Mechanisms Of Myoglobin Induced Injury In Tubular Epithelial Cells And Immunoadsorbent Device Construction

Backgroundst Objective:About 13-50% of rhabdomyolysis is complicated with AKI and myoglobin is the key element. Literatures about myoglobin toxicity mainly focus on oxidative stress, but the results diverse. The excact mechanisms of myoglobin induced kidney injury remains unclear. Apoptosis often occurs in animal models of AKI or cultured cells. Endoplasmic reticulum stress (ERS) has recently been found to play an important role in apoptosis. Myoglobin is able to induce apoptosis in tubular epithelial cells, but whether it is mediated by ERS and the signaling pathways are still unknown. In this study we mainly focus on myoglobin renal toxicity and the ERS mechanisms in myoglobin induced tubular epithelial cell apoptosis, then construct experimental immunoadsorbent devices.Methods:1.Valent states of coordinated iron affect myoglobin toxicity:i)HK-2 cells were incubated with ferrous or ferric myoglobin, respectively, then cell viability was evaluated by MTT assay, cell injury by LDH release test. ii)HK-2 cells were incubated with different doses of ferrous myoglobin to analyse the dose-effect relationships.2.ERS mechanisms in myoglobin induced apoptosis: i)HK-2 cells were incubated with ferrous myoglobin and proteins relating to ERS or apoptosis were analysed by western blot. ii)With IP3R calcium channel blocked by 2-APB, HK-2 cells were incubated with ferrous myoglobin. Then we determined protein expression of Grp78, caspase-4, caspase-9, caspase-3 by western blot and evaluated cell apoptosis by flow cytometry.3.Construct inmmunoadsorbent devices:i)Achieved the purified rabbit anti-rat myoglobin polyclonal antibodies. ii)Prepared immunoadsorbent columns. iii)Verified the adsorption capacity in vitro.Results:1.Ferrous myoglobin cell toxicity:i)HK-2 cells incubated with ferrous myoglobin showed lower viability, more cell injury while ferric myoglobin didn't affect HK-2 cells. ii)Ferrous myoglobin dosage was correlated positively with cell LDH release and apoptosis rate, and inversely with cell viability.2.ERS mediated tubular epithelial cell apoptosis:i)Incubated with 200uM ferrous myoglobin, Grp78 increased and remained at high level in 3-12h while caspase-9 remained high in 12-24h. Caspase-4 was non significant to control. ii)Compared to merely myoglobin incubation,2-APB plus myoglobin incubation showed further more Grp78 expression, inhibited caspase-9, dramatically increased caspase-4, and cell apoptosis wasn't ameliorated.3.Immunoadsorbent devices:i)Synthesized 2 antigen peptides, L-12 and D-12. Linked with KLH and immuned rabbits, obtained 2 polyclonal antibodies, anti-L-12 and anti-D-12 pAb. ii)Determined purity, titer and specificity of the antibodies, then corsslinked anti-D-12 pAb with D-380 resin to prepare adsorbent fillings. Then assemble immunoadsorbent devices. iii)The mimic extracorporeal system verification indicated that the immnoadsorbent column had good function and reusable. FPLC verification showed the removal of myoglobin was significantly more than that of other proteins, i.e., BSA or lysozyme.Conclusions:1.Valent state of cocordinated iron in myoglobin determines its cell toxicity. It is ferrous myoglobin that causes tubular cell injuries and the toxicity is dose-dependent.2.Ferrous myoglobin can induce ERS, then mediates cell apoptosis by caspase-9 dependent mitochondrial pathway. When IP3R calcium channel is blocked, caspase-4 dependent endoplasmic reticulum specific pathway may serve as a substitutive apoptosis pathway.3.With myoglobin polyclonal antibody as the ligand, the immnoadsorbent devices have good adsorption capacity.