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Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca 2+ -dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca 2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria. Patients with diabetic nephropathy suffer from impaired albumin reabsorption by proximal tubular epithelial cells. Here authors use diabetic and transgenic mouse models and in vitro models to show the cause for this lies in the down regulation and internalization of the ion channels, ORAI1-3.

This article summarizes a much lengthier one that appeared in Prevention and Treatment. The earlier article grew out of a project initiated by the Department of Health and Human Services Office of the Assistant Secretary for Planning and Evaluation. The Positive Youth Development Evaluation project described why policy makers, practitioners, and prevention scientists advocated a shift in approach for how youth issues are addressed in this country. The Positive Youth Development Evaluation project sought to define how youth development programs have been defined in the literature and then to locate, through a structured search, strong evaluations of these programs and summarize the outcomes of these evaluations. In the current article, we explain why prevention has shifted from a single problem focus to a focus on factors that affect both positive and problem youth development, describe what is meant by positive youth development, and summarize what we know about the effectiveness of positive youth development programs.

Sight-threatening diabetic retinopathy has been treated with photocoagulation for decades but the mechanisms behind the beneficial clinical effects are poorly understood. One target of irradiation and a potential player in this process is the retinal pigment epithelium (RPE). Here we establish an in vitro model for photocoagulation of human RPE cells. ARPE-19 cells were exposed to photocoagulation and studied at various time points up to 168h. Lesion morphology, necrosis and apoptosis were investigated by light microscopy; LIVE/DEAD staining and measurements of lactate dehydrogenase activity; and TUNEL- and ELISA-based quantification of DNA fragments, respectively. Cell migration and proliferation were explored using docetaxel and mitomycin C; temporal and spatial changes in proliferation were assessed by confocal immunofluorescence of proliferating cell nuclear antigen. Gene expression was measured by qPCR. Photocoagulation of ARPE-19 resulted in denaturation of proteins and reproducible lesion formation. A transient peak in necrosis, followed by a peak in apoptosis was observed in cells within the lesions at 6h and 24h, respectively after photocoagulation. Cell proliferation was depressed during the first hours after photocoagulation, back to control levels at 24h and augmented in the following days. These effects were not limited to cells in the lesions, but also evident in neighbouring cells. Changes in cell proliferation during lesion repair were preceded by changes in cell migration. Altered mRNA expression of genes previously implicated in the regulation of cell proliferation (FOS, IL-1β, IL-8, HMGA2), migration and tissue repairing (TGFBR2, ADAMTS6, TIMP3, CTGF) was observed, as well as increased expression of the alarmin IL33 and the cytoprotective gene HSPA6. Using a laser system and experimental settings that comply with standards used in clinical practice, we have established a suitable model for in vitro photocoagulation of human RPE cells to isolate their contribution to the beneficial effects of laser treatment.

Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis. Streptozotocin (STZ)-induced diabetes in apolipoprotein E(-/-) mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice. Targeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.

Aims/hypothesisEvidence that glucose-dependent insulinotropic peptide (GIP) and/or the GIP receptor (GIPR) are involved in cardiovascular biology is emerging. We hypothesised that GIP has untoward effects on cardiovascular biology, in contrast to glucagon-like peptide 1 (GLP-1), and therefore investigated the effects of GIP and GLP-1 concentrations on cardiovascular disease (CVD) and mortality risk.MethodsGIP concentrations were successfully measured during OGTTs in two independent populations (Malmö Diet Cancer–Cardiovascular Cohort [MDC-CC] and Prevalence, Prediction and Prevention of Diabetes in Botnia [PPP-Botnia]) in a total of 8044 subjects. GLP-1 (n = 3625) was measured in MDC-CC. The incidence of CVD and mortality was assessed via national/regional registers or questionnaires. Further, a two-sample Mendelian randomisation (2SMR) analysis between the GIP pathway and outcomes (coronary artery disease [CAD] and myocardial infarction) was carried out using a GIP-associated genetic variant, rs1800437, as instrumental variable. An additional reverse 2SMR was performed with CAD as exposure variable and GIP as outcome variable, with the instrumental variables constructed from 114 known genetic risk variants for CAD.ResultsIn meta-analyses, higher fasting levels of GIP were associated with risk of higher total mortality (HR[95% CI] = 1.22 [1.11, 1.35]; p = 4.5 × 10−5) and death from CVD (HR[95% CI] 1.30 [1.11, 1.52]; p = 0.001). In accordance, 2SMR analysis revealed that increasing GIP concentrations were associated with CAD and myocardial infarction, and an additional reverse 2SMR revealed no significant effect of CAD on GIP levels, thus confirming a possible effect solely of GIP on CAD.Conclusions/interpretationIn two prospective, community-based studies, elevated levels of GIP were associated with greater risk of all-cause and cardiovascular mortality within 5–9 years of follow-up, whereas GLP-1 levels were not associated with excess risk. Further studies are warranted to determine the cardiovascular effects of GIP per se.

The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes.

ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca 2+ store to Ca 2+ influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25 mM d -glucose), which was accompanied by enhanced store-operated Ca 2+ influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2 Akita /J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca 2+ -calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca 2+ homeostasis and endothelial dysfunction under hyperglycaemia. Key message ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose. Increased expression is confirmed in Akita (Ins2 Akita /J) and STZ diabetic mice and patients. Upregulation mechanism is mediated by Ca 2+ /calcineurin/NFATc3 signalling. High glucose has no direct effects on ORAI1-3 channel activity and channel activation process.

The incretin hormone GIP (glucose-dependent insulinotropic polypeptide) promotes pancreatic β-cell function by potentiating insulin secretion and β-cell proliferation. Recently, a combined analysis of several genome-wide association studies (Meta-analysis of Glucose and Insulin-Related Traits Consortium [MAGIC]) showed association to postprandial insulin at the GIP receptor (GIPR) locus. Here we explored mechanisms that could explain the protective effects of GIP on islet function. Associations of GIPR rs10423928 with metabolic and anthropometric phenotypes in both nondiabetic (N = 53,730) and type 2 diabetic individuals (N = 2,731) were explored by combining data from 11 studies. Insulin secretion was measured both in vivo in nondiabetic subjects and in vitro in islets from cadaver donors. Insulin secretion was also measured in response to exogenous GIP. The in vitro measurements included protein and gene expression as well as measurements of β-cell viability and proliferation. The A allele of GIPR rs10423928 was associated with impaired glucose- and GIP-stimulated insulin secretion and a decrease in BMI, lean body mass, and waist circumference. The decrease in BMI almost completely neutralized the effect of impaired insulin secretion on risk of type 2 diabetes. Expression of GIPR mRNA was decreased in human islets from carriers of the A allele or patients with type 2 diabetes. GIP stimulated osteopontin (OPN) mRNA and protein expression. OPN expression was lower in carriers of the A allele. Both GIP and OPN prevented cytokine-induced reduction in cell viability (apoptosis). In addition, OPN stimulated cell proliferation in insulin-secreting cells. These findings support β-cell proliferative and antiapoptotic roles for GIP in addition to its action as an incretin hormone. Identification of a link between GIP and OPN may shed new light on the role of GIP in preservation of functional β-cell mass in humans.

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-kB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.