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

Modulazione dell'espressione della Serpina Proteasi Nexina 1 (PN1) in sistemi cellulari neurali e gliali

Le serpine (serine proteinase inhibitors) sono una superfamiglia di proteine caratterizzate da un comune ripiegamento e dal meccanismo d'inibizione che è del tipo substrato suicida. Il meccanismo di inibizione consiste nella formazione di un complesso irreversibile tra serpina e proteasi che una volta internalizzato viene degradato. Tra le serpine la Proteasi Nexina-1 (PN1), uno tra i maggiori inibitori della trombina, è presente principalmente nella matrice del sistema nervoso centrale. La sua funzione in questo distretto si pensa sia legato all’inibizione della trombina, che su cellule nervose differenziate causa retrazione dei neuriti. Inoltre, la PN1 è espressa con un particolare pattern spaziale e temporale durante lo sviluppo e l’organogenesi del sistema nervoso centrale, che fa supporre un suo coinvolgimento in questi processi. Per chiarire le funzioni della PN1 sono stati messi a punto un sistema per l’over-espressione inducibile e un sistema per il silenziamento genico di questa proteina. Nel primo caso è stato utilizzato come modello sperimentale il sistema tet-off in cellule PC12, cellule di feocromocitoma di ratto. Le cellule PC12 sono in grado di differenziarsi in neuroni mediante aggiunta di Nerve Growth Factor (NGF) o mediante deprivazione di siero. Il sistema tet-off permette di indurre l’over-espressione della proteina di interesse e studiarne gli effetti sulle cellule. Il plasmide per l’over-espressione inducibile contenente il cDNA della PN1 è stato cotransfettato insieme al plasmide contenente il gene per la resistenza all’igromicina nelle cellule PC12 tet-off. I cloni stabili sono stati isolati ed amplificati. Dall’analisi delle proteine, da lisati totali mediante western blot, e dall’analisi dell’mRNA, mediante RT-PCR semiquantitativa, alcuni dei cloni analizzati sono risultati inducibili. I cloni che hanno mostrato una maggiore produzione di PN1, successivamente all’induzione, sono stati analizzati morfologicamente durante il differenziamento, indotto sia dall’aggiunta di NGF al mezzo, sia da deprivazione di siero. Gli esperimenti condotti in presenza di NGF hanno mostrato un ruolo protettivo, nei confronti delle cellule differenziate, dell’over-espressione di PN1 in caso di aggiunta di trombina al mezzo di coltura. Gli esperimenti condotti in assenza di siero mostrano una diminuzione del numero di cellule differenziate in caso di over-espressione di PN1. Dai campioni analizzati morfologicamente durante il differenziamento sono stati preparati i lisati totali per osservare la produzione di PN1 mediante western blot. Da tale analisi è risultato un aumento di espressione della serpina indipendente dall’induzione del sistema tet-off. Tale aumento, da successivi esperimenti su cellule PC12 wild-type, è risultato dipendente sia dal tempo, sia dalla concentrazione di trombina. Per ottenere il silenziamento genico della PN1, è stato utilizzato l’RNA interference in cellule C6. A questo scopo sono stati clonati nel plasmide pAVU6+27 vari tipi di oligonucleotidi, in grado di attivare il processo di RNA interference, a valle di un promotore per piccoli RNA. I plasmidi ottenuti sono stati transfettati nelle cellule C6. A 48 ore dalla transfezione le cellule sono state raccolte per estrarre RNA totale e lisati totali. Dall’analisi dell’RNA mediante northern blot è risultata una diminuzione di mRNA di PN1 variabile a seconda dei costrutti utilizzati, comunque mai superiore al 60%. Dall’analisi dei lisati, mediante western blot, è risultata una diminuzione della proteina di circa 80% in tre dei campioni analizzati. Sebbene il processo di RNA interference non provochi un’efficiente degradazione del mRNA di PN1, c’è un efficiente blocco della traduzion

Novel applications of lead acetate and flow cytometry methods for detection of sulfur-containing molecules

Hydrogen sulfide (H2S) is the most recently established gaseous vasodilator, enzymatically produced from cysteine metabolism, involved in a number of pathophysiological processes. However, its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur derivatives, thus limiting the data interpretation of its biological roles. We developed new applications of the simple and rapid method to measure H2S release in cell culture systems, based on the lead acetate strip test. This test, previously prevalently used in microbiology, was compared with the agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur represents the major species derived from intracellular H2S. Various fluorescent probes are available for quantitation of H2S derivatives intracellularly. We present here an alternative to the classic imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay. The new application methods for H2S determination appear to be fully suitable for the analysis of H2S release and sulfane sulfur content in biological samples

Zebrafish as a Model of Cardiac Pathology and Toxicity: Spotlight on Uremic Toxins

: Chronic kidney disease (CKD) is an increasing health care problem. About 10% of the general population is affected by CKD, representing the sixth cause of death in the world. Cardiovascular events are the main mortality cause in CKD, with a cardiovascular risk 10 times higher in these patients than the rate observed in healthy subjects. The gradual decline of the kidney leads to the accumulation of uremic solutes with a negative effect on every organ, especially on the cardiovascular system. Mammalian models, sharing structural and functional similarities with humans, have been widely used to study cardiovascular disease mechanisms and test new therapies, but many of them are rather expensive and difficult to manipulate. Over the last few decades, zebrafish has become a powerful non-mammalian model to study alterations associated with human disease. The high conservation of gene function, low cost, small size, rapid growth, and easiness of genetic manipulation are just some of the features of this experimental model. More specifically, embryonic cardiac development and physiological responses to exposure to numerous toxin substances are similar to those observed in mammals, making zebrafish an ideal model to study cardiac development, toxicity, and cardiovascular disease

Uremic Toxin Lanthionine Induces Endothelial Cell Mineralization In Vitro

: Vascular calcification (VC) is a pathological event caused by the unusual deposition of minerals in the vascular system, representing the leading cause of cardiovascular mortality in chronic kidney disease (CKD). In CKD, the deregulation of calcium and phosphate metabolism, along with the effect of several uremic toxins, act as key processes conveying altered mineralization. In this work, we tested the ability of lanthionine, a novel uremic toxin, to promote calcification in human endothelial cell cultures (Ea.hy926). We evaluated the effects of lanthionine, at a concentration similar to that actually detected in CKD patients, alone and under pro-calcifying culture conditions using calcium and phosphate. In pro-calcific culture conditions, lanthionine increased both the intracellular and extracellular calcium content and induced the expression of Bone Morphogenetic Protein 2 (BMP2) and RUNX Family Transcription Factor 2 (RUNX2). Lanthionine treatment, in pro-calcifying conditions, raised levels of tissue-nonspecific alkaline phosphatase (ALPL), whose expression also overlapped with Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1) gene expression, suggesting a possible role of the latter gene in the activation of ALPL. In addition, treatment with lanthionine alone or in combination with calcium and phosphate reduced Inorganic Pyrophosphate Transport Regulator (ANKH) gene expression, a protective factor toward the mineralizing process. Moreover, lanthionine in a pro-calcifying condition induced the activation of ERK1/2, which is not associated with an increase in DKK1 protein levels. Our data underscored a link between mineral disease and the alterations of sulfur amino acid metabolisms at a cell and molecular level. These results set the basis for the understanding of the link between uremic toxins and mineral-bone disorder during CKD progression

Lanthionine, a Novel Uremic Toxin, in the Vascular Calcification of Chronic Kidney Disease: The Role of Proinflammatory Cytokines

Vascular calcification (VC) is a risk factor for cardiovascular events and mortality in chronic kidney disease (CKD). Several components influence the occurrence of VC, among which inflammation. A novel uremic toxin, lanthionine, was shown to increase intracellular calcium in endothelial cells and may have a role in VC. A group of CKD patients was selected and divided into patients with a glomerular filtration rate (GFR) of 2 and ≥45 mL/min/1.73 m2. Total Calcium Score (TCS), based on the Agatston score, was assessed as circulating lanthionine and a panel of different cytokines. A hemodialysis patient group was also considered. Lanthionine was elevated in CKD patients, and levels increased significantly in hemodialysis patients with respect to the two CKD groups; in addition, lanthionine increased along with the increase in TCS, starting from one up to three. Interleukin IL-6, IL-8, and Eotaxin were significantly increased in patients with GFR 2 with respect to those with GFR ≥ 45 mL/min/1.73 m2. IL-1b, IL-7, IL-8, IL-12, Eotaxin, and VEGF increased in calcified patients with respect to the non-calcified. IL-8 and Eotaxin were elevated both in the low GFR group and in the calcified group. We propose that lanthionine, but also IL-8 and Eotaxin, in particular, are a key feature of VC of CKD, with possible marker significance

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-β-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

miRNA-23a modulates sodium-hydrogen exchanger 1 expression: studies in medullary thick ascending limb of salt-induced hypertensive rats

Background. The kidney is the main organ in the pathophys- iology of essential hypertension. Although most bicarbonate reabsorption occurs in the proximal tubule, the medullary thick ascending limb (mTAL) of the nephron also maintains acid–base balance by contributing to 25% of bicarbonate reab- sorption. A crucial element in this regulation is the sodium- hydrogen exchanger 1 (NHE1), a ubiquitous membrane protein controlling intracellular pH, where proton extrusion is driven by the inward sodium ux. MicroRNA (miRNA) expression of hypertensive patients signi cantly di ers from that of normotensive subjects. The aim of this study was to determine the functional role of miRNA alterations at the mTAL level. Methods. By miRNA microarray analysis, we identi ed miRNA expression pro les in isolated mTALs from high sodium intake–induced hypertensive rats (HSD) versus their normotensive counterparts (NSD). In vitro validation was carried out in rat mTAL cells. Results. Five miRNAs involved in the onset of salt-sensitive hypertension were identi ed, including miR-23a, which was bioinformatically predicted to target NHE1 mRNA. Data demonstrated that miRNA-23a is downregulated in the mTAL of HSD rats while NHE1 is upregulated. Consistently, transfec- tion of an miRNA-23a mimic in an mTAL cell line, using a viral vector, resulted in NHE1 downregulation. Conclusion. NHE1, a protein involved in sodium reabsorption at the mTAL level and blood pressure regulation, is upregulated in our model. This was due to a downregulation of miRNA-23a. Expression levels of this miRNA are in uenced by high sodium intake in the mTALs of rats. The downregulation of miRNA- 23a in humans a ected by essential hypertension corroborate our data and point to the potential role of miRNA-23a in the regulation of mTAL function following high salt intake