Reversal by Growth Hormone of Homocysteine-induced Epithelial-to-mesenchymal Transition Through Membrane Raft-redox Signaling in Podocytes

Epithelial-to-Mesenchymal Transition (EMT) is an important pathogenic mechanism mediating glomerular injury or sclerosis in a variety of renal and systemic diseases such as hyperhomocysteinemia (hHcys). The present study was designed to test whether Hcys-induced EMT in podocytes is reversed by growth hormone (GH), a hormone regulating cell differentiation and growth and to explore the cellular and molecular mechanism mediating its action. It was found that Hcys induced significant EMT in podocytes, as shown by marked decreases in slit diaphragm-associated protein P-cadherin and zonula occludens-1 as epithelial markers and by dramatic increases in the expression of mesenchymal markers, fibroblast specific protein-1 and α-smooth muscle actin, which were detected by all examinations via immunocytochemistry, real time RT-PCR and Western blot analysis. When podocytes were treated with GH at 25 ng/mL, however, Hcys failed to induce podocyte EMT. Using electromagnetic spin resonance spectrometry, Hcys-induced superoxide (O(2).(-)) production via NADPH oxidase was found to be significantly inhibited by GH (66%). Functionally, GH was shown to substantially inhibit Hcys-induced increases in the permeability of podocyte monolayers and to block the decrease in podocin expression in these cells. In addition, NADPH oxidase subunit, gp91(phox) and GH receptors aggregated in membrane raft clusters, which produced O(2).(-) in response to Hcys and could be blocked by GH, membrane raft disruptors filipin and MCD or NADPH oxidase inhibitor, apocynin. It is concluded that Hcys-induced podocyte EMT is associated with transmembrane membrane raft-redox signaling and that GH reverses this Hcys-induced EMT protecting podocytes from functional disturbance.