HYPERPHOSPHATEMIA INDUCES INTEGRIN LINKED KINASE EXPRESSION AND LEADS TO CELLULAR SENESCENCE AND AGING: STUDIES IN VIVO AND IN VITRO

NURIA TROYANO; MARÍA DEL NOGAL; INÉS MORA; GEMMA OLMOS; M. LUISA DIEZ-MARQUEZ; M. ALICIA CORTÉS; SUSANA LOPEZ-ONGIL; PALOMA MARTÍN; DIEGO RODRIGUEZ-PUYOL; MARIA P. RUIZ-TORRES

Istanbul

Simposio; 50th ERA-EDTA CONGRESS; 2013

EDTA

Introduction and Aims: Hyperphosphatemia has been related to premature aging in the Klotho deficient mice and in de FGF23 KO mice. Serum phosphate levels inversely correlated with species lifespan. Senescence is a cell cycle arrest promoted by the replicative life of the cells or by stressful stimuli. Mechanisms involved in aging-induced by hyperphosphatemia are actually unknown. Integrin linked kinase (ILK) is a protein involved in the relationship between cells and surrounding extracellular matrix regulating cellular proliferation and survival. It has been previously described that ILK was increased in old animals. Aim: To explore the role of hyperphosphatemia in renal and vascular aging. We propose that serum phosphate levels increases with aging in mice and promote the upregulation of the ILK expression leading to cellular senescence. Methods: We compare 3 months old C57Bl 6 mice with 15 months old mice. We analyze serum phosphate by a colorimetric method and the mRNA expression of Klotho, NaPi2a transporter, 1a-hydroxylase and 24-hydroxylase and ILK by real time RT- PCR in kidney. We analyzed the expression of senescent genes, p53 and p16 by western blot in kidney from these animals . To analyze whether hyperphosphatemia induces ILK expression leading to cellular senescence we used human vascular smooth muscle cells (HVSMC) treated with 10 mM beta-glycerophosphate (BGP) during 24, 48 or 72 hours. Results: We found that 15 month old mice show higher serum phosphate, decreased Klotho expression, increased NaPi2a and 1α-hydroxylase expressions and decreased 24-hydroxylase expression than young animals. Old animals also have a higher expression of ILK. A significant correlation was found between high serum phosphate and increased ILK expression. We found an increase in the expression of senescent genes p53 and p16 in old animals. We found that BGP increases ILK, p53 and p16 expression, assessed by western blot, and induces senescence associated- beta-galactosidase expression evaluated by confocal microscopy with a fluorescent probe. The increase was inhibited using the inhibitor of Pit-1, phosphonoformic acid (PFA) 0.5mM. ILK silenced with small siRNA in HVSMC inhibited the senescence induced by BGP. Conclusions: We conclude that old animals show a decreased expression of Klotho protein disturbing the phosphate homeostasis and leading to the increase in serum phosphate levels. The resulting hyperphosphatemia seem to induce senescence in kidney and vascular cells through the increment of ILK expression.