Myoglobin inhibits proliferation of cultured human proximal tubular (HK-2) cells

Following nephrotoxic injury, renal repair is dependent on tubular regeneration. In the case of myoglobinuric acute renal failure (ARF), persistence of myoglobin within tubular cells, or sublethal injury sustained at the height of exposure to it, might retard this process. To test this hypothesis, a human proximal tubular cell line (HK-2) was cultured for 24 hours in the absence or presence of clinically relevant myoglobin concentrations (0.5, 1, 2, 4 mg/ml). Immediately following myoglobin removal, lethal cell injury (vital dye uptake), lipid peroxidation. and DNA damage (alkaline unwinding assay) were assessed. The extent of cell proliferation was estimated over the next foul days by a tetrazolium 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). Myoglobin induced dose-dependent lipid peroxidation (malondialdehyde generation) 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 induced nearly complete growth arrest. This lasted for approximately three days 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-induced 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 induce transient proximal tubular cell growth arrest, potentially slowing 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.