Ben Caplin

Endogenous Nitric Oxide Synthase Inhibitors in the Biology of Disease Markers, Mediators, and Regulators?

The asymmetric methylarginines inhibit nitric oxide synthesis in vivo by competing with L-arginine at the active site of nitric oxide synthase. High circulating levels of asymmetric dimethylarginine predict adverse outcomes, specifically vascular events but there is now increasing experimental and epidemiological evidence that these molecules, and the enzymes that regulate this pathway, play a mechanistic role in cardiovascular diseases. Recent data have provided insight into the impact of altered levels of these amino acids in both humans and rodents, however these reports also suggest a simplistic approach based on measuring, and modulating circulating asymmetric dimethylarginine alone is inadequate. This review outlines the basic biochemistry and physiology of endogenous methylarginines, examines both the experimental and observational evidence for a role in disease pathogenesis, and examines the potential for therapeutic regulation of these molecules.

Alanine-Glyoxylate Aminotransferase-2 Metabolizes Endogenous Methylarginines, Regulates NO, and Controls Blood Pressure

Objective—Asymmetric dimethylarginine is an endogenous inhibitor of NO synthesis that may mediate cardiovascular disease. Alanine-glyoxylate aminotransferase-2 (AGXT2) has been proposed to degrade asymmetric dimethylarginine. We investigated the significance of AGXT2 in methylarginine metabolism in vivo and examined the effect of this enzyme on blood pressure. Methods and Results—In isolated mouse kidney mitochondria, we show asymmetric dimethylarginine deamination under physiological conditions. We demonstrate increased asymmetric dimethylarginine, reduced NO, and hypertension in an AGXT2 knockout mouse. We provide evidence for a role of AGXT2 in methylarginine metabolism in humans by demonstrating an inverse relationship between renal (allograft) gene expression and circulating substrate levels and an association between expression and urinary concentrations of the product. Finally, we examined data from a meta-analysis of blood pressure genome-wide association studies. No genome-wide significance was observed, but taking a hypothesis-driven approach, there was a suggestive association between the T allele at rs37369 (which causes a valine-isoleucine substitution and altered levels of AGXT2 substrate) and a modest increase in diastolic blood pressure (P=0.0052). Conclusion—Although the effect of variation at rs37369 needs further study, these findings suggest that AGXT2 is an important regulator of methylarginines and represents a novel mechanism through which the kidney regulates blood pressure.

Circulating methylarginine levels and the decline in renal function in patients with chronic kidney disease are modulated by DDAH1 polymorphisms

In patients with chronic kidney disease, high plasma levels of the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine, are thought to contribute to decline in renal function. Here we took a candidate gene approach to determine any causal role of asymmetric dimethylarginine in the progression of chronic kidney disease. The impact of single-nucleotide polymorphisms in the genes encoding the two isoforms of the asymmetric dimethylarginine-degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1 and DDAH2), on enzyme expression, plasma asymmetric dimethylarginine levels, and longitudinal changes in estimated glomerular filtration rate were determined in various patient groups. There was evidence suggesting that the rs17384213 DDAH1 GG genotype was associated with increased expression of its mRNA in kidney allografts. Healthy subjects carrying the rs17384213 G allele had lower plasma asymmetric dimethylarginine, and a similar borderline association was found in patients with chronic kidney disease. This allele, however, was independently associated with a steeper decline in renal function in two separate cohorts of patients with chronic kidney disease. We conclude that polymorphisms in DDAH1 alter the rate of decline of glomerular filtration rate in subjects with chronic kidney disease. Our findings show that DDAH1 modulates plasma asymmetric dimethylarginine and contributes to the decline in renal function. However, it appears that increases in circulating methylarginine did not mediate progressive kidney injury.

Prospective Monitoring of Epstein-Barr Virus DNA in Adult Renal Transplant Recipients During the Early Posttransplant Period: Role of Mycophenolate Mofetil

Background. Epstein-Barr virus (EBV) is associated with posttransplant lymphoproliferative disease. We monitored the incidence of EBV viraemia in adult renal transplant recipients and investigated the association with clinical parameters. Methods. Whole blood from 115 renal transplant patients was tested regularly by quantitative polymerase chain reaction (PCR) assay for EBV DNA during the first 90 days posttransplantation. Results. Sixty four of 115 (56%) patients had detectable EBV DNA in blood (>100 copies/mL) on at least one occasion. The median time to first DNA detection was 15 days post-transplant and median viral load was 598 copies/mL (range 119–53,649 copies/mL). Multivariate Cox-regression analyses showed that patients receiving mycophenolate mofetil (MMF) on the day of transplant had a significantly lower risk of EBV viraemia compared to those who received no MMF (Hazard ratio=0.518, 95% CI 0.307–0.875, p=0.014). Conclusions. EBV viraemia is common during the early posttransplant period in adult renal transplant recipients. Our results suggest a role of MMF in preventing EBV viraemia, however further work is required to identify the mechanism(s) involved.

Dimethylarginine Dimethylaminohydrolase 2 Regulates Nitric Oxide Synthesis and Hemodynamics and Determines Outcome in Polymicrobial Sepsis

Objective—Nitric oxide is a key to numerous physiological and pathophysiological processes. Nitric oxide production is regulated endogenously by 2 methylarginines, asymmetric dimethylarginine (ADMA) and monomethyl-L-arginine. The enzyme that specifically metabolizes asymmetric dimethylarginine and monomethyl-L-arginine is dimethylarginine dimethylaminohydrolase (DDAH). The first isoform dimethylarginine dimethylaminohydrolase 1 has previously been shown to be an important regulator of methylarginines in both health and disease. This study explores for the first time the role of endogenous dimethylarginine dimethylaminohydrolase 2 in regulating cardiovascular physiology and also determines the functional impact of dimethylarginine dimethylaminohydrolase 2 deletion on outcome and immune function in sepsis. Approach and Results—Mice, globally deficient in Ddah2, were compared with their wild-type littermates to determine the physiological role of Ddah2 using in vivo and ex vivo assessments of vascular function. We show that global knockout of Ddah2 results in elevated blood pressure during periods of activity (mean [SEM], 118.5 [1.3] versus 112.7 [1.1] mm Hg; P=0.025) and changes in vascular responsiveness mediated by changes in methylarginine concentration, mean myocardial tissue asymmetric dimethylarginine (SEM) was 0.89 (0.06) versus 0.67 (0.05) &mgr;mol/L (P=0.02) and systemic nitric oxide concentrations. In a model of severe polymicrobial sepsis, Ddah2 knockout affects outcome (120-hour survival was 12% in Ddah2 knockouts versus 53% in wild-type animals; P<0.001). Monocyte-specific deletion of Ddah2 results in a similar pattern of increased severity to that seen in globally deficient animals. Conclusions—Ddah2 has a regulatory role both in normal physiology and in determining outcome of severe polymicrobial sepsis. Elucidation of this role identifies a mechanism for the observed relationship between Ddah2 polymorphisms, cardiovascular disease, and outcome in sepsis.

Peritoneal protein clearance rather than faster transport status determines outcomes in peritoneal dialysis patients.

Faster peritoneal transport has been associated with an increased risk of therapy failure and patient mortality. However, faster transport can the result of many factors. Peritoneal protein clearance (PPC) has been proposed to distinguish faster peritoneal transport attributable to inflammatory conditions, as protein clearance reflects large-pore flow, which increases during inflammation. We followed a cohort of 300 peritoneal dialysis patients, and after adjustments for age and comorbidity, higher PPC was associated with increased risk of death (hazard ratio: 1.81; 95% confidence interval: 1.11 to 2.95), even after patients underwent transplantation or transferred to hemodialysis.

Reduced Renal Methylarginine Metabolism Protects against Progressive Kidney Damage

Nitric oxide (NO) production is diminished in many patients with cardiovascular and renal disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis, and elevated plasma levels of ADMA are associated with poor outcomes. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is a methylarginine-metabolizing enzyme that reduces ADMA levels. We reported previously that a DDAH1 gene variant associated with increased renal DDAH1 mRNA transcription and lower plasma ADMA levels, but counterintuitively, a steeper rate of renal function decline. Here, we test the hypothesis that reduced renal-specific ADMA metabolism protects against progressive renal damage. Renal DDAH1 is expressed predominately within the proximal tubule. A novel proximal tubule-specific Ddah1 knockout (Ddah1(PT-/-)) mouse demonstrated tubular cell accumulation of ADMA and lower NO concentrations, but unaltered plasma ADMA concentrations. Ddah1(PT-/-) mice were protected from reduced kidney tissue mass, collagen deposition, and profibrotic cytokine expression in two independent renal injury models: folate nephropathy and unilateral ureteric obstruction. Furthermore, a study of two independent kidney transplant cohorts revealed higher levels of human renal allograft methylarginine-metabolizing enzyme gene expression associated with steeper function decline. We also report an association among DDAH1 expression, NO activity, and uromodulin expression supported by data from both animal and human studies, raising the possibility that kidney DDAH1 expression exacerbates renal injury through uromodulin-related mechanisms. Together, these data demonstrate that reduced renal tubular ADMA metabolism protects against progressive kidney function decline. Thus, circulating ADMA may be an imprecise marker of renal methylarginine metabolism, and therapeutic ADMA reduction may even be deleterious to kidney function.

Arterial calcification in dialysis patients and transplant recipients.

Calcification of the cardiovascular system is well recognized in patients with chronic kidney disease receiving dialysis, persists following successful kidney transplantation, and is associated with a poor prognosis. Whether deposition of calcium in the arteries of such individuals contributes to excess cardiovascular morbidity and mortality remains uncertain as do the clinical advantages of slowing this process. However, detecting vascular calcification using simple imaging techniques such as plain radiographs or ultrasound can provide valuable information as to the nature of bone disease in patients receiving dialysis and may help to guide therapeutic strategies aimed at preventing the development of secondary hyperparathyroidism. Further advances in our understanding of arterial calcification and its relationship to bone disease are likely to help in the future development of therapies for these interrelated conditions.

VASCULAR CALCIFICATION IN PATIENTS WITH KIDNEY DISEASE: Arterial Calcification in Dialysis Patients and Transplant Recipients

Calcification of the cardiovascular system is well recognized in patients with chronic kidney disease receiving dialysis, persists following successful kidney transplantation, and is associated with a poor prognosis. Whether deposition of calcium in the arteries of such individuals contributes to excess cardiovascular morbidity and mortality remains uncertain as do the clinical advantages of slowing this process. However, detecting vascular calcification using simple imaging techniques such as plain radiographs or ultrasound can provide valuable information as to the nature of bone disease in patients receiving dialysis and may help to guide therapeutic strategies aimed at preventing the development of secondary hyperparathyroidism. Further advances in our understanding of arterial calcification and its relationship to bone disease are likely to help in the future development of therapies for these interrelated conditions.

Galactosylation of IgA1 Is Associated with Common Variation in C1GALT1.

IgA nephropathy (IgAN), an important cause of kidney failure, is characterized by glomerular IgA deposition and is associated with changes in O-glycosylation of the IgA1 molecule. Here, we sought to identify genetic factors contributing to levels of galactose-deficient IgA1 (Gd-IgA1) in white and Chinese populations. Gd-IgA1 levels were elevated in IgAN patients compared with ethnically matched healthy subjects and correlated with evidence of disease progression. White patients with IgAN exhibited significantly higher Gd-IgA1 levels than did Chinese patients. Among individuals without IgAN, Gd-IgA1 levels did not correlate with kidney function. Gd-IgA1 level heritability (h2), estimated by comparing midparental and offspring Gd-IgA1 levels, was 0.39. Genome-wide association analysis by linear regression identified alleles at a single locus spanning the C1GALT1 gene that strongly associated with Gd-IgA1 level (β=0.26; P=2.35×10-9). This association was replicated in a genome-wide association study of separate cohorts comprising 308 patients with membranous GN from the UK (P<1.00×10-6) and 622 controls with normal kidney function from the UK (P<1.00×10-10), and in a candidate gene study of 704 Chinese patients with IgAN (P<1.00×10-5). The same extended haplotype associated with elevated Gd-IgA1 levels in all cohorts studied. C1GALT1 encodes a galactosyltransferase enzyme that is important in O-galactosylation of glycoproteins. These findings demonstrate that common variation at C1GALT1 influences Gd-IgA1 level in the population, which independently associates with risk of progressive IgAN, and that the pathogenic importance of changes in IgA1 O-glycosylation may vary between white and Chinese patients with IgAN.