Hydrodynamics-based delivery of the viral interleukin-10 gene suppresses experimental crescentic glomerulonephritis in Wistar–Kyoto rats

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Abstract

Gene therapy is expected to revolutionize the treatment of kidney diseases. Viral interleukin (vIL)-10 has a variety of immunomodulatory properties. We examined the applicability of vIL-10 gene transfer to the treatment of rats with crescentic glomerulonephritis, a T helper 1 (Th 1) predominant disease. To produce the disease, Wistar–Kyoto rats were injected with a rabbit polyclonal anti-rat glomerular basement membrane antibody. After 3 h, a large volume of plasmid DNA expressing vIL-10 (pCAGGS-vIL-10) solution was rapidly injected into the tail vein. pCAGGS solution was similarly injected into control rats (pCAGGS rats). We confirmed the presence of vector-derived vIL-10 mainly in the liver and observed high serum vIL-10 levels in pCAGGS-vIL-10-injected rats. Compared with the pCAGGS rats, the pCAGGS-vIL-10 rats showed significant therapeutic effects: reduced frequency of crescent formation, decrease in the number of total cells, macrophages, and CD4+ T cells in the glomeruli, decrease in urine protein, and attenuation of kidney dysfunction. Using quantitative real-time polymerase chain reaction, we also observed that this model was Th1-predominant in the glomeruli and that the ratio of the transcripts of CD4, interferon-γ, tumor necrosis factor-α, and monocyte chemotactic protein-1 to the transcripts of glucose-6-phosphate dehydrogenase in the glomeruli were all significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats. These results demonstrate that pCAGGS-vIL-10 gene transfer by hydrodynamics-based transfection suppresses crescentic glomerulonephritis.

Introduction

Crescentic glomerulonephritis (GN) is the nonspecific immune response of the glomerulus to severe inflammation. In this disease, alterations of Bowman's space, which is occupied by fibrin, cells, and matrix materials, lead to crescentic epithelial scar formation and permanent loss of the nephron. The clinical syndrome associated with extensive glomerular crescent formation is called rapidly progressive glomerulonephritis and has a poor renal prognosis in humans.1

Nephrotoxic serum (NTS) nephritis is produced by the administration of a rabbit polyclonal anti-rat glomerular basement membrane (GBM) antibody into Wistar–Kyoto (WKY) rats; this is a well-established experimental model of crescentic GN.2 Within a few hours after the NTS injection, the rats enter the heterologous phase and develop proteinuria. The clinical symptoms of the heterologous phase are usually blended with those of the autologous phase after 5–7 days. In the autologous phase, the host antibodies and cellular sensitivity against the heterologous antibodies fixed to the GBM cause severe GN, which includes the proliferation of glomerular cells, infiltration of lymphocytes and macrophages, intracapillary and extracapillary fibrin deposits, and crescent formation.

Crescentic GN is the manifestation of a T helper 1 (Th 1)-predominant nephritogenic immune response.3 In fact, the administration of Th2 cytokines, interleukin (IL)-10 and/or IL-4, or the inhibition of Th1 cytokines, IL-12 or interferon (IFN)-γ, diminishes nephritogenic immune responses,4,5 and conversely, the administration of Th1 cytokines, or the inhibition of Th2 cytokines exacerbates the severity of GN.6,7,8

IL-10 is produced by macrophages and Th2 cells, and inhibits the synthesis of cytokines by Th1 cells.9,10,11 IL-10 is, therefore, a cytokine of particular interest as a therapeutic agent for Th1-predominant disease. Viral IL-10 (vIL-10), a product encoded by the Epstein–Barr virus BamHI C fragment rightward reading frame 1 (BCRF1) (vIL-10) open reading frame, is homologous to both murine and human IL-10, especially in the coding region of the mature protein sequence.12,13,14 vIL-10 and IL-10 possess many biological properties in common, including activity as a cytokine synthesis inhibitory factor and the ability to downregulate class II major histocompatibility complex (MHC) expression on monocytes.13,14,15 However, vIL-10 lacks most of the immuno-stimulatory properties seen with IL-10.14,16,17 Thus, vIL-10 seems to suppress Th1-mediated immune reactions, and could be an ideal immunosuppressive cytokine.

Gene therapy using vIL-10 has been effective in other animal models.18,19 In a murine collagen-induced arthritis model that is classified as a Th1-predominant model, systemic adenovirus-mediated vIL-10 gene transfer reduced the synovial mRNA expression of tumor necrosis factor-α (TNF-α), IL-1β, and IL-6, and inhibited swelling and redness of the paws when given before disease onset.18 In a murine viral myocarditis model, pCAGGS-vIL-10 gene transfer into the muscles by in vivo electroporation repressed the mRNA expression levels of IFN-γ and inducible nitric oxide synthase in the heart, and improved the survival rates of the animals.19 Thus, vIL-10 gene therapy has been shown to provide a therapeutic effect in other Th1-predominant or inflammatory models.

Gene therapy is expected to revolutionize the treatment of kidney diseases, especially of immunologically mediated GN. Recently, it was reported that high levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of a naked DNA solution into the tail vein. This is known as ‘hydrodynamics-based transfection′.20,21 We demonstrated that this technique is also useful for the delivery of a therapeutic protein into normal rats, using a rat erythropoietin (Epo) expression plasmid vector pCAGGS-Epo.22 Here we tested the effectiveness of vIL-10 gene transfer by hydrodynamics-based transfection for the suppression of crescentic GN in WKY rats. We used the vIL-10 expression plasmid vector pCAGGS-vIL-10, which drives vIL-10 cDNA expression under the CAG (cytomegalovirus immediate-early enhancer/chicken β-actin hybrid) promoter.23

Results

Serum vIL-10 levels

Rats were inoculated with 0.1 ml of NTS per 100 g body weight. After 3 h, in the heterologous phase, using hydrodynamics-based gene transfection, we injected 800 μg of pCAGGS-vIL-10 (pCAGGS-vIL-10 rats; n=7) or pCAGGS plasmid (pCAGGS rats; n=7) into the rats. As expected, the serum vIL-10 levels (Figure 1) in the pCAGGS rats were less than 30 pg/ml, the detection limit of the assay, at each time point. On the other hand, the serum vIL-10 levels in the pCAGGS-vIL-10 rats increased, peaking at 656.1±44.5 ng/ml on day 1, and gradually decreasing to 537.1±25.1 ng/ml on day 4, and 478.0±62.2 ng/ml on day 7. These results indicated that a continuous delivery of vIL-10 for more than 7 days can be achieved in rats by hydrodynamics-based transfection.

Figure 1
figure1

Serum vIL-10 levels after pCAGGS-vIL-10 transfer. On day 0, 0.1 ml of NTS per 100 g body weight was injected into rats followed by an injection of 800 μg of pCAGGS-vIL-10 (pCAGGS-vIL-10 rats) or pCAGGS plasmid (pCAGGS rats) 3 h later. n=7 in each group. Serum vIL-10 levels in the pCAGGS rats were less than 30 pg/ml, the detection limit of the assay, at each time point.

Serum IFN-g, IL-4, and IL-10 levels

To examine the effects of pCAGGS-vIL-10 or pCAGGS on serum IFN-γ, IL-4, and IL-10 levels (Table 1), we performed an ELISA analysis on day 2 before the injection and on day 7 after the injection. The serum levels of IFN-γ, IL-4, and IL-10 in the pCAGGS-vIL-10-injected group (n=7) and the pCAGGS-injected group (n=7), comparing the pre- and postinjection values, were not significantly different. The serum levels of IFN-γ, IL-4, and IL-10 were also not significantly different between the two groups, throughout the experiment period. These results suggested that neither the NTS injection nor the pCAGGS-vIL-10 transfer affected the serum levels of IFN-γ, IL-4, or IL-10.

Table 1 Serum IFN-γ, IL-4, and IL-10 levels

Effect of pCAGGS-vIL-10 transfer on urinary protein

Proteinuria developed in all rats and increased throughout the experiment. There was no difference in the urinary protein level between the pCAGGS-vIL-10 rats (n=7) and the pCAGGS rats (n=7) on day 1 after injection (2.1±1.2 versus 2.6±1.8 mg/day) and on day 4 after injection (6.2±3.0 versus 7.6±3.7 mg/day), respectively. However, the urinary protein level on day 7 after injection was significantly lower in the pCAGGS-vIL-10 rats (n=7) than in the pCAGGS rats (n=7): 46.0±22.5 versus 94.2±25.1 mg/day, respectively (P<0.01). These results indicated that the continuous delivery of vIL-10 decreased the NTS-induced urinary protein excretion (Figure 2).

Figure 2
figure2

Effect of pCAGGS-vIL-10 transfer on proteinuria. Urinary protein excretion in WKY rats given injections of NTS and later pCAGGS-vIL-10 or pCAGGS plasmid was determined. n=7 in each group. **, P<0.01 for comparison with pCAGGS rats. ##, P<0.01, ###, P<0.001, and ####, P < 0.0001 for comparisons with preinjection rats (day −2) within each group. The error bars are not shown for some data points because of low variability.

Effect of pCAGGS-vIL-10 transfer on kidney function

The creatinine clearance (Ccr) levels in the pCAGGS rats (n=7) were markedly decreased, from 0.51±0.12 ml/min/100 g on day 2 before injection to 0.39±0.05 ml/min/100 g on day 7 after injection (P<0.05). On the other hand, the Ccr levels in the pCAGGS-vIL-10 rats (n=7) were not significantly decreased (0.50±0.05 ml/min/100 g on day 2 before injection and 0.46±0.07 ml/min/100 g on day 7 after injection). The Ccr level on day 7 after injection was significantly higher in the pCAGGS-vIL-10 rats than in the pCAGGS rats (P<0.05). These results demonstrated that the pCAGGS-vIL-10 transfer could attenuate the NTS-induced kidney dysfunction (Figure 3).

Figure 3
figure3

Effect of pCAGGS-vIL-10 transfer on Ccr. Ccr in WKY rats given injections of NTS and later pCAGGS-vIL-10 or pCAGGS plasmid. Ccr was measured 2 days before (□) and 7 days after (▪) injection. n=7 in each group. *, P<0.05 for comparison with pCAGGS rats. #, P<0.05 for comparison with preinjection on day −2.

Effect of pCAGGS-vIL-10 transfer on glomerular lesions

The glomerular capillaries of the kidney sections from both the pCAGGS-vIL-10 rats and the pCAGGS rats reacted similarly, showing a linear pattern with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, thus defining the presence of the rabbit polyclonal anti-rat GBM antibody (Figure 4a). Compared with kidney sections from normal rats, it was easy to identify severe proliferative GN in the pCAGGS rats, which showed mononuclear and mesangial cell proliferation, exudative lesions, and crescent formation on day 7 after the injection of NTS. These NTS-induced glomerular injuries were attenuated in the kidney sections from the pCAGGS-vIL-10 rats (Figure 4b). As shown in Figure 5a, the frequency of crescent formation on day 7 after injection was significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats: 30.4±3.9 versus 69.0±3.7% (P<0.0001). The total number of glomerular cells on day 7 after injection was significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats: 54.1±3.3 cells/glomerular cross-section (c/gcs) versus 76.7±4.2 c/gcs, respectively (P<0.0001) (Figure 5b). These results demonstrated that the pCAGGS-vIL-10 transfer could attenuate the NTS-induced glomerular changes.

Figure 4
figure4

(a) Detection of NTS on the glomerular capillaries by immunofluorescence staining of the kidney sections from the pCAGGS and pCAGGS-vIL-10 rats on day 7. The sections of both rat groups were stained similarly in a linear pattern with FITC-conjugated goat anti-rabbit IgG. Original magnification, × 400. (b) Periodic acid-Schiff staining of the kidney sections from the pCAGGS and pCAGGS-vIL-10 rats on day 7. Original magnification, × 400. (c) Detection of glomerular ED1+ cell (macrophage), (d) CD4+ T cells, and (e) CD8+ T cells by immunofluorescence staining of the kidney sections from the pCAGGS and pCAGGS-vIL-10 rats on day 7. Original magnification, × 400.

Figure 5
figure5

(a) Crescent formation, (b) total number of cells, (c) glomerular ED1+ cells (macrophages), (d) CD4+ T cells, and (e) CD8+ T cells in the kidney sections of WKY rats given injections of NTS and later pCAGGS-vIL-10 or pCAGGS plasmid. n=7 in each group. ****, P<0.0001 for comparison with the pCAGGS rats.

Effect of pCAGGS-vIL-10 transfer on glomerular ED1+ cell (macrophage) accumulation

Macrophages are the major effector cells in human and experimental crescentic GN.24,25 To examine the effects of pCAGGS-vIL-10 on the population of macrophages in the glomeruli (Figures 4c and 5c), we performed immunohistochemical analysis of the kidney sections from the rats on day 7 after injection. The number of ED1+ cells in the glomeruli on day 7 after injection was significantly less in the pCAGGS-vIL-10 rats (n=7) than in the pCAGGS rats (n=7): 12.2±3.7 versus 25.7±3.1 c/gcs, respectively (P<0.0001). These results demonstrated that pCAGGS-vIL-10 transfer could inhibit the accumulation of macrophages in the glomeruli.

Effect of pCAGGS-vIL-10 transfer on glomerular CD4+ T cell and CD8+ T cell accumulation

To examine the effects of pCAGGS-vIL-10 on the population of CD4+ T cells and CD8+ T cells in the glomeruli (Figures 4d, e and 5d, e), we performed immunohistochemical analysis on sections from kidneys harvested on day 7 after the injection. The number of CD4+ T cells in the glomeruli was significantly lower in the pCAGGS-vIL-10 rats (n=7) than in the pCAGGS rats (n=7): 0.4±0.1 versus 1.3±0.2 c/gcs, respectively (P<0.0001). However, there was no difference in the number of CD8+ T cells in the glomeruli on day 7 after injection. These results demonstrated that pCAGGS-vIL-10 transfer could inhibit the accumulation of CD4+ T cells, but not CD8+ T cells, in the glomeruli.

Biodistribution of vIL-10 mRNA from injected pCAGGS-vIL-10 on day 7 by quantitative real-time polymerase chain reaction (PCR)

Since injecting plasmid DNA into the tail vein did not permit anatomical targeting of the DNA to the liver, it was important to examine whether the plasmid DNA was transfected into and expressed mainly in the liver, rather than the kidney. Such a finding would indicate that hydrodynamics-based transfection can achieve liver-targeted gene transfer and that circulating vIL-10 mainly produced in the liver brings about therapeutic effects on crescentic GN, which is in the kidney. Normalized levels of the vIL-10 transcripts were calculated as the ratio of vIL-10 transcripts to glucose-6-phosphate dehydrogenase (G6PDH) transcripts. We detected the transgene-derived vIL-10 mRNA by quantitative real-time PCR analysis in the liver, heart, kidney, and lungs of rats that had been given an injection of a 15-ml volume of 800 μg of pCAGGS-vIL-10 (n=3) (Figure 6). The control G6PDH mRNA was detected in all major organs. Among the organs examined, the highest level of vIL-10 gene expression was seen in the liver. Mean values with their s.d.'s were calculated and were presented by standardizing to the mean value of the vIL-10 gene expression in the liver, which was given a value of 1 in the statistical analysis. The transgene expression in the heart (0.037±0.011, P<0.01), kidney (0.0012±0.0001, P<0.01), and lungs (0.0004±0.0001, P<0.01) was much less than in the liver.

Figure 6
figure6

Biodistribution of vIL-10 mRNA in the pCAGGS-vIL-10 rats by quantitative real-time PCR. On day 7 after the injection, rats that had received 800 μg of pCAGGS-vIL-10 were killed and their organs harvested (n=3). Normalized levels of the vIL-10 transcripts were calculated as the ratio of vIL-10 transcripts to G6PDH transcripts. The mean values with their s.d.'s were calculated and standardized to the mean values of the vIL-10 gene expression in the liver, which was given a value of 1 in the statistical analysis. Li, liver; H, heart; K, kidney; Lu, lungs. **, P<0.01 for comparison with the liver.

Quantitative real-time PCR analysis of mRNA expression levels in the glomeruli on days 4 and 7

During the early period after NTS injection, the major renal injury seen in the experimental model is glomerular lesions rather than tubulointerstitial lesions. Therefore, we collected the glomeruli from the rat kidneys using a standard sieving method.26 To clarify the mechanisms underlying NTS-induced GN and the favorable effects of the pCAGGS-vIL-10 transfer, we performed quantitative real-time PCR analysis for the mRNA expression levels of CD4, CD8, IFN-γ, TNF-α, perforin (lytic molecules produced by CD8+ T cells),27 and monocyte chemotactic protein (MCP)-1 in the glomeruli of normal rat kidneys before NTS injection and of kidneys from pCAGGS and pCAGGS-vIL-10 rats on days 4 and 7 after NTS injection (n=5 in each group) (Figure 7). The mean values with their s.d.'s were calculated and standardized to the mean value of each gene expression in the pCAGGS rats on day 4 after injection, which was given a value of 1 in the statistical analysis.

Figure 7
figure7

Quantitative real-time PCR analysis of mRNA expression levels in the glomeruli of normal rat kidneys before NTS injection and on days 4 and 7 after NTS injection. Relative quantities of PCR products from the mRNA of CD4, CD8, IFN-γ, perforin, TNF-α, and MCP-1 were measured with the LightCycler Quick system 330 and normalized to the PCR product from G6PDH mRNA. The mean values with their s.d.'s were calculated and standardized to the mean values of each gene expression in pCAGGS rats on day 4 after injection, which were given a value of 1 in the statistical analysis. n=5 in each group. *, P<0.05 for comparison with pCAGGS rats. #, P<0.05, ##, P<0.01, ###, P<0.001, and ####, P<0.0001 for comparisons with normal rats. The error bars are not shown for some data points because of low variability.

The mRNA expression levels of CD4, CD8, IFN-γ, TNF-α, perforin, and MCP-1 in the glomeruli of the pCAGGS and pCAGGS-vIL-10 rats on days 4 and 7 were significantly higher than in the glomeruli of normal rats before NTS injection. The mRNA expression levels of CD4, IFN-γ, and TNF-α in the glomeruli of pCAGGS-vIL-10 rats were significantly lower than in the glomeruli of pCAGGS rats on days 4 (P<0.05) and 7 (P<0.05) after injection. The MCP-1 mRNA level in the glomeruli of the pCAGGS-vIL-10 rats was significantly lower than in the glomeruli of the pCAGGS rats on day 4 after injection (P<0.05). On the other hand, there were no significant differences in the mRNA levels of CD8 and perforin in the glomeruli of the pCAGGS-vIL-10 and pCAGGS rats on days 4 and 7 after injection. These results suggested that CD4, CD8, IFN-γ, TNF-α, perforin, and MCP-1 in the glomeruli played an important role in experimental crescentic GN, and that pCAGGS-vIL-10 transfer inhibits the glomerular accumulation of CD4+ T cells and the production of IFN-γ, TNF-α, and MCP-1 in the glomeruli.

Th1/Th2 balance

To examine the Th1/Th2 balance (Figure 8) in this model, we performed quantitative real-time PCR analysis for the mRNA expression levels of IFN-γ and IL-4 in the glomeruli and in the spleen, which represents the overall Th1/Th2 balance, of normal rats before NTS injection and of pCAGGS and pCAGGS-vIL-10 rats, on days 4 and 7 after NTS injection (n=5 in each group). The mean values with their s.d.'s were calculated and standardized to the mean value of gene expression in the glomeruli of pCAGGS rats on day 4 after injection, which was given a value of 1 in the statistical analysis. There were no significant differences in the mRNA levels of IFN-γ and IL-4 in the spleen of the normal rats, pCAGGS rats, and pCAGGS-vIL-10 rats on days 4 and 7 after injection. There were no significant differences in the mRNA levels of IL-4 in the glomeruli of the normal rats, pCAGGS rats, and pCAGGS-vIL-10 rats on days 4 and 7 after injection (data not shown). However, the ratio of the IFN-γ/IL-4 mRNA expression levels in the glomeruli of the pCAGGS and pCAGGS-vIL-10 rats on days 4 and 7 was significantly higher than in the glomeruli of normal rats before NTS injection. In addition, the IFN-γ/IL-4 mRNA ratio in the glomeruli of pCAGGS-vIL-10 rats was significantly lower than in the glomeruli of pCAGGS rats on days 4 (P<0.05) and 7 (P<0.01) after injection. These results suggested that experimental crescentic GN has a Th1-predominant immunoresponse in the glomeruli, but not overall, and that pCAGGS-vIL-10 transfer inhibits IFN-γ in the glomeruli.

Figure 8
figure8

Quantitative real-time PCR analysis of IFN-γ/IL-4 mRNA expression levels in the glomeruli and spleen of normal rats before NTS injection and on days 4 and 7 after NTS injection. The mean values with their s.d.'s were calculated and standardized to the mean values of gene expression in the glomeruli of pCAGGS rats on day 4 after injection, which were given a value of 1 in the statistical analysis. n=5 in each group. *, P<0.05, **, P<0.01 for comparisons with pCAGGS rats. ###, P<0.001, and ####, P<0.0001 for comparisons with normal rats. The error bar is not shown for the normal glomeruli point because of low variability.

Detection of vIL-10 protein in the liver

To clarify the level of vIL-10 gene expression in the liver, we delivered 800 μg of pCAGGS or pCAGGS-vIL-10 into the tail vein. Diaminobenzidine (DAB) stained only the liver of pCAGGS-vIL-10 rats (Figure 9), but not the liver of the pCAGGS rats (data not shown). The stained cells were mainly hepatocytes.

Figure 9
figure9

Detection of vIL-10 protein in the liver. DAB-stained rat liver section 1 day after injection with pCAGGS-vIL-10. (a) Original magnification, × 50. (b) Original magnification, × 250.

Liver toxicity evaluation after injection

To determine whether the procedure caused any adverse effects in rats, we measured their serum concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Figure 10). Rats were assigned to four groups: three received injections of 15 ml of Ringer's solution containing either 800 μg of pCAGGS (pCAGGS rats; n=6), 800 μg of pCAGGS-vIL-10 (pCAGGS-vIL-10 rats; n=6) after NTS injection, or no DNA (Ringer's solution rats; n=6), and one group was not injected (control rats; n=6). The serum ALT of the pCAGGS rats, pCAGGS-vIL-10 rats, and Ringer's solution rats sharply increased on day 1 after the injection, returned to normal levels by day 4, and was maintained thereafter. Similarly, the serum AST of the pCAGGS rats, pCAGGS-vIL-10 rats, and Ringer's solution rats abruptly increased on day 1 after the injection and returned to normal levels by day 4 (data not shown).

Figure 10
figure10

Effect of NTS injection and gene transfer on serum ALT. Rats were injected with pCAGGS or pCAGGS-vIL-10 after NTS injection, and Ringer's solution. Control rats were not injected. n=6 in each group. ***, P<0.001 and ****, P<0.0001, for comparisons with normal rats (comparisons were made at each time point). The error bars are not shown for some data points because of low variability.

Histological examination for liver damage by NTS injection and gene transfer

To evaluate the likelihood of liver damage, we analyzed liver sections from rats injected with 15 ml of Ringer's solution containing either 800 μg of pCAGGS or 800 μg of pCAGGS-vIL-10 after NTS injection, and compared them with liver sections from normal, uninjected rats. Compared with the uninjected rats (Figure 11a), no apparent pathological changes were observed in the liver sections of rats killed on day 7 after pCAGGS (Figure 11b) or pCAGGS-vIL-10 injection (Figure 11c). Similarly, no apparent pathological changes were observed in the liver sections of rats killed on day 1 after pCAGGS, or pCAGGS-vIL-10 injection (data not shown).

Figure 11
figure11

Histological examination for liver damage by NTS injection and gene transfer. Liver sections were stained with HE. (a) Liver section of an uninjected rat. Liver section from (b) a pCAGGS and (c) a pCAGGS-vIL-10 rat on day 7. Original magnification, × 50.

Reproducibility of therapeutic effects

To confirm the reproducibility of the therapeutic effects, we performed a second experiment, testing the same parameters as above. The urinary protein level was significantly lower in the pCAGGS-vIL-10 rats (n=6) than in the pCAGGS rats (n=6): 7.8±2.6 versus 27.4±19.7 mg/day, on day 4 after injection (P<0.05), 57.4±6.7 versus 165.8±40.3 mg/day, on day 7 after injection, respectively (P<0.0001) (Figure 12). The Ccr levels in the pCAGGS rats were markedly decreased, from 0.49±0.03 ml/min/100 g on day 2 before injection to 0.37±0.07 ml/min/100 g on day 7 after injection (P<0.01). On the other hand, the Ccr levels in the pCAGGS-vIL-10 rats were not significantly decreased (0.49±0.04 ml/min/100 g on day 2 before injection and 0.48±0.06 ml/min/100 g on day 7 after injection). The Ccr level on day 7 after injection was significantly higher in the pCAGGS-vIL-10 rats than in the pCAGGS rats (P<0.01) (Figure 13). The frequency of crescent formation on day 7 after injection was significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats: 30.4±7.2 versus 70.8±8.6% (P<0.0001) (Figure 14a). The total number of glomerular cells on day 7 after injection was significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats: 57.6±4.3 versus 84.3±8.9 c/gcs, respectively (P<0.0001) (Figure 14b). The number of ED1+ cells in the glomeruli on day 7 after injection was significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats: 11.4±5.9 versus 31.2±4.2 c/gcs, respectively (P<0.0001) (Figure 14c). The number of CD4+ T cells in the glomeruli on day 7 after injection was significantly lower in the pCAGGS-vIL-10 rats than in the pCAGGS rats: 0.5±0.2 versus 1.4±0.3 c/gcs, respectively (P<0.0001) (Figure 14d). Similar to the results in the first experiment, there was no significant difference in the number of CD8+ T cells (Figure 14e). These results showed that the results of the pCAGGS-vIL-10 transfer are reproducible.

Figure 12
figure12

Effect of pCAGGS-vIL-10 transfer on proteinuria in the second experiment. Urinary protein excretion in WKY rats given injections of NTS and later pCAGGS-vIL-10 or pCAGGS plasmid was determined. n=6 in each group. *, P<0.05, and ****, P<0.0001 for comparisons with pCAGGS rats. ##, P<0.01, ###, P<0.001, and ####, P<0.0001 for comparisons with preinjection rats (day −2) within each group. The error bars are not shown for some data points because of low variability.

Figure 13
figure13

Effect of pCAGGS-vIL-10 transfer on Ccr in the second experiment. Ccr in WKY rats given injections of NTS and later pCAGGS-vIL-10 or pCAGGS plasmid. Ccr was measured 2 days before (□) and 7 days after (▪) injection. n=6 in each group. **, P<0.01 for comparison with pCAGGS rats. ##, P<0.01 for comparison with preinjection on day −2.

Figure 14
figure14

(a) Crescent formation, (b) total number of cells, (c) glomerular ED1+ cells (macrophages), (d) CD4+ T cells, and (e) CD8+ T cells in the kidney sections of WKY rats given injections of NTS and later pCAGGS-vIL-10 or pCAGGS plasmid in the second experiment. n=6 in each group. ****, P<0.0001 for comparison with the pCAGGS rats.

Effect of pCAGGS-vIL-10 transfer on circulating mononuclear cells

To examine the effects of pCAGGS-vIL-10 (pCAGGS-vIL-10 rats; n=6) or pCAGGS (pCAGGS rats; n=6) on circulating mononuclear cells, we performed an analysis of the peripheral blood cells on day 2 before injection and on days 4 and 7 after injection, in the second experiment. There was no significant difference in the number of circulating mononuclear cells between day 2 before injection and on days 4 and 7 after injection in the pCAGGS-vIL-10 rats and pCAGGS rats. There was no significant difference in the number of circulating mononuclear cells between pCAGGS-vIL-10 rats and pCAGGS rats on days 4 and 7 (data not shown). These results suggested that neither NTS injection nor pCAGGS-vIL-10 transfer affected the number of circulating mononuclear cells.

Growth

pCAGGS rats and pCAGGS-vIL-10 rats survived the NTS injection and subsequent hydrodynamics-based transfection, developed well, were well nourished, and appeared to have no discomfort.

Discussion

Our present study demonstrates that pCAGGS-vIL-10 gene transfer by hydrodynamics-based transfection suppresses crescentic GN in WKY rats.

Among the various gene delivery systems, plasmid DNA is the simplest. However, there has been no report using gene therapy by plasmid DNA to treat crescentic GN, probably because of this method's low transfection efficiency and low level of expression compared with viral vectors. Nonetheless, we recently showed that this technique is also useful for gene transfer in normal rats, using pCAGGS-Epo.22 We obtained maximal Epo expression when the DNA solution was rapidly injected in a volume of approximately 100 ml/kg body weight. The peak serum Epo levels following the tail vein injection of 800 μg of pCAGGS-Epo were 100-fold greater than the levels reached following the muscle-targeted gene transfer of 400 μg of pCAGGS-Epo by in vivo electroporation.28,29 In the present study, the peak serum vIL-10 levels following the tail vein injection of 800 μg of pCAGGS-vIL-10 were at least three-fold greater than the levels reached following intravenous gene transfer with an adenovirus vector, peaking at 165.4±101 ng/ml 1 day after the gene transfer.30 Thus, our previous and present studies demonstrate that gene transfer into the liver by the hydrodynamics-based transfection method is easily applicable to the rat, which is more than 10 times larger than the mouse.

The administration of recombinant IL-10 prevents macrophage and T-cell infiltration, crescent formation, and proteinuria in crescentic GN.4 However, recombinant cytokines have short half-lives, necessitating frequent administration. To obtain sustained IL-10 levels, a gene therapy system providing continuous delivery may be more effective than an intermittent one. Macrophages transfected with adenovirus expressing IL-4 ex vivo reduced macrophage infiltration and albuminuria in an experimental model of GN.31 However, this type of ex vivo gene transfer is both laborious and expensive because it requires the isolation, growth, and transduction of primary macrophages from each rat. Adenovirus vector, moreover, induces an immune response and causes side effects that render its repeated administration problematic.32,33 Gene therapy by plasmid DNA overcomes these problems.

As in previous reports,20,22 we noted that transgene-derived vIL-10 mRNA could also be detected in the kidney by quantitative real-time PCR. Plasmid DNA can also be delivered to the kidney via the retrograde renal vein route.20,22 Therefore, we needed to take vIL-10 expression in the kidney into account in assessing the favorable effects of gene transfer via tail vein injection on the crescentic GN in WKY rats. Recently, we developed a technique for kidney-targeted plasmid DNA transfer in rats by retrograde renal vein injection. Using a lacZ expression plasmid, pCAGGS-lacZ, we found the transgene expression site to be the fibroblasts near the peritubular (postglomerular) capillaries of the injected kidney by immunoelectron microscopic analysis.34 Taking the renal blood flow into consideration,35 we think that the vIL-10 expressed in the fibroblasts of the kidney entered the peritubular capillaries, and was carried to the glomerulus by the bloodstream along the following pathway: peritubular capillaries, renal vein, systemic circulation, renal artery, afferent arteriole, and, finally, the glomerulus, which is the major injury site. The vIL-10 expressed in the peritubular fibroblasts of the kidney was delivered to the glomerulus via the renal artery following the same route as the vIL-10 expressed in other organs. As shown in Figure 6, the vIL-10 gene expression in the kidney was 0.12±0.01% that of the liver. Thus, the circulating vIL-10 that was mainly produced in the liver probably played a greater role in producing the favorable therapeutic effects on crescentic GN.

In the present study, the overexpression of vIL-10 suppressed crescentic GN, which is classified as a Th1-predominant model. In contrast, continuous administration of IL-10 exacerbated GN in the New Zealand black/white mouse model of lupus, which is classified as a Th2-predominant model.3,36 Many factors complicate the pathogenesis of kidney diseases. To test the involvement of the many recognized candidate genes quickly, the development of a gene transfer method is necessary. The hydrodynamics-based transfection method is simple and allows high-level expression. This technique has the potential to be applicable to the treatment of kidney disease.

In the present study, the suppression of macrophage and CD4+ T cell accumulation, and of the production of IFN-γ, TNF-α, and MCP-1 in the glomeruli, could result in significant therapeutic effects. Many investigators have performed various treatments to reduce these factors in experimental crescentic GN.5,37,38,39 The administration of micro-encapsulated clodronate (dichloromethylene bisphosphonate) for macrophage depletion,37 monoclonal antibodies against CD4,37 IFN-γ,5 and MCP-1,38 and the soluble TNF receptor p55,39 prevent glomerular macrophage infiltration, crescent formation, and proteinuria. In the present study, continuous high-level vIL-10 delivery by hydrodynamics-based transfection suppressed these factors simultaneously. In addition, pCAGGS-vIL-10 transfer did not inhibit normal mRNA expression levels of IFN-γ in the spleen, but inhibited pathological mRNA expression levels of IFN-γ in the glomeruli. Thus, pCAGGS-vIL-10 transfer is suitable for gene therapy in crescentic GN.

vIL-10 and IL-10 can completely prevent the specific proliferative responses of CD4+ T cells by inhibiting the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes, while failing to modulate class I MHC expression on monocytes.15 Although no studies to date have tested the direct effects of vIL-10 on CD8+ T cells, our observations suggest that vIL-10 suppresses CD4+ T cells rather than CD8+ T cells in crescentic GN. This may explain the finding that vIL-10 did not repress perforin, which is produced by CD8+ T cells. A previous study demonstrated that CD8+ T cells play an important role in experimental crescentic GN;40 however, pCAGGS-vIL-10 gene transfer exhibited therapeutic effects without an apparent suppression of CD8+ T cells.

vIL-10 and IL-10 deactivate macrophages, and inhibit the production of TNF-α by macrophages in vitro.9,41 We believe that vIL-10 not only deactivates macrophages via the prevention of IFN-γ production by Th1 cells, but also deactivates macrophages directly, and inhibits the production of TNF-α by macrophages. MCP-1 is released from many types of cells, including macrophages, lymphocytes, platelets, fibroblasts, endothelial cells, epithelial cells,42 and mesangial cells.43 The MCP-1 mRNA level in the glomeruli of the pCAGGS-vIL-10 rats was significantly lower than that in the glomeruli of the pCAGGS rats on day 4 after injection but not on day 7, despite the reduction in the number of macrophages in the glomeruli. These results suggested that vIL-10 could prevent glomerular macrophage accumulation, but it was not able to prevent the production of MCP-1 by macrophages and other cells by day 7.

Similar to the results in our previous study using pCAGGS-lacZ,22 vIL-10 gene expression after pCAGGS-vIL-10 injection was seen in the hepatocytes. Compared with the result in our previous study, the present study demonstrated a lower percentage of vIL-10-transfected cells. This difference may be explained by differences in the methods of staining.

In agreement with our previous study,22 transient increases in serum AST and ALT levels suggested that slight liver damage occurred under our experimental conditions. The damage is principally caused by the large volume of Ringer's solution, given that increases in serum AST and ALT levels were seen in rats with Ringer's solution injections with or without plasmid DNA. In the present experiment, liver injury attributable to gene transfer was not apparent by histological examination of the liver of injected rats.

In the second experiment, we confirmed the reproducibility of the therapeutic effects. The grade of injuries in the second experiment was higher than in the first experiment. There were also differences in the NTS efficiency in the two experiments. Although the levels of the injuries were higher in the second experiment, pCAGGS-vIL-10 gene transfer was effective enough to be used therapeutically.

In conclusion, we have demonstrated that pCAGGS-vIL-10 gene transfer by hydrodynamics-based transfection suppressed crescentic GN in WKY rats. The therapeutic effects were probably attributable to the suppression of the number of macrophages and CD4+ T cells, and of the production of IFN-γ, TNF-α, and MCP-1 in the glomeruli by vIL-10. This method should be useful for the continuous delivery of secretory proteins to examine their biological roles in rat kidney disease models.

Materials and methods

Preparation of rat GBM

In total, 10 8-week-old male Wistar rats (Charles-River Japan Inc., Tokyo, Japan) were used. The 20 kidneys were removed and the cortices were dissected into small pieces. The cortices were then sieved according to a standard sieving method,26 using a series of sieves of 250, 150, and 75 μm (Sansyo, Tokyo, Japan) to isolate the glomeruli, and the GBM was subsequently isolated using the method of Meezan et al.44

Production of NTS containing rabbit polyclonal anti-rat GBM antibodies

In total, three 10-week-old male Japanese white rabbits were purchased from Kitayama Labes Co. (Nagano, Japan). Immediately before the injection, we diluted the rat GBM in 1.5 ml of phosphate-buffered saline (PBS), and then blended it with 1.5 ml of complete Freund's adjuvant (Iatron, Tokyo, Japan).

To generate antisoluble rat GBM antibodies, the rabbits were anesthetized with Nembutal injection, and a total of 3 ml of soluble rat GBM was injected into the back of each rabbit at multiple sites, every 3 weeks. Before the third immunization, we obtained a serum sample from each rabbit, and removed the natural anti-rat antibodies from the sera by absorption with rat red blood cells (RBC). The sera were then injected into Wistar rats (n=2 for each serum sample from the three rabbits). After 24 h, we confirmed that all the rats had developed proteinuria. At 2 weeks after the third immunization of soluble rat GBM, the rabbits were placed under general anesthesia, and serum samples containing the rabbit polyclonal anti-rat GBM antibody – now known to possess the ability to generate NTS – were obtained from the carotid artery of the rabbits and treated with rat RBC. The antiserum from the three rabbits was pooled, heat-inactivated at 56°C for 30 min, and stored at −40°C until used.

Rats

Male WKY rats (6 weeks old) were purchased from Charles-River Japan Inc. After 1 week, rats weighing 150–170 g were used for antiserum administration and gene transfer.

Plasmid DNA

Plasmid pCAGGS-vIL-10 was constructed by inserting vIL-10 cDNA into a unique EcoRI site of the pCAGGS expression vector bearing the CAG promoter.23 The vIL-10 cDNA was derived from pcDSRα-BCRF.45 Plasmids were grown in Escherichia coli DH5α cells, and prepared using a Qiagen EndoFree plasmid Giga kit (Qiagen GmbH, Hilden, Germany), as described previously.28 The empty pCAGGS plasmid was used as a control.

NTS injection

Rats were injected with 0.1 ml/100 g body weight of NTS via the tail vein, using a 26-gauge needle connected to a 1-ml capacity syringe (Terumo, Tokyo, Japan).

Plasmid DNA injection

The plasmid DNA was diluted in 15 ml (approximately 1/10 of body weight) of Ringer's solution (Ohtsuka, Tokushima, Japan) at room temperature.

It is much harder to perform rapid tail vein injection in rats than in mice because of the thick skin that covers the rats' tail vein and the fact that a higher injection pressure against vascular resistance is required. To cope with these problems, we use a SURFLO® winged injection set (Terumo), consisting of a 23-gauge winged needle 19-mm long and 0.65 mm in diameter, with an external tube 30-cm long and 1.1 mm in diameter.

At 3 h after the NTS injection, 800 μg of either pCAGGS-vIL-10 (pCAGGS-vIL-10 rats) or pCAGGS plasmid (pCAGGS rats) was injected into the tail vein through the winged needle connected to a 25-ml capacity syringe (Terumo), with a <10 s injection time. We based the amount of injected DNA on the results of our previous study of pCAGGS-Epo delivery to the rat liver via tail vein injection: a dose–response relation between serum Epo levels and the amount of injected DNA was seen for up to 800 μg of DNA.22

Serum vIL-10 assay

Rats were placed under general anesthesia, and blood samples were obtained from the heart according to the method of Ohwada.46 Serum vIL-10 was measured by ELISA as follows. Plates (96-wells) were coated with 2 μg/ml of rat anti-human and anti-vIL-10 monoclonal antibody (PharMingen, Carlsbad, CA, USA) at 4°C overnight, washed with PBS containing 0.05% Tween 20 (PBS/Tween), and blocked by incubation with PBS containing 1% bovine serum albumin at room temperature for 1 h. After washing with PBS/Tween, appropriately diluted samples were added to the wells. Plates were incubated at 4°C overnight and washed with PBS/Tween. Biotinylated rat anti-human and anti-vIL-10 monoclonal antibody (2 μg/ml) (PharMingen) was added to the wells and the plates were incubated at room temperature for 3 h with agitation. After washing with PBS/Tween, diluted streptavidin–horseradish peroxidase conjugate was added to the wells. The plates were incubated at room temperature for 30 min and washed with PBS/Tween. Substrate (o-phenylenediamine) was added to the wells and absorbance at 492 nm was measured on a Multiskan MS-UV microplate reader (Labsystems, Helsinki, Finland). Recombinant vIL-10 (PharMingen) was used as a standard and the linear range of this ELISA system was from 30 to 2000 pg/ml.19

Serum IFN-γ, IL-4, and IL-10 assay

Rat serum IFN-γ, IL-4, and IL-10 levels were measured using rat ELISA kits (Amersham, Little Chalfont, UK).

Urinary protein measurement

Urine was collected for 24 h from rats housed in metabolic cages (Okazaki Sangyo, Tokyo, Japan). The amount of urinary excretion for 24 h was determined with Micro TP-Test Wako reagent (Wako, Osaka, Japan) on an H-7150 automated analyzer (Hitachi, Tokyo, Japan).

Kidney function

Creatinine (Cr), which is an endogenous marker for kidney function, was measured with creatinine reagent (Daiya Shiyaku, Tokyo, Japan) using an AU 5232 automated analyzer (Olympus, Tokyo, Japan). The Ccr closely resembles the true glomerular filtration rate. Cr is largely produced from the metabolism of muscle and is excreted in the urine. Therefore, we calculated the Ccr value using the following equation: Ccr (ml/min/100 g)=Ucr (mg/dl) × V (ml/min)/Scr (mg/dl), where Ccr is the corrected value per 100 g of rat body weight,47 Ucr and Scr are the concentrations of Cr in the urine and serum, respectively, and V is the urine flow rate.48

Light microscopy

The kidneys were harvested on day 7 after the gene transfer, fixed in 10% buffered formaldehyde, embedded in paraffin, and processed for routine light microscopy. Sections 2 μm thick were stained with periodic acid-Schiff reagent, and 100 equatorially sectioned glomeruli were assessed per rat. Crescent formation was expressed as the number of crescentic glomeruli per 100 glomeruli (%). The total number of cells in the glomeruli was expressed as c/gcs.

Immunohistochemical staining

The kidneys were harvested on day 7 after gene transfer, embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical, Tokyo, Japan), and frozen in a mixture of dry ice and acetone. Sections 2 μm thick were cut with a cryostat and placed on glass slides coated with 3-amino-propyltriethoxysilane. The sections were rinsed in PBS for 15 min, and fixed in absolute acetone for 10 min. FITC-conjugated goat anti-rabbit IgG (Cappel, Malverne, PA, USA) was used for direct immunofluorescence. The presence of macrophages was examined immunohistochemically on frozen tissue specimens using a mouse monoclonal antibody against rat macrophage (ED1, IgG1; Serotec, Oxford, UK) and FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL, USA). The presence of CD4+ T cells and CD8+ T cells was examined immunohistochemically on frozen tissue specimens using a mouse monoclonal antibody against rat CD4 (OX-38, IgG2a; PharMingen) and CD8 (OX-8, IgG1; Serotec), and FITC-conjugated goat anti-mouse IgG2a and IgG1 (Southern Biotechnology Associates). A total of 30 equatorially sectioned glomeruli were assessed and the results were expressed as c/gcs.

Quantitative real-time PCR analysis for vIL-10 mRNA in major organs

On day 7 after the injection, rats that had received 800 μg of pCAGGS-vIL-10 were killed and the livers harvested. The total RNA of the liver sample was isolated with Isogen (Nippon Gene, Tokyo, Japan) and used for the synthesis of first-strand cDNA using Moloney Murine Leukemia Virus reverse transcriptase (RT; Gibco BRL, Rockville, MD, USA) and random hexamers (Promega, Madison, WI, USA). The RT product was amplified by PCR with Taq DNA polymerase (Promega) and specific primers as follows: vIL-10 backward primer, 5′-IndexTermACGACTGAAGGCATCTCTTAG-3′; vIL-10 forward primer, 5′-IndexTermTCTGACTGACCGCGTTACTC-3′; rat housekeeping gene, G6PDH, backward primer, 5′-IndexTermTTCTTGGTCATCATCTTGGTGTAT-3′; and G6PDH forward primer, 5′-IndexTermTATCTCAGAGGTGGAAACTGACAA-3′. We designed the vIL-10 forward primer to hybridize with the sequence immediately downstream of the transcriptional start site of the CAG promoter. There is an intron between the CAG promoter and the vIL-10 cDNA in pCAGGS-vIL-10. The primer set for the detection of the vIL-10 mRNA encompasses this intron, allowing us to distinguish any possible PCR products from contaminating plasmid DNA or genomic DNA. The primer set for the detection of G6PDH mRNA was also designed to span introns. The lengths of the expected products were 210 bp for vIL-10 mRNA and 342 bp for G6PDH mRNA. These PCR products were directly inserted into the pGEM-T Easy Vector (Promega), to create pGEM-T-vIL-10 or pGEM-T-G6PDH, respectively. These plasmids were grown in E. coli DH5α cells and prepared using a QIAprep Spin Miniprep kit (Qiagen) for use as the external standard.

To determine the levels of transgene-derived vIL-10 mRNA in major organs including the brain, lungs, heart, liver, kidney, spleen, muscle, skin, and testes, we performed quantitative real-time PCR analysis using the LightCycler Quick system 330 (Roche Diagnostics, Mannheim, Germany).22 The PCR was performed with LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics), according to the manufacturer's protocol, and using the primer pairs described above. The PCR reaction consisted of 95°C for 10 min, then a total of 40 cycles of 95°C briefly, 62°C for 10 s, and 72°C for 13 s with a transition rate of 20°C/s between temperature plateaus. The data were quantified using the LightCycler analysis software (version 3, Roche Diagnostic). A standard curve for the vIL-10 plasmid was created, and the standard error was <0.001. The results were expressed initially as the number of target molecules/2μl cDNA. To standardize the results for variability in RNA and cDNA quantity and quality, we quantified the total number of G6PDH transcripts in each sample as an internal control. Normalized levels of the vIL-10 transcripts were calculated as the ratio of the number of vIL-10 transcripts to G6PDH transcripts. We confirmed the RT-PCR products by 4% agarose gel electrophoresis.

RNA extraction from glomeruli and spleen and quantitative real-time PCR analysis

To create the plasmids used for the external standard, we obtained the total RNA of the glomeruli samples from rat kidney on day 4 after NTS injection and constructed the plasmids as described above. We used specific primers as follows: CD4 backward primer, 5′-IndexTermTCACAGGTCAAAGTGTTGCTGTCGG-3′; CD4 forward primer, 5′-IndexTermTGCGAGCTGGAGAACAAGAAAGAGG-3′; CD8 backward primer, 5′-IndexTermTGTCAAGCCTTTCTGGGTCTTTGGG-3′; CD8 forward primer, 5′-IndexTermTTCAGACTCCTTCATCCCTGCTGG-3′; IFN-γ backward primer, 5′-IndexTermCCTTAGGCTAGATTCTGGTGACAGC-3′; IFN-γ forward primer, 5′-IndexTermATCTGGAGGAACTGGCAAAAGGACG-3′; TNF-α backward primer, 5′-IndexTermACTCCAGCTGCTCCTCTGCT-3′; TNF-α forward primer, 5′-IndexTermATGGGCTCCCTCTCATCAGT-3′; perforin backward primer, 5′-IndexTermCCATCCAGGGTCAGCTGACAGGTA-3′; perforin forward primer, 5′-IndexTermCGGCTCACACTGCCAGCGTAATGT-3′; MCP-1 backward primer, 5′-IndexTermTATGGGTCAAGTTCACATTCAAAG-3′; MCP-1 forward primer, 5′-IndexTermCTGTCTCAGCCAGATGCAGTTAAT-3′; IL-4 backward primer, 5′-IndexTermTTTCAGTGTTGTGAGCGTGGA-3′, and IL-4 forward primer, 5′-IndexTermATGCACCGAGATGTTTGTACC-3′. The primer sets for the detection of these mRNAs were also designed to span introns. The lengths of the expected products were 656, 428, 288, 555, 450, 301, and 238bp for CD4 mRNA, CD8 mRNA, IFN-γ mRNA, TNF-α mRNA, perforin mRNA, MCP-1 mRNA, and IL-4 mRNA, respectively. These PCR products were directly inserted into the pGEM-T Easy Vector, to create pGEM-T-CD4, pGEM-T-CD8, pGEM-T-IFN-γ, pGEM-T-TNF-α, pGEM-T-perforin, pGEM-T-MCP-1, and pGEM-T-IL-4. These plasmids were also prepared as described above and used for the external standard.

To determine the amounts of mRNA of these genes in the glomeruli of normal rat kidneys before NTS injection and on days 4 and 7 after NTS injection, we performed quantitative real-time PCR analysis as described above. In addition, we performed this method for the mRNA expression levels of IFN-γ and IL-4 in the spleen.

vIL-10 detection in the liver using immunohistochemistry

To examine the transgene expression in the liver, we analyzed the liver of rats that received 800 μg of pCAGGS-vIL-10 or pCAGGS via the tail vein. The livers were harvested on day 1 after the gene transfer, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections 2 μm thick were dewaxed and dehydrated. They were then incubated with 0.6% H2O2 in methanol for 30 min to eliminate endogenous peroxidase activity. After being washed with PBS, they were blocked by incubation with PBS containing 1% bovine serum albumin at room temperature for 1 h. After another wash with PBS, they were incubated with biotinylated rat anti-human and anti-vIL-10 monoclonal antibody (2 μg/ml) (PharMingen) at room temperature for 3 h. After being washed with PBS, they were incubated with diluted streptavidin–horseradish peroxidase conjugate (PharMingen) at room temperature for 30 min and washed with PBS. The sections were incubated with a DAB Substrate-Chromogen solution (Dako, Carpinteria, CA, USA). The cellular nuclei of the sections were counterstained with hematoxylin.

Liver function

Serum concentrations of AST and ALT were measured by an AU 5232 automated analyzer (Olympus, Tokyo, Japan).

Liver. histology

We harvested the liver of rats that received a pCAGGS-vIL-10 injection on days 1 and 7 after the gene transfer, and the liver from normal, uninjected rats. Livers were fixed in 10% buffered formaldehyde, embedded in paraffin, and processed for routine light microscopy. Sections 2 μm thick were stained with hematoxylin and eosin (HE) for the detection of possible tissue injury caused by the gene transfer procedure.

Mononuclear cell count

Total white blood cell (WBC) count was measured with a Sysmex SE-9000 (Sysmex, Hyogo, Japan). We made a smear and stained it with Wright–Giemsa stain. The mononuclear cell count was calculated from the ratio of mononuclear cells per 100 WBC.

Statistical analysis

Data are presented as the mean values±standard deviation of the mean. All data were analyzed using the StatView statistical program for Macintosh (SAS, Cary, NC, USA). Statistical significance was evaluated by the unpaired t test. Correlation was assessed by linear regression analysis. P values of < 0.05 were considered to be of significance.

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Acknowledgements

We are grateful to Ms Keiko Yamagiwa and Mr Naofumi Imai of the Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, for technical assistance.

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Correspondence to H Maruyama.

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Higuchi, N., Maruyama, H., Kuroda, T. et al. Hydrodynamics-based delivery of the viral interleukin-10 gene suppresses experimental crescentic glomerulonephritis in Wistar–Kyoto rats. Gene Ther 10, 1297–1310 (2003) doi:10.1038/sj.gt.3301988

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Keywords

  • gene therapy hydrodynamics-based transfection
  • viral IL-10
  • crescentic glomerulonephritis

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