Exploring Key Challenges of Understanding the Pathogenesis of Kidney Disease in Bardet–Biedl Syndrome

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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

Nephroplex: a kidney-focused NGS panel highlights the challenges of PKD1 sequencing and identifies a founder BBS4 mutation

Background: Genetic testing of patients with inherited kidney diseases has emerged as a tool of clinical utility by improving the patients' diagnosis, prognosis, surveillance and therapy. Methods: The present study applied a Next Generation Sequencing (NGS)-based panel, named NephroPlex, testing 115 genes causing renal diseases, to 119 individuals, including 107 probands and 12 relatives. Thirty-five (poly)cystic and 72 non (poly)cystic individuals were enrolled. The latter subgroup of patients included Bardet-Biedl syndrome (BBS) patients, as major components. Results: Disease-causing mutations were identified in 51.5 and 40% of polycystic and non-polycystic individuals, respectively. Autosomal dominant polycystic kidney disease (ADPKD) patients with truncating PKD1 variants showed a trend towards a greater slope of the age-estimated glomerular filtration rate (eGFR) regression line than patients with (i) missense variants, (ii) any PKD2 mutations and (iii) no detected mutations, according to previous findings. The analysis of BBS individuals showed a similar frequency of BBS4,9,10 and 12 mutations. Of note, all BBS4-mutated patients harbored the novel c.332+1G>GTT variant, which was absent in public databases, however, in our internal database, an additional heterozygote carrier was found. All BBS4-mutated individuals originated from the same geographical area encompassing the coastal provinces of Naples. Discussion: In conclusion, these findings indicate the potential for a genetic panel to provide useful information at both clinical and epidemiological levels

Metabolomic fingerprinting of renal disease progression in Bardet-Biedl syndrome reveals mitochondrial dysfunction in kidney tubular cells.

Chronic kidney disease (CKD) is a major clinical sign of patients with Bardet-Biedl syndrome (BBS), especially in those carrying BBS10 mutations. Twenty-nine patients with BBS and 30 controls underwent a serum-targeted metabolomic analysis. In vitro studies were conducted in two kidney-derived epithelial cell lines, where Bbs10 was stably deleted (IMCD3-Bbs10-/-cells) and over-expressed. The CKD status affected plasmatic metabolite fingerprinting in both patients with BBS and controls. Specific phosphatidylcholine and acylcarnitines discriminated eGFR decline only in patients with BBS. IMCD3-Bbs10-/ cells displayed intracellular lipidaccumulation, reduced mitochondrial potential membrane and citrate synthase staining. Mass-Spectrometry-based analysis revealed that human BBS10 interacted with six mitochondrial proteins, in vitro. In conclusion, renal dysfunction correlated with abnormal phosphatidylcholine and acylcarnitines plasma levels in patients with BBS; in vitro, Bbs10 depletion caused mitochondrial defects while human BBS10 interacted with several mitochondria-related proteins, suggesting an unexplored role of this protein

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

Zebrafish, a Novel Model System to Study Uremic Toxins: The Case for the Sulfur Amino Acid Lanthionine

The non-proteinogenic amino acid lanthionine is a byproduct of hydrogen sulfide biosynthesis: the third endogenous vasodilator gas, after nitric oxide and carbon monoxide. While hydrogen sulfide is decreased in uremic patients on hemodialysis, lanthionine is increased and has been proposed as a new uremic toxin, since it is able to impair hydrogen sulfide production in hepatoma cells. To characterize lanthionine as a uremic toxin, we explored its effects during the early development of the zebrafish (Danio rerio), a widely used model to study the organ and tissue alterations induced by xenobiotics. Lanthionine was employed at concentrations reproducing those previously detected in uremia. Light-induced visual motor response was also studied by means of the DanioVision system. Treatment of zebrafish embryos with lanthionine determined acute phenotypical alterations, on heart organogenesis (disproportion in cardiac chambers), increased heart beating, and arrhythmia. Lanthionine also induced locomotor alterations in zebrafish embryos. Some of these effects could be counteracted by glutathione. Lanthionine exerted acute effects on transsulfuration enzymes and the expression of genes involved in inflammation and metabolic regulation, and modified microRNA expression in a way comparable with some alterations detected in uremia. Lanthionine meets the criteria for classification as a uremic toxin. Zebrafish can be successfully used to explore uremic toxin effects

The role of the intestinal microbiota in uremic solute accumulation : a focus on sulfur compounds

The gut microbiota is considered to be a novel important factor to take into account in the pathogenesis of chronic kidney disease and uremia. Much attention has been paid to specific uremic retention solutes of microbial origin, such as indoxyl sulfate, p-cresyl sulfate, and trimethylamine-N-oxide. However, other novel less well studied compounds, such as hydrogen sulfide and related sulfur metabolites (sulfane sulfur, lanthionine, etc.), should be included in a more comprehensive appraisal of this topic, in light of the potential therapeutic opportunities for the future

Proteomics and metabolomics studies exploring the pathophysiology of renal dysfunction in autosomal dominant polycystic kidney disease and other ciliopathies

The primarycilium(PC)wasconsideredasa vestigial organelle withnosignificantphysiologicalimportance,untilthediscovery that PC perturbation disturbs several signalling pathways and results in the dysfunction of a variety of organs. Genetic studies have demonstrated that mutations affecting PC proteins or its anchoring structure, the basal body, underlie a class of human disorders (known as ciliopathies) characterized by a constella- tion of clinical signs. Further investigations have demonstrated that the PC is involved in a broad range of biological processes, inbothdevelopingandmaturetissues.Kidneydiseaseisacom- monclinicalfeatureofciliadisorders,supportingthehypothesis of a crucial role of the PC in kidneyhomoeostasis. Clinical pro- teomics and metabolomics are an expanding research area. Interestingly, the application of these methodologies to the analysis of urine, a biological sample that can be collected in a non-invasive fashion and possibly in large amounts, makes these studies feasible also in patients. The present article describes the most recent proteomic and metabolomic studies exploring kidney dysfunction in the setting of ciliopathies, showing the potential of these methodologies in the elucidation ofdiseasepathophysiologyandinthediscoveryofbiomarkers