Endoplasmic Reticulum Stress In The Podocytes Regulation On Calcineurin Expression

BackgroundDiabetic nephropathy (DN) is the second cause of end stage renal disease in China today. The pathogenesis of DN has not been understood due to its complicated pathways. Intensive investigation will guide more efficient clinical treatment strategy. Calcineurin (CaN) is a kind of calcium-dependent phosphatase. It can directly regulate the stability of podocyte’s skeleton, which is thought the mainstay to prevent podocyte injury and onset of proteinuria. The endoplamic reticulum (ER) is the organelle where secretory and membrane proteins are folded. The accumulation of unfolded and misfolded proteins constitutes a form of cellular stress termed endoplasmic reticulum stress (ERS). Recently, it is found that under endoplasmic reticulum stress (ERS), the activity of calcineurin is upregulated. Paltimate is a causative factor of ERS in DM, while ursodeoxycholic acid (UDCA) can alleviate ERS by preventing the phosphorylation of ERS-sensitive proteins PER-like ER kinase (PERK) and inositol-requiring enzyme1α (IRE-1α), and further suppress the activation of ERS-dependent c-Jun N-terminal kinase (JNK). DN is clinically characterized by proteinuria and pathologically by glomerular hypertrophy and loss of podocyte and foot process effacement. Our previous work showed that ERS in podocytes preceded the onset of microalbuminuria in db/db mice, an animal model of diabetic nephropathy. Therefore, further exploration of the regulation of CaN by ERS in podocytes will extend the understandings of microalbuminuria onset in DN and guide the treatment to alleviate the ERS or regulate the activity of CaN.ObjectiveTo investigate the regulation of calcineurin by ERS in podocytes in vitro and in vivo..MethodsThe expressions of calcineurin and synaptopodin were observed in C57BLKS db/db mice at the age of6,9and12weeks. Permanent mice podocytes in culture stimulated by paltimate with or without UDCA were studied. The expression of CaN in podocytes incubated with different concentrations of paltimate was quantitatively determined by Real-time PCR. The changes of calcineurin incubated with paltimate with or without UDCA were analyzed by confocal microscopy and western blotting analysis. The expression of synaptopodin in ERS was also assessed by western blotting.Results(1) As urine protein increased, the expression of CaN was up-regulated in the podocytes of C57BLKS db/db mice and the expression of synaptopodin was down-regulated under ERS.(2) As the concentration of paltimate increased from250μM to500μM, mRNA of CaN was upregulated (P<0.05). By confocal microscopy, the fluorescence intensity of CaN was increased in paltimate treatment group. Co-incubation with paltimate and UDCA, the fluorescence intensity of CaN in podocytes was deceased (P<0.05). The similar results were shown by western blotting.Conclusions(2)The expression of calcineurin was up-regulated in the glomerulus of C57BLKS db/db mice while that of synaptopodin was down-regulated.(1) The expression of calcineurin in podocyte was up-regulated under ERS while ERS pathway inhibitor, UDCA could inhibit it, suggesting ERS pathway induced podocyte injury worth to be further investigated to prevent podocyte injury from DN.