MYOGLOBIN INHIBITS PROLIFERATION OF CULTURED HUMAN PROXIMAL TUBULAR (HK-2) CELLS

Following nephrotoxic injury, renal repair is dependent on tubular reg eneration. In the case of myoglobinuric acute renal failure (ARF), per sistence of myoglobin within tubular cells, or sublethal injury sustai ned at tile height of exposure to it, might retard this process. To te st this hypothesis, a human proximal tubular cell line (HK-2) was cult ured for 24 hours in the absence or presence of clinically relevant my oglobin concentrations (0.5, 1, 2, 4 mg/ml). Immediately following myo globin removal, lethal cell injury (vital dye uptake), lipid peroxidat ion. and DNA damage (alkaline unwinding assay) were assessed. The exte nt of cell proliferation was estimated over the next foul days by a te trazolium based (MTT) assay and by determining total intracellular LDH . Myoglobin's effects on protein and DNA synthesis were also assessed (S-35-methionine and bromodeoxyuridine incorporation, respectively). M yoglobin induced dose-dependent lipid peroxidation (malondialdehyde ge neration) and cell death (up to 80% vital dye uptake with the 4 mg/ml challenge). Although 1 mg/ml myoglobin caused no cell death, it induce d nearly complete growth arrest. This lasted for approximately three d ays following myoglobin removal from the media. Neither of two control proteins (albumin: lysozyme) nor a second nephrotoxin (gentamicin; 1 mg!ml) reproduced this effect. The 1 mg/ml myoglobin challenge caused an 80 to 90% depression in protein and DNR synthesis. It also induced significant DNA damage, as assessed by the alkaline unwinding assay (P < 0.01). Iron chelation therapy (defetoxamine) mitigated myoglobin-in duced cell killing. However, its addition following myoglobin loading worsened HK-2 outgrowth by exerting a direct anti-proliferative effect . These results indicate that: (i) sublethal myoglobin toxicity can in duce transient proximal tubular cell growth arrest, potentially slowin g recovery from ARF: (2) this effect correlates with, and could result from, heme-induced DNA damage and a blockade in DNA/protein synthesis , and (3) deferoxamine can inhibit proximal tubular cell proliferation . This possibility needs to be considered in designing clinical trials viith DFO for myohemoglobinuric ARF.