Association of urine and plasma ADMA with atherosclerotic risk in DKD cardiovascular disease risk in diabetic kidney disease: findings from the Chronic Renal Insufficiency Cohort (CRIC) study.

BACKGROUND: Chronic kidney disease (CKD) is associated with atherosclerotic cardiovascular disease (ASCVD) risk, especially among those with diabetes. Altered metabolism of solutes that accumulate in CKD [asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and trimethylamine N-oxide (TMAO)] may reflect pathways linking CKD with ASCVD. METHODS: This case-cohort study included Chronic Renal Insufficiency Cohort participants with baseline diabetes, estimated glomerular filtration rate <60 mL/min/1.73 m2, and without prior history for each outcome. The primary outcome was incident ASCVD (time to first myocardial infarction, stroke or peripheral artery disease event) and secondary outcome was incident heart failure. The subcohort comprised randomly selected participants meeting entry criteria. Plasma and urine ADMA, SDMA and TMAO concentrations were determined by liquid chromatography-tandem mass spectrometry. Associations of uremic solute plasma concentrations and urinary fractional excretions with outcomes were evaluated by weighted multivariable Cox regression models, adjusted for confounding covariables. RESULTS: Higher plasma ADMA concentrations (per standard deviation) were associated with ASCVD risk [hazard ratio (HR) 1.30, 95% confidence interval (CI) 1.01-1.68]. Lower fractional excretion of ADMA (per standard deviation) was associated with ASCVD risk (HR 1.42, 95% CI 1.07-1.89). The lowest quartile of ADMA fractional excretion was associated with greater ASCVD risk (HR 2.25, 95% CI 1.08-4.69) compared with the highest quartile. Plasma SDMA and TMAO concentration and fractional excretion were not associated with ASCVD. Neither plasma nor fractional excretion of ADMA, SDMA and TMAO were associated with incident heart failure. CONCLUSION: These data suggest that decreased kidney excretion of ADMA leads to increased plasma concentrations and ASCVD risk.

Association of Uremic Solutes With Cardiovascular Death in Diabetic Kidney Disease.

RATIONALE & OBJECTIVE: Cardiovascular disease (CVD) is a major cause of mortality among people with diabetic kidney disease (DKD). The pathophysiology is inadequately explained by traditional CVD risk factors. The uremic solutes trimethylamine-N-oxide (TMAO) and asymmetric and symmetric dimethylarginine (ADMA, SDMA) have been linked to CVD in kidney failure with replacement therapy (KFRT), but data are limited in populations with diabetes and less severe kidney disease. STUDY DESIGN: Observational cohort. SETTINGS & PARTICIPANTS: Random subcohort of 555 REGARDS (Reasons for Geographic and Racial Differences in Stroke) study participants with diabetes and estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 at study entry. EXPOSURE: ADMA, SDMA, and TMAO assayed by liquid chromatography-mass spectrometry in plasma and urine. OUTCOME: Cardiovascular mortality (primary outcome); all-cause mortality and incident KFRT (secondary outcomes). ANALYTICAL APPROACH: Plasma concentrations and ratios of urine to plasma concentrations of ADMA, SDMA, and TMAO were tested for association with outcomes. Adjusted Cox regression models were fitted and hazard ratios of outcomes calculated per standard deviation and per doubling, and as interquartile comparisons. RESULTS: The mean baseline eGFR was 44 mL/min/1.73 m2. Cardiovascular death, overall mortality, and KFRT occurred in 120, 285, and 89 participants, respectively, during a mean 6.2 years of follow-up. Higher plasma ADMA and SDMA (HRs of 1.20 and 1.28 per 1-SD greater concentration), and lower ratios of urine to plasma concentrations of ADMA, SDMA, and TMAO (HRs per halving of 1.53, 1.69, and 1.38) were associated with cardiovascular mortality. Higher plasma concentrations of ADMA, SDMA, and TMAO (HRs of 1.31, 1.42, and 1.13 per 1-SD greater concentration) and lower urine to plasma ratios of ADMA, SDMA, and TMAO (HRs per halving of 1.34, 1.37, and 1.26) were associated with all-cause mortality. Higher plasma ADMA and SDMA were associated with incident KFRT by categorical comparisons (HRs of 2.75 and 2.96, comparing quartile 4 to quartile 1), but not in continuous analyses. LIMITATIONS: Single cohort, restricted to patients with diabetes and eGFR < 60 mL/min/1.73 m2, potential residual confounding by GFR, no dietary information. CONCLUSIONS: Higher plasma concentrations and lower ratios of urine to plasma concentrations of uremic solutes were independently associated with cardiovascular and all-cause mortality in DKD. Associations of ratios of urine to plasma concentrations with mortality suggest a connection between renal uremic solute clearance and CVD pathogenesis.

Effect of Oat ß-Glucan Supplementation on Chronic Kidney Disease: A Feasibility Study.

OBJECTIVE: Dietary supplementation with grains containing high ß-glucan fiber has been shown to attenuate the progression of chronic kidney disease (CKD) and vascular calcification in animal models. The aim of this study was to investigate the feasibility of consuming an oat ß-glucan supplement and to assess its effects on certain uremic toxins and markers of mineral metabolism in patients with CKD. DESIGN: This is a 20-week, nonrandomized, single-center, pretest-posttest study. Twenty-eight subjects with CKD stages 3-4 were enrolled. The mean age was 67.6 ± 8.9 years, and the mean estimated glomerular filtration rate was 35 ± 14 mL/min/1.73 m2. Subjects received a dietary supplement containing 3 g of oat ß-glucan per day for 12 weeks. The 4-week period before the start of the intervention was used as a baseline comparison for each subject. The primary outcome was pre-post supplement changes in plasma levels of two uremic toxins: trimethylamine N-oxide (TMAO) and asymmetric dimethylarginine. Secondary outcomes were pre-post supplement changes in serum calcium, phosphorus, and Klotho levels. Repeated-measures analysis of variance was used to test the differences in outcomes over the three-month-long intervention. RESULTS: Serum levels of TMAO decreased by a median of -17% (interquartile range: -46%, 7%) at the end of the intervention. A nonstatistically significant change was observed for asymmetric dimethylarginine (median -0.6% [-12%, 20%]) and serum Klotho (median -3% [-8%, 7%]). There were no changes in serum levels of calcium and phosphorus. One month after discontinuation of ß-glucan therapy, TMAO levels increased by a median of 16% (-12%, 36%) but remained slightly below the pretreatment levels. Eight subjects experienced side effects and discontinued the treatment. CONCLUSION: A diet supplemented with ß-glucan is safe and potentially efficacious in lowering serum concentrations of TMAO in patients with CKD. Larger trials with longer follow-up times are needed to determine whether such reductions translate into clinical benefits.

A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis.

The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

A bifunctional enzyme that has both monoacylglycerol acyltransferase and acyl hydrolase activities.

Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX(4)D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions.

Diabetes Modulates Iodothyronine Deiodinase 2 Expression in the Mouse Retina: A Role for Thyroid Hormone in the Pathogenesis of Diabetic Retinopathy.

Purpose: Clinical investigations associate hypothyroidism with an increased risk for microvascular complications, yet the mechanism by which thyroid hormone regulates the development of diabetic retinopathy is not clearly understood. We investigated the role of iodothyronine deiodinase 2 (DIO2) in the pathogenesis of diabetic retinopathy. Methods: Retinas from streptozotocin-induced diabetic and nondiabetic mice were evaluated by RNA sequencing, RT-PCR, and immunostaining. Media and cell lysates from mouse retinal microvascular endothelial cells and retinal astrocytes exposed to physiologic (5 mM) and high glucose (25 mM) containing media were assessed by liquid chromatography-tandem mass spectrometry to measure tetraiodothyronine (T4) and tri-iodothyronine (T3) concentrations and by Western blot analysis to determine the relationship of T4/T3 to oxidative stress and inflammatory mediators. Cell death was determined by Trypan Blue exclusion assay. Results: At 12 weeks of diabetes duration, retinas from diabetic mice compared with nondiabetic mice demonstrated a significant decrease in Dio2 transcripts and Dio2 gene and protein (P < 0.05) expression. When cultured in the presence of high glucose, both mouse retinal astrocytes and microvascular endothelial cells demonstrated a significant reduction of DIO2 protein compared with cells cultured in physiologic glucose. High glucose inhibited generation of T3, leading to a significantly increased T4/T3 (P < 0.0079). Supplementation of cells with T3, but not T4, prevented the high glucose-induced rise in endothelial nitric oxide synthase, intercellular cell adhesion molecule 1, and endothelial cell death (P < 0.0079). Conclusions: Decreased intraretinal T3 owing to diabetes-induced loss of DIO2 may lead to dysfunction and death of cells in the retina, thereby contributing to the pathogenesis of early diabetic retinopathy.

Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase.

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX(4)D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

Ventricular arrhythmias in mouse models of diabetic kidney disease.

Chronic kidney disease (CKD) affects more than 20 million people in the US, and it is associated with a significantly increased risk of sudden cardiac death (SCD). Despite the significance, the mechanistic relationship between SCD and CKD is not clear and there are few effective therapies. Using optical mapping techniques, we tested the hypothesis that mouse models of progressive diabetic kidney disease (DKD) exhibit enhanced ventricular arrhythmia incidence and underlying arrhythmia substrates. Compared to wild-type mice, both Leprdb/db eNOS-/- (2KO) and high fat diet plus low dose streptozotocin (HFD + STZ) mouse models of DKD experienced sudden death and greater arrhythmia inducibility, which was more common with isoproterenol than programmed electrical stimulation. 2KO mice demonstrated slowed conduction velocity, prolonged action potential duration (APD), and myocardial fibrosis; both 2KO and HFD + STZ mice exhibited arrhythmias and calcium dysregulation with isoproterenol challenge. Finally, circulating concentrations of the uremic toxin asymmetric dimethylarginine (ADMA) were elevated in 2KO mice. Incubation of human cardiac myocytes with ADMA prolonged APD, as also observed in 2KO mice hearts ex vivo. The present study elucidates an arrhythmia-associated mechanism of sudden death associated with DKD, which may lead to more effective treatments in the vulnerable DKD patient population.

Cholesterol efflux capacity assay using immobilized liposomes and apolipoprotein B-depleted serum.

Cholesterol efflux capacity (CEC), an important functional step in reverse cholesterol transport, is the main anti-atherosclerotic function of high-density lipoprotein (HDL). Assays that improve the determination of CEC ex vivo for clinical applications are constantly explored. In the accompanying article, Horiuchi et al. (Biosci. Rep. (2019) 39(4), BSR20190213) evaluate the availability of apolipoprotein B-depleted serum for CEC assays. Using their recently developed immobilized liposome-bound gel beads (ILG) method, Horiuchi et al. demonstrate that apolipoprotein B-depleted serum obtained with poly ethylene glycol precipitation enables CEC assays to be easily and accurately introduced into laboratory medicine.