NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in retinal endothelial cells (ECs) and is upregulated under hyperglycemic and hypoxic conditions. However, the role of endothelial Nox4 upregulation in long-term retinal blood vessel damage in diabetic retinopathy (DR) remains undefined. Here, we attempted to address this question using humanized EC-specific Nox4 transgenic (hNox4EC-Tg) and EC-specific Nox4 knockout (Nox4EC-KO) mouse models. Our results show that, hNox4EC-Tg mice at age of 10-12 months exhibited increased tortuosity of retinal blood vessels, focal vascular leakage, and acellular capillary formation. In vitro study revealed enhanced apoptosis in brain microvascular ECs (BMECs) derived from hNox4EC-Tg mice, concomitant with increased mitochondrial ROS, elevated lipid peroxidation, decreased mitochondrial membrane potential (ΔΨm), and reduced mitochondrial respiratory function. In contrast, EC-specific deletion of Nox4 decreased mitochondrial ROS generation, alleviated mitochondrial damage, reduced EC apoptosis, and protected the retina from acellular capillary formation and vascular hyperpermeability in a streptozotocin-induced diabetes mouse model. These findings suggest that sustained upregulation of Nox4 in the endothelium contributes to retinal vascular pathology in diabetes, at least in part, through impairing mitochondrial function. Normalization of Nox4 expression in ECs may provide a new approach for prevention of vascular injury in DR.

NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidases playing an important role in many biological processes. Previously we have shown that Nox4 is highly expressed in retinal blood vessels and is upregulated in oxygen-induced retinopathy (OIR). However, the exact role of endothelial Nox4 in retinal angiogenesis remains elusive. Herein, using endothelial cell (EC)-specific Nox4 knockout (Nox4EC−KO) mice, we investigated the impact of endothelial Nox4 deletion on retinal vascular development and pathological angiogenesis during OIR. Our results show that deletion of Nox4 in ECs led to retarded retinal vasculature development with fewer, blunted-end tip cells and sparser, dysmorphic filopodia at vascular front, and reduced density of vascular network in superficial, deep, and intermediate layers in postnatal day 7 (P7), P12, and P17 retinas, respectively. In OIR, loss of endothelial Nox4 had no effect on hyperoxia-induced retinal vaso-obliteration at P9 but significantly reduced aberrant retinal neovascularization at P17 and decreased the deep layer capillary density at P25. Ex vivo study confirmed that lack of Nox4 in ECs impaired vascular sprouting. Mechanistically, loss of Nox4 significantly reduced expression of VEGF, p-VEGFR2, integrin αV, angiopoietin-2, and p-ERK1/2, attenuating EC migration and proliferation. Taken together, our results indicate that endothelial Nox4 is important for retinal vascular development and contributes to pathological angiogenesis, likely through regulation of VEGF/VEGFR2 and angiopoietin-2/integrin αV/ERK pathways. In addition, our study suggests that endothelial Nox4 appears to be essential for intraretinal revascularization after hypoxia. These findings call for caution on targeting endothelial Nox4 in ischemic/hypoxic retinal diseases.

OBJECTIVE:Glomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology. METHODS:We studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice. RESULTS:We demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3' untranslated region (3'-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of invitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3'-UTR in response to HG. The invivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function. CONCLUSIONS:We established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.

The mechanism by which TSC2 inactivation or deficiency contributes to the pathology of tuberous sclerosis complex (TSC) is not fully clear. We show that renal angiomyolipomas from TSC patients and kidney cortex from Tsc2 +/− mice exhibit elevated levels of reactive oxygen species (ROS). Downregulation of tuberin (protein encoded by TSC2 gene) in renal proximal tubular epithelial cells significantly increased ROS concomitant with enhanced Nox4. Similarly, we found elevated levels of Nox4 in the renal cortex of Tsc2 +/− mice and in the renal angiomyolipomas from TSC patients. Tuberin deficiency is associated with activation of mTORC1. Rapamycin, shRNAs targeting raptor, or inhibition of S6 kinase significantly inhibited the expression of Nox4, resulting in attenuation of production of ROS in tuberin-downregulated proximal tubular epithelial cells. In contrast, activation of mTORC1 increased Nox4 and ROS. These results indicate that Nox4 may be a potential target for tuberin-deficiency-derived diseases. Using a xenograft model from tuberin-null tubular cells in nude mice, both anti-sense Nox4 and GKT137831, a specific inhibitor of Nox1/4, significantly inhibited the tumor growth. Thus, our results demonstrate the presence of an antagonistic relationship between tuberin and Nox4 to drive oncogenesis in the tuberin deficiency syndrome and identify Nox4 as a target to develop a therapy for TSC.

In vitro models for the investigation of renal vascular development are limited. We previously showed that isolated metanephric mesenchymal (MM) and ureteric bud (UB) cells grown in three-dimensional (3D) matrices formed organoids that consisted of primitive vascular structures surrounding a polarized epithelium. Here, we examined the potential of two principal effectors of vasculogenesis, vascular endothelial growth factor A (VEGF-A), and platelet-derived growth factor B chain (PDGF-BB), to stimulate MM cell differentiation. The results showed that MM cells possess angioblast characteristics by expressing phenotypic markers for endothelial and mesenchymal cells. UB cells synthesize VEGF-A and PDGF-BB proteins and RNA, whereas the MM cells express the respective cognate receptors, supporting their role in directional induction of vasculogenesis. VEGF-A stimulated proliferation of MM cells in monolayer and in 3D sponges but did not affect MM cell migration, organization, or vasculogenesis. However, PDGF-BB stimulated MM cell proliferation, migration, and vasculogenesis in monolayer and organization of the cells into primitive capillary like assemblies in 3D sea sponge scaffolds in vitro. A role for PDGF-BB in vasculogenesis in the 3D MM/UB co-culture system was validated by direct interference with PDGF-BB or PDGF receptor-beta cell interactions to implicate PDGF-BB as a primary effector of MM cell vasculogenesis. Thus, MM cells resemble early renal angioblasts that may provide an ideal platform for the investigation of renal vasculogenesis in vitro.

This corrects the article DOI: 10.1038/sdata.2017.179.

Objective: Type 2 diabetes (T2D) is the primary case of chronic kidney disease (CKD). Inflammation is associated with metabolic dysregulation in patients with T2D and CKD. Tryptophan (TRP) metabolism may have relevance to the CKD outcomes and associated symptoms. We investigated the relationships of TRP metabolism with inflammatory markers in patients with T2D and CKD. Methods: Data were collected from a well-characterized cohort of type 2 diabetic individuals with all stages of CKD, including patients on hemodialysis. Key TRP metabolites (kynurenine [KYN], kynurenic acid [KYNA], and quinolinic acid [QA]), proinflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]), and C-reactive protein were measured in plasma. The KYN/TRP ratio was utilized as a surrogate marker for indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity. Results: There was a significant inverse association between circulating TRP level and stages of CKD ( P < 0.0001). Downstream bioactive TRP metabolites KYN, KYNA, and QA were positively and robustly correlated with the severity of kidney disease ( P < 0.0001). In multiple linear regression, neither TNF-α nor IL-6 was independently related to KYN/TRP ratio after adjusting for estimated glomerular filtration rate (eGFR). Only TNF-α was independently related to KYN after taking into account the effect of eGFR. Conclusions: Chronic kidney disease secondary to T2D may be associated with accumulation of toxic TRP metabolites due to both inflammation and impaired kidney function. Future longitudinal studies to determine whether the accumulation of KYN directly contributes to CKD progression and associated symptoms in patients with T2D are warranted.

High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (H2S) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether H2S inhibits high glucose-induced expression of NOX4 and matrix protein and whether H2S and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of H2S. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by N(ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, H2S recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters H2S and NO and their interaction may serve as therapeutic targets in diabetic kidney disease.

Type 2 diabetes (T2D) affects more than 415 million people worldwide, and its costs to the health care system continue to rise. To identify common or rare genetic variation with potential therapeutic implications for T2D, we analyzed and replicated genome-wide protein coding variation in a total of 8,227 individuals with T2D and 12,966 individuals without T2D of Latino descent. We identified a novel genetic variant in the IGF2 gene associated with ∼20% reduced risk for T2D. This variant, which has an allele frequency of 17% in the Mexican population but is rare in Europe, prevents splicing between IGF2 exons 1 and 2. We show in vitro and in human liver and adipose tissue that the variant is associated with a specific, allele-dosage–dependent reduction in the expression of IGF2 isoform 2. In individuals who do not carry the protective allele, expression of IGF2 isoform 2 in adipose is positively correlated with both incidence of T2D and increased plasma glycated hemoglobin in individuals without T2D, providing support that the protective effects are mediated by reductions in IGF2 isoform 2. Broad phenotypic examination of carriers of the protective variant revealed no association with other disease states or impaired reproductive health. These findings suggest that reducing IGF2 isoform 2 expression in relevant tissues has potential as a new therapeutic strategy for T2D, even beyond the Latin American population, with no major adverse effects on health or reproduction.

To identify novel coding association signals and facilitate characterization of mechanisms influencing glycemic traits and type 2 diabetes risk, we analyzed 109,215 variants derived from exome array genotyping together with an additional 390,225 variants from exome sequence in up to 39,339 normoglycemic individuals from five ancestry groups. We identified a novel association between the coding variant (p.Pro50Thr) in AKT2 and fasting plasma insulin (FI), a gene in which rare fully penetrant mutations are causal for monogenic glycemic disorders. The low-frequency allele is associated with a 12% increase in FI levels. This variant is present at 1.1% frequency in Finns but virtually absent in individuals from other ancestries. Carriers of the FI-increasing allele had increased 2-h insulin values, decreased insulin sensitivity, and increased risk of type 2 diabetes (odds ratio 1.05). In cellular studies, the AKT2-Thr50 protein exhibited a partial loss of function. We extend the allelic spectrum for coding variants in AKT2 associated with disorders of glucose homeostasis and demonstrate bidirectional effects of variants within the pleckstrin homology domain of AKT2.

BACKGROUND:The presence of population structure in a sample may confound the search for important genetic loci associated with disease. Our four samples in the Family Investigation of Nephropathy and Diabetes (FIND), European Americans, Mexican Americans, African Americans, and American Indians are part of a genome- wide association study in which population structure might be particularly important. We therefore decided to study in detail one component of this, individual genetic ancestry (IGA). From SNPs present on the Affymetrix 6.0 Human SNP array, we identified 3 sets of ancestry informative markers (AIMs), each maximized for the information in one the three contrasts among ancestral populations: Europeans (HAPMAP, CEU), Africans (HAPMAP, YRI and LWK), and Native Americans (full heritage Pima Indians). We estimate IGA and present an algorithm for their standard errors, compare IGA to principal components, emphasize the importance of balancing information in the ancestry informative markers (AIMs), and test the association of IGA with diabetic nephropathy in the combined sample. RESULTS:A fixed parental allele maximum likelihood algorithm was applied to the FIND to estimate IGA in four samples: 869 American Indians; 1385 African Americans; 1451 Mexican Americans; and 826 European Americans. When the information in the AIMs is unbalanced, the estimates are incorrect with large error. Individual genetic admixture is highly correlated with principle components for capturing population structure. It takes ~700 SNPs to reduce the average standard error of individual admixture below 0.01. When the samples are combined, the resulting population structure creates associations between IGA and diabetic nephropathy. CONCLUSIONS:The identified set of AIMs, which include American Indian parental allele frequencies, may be particularly useful for estimating genetic admixture in populations from the Americas. Failure to balance information in maximum likelihood, poly-ancestry models creates biased estimates of individual admixture with large error. This also occurs when estimating IGA using the Bayesian clustering method as implemented in the program STRUCTURE. Odds ratios for the associations of IGA with disease are consistent with what is known about the incidence and prevalence of diabetic nephropathy in these populations.

Reactive oxygen species ( ROS ) are an important endogenous source of DNA damage and oxidative stress in all cell types. Deficiency in tuberin resulted in increased oxidative DNA damage in renal cells. In this study, the role of tuberin in the regulating of ROS and NADPH oxidases was investigated. Formation of ROS and activity of NADPH oxidases were significantly higher in mouse embryonic fibroblasts and in primary culture of rat renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. In addition, expression of NADPH oxidase (Nox)1, Nox2, and Nox4 (Nox isoforms) was higher in mouse embryonic fibroblasts and renal proximal tubular epithelial tuberin‐deficient cells compared to wild‐type cells. Furthermore, activity levels of NADPH oxidases and protein expression of all Nox isoforms were higher in the renal cortex of rat deficient in tuberin. However, treatment of tuberin‐deficient cells with rapamycin showed significant decrease in protein expression of all Nox. Significant increase in protein kinase C β II expression was detected in tuberin‐deficient cells, whereas inhibition of protein kinase C β II by bisindolylmaleimide I resulted in decreased protein expression of all Nox isoforms. In addition, treatment of mice deficient in tuberin with rapamycin resulted in significant decrease in all Nox protein expression. Moreover, protein and mRNA expression of all Nox were highly expressed in tumor kidney tissue of patients with tuberous sclerosis complex compared to control kidney tissue of normal subjects. These data provide the first evidence that tuberin plays a novel role in regulating ROS generation, NADPH oxidase activity, and Nox expression that may potentially be involved in development of kidney tumor in patients with tuberous sclerosis complex.

The genetic architecture of common traits, including the number, frequency, and effect sizes of inherited variants that contribute to individual risk, has been long debated. Genome-wide association studies have identified scores of common variants associated with type 2 diabetes, but in aggregate, these explain only a fraction of the heritability of this disease. Here, to test the hypothesis that lower-frequency variants explain much of the remainder, the GoT2D and T2D-GENES consortia performed whole-genome sequencing in 2,657 European individuals with and without diabetes, and exome sequencing in 12,940 individuals from five ancestry groups. To increase statistical power, we expanded the sample size via genotyping and imputation in a further 111,548 subjects. Variants associated with type 2 diabetes after sequencing were overwhelmingly common and most fell within regions previously identified by genome-wide association studies. Comprehensive enumeration of sequence variation is necessary to identify functional alleles that provide important clues to disease pathophysiology, but large-scale sequencing does not support the idea that lower-frequency variants have a major role in predisposition to type 2 diabetes.

BACKGROUND/AIM:Toll-like receptor 4 (TLR4) is one of the regulators of the innate immune response. Genetic variations in TLR4 have been associated with inflammatory diseases, including type 2 diabetes. However, to our knowledge, there are no reports on the role of variations in TLR4 in chronic kidney disease susceptibility. The objective of this study is to determine whether the genetic variants in TLR4 are associated with the estimated glomerular filtration rate (eGFR), a measure of renal function.METHODS:To evaluate the association between TLR4 variants and eGFR, we used data obtained from 434 Mexican American participants from the San Antonio Family Diabetes/Gallbladder Study. GFR was estimated using the Modification of Diet in Renal Disease formula. The Asp(299)Gly (rs4986790) and Thr(399)Ile (rs4986791) variants of TLR4 were genotyped using the TaqMan assay. Association analyses between genotypes and eGFR were performed using the measured genotype approach.RESULTS:Of the two genetic markers examined for association, only the Asp(299)Gly variant of TLR4 exhibited a nominally significant association with eGFR (p = 0.025) after accounting for the covariate effects of age and sex terms, diabetes, duration of diabetes, systolic blood pressure, body mass index, and antihypertensive treatment. Carriers of Gly299 had significantly decreased eGFR values. Although, the Thr(399)Ile variant failed to exhibit a statistically significant association with eGFR, the carriers of Ile399, however, showed a trend towards decrease in eGFR.CONCLUSION:We show for the first time that Asp(299)Gly variants of TLR4 are associated with decrease in renal function in Mexican Americans.

AIMS:To study the potential pathogenic significance of the coexistence of membranous nephropathy, cerebellar degeneration and anti-glutamic acid decarboxylase (GAD) autoantibodies in patients with diabetes.METHODS:We performed a direct immunocytochemistry on human kidney slides, electron microscopy on human kidney biopsy, direct immunofluorescence on human kidney biopsy. Baboon and rat kidney cell lines were fractionated and subjected to western blotting with antibodies to GAD.RESULTS:In this patient we demonstrate the presence of autoantibodies to GAD, which is highly enriched in podocytes plasma membrane and tubular cells of the kidney as well as sub-endothelial IgG and complement C3 deposits in the glomerular basement membrane (GBM).CONCLUSIONS:We hypothesize the existence in this patient of a common autoimmune pathogenic mechanism with GAD as the autoantigenic determinant, underlying cerebellar degeneration and membranous nephropathy.

Diabetic kidney disease (DKD) is the most common etiology of chronic kidney disease (CKD) in the industrialized world and accounts for much of the excess mortality in patients with diabetes mellitus. Approximately 45% of U.S. patients with incident end-stage kidney disease (ESKD) have DKD. Independent of glycemic control, DKD aggregates in families and has higher incidence rates in African, Mexican, and American Indian ancestral groups relative to European populations. The Family Investigation of Nephropathy and Diabetes (FIND) performed a genome-wide association study (GWAS) contrasting 6,197 unrelated individuals with advanced DKD with healthy and diabetic individuals lacking nephropathy of European American, African American, Mexican American, or American Indian ancestry. A large-scale replication and trans-ethnic meta-analysis included 7,539 additional European American, African American and American Indian DKD cases and non-nephropathy controls. Within ethnic group meta-analysis of discovery GWAS and replication set results identified genome-wide significant evidence for association between DKD and rs12523822 on chromosome 6q25.2 in American Indians (P = 5.74x10-9). The strongest signal of association in the trans-ethnic meta-analysis was with a SNP in strong linkage disequilibrium with rs12523822 (rs955333; P = 1.31x10-8), with directionally consistent results across ethnic groups. These 6q25.2 SNPs are located between the SCAF8 and CNKSR3 genes, a region with DKD relevant changes in gene expression and an eQTL with IPCEF1, a gene co-translated with CNKSR3. Several other SNPs demonstrated suggestive evidence of association with DKD, within and across populations. These data identify a novel DKD susceptibility locus with consistent directions of effect across diverse ancestral groups and provide insight into the genetic architecture of DKD.