Smad7 Gene Therapy Ameliorates an Autoimmune Crescentic Glomerulonephritis in Mice

Modified Ultrasound-Mediated Gene Transfer of Inducible Smad7 Gene-Bearing Microbubbles into the Kidney of Mice

Preparation of a mixture of doxycycline-regulated pTRE-m2Smad7–expressing plasmids and gene transfer into the kidney of mouse using the ultrasound-microbubble–mediated technique were used as described previously (13–15), with modification. Briefly, for achievement of doxycycline-induced (a tetracycline derivative) Smad7 transgene expression, a mixed solution that contained 200 μg of pTRE-m2Smad7 and Tet-On plasmids and Optison (echocardiographic contrast microbubbles; Mallinckrodt, St. Louis, MO) at the ratio of 1:1 (vol:vol) in 300 μl was co-transfected into the kidney via the tail vein followed by ultrasound treatment transcutaneously on the back of both sides of kidney location (Sonopuls 590, 1 MHz; Ernaf-Nonius, Delft, Netherlands). Ultrasound was performed immediately after injection with a continuous-wave output of 1 MHZ for 30 s on one side, and then 30 s on the other side, for a total of 10 min. After ultrasound treatment, 200 μg of doxycycline (Sigma, St. Louis, MO) was injected intraperitoneally, followed by the addition of doxycycline in the daily drinking water (200 μg/ml).

For determination of the efficacy of ultrasound-microbubble–mediated gene transfer into the kidney of the mice, groups of six normal mice were given a mixture of Smad7 plasmids (200 μg per mouse) and Optison via the tail vein, followed immediately by ultrasound treatment as described. The mice were killed at days 1, 7, 14, and 28 after Smad7 gene transfer, and their kidneys were collected for examination of Smad7 mRNA expression by real-time PCR and Smad7 protein by Western blot analysis and the expression of Smad7 by immunohistochemistry (IHC) with the flag m2 mAb (Sigma).

Establishment of ACGN Model

A murine ACGN model was induced in 7- to 8-wk-old (C57BL/6 × DBA/2J) F1 hybrid mice by giving DBA/2J donor lymphocytes as described previously (17). Briefly, cell suspensions that contained a mixture of donor cells from the thymus, spleen, and lymph nodes (originating from the neck, axillary, and inguinal regions) were injected intravenously three times at 3- to 4-d intervals. Immediately after the induction, mice were randomly assigned into groups of 10 and received either Smad7 or empty vectors. In addition, 10 (C57BL/6 × DBA/2J) F1 hybrid mice were used as normal control. All mice were killed at week 9 after disease induction. Renal tissues and blood and urine samples were collected for analysis as described next. Two weeks after the induction of ACGN model, the mice were treated biweekly with the ultrasound-mediated mixture (0.3 ml) of pTRE-m2Smad7/Tet-on plasmids plus Optison or the mixture of empty vectors and Optison via the tail vein as described and killed at week 9.

Clinical and Pathologic Evaluation

As described previously (18), 24-h urine samples were collected in metabolic cages at week 0 and weekly for the determination of urinary levels of protein by Pierce BCA protein assay kit (Perbio Science, Etten-Leur, Netherlands). Mouse serum was collected at week 9 for serum urea nitrogen and creatinine determination.

For histopathology, the tissues were fixed in 10% buffer formalin and embedded in paraffin. Sections (4 μm) were stained with hematoxylin and eosin. A semiquantitative evaluation for staining was performed as described previously (17).

For immunofluorescence (IF), frozen sections were cut, air-dried, fixed in acetone for 5 min at room temperature, and incubated with FITC-conjugated goat anti-mouse IgG and C3 (Cappel; Organon Teknika, Durham, NC). A semiquantitative evaluation for staining was performed as described previously (18).

For IHC, formalin-fixed, paraffin-embedded tissue sections (4 μm) were stained with biotin-labeled mouse anti–α-smooth muscle actin (anti–α-SMA; Neomarkers, Fremont, CA), goat anti–collagen IV (Southern Biotech, Birmingham, AL), rabbit anti-pSmad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit anti–pNF-κB p65 (Cell Signaling, Beverly, MA) antibodies, using a microwave heating procedure as described previously (19). In addition, methyl Carnoy's solution–fixed paraffin sections were stained with the mouse anti–flag m2 mAb (Sigma), rabbit anti–TGF-β (Santa Cruz Biotechnology), goat anti–collagen I and III (Southern Biotech), IL-1β (R&D Systems, Minneapolis, MN), or rat anti-F4/80 (macrophage marker; Serotec, Raleigh, NC) antibodies. For studying Smad7, monocyte chemoattractant protein-1 (MCP-1), IL-6, and inducible nitric oxide synthase (iNOS) expression, frozen sections were fixed in acetone for 5 min and then incubated with goat anti-Smad7 (Santa Cruz Biotechnology), goat anti–MCP-1 (Santa Cruz Biotechnology), rat anti–IL-6 (R&D Systems), or rabbit anti-iNOS (BD Pharmingen, San Diego, CA) antibodies. For studying CD4⁺ (T helper cell marker), tissues were fixed in periodate-lysine paraformaldehyde as described previously (20), and tissue sections were labeled with the rat anti-CD4⁺ antibody (BioLegend, San Diego, CA). The horseradish peroxidase–conjugated rabbit anti-goat (Dako, Carpinteria, CA), swine anti-rabbit (Dako), Vector M.O.M. kit (Vector Laboratories, Burlingame, CA), or streptavidin-biotin peroxidase system (Dako) were then applied to the sections for 1 h. Sections were counterstained with hematoxylin or methyl green. Semiquantitative evaluations for staining were performed as described previously (15,17,18).

Real-Time PCR Analysis

Total kidney RNA was extracted with TriZOL reagents (Invitrogen, Carlsbad, CA) from cortical kidney. For first-strand cDNA synthesis, 1.5 μg of total RNA was used in a single-round reverse transcriptase reaction. The reaction mixture consisted of 0.9 μl of Oligo (dT) 12 to 18 primer, 1.0 mM deoxyribonucleotide triphosphate (dNTP), 1× first-strand buffer, 0.4 mM dithiothreitol, 80 U of RNaseout recombinant ribonuclease inhibitor, and 300 U of superscript II RNase H (Invitrogen). Real-time PCR was performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). All of the probes and primers were Assays-on-Demand Gene expression products (Applied Biosystems). Real-time PCR reactions were using 10 μl of cDNA, 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems), and 1.25 μl of the specific probe/primer mixed in a total volume of 25 μl. The thermal cycler conditions were as follows: 2 min at 50°C, 10 min at 95°C, 40 cycles of denaturation (15 s at 95°C), and combined annealing/extension (1 min at 60°C). Housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used as internal standard.

Western Blot Analysis

Each protein sample was run on a 12% SDS-PAGE gel. The gel was electroblotted onto polyvinylidene difluoride nitrocellulose membrane (Amersham Int., Buckinghamshire, UK); incubated for 1 h in 20 ml of blocking buffer (Tris-buffered saline that contained 5% skim milk); and incubated with goat anti-Smad7 (Santa Cruz Biotechnology), goat anti-Smad2 (Santa Cruz, Biotechnology), rabbit anti-pSmad2 (Biosource, Camarillo, CA), rabbit anti-Smad3 (Zymed, San Francisco, CA), or rabbit anti-pSmad3 (Biosource) antibodies at 4°C overnight. The antibody was specific for pSmad3 as described previously (21,22). After washing, the membrane was incubated with horseradish peroxidase–conjugated rabbit anti-goat or goat anti-rabbit (Pierce, Rockford, IL) antibodies for 1 h at room temperature. The membrane-bound antibody detected was incubated with chemiluminescent reagent plus (PerkinElmer Life Sciences, Boston, MA) and captured on x-ray film.

ELISA

The TGF-β1 protein levels in renal tissue were measured using the commercial ELISA kits (R&D Systems), according to the manufacturer's instructions. Briefly, protein samples were acidified with 1 N HCl and neutralized with 1.2 N NaOH/0.5 M HEPES to assay for the amount of TGF-β1. The absorbance was determined at 450 nm using an ELISA plate reader (Bio-Tek, Winooski, VT).

NF-κB p65 was measured in renal tissue nuclear protein extracts using Trans-AM ELISA assay kits (Active Motif, Carlsbad, CA), according to the manufacturer's instructions. Briefly, nuclear proteins were extracted using a nuclear extract kit (Active Motif) and were measured using a Pierce BCA protein assay kit (Perbio Science, Bezons, France). The absorbance was determined at 450 nm using an ELISA plate reader (Bio-Tek).

Statistical Analyses

Values were presented as means ± SEM. Individual experimental group means of data were compared with controls using t test. P < 0.05 was considered statistically significant.

Ultrasound-Microbubble–Mediated Gene Transfer to the Kidney

As shown in Figure 1, Smad7 mRNA was rapidly increased at day 1 (P < 0.05) and then declined to normal levels by day 14 (Figure 1A) after ultrasound treatment. Western blot analysis showed that Smad7 protein expression was increased at day 1, peaked at day 7 (P < 0.005), and became NS by day 14 (Figure 1B). The flag m2 Smad7 gene transfection rate and transgene expression were further detected by IHC with the anti–flag m2 mAb. As shown in Figure 1C (day 0), whereas the anti–flag m2 mAb did cross-react to normal tubular cells as a result of the antibody cross-reactivity, it was negative in glomeruli as described previously in the normal kidney (13). Thus, the Smad7 gene transfection rate and transgene expression were evaluated in the glomerulus. As shown in Figure 1C (days 7, 14, and 28), ultrasound-mediated Smad7 gene transfer resulted in a marked increase in Smad7 transgene expression as identified by the anti–flag m2–positive cells in the majority of glomerular cells, peaked at day 7, and then declined from day 14 onward. Therefore, to maintain a high level of Smad7 within the kidney, Smad7 gene transfer was repeatedly given every 14 d after the previous treatment.

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Figure 1.

Smad7 transgene expression in mouse kidney by modified ultrasound-microbubble–mediated gene transfer. (A) mRNA levels of Smad7 in the kidney as detected by real-time PCR. (B) Protein levels of Smad7 in the kidney as detected by Western blot analysis. (C) Immunohistochemistry (IHC) with flag m2 mAb. Each point represents the mean ± SEM for groups of six mice. *P < 0.05, ***P < 0.005 versus day 0. Magnification, ×200.

Smad7 Gene Transfer Improves Proteinuria, Renal Function, and Pathology

All ACGN mice survived until they were killed, although mice that were treated with empty vector showed moderate or severe ascites. As shown in Figure 2, ACGN mice that were treated with empty vectors developed progressive renal injury as demonstrated by an increase in proteinuria over weeks 3 to 9 after ACGN induction. In contrast, mice that were treated with Smad7 exhibited a significant reduction of proteinuria over weeks 6 to 9 (Figure 2A). This was associated with a significant reduction in both serum levels of serum urea nitrogen (Figure 2B) and creatinine (Figure 2C). Histologically, as shown in Figure 3, diseased mice that were treated with the empty vectors revealed extensive crescent formation, glomerulosclerosis, and remarkable interstitial mononuclear cell infiltration (mainly periglomerular; Figure 3B). In contrast, as shown in Figure 3, C and J, ACGN mice that were treated with Smad7 substantially inhibited crescent formation, glomerulosclerosis, and interstitial mononuclear cell infiltration (all P < 0.01, respectively). It is interesting that there was no significant difference in the intensity of IgG and C3 deposition in the glomerulus between the empty vector and the Smad7-treated ACGN mice (Figure 3, D through I and K), indicating that treatment with Smad7 had no effect on immune complex deposition.

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Figure 2.

Effects of Smad7 gene therapy on proteinuria and renal function. (A) Time-course studies of 24-h proteinuria. (B) Serum levels of serum urea nitrogen. (C) Serum levels of creatinine. Mice were killed at week 9 after disease induction. Data are means ± SEM for groups of 10 mice. Each arrow indicates the time of gene transfer with Smad7 or empty vector. ACGN, autoimmune crescentic glomerulonephritis. *P < 0.05; **P < 0.01.

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Figure 3.

Effects of Smad7 gene therapy on renal pathology and immune complex deposition in the glomeruli. Mice were killed at week 9 after disease induction. Hematoxylin and eosin (H&E) stain: Normal control (A), empty vector-treated mice with ACGN (B), Smad7-treated mice with ACGN (C). Immunofluorescence (IF) with IgG: Normal control (D), empty vector–treated mice with ACGN (E), Smad7-treated mice with ACGN (F). IF with C3: Normal control (G), Empty vector–treated mice with ACGN (H), Smad7-treated mice with ACGN (I). (J) Semiquantitative analysis of H&E. (K) Semiquantitative analysis of IF. Each bar represents the mean ± SEM for a group of 10 mice. **P < 0.01; ***P < 0.005; ^#Not detectable. Magnification, ×400.

Renal Fibrosis Is Inhibited by Treatment with Smad7

We first examined the therapeutic effect of Smad7 on renal fibrosis. As shown in Figure 4A, real-time PCR showed that Smad7 treatment resulted in a significant increase in the level of renal Smad7 mRNA. All fibrogenic markers including mRNA were upregulated in ACGN that were mice treated with empty vector. In contrast, this was associated with the marked inhibition of α-SMA (Figure 4B), collagen I (Figure 4C), collagen III (Figure 4D), and collagen IV (Figure 4E) mRNA expression. Similarly, IHC demonstrated that increased expression of α-SMA and collagens I, III, and IV within the diseased kidney of ACGN mice that were treated with empty vectors was inhibited by gene transfer with Smad7 (Figure 5).

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Figure 4.

Effects of Smad7 gene therapy on mRNA expression of renal fibrogenic markers by real-time PCR. Mice were killed at week 9 after disease induction. (A) Smad7 mRNA. (B) α-Smooth muscle actin (α-SMA) mRNA. (C) Collagen I mRNA. (D) Collagen III mRNA. (E) Collagen IV mRNA. Each bar represents the mean ± SEM for a group of 10 mice that were treated with Smad7 (▒), empty vector (▪), or normal control (□). *P < 0.05; **P < 0.01; ***P < 0.005.

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Figure 5.

Effects of Smad7 gene therapy on protein expression of renal fibrogenic markers by IHC. Mice were killed at week 9 after disease induction. (A through C) Anti–α-SMA. (D through F) Anti–collagen I. (G through I) Anti–collagen III. (J through L) Anti–collagen IV. Magnification, ×400.

Blockade of the TGF-β/Smad2/3 Signaling Is a Mechanism by Which Smad7 Inhibits Renal Fibrosis

We next investigated the mechanisms by which Smad7 inhibits renal fibrosis in ACGN. As shown in Figure 6, A and B, real-time PCR and ELISA analyses showed that compared with normal control mice, renal TGF-β1 mRNA and protein were significantly upregulated in ACGN mice that were treated with empty vectors (P < 0.05), which resulted in a significant increase in phosphorylation of Smad3 (Figure 6, C and D) and Smad2. However, overexpression of Smad7 abrogated an increase in both TGF-β1 mRNA (Figure 6A) and protein (Figure 6B), thereby blocking Smad3 (Figure 6, C and D) and Smad2 activations. To confirm the expression of these proteins in renal tissues, we performed IHC. As shown in Figure 7, A through C, both normal control and empty vector–treated ACGN mice exhibited a weak Smad7 staining in both glomeruli and tubulointerstitium (Figure 7, A and B). In contrast, ACGN mice that were treated with Smad7 showed a moderate to strong Smad7 protein expression within kidney, particularly in the glomeruli and tubulointerstitial tissues (Figure 7C). For detection of the transfected Smad7 gene expression within the kidney, a monoclonal anti–flag m2 antibody was applied. As shown in Figure 7, D through F, whereas mice that were treated with Smad7 revealed a moderate to strong positivity for the flag m2 in both the glomerulus and tubulointerstitial tissues (Figure 7F), both normal control and diseased mice that were treated with empty vectors showed negative signals for the flag m2 in the glomerulus, although the tubular staining remained strong (Figure 7, D and E). The latter may be associated with the cross-reactivity of the anti–flag m2 antibody to an isoform of Mg²⁺-dependent protein phosphatase as previously reported (13).

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Figure 6.

Effects of Smad7 gene therapy on renal TGF-β1 expression and Smad2/3 activation. Mice were killed at week 9 after disease induction. (A) TGF-β1 mRNA levels of kidney as detected by real-time PCR. (B) TGF-β1 protein levels of kidney as detected by ELISA. (C) Smad7 and phosphorylated Smad3 as detected by Western blot analysis. (D) Semiquantitative analysis of Western blot analysis. Compared with normal control mice, Smad7 expression is slightly reduced in the empty vector–treated ACGN mice, whereas gene transfer increases renal Smad7, which substantially inhibits Smad3 phosphorylation. Representative experiment was shown for Western blot analysis. Each lane represents one mouse kidney, and each bar represents the mean ± SEM for a group of 10 mice that were treated with Smad7 (▒), empty vector (▪), or normal control (□). *P < 0.05; **P < 0.01.

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Figure 7.

Effects of Smad7 gene therapy on renal TGF-β/Smad2/3 expression by IHC. Mice were killed at week 9 after disease induction. (A through C) Anti-Smad7. (D through F) Anti–flag m2. (G through I) Anti-phosphorylated Smad2/3. (J through L) Anti–TGF-β1. Magnification, ×400.

By IHC, low levels of TGF-β1 and phosphorylated Smad2/3 within the nuclei were found in the normal control kidney with moderate Smad7 expression (Figure 7, A, D, G, and J). However, TGF-β1 was upregulated in the diseased mice that were treated with empty vectors (Figure 7K), resulting in a marked activation of Smad2/3 (Figure 7H), whereas renal Smad7 remained lower (Figure 7, B and E). In contrast, gene transfer with Smad7 resulted in a marked upregulation of Smad7, which was associated with inhibition of renal TGF-β1 expression and Smad2/3 activation (Figure 7, C, F, I, and L).

Smad7 Gene Transfer Inhibits Renal Inflammation in ACGN

It is known that T cell (23,24) or macrophage infiltration (25–27) may play an important role in the development and progression of crescentic glomerulonephritis. In the context of renal immune injury, we first tested whether overexpression of Smad7 inhibits renal inflammation in mouse with ACGN. IHC showed that a profound T cell and macrophage infiltration was noted in the kidney of ACGN mice that were treated with empty vectors (Figure 8, B, E, and G), which was abrogated by Smad7 treatment (Figure 8, C, F, and G). It seemed that the effect of Smad7 overexpression was dominant in blocking monocyte/macrophage over T cell infiltrates. Further studies with real-time PCR showed that inhibition of T cell and macrophage infiltration was associated with suppression of inflammatory cytokines (IL-1β and, IL-6; Figure 9, A and B), adhesion molecule and chemokine (ICAM-1 and MCP-1; Figure 9, C and D), and iNOS mRNA expression (Figure 9E). Although Smad7 treatment resulted in a great suppression of inflammatory cytokines in ACGN mice, MCP-1 levels remained high when compared with normal control mice (Figure 9C). Similarly, Smad7 treatment inhibited the protein levels of IL-1β, IL-6, MCP-1, iNOS, and ICAM-1 in the glomerulus of the ACGN model as demonstrated by IHC (Figure 10).

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Figure 8.

Effects of Smad7 gene therapy on renal T cell and macrophage infiltration by IHC. Mice were killed at week 9 after disease induction. (A through C) Anti-CD4⁺ T cells. (D through F) Anti-F4/80 (macrophages). (G) Semiquantitative analysis. ***P < 0.005. Magnification, ×400.

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Figure 9.

Effects of Smad7 gene therapy on renal inflammatory mRNA gene expression by real-time PCR. Mice were killed at week 9 after disease induction. (A) IL-1β mRNA. (B) IL-6 mRNA. (C) Monocyte chemoattractant protein-1 (MCP-1) mRNA. (D) Intercellular adhesion molecule-1 (ICAM-1) mRNA. (E) Inducible nitric oxide synthase (iNOS) mRNA. Each bar represents the mean ± SEM for a group of 10 mice that were treated with Smad7 (▒), empty vector (▪), or normal control (□). **P < 0.01; ***P < 0.005; ^&P < 0.05 versus normal control.

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Figure 10.

Effects of Smad7 gene therapy on renal inflammatory cytokine (IL-1β and IL-6), MCP-1, iNOS, and NF-κB p65 expression. Mice were killed at week 9 after disease induction. (A through C) Anti–IL-1β. (D through F) Anti–IL-6. (G through I) Anti–MCP-1. (J through L) Anti-iNOS. (M through O) Anti–NF-κB p65. (P) Semiquantitative analysis of NF-κB p65 (IHC). (Q) NF-κB p65 as detected by ELISA (renal tissue nuclear protein extracts). Each bar represents the mean ± SEM for a group of 10 mice treated with Smad7 (▒), empty vector (▪), or normal control (□). **P < 0.01; ***P < 0.005. Magnification, ×400 each.

Blockade of NF-κB Activation Is a Key Mechanism by Which Smad7 Inhibits Renal Inflammation

We further investigated whether inhibition of NF-κB activation is a central mechanism whereby overexpression of Smad7 inhibits renal inflammation in the ACGN model. As shown in Figure 10, NF-κB p65 was markedly activated in the diseased mice that were treated with empty vectors, compared with the normal control kidney, as evident by its nuclear location in the glomeruli and tubulointerstitium as well as in mononuclear cells that infiltrated the periglomerular regions (Figure 10, M, N, and P). In contrast, NF-κB p65 activation was virtually blocked by gene transfer of Smad7 (Figure 10, O and P). Consistent with the IHC, ELISA assay for renal tissue nuclear protein extracts also demonstrated that the Smad7-treated ACGN mice produced a significant inhibition of NF-κB p65 activation, compared with the empty vector–treated ACGN mice (16.46 ± 2.48 versus 40.92 ± 11.10 ng/ml; P < 0.01; Figure 10Q).