Alessandra F. Perna

Hydrogen sulphide-generating pathways in haemodialysis patients: a study on relevant metabolites and transcriptional regulation of genes encoding for key enzymes

BACKGROUND Hydrogen sulphide, H(2)S, is the third endogenous gas with putative cardiovascular properties, after nitric oxide and carbon monoxide. H(2)S is a vasorelaxant, while H(2)S deficiency is implicated in the pathogenesis of hypertension and atherosclerosis. Cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (MPS) catalyze H(2)S formation, with different relative efficiencies. Chronic kidney disease (CKD) is characterized by elevation of both plasma homocysteine and cysteine, which are substrates of these enzymes, and by a high prevalence of hypertension and cardiovascular mortality, particularly in the haemodialysis stage. It is possible that the H(2)S-generating pathways are altered as well in this patient population. METHODS Plasma H(2)S levels were measured with a common spectrophotometric method. This method detects various forms of H(2)S, protein-bound and non-protein-bound. Blood sulphaemoglobin, a marker of chronic exposure to H(2)S, was also measured, as well as related sulphur amino acids, vitamins and transcriptional levels of relevant genes, in haemodialysis patients and compared to healthy controls. RESULTS Applying the above-mentioned methodology, H(2)S levels were found to be decreased in patients. Sulphaemoglobin levels were significantly lower as well. Plasma homocysteine and cysteine were significantly higher; vitamin B(6), a cofactor in H(2)S biosynthesis, was not different. H(2)S correlated negatively with cysteine levels. CSE expression was significantly downregulated in haemodialysis patients. CONCLUSIONS Transcriptional deregulation of genes encoding for H(2)S-producing enzymes is present in uraemia. Although the specificity of the method employed for H(2)S detection is low, the finding that H(2)S is decreased is complemented by the lower sulphhaemoglobin levels. Potential implications of this study relate to the pathogenesis of the uraemic syndrome manifestations, such as hypertension and atherosclerosis.

Hyperhomocysteinemia in uremia--a red flag in a disrupted circuit.

Hyperhomocysteinemia is an independent cardiovascular risk factor, according to most observational studies and to studies using the Mendelian randomization approach, utilizing the common polymorphism C677T of methylene tetrahydrofolate reductase. In contrast, the most recent secondary preventive intervention studies, in the general population and in chronic kidney disease (CKD) and uremia, which are all negative (with the possible notable exception of stroke), point to other directions. However, all trials use folic acid in various dosages as a means to reduce homocysteine levels, with the addition of vitamins B6 and B12. It is possible that folic acid has negative effects, which offset the benefits; alternatively, homocysteine could be an innocent by‐stander, or a surrogate of the real culprit. The latter possibility leads us to the search for potential candidates. First, the accumulation of homocysteine in blood leads to an intracellular increase of S‐adenosylhomocysteine (AdoHcy), a powerful competitive methyltransferase inhibitor, which by itself is considered a predictor of cardiovascular events. DNA methyltransferases are among the principal targets of hyperhomocysteinemia, as studies in several cell culture and animal models, as well as in humans, show. In CKD and in uremia, hyperhomocysteinemia and high intracellular AdoHcy are present and are associated with abnormal allelic expression of genes regulated through methylation, such as imprinted genes, and pseudoautosomal genes, thus pointing to epigenetic dysregulation. These alterations are susceptible to reversal upon homocysteine‐lowering therapy obtained through folate administration. Second, it has to be kept in mind that homocysteine is mainly protein‐bound, and its effects could be linked therefore to protein homocysteinylation. In this respect, increased protein homocysteinylation has been found in uremia, leading to alterations in protein function.

Plasma Protein Aspartyl Damage Is Increased in Hemodialysis Patients: Studies on Causes and Consequences

Plasma proteins in hemodialysis patients display a significant increase in deamidated/isomerized Asx (asparagine and aspartic acid) content, a marker of protein fatigue damage. This has been linked to the toxic effects of hyperhomocysteinemia in uremic erythrocytes; however, treatment aimed at abating homocysteine levels did not lead to significant reductions in plasma protein damage. The hypothesis that lack of reduction in protein damage could be due to protein increased intrinsic instability, as result of interference with the uremic milieu rather than to hyperhomocysteinemia, was put forward. The deamidated/isomerized Asx content of normal plasma incubated with several uremic toxins for 24 h, 72 h, and 7 d was measured, identifying a group of toxins that were able to elicit this kind of damage. Uremic toxins were also incubated with purified human albumin, and dose-response experiments with the two most toxic agents in terms of protein damage (guanidine and guanidinopropionic acid) were carried out. The effect of the hemodialysis procedure on protein damage was evaluated. For investigating also the consequences of these alterations, human albumin was treated in vitro to produce an increase in deamidated/isomerized Asx residues, and the effects of albumin deamidation on protein binding were evaluated. Among the uremic toxins that are able to elicit protein damage, guanidine produced a dose-dependent increase in protein damage. No difference was found after a hemodialysis session. Deamidated albumin shows normal binding capacity to warfarin, salicylic acid, or diazepam but reduced binding to homocysteine. In conclusion, uremic toxins, especially guanidine, display an ability to induce significant protein damage, which can in turn have functional consequences.

Hyperhomocysteinemia and macromolecule modifications in uremic patients.

Abstract Hyperhomocysteinemia is present in the majority of well-nourished chronic renal failure and uremic patients. Most observations reported in the literature come from studies carried out in end-stage renal disease patients treated with hemodialysis. The underlying mechanisms of the toxic effects of homocysteine in uremia related to cardiovascular disease and other disturbances are still under scrutiny. As a consequence, macromolecules (i.e., proteins and DNA) have been found to be altered to various extents. One of the mechanisms of homocysteine toxicity is related to the action of its metabolic precursor, S-adenosylhomocysteine, a powerful methyltransferase competitive inhibitor. Disruption of DNA methylation has been demonstrated to occur as a result of hyperhomocysteinemia, and/or is associated with vascular damage. DNA hypomethylation has been found in the mononuclear cell fraction of uremic patients with hyperhomocysteinemia. Proteins are also targets of homocysteine-dependent molecular damage. The formation of oxidative products with free cysteinyl residue thiol groups has been demonstrated to occur in blood. The latter also represents a mechanism for the transport of homocysteine in plasma. In addition, homocysteine thiolactone has been shown to react with free amino groups in proteins to form isopeptide bonds, in particular at the lysine residue level. Another type of isopeptide bond in proteins may result from the deamidation and isomerization of asparaginyl residues, yielding abnormal isoaspartyl residues, which have been demonstrated to be increased in uremic patients. Folate treatment exerts a partial, but significant, homocysteine-lowering effect in uremic patients and has been shown to improve the changes in macromolecules induced by high homocysteine levels. In conclusion, both DNA and proteins are structurally modified in uremia as a consequence of high homocysteine levels. The role of these macromolecule changes in inducing the clinical complications of hyperhomocysteinemia in these patients, although still conjectural in some respects, is at present sustained by several pieces of evidence.

Hydrogen Sulfide, a Toxic Gas with Cardiovascular Properties in Uremia: How Harmful Is It?

Hydrogen sulfide (H2S) is a poisonous gas which can be lethal. However, it is also produced endogenously, thus belonging to the family of gasotransmitters along with nitric oxide and carbon monoxide. H2S is in fact involved in mediating several signaling and cytoprotective functions, for example in the nervous, cardiovascular, and gastrointestinal systems, such as neuronal transmission, blood pressure regulation and insulin release, among others. When increased, it can mediate inflammation and apoptosis, with a role in shock. When decreased, it can be involved in atherosclerosis, hypertension, myocardial infarction, diabetes, sexual dysfunction, and gastric ulcer; it notably interacts with the other gaseous mediators. Cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase are the principal enzymes involved in H2S production. We have recently studied H2S metabolism in the plasma of chronic hemodialysis patients and reported that its levels are significantly decreased. The plausible mechanism lies in the transcription inhibition of the cystathionine γ-lyase gene. The finding could be of importance considering that hypertension and high cardiovascular mortality are characteristic in these patients.

Hydrogen Sulfide, the Third Gaseous Signaling Molecule With Cardiovascular Properties, Is Decreased in Hemodialysis Patients

Hydrogen sulfide, H(2)S, is the third endogenous gas with cardiovascular properties, after nitric oxide and carbon monoxide. H(2)S is a potent vasorelaxant, and its deficiency is implicated in the pathogenesis of hypertension and atherosclerosis. Cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfurtransferase catalyze H(2)S formation. Chronic kidney disease is characterized by high prevalence of hyperhomocysteinemia, hypertension, and high cardiovascular mortality, especially in hemodialysis patients. H(2)S levels are decreased in hemodialysis patients through transcriptional deregulation of genes encoding for the H(2)S-producing enzymes. Potential implications relate to the pathogenesis of the manifestations of the uremic syndrome, such as hypertension and atherosclerosis.

Hyperhomocysteinemia in Chronic Renal Failure: Alternative Therapeutic Strategies

Chronic renal failure and uremia represent states wherein high blood levels of homocysteine, a cardiovascular risk factor, are largely resistant to folate therapy. Indeed, normalization of homocysteine levels through vitamin administration is rarely achieved in this population, and this fact could explain, among other causes, the negative results of intervention trials designed to lower cardiovascular risk. Dialysis itself lowers homocysteine levels, albeit transitorily. N-acetylcysteine therapy could induce an additional decrease in homocysteine removal during dialysis, thus representing an alternative approach in the attempt to lower cardiovascular risk in these patients.

Plasma protein homocysteinylation in uremia

Abstract Protein homocysteinylation is proposed as one of the mechanisms of homocysteine toxicity. It occurs through various means, such as the post-biosynthetic acylation of free amino groups (protein-N-homocysteinylation, mediated by homocysteine thiolactone) and the formation of a covalent -S-S- bond found primarily with cysteine residues (protein-S-homocysteinylation). Both protein modifications are a cause of protein functional derangements. Hemodialysis patients in the majority of cases are hyperhomocysteinemic, if not malnourished. Protein-N-homocysteinylation and protein-S-homocysteinylation are significantly increased in hemodialysis patients compared to controls. Oral folate treatment normalizes protein-N-homocysteinylation levels, while protein-S-homocysteinylation is significantly reduced. Albumin binding experiments after in vitro homocysteinylation show that homocysteinylated albumin is significantly altered at the diazepam, but not at the warfarin and salicilic acid binding sites. Clin Chem Lab Med 2007;45:1678–82.

The Sulfur Metabolite Lanthionine: Evidence for a Role as a Novel Uremic Toxin

Lanthionine is a nonproteinogenic amino acid, composed of two alanine residues that are crosslinked on their β-carbon atoms by a thioether linkage. It is biosynthesized from the condensation of two cysteine molecules, while the related compound homolanthionine is formed from the condensation of two homocysteine molecules. The reactions can be carried out by either cystathionine-β-synthase (CBS) or cystathionine-γ-lyase (CSE) independently, in the alternate reactions of the transsulfuration pathway devoted to hydrogen sulfide biosynthesis. Low plasma total hydrogen sulfide levels, probably due to reduced CSE expression, are present in uremia, while homolanthionine and lanthionine accumulate in blood, the latter several fold. Uremic patients display a derangement of sulfur amino acid metabolism with a high prevalence of hyperhomocysteinemia. Uremia is associated with a high cardiovascular mortality, the causes of which are still not completely explained, but are related to uremic toxicity, due to the accumulation of retention products. Lanthionine inhibits hydrogen sulfide production in hepatoma cells, possibly through CBS inhibition, thus providing some basis for the biochemical mechanism, which may significantly contribute to alterations of metabolism sulfur compounds in these subjects (e.g., high homocysteine and low hydrogen sulfide). We therefore suggest that lanthionine is a novel uremic toxin.

Hydrogen sulfide increases after a single hemodialysis session

To the Editor: Hydrogen sulfide, H2S, the third endogenous gas with cardiovascular properties after nitric oxide and carbon monoxide, is a newly recognized vasorelaxant, and H2S deficiency is involved in the pathogenesis of hypertension and atherosclerosis.1 We demonstrated that in hemodialysis patients, H2S levels are decreased through transcriptional deregulation of genes encoding H2S-producing enzymes.2 In fact, gene expression of cystathionine γ-lyase (CSE), an enzyme implicated in H2S formation, is downregulated in these patients.2 We measured H2S levels before and after a single standard 4-h hemodialysis session in 131 patients, in two dialysis sessions separated by 1 month. Measurement was performed by a slight modification of our previously published method.2 Basically, a 20-min incubation period was added before deproteinization. Homocysteine is a main direct H2S precursor in vivo, which functions through the catalytic activity of either cystathionine β-synthase or CSE (see Perna et al.3 for a review). Plasma total homocysteine was also measured by using high-performance liquid chromatography separation and fluorescence detection.4