Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan1
Division of Molecular Virology, Institute for Genetic Medicine, Hokkaido University, Hokkaido, Japan2
Immunology Division and Division of Molecular Virology, Jichi Medical School, Tochigi-Ken, Japan3
Author for correspondence: Yoichiro Iwakura. Fax +81 3 5449 5430. e-mail iwakura{at}ims.u-tokyo.ac.jp
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Abstract |
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Introduction |
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In addition to blood transfusion transmission, TTV seems to be non-parentally transmitted via the faecaloral route, as TTV DNA has been detected in both faeces (Okamoto et al., 1998 ) and saliva (Ross et al., 1999
). TTV DNA was found also in breast milk, indicating post-natal transmission of TTV (Kazi et al., 2000
). These routes of TTV transmission could contribute to the high prevalence of TTV infections in the general population worldwide. Recent epidemiological studies have shown that TTV infection is very frequent, with a high number of asymptomatic individuals. The high rate of asymptomatic TTV infection in the population has suggested that TTV probably does not cause hepatitis (Allain, 2000
; Nishiguchi et al., 2000
). However, TTV has an extremely wide divergence in the sequence of its genome (Hijikata et al., 1999
) and is classified into at least 16 subtypes, according to differences in the genome sequence of more than 30% from one another (Okamoto et al., 1999
). Therefore, it is possible that some of the genotypes may be capable of causing hepatitis (Okamoto et al., 1999
; Okamura et al., 2000
; Sugiyama et al., 2000
). Moreover, TTV-related virus (Hijikata et al., 1999
; Khudyakov et al., 2000
) and TTV-like mini virus (Takahashi et al., 2000
) were discovered recently, suggesting that these viruses, which were not distinguished previously from TTV, may be involved in the development of the disease.
The TTV genome has a GC-rich region that contains a potential stemloop structure, which is highly conserved among TTV genotypes. This region appears to be involved in virus replication and acts as the origin of replication (Miyata et al., 1999 ). It was reported that there are five open reading frames (ORFs) in the TTV genome, although we have found an additional ORF, ORF6, by a computer search (Fig. 1A
). Some of the transcripts of TTV were spliced in a process by which ORF2 was linked to either ORF4 or ORF5, tentatively designated ORF2-4 and ORF2-5, respectively (Kamahora et al., 2000
). However, the roles of each ORF gene product have not been clarified yet.
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Methods |
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RNA analysis.
Total RNA was extracted using the acid guanidium thiocyanatephenolchloroform method (Chomczynski & Sacchi, 1987 ). mRNA was purified from total RNA using the QuickPrep Micro mRNA Purification kit (Pharmacia). For Northern blot analysis, 2 µg mRNA was electrophoresed in a 1·2% agarose gel containing 50% formaldehyde and blotted onto a Gene Screen Plus membrane (Du Pont NEN). After UV cross-linking, the membrane was hybridized with a 32P-labelled DNA probe at 42 °C overnight. Autoradiograms were developed and the radioactivity of each band was quantified using the BAS 2000 BioImage analyser (Fuji).
Biochemical analysis of mouse sera.
To determine the levels of serum albumin (Alb), serum creatinine (Cr), blood urea nitrogen (BUN) and total serum cholesterol (T-chol), 10 µl of sera from 4-week-old wild-type and LT1 mice and a 2-month-old Snd15 mouse were applied onto DRI-CHEM slides (Fuji) and measured using the DRI-CHEM 5500V (Fuji).
Histological analysis.
The kidneys from 4-week-old wild-type and LT1 mice and a 2-month-old Snd15 mouse were fixed in 10% formalinPBS, paraffin-embedded and sectioned at a thickness of 2 µm, according to standard procedures. The sections were stained with haematoxylin and eosin (H&E) or periodic acidSchiff (PAS). For electron microscopy, tissues were fixed with 1% glutaraldehyde in PBS, embedded in Epon and sliced at a thickness of 70 nm. The ultra-thin sections were stained with uranyl acetate and lead citrate. All samples were examined under a JEOL 1200EX electron microscope.
Immunohistochemistry.
Paraffin-embedded sections (2 µm) were immunostained against proliferating cell nuclear antigen (PCNA) using the Rabbit ImmunoCruz Staining system (Santa Cruz Biotechnology), according to the manufacturers instructions. In brief, the deparaffined sections were incubated with 2·5 µg/ml of rabbit anti-PCNA polyclonal antibody (Santa Cruz Biotechnology) in 5% normal goat serum in PBS for 1·5 h at room temperature. Then, the sections were incubated with biotinylated secondary antibody and subsequently incubated with streptavidinhorseradish peroxidase. The sections were counterstained with methyl green.
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Results |
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The donor and acceptor sites of the 1·1 kb transcript were identical to those of ORF2-5 (Fig. 1A and Fig. 3C
). The reading frame of the spliced form of ORF1 was switched from frame 1 to frame 2 at the splicing junction. The putative protein encoded by the spliced form of ORF1 was highly basic, with an arginine-rich domain at the N-terminal end and a serine-rich domain at the C-terminal end (Fig. 3D
).
Renal failure of LT1 mice
Since LT1 mice showed an accumulation of ascites, we suspected kidney function failure in these mice and, thus, measured the levels of serum Cr, serum Alb, BUN and T-chol. As shown in Table 3, the levels of serum Cr and BUN from LT1 mice were significantly higher than those of wild-type mice. This suggested that the renal function of LT1 mice was impaired, resulting in uraemia. The level of BUN in the Snd15 mouse was also high, but 6-fold less than that seen in LT1 mice. In LT1 mice, the level of serum Alb was very low, while it was normal in the Snd15 mouse. Although proteinuria could not be determined precisely because of poor urination, an approximate level of proteinuria was estimated using urinalysis paper. The level of proteinuria in LT1 mice was at least 3-fold higher than that seen in wild-type mice, but the level of proteinuria in the Snd15 mouse was normal (data not shown). Since nephrotic syndrome is characterized by massive proteinuria and hypoalbuminaemia, these results indicated that LT1 mice developed nephrotic syndrome. This diagnosis was supported by finding high levels of serum T-chol in LT1 mice, which is another feature of nephrotic syndrome. On the other hand, serum ALT levels in LT1 mice and Snd15 were normal, suggesting that these transgenic mice did not develop hepatitis (data not shown). In fact, the livers of LT1 mice and Snd15 were examined under light microscopy and no abnormality was observed (data not shown).
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It is accepted generally that excessive proliferation of renal epithelial cells is involved in the development of various human kidney diseases. Because of this, we examined the proliferation of renal cells using PCNA as a proliferating cell marker. In the kidney cortex of LT1 mice and Snd15, some of the nuclei in distal and proximal tubular cells were stained deeply when stained with an anti-PCNA antibody (Fig. 4H, I
). The number of PCNA-positive tubular epithelial cells was 10- and 3-fold higher in LT1 mice and Snd15, respectively, when compared with wild-type mice (negative control, data not shown). A result of particular interest is that 1 out of 10 glomeruli from the Snd15 mouse had PCNA-positive cells (Fig. 4I
), which were not found in the glomeruli of LT1 and wild-type mice.
Electron microscope analysis of renal epithelial cells
In order to investigate the kidney changes in more detail, electron microscope analysis was performed on LT1 and wild-type mice. The results showed that the number of mitochondria and microvilli in the proximal tubule cells from LT1 mice was decreased significantly, compared with normal levels in wild-type mice (compare Fig. 5B with A
). Mitochondria were distributed diffusely in the cytoplasm of the proximal tubule cells. There were also fewer endocytotic vesicles in the cytoplasm of proximal tubular cells as compared to those in the wild-type mouse. In addition, necrotic profiles were observed in some of the proximal tubular cells from the LT1 mouse. These observations suggest that the function of the proximal tubule cells is impaired in LT1 mice. As shown in Fig. 5(D)
, the glomeruli of the LT1 mouse showed severe abnormalities. The profile of podocytes was extremely swollen and appeared immature. The normal shape of a mature podocyte is very flat (Fig. 5C
), like a capsule cell. The foot processes in the LT1 mouse were completely absent. Electron-dense deposits, which denote immune-complex deposition, were not detected in the glomeruli of the LT1 mouse.
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Discussion |
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Another transgenic mouse, Snd15, which presented a lower level of ORF1 gene expression (1/20 compared with LT1 mice), also showed similar, but milder, changes in the renal epithelial cells. All of the capsule cells in the Snd15 mouse were enlarged abnormally and filled Bowmans space. Since the normal morphology of the podocyte and capsule cell changes from a cuboid shape to a very flat shape during the maturation process, the enlarged shape of the capsule cells in the Snd15 mouse also suggests that these cells are immature. In both LT1 mice and Snd15, vacuolar degeneration was confirmed in some of the proximal tubular cells. Furthermore, the number of proliferating cells in the renal epithelia was increased significantly in both of these mouse types, as determined by the number of PCNA-positive cells. This observation suggests that immature cells are increased in this transgenic mouse, because cells that have completed terminal differentiation tend to stay at the G0 phase and proliferative activity is low relative to that of immature cells. These results suggest that the expression of the TTV ORF1 gene in mice affects the maturation of renal epithelial cells in a dose-dependent manner. Since two independent founder mice and their derivatives developed similar renal epithelial cell abnormalities, it is clear that these abnormalities are not caused by the effects of the transgene insertion into the host chromosome, but, rather, by the toxic effects of the ORF1 gene product.
It is known that immunological mechanisms are involved in the development of various kidney diseases. The electron microscope study showed that the electron-dense deposits, which denote immune-complex deposition, were not found in the glomeruli of LT1 mice. Immune-complex deposition in the glomeruli was also examined by immunofluorescent staining and no significant difference between the transgenic and wild-type glomeruli was observed (data not shown), suggesting that immunological mechanisms were not involved in the generation of renal abnormalities in our transgenic mice. This is consistent with the observations from histological analysis in which no inflammatory cell infiltration was found in the kidneys of these transgenic mice.
The most prominent characteristic of LT1 mice is a dysplasia of renal epithelial cells, including podocytes, capsule cells and proximal tubule cells, while the other parts of the kidney and other tissues were histologically normal. During the development of the kidney, podocytes, capsule cells and distal and proximal tubule cells arise from common precursor cells called induced metanephrotic mesenchymal cells (Horster et al., 1999 ). After induction by the uretic bud, the precursor cells aggregate and transmit themselves to the epithelial lineage, which differentiates terminally to podocytes, capsule cells and distal and proximal tubule cells. This transmission is known as mesenchymalepithelial transmission (MET). The results presented here suggest strongly that expression of the TTV ORF1 gene impairs specifically the processes of terminal differentiation of precursor cells to podocytes, capsule cells and distal and proximal tubule cells after MET and keeps them at the immature stage. The abnormalities of the Snd15 mouse were also restricted to the cells derived from the precursor cells.
Since TTV was isolated originally from a patient with post-transfusion hepatitis of unknown aetiology, most pathoepidemiological studies of TTV have focused on the association of this virus with hepatitis. There are only a few reports that have investigated the correlation between TTV and kidney disease, although it is well known that the co-prevalence of TTV infection was significantly greater in haemodialysis patients (Campo et al., 2000 ; Gallian et al., 1999
; Maggi et al., 1999
; Martinez et al., 2000
). Therefore, we think it is important to investigate the potential effects of TTV infection on the progression of the disease in haemodialysis patients and patients with kidney disease.
Kamahora et al. (2000) examined the transcriptional profile of TTV in COS-1 cells and detected 3·0, 1·2 and 1·0 kb mRNAs corresponding to ORF1, ORF2-4 and ORF2-5 mRNA, respectively. These mRNAs were transcribed from a single promoter and were spliced alternatively. All of the mRNAs were observed in the bone marrow cells of a patient with acute myeloblastic leukaemia (Okamoto et al., 2000a
). The splicing sites of the spliced form of ORF1 found in our transgenic mice are identical to those of ORF2-5 and the complete sequence encoded by the spliced form of ORF1 lies completely within the 1·0 kb TTV mRNA, which is detected in COS-1 cells and in leukaemia patients. Because of the lack of reliable antibodies against TTV proteins, it has not been shown that the spliced product of ORF1 nor the other products of the other TTV genes are produced in infected cells.
The putative product encoded by the spliced form of ORF1 has an arginine-rich domain at the N-terminal end and a serine-rich domain at the C-terminal end. The arginine-rich domain of the putative product shows 63% identity to protamine, which binds to chromosomes and tightly condenses them (Wouters-Tyrou et al., 1991 ). The positive charge of the arginine-rich domain in protamine is essential to bind and neutralize the negative charges of the DNA strands (Shiffman et al., 1978
). It is accepted generally that the binding of protamine inactivates gene transcription by the process of chromosome condensation, because the condensed chromosome tends to prevent transcriptional factors from accessing the target sites (Mahlknecht & Hoelzer, 2000
). We speculate that the putative product binds to the host chromosomes using the arginine-rich domain and then alters the secondary structure of the DNA strands around the binding sites. As a result, the regulation of gene transcription around the binding sites may be affected. Some of the genes affected by the putative product may be important for the differentiation of renal epithelial cells, thus resulting in the phenotypes observed in our mice.
Although the association between TTV infection and hepatitis has been suspected from prior epidemiological studies (Okamoto et al., 1999 ; Okamura et al., 2000
; Sugiyama et al., 2000
), in our transgenic mice, the liver was histologically and serochemically normal. Nevertheless, this does not exclude the possibility that the putative product of the spliced form of the TTV ORF1 gene is involved in the development of liver disease. This is because TTV ORF1 has been expressed from the early embryonic stage in our mice and, therefore, immunological tolerance to the transgene product may be established in our transgenic mice already. Further studies are required to elucidate the pathogenicity of TTV.
Taken together, we have shown that expression of the TTV ORF1 gene affects the differentiation of renal epithelial cells. The results of this study may provide new insights for the investigation of TTV pathogenicity.
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Acknowledgments |
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Footnotes |
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References |
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Received 23 May 2001;
accepted 17 September 2001.