title: Lysine triggers apoptosis through a NADPH oxidase-dependent mechanism in human renal tubular cells.
abstract: Progressive chronic kidney disease (CKD) is common in lysinuric protein intolerance (LPI), a primary inherited aminoaciduria characterized by massive Lysine excretion in urine. However, by which mechanisms Lysine may cause kidney damage to tubule cells is still not understood. This study determined whether Lysine overloading of human proximal tubular cells (HK-2) in culture enhances apoptotic cell loss and its associated mechanisms. Overloading HK-2 with Lysine levels reproducing those observed in urine of patients affected by LPI (10 mM) increased apoptosis (+30%; p < 0.01 vs.C), as well as Bax and Apaf-1 expressions (+30-50% p < 0.05), while downregulated Bcl-2 (-40% p < 0.05). Apoptosis induced by high Lysine was no longer observed after addition of caspase-9 and caspase-3 inhibitors while caspase-8 inhibitor had no protective effect. High Lysine induced elevations in ROS generation and NADPH oxidase subunits mRNAs (p22 (phox) +106 ± 23%, p67 (phox) +108 ± 22% and gp91 (phox) +75 ± 4% p < 0.05-0.01). In addition, the NADPH oxidase inhibitor DPI prevented both ROS production and apoptosis. Treating HK-2 with antioxidants, such as Cysteine and its analog, N-acetyl-L-cysteine (NAC), rescued the HK-2 from apoptosis induced by Lysine. In summary, our data show that high Lysine in vitro increases the permissiveness of proximal tubule kidney cells to apoptosis by triggering a pathway involving NADPH oxidase signaling. This event may represent a key cellular effect in the increasing the susceptibility of human tubular cells to apoptosis when the tubules cope with a high Lysine load. This effect is instrumental to renal damage and disease progression in patients with LPI.
authors.affiliation: Department of Internal Medicine, Nephrology Division, Genoa University, IRCSS Azienda Ospedaliera Universitaria San Martino - IST, Viale Benedetto XV,6, 16132, Genoa, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Verzola
authors.forename: Annamaria
authors.initials: A
authors.lastname: Famà
authors.forename: Barbara
authors.initials: B
authors.lastname: Villaggio
authors.forename: Maia
authors.initials: M
authors.lastname: Di Rocco
authors.forename: Alchiede
authors.initials: A
authors.lastname: Simonato
authors.forename: Elena
authors.initials: E
authors.lastname: D'Amato
authors.forename: Fabio
authors.initials: F
authors.lastname: Gianiorio
authors.forename: Giacomo
authors.initials: G
authors.lastname: Garibotto
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000150:complications / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D061945:Caspase Inhibitors / Q000494:pharmacology; D002460:Cell Line; D018450:Disease Progression; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D008239:Lysine / Q000378:metabolism* / Q000633:toxicity*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D017382:Reactive Oxygen Species / Q000378:metabolism; D051436:Renal Insufficiency, Chronic / Q000209:etiology / Q000378:metabolism / Q000473:pathology
chemical_list: D000975:Antioxidants; D061945:Caspase Inhibitors; D021122:Protein Subunits; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; D008239:Lysine