increased 8-isoprostanes in healthy adults, and vascular cell adhesion molecule-1 (VCAM-1) in patients with diabetes. Markers of CKD were not widely assessed. The evidence presented indicates that a high AGE diet may contribute to risk factors associated with chronic disease, such as inflammation and oxidative stress, however, due to a lack of high quality randomised trials, more research is required.

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.

Deficiency in Apoptosis-Inducing Factor Recapitulates Chronic Kidney Disease via Aberrant Mitochondrial Homeostasis.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease.

Direct Endothelial Nitric Oxide Synthase Activation Provides Atheroprotection in Diabetes-Accelerated Atherosclerosis.

Patients with diabetes have an increased risk of developing atherosclerosis. Endothelial dysfunction, characterized by the lowered bioavailability of endothelial NO synthase (eNOS)-derived NO, is a critical inducer of atherosclerosis. However, the protective aspect of eNOS in diabetes-associated atherosclerosis remains controversial, a likely consequence of its capacity to release both protective NO or deleterious oxygen radicals in normal and disease settings, respectively. Harnessing the atheroprotective activity of eNOS in diabetic settings remains elusive, in part due to the lack of endogenous eNOS-specific NO release activators. We have recently shown in vitro that eNOS-derived NO release can be increased by blocking its binding to Caveolin-1, the main coat protein of caveolae, using a highly specific peptide, CavNOxin. However, whether targeting eNOS using this peptide can attenuate diabetes-associated atherosclerosis is unknown. In this study, we show that CavNOxin can attenuate atherosclerotic burden by ∼84% in vivo. In contrast, mice lacking eNOS show resistance to CavNOxin treatment, indicating eNOS specificity. Mechanistically, CavNOxin lowered oxidative stress markers, inhibited the expression of proatherogenic mediators, and blocked leukocyte-endothelial interactions. These data are the first to show that endogenous eNOS activation can provide atheroprotection in diabetes and suggest that CavNOxin is a viable strategy for the development of antiatherosclerotic compounds.

Ebselen by modulating oxidative stress improves hypoxia-induced macroglial Müller cell and vascular injury in the retina.

Oxidative stress is an important contributor to glial and vascular cell damage in ischemic retinopathies. We hypothesized that ebselen via its ability to reduce reactive oxygen species (ROS) and augment nuclear factor-like 2 (Nrf2) anti-oxidants would attenuate hypoxia-induced damage to macroglial Müller cells and also lessen retinal vasculopathy. Primary cultures of rat Müller cells were exposed to normoxia (21% O2), hypoxia (0.5% O2) and ebselen (2.5 µM) for up to 72 h. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice while control mice were housed in room air. Mice received vehicle (saline, 5% dimethyl sulfoxide) or ebselen (10 mg/kg) each day between postnatal days 6-18. In cultured Müller cells, flow cytometry for dihydroethidium revealed that ebselen reduced the hypoxia-induced increase in ROS levels, whilst increasing the expression of Nrf2-regulated anti-oxidant genes, heme oxygenase 1, glutathione peroxidase-1, NAD(P)H dehydrogenase quinone oxidoreductase 1 and glutamate-cysteine ligase. Moreover, in Müller cells, ebselen reduced the hypoxia-induced increase in protein levels of pro-angiogenic and pro-inflammatory factors including vascular endothelial growth factor, interleukin-6, monocyte chemoattractant-protein 1 and intercellular adhesion molecule-1, and the mRNA levels of glial fibrillary acidic protein (GFAP), a marker of Müller cell injury. Ebselen improved OIR by attenuating capillary vaso-obliteration and neovascularization and a concomitant reduction in Müller cell gliosis and GFAP. We conclude that ebselen protects against hypoxia-induced injury of retinal Müller cells and the microvasculature, which is linked to its ability to reduce oxidative stress, vascular damaging factors and inflammation. Agents such as ebselen may be potential treatments for retinopathies that feature oxidative stress-mediated damage to glia and the microvasculature.

Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets.

Type 1 diabetes (T1D) is the result of an autoimmune assault against the insulin-producing pancreatic ß-cells, where chronic local inflammation (insulitis) leads to ß-cell destruction. T cells and macrophages infiltrate into islets early in T1D pathogenesis. These immune cells secrete cytokines that lead to the production of reactive oxygen species (ROS) and T-cell invasion and activation. Cytokine-signaling pathways are very tightly regulated by protein tyrosine phosphatases (PTPs) to prevent excessive activation. Here, we demonstrate that pancreata from NOD mice with islet infiltration have enhanced oxidation/inactivation of PTPs and STAT1 signaling compared with NOD mice that do not have insulitis. Inactivation of PTPs with sodium orthovanadate in human and rodent islets and ß-cells leads to increased activation of interferon signaling and chemokine production mediated by STAT1 phosphorylation. Furthermore, this exacerbated STAT1 activation-induced cell death in islets was prevented by overexpression of the suppressor of cytokine signaling-1 or inactivation of the BH3-only protein Bim. Together our data provide a mechanism by which PTP inactivation induces signaling in pancreatic islets that results in increased expression of inflammatory genes and exacerbated insulitis.