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

Plasma proteins containing damaged L-isoaspartyl residues are increased in uremia: Implications for mechanism

Plasma proteins containing damaged L-isoaspartyl residues are increased in uremia: Implications for mechanism.BackgroundSeveral alterations of protein structure and function have been reported in uremia. Impairment of a transmethylation-dependent protein repair mechanism possibly related to a derangement in homocysteine metabolism is also present in this condition, causing erythrocyte membrane protein damage. Homocysteine may affect proteins via the accumulation of its parent compound S-adenosylhomocysteine (AdoHcy), a powerful in vivo methyltransferase inhibitor. However, since plasma homocysteine is mostly protein bound, a direct influence on protein structures cannot be ruled out. We measured the levels of L-isoaspartyl residues in plasma proteins of uremic patients on hemodialysis. These damaged residues are markers of molecular age, which accumulate when transmethylation-dependent protein repair is inhibited and/or protein instability is increased.MethodsL-isoaspartyl residues in plasma proteins were quantitated using human recombinant protein carboxyl methyl transferase (PCMT). Plasma concentrations of homocysteine metabolites were also measured under different experimental conditions in hemodialysis patients.ResultsThe concentration of damaged plasma proteins was increased almost twofold compared to control (controls 147.83 ± 17.75, uremics 282.80 ± 26.40 pmol of incorporated methyl groups/mg protein, P < 0.003). The major protein involved comigrated with serum albumin. Although hyperhomocysteinemia caused a redistribution of thiols bound to plasma proteins, this mechanism did not significantly contribute to the increase in isoaspartyl residues. The S-adenosylmethionine (AdoMet)/AdoHcy concentration ratio, an indicator of the flux of methyl group transfer, was altered. This ratio was partially corrected by folate treatment (0.385 ± 0.046 vs. 0.682 ± 0.115, P < 0.01), but protein L-isoaspartate content was not.ConclusionsPlasma protein damage, as determined by protein L-isoaspartyl content, is increased in uremia. This alteration is to be ascribed to an increased protein structural instability, rather than the effect of hyperhomocysteinemia

Uremic Toxin Lanthionine Interferes with the Transsulfuration Pathway, Angiogenetic Signaling and Increases Intracellular Calcium

(1) The beneficial effects of hydrogen sulfide (H2S) on the cardiovascular and nervous system have recently been re-evaluated. It has been shown that lanthionine, a side product of H2S biosynthesis, previously used as a marker for H2S production, is dramatically increased in circulation in uremia, while H2S release is impaired. Thus, lanthionine could be classified as a novel uremic toxin. Our research was aimed at defining the mechanism(s) for lanthionine toxicity. (2) The effect of lanthionine on H2S release was tested by a novel lead acetate strip test (LAST) in EA.hy926 cell cultures. Effects of glutathione, as a redox agent, were assayed. Levels of sulfane sulfur were evaluated using the SSP4 probe and flow cytometry. Protein content and glutathionylation were analyzed by Western Blotting and immunoprecipitation, respectively. Gene expression and miRNA levels were assessed by qPCR. (3) We demonstrated that, in endothelial cells, lanthionine hampers H2S release; reduces protein content and glutathionylation of transsulfuration enzyme cystathionine--synthase; modifies the expression of miR-200c and miR-423; lowers expression of vascular endothelial growth factor VEGF; increases Ca2+ levels. (4) Lanthionine-induced alterations in cell cultures, which involve both sulfur amino acid metabolism and calcium homeostasis, are consistent with uremic dysfunctional characteristics and further support the uremic toxin role of this amino acid

Polymorphism of Beta2-Adrenoceptor and Regular Use of Formoterol in Asthma: Preliminary Results

Polymorphism at codon 16 of the beta2-adrenoceptor (beta2-AR) affects the responsiveness to salmeterol in asthmatics. Data concerning formoterol are more controversial in the literature. The aim of this study was to verify whether homozygous for arginine-16 (ArgArg16) and homozygous for glycine-16 (GlyGly16) genotypes differently influence the long-term responsiveness to formoterol. Twenty-nine patients with mild-to-moderate asthma, in stable clinical conditions, underwent genotyping at codon 16 of the beta2-AR by RFLP-PCR assay. The effects of a 4-week monotherapy with formoterol (12 μg BID) were tested on the peak expiratory flow (PEF) variability and the forced expiratory volume in 1 sec (FEV1) slope of the dose-response curve to salbutamol. Variability in PEF significantly increased during the 4-week treatment period in 14 patients with GlyGly16, but not in 15 patients with ArgArg16 and ArgGly16 (P=0.032). The FEV1 slope of the dose-response curve to salbutamol decreased after the 4-week treatment period in GlyGly16, but not in pooled ArgArg16 and ArgGly16 patients. This study provides preliminary evidence that tolerance to formoterol develops more frequently in asthmatics with GlyGly16 genotype. If confirmed in a larger population, this finding might be useful in choosing the bronchodilator therapy on the basis of genetic polymorphism of the beta2-AR

Application of a Clinical Workflow May Lead to Increased Diagnostic Precision in Hereditary Spastic Paraplegias and Cerebellar Ataxias: A Single Center Experience

The molecular characterization of Hereditary Spastic Paraplegias (HSP) and inherited cerebellar ataxias (CA) is challenged by their clinical and molecular heterogeneity. The recent application of Next Generation Sequencing (NGS) technologies is increasing the diagnostic rate, which can be influenced by patients\u2019 selection. To assess if a clinical diagnosis of CA/HSP received in a third-level reference center might impact the molecular diagnostic yield, we retrospectively evaluated the molecular diagnostic rate reached in our center on 192 unrelated families (90 HSP and 102 CA) (i) before NGS and (ii) with the use of NGS gene panels. Overall, 46.3% of families received a genetic diagnosis by first-tier individual gene screening: 43.3% HSP and 50% spinocerebellar ataxias (SCA). The diagnostic rate was 56.7% in AD-HSP, 55.5% in AR-HSP, and 21.2% in sporadic HSP. On the other hand, 75% AD-, 52% AR- and 33% sporadic CA were diagnosed. So far, 32 patients (24 CA and 8 HSP) were further assessed by NGS gene panels, and 34.4% were diagnosed, including 29.2% CA and 50% HSP patients. Eleven novel gene variants classified as (likely) pathogenic were identified. Our results support the role of experienced clinicians in the diagnostic assessment and the clinical research of CA and HSP even in the next generation era

Homocysteinylated Albumin Promotes Increased Monocyte-Endothelial Cell Adhesion and Up-Regulation of MCP1, Hsp60 and ADAM17

RATIONALE:The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties. OBJECTIVE:Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels. METHODS AND RESULTS:Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2. CONCLUSIONS:Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling

Integrin Beta 1 Is Crucial for Urinary Concentrating Ability and Renal Medulla Architecture in Adult Mice

Integrins are heterodimers anchoring cells to the surrounding extracellular matrix (ECM), an active and complex process mediating a series of inside-out and outside-in stimuli regulating cellular turn-over, tissue growth and architecture. Itgb1 is the main subunit of the renal integrins and it is critical for renal development. This study aims to investigate the role of Itgb1 in the adult renal epithelial cells by knocking down Itgb1 in PAX8 expressing cells. Itgb1-Pax8 cKO mice develop a progressively worsening proteinuria and renal abnormalities leading to severe renal failure and hypertension. This phenotype is also associated with severe dysfunction of distal nephron and polyuria. To further investigate whether distal nephron involvement was primarily related to Itgb1 suppression or secondary to renal failure, an Itgb1-AQP2 cKO mouse model was generated. These mice lack Itgb1 expression in AQP2 expressing cells. They do not show any developmental alteration, but 1 month old mice are resistant to dDAVP administration and finally, at 2 months of age, they develop overt polyuria. This phenotype is due to primary collecting duct (CD) cells anoikis. The entire architecture of the outer medulla is altered, with loss of the typical organization pattern of vascular and tubular bundles alternation. Indeed, even though not primarily affected by genetic ablation, the TAL is secondarily affected in this model. It is sufficient to suppress Itgb1 expression in the CD in order to stimulate proliferation and then disappearance of neighboring TAL cells. This study shows that cell to cell interaction through the ECM is critical for architecture and function maintenance of the outer medulla and that Itgb1 is crucial for this process

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor