Gene expression profile of transgenic mouse ... - Wiley Online Library

1 downloads 0 Views 201KB Size Report
Jan 9, 2006 - Gene Expression Profile of Transgenic Mouse. Kidney Reveals Pathogenesis of Hepatitis. B Virus Associated Nephropathy. {. J. Ren,1 L. Wang ...
Journal of Medical Virology 78:551–560 (2006)

Gene Expression Profile of Transgenic Mouse Kidney Reveals Pathogenesis of Hepatitis B Virus Associated Nephropathy{ J. Ren,1 L. Wang,2 Z. Chen,1 Z.M. Ma,1 H.G. Zhu,3 D.L. Yang,4 X.Y. Li,4 B.I. Wang,4 J. Fei,2 Z.G. Wang,2 and Y.M. Wen1* 1

Key laboratory of Medical Molecular Virology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai, China 2 Shanghai Research Center for Model Organisms, Shanghai, China 3 Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China 4 Division of Clinical Immunology, Tongji Hospital, Tongji Medical College, HUST, Shanghai, China

Hepatitis B virus (HBV)-associated nephritis has been reported worldwide. Immune complex deposition has been accepted as its pathogenesis, although the association between the presence of local HBV DNA and viral antigen and the development of nephritis remains controversial. To understand better the roles played by HBV protein expression in the kidney, the global gene expression profile was studied in the kidney tissue of a lineage of HBV transgenic mouse (#59). The mice expressed HBsAg in serum, and HBsAg and HBcAg in liver and kidney, but without virus replication. Full-length HBV genome (adr subtype, C genotype) isolated from a chronic HBV carrier was used to establish the transgenic mice #59. Similarly manipulated mice that did not express HBV viral antigens served as controls. Southern blotting, hybridization with HBV probe, and immuno-histochemical staining were used to study HBV gene expression. mRNA extracted from the kidney tissue was analyzed using Affymetrix microarrays. HBsAg and HBcAg were located mainly in the cytoplasm of tubular epithelium. Altogether 520 genes were ‘‘upregulated’’ more than twofold and 76 genes ‘‘down-regulated’’ more than twofold in the kidney. The complement activation, blood coagulation, and acute-phase response genes were markedly ‘‘up-regulated’’. Compared to the controls, the level of serum C3 protein was decreased in #59 mice, while the level of C3 protein from kidney extract was increased. Results indicate that expression of HBsAg and HBcAg in tubular epithelial cells of the kidney per se can upregulate complement-mediated inflammatory gene pathways, in addition to immune complex formation. J. Med. Virol. 78:551–560, 2006. ß 2006 Wiley-Liss, Inc.

ß 2006 WILEY-LISS, INC.

KEY WORDS: hepatitis B virus; nephropathy; gene expression; mRNA array; transgenic mice

INTRODUCTION Epidemiological studies have shown that chronic carriage of hepatitis B virus (HBV) leads to the development of nephritic syndrome, which is particularly common in children in HBV endemic regions. The presence of immune complexes strongly suggests that the pathogenesis of HBV-associated nephropathy is complex mediated [Combes et al., 1971]. However, other studies showed that the persistence of HBV genes and expression of HBV viral antigens in kidney tissues per se might induce pathological changes and chronic immunological injury [Ohba et al., 1997; Wang et al., 2003]. In addition, genetic factors such as HLA class I and II genes have been implicated in the predisposition to membranous nephropathy, indicating that host factors also could be involved in the pathogenesis of HBV-associated nephropathy [Bhimma et al., 2002]. To date, at least eight genotypes of HBV (A–H) have been described [Stuyver et al., 2000; Norder et al., 2004], and mutations {

J. Ren and L. Wang contributed equally to this work. Grant sponsor: National Nature Science Foundation of China; Grant number: 30530040; Grant sponsor: Shanghai Municipal Science and Technology Commission; Grant number: 05JC14008; Grant sponsor: State Basic Research Fund (863); Grant number: 2004AA2Z3970. *Correspondence to: Dr. Y.M. Wen, Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, 200032 Shanghai, China. E-mail: [email protected] Accepted 9 January 2006 DOI 10.1002/jmv.20575 Published online in Wiley InterScience (www.interscience.wiley.com)

552

Ren et al.

in the genome of HBV may affect the replication competency as well as the immunogenicity of different HBV strains [Wen, 2004]. It is difficult to analyze the interactions between HBV and the kidney tissue of patients who have different genetic backgrounds and have been infected with different genotypes of HBV. A transgenic mouse lineage with known genetic background and well-characterized HBV isolate would serve as a good model to study the interactions between HBV and the kidney tissue in vivo. In the present work, a lineage of HBV transgenic mouse (#59) that has been established using a clone from a full-length sequenced HBV isolate (GenBank accession number AF461363) is described. Result showed that the expression of HBsAg and HBcAg was high in the tubular epithelium of the kidney. Comparing the global gene expression profile of the kidney from #59 HBV transgenic mice with that of their siblings using Affymetrix Mouse 430A microarray, there was marked up-regulation of complement activation, blood coagulation, and acute-phase response genes in the kidney tissue of the #59 mice. MATERIALS AND METHODS Cloning of HBV Genome The full-length HBV isolate used to generate the transgenic mouse lineage was from the serum of a HBV carrier positive for HBsAg and HBeAg, cloned into vector pUC18 by the method described by Gunther et al. [1995] and one clone (#25-8, GenBank accession number AF461363) was sequenced using primers as previously reported [Lin et al., 2001]. This HBV clone is adr serotype. It is 3,215 base pairs in length and according to Norder’s HBV genotype system, belongs to genotype C, subgenotype C1. Neither core promoter mutations nor precore stop codon mutation was detected, and the phenotype is HBeAg positive. Full-length HBV DNA from clone #25-8 was released using restriction enzyme SapI, linearized and purified. This HBV genome did not contain any exogenous genes or promoter and was used for microinjection. Generation of HBV Transgenic Mice and Genotype Analysis of the HBV Transgenic Mice HBV transgenic mice were generated in Shanghai Research Center for Model Organisms by the routine microinjection of linearized HBV DNA of clone #25-8 into fertilized eggs of C57BL/6J mice. The microinjection and embryo manipulation were carried out according to methods described previously [Gordon and Ruddle, 1986; Kupriyanov et al., 1998]. Both the donor and recipient mice were F1 hybrid mice (CBA  C57BL/ 6J). Heterozygous transgenic mice were backcrossed for six generations in C57BL/6J background mice, and were used at 24 weeks of age. The transgenic mice were genotyped by PCR using primer pairs designed according to the conserved sequence of HBV strain AF461363. Mice that were positive by PCR were tested further by Southern blot, linker-PCR, and real-time quantitative J. Med. Virol. DOI 10.1002/jmv

PCR to determine the detail of HBV genome integration. These transgenic mice were assayed for expression of HBsAg in their sera. The follow-up duration for each generation of #59 transgenic mice was between 8 and 15 months. Assays for Serum HBV DNA, HBsAg, HBeAg, Anti-HBs, and Anti-HBe in Transgenic Mice Venous bloods were collected from eye sockets of mice, and the sera were tested for HBsAg, HBeAg, anti-HBs, and anti-HBe by ELISA kits (Kehua Co., Shanghai, China). For detection of anti-HBs or anti-HBe which might be present as circulating immune complexes, modified precipitation with polyethylene glycol method was used [Yu et al., 1986]. In short, pooled serum samples were first precipitated with polyethylene glycol, washed and then dissociated in PBS, which was assayed for anti-HBs and anti-HBe by ELISA. Serum HBsAg pooled from each generation of positive transgenic mice were further quantified by Abbott kits. To monitor whether virus replication occurred, PCR was used to detect serum HBV DNA in successive generations of mice. Expression of HBsAg and HBcAg in Transgenic Mice Tissues The liver, kidney, spleen, and heart tissues were collected separately from transgenic mice that expressed HBsAg in their sera. Immuno-histochemical staining for HBsAg and HBcAg (Dako, Glostrup, Denmark) was carried out on sections from these tissues. Sections of liver tissues from HBV-infected human patients served as positive controls, and normal C57BL/6J sibling mice of the same age served as negative controls. Extracts of pooled tissues from #59 were centrifuged and the supernatants assayed for HBsAg by ELISA (Kehua Co., Shanghai, China). Microarray of Kidney Tissues from Transgenic Mice Three HBV genotype-positive transgenic mice expressing serum HBsAg at 24 weeks old and three siblings that had been inoculated with the same recombinant DNA but were HBV genotype negative were used in microarray study. After euthanasia, the left kidney of each mouse was dissected aseptically and was snap frozen in liquid nitrogen. The total RNA of each kidney was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) and the total RNA from each of the three transgenic mice was pooled in equal amounts. The pooled RNA from #59 mice and the control mice was used to hybridize with the Affymetrix (Santa Clara, CA) Mouse 430A slide. The cDNA synthesis, labeling, microarray hybridization, slide scanning, data acquisition, and analysis were conducted on the Affymetrix GeneChip1 Instrument System and web-based analytic protocol by the technical agents of Affymetrix.

Gene Expression Profile of HBV Transgenic Mouse Kidney

553 TABLE I. Quantification of HBsAg From Pooled Serum Samples of Each Generation of Transgenic Mice

Real-Time Quantitative RT-PCR Assay To confirm the results of microarray, eight genes (complement component 3, C-reactive protein, FBJ osteosarcoma oncogene, mannose binding lectin, mitochondrial ribosomal protein L15, plasminogen, serum amyloid A 1 and 2), were randomly selected and primers were designed according to the sequences of these genes for real-time quantitative RT-PCR assay. The assay protocol has been described previously [Fann et al., 2005]. The house-keeping gene GAPDH was used for normalization. Among these eight genes, six were upregulated and two were down-regulated. Dot Blot Immunoassay of Complement C3 Component in Mouse Serum and Kidney Tissue Extract To study whether the levels of C3 in the serum and in the kidney tissues of transgenic mice coincided with the up-regulation of C3 expression shown by microarray and real-time PCR, dot blot immunoassay was carried out. Mouse serum samples were used in serial 10-fold dilution, while supernatants of kidney homogenates were used in a dot blot assay. After centrifugation at 16,000g for 5min, the protein content of supernatant from the kidney homogenate was determined by Bradford assay. Each kidney extract sample was used at 170 ng protein per ml and at 10-fold serial dilutions. One ml of diluted mouse serum or kidney extract was added onto a nitrocellulose membrane and kept at 48C overnight. The dot blot was blocked with 10% skim milk in PBS prior to incubation with 1:10 diluted rabbit antimouse C3 polyclonal antibody (Hycult biotech, Uden, The Netherlands) at room temperature for 2 hr. After being washed, the blot was incubated in 1:200 HRPlabeled second antibody (goat anti-rabbit IgG, PuFei, Shanghai, China) at room temperature for 2 hr. Finally the substrate solution (O-dianisidine tetrazotized dissolved in 1 ml of methanol, mixed with 5 ml PBS and 5 ml 30% H2O2) was added. Positive signals were shown by insoluble purple dots on the blot.

Generation a

HBsAg (ng/ml)

1

2

3

4

5

6

7

109.8 196.5 419.7 351.3 131.6 191.1 298.6

a

Serum samples from 5  6 transgenic mice of each generation were pooled for quantitative assay.

was intact and functional (data not shown). In the #59 lineage, 25–40% transgenic mice were persistently serum HBsAg positive from F1 to F7. Pooled serum samples from each generation of HBsAg-positive transgenic mice were assayed by Abbott quantification kits and are shown in Table I. The longest duration of followup for serum HBsAg in #59 was 15 months. The fluctuation of the titer of serum HBsAg during the life span of one generation of #59 mice is shown in Figure 1. Expression of HBsAg and HBeAg, HBcAg in #59 Lineage Transgenic Mice In the serum of #59 transgenic mice, only HBsAg was positive; no serum HBeAg anti-HBs, anti-HBe, or antiHBc was detected. No anti-HBs or anti-HBe was detected in the polyethylene glycol precipitates and no HBV DNA was detected in serum. HBsAg was detected in the cytoplasm of a few dispersed hepatocytes, while a substantial amount of the tubule epithelial cells of the kidney tissue were HBsAg positive (Fig. 2). HBcAg was detected in the cytoplasm of hepatocytes and tubule epithelial cells in the kidney (Fig. 3). Neither HBsAg nor HBcAg was detected in the spleen or cardiac tissue of #59 mice. No infiltration of cells, or other pathological changes was observed in the liver and kidney of #59 by histological studies. Microarray Analysis The Affymetrix Mouse 430A slide, which contains 22,690 probe sets (among which 12,440 probe sets were

RESULTS Establishment of HBsAg-Positive Transgenic Mice In total, 538 fertilized eggs were microinjected with HBV clone #25-8 DNA. After transferred into pseudopregnant mice, 62 mice were born and genotyped. Of the seven founder mice that were HBV DNA positive, two (#10 & #59) were positive for serum HBsAg. Transgenic mouse lineage #59 was established by backcrossing positive founder mice with wild-type C57BL/6J mice. Further characterization of #59 lineage mice was done using Southern blot, linker-PCR, and real-time quantitative PCR. Results showed that a single copy linearized HBV genome (cut at HBV nucleic acid 1806) was integrated into mouse chromosome 9 in the second exon of an unknown gene AI604832. PCR and RT-PCR analysis indicated that the other allele of AI604832

Fig. 1. Follow-up study of serum HBsAg titer during the life span of lineage #59 transgenic mice of generation 2. The serum HBsAg titer of transgenic mouse lineage #59 was determined by ELISA assay from month 2 to 15. The average OD values (n ¼ 211) of the assays are shown in the Y-axis and standard deviations are shown as the error bar. OD value 0.1 was the cutoff value for HBsAg positive.

J. Med. Virol. DOI 10.1002/jmv

554

Ren et al.

Fig. 2. Immuno-histochemical staining of HBsAg in the liver and kidney sections of #59 transgenic mice. Sections of liver (A) or kidney (B) tissues from #59 transgenic mice were stained for HBsAg. Sections were stained with biotinylated mouse monoclonal anti-HBs antibody, washed, and incubated with avidin-labeled horseradish peroxidase at

378C for 25 min. DAB-H2O2 was added until color appeared and counter-stained with hematoxylin-eosin. A: Liver HBsAg (200): two positive-stained cells are shown distributed near the central vein. B: Kindey HBsAg (200): most positive-stained cells are the epithelia in the cortex of kidney tubules.

annotated with biological function), was used for microarray analysis. After hybridization and computer-based analysis compared with the control mice, 572 probe sets, (corresponding to 520 genes), were upregulated more than twofold; while 86 probe sets corresponding to 76 genes were down-regulated more than twofold in the kidney tissue of transgenic mice. This indicates that there were far more genes being upregulated than down-regulated. The highest up-regulated gene expression reached 512-fold, while the most that a gene down-regulated was 8-fold. A good correlation was shown between the microarray assay and real-time PCR of the eight selected genes for confirmation (Table II). Based on the annotation system of Affymetrix, the up- and down-regulated genes detected in #59 kidney were categorized into the following functional groups (with some overlaps between categories): cell growth and/or maintenance (123 genes); immune responses (62 genes); cell communication (45 genes); development (31 genes); and metabolism (201 genes). The genes in the last group could be further classified as biosynthesis (48 genes), regulation of metabolism (26 genes), nucleic acid metabolism (30 genes), organic acid metabolism (30 genes), amino acid and derivative metabolism (21 genes)

lipid metabolism (36 genes), and catabolism (43 genes) (detailed data available upon request). The most striking finding from the microarray data was that, numerous genes involved in the biological pathways of complement activation, blood coagulation, and acute-phase response were significantly up-regulated in the kidney of transgenic mice. Specifically, 42% (22 out of 52) complement activation-related genes (probe sets), 53% (15 out of 28) of the acute-phase response-related gene, and 35% (19 out of 54) of the blood coagulation-related genes were up-regulated (Table IIIA–C). By real-time PCR assay, C-reactive proteins gene expression was up-regulated to 1,000-fold, while complement component 3, plasminogen and fibrinogen genes were also significantly up-regulated (Table II). Aside from these genes being up-regulated, immunoglobulin heavy chain genes involved in antigen binding (Igh-4, Igh-1, IgK-V8, igh-Vj558) and the MHC1 receptor activating gene (H2-Q10), interleukin-1 receptor gene, and chemokine cc19 genes were all upregulated. Besides, the apolipoprotein genes involved in lipid transportation and genes of the cytochrome P450 family, which was described as involved in electron transport and oxidase-reductase activities, were also significantly up-regulated (Table II).

Fig. 3. Immuno-histochemical staining of HBcAg in the liver and kidney sections of #59 transgenic mice. Sections of liver (A) or kidney (B) tissues from #59 transgenic mice were stained for HBcAg. Staining procedures were similar to Figure 2, while the first antibody was rabbit anti-HBc and the second antibody was biotinylated goat anti-rabbit serum. A: HBcAg-positive cells are sparsely dispersed in the liver. B: HBcAg-positive cells are found in a number of epithelial cells of the tubules in the kidney.

J. Med. Virol. DOI 10.1002/jmv

Gene Expression Profile of HBV Transgenic Mouse Kidney TABLE II. Eight Selected Genes From the Kidney of #59 Mice Assayed by Microarray and Real-Time PCR Gene

Microarraya

Real-time PCRa

32 48 60 8 128 6

251 >1,000 138 5 192 1

446 60

1,000 287

Complement component 3 (C3) C-reactive protein (CRP) Fibrinogen (FGG) FBJ osteosarcoma oncogene (Fos) Mannose binding lectin (MBL2) Mitochondrial ribosomal protein L15 (Mrpl15) Plasminogen (PLG) Serum amyloid A 1 and 2 (Saa1 and 2) a

The numbers represent the folds of mRNA of the genes being up- or down-regulated.

Dot Blot Immunoassay of Complement C3 Component in Mouse Serum and Kidney Tissue Extract The relative C3 levels in the mouse serum and kidney tissue extracts from three individual transgenic mice and three control mice are shown by dot blot immunoassay in Figure 4. Compared to the control mice, C3 concentration was markedly lower in the serum of two transgenic mice and lower in one transgenic mouse. C3 was higher in the kidney extracts of all three transgenic mice. DISCUSSION HBV-associated nephropathy is common in HBVendemic areas, and this extrahepatic clinical manifestation of HBV is of great concern. The clinical features, pathology, and pathogenesis of HBV-associated nephropathy have recently been reviewed [Bhimma and Coovadia, 2004]. Though the most widely accepted mechanism for the pathogenesis of HBV-associated nephropathy is the deposition of immune complexes of HBV antigen and antibodies [Brzosko et al., 1974; Hirose et al., 1984; Lai et al., 1991], some authors have reported a high incidence of persistence of HBV DNA and HBV antigens in the renal tissues, and have claimed that the local expression of viral antigens may also contribute in the pathogenesis of HBV-associated nephropathy. Dejean et al. [1984] reported the detection of HBV DNA in the pancreas, kidney, and skin of HBV carriers. Lin [1993] detected HBV DNA in the kidney cells and speculated that viral pathogenesis could be involved in HBV-associated membranous nephropathy. Zhou et al. [1995] reported that in 93.3% of HBV DNApositive renal tissues there was HBcAg in renal tubules and/or glomeruli. They suggested that when HBV antigens were persistently expressed in the kidney tissue, it might cause cellular pathological changes and chronic immunopathogenicity. Moreover, by in situ hybridization, HBV DNA was positive in 95.3% from renal biopsies in Chinese children with HBV-associated glomerulonephritis [He et al., 1998], and the presence of viral transcripts in glomerular cells and renal tubular epithelia supported an etiological role of HBV protein

555

expression in some HBV-associated nephropathy [Lai et al., 1996]. In duck hepatitis B and in woodchuck hepatitis virus infected animals, hepadnaviruses have been detected in the kidney and spleen tissues and peripheral blood mononuclear cells [Freiman et al., 1988; Korba et al., 1989]. Although the origin and the mechanisms of HBV infection of extrahepatic tissues have not been fully elucidated, the possible role of extrahepatic HBV as a reservoir has been considered in the re-appearance of HBV infection after liver transplantation in patients. To date, a number of lineages of HBV transgenic mice have been established. Many important observations regarding viral protein expression, virus replication, viral pathogenesis, immune responses, virus-host interactions, and HBV-associated hepatocellular carcinogenesis have been reported in this well-defined inbred small animal model [Babinet et al., 1985; Araki et al., 1989; Chisari et al., 1989; Kim et al., 1991; Cavanaugh et al., 1997; Hildt et al., 2002]. However, these studies have concentrated on the liver tissue. In HBV transgenic mouse lineage 1.3.32 (official designation, Tg [1.3 genome] Chi 32), though HBV replicated at high levels in the liver and kidney without any evidence of cytopathology, only small amounts of HBsAg were detectable in the cytoplasm of centrilobular hepatocytes and HBsAg was reported as positive in the kidney, but with no detailed description of the location of this antigen [Guidotti et al., 1995]. In this study, lineage #59 transgenic mice not only expressed HBsAg and HBcAg in the liver, but also expressed the two antigens in the kidney, mainly in the tubular epithelium. The expression of HBsAg and HBcAg in the kidney makes it possible to use this model to study the interaction between HBV and kidney cells. Besides, because in lineage #59 the integration of HBV gene was interrupted at nucleotide 1806, while the initiation site of the HBeAg mRNA is 1785, this lineage cannot express HBeAg. Since HBV replication needs the 3.5 kb pregenomic RNA [Will et al., 1987], and only the linearized full-length HBV genome construct was used to establish lineage #59 transgenic mouse, no replication of HBV would occur. Results gained from the microarray study in this mouse lineage, therefore, only reflect the interactions between the viral proteins and host cells. Microarray analysis showed that the number of expressed genes up-regulated in the kidney was much higher than the number of genes being down-regulated (520 vs. 76). Since this transgenic mouse lineage does not replicate HBV, and no HBV protein antibody complex was detected, the locally expressed HBsAg and HBcAg appeared to initiate changes in the cellular gene expression in the kidney. In this study, the most strikingly up-regulated genes were genes coding for the C reactive protein, the complement C3 component and acute-phase response. Apart from confirming the upregulated expression by real-time PCR, the C3 protein in the serum and kidney tissues of #59 transgenic mice was also measured by dot blot immunoassay. Compared to the control mice, the serum C3 level was much lower in J. Med. Virol. DOI 10.1002/jmv

Complement component 1, q subcomponent, gamma polypeptide

Complement component 1, r subcomponent

Component 2 (within H-2S)

Complement component 3

Complement component 4 (within H-2S)

Component 4 binding protein Complement component 6 Complement component 8, alpha polypeptide Complement component 8, beta subunit

Complement component 9

Complement component factor h Complement component factor i

Hemolytic complement

Immunoglobulin heavy chain 1a (serum IgG2a)

Immunoglobulin heavy chain 4 (serum IgG1)

Mannan binding lectin serine protease 1

Mannose binding lectin, serum (C)

Serine (or cysteine) proteinase inhibitor, clade G, member 1

1417009_at

1416051_at

1423954_at

1418021_at

1418037_at 1449308_at 1428012_at

1427472_a_at

1422815_at

1425633_at 1418724_at

1419407_at

1425385_a_at

1425247_a_at

1419677_at

1418787_at

1416625_at

Gene name

1449401_at

Affymetrix probe set ID

J. Med. Virol. DOI 10.1002/jmv

Serping1

Mbl2

Masp1

Igh-4

Igh-1a

Hc

Cfh Cfi

C9

C8b

C4bp C6 C8a

C4

C3

C2

C1r

C1qg

Gene symbol

Complement activation, classical pathway

Complement activation, classical pathway Defense response to pathogenic bacteria Humoral defense mechanism (sensu Vertebrata) Complement activation, classical pathway Proteolysis and peptidolysis Complement activation, classical pathway Phosphate transport Blood coagulation

Antigen processing Complement activation, classical pathway Early endosome to late endosome transport Endosome to lysosome transport Humoral defense mechanism (sensu Vertebrata) Antibody-dependent cellular cytotoxicity

Complement activation, alternative pathway Complement activation, classical pathway Cytolysis Complement activation, alternative pathway Complement activation, classical pathway Complement activation, alternative pathway Complement activation, classical pathway Proteolysis and peptidolysis Complement activation, alternative pathway Complement activation, classical pathway Inflammatory response Antibody-dependent cellular cytotoxicity

Phosphate transport Complement activation, classical pathway Proteolysis and peptidolysis Complement activation, classical pathway Proteolysis and peptidolysis Complement activation, alternative pathway Complement activation, classical pathway Complement activation Humoral defense mechanism (sensu Vertebrata) Complement activation, classical pathway Complement activation Complement activation

Complement activation, classical pathway

Biological function

TABLE IIIA. Genes Involved in Complement Activation Up-Regulated

4

128

13

8

10

111

5 30

39

84

362 13 18

14

32

2

4

2

Fold changes in microarray

556 Ren et al.

Coagulation factor X

Coagulation factor XI

Coagulation factor II

Coagulation factor V

Coagulation factor VII

Coagulation factor IX Fibrinogen, B beta polypeptide Fibrinogen, gamma polypeptide kininogen 1

Macrophage stimulating 1 (hepatocyte growth factor-like)

Plasminogen

Protein Z, vitamin K-dependent plasma glycoprotein

Serine (or cysteine) proteinase inhibitor, clade C (antithrombin), member 1 Serine (or cysteine) proteinase inhibitor, clade D, member 1 Serine (or cysteine) proteinase inhibitor, clade G, member 1

1418992_at

1451788_at

1418897_at

1418907_at

1419321_at

1427393_at 1428079_at 1416025_at 1416676_at

1418267_at

1416729_at

1431721_a_at

1417909_at

1416625_at

1418680_at

Cytochrome P450, family 4, subfamily v, polypeptide 3 kallikrein B, plasma 1

Gene name

1449034_at

Affymetrix probe set ID

Serping1

Serpind1

Serpinc1

Proz

Plg

Mst1

F9 Fgb Fgg Kng1

F7

F5

F2

F11

Complement activation Complement activation, classical pathway

Blood coagulation

Blood coagulation

Proteolysis and peptidolysis Blood coagulation

Embryo implantation Proteolysis and peptidolysis Induction of apoptosis Myogenesis Negative regulation of angiogenesis Negative regulation of blood coagulation Blood coagulation

Inflammatory response Negative regulation of blood coagulation Blood coagulation Proteolysis and peptidolysis Blood coagulation Proteolysis and peptidolysis Acute-phase response Blood coagulation Blood coagulation Cell adhesion Blood coagulation Metabolism Blood coagulation Blood coagulation Blood coagulation Blood coagulation Inflammatory response Regulation of blood pressure Blood coagulation

Klkb1 F10

Blood coagulation

Biological function

Cyp4v3

Gene symbol

TABLE IIIB. Genes Associated Within Blood Coagulation Up-Regulated

4

23

74

6

446

5

169 478 60 4

49

5

128

4

45

10

Fold changes in microarray

Gene Expression Profile of HBV Transgenic Mouse Kidney 557

J. Med. Virol. DOI 10.1002/jmv

J. Med. Virol. DOI 10.1002/jmv

Haptoglobin

Hemopexin Orosomucoid 1 Serum amyloid A 1 Serum amyloid A 2 Serum amyloid A 2 Serum amyloid A 3 Serum amyloid A 4 Serine (or cysteine) proteinase inhibitor, clade A, member 1a Serine (or cysteine) proteinase inhibitor, clade A, member 1a Serine (or cysteine) proteinase inhibitor, clade A, member 1b

1448881_at

1423944_at 1451054_at 1419075_s_at

1449321_x_at

1451513_x_at

1448680_at

Serine (or cysteine) proteinase inhibitor, clade A, member 1b Serine (or cysteine) proteinase inhibitor, clade A, member 1d Serine (or cysteine) proteinase inhibitor, clade A, member 1a Serine (or cysteine) proteinase inhibitor, clade A, member 1e

Fibronectin 1

1426642_at

1449326_x_at 1450826_a_at 1419318_at 1420553_x_at

C-reactive protein, petaxin related Coagulation factor II

Gene name

1421946_at 1418897_at

Affymetrix probe set ID

Serpina1e

Serpina1a

Serpina1d

Serpina1b

Serpina1b

Serpina1a

Hpxn Orm1 Saa1 Saa2 Saa2 Saa3 Saa4 Serpina1a

Hp

Fn1

Crp F2

Gene symbol

Acute-phase response Acute-phase response, blood coagulation, proteolysis and peptidolysis Acute-phase response, cell adhesion Metabolism, heparin binding Acute-phase response, proteolysis and peptidolysis, chymotrypsin activity Acute-phase response, transport Acute-phase response, transporter activity Acute-phase response Lipid transporter activity Acute-phase response lipid transporter activity Acute-phase response lipid transporter activity Acute-phase response lipid transporter activity Acute-phase response serine-type endopeptidase inhibitor activity Acute-phase response serine-type endopeptidase inhibitor activity Acute-phase response endopeptidase inhibitor activity Serine-type endopeptidase inhibitor activity Acute-phase response endopeptidase inhibitor activity Serine-type endopeptidase inhibitor activity

Biological function

TABLE IIIC. Acute-Phase Responses Genes Up-Regulated

56

79

104

15 3 42 20

676 104 60

52

6

48 128

Fold changes in microarray

558 Ren et al.

Gene Expression Profile of HBV Transgenic Mouse Kidney

Fig. 4. Dot blot immunoassay of complement C3 component in mouse serum and kidney tissue extract. Levels of C3 were examined in three individual #59 transgenic mice and three control mice (C57). Serum and kidney tissue extract samples were loaded onto nitrocellulose membranes and assayed by polyclonal antibody against mouse C3 complement. The density of the signals of dots reflects the levels of C3 in samples. Serum C3 was markedly lower in two transgenic mice and lower in one transgenic mouse, while C3 from kidney extracts was higher in transgenic mice.

#59 mice, indicating that C3 might have bound to certain tissues in vivo. The higher level of C3 detected in the kidney tissue extract supports the concept that the upregulated C reactive protein, C3 and other acute-phase response genes in the kidney tissues could have initiated deposition of C3 proteins in the kidney. Attempt to detect C3 in renal tissues by immuno-histochemical staining was not successful, probably, due to the low sensitivity of the method used. The microarray study of the kidney of #59 transgenic mice, for the first time, revealed the role of HBV protein on regulating cellular gene expression in an animal model. Alterations in the expression of cellular genes strongly suggest that local persistent expression of viral proteins could contribute to the pathogenesis of HBVassociated nephropathy. However, while no transcription regulator function has been identified in the small S protein, other possible mechanisms such as, the insertion of HBV enhancers, response elements and viral promoters into host cell genome, and the expression of HBxAg might also participate in modulating host cell gene expression. ACKNOWLEDGMENTS We thank Dr. Philip Mortimer for commenting and editing this article, and Dr. MY Guo for helpful discussion with. REFERENCES Araki K, Miyazaki J, Hino O, Tomita N, Chisaka O, Matsubara K, Yamamura K. 1989. Expression and replication of hepatitis B virus genome in transgenic mice. Proc Natl Acad Sci USA 86: 207–211. Babinet C, Farza H, Morello D, Hadchouel M, Pourcel C. 1985. Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice. Science 230:1160–1163. Bhimma R, Coovadia HM. 2004. Hepatitis B virus-associated nephropathy. Am J Nephrol 24:198–211. Bhimma R, Hammond MG, Coovadia HM, Adhikari M, Connolly CA. 2002. HLA class I and II in black children with hepatitis B virus-associated membranous nephropathy. Kidney Int 61:1510– 1515. Brzosko WJ, Krawczynski K, Nazarewicz T, Morzycka M, Nowoslawski A. 1974. Glomerulonephritis associated with hepatitis-B surface antigen immune complexes in children. Lancet 2:477–482.

559 Cavanaugh VJ, Guidotti LG, Chisari FV. 1997. Interleukin-12 inhibits hepatitis B virus replication in transgenic mice. J Virol 71:3236– 3243. Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, Sell S, Pinkert CA, Brinster RL, Palmiter RD. 1989. Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59:1145–1156. Combes B, Shorey J, Barrera A, Stastny P, Eigenbrodt EH, Hull AR, Carter NW. 1971. Glomerulonephritis with deposition of Australia antigen-antibody complexes in glomerular basement membrane. Lancet 2:234–237. Dejean A, Luggasy C, Zafram S, Tiollais P. Brechot C. 1984. Detection of hepatitis B virus DNA in pancreas, kidney and skin of two human carriers of the virus. J Gen Virol 65:651–655. Fann M, Chiu WK, Wood WH III, Levine BL, Becker KG, Weng NP. 2005. Gene expression characteristics of CD28null memory phenotype CD8þ T cells and its implication in T-cell aging. Immunol Rev 205:190–206. Freiman JS, Gilbert AR, Dixon RJ, Holmes M, Gowan EJ, Burrell CJ, Will EJ, Cossart YE. 1988. Experimental Duck hepatitis B virus infection: Pathology and evolution of hepatic and extrahepatic infection. Hepatology 8:507–513. Gordon K, Ruddle FH. 1986. Gene transfer into mouse embryos. Dev Biol (NY 1985) 4:1–36. Guidotti LG, Matzke B, Schaller H, Chisari FV. 1995. High-level hepatitis B virus replication in transgenic mice. J Virol 69:6158– 6169. Gunther S, Li BC, Miska S, Kruger DH, Meisel H, Will H. 1995. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol 69: 5437–5444. He XY, Fang LJ, Zhang YE, Sheng FY, Zhang XR, Guo MY. 1998. In situ hybridization of hepatitis B DNA in hepatitis B-associated glomerulonephritis. Pediatr Nephrol 12:117–120. Hildt E, Munz B, Saher G, Reifenberg K, Hofschneider PH. 2002. The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice. Embo J 21:525–535. Hirose H, Udo K, Kojima M, Takahashi Y, Miyakawa Y, Miyamoto K, Yoshizawa H, Mayumi M. 1984. Deposition of hepatitis B e antigen in membranous glomerulonephritis: Identification by F(ab’)2 fragments of monoclonal antibody. Kidney Int 26:338–341. Kim CM, Koike K, Saito I, Miyamura T, Jay G. 1991. HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature 351:317–320. Korba BE, Cote PJ, Wells FV, Baldwin B, Popper H, Purcell RH. Tennet BC, Gerin JL. 1989. Natural history of woodchuck hepatitis virus infections during the course of experimental infection. Moleuclar virological features of the liver and lymphoid tissues. J Vriol 63: 1360–1372. Kupriyanov S, Zeh K, Baribault H. 1998. Double pronuclei injection of DNA into zygotes increases yields of transgenic mouse lines. Transgenic Res 7:223–226. Lai KN, Li PK, Lui SF, Au TC, Tam JS, Tong KL, Lai FM. 1991. Membranous nephropathy related to hepatitis B virus in adults. N Engl J Med 324:1457–1463. Lai KN, Ho RT, Tam JS, Lai FM. 1996. Detection of hepatitis B virus DNA and RNA in kidneys of HBV related glomerulonephritis. Kidney Int 50:1965–1977. Lin CY. 1993. Hepatitis B virus deoxyribonucleic acid in kidney cells probably leading to viral pathogenesis among hepatitis B virus associated membranous nephropathy patients. Nephron 63:58–64. Lin X, Qian GS, Lu PX, Wu L, Wen YM. 2001. Full-length genomic analysis of hepatitis B virus isolates in a patient progressing from hepatitis to hepatocellular carcinoma. J Med Virol 64:299– 304. Norder H, Courouce AM, Coursaget P, Echevarria JM, Lee SD, Mushahwar IK, Robertson BH, Locarnini S, Magnius LO. 2004. Genetic diversity of hepatitis B virus strains derived worldwide: Genotypes, subgenotypes, and HBsAg subtypes. Intervirology 47:289–309. Ohba S, Kimura K, Mise N, Konno Y, Suzuki N, Miyashita K, Tojo A, Hirata Y, Uehara Y, Atarashi K, Goto A, Omata M. 1997. Differential localization of s and e antigens in hepatitis B virusassociated glomerulonephritis. Clin Nephrol 48:44–47.

J. Med. Virol. DOI 10.1002/jmv

560 Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R. 2000. A new genotype of hepatitis B virus: Complete genome and phylogenetic relatedness. J Gen Virol 81:67–74. Wang NS, Wu ZL, Zhang YE, Guo MY, Liao LT. 2003. Role of hepatitis B virus infection in pathogenesis of IgA nephropathy. World J Gastroenterol 9:2004–2008. Wen YM. 2004. Structural and functional analysis of full-length hepatitis B virus genomes in patients: Implications in pathogenesis. J Gastroenterol Hepatol 19:485–489.

J. Med. Virol. DOI 10.1002/jmv

Ren et al. Will H, Reiser W, Weimer T, Pfaff E, Buscher M, Sprengel R, Cattaneo R, Schaller H. 1987. Replication strategy of human hepatitis B virus. J Virol 61:904–911. Yu JR, Lennette ET, Karpatkin S. 1986. Anti-F(ab’) 2 antibodies in thrombocytopenic patients at risk for acquired immunodeficiency syndrome. J Clin Invest 77:1756–1761. Zhou S, Zhang Y, Fang L. 1995. [A study on the appearance of hepatitis B virus markers in renal tissue of glomerulonephritis]. Zhonghua Bing Li Xue Za Zhi 24:296–299 (in Chinese).