The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells

Background. Activation of urinary complement proteins in situ by proximal tubular epithelial cells (PTEC) may contribute to the mediation of tubulointerstitial injury ill patients with significant proteinuria. However, the mechanism involved is unclear, and the role of changes in urinary pH and in the concentrations of urea or ammonia requires further clarification. Methods. The protein fraction of urine samples from nine patients with proteinuria > 1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.5-8.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition. Results. Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components. Conclusions. These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a 'protected site' on their surface, rather than by the activation of C3 by convertase-like protease(s).