Cellular Distribution of NDRG1 Protein in the Rat Kidney and Brain During Normal Postnatal Development
Department of Neuropathology, Neurological Institute (YW,AF,TI) and Department of Medical Biochemistry (KM,WM,MK), Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Correspondence to: Yoshinobu Wakisaka, MD, Dept. of Neuropathology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. E-mail: w-yoshi{at}np.med.kyushu-u.ac.jp
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Summary |
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(J Histochem Cytochem 51:15151525, 2003)
Key Words: NDRG1 differentiation brain kidney cellular localization immunohistochemistry Western blotting
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Introduction |
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Previous studies regarding expression of the NDRG family have mostly been at the mRNA level. NDRG1 mRNA was reported to be ubiquitously expressed in human and rat heart, brain, kidney, placenta, lung, liver, skeletal muscle, and pancreas. The kidney was the organ with the most abundant NDRG1 mRNA. (Kokame et al. 1996; van Belzen et al. 1997
; Nishie et al. 2001
; Zhou et al. 2001
; Lachat et al. 2002
). NDRG2 mRNA was strongly expressed in brain, heart, and skeletal muscle, while NDRG3 mRNA was strongly expressed in brain. NDRG2 and NDRG3 also showed ubiquitous weak expression in other organs. NDRG4 mRNA was selectively expressed in brain and heart. In particular, NDRG4-B, which is one of the three isoforms of NDRG4, was expressed only in brain (Zhou et al. 2001
). Therefore, these results suggest the specific importance of the NDRG family in the brain. On the other hand, there are only a couple of studies on NDRG1 protein expression in vivo (van Belzen et al. 1997
; Cangul et al. 2002
; Lachat et al. 2002
). These studies have been carried out only on human surgical specimens. Although high expression of NDRG1 protein was noted not only in a variety of cancer cells but also in normal kidney, NDRG1 protein has not been observed in normal brain tissues. Moreover, although NDRG1 had been thought to be involved in cell differentiation, there are only two studies on NDRG1 mRNA expression during embryonal development (Okuda and Kondoh 1999
; Shimono et al. 1999
) and no studies on NDRG1 protein expression during postnatal development.
To study NDRG1 expression at the protein level during postnatal development in rat kidney and brain, we raised an antiserum against a repeated sequence in the C-terminal region of NDRG1. This antiserum was specific for NDRG1 because the tandem repeats are characteristic of NDRG1 and are not present in other members of the NDRG family (Zhou et al. 2001; Qu et al. 2002
). Here we demonstrate that NDRG1 protein exists in brain as well as kidney, and that regional and cell type changes in NDRG1 expression occur during postnatal rat development. Moreover, we suggest the possibility that NDRG1 forms a polymer in vivo.
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Materials and Methods |
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Production of Anti-NDRG1 Antisera
A cysteine-conjugated peptide corresponding to the internal sequence of human NDRG1 (TSEGTRSRSC), which corresponded to the tandem repeat region (Zhou et al. 2001; Qu et al. 2002
), was synthesized. The terminal cysteine residue was coupled with keyhole limpet hemocyanin (KLH) and then used to immunize rabbits (Masuda et al. 2003
).
Immunohistochemistry
Paraffin-embedded sections from rats at each time period were used for histological examination. Coronal sections of the cerebrum at the level of the caudate putamen and the dorsal hippocampi, and sagittal sections of the cerebellum and the kidney, were routinely stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was carried out using the labeled biotinstreptavidin method. Paraffin-embedded sections were cut at 5-µm thickness, then deparaffinized in xylene, hydrated in ethanol, and incubated with 0.3% H2O2 in absolute methanol for 30 min at room temperature to inhibit endogenous peroxidase. After washing with Tris-HCl buffer (50 mM Tris-HCl, pH 7.6), the sections were incubated at 4C overnight with a rabbit polyclonal antiserum against NDRG1 at a dilution of 1:2000 in PBS containing 5% normal goat serum. The sections were then sequentially incubated with a biotinylated secondary antibody diluted 1:200 and a peroxidase-conjugated streptavidin-biotin complex diluted 1:100 (Amersham; Poole, UK). The colored reaction product was developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution. The sections were lightly counterstained with hematoxylin.
Because the immunocytochemical localization of B-crystallin in rat kidney is restricted to the zonal structures (Iwaki et al. 1990
,1991
), double labeling with the anti-NDRG1 antiserum and an anti-
B-crystallin antibody was conducted on P21 and P42 kidney sections. The sections were rehydrated and endogenous peroxidase activity was blocked with 0.3% H2O2 in absolute methanol for 30 min. After washing with Tris-HCl buffer, the sections were incubated with the anti-NDRG1 antiserum overnight at 4C. The bound antiserum was visualized by the biotinstreptavidin method using 3-amino-9-ethylcarbazole (AEC; Vector Laboratories, Burlingame, CA) as the chromogen, which yields a brownish-red product. After the immunoreactive structures were photographed, the AEC sections were destained in 99% ethanol, completely immersed in 0.01 M citrate buffer, pH 6.0, and autoclaved at 121C for 10 min to remove the immunoreaction products. The sections were then incubated at 4C overnight with a polyclonal rabbit antibody against
B-crystallin (Iwaki et al. 1989
), and the immunoreactivity was visualized by the biotinstreptavidin method using AEC. After the immunoreactive structures were photographed, sections were decolorized and the immunoreaction products were removed using the methods described above. H&E staining was then performed.
For double immunofluorescence of brain sections, specimens from P21 rats were incubated with combinations of the polyclonal antiserum against NDRG1 and a monoclonal antibody against GFAP (clone GA5; Novocastra Laboratories, Newcastle, UK) at 4C overnight. The immunoreactivities were visualized with FITC-conjugated anti-mouse IgG and Texas red-conjugated anti-rabbit IgG (Amersham), and the sections were observed under a confocal laser microscope (LSM-GB200; Olympus, Tokyo, Japan).
Western Blotting
Fresh frozen samples from the kidneys and cerebrums of P1, P3, P7, P8, P10, P14, P21, P28, P35, and P42 rats were homogenized in a protein lysis buffer, consisting of 20 mM Tris-HCl (pH 7.4) containing 10% sucrose and protease inhibitors (protease inhibitor cocktail, Complete Mini; Roche Diagnostic, Mannheim, Germany), and centrifuged at 800 x g for 10 min at 4C. The supernatants were collected and the protein concentrations were determined using the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL) with bovine serum albumin as the standard. Protein samples were denatured in electrophoresis sample buffer (62.5 mM Tris-HCl, pH 6.8, 5% SDS, 5% glycerol, 3 mM EDTA, and 0.02% bromophenol blue) with or without a reducing agent (4% 2-mercaptoethanol), and each sample (10 µg/lane) was separated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (PDVF; Millipore, Bedford, MA). After blocking with 5% non-fat dry milk with 0.05% Tween-20 in Tris-buffered saline for 1 hr at RT, the membranes were incubated overnight with the anti-NDRG1 antiserum (1:2000) in the blocking buffer, then washed and incubated with a peroxidase-conjugated secondary antibody (1:20,000; Chemicon, Temecula, CA) for 1 hr. The immunoreactive proteins were visualized with enhanced chemiluminescence (ECL; Amersham).
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Results |
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Discussion |
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Expression of NDRG1 Protein in Developing Rat Brain and Kidney
The present study revealed the existence of NDRG1 protein, not only in postnatal rat kidney but also in brain, by both IHC and Western blotting. The immunoreactivity observed in the kidney was almost consistent with previous findings concerning NDRG1 mRNA expression and NDRG1 protein localization in the kidney (Cangul et al. 2002; Lachat et al. 2002
). However, the immunoreactivity in the brain was not comparable with previous studies. High expression of NDRG1 protein was found in brain tumor tissue, while immunoreactivity for the NDRG1 antibody has not previously been recognized in normal adult human brain (Cangul et al. 2002
; Lachat et al. 2002
). The difference between our study and previous reports concerning the NDRG1 protein expression in normal brain tissue may be due to a difference in species, maturity, antibodies, or tissue preparations. However, a difference due to species specificity is unlikely because NDRG1 is highly conserved in different mammals, as revealed by database searches and phylogenetic analysis (Qu et al. 2002
). In addition, because NDRG1 protein was evident in P42 rat brain and high NDRG1 protein expression was also recognized in adult rat brain analyzed by IHC and Western blotting (data not shown), a difference due to brain maturity also appears unlikely. Therefore, we consider that the NDRG1 protein expressed in rat brain in our study is mainly due to the immunological characteristics of our NDRG1 antiserum or the tissue preparation. Because the Western blotting in our study revealed a strong signal at the expected molecular weight of 43 kD, our antiserum seemed to be highly specific for NDRG1.
Altered Localization of NDRG1 Protein in Postnatal Rat Kidney
In the IHC study, we found that the localization of NDRG1 protein altered around the end of the second postnatal week. In the kidney, we found strong immunoreactivity for NDRG1 in the proximal convoluted tubules of the cortical region during the first 2 weeks which disappeared by P28, whereas NDRG1 immunoreactivity was gradually recognized in the collecting ducts in the outer stripe of the medulla from P10. Neonatal rat kidneys are immature and nephrogenesis continues after birth, proceeding in a centrifugal pattern. The juxtamedullary convoluted tubules are the most highly developed at birth, and there is a progression of maturation of convoluted tubules towards the periphery during the postnatal period (Edwards et al. 1981). Although the collecting ducts in the medulla are immature in the early postnatal stage, they are rapidly elongated from at least the end of the second postnatal week (Edwards et al. 1981
; Nigam et al. 1996
). Functionally, the activity of most of the transporting systems related to the acidbase equilibrium are activated gradually after birth. The enzyme aromatic L-amino-acid decarboxylase (AADC), which is used as a marker of proximal convoluted tubule differentiation, exists only on the proximal convoluted tubules in the deep cortex during the first postnatal week and is gradually recognized on the proximal convoluted tubules in the outer cortex starting at P8 (Nigam et al. 1996
). In addition, with respect to the collecting ducts in the medulla, the responsiveness to arginine vasopressin and the activity of Na+,K+-ATPase rapidly increase after the beginning of the third postnatal week (Edwards et al. 1981
; Nigam et al. 1996
). Therefore, the strong NDRG1 expression recognized on the proximal convoluted tubules in the cortex during the first 2 postnatal weeks appears to reflect the morphological and functional differentiation of the proximal convoluted tubules. Similarly, the immunoreactivity of the collecting ducts in the medulla detected from the end of the second postnatal week would be due to the differentiation and maturation of the collecting ducts.
Altered Localization of NDRG1 Protein in Postnatal Rat Brain
In the brain, we found strong immunoreactivity for NDRG1 on the hippocampal pyramidal neurons from P8 to P10, which disappeared by P14, whereas NDRG1 staining was gradually recognized on the astrocytes in the hippocampus, neocortex, and cerebellum from the end of the second postnatal week.
Hippocampal pyramidal neurons undergo morphological and functional differentiation during P1P12. The appearance of pyramidal neurons changes from oval cells with packed nuclei and scant cytoplasm to large triangular cells during the early postnatal stage, occurring abruptly between P7 and P11 (LópezGallardo and Prada 2001). In addition, synaptogenesis occurs during the first 2 postnatal weeks. The functional maturation of the GABAergic and excitatory glutamatergic synaptic transmissions onto the hippocampal pyramidal neurons takes place during this period (Scheetz and ConstantinePaton 1994
; Gubellini et al. 2001
; Khazipov et al. 2001
). The numbers of GABA and NMDA receptor sites increase rapidly after birth. They are greatest during the second postnatal week, after which there is a decrease to the adult level, which is attained at the end of the third postnatal week (Tasker 2001
). Moreover, the neuronal glutamate transporter EAAC1 is also enriched in pyramidal neurons between P5 and P16 (Rothstein et al. 1994
; Furuta et al. 1997
). Therefore, the transient strong expression of NDRG1 on hippocampal pyramidal neurons in the early developmental stage may reflect the metabolic changes in the immature brain.
Development of astrocytes begins at the postnatal stage. During the maturation of glial cells, vimentin (VIM)-positive radial glial cells transform into GFAP-immunoreactive astrocytes. The radial glial cells disappear by P21 with the greatest rate of disappearance between P8 and P15 (Miller and Robertson 1993; Munekawa et al. 2000
). Between birth and P20, GFAP is increased to the adult pattern in mature-shaped astrocytes (Pixley and de Vellis 1984
; Kaur et al. 1989
; DomaradzkaPytel et al. 2000
). Therefore, the strong NDRG1 expression recognized on the hippocampal pyramidal cells from P8 to P10 and on the astrocytes from the end of the second postnatal week appears to reflect their morphological and functional differentiation.
Interestingly, we found NDRG1-immunopositive astrocytes only in the regions where neurons existed, and the astrocytes in the white matter were hardly immunoreactive for NDRG1. This distribution of NDRG1-immunoreactive astrocytes resembles that of glutamate transporter GLAST- and GLT-1-immunoreactive astrocytes (Lehre et al. 1995; Furuta et al. 1997
). Moreover, the processes of Bergmann glia, which covered the dendrites of Purkinje cells, were also immunoreactive for the NDRG1 antiserum. These findings suggest that, although the exact function of NDRG1 remains unresolved, the NDRG1 protein of astrocytes may have a certain role in neuron survival.
Formation of the NDRG1 Polymer as a Functional Molecule
NDRG1 protein is a 43-kD molecule overexpressed under several conditions, such as severe hypoxia. In this study, the Western blotting using a specific antibody for NDRG1 under reduced conditions showed the existence of an immunoreactive band around 43 kD. Therefore, the molecular weight of a single polypeptide of NDRG1 was confirmed to be 43 kD. However, in the cerebral samples we recognized other bands around 129 and 172 kD, which may correspond to the trimeric and tetrameric bands. Therefore, we consider that the NDRG1 antiserum also specifically recognized these bands. Because NDRG1 is a cysteine-rich protein having eight cysteines in its amino acid sequence (Qu et al. 2002), we suspected that the NDRG1 protein in vivo formed a polymer or protein complex by disulfide bonding. The Western blotting against kidney and cerebral samples in the absence of a reducing agent in order not to cleave disulfide bonds revealed that an NDRG1-immunoreactive band only existed at around 215 kD during the early postnatal stage. With development, this 215-kD band gradually disappeared and bands around 43 kD in kidney samples and around 129 and 172 kD in cerebrum samples increased from the end of the second postnatal week. The time when the molecular weight change was recognized by Western blotting agreed with the time when the change in NDRG1 localization was detected by IHC. Therefore, in both kidney and brain, NDRG1 protein as a functional molecule in vivo may change its molecular weight during postnatal development. NDRG1 protein may form pentamers in the proximal and intermediate convoluted tubules of the kidney cortex and in hippocampal pyramidal cells during the early postnatal stage. NDRG1 protein would exist as a monomer in the collecting ducts of the renal medulla and may also form trimers in astrocytes after the end of the second postnatal week. These different immunostaining and Western blotting patterns suggest the existence of diverse molecular forms of NDRG1 in postnatal rat development. However, there still remain some limitations to the interpretation of our results. First, we could not exclude the possibility that the NDRG1 antiserum crossreacts with other proteins and the possibility that NDRG1 has unknown spliced form variants because of insensitivity of 129- and 172-kD bands to the reducing reagent. Second, because our protein lysis buffer did not contain any detergents, such as Triton X-100, Western blotting against kidney samples may not include the results of NDRG1 protein existing in the membrane fraction. Further studies are required to clarify these points and to explore the biological function of NDRG1.
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Acknowledgments |
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Footnotes |
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