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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., Here, we present studies where we have selectively over‐expressed the gene encoding for OST48 within podocytes, a cell type intimately involved in the pathology of DKD. In this novel study, despite facilitating renal clearance of AGEs, overexpression of podocyte OST48 resulted in a decline in kidney function and significant glomerular damage and dysfunction exacerbated by diabetes. We have also identified for the first time the proteomic profiles detailing the mechanisms driving the decline in GFR and provided ultra‐resolution imaging visualizing the selective binding of AGE‐OST48 within the podocytes.

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.

This study shows how highly processed foods can cause innate immune inflammation that promotes chronic microvascular disease., 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.

Signaling via the receptor of advanced glycation end products (RAGE)—though complex and not fully elucidated in the setting of diabetes—is considered a key injurious pathway in the development of diabetic nephropathy (DN). We report here that RAGE deletion resulted in increased expression of fibrotic markers (collagen I and IV, fibronectin) and the inflammatory marker MCP-1 in primary mouse mesangial cells (MCs) and in kidney cortex. RNA sequencing analysis in MCs from RAGE−/− and wild-type mice confirmed these observations. Nevertheless, despite these gene expression changes, decreased responsiveness to transforming growth factor-β was identified in RAGE−/− mice. Furthermore, RAGE deletion conferred a more proliferative phenotype in MCs and reduced susceptibility to staurosporine-induced apoptosis. RAGE restoration experiments in RAGE−/− MCs largely reversed these gene expression changes, resulting in reduced expression of fibrotic and inflammatory markers. This study highlights that protection against DN in RAGE knockout mice is likely to be due in part to the decreased responsiveness to growth factor stimulation and an antiapoptotic phenotype in MCs. Furthermore, it extends our understanding of the role of RAGE in the progression of DN, as RAGE seems to play a key role in modulating the sensitivity of the kidney to injurious stimuli such as prosclerotic cytokines.

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.

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.

Defects in the activity of enzyme complexes of the mitochondrial respiratory chain are thought to be responsible for several disorders, including renal impairment. Gene mutations that result in complex I deficiency are the most common oxidative phosphorylation disorders in humans. To determine whether an abnormality in mitochondrial complex I per se is associated with development of renal disease, mice with a knockdown of the complex I gene, Ndufs6 were studied.Ndufs6 mice had a partial renal cortical complex I deficiency; Ndufs6gt/gt, 32% activity and Ndufs6gt/+, 83% activity compared with wild-type mice. Both Ndufs6gt/+ and Ndufs6gt/gt mice exhibited hallmarks of renal disease, including albuminuria, urinary excretion of kidney injury molecule-1 (Kim-1), renal fibrosis, and changes in glomerular volume, with decreased capacity to generate mitochondrial ATP and superoxide from substrates oxidized via complex I. However, more advanced renal defects in Ndufs6gt/gt mice were observed in the context of a disruption in the inner mitochondrial electrochemical potential, 3-nitrotyrosine-modified mitochondrial proteins, increased urinary excretion of 15-isoprostane F2t, and up-regulation of antioxidant defence. Juvenile Ndufs6gt/gt mice also exhibited signs of early renal impairment with increased urinary Kim-1 excretion and elevated circulating cystatin C.We have identified renal impairment in a mouse model of partial complex I deficiency, suggesting that even modest deficits in mitochondrial respiratory chain function may act as risk factors for chronic kidney disease.These studies identify for the first time that complex I deficiency as the result of interruption of Ndufs6 is an independent cause of renal impairment.

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.

Glycaemic control, reduction of blood pressure using agents that block the renin-angiotensin system and control of dyslipidaemia are the major strategies used in the clinical management of patients with diabetes mellitus. Each of these approaches interrupts a number of pathological pathways, which directly contributes to the vascular complications of diabetes mellitus, including renal disease, blindness, neuropathy and cardiovascular disease. However, research published over the past few years has indicated that many of the pathological pathways important in the development of the vascular complications of diabetes mellitus are equally relevant to the initiation of diabetes mellitus itself. These pathways include insulin signalling, generation of cellular energy, post-translational modifications and redox imbalances. This Review will examine how the development of diabetes mellitus has come full circle from initiation to complications and suggests that the development of diabetes mellitus and the progression to chronic complications both require the same mechanistic triggers.

Cardiovascular benefits of ubiquinone have been previously demonstrated, and we administered it as a novel therapy in an experimental model of type 2 diabetic nephropathy. db/db and dbH mice were followed for 10 weeks, after randomization to receive either vehicle or ubiquinone (CoQ10; 10mg/kg/day) orally. db/db mice had elevated urinary albumin excretion rates and albumin:creatinine ratio, not seen in db/db CoQ10-treated mice. Renal cortices from db/db mice had lower total and oxidized CoQ10 content, compared with dbH mice. Mitochondria from db/db mice also contained less oxidized CoQ10(ubiquinone) compared with dbH mice. Diabetes-induced increases in total renal collagen but not glomerulosclerosis were significantly decreased with CoQ10 therapy. Mitochondrial superoxide and ATP production via complex II in the renal cortex were increased in db/db mice, with ATP normalized by CoQ10. However, excess renal mitochondrial hydrogen peroxide production and increased mitochondrial membrane potential seen in db/db mice were attenuated with CoQ10. Renal superoxide dismutase activity was also lower in db/db mice compared with dbH mice. Our results suggest that a deficiency in mitochondrial oxidized CoQ10 (ubiquinone) may be a likely precipitating factor for diabetic nephropathy. Therefore CoQ10 supplementation may be renoprotective in type 2 diabetes, via preservation of mitochondrial function.

Background. Advanced glycation end-products (AGEs) and their receptors are prominent contributors to diabetic kidney disease.Methods. Flow cytometry was used to measure the predictive capacity for kidney impairment of the AGE receptors RAGE, AGE-R1, and AGE-R3 on peripheral blood mononuclear cells (PBMCs) in experimental models of type 2 diabetes (T2DM) fed varied AGE containing diets and in obese type 2 diabetic and control human subjects.Results. Diets high in AGE content fed to diabetic mice decreased cell surface RAGE on PBMCs and in type 2 diabetic patients with renal impairment (RI). All diabetic mice had elevated Albumin excretion rates (AERs), and high AGE fed dbdb mice had declining Glomerular filtration rate (GFR). Cell surface AGE-R1 expression was also decreased by high AGE diets and with diabetes in dbdb mice and in humans with RI. PBMC expression of AGE R3 was decreased in diabetic dbdb mice or with a low AGE diet.Conclusions. The most predictive PBMC profile for renal disease associated with T2DM was an increase in the cell surface expression of AGE-R1, in the context of a decrease in membranous RAGE expression in humans, which warrants further investigation as a biomarker for progressive DN in larger patient cohorts.

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.

Background— Low plasma high-density lipoprotein (HDL) is associated with elevated cardiovascular risk and aspects of the metabolic syndrome. We hypothesized that HDL modulates glucose metabolism via elevation of plasma insulin and through activation of the key metabolic regulatory enzyme, AMP-activated protein kinase, in skeletal muscle. Methods and Results— Thirteen patients with type 2 diabetes mellitus received both intravenous reconstituted HDL (rHDL: 80 mg/kg over 4 hours) and placebo on separate days in a double-blind, placebo-controlled crossover study. A greater fall in plasma glucose from baseline occurred during rHDL than during placebo (at 4 hours rHDL=−2.6±0.4; placebo=−2.1±0.3mmol/L; P =0.018). rHDL increased plasma insulin (at 4 hours rHDL=3.4±10.0; placebo= −19.2±7.4 pmol/L; P =0.034) and also the homeostasis model assessment β-cell function index (at 4 hours rHDL=18.9±5.9; placebo=8.6±4.4%; P =0.025). Acetyl-CoA carboxylase β phosphorylation in skeletal muscle biopsies was increased by 1.7±0.3-fold after rHDL, indicating activation of the AMP-activated protein kinase pathway. Both HDL and apolipoprotein AI increased glucose uptake (by 177±12% and 144±18%, respectively; P Conclusions— rHDL reduced plasma glucose in patients with type 2 diabetes mellitus by increasing plasma insulin and activating AMP-activated protein kinase in skeletal muscle. These findings suggest a role for HDL-raising therapies beyond atherosclerosis to address type 2 diabetes mellitus.

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.

OBJECTIVE—Excessive production of reactive oxygen species (ROS) via NADPH oxidase has been implicated in the pathogenesis of diabetic nephropathy. Since NADPH oxidase activation is closely linked to other putative pathways, its interaction with changes in protein kinase C (PKC) and increased advanced glycation was examined. RESEARCH DESIGN AND METHODS—Streptozotocin-induced diabetic or nondiabetic Sprague Dawley rats were followed for 32 weeks, with groups randomized to no treatment or the NADPH oxidase assembly inhibitor apocynin (15 mg · kg−1 · day−1; weeks 16–32). Complementary in vitro studies were performed in which primary rat mesangial cells, in the presence and absence of advanced glycation end products (AGEs)-BSA, were treated with either apocynin or the PKC-α inhibitor Ro-32-0432. RESULTS—Apocynin attenuated diabetes-associated increases in albuminuria and glomerulosclerosis. Circulating, renal cytosolic, and skin collagen–associated AGE levels in diabetic rats were not reduced by apocynin. Diabetes-induced translocation of PKC, specifically PKC-α to renal membranes, was associated with increased NADPH-dependent superoxide production and elevated renal, serum, and urinary vascular endothelial growth factor (VEGF) concentrations. In both diabetic rodents and in AGE-treated mesangial cells, blockade of NADPH oxidase or PKC-α attenuated cytosolic superoxide and PKC activation and increased VEGF. Finally, renal extracellular matrix accumulation of fibronectin and collagen IV was decreased by apocynin. CONCLUSIONS—In the context of these and previous findings by our group, we conclude that activation of NADPH oxidase via phosphorylation of PKC-α is downstream of the AGE–receptor for AGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.