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.
Subject(s)
Apoptosis/drug effects , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Lysine/metabolism , Lysine/toxicity , NADPH Oxidases/metabolism , Amino Acid Metabolism, Inborn Errors/complications , Amino Acid Metabolism, Inborn Errors/etiology , Amino Acid Metabolism, Inborn Errors/metabolism , Amino Acid Metabolism, Inborn Errors/pathology , Antioxidants/pharmacology , Apoptosis/physiology , Caspase Inhibitors/pharmacology , Cell Line , Disease Progression , Gene Expression/drug effects , Humans , Kidney Tubules, Proximal/pathology , Membrane Potential, Mitochondrial/drug effects , NADPH Oxidases/chemistry , NADPH Oxidases/genetics , Protein Subunits , Reactive Oxygen Species/metabolism , Renal Insufficiency, Chronic/etiology , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/pathologyABSTRACT
The incidence and the rate of progression of chronic kidney diseases (CKD) are for most diseases greater in men than in age-matched women. We have previously shown that testosterone (T) promotes the apoptosis of proximal tubule kidney cells. To better understand the downstream signaling process associated with T-induced apoptosis, we examined the involvement of c-Jun amino terminal kinase (JNK) in a human proximal tubule cell line (HK-2) exposed to T: JNK and its downstream effector c-Jun were rapidly phosphorylated. By blocking androgen receptor, JNK phosphorylation was reduced and 17beta-Estradiol treatment had no effect on it. Similarly, pre-treatment with the JNK inhibitor SP600125 prevented the T-induced apoptosis, the phosphorylation of c-Jun and the upregulation of the Fas/FADD pathway. These data show that the JNK/c-Jun pathway is directly regulated by androgens in vitro and highlight a potential mechanism explaining the reported gender differences in the progression of renal diseases.
