Abstract
The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, the authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa immunoreactive band (~90 kDa) was only present in wild-type kidney membranes and not in kidney knockout or wild-type brain membranes. In the water-soluble fraction of wild-type and knockout brains, another band (~50 kDa) was observed; this band was not detected in the kidney. Light and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed immunolabeling of kidney tubules in wild-type but not knockout mice. In the brain, labeling of presynaptic nerve terminals was present also in NaPi-IIa-deficient mice. This labeling pattern was also produced by the NaPi-IIa preimmune serum. The authors conclude that the polyclonal antiserum is specific toward NaPi-IIa in the kidney, but in the brain, immunolabeling is caused by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest and underlines the utility of specificity testing using preimmune sera.
The inorganic Na+-dependent phosphate co-transporter NaPi-IIa belongs to the solute carrier family 34 (SLC34; Murer et al. 2004). NaPi-IIa was originally cloned from rat kidney cortex (Magagnin et al. 1993), and immunocytochemical examination showed that it was located in microvilli of the brush-border membrane of proximal tubules (Custer et al. 1994). Although NaPi-IIa mRNA expression was initially only found in the kidney and not, for instance, in the brain (Magagnin et al. 1993), NaPi-IIa expression at the protein and mRNA levels was later detected by immunohistochemistry and RT-PCR in the brain (Hisano et al. 1997). We hypothesized that NaPi-IIa may be localized to synaptic vesicles, where it, in congruence with other Na+/phosphate transporters such as the vesicular glutamate transporters, could function as a vesicular transporter of organic molecules. Indeed, in initial immunoelectron microscopic experiments using an antibody known to specifically recognize NaPi-IIa in the kidney (Custer et al. 1994), we detected NaPi-IIa immunolabeling over synaptic vesicle clusters in the rat hippocampus. To verify the specificity of this labeling, we examined tissue from NaPi-IIa knockout and wild-type brain and kidney. We describe some of the difficulties in determining the specificity of an antibody using tissue from knockout animals.
Materials and Methods
Animals and Tissue Preparation
Slc34a1−/− mice, kindly provided by H.S. Tenenhouse, was generated by deleting exons VI–X of the slc34A1 gene (described in detail in Beck et al. 1998). For light and electron microscopy, adult knockout mice and wild-type littermates were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and fixed by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains and kidneys were removed and placed in phosphate buffer. For light microscopic immunohistochemistry, the tissue was cryoprotected in 30% sucrose and cut in 40-µm-thick parasagittal (brain) or horizontal (kidney) sections and processed for immunofluorescence as described below. For immunoelectron microscopy, tissue specimens of the hippocampal CA1 region and renal cortex were dissected and embedded in Lowicryl HM20 resin as described (Bergersen et al. 2008). Ultrathin sections were cut from the embedded tissue, placed on Ni mesh grids, and subjected to postembedding immunogold labeling as described below. For Western blotting, after decapitation, the brains and kidneys of knockout and wild-type mice were quickly dissected, immediately frozen in liquid nitrogen, and stored at −80C. Animal experiments were performed in accordance with the guidelines of the Norwegian Committees on Animal Experimentation (Norwegian Animal Welfare Act and European Communities Council Directive of November 24, 1986 [86/609/EEC]).
NaPi-IIa Antiserum
The polyclonal antiserum against the NaPi-IIa protein was raised in rabbit against a peptide (MMSYSERLGGPAV) derived from the N-terminal region of the rat NaPi-IIa protein (Custer et al. 1994). The antibody labels the luminal membrane of renal proximal tubules by immunofluorescence, and by Western blotting, it detects a band of ~90 kDa in the renal brush-border membrane fraction obtained from rat or wild-type mice (Custer et al. 1994; Beck et al. 1998). This band is absent in mice lacking NaPi-IIa (Beck et al. 1998). NaP-IIa preimmune serum was obtained from the rabbits before immunization with the NaPi-IIa peptide.
Immunofluorescence
Free-floating tissue sections were sequentially incubated in blocking buffer (phosphate-buffered saline [PBS] with 3% normal goat serum, 0.5% bovine serum albumin, and 0.5% Triton X-100) for 2 hr, anti-NaPi-IIa 1:1000 in blocking buffer overnight at room temperature, PBS 6 × 10 min, goat anti-rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) 1:1000 in blocking buffer for 2 hr, and PBS 3 × 10 min. After mounting, sections were covered with Prolong Gold (Invitrogen) and examined using the epifluorescence mode of a Zeiss LSM 5 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). For immunofluorescence microscopy, one pair of NaPi-IIa knockout and wild-type mice was analyzed.
Postembedding Immunogold Labeling
Ultrathin sections of hippocampal and renal cortex tissue were subjected to postembedding immunogold labeling essentially as described (Bergersen et al. 2008). In brief, sections were incubated in 50 mM glycine in Tris-buffered saline (0.3% NaCl, pH 7.4) with 0.1% Triton X-100 (TBST) for 10 min, followed by blocking buffer (TBST supplemented with 2% human serum albumin) for 10 min and primary antibody solution (anti-NaPi-IIa 1:300 or NaP-IIa preimmune serum 1:300 in blocking buffer) overnight at 4C. After rinsing in TBST, the sections were incubated in blocking buffer for 10 min and for 1 hr in goat anti-rabbit coupled to 10 nm colloidal gold (British Biocell, Cardiff, UK) diluted 1:20 in blocking buffer. The sections were rinsed in H2O and counterstained with uranyl acetate and lead citrate before examination in a Tecnai 12 (FEI) electron microscope. For immunogold electron microscopy, another pair of NaPi-IIa knockout and wild-type mice than that used for immunofluorescence microscopy was used.
Western Blotting
Brain and kidney tissue was homogenized in water containing 5 mM EDTA and 1 mM PMSF (freshly dissolved in DMSO). Then the homogenate was centrifuged at 39,000 × g (18,000 rpm) for 10 min at 4C, before the pellet was dissolved in SDS–solubilization buffer (1% SDS with 150 mM NaCl and 10 mM NaPi [pH 7.4]), to which 1 mM PMSF was added. This mixture was incubated for 10 min at room temperature and centrifuged at 1000 × g for 10 min at 15C to remove unsolubilized material. The water-soluble supernatant fraction and SDS-soluble pellet fraction were subjected to SDS-PAGE and Western blotting according to standard protocol. The blots were incubated overnight at 4C in anti-NaPi-IIa (final dilution 1:3000) in Tris-buffered saline (pH 7.4) with 1% BSA and 0.05% Tween-20. After rinsings, the blots were incubated in secondary anti-rabbit IgG peroxidase conjugated antibodies (dilution 1:10,000; Chemicon, Temecula, CA) in Tris-buffered saline (pH 7.4) with 4% dry milk powder 0.05% Tween-20. The blots were developed with SuperSignal West Dura Extended Duration Substrate (Thermo Scentific) and analyzed in a Fuji LAS-3000 luminescence scanner (Fujifilm, Tokyo, Japan). The Western blots were made from homogenates of 3 NaPi-IIa knockout and 3 wild-type mice. The Western blots were performed twice and gave similar results in both instances.
Results
Immunocytochemistry
Tissue sections were immunofluorescence labeled with the NaPi-IIa antiserum and examined by epifluorescence microscopy. The luminal part of kidney proximal tubules of wild-type mice was strongly immunolabeled in accordance with previous reports (Custer et al. 1994), whereas NaPi-IIa-deficient mice showed no such labeling (Fig. 1A,B). Immunofluorescently stained brain sections from wild-type mice showed scattered labeled neuronal cell bodies and a dotted neuropil staining. However, the immunolabeling pattern observed in brain tissue obtained from knockout mice was indistinguishable from that of the wild-type mice (Fig. 1C,D). Postembedding immunogold labeling and electron microscopy were used to characterize the distribution of immunolabeling at the ultrastructural level. In ultrathin sections of kidney cortex from wild-type mice, NaPi-IIa immunolabeling was predominantly situated in the apical microvilli of proximal tubules (Fig. 2A), whereas only faint immunolabeling was observed in the proximal tubular microvilli of mice lacking NaPi-IIa (Fig. 2B). In the CA1 region of the hippocampus, a conspicuous immunolabeling over presynaptic nerve terminals, apparently associated with synaptic vesicle clusters, was found both in knockout and wild-type mice (Fig. 2C,D). However, this labeling pattern was also produced by the NaPi-IIa preimmune serum (Fig. 2E,F).
(A, B) Immunofluorescent staining of NaPi-IIa in kidney proximal tubules from wild-type (A, WT) and NaPi-IIa-deficient (B, KO) mice. (C, D) NaPi-IIa immunofluorescence labeling in the cerebral cortex from wild-type (C) and NaPi-IIa-deficient (D) mice. Insets show punctate staining of neuropil. Arrowheads indicate immunolabeled cell bodies. Scale bar in D, 20 µm, valid for all panels. WT, wild-type; KO, knockout.
(A, B) Postembedding immunogold labeling of NaPi-IIa in kidney proximal tubules from wild-type (A, WT) and NaPi-IIa-deficient (B, KO) mice. (C–F) Examples of immunogold labeling produced by the anti-NaPi-IIa antibodies (C, D) and the NaPi-IIa preimmune serum (E, F). Note that presynaptic terminals (t) forming asymmetric synapses with dendritic spines (s) in the stratum radiatum of hippocampal CA1 from wild-type (C, E) and NaPi-IIa-deficient (D, F) mice are labeled. Scale bar in F, 200 nm, valid for all panels. WT, wild-type; KO, knockout.
Immunoblotting
Next, we made Western blots of membrane and cytosolic fractions of brain and kidney homogenates using the NaPi-IIa antiserum. In wild-type kidney, we detected a single strong band at about 90 kDa in the SDS-soluble membrane fraction, but no substantial bands were evident in the cytosolic (water-soluble) fraction (Fig. 3). This was as expected considering that NaPi-IIa is a membrane protein. No bands were observed in either membrane or cytosolic fractions from the kidney of NaPi-IIa knockout mice. However, in whole-brain homogenates, a distinct band at about 50 kDa was present in the cytosolic fraction but not in the membrane fraction in both NaPi-IIa knockout and wild-type mice (Fig. 3). Notably, this 50-kDa band was not detected in the water-soluble kidney fraction and only very faintly so (compared to the major band at 90 kDa) in the membrane fraction of wild-type and knockout kidney homogenate. As this band was present also in knockout tissue, it is unlikely to represent a breakdown product of the full-length NaPi-IIa protein but rather corresponds to an unrelated, cytosolic protein for which the antiserum is specific. Importantly, this second protein appears to not be expressed to an appreciable extent in the kidney, resulting in immunolabeling that is highly specific for NaPi-IIa in this tissue.
Western blot of NaPi-IIa of SDS-soluble membrane fractions and water-soluble cytosolic fractions of kidney (K) and brain (B) homogenate obtained from wild-type (WT) and NaPi-IIa-deficient (knockout [KO]) mice. Arrow indicates the NaPi-IIa reactive band.
Discussion
The original aim of this study was to investigate whether the Na+/inorganic phosphate transporter NaPi-IIa, which is essential for retention of inorganic phosphate in the kidney (Beck et al. 1998), is present in synaptic vesicles in the central nervous system, where, we hypothesized, it could function to transport organic ions over the vesicle membrane. Initial support for this hypothesis was obtained by immunohistochemical labeling of NaPi-IIa in rat (not shown) and wild-type mice, which showed punctate staining at the light microscopic level and presynaptic labeling that appeared associated with synaptic vesicles at the electron microscopic level. Furthermore, the polyclonal NaPi-IIa antiserum used produced no detectable immunofluorescent staining and only faint postembedding immunogold labeling in kidney sections from mice lacking NaPi-IIa. However, when attempting to corroborate the specificity of the immunohistochemical reaction in the brain using nervous tissue from knockout animals, neither the distribution nor strength of immunolabeling differed from that observed in brain tissue of wild-type mice. Thus, the antiserum specifically detects NaPi-IIa in the kidney but not in the brain.
The failure to detect NaPi-IIa in the brain in the present study is consistent with the study by Magagnin et al. (1993), who did not detect NaPi-IIa mRNA by Northern blotting in brain tissue, but appears at odds with the work of Hisano et al. (1997), who used the more sensitive RT-PCR technique and reported NaPi-IIa expression in the rat brain. Using an antibody directed to the C-terminus of NaPi-IIa, they further concluded by immunocytochemistry that NaPi-IIa is confined to subsets of neuronal somata and fibers and of ependymal and endothelial cells (Hisano et al. 1997). However, because NaPi-IIa-deficient animals were not available, the immunocytochemical signal could not be validated. Here, we have used an antibody raised against the N-terminal part of NaPi-IIa that produced a quite different labeling pattern in the brain. Although a weak NaPi-IIa expression may have been masked by a stronger expression of the unrelated cross-reacting protein in our immunohistochemical experiments, our inability to detect NaPi-IIa in the brain also by Western blotting suggests that this transporter is not expressed, or only very weakly so, in the mouse brain.
It should be noted in parallel experiments of rat brain tissue (not shown) the NaPi-IIa antiserum produced a 50-kDa band on Western blots of whole-brain homogenates and isolated synaptic vesicles, as well as the same hippocampal immunogold labeling as that observed in the mouse. Thus, we think that the cross-reacting protein is present across different species.
The present data show that antibodies specific and selective for a particular protein in one type of tissue are not necessarily so in other tissue types, underlining the importance that specificity testing of antibodies should be performed in the tissue that is the subject of the study. Therefore, using as a specificity control system cell cultures transfected for the protein of interest is not sufficient to rule out cross-reactivity. Antibodies should, whenever possible, be tested, in knockout tissue that is as identical as possible to that under immunohistochemical investigation (Holmseth et al. 2006).
When animals are injected with an antigen, multiple B cells are usually activated, resulting in a number of different cell clones producing different amounts of antibodies to the same antigen. The desired antibodies are of course those that only recognize the antigen of interest, and a good antiserum contains high levels of these. However, antibodies also recognizing other molecules are usually produced. Some of these antibody clones are polyreactive—that is, they are directed to several distinct epitopes (Kramer et al. 1997; Danbolt et al. 1998), often with high affinity against one epitope and lower affinities against others (Mouquet et al. 2010). This means that also monoclonal antibodies can produce cross-reactive immunohistochemical labeling, like that observed in this study. Thus, specificity testing should be just as rigorous for monoclonal as for polyclonal antibodies.
Another question is whether we could have discovered that the NaPi-IIa labeling of brain tissue was unspecific without using the NaPi-IIa knockout tissue. In wild-type and NaPi-IIa knockout hippocampus, the labeling pattern produced by the preimmune serum and the NaPi-II antiserum was similar. This is strong evidence that the cross-reacting antibodies were present in the serum before the immunization procedure with the specific NaPi-IIa antigen, underscoring the usefulness of performing controls with preimmune sera. Peptide absorption controls can also give certain indications about antibody specificity. If a peptide used for producing an antiserum does not block the immunolabeling, this may indicate that the cross-reacting antibodies are not those that bind to the desired antigen (which, given the labeling produced by the preimmune serum, is presumably the case here). If, however, the antigenic peptide blocks the labeling, this does not necessarily mean that the labeling is specific for the transporter because the cross-reaction could be produced by polyreacting antibodies (see Holmseth et al. 2005; Holmseth et al. 2006). In sum, the present results highlight the importance of performing appropriate specificity controls of antibody reactivities (Saper and Sawchenko 2003; Holmseth et al. 2005; Holmseth et al. 2006). The NaPi-IIa antibodies are an example of a situation where antibodies specific for a protein in one tissue are not selectively specific for that protein in another tissue. The best way of ascertaining that an immunohistochemical signal in a given tissue results from specific antibody binding of the antigen of interest is by observing that this signal is absent in the same tissue lacking the antigen.
Footnotes
The author(s) declared no potential conflicts of interest with respect to the authorship and/or publication of this article.
The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article: This work was funded by grants from the Research Council of Norway, including from FUGE (183727; NCD).