Effect of Insulin Glargine and n-3FA on Carotid Intima-Media Thickness in People With Dysglycemia at High Risk for Cardiovascular Events: The Glucose Reduction and Atherosclerosis Continuing Evaluation Study (ORIGIN-GRACE)

OBJECTIVE

To evaluate the effects of insulin glargine and n-3 polyunsaturated fatty acid (n-3FA) supplements on carotid intima-media thickness (CIMT).

RESEARCH DESIGN AND METHODS

We enrolled 1,184 people with cardiovascular (CV) disease and/or CV risk factors plus impaired fasting glucose, impaired glucose tolerance, or early type 2 diabetes in a randomized multicenter 2 × 2 factorial design trial. Participants received open-label insulin glargine (targeting fasting glucose levels ≤5.3 mmol/L [95 mg/dL]) or standard glycemic care and double-blind therapy with a 1-g capsule of n-3FA or placebo. The primary trial outcome was the annualized rate of change in maximum CIMT for the common carotid, bifurcation, and internal carotid artery segments. Secondary outcomes were the annualized rates of change in maximum CIMT for the common carotid and the common carotid plus bifurcation, respectively. Baseline followed by annual ultrasounds were obtained during a median follow-up of 4.9 years.

RESULTS

Compared with standard care, insulin glargine reduced the primary CIMT outcome, but the difference was not statistically significant (difference = 0.0030 ± 0.0021 mm/year; P = 0.145) and significantly reduced the secondary CIMT outcomes (differences of 0.0033 ± 0.0017 mm/year [P = 0.049] and 0.0045 ± 0.0021 mm/year [P = 0.032], respectively). There were no differences in the primary and secondary outcomes between the n-3FA supplement and placebo groups.

CONCLUSIONS

In people with CV disease and/or CV risk factors and dysglycemia, insulin glargine used to target normoglycemia modestly reduced CIMT progression, whereas daily supplementation with n-3FA had no effect on CIMT progression.Atherosclerosis is the major cause of death and disability in people with type 2 diabetes and lesser degrees of dysglycemia (1,2). Large epidemiological studies show consistent independent associations between glycemia and cardiovascular (CV) risk (1–4), and the metabolic abnormalities associated with dysglycemia promote atherosclerosis (5). Exogenous insulin can provide effective glycemic control, but its effects on atherosclerosis are unknown. Moreover, some studies suggest possible proatherogenic effects (6,7).Essential long-chain n-3 polyunsaturated fatty acids (n-3FA) may have beneficial effects on atherosclerosis (8). Higher intake of fish or n-3FA supplements is associated with lower rates of coronary heart disease and death (9,10) and lower atherosclerotic burden (11,12), and some, but not all, previous trials reported reduced CV events in patients receiving n-3FA supplements (13–16). The effects of these supplements on human atherosclerosis progression were evaluated in a few small studies, which were inconclusive (17–21).Therefore, we evaluated the effects of insulin glargine and n-3FA supplements on carotid intima-media thickness (CIMT) in people with dysglycemia and additional risk factors for atherosclerosis progression in a substudy of the Outcome Reduction with an Initial Glargine Intervention (ORIGIN) trial (22–24).     

Platelet Redox Balance in Diabetic Patients With Hypertension Improved by n-3 Fatty Acids

OBJECTIVE

Patients with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease, largely as a result of defective production of cardioprotective nitric oxide and a concomitant rise in oxidative stress. Dietary interventions that could reverse this trend would be extremely beneficial. Here we investigated whether dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation positively affected platelet nitroso-redox imbalance.

RESEARCH DESIGN AND METHODS

We randomized hypertensive T2DM patients (T2DM HT; n = 22) and age-and-sex matched hypertensive study participants without diabetes (HT alone; n = 23) in a double-blind, crossover fashion to receive 8 weeks of n-3 PUFAs (1.8 g eicosapentaenoic acid and 1.5 g docosahexaenoic acid) or identical olive oil capsules (placebo), with an intervening 8-week washout period. Platelet nitrite and superoxide were measured and compared before and after treatment; 8-isoprostane was determined by ELISA and subcellular compartmentalization of the NAD(P)H oxidase subunit p47-phox examined by Western blotting.

RESULTS

The n-3 PUFA supplementation reduced 8-isoprostane and superoxide levels in platelets from T2DM HT, but not HT alone, participants, without effect on nitrite production. This coincided with a significant decrease in p47-phox membrane localization and a similar reduction in superoxide to that achieved with apocynin. At baseline, a subcohort of T2DM HT and HT alone participants showed evidence of nitric oxide synthase (NOS)–derived superoxide production, indicating defective enzymatic activity. This was reversed significantly in T2DM HT participants after treatment, demonstrating improved NOS function.

CONCLUSIONS

Our finding that n-3 PUFAs diminish platelet superoxide production in T2DM HT patients in vivo suggests a therapeutic role for these agents in reducing the vascular-derived oxidative stress associated with diabetes.Dietary interventions that could reduce the risk of development of cardiovascular disease (CVD) in patients with type 2 diabetes mellitus (T2DM) would clearly be advantageous instead of expensive pharmacological treatments. There is now considerable evidence demonstrating an association between enhanced oxidative stress and the incidence of CVD. In healthy vasculature, oxidative stress is kept in check by nitric oxide (NO) derived from endothelial NO synthase (NOS). Endothelial- and platelet-derived NO plays a pivotal role in maintaining normal vascular homeostasis and has atheroprotective effects, inhibiting smooth muscle cell proliferation, leukocyte adhesion, and aggregation of platelets (1–3). Diabetic vascular disease, in contrast, is characterized by altered NO bioavailability resulting from an increase in the amount of the NO scavenger superoxide (O₂⁻), derived predominantly from NAD(P)H oxidase or from uncoupled endothelial NOS (1,4). Together, these alterations lead to endothelial dysfunction, enhanced platelet activity, and an increased risk of thrombosis (5–7). Despite a large body of evidence linking dysregulated NO production and enhanced oxidative stress to vascular disease, however, clinical studies with well-characterized antioxidant vitamins, such as vitamins C and E, have proved disappointing (8). The inability of antioxidants to be delivered and retained in the vascular bed may explain these results. Moreover, with increasing evidence of the importance of free radicals in normal physiological function, a new paradigm is emerging that favors controlled free radical production. In this model, a therapeutically beneficial rebalancing of NO relative to O₂⁻ production is sought, rather than the complete suppression of free radical production, such as occurs after antioxidant administration (9,10). In this regard, we have recently shown that n-3 polyunsaturated fatty acids (n-3 PUFAs), the major constituents of oily fish, can modulate the nitroso-redox balance by increasing vasculoprotective NO and decreasing O₂⁻ production in endothelial cells (11). This finding may be extremely valuable for diabetic patients whose disease manifests as a high oxidative status. Furthermore, fish oil–derived n-3 PUFAs may also offer several other advantages. Importantly, they are easily absorbed and retained within cellular membranes, where they can act as a readily available supply of free radical–modifying agents. This bioavailability, coupled with their ability to specifically modulate the function of enzymes producing reactive oxygen species (ROS) and reactive nitrogen species, such as NAD(P)H and NOS, suggests that, far from simply being free radical scavengers like other antioxidants, n-3 PUFAs may be therapeutically superior.The health benefits of high oily fish consumption were first recognized in the Greenland Inuit, in whom n-3 PUFA consumption was correlated with a lower CVD mortality rate. Since that discovery, a substantial amount of evidence has accumulated to show that the high content of n-3 PUFAs, particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are responsible for these health benefits. The n-3 PUFAs are believed to exert their therapeutic benefits through a variety of mechanisms, including a reduction in antithrombotic and antiarrhythmic actions and improved endothelial function (12–14). Few studies, however, have investigated the potential benefits of n-3 PUFA treatment for diabetic patients (15–17).Platelets contain all the components of a functioning NO-producing pathway, including the enzyme necessary for the production of the cofactor tetrahydrobiopterin (18,19), and are therefore capable of producing NO in response to platelet-specific agonists (20,21). In addition, they also contain all the subunits required for the assembly of the NAD(P)H oxidase system (7,22). Platelets thus represent an accessible cellular model for functional studies to explore free radical activity in relation to changes in NAD(P)H oxidase and NOS activity (7,20,22). Accordingly, in the current study we examined platelets to investigate the effects of n-3 PUFAs on NO and O₂⁻ bioactivities and on oxidative stress in a diabetic population.     

Microalbuminuria in Type 1 Diabetes Is Associated With Enhanced Excretion of the Endocytic Multiligand Receptors Megalin and Cubilin

OBJECTIVE

Proteinuria is the hallmark of diabetic nephropathy; yet, glomerular histology does not fully explain mechanisms contributing to proteinuria. Our objective was to identify proteins in the urine of individuals with type 1 diabetes and microalbuminuria that might implicate a mechanistic pathway operative in proteinuria.

RESEARCH DESIGN AND METHODS

Using a GeLC/MS platform proteomics approach, we compared the urine proteome from 12 healthy nondiabetic individuals, 12 subjects with type 1 diabetes yet normal urinary albumin excretion rates, and 12 subjects with type 1 diabetes and microalbuminuria (type 1 diabetes + microalbuminuria).

RESULTS

The abundance of megalin and cubilin, two multiligand receptors expressed in kidney proximal tubule cells and involved with the reuptake of filtered albumin and megalin/cubilin ligands, was significantly increased in type 1 diabetes + microalbuminuria urine, compared with both nonalbuminuric groups.

CONCLUSIONS

Aberrant shedding of megalin and cubilin could contribute to albuminuria in diabetes and to deficiency states of important vitamins and hormones.Excess urinary albumin excretion (UAE) (30–299 mg/day), termed microalbuminuria, portends incipient diabetic nephropathy. Mechanisms contributing to proteinuria in diabetic nephropathy are incompletely understood but likely involve pathology within the glomerulus, including endothelial cell injury, glomerular basement membrane thickening, loss of slit diaphragm veracity, and podocytopenia (1). Additionally, in the proximal tubule (PT), decreased protein reabsorption likely occurs (1,2); data from diabetic animals suggest that altered PT handling and diminished albumin retrieval contribute to albuminuria (3,4). Because ∼70% of the urinary proteome originates from kidney or genitourinary tissues (5,6), we used the GeLC/MS platform proteomics approach to compare the urine proteome from 1) nondiabetic individuals, 2) subjects with type 1 diabetes yet normal UAE, and 3) subjects with type 1 diabetes and microalbuminuria (type 1 diabetes + microalbuminuria), so as to identify proteins that might implicate a mechanistic pathway operative in proteinuria.     

A Double-Blind, Randomized, Placebo-Controlled Clinical Trial on Benfotiamine Treatment in Patients With Diabetic Nephropathy

OBJECTIVE

To investigate the effect of benfotiamine on urinary albumin excretion (UAE) and the tubular damage marker kidney injury molecule-1 (KIM-1) in patients with type 2 diabetes and nephropathy.

RESEARCH DESIGN AND METHODS

Patients with type 2 diabetes and UAE equivalent to 15–300 mg/24 h, despite ACE inhibitors (ACE-Is) or angiotensin receptor blockers (ARBs), were randomly assigned to 12 weeks of benfotiamine (900 mg/day) (n = 39) or placebo (n = 43).

RESULTS

Compared with placebo, benfotiamine treatment resulted in significant improvement of thiamine status (P < 0.001). Benfotiamine treatment did not significantly decrease 24-h UAE or 24-h KIM-1 excretion.

CONCLUSIONS

In patients with type 2 diabetes and nephropathy, high-dose benfotiamine treatment for 12 weeks in addition to ACE-Is or ARBs did not reduce UAE or KIM-1 excretion, despite improvement of thiamine status.The pathophysiology of diabetic nephropathy includes albuminuria as a consequence of glomerular endothelial damage and further progression due to tubulointerstitial inflammation and fibrosis (1,2). Despite protective treatment with ACE inhibitors (ACE-Is) and angiotensin receptor blockers (ARBs), many patients progress to end-stage renal disease (3).Thiamine and benfotiamine have been proposed as protective agents for diabetes complications (4,5). Benfotiamine is a lipophilic thiamine derivative with high bioavailability (6). In animal studies, both compounds had beneficial effects on microvascular complications, including diabetic nephropathy (5,7).We investigated whether benfotiamine results in reduction in urinary albumin excretion (UAE) or tubulointerstitial damage markers in patients with type 2 diabetes and increased UAE despite ACE-Is or ARBs.     

Highly Purified Eicosapentaenoic Acid Increases Interleukin-10 Levels of Peripheral Blood Monocytes in Obese Patients With Dyslipidemia

OBJECTIVE

It has recently been highlighted that proinflammatory (M1) macrophages predominate over anti-inflammatory (M2) macrophages in obesity, thereby contributing to obesity-induced adipose inflammation and insulin resistance. A recent clinical trial revealed that highly purified eicosapentaenoic acid (EPA) reduces the incidence of major coronary events. In this study, we examined the effect of EPA on M1/M2-like phenotypes of peripheral blood monocytes in obese dyslipidemic patients.

RESEARCH DESIGN AND METHODS

Peripheral blood monocytes were prepared from 26 obese patients without and 90 obese patients with dyslipidemia. Of the latter 90 obese patients with dyslipidemia, 82 patients were treated with or without EPA treatment (1.8 g daily) for 3 months.

RESULTS

Monocytes in obese patients with dyslipidemia showed a significantly lower expression of interleukin-10 (IL-10), an M2 marker, than those without dyslipidemia. EPA significantly increased serum IL-10 and EPA levels, the EPA/arachidonic acid (AA) ratio, and monocyte IL-10 expression and decreased the pulse wave velocity (PWV), an index of arterial stiffness, compared with the control group. After EPA treatment, the serum EPA/AA ratio was significantly correlated with monocyte IL-10 expression. Only increases in monocyte IL-10 expression and serum adiponectin were independent determinants of a decreased PWV by EPA. Furthermore, EPA significantly increased the expression and secretion of IL-10 in human monocytic THP-1 cells through a peroxisome proliferator–activated receptor (PPAR)γ-dependent pathway.

CONCLUSIONS

This study is the first to show that EPA increases the monocyte IL-10 expression in parallel with decrease of arterial stiffness, which may contribute to the antiatherogenic effect of EPA in obese dyslipidemic patients.The monocyte–macrophage system plays a role in the pathogenesis of obesity and atherosclerotic disease (1,2). This system shows at least two distinct phenotypes of differentiation: proinflammatory (M1) and anti-inflammatory (M2) (3). It has been reported that, in obese adipose tissue, macrophage accumulation is increased, and proinflammatory M1 macrophages predominate over anti-inflammatory M2 macrophages, thereby contributing to obesity-induced adipose inflammation and insulin resistance (4–6). The expression of both M1 and M2 markers is detected in peripheral blood mononuclear cells and even in atherosclerotic plaques (7,8). We and others also provided evidence for the inflammatory state and unfavorable M1/M2-like phenotypes of peripheral blood monocytes in obese diabetic patients (9,10). In particular, interleukin-10 (IL-10), an anti-inflammatory cytokine and M2 marker, might be involved in M2 macrophage recruitment, thus contributing to reducing inflammation and improving the insulin signal (5,11).In epidemiological and clinical trials, fish oil and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) were found to reduce the incidence of coronary heart disease (12). A large-scale, prospective, randomized clinical trial, the Japan Eicosapentaenoic Acid Lipid Intervention Study (JELIS), demonstrated that highly purified eicosapentaenoic acid (EPA), a specific n-3 PUFA used clinically to treat dyslipidemia, significantly reduces the incidence of major coronary events via cholesterol-independent mechanisms (13). As antiatherogenic effects, we previously demonstrated that EPA reduces atherogenic lipoproteins and C-reactive protein (CRP), an inflammatory marker, as well as the pulse wave velocity (PWV), an index of arterial stiffness, and increases the secretion of adiponectin, the only established adipocytokine with anti-inflammatory and antiatherogenic properties, in obese patients (14–16). We also reported that EPA markedly inhibits LPS-induced monocyte adhesion to the aortic endothelium in parallel with the suppression of endothelial adhesion molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 (17). Several studies showed that dietary n-3 PUFAs and EPA inhibit the ability of macrophages to secrete several effector molecules that may be involved in the pathogenesis of atherosclerosis (18,19). Given these protective effects of n-3 PUFAs and EPA on the monocyte–macrophage system (17–19), it is tempting to speculate on the beneficial effect of EPA on the M1/M2-like phenotypes of peripheral blood monocytes in obese patients during the progression of atherosclerosis; however, no direct evidence for such an effect of EPA has been established. EPA can be metabolized to anti-inflammatory eicosanoids and also competitively inhibits the production of arachidonic acid (AA), an n-6 PUFA, and inflammatory eicosanoids derived from AA, which is the precursor of important molecules involved in inflammation and atherosclerotic process (20). Subanalysis of JELIS and other studies suggested that a decreased serum EPA/AA ratio is significantly associated with the incidence of cardiac death and myocardial infarction and the coronary plaque score (21,22).In this study, we demonstrate for the first time that EPA increased IL-10 RNA expression in peripheral blood monocytes of obese patients with dyslipidemia in parallel with the decrease of arterial stiffness. In addition, the serum EPA/AA ratio after EPA treatment was significantly correlated with IL-10 RNA expression of monocytes. Furthermore, EPA enhanced the expression level of IL-10 RNA through peroxisome proliferator–activated receptor (PPAR)γ in vitro. As EPA reduced the risk of major coronary events in a large-scale, prospective, randomized clinical trial (13), this study provides important insight into its therapeutic implications for obesity-related metabolic sequelae and cardiovascular disease.     

Long-Term Safety and Efficacy of Empagliflozin,Sitagliptin, and Metformin: An active-controlled,parallel-group,randomized, 78-week open-label extension study in patients with type 2 diabetes

OBJECTIVE

To investigate the long-term safety and efficacy of empagliflozin, a sodium glucose cotransporter 2 inhibitor; sitagliptin; and metformin in patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS

In this randomized, open-label, 78-week extension study of two 12-week, blinded, dose-finding studies of empagliflozin (monotherapy and add-on to metformin) with open-label comparators, 272 patients received 10 mg empagliflozin (166 as add-on to metformin), 275 received 25 mg empagliflozin (166 as add-on to metformin), 56 patients received metformin, and 56 patients received sitagliptin as add-on to metformin.

RESULTS

Changes from baseline in HbA_1c at week 90 were −0.34 to −0.63% (−3.7 to −6.9 mmol/mol) with empagliflozin, −0.56% (−6.1 mmol/mol) with metformin, and −0.40% (−4.4 mmol/mol) with sitagliptin. Changes from baseline in weight at week 90 were −2.2 to −4.0 kg with empagliflozin, −1.3 kg with metformin, and −0.4 kg with sitagliptin. Adverse events (AEs) were reported in 63.2–74.1% of patients on empagliflozin and 69.6% on metformin or sitagliptin; most AEs were mild or moderate in intensity. Hypoglycemic events were rare in all treatment groups, and none required assistance. AEs consistent with genital infections were reported in 3.0–5.5% of patients on empagliflozin, 1.8% on metformin, and none on sitagliptin. AEs consistent with urinary tract infections were reported in 3.8–12.7% of patients on empagliflozin, 3.6% on metformin, and 12.5% on sitagliptin.

CONCLUSIONS

Long-term empagliflozin treatment provided sustained glycemic and weight control and was well tolerated with a low risk of hypoglycemia in patients with type 2 diabetes.Type 2 diabetes is characterized by insulin resistance and progressive deterioration of β-cell function (1). Metformin is the recommended first-line antidiabetes agent for patients with type 2 diabetes (2). However, in order to achieve and maintain glycemic control as the disease progresses, patients often require therapies in addition to metformin (2,3).Despite the availability of a number of antihyperglycemic agents, the side effects associated with existing treatments and their gradual loss of efficacy over time (2,3) mean that many patients with type 2 diabetes do not reach therapeutic goals (3,4). In addition, treatment is often complicated by common comorbidities of type 2 diabetes such as obesity and hypertension, which are not addressed by existing oral antidiabetes agents (5–7).Inhibition of sodium glucose cotransporter 2 (SGLT2), located in the proximal tubule of the kidney, represents an approach for the treatment of type 2 diabetes that is independent of β-cell function and insulin resistance (8,9). SGLT2 mediates most of renal glucose reabsorption, and inhibition of this transporter leads to reduced reabsorption of filtered glucose and increased urinary glucose excretion (8,10), resulting in reduced plasma glucose levels in patients with type 2 diabetes (8–10). In addition, this mechanism leads to weight loss owing to the loss of calories via urinary glucose excretion (8,11).Empagliflozin is a potent and selective inhibitor of SGLT2 (12), which in patients with type 2 diabetes causes urinary glucose excretion of up to 90 g/day (13). In two placebo- and active-controlled, dose-finding trials, treatment with empagliflozin for 12 weeks in patients with type 2 diabetes was generally well tolerated and resulted in placebo-corrected reductions in HbA_1c of up to 0.72% (7.9 mmol/mol) and placebo-corrected reductions in weight of up to 1.7 kg (14,15). In these studies, reductions in HbA_1c were comparable to those of the active comparators metformin and sitagliptin (14,15). The objective of this study was to assess the long-term safety and efficacy of empagliflozin, sitagliptin, and metformin in a 78-week, open-label extension study of two dose-finding trials.     

TRAF-interacting Protein (TRIP): A Novel Component of the Tumor Necrosis Factor Receptor (TNFR)- and CD30-TRAF Signaling Complexes That Inhibits TRAF2-mediated NF-κB Activation

Through their interaction with the TNF receptor–associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and characterized a novel component of the receptor–TRAF signaling complex, designated TRIP (TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-κB activation that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a receptor–proximal regulator that may influence signals responsible for cell activation/proliferation and cell death induced by members of the TNFR superfamily.Members of the TNF receptor (TNFR)¹ superfamily play important roles in the induction of diverse signals leading to cell growth, activation, and apoptosis (1). Whether the signals induced by a given receptor leads to cell activation or death is, however, highly cell-type specific and tightly regulated during differentiation of cells. For example, the TNFRs can exert costimulatory signals for proliferation of naive lymphocytes but also induce death signals required for deletion of activated T lymphocytes (1). The cytoplasmic domains of these receptors lack intrinsic catalytic activity and also exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNFR1 that share a significant homology within an 80–amino acid region of their cytoplasmic tails (called the “death domain”; 2, 3). Therefore, it is suggested that the TNFR family members can initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor complex (1).Indeed, several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily (4–19). Recent biochemical and molecular studies showed that a class of signal-transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains (2, 3, 6, 12, 17, 20). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD (MORT1)/RIP through the interactions of their respective death domains (2, 3, 6, 12, 17, 20, 21). Clustering of these signal transducers leads to the recruitment of FLICE/ MACH, and subsequently, to cell death (13, 14).The TNFR family members can also recruit a second class of signal transducers called TRAFs (TNFR-associated factor), some of which are responsible for the activation of NF-κB or JNK (9, 20, 22). TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30, or LT-βR (4, 5, 10, 11, 15, 23–27). These receptors interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails, but do not interact with the death domain containing proteins (4, 5, 15, 24–27). To date, five members of the TRAF family have been identified as signaling components of the TNFR family members. All TRAF members contain a conserved TRAF domain, ∼230 amino acids in length, that is used for either homo- or heterooligomerization among the TRAF family, for interactions with the cytoplasmic regions of the TNFR superfamily, or for interactions with downstream signal transducers (4, 5, 8, 10, 11, 19, 23–25, 28). In addition to the TRAF domain, most of the TRAF family members contain an NH₂-terminal RING finger and several zinc finger structures, which appear to be important for their effector functions (4, 5, 10, 11, 23–25).Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2 (4), was subsequently shown to mediate NF-κB activation induced by two TNF receptors, CD40 and CD30 (9, 28–30). TRAF5 was also implicated in NF-κB activation mediated by LTβR (10), whereas TRAF3 (also known as CRAF1, CD40bp, or LAP1; references 5, 11, 24, and 25) was shown to be involved in the regulation of CD40-mediated CD23 upregulation in B cells (5). The role of other TRAF members in the TNFR family–mediated signal transduction is not clear. They may possess some effector functions as yet to be revealed, or work as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the cellular inhibitor of apoptosis protein (c-IAP) family to the TNFR2-signaling complex (7). In addition to the signal transduction by the TNFR family members, TRAFs may regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of IL-1 receptor (IL1R)–signaling complex, in which it mediates the activation of NF-κB by IL-1R (31). Since TRAFs form homo- or heterooligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers. Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, it needs to be determined whether TRAF proteins will also mediate the apoptotic signals induced by the “death-domain-less” members of the TNFR superfamily (1, 27, 32–36).Here we report the isolation and characterization of a novel component of the TNFR superfamily/TRAFs signaling complex, named TRIP (TRAF-interacting protein). TRIP associates with the receptor/TRAF signaling complex, and inhibits the TRAF2-mediated NF-κB activation. Biochemical studies indicate that TRIP associates with the TNFR2 or CD30 receptor complex via its interaction with TRAF proteins, suggesting a model which can explain why the ligation of these receptors can promote different cell fates: proliferation or death.     

Urinary Liver-Type Fatty Acid–Binding Protein and Progression of Diabetic Nephropathy in Type 1 Diabetes

OBJECTIVE

Diabetic nephropathy (DN) has mainly been considered a glomerular disease, although tubular dysfunction may also play a role. This study assessed the predictive value for progression of a tubular marker, urinary liver-type fatty acid–binding protein (L-FABP), at all stages of DN.

RESEARCH DESIGN AND METHODS

At baseline, 1,549 patients with type 1 diabetes had an albumin excretion rate (AER) within normal reference ranges, 334 had microalbuminuria, and 363 had macroalbuminuria. Patients were monitored for a median of 5.8 years (95% CI 5.7–5.9). In addition, 208 nondiabetic subjects were studied. L-FABP was measured by ELISA and normalized with urinary creatinine. Different Cox proportional hazard models for the progression at every stage of DN were used to evaluate the predictive value of L-FABP. The potential benefit of using L-FABP alone or together with AER was assessed by receiver operating characteristic curve analyses.

RESULTS

L-FABP was an independent predictor of progression at all stages of DN. As would be expected, receiver operating characteristic curves for the prediction of progression were significantly larger for AER than for L-FABP, except for patients with baseline macroalbuminuria, in whom the areas were similar. Adding L-FABP to AER in the models did not significantly improve risk prediction of progression in favor of the combination of L-FABP plus AER compared with AER alone.

CONCLUSIONS

L-FABP is an independent predictor of progression of DN irrespective of disease stage. L-FABP used alone or together with AER may not improve the risk prediction of DN progression in patients with type 1 diabetes, but further studies are needed in this regard.Diabetic nephropathy (DN) affects ∼30% of all patients with type 1 diabetes. It is also the most severe diabetes complication because it is associated with progression to end-stage renal disease (ESRD) and a high risk of premature death (1,2).Early screening and detection is essential for the prevention of DN and is currently based on the measurement of the urinary albumin excretion rate (AER) (3). An increased AER is regarded as a marker of glomerular injury, and its early diagnosis makes intervention possible before renal function starts to decline, as reflected by an impaired glomerular filtration rate (GFR). However, AER has some limitations, at both the early and the late stages of disease (4–6).Although DN has long been considered a glomerular disease, tubulointerstitial injury has also been demonstrated to play a role in the pathogenesis (7). In this context, it is attractive to study molecules that are linked to tubular dysfunction. These molecules may serve as potential new markers for DN and may also provide additional information about clinical course or prognosis that may enable an earlier diagnosis and means to better tailor the treatment.Urinary liver-type fatty acid–binding protein (L-FABP) is mainly regarded as a urinary tubular biomarker associated with structural and functional kidney damage (8). Urinary levels of L-FABP are not influenced by its serum levels because urinary L-FABP originates mainly from the tubular cells (9). This biomarker is elevated in the early stages of diabetes but is also influenced by lipid-lowering medication and angiotensin II receptor antagonists (10–12). Urinary L-FABP predicts adverse outcomes in acute kidney injury and progression of chronic kidney disease of nondiabetic causes (13–15). It is of note that urinary L-FABP has been linked to DN in patients with type 2 diabetes and has furthermore been suggested to be a predictor of progression to microalbuminuria in patients with type 1 diabetes (16,17). However, whether L-FABP would be a more sensitive marker of DN than AER or whether its predictive role is solely confined to the progression of the disease process is not yet known. Therefore, the aim of the current study is to investigate if baseline levels of L-FABP predict the development of DN and its progression at any stage of the disease and if the use of L-FABP alone or together with AER adds a benefit compared with current standard testing by AER.     

Significance of urinary full-length and ectodomain forms of megalin in patients with type 2 diabetes

OBJECTIVE

Megalin, an endocytic receptor in proximal tubule cells, is involved in the mechanisms of albuminuria in diabetic nephropathy (DN). To develop efficient novel biomarkers associated with the pathogenesis of DN, we investigated urinary megalin excretion in type 2 diabetes.

RESEARCH DESIGN AND METHODS

Sandwich enzyme-linked immunosorbent assay systems were established with monoclonal antibodies against the NH₂ (amino [A]-megalin assay) and COOH (C-megalin assay) termini of megalin to analyze urinary forms of megalin in 68 patients with type 2 diabetes.

RESULTS

The A-megalin assay mainly detected a megalin ectodomain form in the soluble urinary fraction, whereas the C-megalin assay identified a full-length form in both soluble and insoluble fractions. Urinary C-megalin levels were significantly high in patients with normoalbuminuria, were elevated in line with increased albuminuria, and showed a better association with estimated glomerular filtration rate (eGFR) (<60 mL/min/1.73 m²) than did urinary albumin. In contrast, urinary A-megalin levels were increased in patients with normo- and microalbuminuria but not in those with macroalbuminuria. Urinary C-megalin levels were also positively associated with plasma inorganic phosphate and negatively with hemoglobin levels in those showing no features of bleeding and not taking vitamin D analogs, phosphate binders, or erythropoiesis-stimulating agents.

CONCLUSIONS

Urinary full-length megalin excretion as measured by the C-megalin assay is well associated with reduced eGFR and linked to the severity of DN, phosphate dysregulation, and anemia, whereas urinary excretion of megalin ectodomain as measured by the A-megalin assay may be associated with distinctive mechanisms of earlier DN in type 2 diabetes.Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD) and end-stage kidney disease and is highly associated with the development of cardiovascular disease (CVD) (1). Albuminuria has been generally used as a biomarker for DN, but its clinical relevance as a surrogate outcome in CKD has not been confirmed (2).Phenotypic changes in proximal tubule cells (PTCs) are initial signs of DN (3,4), and consequent tubulointerstitial damage plays a central role in the progression to end-stage kidney disease (5). Furthermore, PTC dysfunction is likely to be involved in the development of CVD in diabetes (6). However, efficient biomarkers associated with the mechanism of PTC dysfunction in diabetes have not been established.Megalin is a large (∼600 kDa) glycoprotein member of the LDL receptor family (7,8) that is expressed at the apical membranes of PTCs (9). Its extracellular region consists of 4,398 amino acids (in humans) and consists of four clusters of cysteine-rich repeats, the so-called ligand-binding domains (LBDs) 1–4. The extracellular region is followed by a single transmembrane segment and a cytoplasmic tail (CT) of 209 amino acids. Megalin plays a critical role in the reabsorption of glomerular-filtered substances, including albumin and low-molecular-weight proteins (9). In addition, megalin may be involved in signal transduction via regulated intramembrane proteolysis (RIP) in PTCs (10,11).Impairment of megalin function has been suggested even in the early stages of diabetes in animal models (12,13) and patients (14–16). Therefore, dysfunction of megalin is likely to be associated with the development of proteinuria/albuminuria in diabetic patients.Megalin is known to be shed into urine, as was first shown by immunoblotting of human urine samples (17). Increased urinary megalin excretion, evaluated by gel-based liquid chromatography–mass spectrometry, was suggested in microalbuminuric patients with type 1 diabetes (18). However, more qualitative and quantitative analyses of urinary megalin excretion are required to clarify its association with the pathogenesis of DN and its usefulness as a biomarker.In this study, we therefore developed enzyme-linked immunosorbent assay (ELISA) systems to investigate urinary megalin forms and quantities and evaluate their clinical significance as novel biomarkers for DN and related disorders in patients with type 2 diabetes.     

Association Between Urinary Type IV Collagen Level and Deterioration of Renal Function in Type 2 Diabetic Patients Without Overt Proteinuria

OBJECTIVE

Cross-sectional studies have reported increased levels of urinary type IV collagen in diabetic patients with progression of diabetic nephropathy. The aim of this study was to determine the role of urinary type IV collagen in predicting development and progression of early diabetic nephropathy and deterioration of renal function in a longitudinal study.

RESEARCH DESIGN AND METHODS

Japanese patients with type 2 diabetes (n = 254, 185 with normoalbuminuria and 69 with microalbuminuria) were enrolled in an observational follow-up study. The associations of urinary type IV collagen with progression of nephropathy and annual decline in estimated glomerular filtration rate (eGFR) were evaluated.

RESULTS

At baseline, urinary type IV collagen levels were higher in patients with microalbuminuria than in those with normoalbuminuria and correlated with urinary β₂-microglobulin (β = 0.57, P < 0.001), diastolic blood pressure (β = 0.15, P < 0.01), eGFR (β = 0.15, P < 0.01), and urinary albumin excretion rate (β = 0.13, P = 0.01) as determined by multivariate regression analysis. In the follow-up study (median duration 8 years), urinary type IV collagen level at baseline was not associated with progression to a higher stage of diabetic nephropathy. However, the level of urinary type IV collagen inversely correlated with the annual decline in eGFR (γ = −0.34, P < 0.001). Multivariate regression analysis identified urinary type IV collagen, eGFR at baseline, and hypertension as factors associated with the annual decline in eGFR.

CONCLUSIONS

Our results indicate that high urinary excretion of type IV collagen is associated with deterioration of renal function in type 2 diabetic patients without overt proteinuria.Diabetic nephropathy is characterized structurally by accumulation of mesangial matrix and thickening of the basement membrane in the glomeruli (1), as well as renal tubular hypertrophy and associated basement membrane alterations in the tubulointerstitium, which precede tubulointerstitial fibrosis (2). These abnormalities are associated with renal overproduction of the extracellular matrix proteins such as type IV collagen (3). We and others reported previously that the production of type IV collagen is enhanced in glomerular mesangial cells (4,5), podocytes (6), and proximal tubular cells (7,8) cultured under a high-glucose condition. Cross-sectional studies reported a stepwise increase in urinary type IV collagen excretion in diabetic patients, in parallel with increased levels of urinary albumin (9–14). Thus, a high level of urinary type IV collagen has been proposed as a marker of development and progression of early diabetic kidney disease. However, there is little information on whether the increase in type IV collagen excretion in urine is a predictor of progression of diabetic nephropathy or deterioration of renal function in type 2 diabetic patients.The aim of this study was to determine whether the urinary levels of type IV collagen can predict the progression of early diabetic kidney disease. For this purpose, we conducted a prospective observational follow-up study involving type 2 diabetic patients with normoalbuminuria and microalbuminuria and investigated the relationship between urinary levels of type IV collagen and the progression of diabetic nephropathy stage as well as the annual decline in estimated glomerular filtration rate (eGFR) during the follow-up period.     

The Acute Effect of Clamped Hyperglycemia on the Urinary Excretion of Inflammatory Cytokines/Chemokines in Uncomplicated Type 1 Diabetes: A pilot study

OBJECTIVE

Acute glycemic variability contributes to diabetic complications potentially through induction of inflammation. Our objective was to determine whether acute hyperglycemia affects urinary secretion of inflammatory cytokines/chemokines in humans with uncomplicated type 1 diabetes.

RESEARCH DESIGN AND METHODS

Blood pressure, renal hemodynamics (inulin and paraaminohippurate clearances), and urine samples were obtained after 6 h of clamped euglycemia (4–6 mmol/l) and hyperglycemia (9–11 mmol/l) on two consecutive days in subjects with type 1 diabetes (n = 25). Forty-two urinary cytokines/chemokines were measured using a Luminex platform.

RESULTS

Clamped hyperglycemia produced an expected increase in glomerular filtration rate (131 ± 4 to 148 ± 8 ml/min/1.73 m²). Clamped hyperglycemia was associated with significant increases in urinary eotaxin, fibroblast growth factor-2, granulocyte-macrophage colony-stimulating factor, interferon-α 2, interleukin-2 and -12, monocyte chemoattractant protein-3, macrophage-derived chemokine, macrophage inflammatory protein-1α, platelet-derived growth factor, tumor necrosis factor-α, and CD40 ligand (P < 0.05).

CONCLUSIONS

Acute hyperglycemia results in increased urinary excretion of inflammatory cytokines/chemokines in humans with uncomplicated type 1 diabetes, and this may contribute to kidney injury.Emerging evidence suggests that acute glycemic excursions may significantly contribute to microvascular end-organ injury in patients with diabetes, independent of long-term glycemic control (1). While the mechanisms accounting for this observation are not completely understood, evidence suggests that acute hyperglycemia affects systemic and renal microvascular hemodynamic function and activates systemic inflammatory pathways (2–6). The effect of acute clamped hyperglycemia on renal inflammatory mediators, however, is not completely understood (7–9). Accordingly, our goal was to characterize the earliest effects of acute hyperglycemia on the activation of renal inflammatory pathways in patients with uncomplicated type 1 diabetes in order to better understand how acute glycemic excursions may independently contribute to long-term kidney injury.     

Adverse Outcomes After Noncardiac Surgery in Patients With Diabetes: A nationwide population-based retrospective cohort study

OBJECTIVE

To investigate whether diabetes affects perioperative complications or mortality and to gauge its impact on medical expenditures for noncardiac surgeries.

RESEARCH DESIGN AND METHODS

With the use of reimbursement claims from the Taiwan National Health Insurance system, we performed a population-based cohort study of patients with and without diabetes undergoing noncardiac surgeries. Outcomes of postoperative complications, mortality, hospital stay, and medical expenditures were compared between patients with and without diabetes.

RESULTS

Diabetes increased 30-day postoperative mortality (odds ratio 1.84 [95% CI 1.46–2.32]), particularly among patients with type 1 diabetes or uncontrolled diabetes and patients with preoperative diabetes-related comorbidities, such as eye involvement, peripheral circulatory disorders, ketoacidosis, renal manifestations, and coma. Compared with nondiabetic control patients, coexisting medical conditions, such as renal dialysis (5.17 [3.68–7.28]), liver cirrhosis (3.59 [2.19–5.88]), stroke (2.87 [1.95–4.22]), mental disorders (2.35 [1.71–3.24]), ischemic heart disease (2.08 [1.45–2.99]), chronic obstructive pulmonary disease (1.96 [1.29–2.97]), and hyperlipidemia (1.94 [1.01–3.76]) were associated with mortality for patients with diabetes undergoing noncardiac surgery. Patients with diabetes faced a higher risk of postoperative acute renal failure (3.59 [2.88–4.48]) and acute myocardial infarction (3.65 [2.43–5.49]). Furthermore, diabetes was associated with prolonged hospital stay (2.30 [2.16–2.44]) and increased medical expenditures (1.32 [1.25–1.40]).

CONCLUSIONS

Diabetes increases postoperative 30-day mortality, complications, and medical expenditures in patients undergoing in-hospital noncardiac surgeries.Diabetes is a common chronic disease that causes widespread disability and death, with a global prevalence of 2.8% in 2000 and an estimated prevalence of 4.4% in 2030 (1). In the U.S., the national burden of diabetes was estimated to be $245 billion in 2012 (2). The epidemiology, pathogenesis, prevention, and treatment of diabetes have been well established over the past 2 centuries (3).Diabetes is an independent determinant of increased risk of perioperative complications and mortality in cardiovascular surgeries (4,5), yet how extensively diabetes affects postoperative mortality and complications in noncardiac surgeries has not been determined. Some studies indicated that survival outcomes and perioperative complications in noncardiac surgeries do not differ between patients with and without diabetes (6,7), whereas other research showed conflicting data about whether diabetes increased perioperative complications, mortality, hospital stay, and health care expenditures (8–16).Previous studies were limited by several factors, including a focus on a single type of noncardiac surgery (6,8,10,12,14), small sample size (6,7,9,13), inappropriate selection of nondiabetes control subjects (6–16), inadequate adjustment for potential confounders (7,9–12,15), and reporting of a single outcome after surgery (10,16). It remains unclear whether coexisting medical conditions, types of diabetes, glycemic control, and diabetes-related comorbidities affect postoperative outcomes in patients with diabetes.This study used Taiwan National Health Insurance Program reimbursement claims to investigate postoperative complications, 30-day mortality, length of hospital stay, and medical expenditures after adjustment by propensity score-matched pair method in patients with diabetes undergoing noncardiac surgeries. We also investigated the impact of coexisting medical conditions and diabetes-related comorbidities on postoperative 30-day mortality among patients with diabetes.     

Eubaric hyperoxia: controversies in the management of acute traumatic brain injury

Controversy exists on the role of hyperoxia in major trauma with brain injury. Hyperoxia on arterial blood gas has been associated with acute lung injury and pulmonary complications, impacting clinical outcome. The hyperoxia could be reflective of the physiological interventions following major systemic trauma. Despite the standard resuscitation of patients with acute traumatic brain injury, up to 60% demonstrate low brain oxygen upon admission to the ICU. While eubaric hyperoxia has been beneficial in experimental studies, clinical brain oxygen protocols incorporating intracranial pressure control, maintenance of cerebral perfusion pressure, and the effective use of fraction of inspired oxygen adjustments to maintain cerebral oxygenation levels >20 to 25 mmHg have demonstrated mortality reductions and improved clinical outcomes. The risk of low brain oxygen is most acute in the first 24 to 48 hours after injury. The administration of a high fraction of inspired oxygen (0.6 to 1.0) in the emergency room may be justifiable until ICU admission for the placement of invasive neurocritical care monitoring systems. Thereafter, fraction of inspired oxygen levels need to be careful titrated to prevent low brain oxygen levels.One of the major advantages of bureaucratic institutionalized national healthcare systems is the ability to analyze treatment pdigms implemented across a wide population, providing a comprehensive overview of clinical care with the view to improve quality and outcome. In a retrospective interrogation of a prospectively collected database, Raj and colleagues demonstrate that the presence of arterial hyperoxia within the first 24 hours is not a predictive marker of neurological outcome in moderate to severe traumatic brain injury (TBI) [1].The human has evolved into a complex adaptive system that is dynamic, exhibiting nonlinear relationships, and is acutely affected by many physiological systems [2]. Although trauma scoring systems (for example, injury severity score, revised trauma score and probability of survival) or physiological scoring methods (for example, Acute Physiology and Chronic Health Evaluation) employ a multitude of pmeters, the use of a single physiological pmeter – for example, arterial hyperoxia [1] – is not predictive for outcome. The hyperoxia detected could be reflective of the therapeutic intervention associated with major trauma, and the subsequent treatment and clinical course would vary after the initial arterial blood oxygen measurement since <50% of patients underwent intracranial pressure monitoring [1]. Even when intracranial pressure monitoring is implemented, a high mortality is observed. Rockswold and colleagues reported a mortality of 42% in the control group for severe TBI, similar to the publication by Raj and colleagues (39%) [1,3], but the addition of hyperoxia results in a significant relative risk reduction for mortality [3], similar to other published studies [4,5].Early initiation of brain oxygen protocols – that is, intracranial pressure/cerebral perfusion pressure/partial pressure of brain tissue oxygen (PbtO₂) [5-7] and PbtO₂ critical care guide [4,8-10] – have shown mortality reductions [4,5] and improved patient neurological outcome [4], in contrast to delayed implementation, which may not translate into outcome benefits [11]. The addition of oxygen as a therapeutic tool would necessitate multimodality invasive neurological monitoring in all patients with severe TBI since the fraction of inspired oxygen and airway pressures (that is, positive end-expiratory pressure or pressure support) would need to be carefully titrated against PbtO₂ levels.The deleterious effects of eubaric hyperoxia (that is, adult respiratory distress syndrome, atelectasis, ventilation/perfusion mismatch) have been postulated, which may be due to concomitant pulmonary contusion, aspiration or even high ventilator pressures [12], but have not been substantiated by clinical experience [3-5,9]. Indeed, eubaric hyperoxia – needed primarily in the first 48 hours [4] – immediately improves PbtO₂, reducing lactate levels and infarct size, and exhibits neuroprotective properties [13,14]. High airway pressure of barotraumas, previously implicated in lung injury [12], has been refuted by Rockswold and colleagues who demonstrated the safety and efficacy of eubaric and hyperbaric oxygen therapy in the management of acute TBI in a randomized control study. Specifically, bronchial alveolar lavage revealed no differences in interleukin levels and, rather than toxicity, microdialysis data revealed the neuroprotective effects of oxygen in both the normal and the injured brain [3].In conclusion, one has to differentiate between hyperoxia (detected in laboratory tests) with its potential association with clinical outcome, and the use of eubaric or hyperbaric oxygen as a therapeutic tool. In the former, a single episode of hyperoxia may have shown an association with mortality as reported in this study [1], but it would rarely prove causality. In the latter, the high therapy intensity level employed with brain oxygen protocols, of which eubaric hyperoxia is critical, requires invasive monitoring with careful goal-directed oxygen titration. The emerging clinical experience demonstrates that hyperoxia is safe and beneficial to the brain, and does not injure the lung as previously feared [3].

Abbreviations

PbtO2: Partial pressure of brain tissue oxygen; TBI: Traumatic brain injury

Competing interests

PKN served as a scientific advisor for Integra Neurosciences, Plainsboro, NJ, USA, who manufactured the neuromonitoring devices between 2001 and 2010.     

The association between dietary sodium intake, ESRD, and all-cause mortality in patients with type 1 diabetes

OBJECTIVE

Many guidelines recommend reduced consumption of salt in patients with type 1 diabetes, but it is unclear whether dietary sodium intake is associated with mortality and end-stage renal disease (ESRD).

RESEARCH DESIGN AND METHODS

In a nationwide multicenter study (the FinnDiane Study) between 1998 and 2002, 2,807 enrolled adults with type 1 diabetes without ESRD were prospectively followed. Baseline urinary sodium excretion was estimated on a 24-h urine collection. The predictors of all-cause mortality and ESRD were determined by Cox regression and competing risk modeling, respectively.

RESULTS

The median follow-up for survival analyses was 10 years, during which 217 deaths were recorded (7.7%). Urinary sodium excretion was nonlinearly associated with all-cause mortality, such that individuals with the highest daily urinary sodium excretion, as well as the lowest excretion, had reduced survival. This association was independent age, sex, duration of diabetes, the presence and severity of chronic kidney disease (CKD) (estimated glomerular filtration rate [eGFR] and log albumin excretion rate), the presence of established cardiovascular disease, and systolic blood pressure. During follow-up, 126 patients developed ESRD (4.5%). Urinary sodium excretion was inversely associated with the cumulative incidence of ESRD, such that individuals with the lowest sodium excretion had the highest cumulative incidence of ESRD.

CONCLUSIONS

In patients with type 1 diabetes, sodium was independently associated with all-cause mortality and ESRD. Although we have not demonstrated causality, these findings support the calls for caution before applying salt restriction universally. Clinical trials must be performed in diabetic patients to formally test the utility/risk of sodium restriction in this setting.Blood pressure control is a key target for the prevention and management of the complications of type 1 diabetes. To this end, many treatment guidelines have promoted the potential utility of dietary salt restriction in patients with diabetes (1,2), as a means to reduce blood pressure levels and, with it, potentially modify the risk and severity of complications. However, any restriction in dietary sodium intake is also associated with activation of the sympathetic nervous system (3) and the renin-angiotensin-aldosterone system (4) and increased LDL cholesterol (4). Some studies have also suggested that salt restriction reduces insulin sensitivity in patients with type 2 diabetes (5) and healthy human volunteers (6). In the context of type 1 diabetes, and especially in patients with chronic kidney disease (CKD) who have the greatest risk of adverse outcomes (7,8), activation of these pathways may offset or overshadow any gains achieved from modest and transient blood pressure lowering. Hence, in this study we aimed to determine the association between dietary salt intake and 1) all-cause mortality and 2) end-stage renal disease (ESRD) in 2,807 patients with type 1 diabetes from the large nationwide multicenter cohort of Finnish adults with type 1 diabetes (the Finnish Diabetic Nephropathy [FinnDiane] Study) (7,9).     

A New Mechanism of NK Cell Cytotoxicity Activation: The CD40–CD40 Ligand Interaction

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2–activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBLderived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2–activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.NK cells represent a distinct lineage of lymphocytes that are able to kill a variety of tumor (1), virus-infected (2), bone marrow transplanted (3), and allogeneic target cells (4). NK cells do not express T cell receptors or immunoglobulins and are apparently normal in mice with defects in the recombinase machinery (5, 6).Our knowledge about NK cell specificity has increased considerably in the last years. NK cells can probably interact with target cells by a variety of different cell surface molecules, some involved in cell adhesion, some activating the NK cytolytic program (7, 8), and other ones able to inhibit this activation by negative signaling (as reviewed in reference 9).A common feature of several inhibitory NK receptors is the capability to bind MHC class I molecules (10, 11), as predicted by the effector inhibition model within the missing self hypothesis of recognition by NK cells (12–14). Interestingly, the MHC class I receptors identified so far belong to different gene families in mouse and man; these are the p58/p70/NKAT or killer cell inhibitory receptors (KIR)¹ of the immunoglobulin superfamily in man and the Ly49 receptors of the C-type lectin family in the mouse. There is also evidence that MHC class I molecules can be recognized as triggering signals in NK cells of humans, rats as well as mice (13). The inhibitory receptors allow NK cells to kill tumor or normal cell targets with deficient MHC class I expression (12, 14). This does not exclude that other activating pathways can override inhibition by MHC class I molecules (15) and, even in their absence, there must be some activating target molecules that initiate the cytolytic program. Several surface molecules are able to mediate positive signals in NK cells. Some of these structures, like NKRP1 (16), CD69 (17), and NKG2 (18) map to the NK complex region (NKC) of chromosome 6 in mice and of chromosome 12 in humans (13). CD2 (19) and CD16 (20) molecules can also play a role in the activation pathway.NK cells resemble T cells in many respects, both may arise from an immediate common progenitor (21, 22), and share the expression of several surface molecules (23). NK cells produce cytokines resembling those secreted by some helper T cell subsets (24) and contain CD3 components in the cytoplasm (21). The expression of some surface structures, involved in TCR-dependent T cell costimulation, like CD28 in human (25), has been described on NK cells, but the functional relevance of these molecules for NK activation processes has not been fully established.Another T cell molecule of interest is CD40L, which interacts with CD40, a 50-kD membrane glycoprotein expressed on B cells (26), dendritic cells (27), and monocytes (28). CD40 is a member of the tumor necrosis factor/nerve growth factor receptor family (29) which includes CD27 (30), CD30 (31), and FAS antigen (32). Murine and human forms of CD40L had been cloned and found to be membrane glycoproteins with a molecular mass of ∼39 kD induced on T cells after activation (33). Also mast cells (34), eosinophils (35), and B cells (36) can be induced to express a functional CD40L. The CD40L–CD40 interaction has been demonstrated to be necessary for T cell–dependent B cell activation (33, 37). Mutations in the CD40L molecule cause a hyper-IgM immunodeficiency condition in man (38, 39, 40). On the other hand, CD40–CD40L interactions also orchestrate the response of regulatory T cells during both their development (41, 42) and their encounter with antigen (43, 44).NK cells have also been suggested to play a role in B cell differentiation and immunoglobulin production (45). Therefore, it was of interest to investigate whether NK cells could use a CD40-dependent pathway in their interactions with other cells. Therefore, we have investigated the ability of target cells expressing CD40 to induce activation of NK cytotoxicity.     

Infancy-Onset Dietary Counseling of Low-Saturated-Fat Diet Improves Insulin Sensitivity in Healthy Adolescents 15–20 Years of Age: The Special Turku Coronary Risk Factor Intervention Project (STRIP) study

OBJECTIVE

We reported previously that low-saturated-fat dietary counseling started in infancy improves insulin sensitivity in healthy children 9 years of age. The aim of this study was to evaluate the effect of lifelong dietary counseling on insulin sensitivity in healthy adolescents between 15 and 20 years of age. In addition, we examined dietary fiber intake and the polyunsaturated fatty acid (PUFA) + monounsaturated (MUFA)-to-saturated fatty acid (SFA) ratio in the intervention and control adolescents and the association of these dietary factors with homeostasis model of insulin resistance (HOMA-IR).

RESEARCH DESIGN AND METHODS

The study comprised adolescents participating in the randomized, controlled Special Turku Coronary Risk Factor Intervention Project (STRIP) study, which aims to guide the study participants toward a diet beneficial for cardiovascular health. HOMA-IR was assessed annually between 15 and 20 years of age (n = 518; intervention, n = 245; control, n = 273), along with diet, BMI, pubertal status, serum cotinine concentrations, and physical activity. Dietary counseling was given biannually during the follow-up.

RESULTS

HOMA-IR was lower (7.5% on average) in the intervention group than in the control group between 15 and 20 years of age (P = 0.0051). The intervention effect was similar in girls and boys. The PUFA+MUFA-to-SFA ratio was higher (P < 0.0001) and the dietary fiber (g/MJ) intake was higher (P = 0.0058) in the intervention group compared with the control group. There was no association between the PUFA+MUFA-to-/SFA ratio and HOMA-IR, whereas dietary fiber intake (g/MJ) was associated with HOMA-IR in girls (P < 0.0001).

CONCLUSIONS

Dietary counseling initiated in infancy and maintained until 20 years of age was associated with improved insulin sensitivity in adolescents.Insulin sensitivity is related to the life-long pathogenesis of atherosclerosis (1–3). Abnormally low insulin sensitivity (i.e., insulin resistance) is essential in the pathogenesis of the metabolic syndrome and related chronic diseases such as type 2 diabetes mellitus (4–7). The homeostasis model assessment of insulin resistance (HOMA-IR), the combined outcome of serum insulin and glucose levels, is a proxy measure of insulin sensitivity commonly used in epidemiological studies (8–10). High-fiber and high-polyunsaturated fatty acid (PUFA), high-monounsaturated fatty acid (MUFA), and low-saturated fatty acid (SFA) fat diets are hypothesized to improve insulin sensitivity (5,11–16). Dietary fat intervention studies in adults have shown the beneficial effect of higher unsaturated and lower SFA intake on insulin sensitivity (17,18).This study analyzed data from the Special Turku Coronary Risk Factor Intervention Project for Children (STRIP). STRIP is a prospective, randomized trial that was started in infancy and aims to guide the study children toward a diet beneficial for cardiovascular health (19). Children in the intervention group have had a lower SFA intake from 7 months to 19 years of age (20) and a higher dietary fiber intake studied until 9 years of age (21). A favorable intervention effect has been found on serum LDL-cholesterol concentrations through infancy to adulthood (20). In addition, we have previously reported that the dietary counseling improved insulin sensitivity at 9 years of age (14). Similarly, in the Dietary Intervention Study for Children (DISC) examining hypercholesterolemic children, benefits of the low-fat and high-fiber dietary intervention given in childhood/adolescence on glycemic control were evident later in adulthood (15). Studies on the effect of dietary intervention on insulin sensitivity and the association of dietary fiber intake and quality of dietary fat with HOMA-IR among repeatedly studied healthy adolescents are, however, nonexisting. We therefore hypothesized that the dietary counseling given in STRIP would be associated with improved insulin sensitivity and that this effect would be explained by differences in fiber intake and/or quality of dietary fat.     

Acute kidney injury in burns: a story of volume and inflammation

Acute kidney injury occurs in approximately one-quarter to one-third of patients with major burn injury. Apart from the usual suspects – such as older age, severity of burn injury, sepsis and multiple organ dysfunction – volume overload probably has an important role in the pathogenesis of acute kidney injury.Steinvall and collaborators present the third study on acute kidney injury (AKI) defined by the RIFLE classification in patients with major burn injury [1]. AKI was formerly only considered relevant when there was a need for renal replacement therapy. We now know that moderate decreased kidney function also has an impact on patient outcomes [2]. Only since the first consensus definition for AKI, however – the RIFLE classification [3], which was modified later into the AKI staging system [4] – are we able to truly evaluate the epidemiology of AKI in diverse cohorts of patients. AKI has a population incidence greater than that of acute respiratory distress syndrome, and is comparable with that of sepsis [5]. The incidence rate in a general intensive care unit is on average 30% to 40%, but this rate varies according to the specific cohort.Despite the limitation that the study by Steinvall and colleagues includes only 127 patients with major burns, the study has several strengths. The authors present a very thorough evaluation of AKI, including many possible confounders for AKI. The cohort of patients also seems representative for burn unit patients in the western world [1].What did these studies learn, and how does the study of Steinvall and colleagues relate to the other two studies on this subject – those by Lopes and colleagues (n = 126) [6] and by Coca and colleagues (n = 304) [7]? Importantly, all three studies confirmed findings in other cohorts that increasing RIFLE class was associated with a stepwise increase of mortality. There was a large difference, however, in the incidence of AKI between the studies of Coca and colleagues and of Steinvall and colleagues (26.6% and 24.4%, respectively) compared with that of Lopes and colleagues (35.7% incidence). This difference cannot be explained by differences in baseline characteristics, such as age and total burned surface area. Other explanations should therefore be explored.The study by Lopes and colleagues classifies patients according to the original RIFLE classification, on both urine output and serum creatinine concentration [6]. This is in contrast to the studies by Steinvall and colleagues and Coca and colleagues, which only use serum creatinine [1,7]. Especially in burn patients, the serum creatinine concentration may underestimate kidney function. The cornerstone in acute burn care therapy is large-volume resuscitation to compensate for the massive fluid losses and decreased effective circulating volume. This may lead to hemodilution, and to false low serum creatinine concentrations that do not reflect true kidney function. Catabolism, leading to loss of muscle mass, may also contribute to low serum concentrations. As the muscles are the source of creatinine, less muscle mass will result in lower serum creatinine concentrations for the same glomerular filtration rate [8]. In other words, the two studies that only used creatinine criteria may have underestimated the true incidence of AKI.Steinvall and colleagues also report interesting data on the occurrence of AKI in relation to other organ dysfunctions. They found that approximately one-half of patients, especially those with more severe burn injury, developed AKI during the first week; the other half developed AKI during the next week. AKI was preceded, however, by other organ dysfunctions or sepsis in the majority of patients [1]. In burn injury, decreases of effective circulating volume are maximal during the first 8 hours. Apparently, the burn shock resuscitation schedule used was successful in preventing AKI in this very early phase of burn shock. So when burn shock is not the cause of AKI, what else is?This question brings us to the shift in paradigm on the pathophysiology of AKI. While formerly hypoperfusion and ischemia of the kidneys were thought the main causes of AKI in sepsis, there are now more data indicating that renal perfusion is not decreased in sepsis [9]. Instead, inflammation and apoptosis are probably playing an important role [10]. Data on renal perfusion in burn injury are lacking, but the present findings suggest that renal ischemia is also less relevant, at least in the acute phase of burn injury. Furthermore, major burn trauma patients differ from other intensive care trauma patients by experiencing an inflammatory response that is often more severe, and lasts much longer, compared with other trauma patients [11].Another factor that may contribute to AKI in a second stage after burn shock is that volume resuscitation leads to development of intra-abdominal hypertension and abdominal compartment syndrome [12,13].This brings us to the issue of the optimal burn resuscitation schedule. Most units use Ringer''s lactate and the Parkland resuscitation schedule (4 ml/body weight (kg)/% total burned surface area). Most patients receive more fluids, however – even volumes up to 6 ml/kg/total burned surface area have been reported [14]. Other resuscitation endpoints, or other types of fluids – such as hypertonic saline or colloids – may decrease the volume, decrease the incidence of intra-abdominal hypertension and abdominal compartment syndrome, and decrease AKI [15-17].In conclusion, AKI is also an important complication in burn patients as it is frequent and it is associated with mortality. Inflammation and volume overload play an important role in the pathogenesis of AKI. After decades of care for burn patients, therefore, we definitely need good studies into the optimal volume resuscitation strategy.     

Extended Family History of Type 1 Diabetes and Phenotype and Genotype of Newly Diagnosed Children

OBJECTIVE

To determine the frequency of newly diagnosed diabetic children with first- and second-degree relatives affected by type 1 diabetes and to characterize the effects of this positive family history on clinical markers, signs of β-cell autoimmunity, and HLA genotype in the index case.

RESEARCH DESIGN AND METHODS

Children (n = 1,488) with type 1 diabetes diagnosed under 15 years of age were included in a cross-sectional study from the Finnish Pediatric Diabetes Register. Data on family history of diabetes and metabolic decompensation at diagnosis were collected using a questionnaire. Antibodies to β-cell autoantigens (islet cell antibodies, insulin autoantibodies, GAD antibodies, and antibodies to the islet antigen 2 molecule) and HLA genotypes were analyzed.

RESULTS

A total of 12.2% of the subjects had a first-degree relative with type 1 diabetes (father 6.2%, mother 3.2%, and sibling 4.8%) and 11.9% had an affected second-degree relative. Children without affected relatives had lower pH (P < 0.001), higher plasma glucose (P < 0.001) and β-hydroxybutyrate concentrations (P < 0.001), a higher rate of impaired consciousness (P = 0.02), and greater weight loss (P < 0.001). There were no differences in signs of β-cell autoimmunity. The familial cases carried the HLA DR4-DQ8 haplotype more frequently than sporadic cases (74.0 vs. 67.0%, P = 0.02).

CONCLUSIONS

When the extended family history of type 1 diabetes is considered, the proportion of sporadic diabetes cases may be reduced to <80%. A positive family history for type 1 diabetes associates with a less severe metabolic decompensation at diagnosis, even when only second-degree relatives are affected. Autoantibody profiles are similar in familial and sporadic type 1 diabetes, suggesting similar pathogenetic mechanisms.Familial clustering of type 1 diabetes is a conspicuous feature; the risk of developing type 1 diabetes is 8–15-fold higher in first-degree relatives (1–6) and twofold in second-degree relatives (1,7). Despite this, the vast majority of children are diagnosed with the sporadic form of diabetes. The proportion of children with an affected first-degree relative at the time of diagnosis is ∼10–12% (7–13), and after decades of follow-up, this frequency increases to >20% (8,14,15). Fathers transmit the disease to their offspring more often than mothers (3,16). Accordingly, at diagnosis, 4–7% of children have a father with type 1 diabetes whereas only 1.5–3% have an affected mother (7–12,17). Fewer reports exist on type 1 diabetes in the extended family. Depending on the definition of second-degree relatives and length of time from the diagnosis of the index case, 5–16% of children with type 1 diabetes have an affected second-degree relative (1,5,11,17–19).Familial and sporadic type 1 diabetes have been suggested to differ in terms of pathogenetic mechanisms (20,21). The risk-associated HLA genotypes have been observed more often in familial type 1 diabetes (8,20,22,23), although not all studies have found significant differences (24). Two studies have noticed no differences in diabetes-associated autoantibodies, e.g., insulin autoantibodies (IAAs) (8), GAD antibodies (GADAs) (8), or islet cell antibodies (ICAs) (8,20). A recent study from Israel reported, however, higher frequencies of IAAs and a higher number of positive antibody responses among familial cases (13). In families with prior experience of type 1 diabetes in a first-degree relative, the clinical status of the child at diagnosis is less severe (8,13,21).Data on the possible pathogenetic differences between familial and sporadic type 1 diabetes are still inconsistent and based on a positive family history in first-degree relatives only. To further our understanding of familial clustering of type 1 diabetes, we used data from the large, nationwide Finnish Pediatric Diabetes Register for a cross-sectional observational study. Since the knowledge of the effects of an extended family history on the diabetes of the index case is lacking, we included information on second-degree relatives (grandparents and siblings of parents). β-Cell autoimmunity, metabolic decompensation at diagnosis, and HLA genetics were compared in children with familial or sporadic type 1 diabetes. We postulated to see a stronger genetic susceptibility to type 1 diabetes and a milder metabolic decompensation in children with a positive family history for type 1 diabetes, whereas no differences were expected in the autoantibody profile.     

Prospective Association Between Inflammatory Markers and Progression of Coronary Artery Calcification in Adults With and Without Type 1 Diabetes

OBJECTIVE

The role of inflammation in the increased risk of cardiovascular disease in type 1 diabetes is unclear. We examined the association of inflammation and progression of coronary artery calcification (CAC)—a marker of subclinical atherosclerosis—in adults with and without type 1 diabetes.

RESEARCH DESIGN AND METHODS

A nested case-control study was performed within the prospective cohort of the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. Participants underwent two CAC measurements ∼2.5 years apart. Case subjects (n = 204) were those with significant progression of CAC. Control subjects (n = 258) were frequency-matched to case subjects on diabetes status, sex, age, and baseline CAC status. Inflammatory marker assessments were performed on stored blood samples from baseline. A principal components analysis (PCA) was performed and a composite score derived from that analysis. The composite score was constructed by assigning a value of 1 for each PCA component where at least one of the markers exceeded the 75th percentile (range 0–4). Conditional logistic regression was used for the matching strategy.

RESULTS

The first two components of the PCA were modestly (odds ratio 1.38 [95% CI 1.08–1.77] and 1.27 [1.02–1.59], respectively) associated with CAC progression after adjustment for other risk factors. The composite score was more strongly associated with CAC progression for those with elevated markers in three or four of the principal components compared with those with none.

CONCLUSIONS

Measures of inflammation were associated with progression of CAC in a population of adults with and without type 1 diabetes.Individuals with type 1 diabetes are at a dramatically increased risk of cardiovascular disease (CVD) compared with those without diabetes (1). Inflammation is a factor in the pathogenesis of atherosclerosis (2) and is elevated in type 1 diabetes (3,4), but the role it may play in the increased risk of CVD in type 1 diabetes is unclear.Increased levels of high-sensitivity C-reactive protein (hsCRP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α have been shown to be associated with microvascular complications in type 1 diabetes (5–8); however, these cross-sectional studies do not prove causality. Large prospective studies have so far suggested that measures of inflammation are associated with microvascular and macrovascular complications, but the inflammatory markers measured have been limited. An analysis of data collected from the Pittsburgh Epidemiology of Diabetes Complications (EDC) study found that elevated hsCRP levels were associated with an increased risk of coronary artery disease (CAD) during 18 years of follow-up, particularly in certain haptoglobin genotypes (9). The Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications study found that plasma fibrinogen and soluble cell adhesion molecules were associated with the progression in carotid intimal-medial thickness and diabetic nephropathy, respectively, but no association was found for either of these outcomes with hsCRP (10).Coronary artery calcification (CAC), a subclinical measure of CVD, predicts cardiac events and has been a valuable tool for quantifying the burden of atherosclerosis (11–13). Short-term progression of CAC predicts all-cause mortality (14) independently of the baseline CAC levels. Associations between inflammatory markers and the amount of CAC have been reported for the general population (15,16), but few studies have looked at the association between inflammation and the progression of CAC (17). One study found that plasma homocysteine predicted progression of CAC, but a significant association was not found with CRP (18). In the Multi-Ethnic Study of Atherosclerosis, fibrinogen and CRP were not associated with incident CAC after adjusting for BMI (19). Analyses of data from the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study found significant relationships between soluble IL-2 receptor (sIL-2R), fibrinogen, and progression of CAC (20,21).The goal of this study was to examine the prospective association of markers of inflammation with the progression of CAC over time in adults with and without type 1 diabetes.