| Summary: | 碩士 === 國立清華大學 === 生物技術研究所 === 89 === Previous studies had provided evidence that exposure of renal epithelial cells to oxalate and calcium oxalate monohydrate crystals induced free radicals generation, lipid peroxidation and injured the renal cells. Since oxidant and antioxidant balance was likely to play a critical role, we wanted to demonstrated the effect of Tamm-Forsfall protein (THP), which the most abundant glycoprotein in the urine, on the production of free radicals and injury to MDCK cells from exposure to oxalate (Ox) or Ox + calcium oxalate monohydrate (COM) crystals. On the other hand, we also wanted to illustrate that whether or not deglycosylation of THP (D-NANA) would influence the rules of this important protein. MDCK cells were grown in monolayers and exposed to 1.0 mM Ox or 1.0 mM Ox + 500 mg/ml COM crystal with THP or D-NANA in serial concentrations (100, 200, and 500nM) for 120 and 240 minutes. Superoxide radicals were measured in the presence or absence of THP or D-NANA. We measured the release of lactate dehydrogenase (LDH) as a marker for cell injury, malondialdehyde (MDA) as a marker of lipid peroxidation, and the activity of cytosolic caspase 3 as a marker of early stage of cell apoptosis. We also used the phase contrast microscopy to observe the CaOx crystal retention on the MDCK cell membranes in different treatments. Exposure of MDCK cells to Ox resulted in a significant increase in release LDH and MDA, which were further elevated when COM crystals were added. In MDCK cells, the addition of THP at 500nM significantly reduced the superoxide radical generation release of LDH and increase of MDA. But add the D-NANA protein to the MDCK cell, no matter what concentration of D-NANA could not reduce the superoxide radical generation, release of LDH and increase of MDA. Results of the present studies indicated that the both Ox and COM crystals are injurious the MDCK cells in releasing LDH and increasing MDA in the cells and the injury is associated with generation of free radicals and triggering the initiation of cell apoptosis. THP, like the free radical scavengers, could reduce the free radicals and provide significant protection at a critical concentration 500nm. The deglycosylated THP could not protect the MDCK cells from Ox-induced injury. The lower content of THP or non-glycosylation of THP in the urine may be the important reason for the initiation of urolithiasis pathogenesis.
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