Use of monoclonal antibodies to assess expression of anaphylatoxin receptors in tubular epithelial cells of human, murine and rat kidneys

To assess published evidence of anaphylatoxin receptor expression in renal tubular epithelial cells, monoclonal antibodies (mAbs) against human, mouse and rat receptors for C5a and C3a (C5aR, C3aR) were raised using receptor-expressing transfectants as immunogens. Applying these reagents in immunohistochemistry, we observed that mAbs with reactivities against three distinct epitopes of human C5aR N-terminus recognized tissue macrophages but not at all renal tubular epithelial cells. These findings were surprising, as strong tubular staining had been previously demonstrated by mAbs raised against a synthetic N-terminal C5aR peptide. To extend our study to mammalian kidneys, renal specimens from normal rats as well as LPS-treated Balb/c and MRL/lpr mice, which suffered from lupus-type nephritis, were examined. Similar to humans, mAbs against murine or rat C5aR strongly recognized infiltrating leukocytes in situ whereas tubular epithelial cells remained negative. As a mAb has been previously used to document C3aR expression in renal tubular epithelial cells, kidney specimens were examined using newly established mAbs against different epitopes of human, murine and rat C3aR. In contrast to published evidence, C3aR was detectable exclusively in interstitial leukocytes but not in epithelial tubular cells of normal and diseased tissues. Taken together, our findings question a direct involvement of tubular epithelial cells in anaphylatoxin-mediated renal inflammation. Furthermore, as we demonstrate in the case of anaphylatoxin receptors, cross-reactivities of mAbs may constitute as yet underestimated pitfalls in immunohistochemical antigen detection.