The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression associated with IgAN by analyzing the diseased kidneys on cDNA microarrays. In particular, we showed an enhanced expression of several genes regulating the cell cycle and proliferation, including growth factors and their receptors, as well as endothelial differentiation gene-5 (EDG5), a receptor for sphingosine 1-phosphate (SPP). One of the growth factors, platelet-derived growth factor (PDGF) induces a marked upregulation of EDG5 in proliferative mesangial cells, and promotes cell proliferation synergistically with SPP. The genomic approach allows us to identify families of genes involved in a process, and can indicate that enhanced PDGF-EDG5 signaling plays an important role in the progression of IgAN.
IgA nephropathy (IgAN) is the most common non-diabetic, glomerulopathy worldwide, and of those diagnosed as having IgAN as many as 20–30% will suffer eventual kidney failure, and will require life saving dialysis and/or a kidney transplant.1 The pathological features of IgAN include hematuria, high levels of circulating IgA, abnormal glomerular deposition of IgA, complement C3, fibronectin and collagen, and mesangial cell proliferation. In order to explore the underlying molecular basis of IgAN, for which there is as yet no effective therapy, a valid animal model that displays the clinical and pathological features of human IgAN is required.
An outbred mouse strain, ddY, has been reported to spontaneously develop mesangioproliferative glomerulonephritis with a severe deposition of IgA. By selective breeding for high serum IgA levels from outbred ddY mice, a mouse strain of IgAN (HIGA: high serum IgA) was established.2 This strain shows consistently high serum IgA levels, and various mesangial lesions are observed microscopically, from mild mesangial cell proliferation to marked mesangial expansion accompanied by extracellular matrix accumulation.3 HIGA mice, therefore, show almost all of the clinical and pathological features of human IgAN, indicating that this is a valid animal model for human IgAN.
To gain insight into the underlying molecular basis of IgAN, we have used cDNA microarrays to assess changes in gene expression in the kidneys of HIGA mice. As the genetic background of HIGA and ddY mice is considered to be similar, we used ddY mice as controls to examine the gene expression specifically related to IgAN. This genomic approach allows us to identify families of genes involved, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes. Based on the expression profile obtained, we found an enhanced PDGF-EDG cascade that may play a potentially important role in the proliferation of kidney mesangial cells.
cDNA Microarray Analysis in HIGA Mice
The weight of the kidneys of HIGA mice was about the same as that of ddY mice at 6 weeks of age (292 ± 6.1 vs 278 ± 7.5 mg, n = 4), whereas at the age of 25 weeks the kidney weight of HIGA mice was significantly greater than that of ddY mice (465 ± 15.2 vs 395 ± 9.5 mg, n = 4, P < 0.01). In HIGA mice, mesangial cell proliferation can be observed between about 10 and 25 weeks of age, and the mesangial matrix expands significantly after the age of 40 weeks.3 We therefore examined HIGA and control ddY mice before and after the pathological changes become microscopically observable, at age 6 and 25 weeks, respectively. In histological studies, no pathologic features were detected at 6 weeks of age (Figure 1). At 25 weeks of age, in contrast, the mesangial cells had markedly proliferated, and the Bowman's space and glomerular capillaries had been narrowed by expansion of the mesangial matrix (Figure 1).
To investigate the molecular mechanism of IgAN, we generated microarrays with a normalized kidney cDNA library to assess changes in gene expression in the kidneys of HIGA mice. The differentially expressed genes revealed by microarray analysis included those that encode extracellular matrix proteins (eg, collagen), ion channel and transporters (eg, OAT-K1, ABC2, Kir5.1), and transcription factors (eg, STAT5A, Mist1). Among genes showing significantly enhanced expression are growth factors and their receptors, and molecules associated with cell growth, proliferation, and cellular signaling (Table 1). One of the major sets of genes is the growth factors and their receptors, and all growth factors listed in Table 1 are reported to have a potent mitogenic effect on mesangial cells.4,5,6,7,8,9 Another set of genes are G-protein-coupled receptors (GPCRs), including receptors for a chemoattractive lipid mediator leukotriene B4,10,11 a 5-hydroxytryptamine receptor (5HT-2)12 and H218,13 otherwise known as the SPP receptor EDG5 which is involved in cell proliferation, cell migration, and apoptosis.14
Analysis of EDG Subtype by RT-PCR in the Kidney of HIGA Mice
To date, eight closely related GPCRs of the EDG family have been identified as high affinity receptors for SPP (EDG1, 3, 5, 6 and 8) and lysophosphatidic acid (EDG2, 4, and 7).15,16,17 As these receptors show high sequence homology, we next examined changes in the expression levels of each EDG subtype by RT-PCR using subtype-specific primers. At age 6 weeks, EDG5 and EDG6 were elevated 2.9- and 4.4-fold, respectively in HIGA mice compared to ddY mice (Figure 2). Moderate upregulation of EDG2 and EDG3 was also observed. On the other hand, we found no significant change in the expression of each EDG subtype at the age of 25 weeks (Figure 2), suggesting that EDG upregulation occurs in the early stages of IgAN.
Semi-quantitative RT-PCR Analysis of Growth Factors and Their Receptors in the Kidney of HIGA Mice
Next, we conducted semi-quantitative RT-PCR analysis of growth factors and their receptors to show gene expression of each molecule in more detail. This analysis confirmed an increased expression of genes encoding PDGF, bFGF and EGF, as well as their receptor subtypes, in the kidney of HIGA mice, in particular at 6 weeks in HIGA mice (Figure 3), suggesting that growth factor signaling is enhanced even at the early stage of IgAN.
Effects of Growth Factors and SPP on Cultured Rat Mesangial Cells
Growth factors, such as PDGF-BB, have been shown to activate sphingomyelinase and ceramidase in mesangial cells, and this results in de novo SPP synthesis.18 As enhanced expression of PDGF and PDGF receptors was observed in the kidney of HIGA mice, we hypothesized that PDGF may affect not only the synthesis of SPP but also the expression of the SPP receptors in mesangial cells. Using cultured rat mesangial cells, we first confirmed the significant proliferative response to PDGF-BB (10 ng ml−1), bFGF (10 ng ml−1) and EGF (20 ng ml−1) as previously reported8,9 (Figure 4). Among EDG subtypes of which expression was confirmed in the kidney (EDG1–7), our RT-PCR analysis showed that only EDG2, EDG3, and EDG5 were expressed in mesangial cells (data not shown); hence, we further examined the effects of the growth factors on these three EDG subtypes. As summarized in Table 2, PDGF-BB (10 ng ml−1) had significantly induced the expression only of EDG5 after 2 h of stimulation. Furthermore, we examined the effects of SPP on mesangial cell proliferation. As shown in Figure 5, SPP (5 or 10 μM) alone significantly induced mesangial cell proliferation in a dose-dependent manner. Moreover, SPP exhibited a significantly positive effect on PDGF (40 ng ml−1)-induced cell proliferation, indicating that SPP and PDGF promoted proliferation of mesangial cells synergistically (Figure 5). The synergistic effect of SPP was also observed with 10 ng ml−1 PDGF treatment (data not shown).
In general, DNA microarrays are facilitating systematic exploration of gene expression on a genome-wide scale. Working with expanded sets of genes, more complete in number and functional characterization than hitherto, should yield a wealth of information about a variety of physiological and pathological states. Comparisons of gene expression changes among different models also contribute greater understanding of the relationship between genes and disease. Here, this analysis was used to look at complex changes in gene expression in an experimental mouse model of IgAN, as they develop typical histological profiles. Based on the microarray results, we found a marked upregulation of EDG5 in the kidney of HIGA. These results are in good agreement with clinical observations of the enhanced expression of PDGF and PDGF receptors in the kidneys of IgAN patients.19,20,21,22
Besides the growth factors and their receptors, and GPCRs, our microarray analysis showed an enhanced expression of molecules associated with cell growth and proliferation (Table 1). Cyclin D1 and cyclin G are essential regulatory factors in the progression of the cell cycle from G0 through G1 and S phase,23 and TAPA-1 plays an important role in the regulation of cell growth.24 Bcl-2 is well-known anti-apoptotic protein. Recent studies showed that bcl-2 expression was upregulated in the kidney of mesangial proliferative nephritis25 and redox signals induced bcl-2 accumulation in mesangial cells.26 Another two genes are anti-proliferative factors in growth control and differentiation.27,28 This apparently conflicting expression profile may be interpreted as a mechanism of self-defense against abnormal cell proliferation in the kidney of HIGA mouse.
Using cultured rat mesangial cells, we conducted in vitro experiments to verify the microarray results. As our microarray analysis was performed with RNA extracted from whole kidney without perfusion and without microdissection, the expression profile obtained could not exclude the possible contamination of the circulating blood cells. In fact, our RT-PCR study in the kidney of HIGA mouse showed an elevated expression of not only EDG5 but also EDG6. As EDG6 is a predominant SPP receptor subtype in platelets,29 these results may indicate that the mRNA used might have contained EDG6 transcript derived from infiltrating platelets. Additionally, mRNA for platelet factor 4, which is known to be specifically expressed in platelets, was amplified from the same preparation, which supports this possibility (data not shown).
Our present study highlights the role of EDG5 in IgAN. EDG5 has been recently reported to be involved in multiple signaling pathways,30 and mediating SPP-induced cell proliferation and the anti-apoptotic effect in rat hepatoma cells;14 however, the function of EDG5 is much still uncertain. The present study for the first time showed that PDGF upregulates expression of EDG5 in proliferative mesangial cells. Interestingly, our study further showed that SPP (ligand for EDG5) promotes mesangial cell proliferation synergistically with PDGF. Taken together with the recently accumulating experimental evidence, our present study may indicate a positive amplification loop of PDGF-EDG cascade in the mesangial cell proliferation. As summarized in Figure 6, when stimulated with PDGF, mesangial cells produce and release PDGF, and concomitantly the expression of PDGF receptor on the cell surface is increased in both an autocrine and a paracrine manner.31,32 Also, Coroneos et al previously reported that PDGF can activate sphingomyelinase and ceramidase in mesangial cells, which results in an increased synthesis of SPP.18 Besides the increased release of SPP from mesangial cells, it can also be released from the activated and/or aggregated platelets.29,33,34,35 The enhanced expression of EDG5 by PDGF as shown in the present study, and the augmented production of its ligand SPP by PDGF18 can synergistically promote the mesangial cell proliferation. In the kidney of HIGA mice, the PDGF-SPP-EDG pathway might act in a positive feedback loop to amplify the cascade of events linking growth factor signaling to cellular proliferative activity (Figure 6).
In summary, the presented results provide the first look on the complex pattern of changes in a disease state by employing microarray and associated analyses. Much should be learned by examining gene expression changes in HIGA mice treated by genetic manipulation and/or pharmacological agents (such as glucocorticoids and cytotoxic drugs) that show beneficial effects. As exemplified in the present study, the combination of a genome-wide search and cell biology research in animal models of human diseases would be a potentially useful functional genomic approach in the diagnosis and prognosis of the disease, and to accelerate the identification of novel therapeutic targets.
MATERIALS AND METHODS
Mice were maintained under normal laboratory conditions. Female ddY and HIGA mice at age 6 and 25 weeks were used in the experiments. The National Children's Medical Research Center Committee on Animal Welfare approved all animal protocols.
From female HIGA mice aged 6 or 25 weeks, kidneys were removed and embedded in paraffin. The paraffin sections were stained with the periodic acid-Schiff reagent and examined by light microscopy.
Rat Mesangial Cell Culture and Treatment
Rat glomerular mesangial cells were cultured and cloned by standard protocols as described elsewhere.36 Cells were grown in RPMI 1640 supplemented with 10% (v/v) fetal calf serum (FCS). Mesangial cells were serum-starved for 16–24 h, and treated with or without growth factors or SPP in serum-free RPMI 1640. For treatment with SPP, serum-free RPMI 1640 containing 0.4% FAF-BSA was used. We used the XTT assay kit (Roche Diagnostics, Tokyo, Japan) to assess mesangial cell proliferation. Total RNA was isolated using Isogen (Nippon Gene, Toyama, Japan).
Construction of Microarray with a Normalized Kidney cDNA Library
‘Normalization’ is a procedure that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species, and the cDNA library applied by this procedure is ‘a normalized cDNA library’. Ordinary cDNA libraries originated from tissues contain a high frequency of undesirable clones because of redundancy of mRNA species in the cell. To get rid of the redundancy from ordinary cDNA libraries (‘normalization’), we used an approach of ‘subtractive library’ reported by Bonaldo et al37 with minor modifications. Briefly, we hybridized in vitro synthesized DNA driver from an entire library with the library itself in the form of a single-stranded tracer. Single-stranded DNA circles were separated and conversed to double-stranded plasmids. Plasmid was electroporated into DH10B bacteria cells (Life Technologies, Gaithersburg, MD, USA), and we used bacterial transformants as a normalized cDNA library. This method has been described in more detail by Katsuma et al.38 Bacterial clones (total 4224 clones) were randomly isolated from a normalized kidney cDNA library. Partial sequencing of the clones indicated that there were about 3000 distinct sequences, which is a rough estimate of the number of distinct genes printed.
Preparation of Target DNA for cDNA Microarray
Two micrograms of each Poly (A)+ RNA prepared from mouse kidneys were reverse transcribed with Cy3- or Cy5-conjugated dUTP (Amersham Pharmacia Biotech, Uppsala, Sweden) using Superscript II reverse transcriptase (Life Technologies). After a 2-h incubation, labeled probes were concentrated in a Microcon filter device (Millipore, Bedford, MA, USA), diluted in 10 μl hybridization solution (3.4× SSC containing 0.3% SDS, 20 μg polyA DNA, and 20 μg yeast tRNA), and applied to the microarray.
Hybridization and Scanning
We hybridized labeled probes against cDNA microarrays with overnight incubation at 65°C. After removing the cover glasses, the slides were washed twice with 2× SSC containing 0.5% SDS for 5 min at room temperature, once with 0.2× SSC containing 0.5% SDS at 40°C for 3 min, and finally with 0.2× SSC for 3 min. Slides were dried by centrifugation at low speed. Hybridization images were scanned by ScanArray 5000 (GSI Lumonics, Billerica, MA, USA) and signal intensities were quantified by Array Gauge (FUJIFILM, Tokyo, Japan).
Data Analysis of cDNA Microarray
Data analysis of the cDNA microarray was performed as described elsewhere.39 Briefly, Cy3:Cy5 intensity ratios from each clone were calculated and subsequently standardized to ratios of overall signal intensity in each hybridization. We defined differentially expressed clones as positives when ratios of Cy3-labeled HIGA mRNA vs Cy5-labeled ddY mRNA were statistically different (t-test, P < 0.05) from those of Cy3-labeled ddY mRNA vs Cy5-labeled ddY mRNA. At least two hybridizations were performed in four different animals at age 6 and 25 weeks.
mRNA prepared from mouse kidneys or rat cultured mesangial cells was reverse transcribed, diluted and used for PCR. We amplified cDNA fragments for 15–25 cycles and determined the exponential phase to allow semi-quantitative analysis of each reaction. Negative controls without RT were routinely included in each reaction. PCR primers were designed on the basis of the reported cDNA sequences or expressed-sequence tag (EST) sequences. Densitometric analysis was performed with NIH image software. β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used to standardize the mRNA levels of target genes.
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We thank Drs S Stojilkovic (National Institute of Child Health and Human Development, National Institutes of Health) and GE Smyth (Research Laboratories, Nippon Shinyaku Co) for critical reading of the manuscript. This work was supported in part by research grants from the Scientific Fund of the Ministry of Education, Science, and Culture of Japan, the Japan Health Science Foundation and Ministry of Human Health and Welfare, the Organization for Pharmaceutical Safety and Research (OPSR), and a Grant for Liberal Harmonious Research Promotion System from the Science and Technology Agency.
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Katsuma, S., Shiojima, S., Hirasawa, A. et al. Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF–EDG5 cascade. Pharmacogenomics J 1, 211–217 (2001) doi:10.1038/sj.tpj.6500043
- cDNA microarray
- IgA nephropathy
- endothelial differentiation gene
- platelet-derived growth factor
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