Abstract
Dr. Graham Betton (this issue) offered a valid critique of our immunohistochemistry (IHC) evaluation of the tissue/cell origin of renal papillary antigen-1 Zhang et al. (2008a), a collecting duct (CD) injury marker, and raised a very important concern related to studies employing IHC in general. He correctly pointed out that our study did not include a relevant negative control for the renal papillary antigen-1 (RPA-1) monoclonal antibody, that is, a matched isotype IgG, thus leading to potential false-positive staining of necrotic proximal tubular cells. Dr. Betton’s concerns prompted us to undertake additional IHC experiments, which we report herein (Table 1and Figure 1).
Our examination of RPA-1 IHC resulted in part from a finding in earlier studies we conducted on the appearance of several novel and traditional biomarkers in urine in response to various nephrotoxicants. In one study (Shaw et al. 2007), we observed a dose-related increase in RPA-1 in the urine of rats treated with gentamicin (Gen), well known as a classic proximal tubule toxicant. Because of this somewhat unexpected finding, we conducted IHC analyses using kidney tissue sections from the same rats to determine the cell-specific origin of RPA-1.
In the IHC analysis by Zhang et al. (2008a), we observed intense positive staining (high degree of dark brown color) using the mAb specific for RPA-1 in CD of the renal medulla and cortex in rats treated with saline, Gen, mercury (Hg) or chromium (Cr), which indicated that the RPA-1 mAb bound to the specific antigen RPA-1, localized in the CD. We also observed moderate brown staining in necrotic cells of proximal tubules. Whether the moderate brown staining in tissue sites other than those (i.e., CD) containing the primary antigen represented binding of the anti-RPA-1 to an RPA-1–like substance or nonspecific binding remained unanswered, particularly since an appropriate negative control, matched isotype IgG, was not included in IHC experiments, as suggested by Dr. Betton. Therefore, we repeated the experiments using such a control to attempt to resolve this question. Since mouse non-specific IgG isotype matched control for mAb was/is not available from Biotrin International, we employed a mouse IgG1from another source (DAKO, Carpinteria, CA, USA). We also repeated the concurrent positive staining experiments for RPA-1 (Table 1and Figure 1).
In the positive staining experiments, we confirmed the intense dark brown staining indicative of a specific reaction with constitutive RPA-1 in CD located in the papilla, medulla, and cortex. Additionally, in the same experiments, we confirmed that moderate brown staining was located in necrotic proximal tubular cells. This signal, observed in the necrotic cells, does not necessarily imply the presence of the same antigen (RPA-1) as in the CD, but it could represent nonspecific reactivity, since it is presently unknown whether RPA-1 antigens are inducible in proximal tubules. In negative control staining experiments using the mouse IgG1, light brown staining of necrotic cells was observed, which may be indicative of nonspecific binding of the mouse IgG1to necrotic tissue (Table 1and Figure 1). It should be noted, however, that the immunostaining intensity in necrotic proximal tubular cells from the positive staining experiments using RPA-1 mAb was higher than the intensity observed for the same cells in the negative staining experiments using mouse control IgG1. This difference in signal intensity suggests that while nonspecific binding is occurring, there is also the possibility of RPA-1–like substance being present in these necrotic cells. The reasons for the possible RPA-1 reactivity in necrotic proximal tubular cells in the positive staining experiments are not completely known, but it may be a result of anti-RPA-1 cross-reactivity with unknown antigenic determinants (epitopes) exposed in the necrotic cells (Boenisch 2006).
Whether the potential RPA-1 cross-reactivity in necrotic proximal tubular cells in Gen-, Hg-, or Cr-treated rats has immunohistochemical as well as histopathological implications remains an open question for further investigation. This cross-reactivity was evident in necrotic cells in Gen-, Cr-, or Hg-treated rats, but it was absent in normal proximal tubular cells in the same rats, as well as in saline-treated rats (Table 1and Figure 1). Moreover, the positive staining intensity was correlated with the severity of proximal tubular cell necrosis (Zhang et al. 2008a.). Although further work is needed to confirm these results and to determine its mechanistic basis, we hope that the IHC results observed in the present study will arouse other investigators’ attention to this possible RPA-1 cross-reactivity. Other studies in our laboratory have also demonstrated possible cross-reactivity of RPA-1 in injured proximal tubular cells in kidneys of rats treated with doxorubicin or mitoxantrone (Zhang et al. 2008b). Finally, our results of no or little CD injury, proximal tubular cell necrosis, and potential RPA-1 cross-reactivity raise the question of the cellular origin of RPA-1, which is elevated in urine after treatment with agents that damage the proximal tubules.