Background: In proteinuria, proximal tubular epithelial cells (PTECs) are exposed to abnormally high protein concentrations, eventually leading to tubular atrophy and end-stage renal disease. The mode of cell death leading to tubular atrophy in proteinuria has not been fully established. This study examines the role of protein overload on apoptosis, necrosis and cell proliferation in primary cultures of human PTECs using plasma protein fractions representative of selective and non-selective proteinuria. The involvement of the Fas/Fas ligand (FasL) system was also investigated.
Methods: Plasma was collected from healthy volunteers and fractionated into albumin-rich (30-100 kDa), high molecular weight (100-440 kDa) and combined (30-440 kDa) fractions. PTECs were exposed to 10 mg/ml of the protein fractions for 24, 48 and 72 h. Apoptosis was measured using fluorescein isothiocyanate (FITC)-annexinV and TUNEL. Necrosis was measured using propidium iodide, metabolic activity by MTT and cell proliferation by bromodeoxyuridine incorporation. Fas and FasL expression was analysed by western blotting.
Results: Exposure to the 100-440 and 30-440 kDa fractions produced significant increases in apoptosis at all time points, whereas PTECs exposed to the 30-100 kDa fraction were not significantly different from control cells. There were no changes in the rates of necrosis as a result of protein loading. A significant reduction in metabolic activity was observed in PTECs exposed to the 100-440 and 30-440 kDa fractions, but not to the 30-100 kDa fraction. Cell proliferation was significantly reduced by 24 h in cells exposed to the 100-440 and 30-440 kDa fractions. By 48 and 72 h, all the three fractions had inhibited cell proliferation. PTECs exposed to the 100-440 and the 30-440 kDa fractions showed a significant upregulation in the expression of Fas and FasL. Overall, the high molecular weight fraction was more 'toxic' than the albumin-rich or combined fraction.
Conclusion: Increased apoptosis and decreased cell proliferation are the major mechanisms of cell death in human PTECs in response to protein overload. These effects may be mediated at least in part by overexpression of the Fas/FasL system. The severity of such changes is largely determined by the high molecular weight fraction (100-440 kDa) rather than the albumin-rich fraction.