Smad7 Gene Therapy Ameliorates an Autoimmune Crescentic Glomerulonephritis in Mice
Shuk-Man Ka*,
Xiao-Ru Huang,
Hui-Yao Lan,
Pei-Yi Tsai,
Shun-Min Yang,
Hao-Ai Shui and
Ann Chen*
* Department of Pathology, Tri-Service General Hospital, Graduate Institute of Medical Sciences, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China; and Center for Inflammatory Diseases and Molecular Therapies, University of Hong Kong Li Kat Shing Faculty of Medicine, Hong Kong
Address correspondence to: Dr. Ann Chen, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Gung Road, Taipei, Taiwan, ROC. Phone: +886-2-8792-7008; Fax: +886-2-8792-7009; E-mail: doc31717{at}ndmctsgh.edu.tw
Received for publication August 25, 2006.
Accepted for publication March 21, 2007.
Autoimmune crescentic glomerulonephritis is characterized bysevere immune response with glomerular crescentic formationand fibrosis in the kidney. Recent studies indicate that overexpressionof renal Smad7 attenuates both renal fibrosis and inflammationin rat remnant kidney. However, little attention has been paidto the potential role of TGF-/Smad signaling in autoimmune kidneydisease. This study tested the hypothesis that blocking TGF-signaling by overexpression of Smad7 may have a therapeuticeffect in a mouse model of autoimmune crescentic glomerulonephritisthat was induced in C57BL/6 x DBA/2J F1 hybrid mice by givingDBA/2J donor lymphocytes. Smad7 gene was transfected into thekidney using the ultrasound-microbubble–mediated system.Results showed that overexpression of Smad7 blocked both renalfibrosis and inflammatory pathways in terms of Smad2/3 and NF-Bactivation (P < 0.01), thereby inhibiting -smooth muscleactin; collagen I, III, and IV accumulation; and expressionof inflammatory cytokines (IL-1 and IL-6), adhesion molecule/chemokine(intercellular adhesion molecule-1, monocyte chemoattractantprotein-1), and inducible nitric oxide synthase (all P <0.01). Leukocyte infiltration (CD4+ cells and macrophages) wasalso suppressed (P < 0.005). Severe histologic damage (glomerularcrescent formation and tubulointerstitial injury) and functionalinjury including proteinuria were significantly improved (allP < 0.05). This study provides important evidence that overexpressionof Smad7 may have therapeutic potential for autoimmune kidneydisease.
Autoimmune crescentic glomerulonephritis (ACGN) is an extremelyprogressive from of glomerulonephritis and pathologically fallsunder the group of crescentic glomerulonephritis (1). Unfortunately,the current therapy in crescentic glomerulonephritis, includingACGN, is relatively ineffective and nonspecific with poor clinicaloutcomes (2–4).
TGF- is a fundamental growth factor and cytokine in fibrogenesisand inflammation (5,6). TGF- has been shown to exert its biologiceffects by signaling through a heteromeric receptor complexof the type I and type II receptors to activate the downstreamintracellular substrates Smad2 and Smad3 (7,8). In addition,TGF- can induce its downstream inhibitory Smad7, which in turninhibits Smad2/3 phosphorylation via the negative feedback mechanisms(9,10). In the context of immune and inflammation, TGF- playsa critical role in negatively modulating the immune and inflammatoryresponse (11). This is demonstrated by the observations thatmice that lack TGF- develop uncontrollable systemic inflammationand die in 3 wk after birth (12). It should be noted that theblockade of TGF- by targeting the upstream of its signalingmay cause inflammation, although fibrosis response is inhibited.This concern has been addressed by recent studies that blockadeof downstream of the TGF-/Smad signaling pathway by overexpressingSmad7 is able to inhibit both renal fibrosis and inflammationvia the mechanisms of blocking both Smad2/3 and NF-B signalingpathways in rat remnant kidney and obstructive kidney disease(13–16). However, it remains unknown whether blockadeof the TGF- pathway by Smad7 can inhibit immunologically inducedkidney disease.
Recently, we demonstrated that a murine chronic graft versushost disease can progress to an experimental ACGN that is characterizedby extensive crescent formation in up to 80% of the glomeruli(17). This study tested whether blocking TGF- signaling by overexpressionof Smad7 is able to inhibit progressive renal injury that isinduced immunologically in experimental ACGN. In addition, weextended these findings by presenting a safe, noninvasive, effective,and controllable gene therapy method for treatment of kidneydisease with progressive renal injury in a murine ACGN modelby transferring a doxycycline-regulated Smad7 using a modifiedultrasound-microbubble gene transfer–mediated system.
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–expressingplasmids and gene transfer into the kidney of mouse using theultrasound-microbubble–mediated technique were used asdescribed previously (13–15), with modification. Briefly,for achievement of doxycycline-induced (a tetracycline derivative)Smad7 transgene expression, a mixed solution that contained200 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 wasco-transfected into the kidney via the tail vein followed byultrasound treatment transcutaneously on the back of both sidesof kidney location (Sonopuls 590, 1 MHz; Ernaf-Nonius, Delft,Netherlands). Ultrasound was performed immediately after injectionwith 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. Afterultrasound treatment, 200 µg of doxycycline (Sigma, St.Louis, MO) was injected intraperitoneally, followed by the additionof doxycycline in the daily drinking water (200 µg/ml).
For determination of the efficacy of ultrasound-microbubble–mediatedgene transfer into the kidney of the mice, groups of six normalmice were given a mixture of Smad7 plasmids (200 µg permouse) and Optison via the tail vein, followed immediately byultrasound treatment as described. The mice were killed at days1, 7, 14, and 28 after Smad7 gene transfer, and their kidneyswere collected for examination of Smad7 mRNA expression by real-timePCR and Smad7 protein by Western blot analysis and the expressionof 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 xDBA/2J) F1 hybrid mice by giving DBA/2J donor lymphocytes asdescribed previously (17). Briefly, cell suspensions that containeda mixture of donor cells from the thymus, spleen, and lymphnodes (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 assignedinto groups of 10 and received either Smad7 or empty vectors.In addition, 10 (C57BL/6 x DBA/2J) F1 hybrid mice were usedas normal control. All mice were killed at week 9 after diseaseinduction. Renal tissues and blood and urine samples were collectedfor analysis as described next. Two weeks after the inductionof ACGN model, the mice were treated biweekly with the ultrasound-mediatedmixture (0.3 ml) of pTRE-m2Smad7/Tet-on plasmids plus Optisonor the mixture of empty vectors and Optison via the tail veinas described and killed at week 9.
Clinical and Pathologic Evaluation
As described previously (18), 24-h urine samples were collectedin metabolic cages at week 0 and weekly for the determinationof urinary levels of protein by Pierce BCA protein assay kit(Perbio Science, Etten-Leur, Netherlands). Mouse serum was collectedat week 9 for serum urea nitrogen and creatinine determination.
For histopathology, the tissues were fixed in 10% buffer formalinand embedded in paraffin. Sections (4 µm) were stainedwith hematoxylin and eosin. A semiquantitative evaluation forstaining 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 incubatedwith FITC-conjugated goat anti-mouse IgG and C3 (Cappel; OrganonTeknika, Durham, NC). A semiquantitative evaluation for stainingwas performed as described previously (18).
For IHC, formalin-fixed, paraffin-embedded tissue sections (4µm) were stained with biotin-labeled mouse anti–-smoothmuscle actin (anti–-SMA; Neomarkers, Fremont, CA), goatanti–collagen IV (Southern Biotech, Birmingham, AL), rabbitanti-pSmad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA), orrabbit anti–pNF-B p65 (Cell Signaling, Beverly, MA) antibodies,using a microwave heating procedure as described previously(19). In addition, methyl Carnoy's solution–fixed paraffinsections were stained with the mouse anti–flag m2 mAb(Sigma), rabbit anti–TGF- (Santa Cruz Biotechnology),goat anti–collagen I and III (Southern Biotech), IL-1(R&D Systems, Minneapolis, MN), or rat anti-F4/80 (macrophagemarker; Serotec, Raleigh, NC) antibodies. For studying Smad7,monocyte chemoattractant protein-1 (MCP-1), IL-6, and induciblenitric oxide synthase (iNOS) expression, frozen sections werefixed in acetone for 5 min and then incubated with goat anti-Smad7(Santa Cruz Biotechnology), goat anti–MCP-1 (Santa CruzBiotechnology), rat anti–IL-6 (R&D Systems), or rabbitanti-iNOS (BD Pharmingen, San Diego, CA) antibodies. For studyingCD4+ (T helper cell marker), tissues were fixed in periodate-lysineparaformaldehyde as described previously (20), and tissue sectionswere labeled with the rat anti-CD4+ antibody (BioLegend, SanDiego, CA). The horseradish peroxidase–conjugated rabbitanti-goat (Dako, Carpinteria, CA), swine anti-rabbit (Dako),Vector M.O.M. kit (Vector Laboratories, Burlingame, CA), orstreptavidin-biotin peroxidase system (Dako) were then appliedto the sections for 1 h. Sections were counterstained with hematoxylinor methyl green. Semiquantitative evaluations for staining wereperformed 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 reversetranscriptase reaction. The reaction mixture consisted of 0.9µl of Oligo (dT) 12 to 18 primer, 1.0 mM deoxyribonucleotidetriphosphate (dNTP), 1x first-strand buffer, 0.4 mM dithiothreitol,80 U of RNaseout recombinant ribonuclease inhibitor, and 300U of superscript II RNase H (Invitrogen). Real-time PCR wasperformed on an ABI Prism 7700 Sequence Detection System (AppliedBiosystems, Foster City, CA). All of the probes and primerswere Assays-on-Demand Gene expression products (Applied Biosystems).Real-time PCR reactions were using 10 µl of cDNA, 12.5µl of TaqMan Universal PCR Master Mix (Applied Biosystems),and 1.25 µl of the specific probe/primer mixed in a totalvolume of 25 µl. The thermal cycler conditions were asfollows: 2 min at 50°C, 10 min at 95°C, 40 cycles ofdenaturation (15 s at 95°C), and combined annealing/extension(1 min at 60°C). Housekeeping gene glyceraldehyde-3-phosphatedehydrogenase was used as internal standard.
Western Blot Analysis
Each protein sample was run on a 12% SDS-PAGE gel. The gel waselectroblotted onto polyvinylidene difluoride nitrocellulosemembrane (Amersham Int., Buckinghamshire, UK); incubated for1 h in 20 ml of blocking buffer (Tris-buffered saline that contained5% skim milk); and incubated with goat anti-Smad7 (Santa CruzBiotechnology), 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 forpSmad3 as described previously (21,22). After washing, the membranewas incubated with horseradish peroxidase–conjugated rabbitanti-goat or goat anti-rabbit (Pierce, Rockford, IL) antibodiesfor 1 h at room temperature. The membrane-bound antibody detectedwas incubated with chemiluminescent reagent plus (PerkinElmerLife Sciences, Boston, MA) and captured on x-ray film.
ELISA
The TGF-1 protein levels in renal tissue were measured usingthe commercial ELISA kits (R&D Systems), according to themanufacturer's instructions. Briefly, protein samples were acidifiedwith 1 N HCl and neutralized with 1.2 N NaOH/0.5 M HEPES toassay for the amount of TGF-1. The absorbance was determinedat 450 nm using an ELISA plate reader (Bio-Tek, Winooski, VT).
NF-B p65 was measured in renal tissue nuclear protein extractsusing Trans-AM ELISA assay kits (Active Motif, Carlsbad, CA),according to the manufacturer's instructions. Briefly, nuclearproteins were extracted using a nuclear extract kit (ActiveMotif) and were measured using a Pierce BCA protein assay kit(Perbio Science, Bezons, France). The absorbance was determinedat 450 nm using an ELISA plate reader (Bio-Tek).
Statistical Analyses
Values were presented as means ± SEM. Individual experimentalgroup 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 day1 (P < 0.05) and then declined to normal levels by day 14(Figure 1A) after ultrasound treatment. Western blot analysisshowed 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 expressionwere further detected by IHC with the anti–flag m2 mAb.As shown in Figure 1C (day 0), whereas the anti–flag m2mAb did cross-react to normal tubular cells as a result of theantibody cross-reactivity, it was negative in glomeruli as describedpreviously in the normal kidney (13). Thus, the Smad7 gene transfectionrate and transgene expression were evaluated in the glomerulus.As shown in Figure 1C (days 7, 14, and 28), ultrasound-mediatedSmad7 gene transfer resulted in a marked increase in Smad7 transgeneexpression as identified by the anti–flag m2–positivecells in the majority of glomerular cells, peaked at day 7,and then declined from day 14 onward. Therefore, to maintaina high level of Smad7 within the kidney, Smad7 gene transferwas repeatedly given every 14 d after the previous treatment.
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, x200.
Smad7 Gene Transfer Improves Proteinuria, Renal Function, and Pathology
All ACGN mice survived until they were killed, although micethat were treated with empty vector showed moderate or severeascites. As shown in Figure 2, ACGN mice that were treated withempty vectors developed progressive renal injury as demonstratedby an increase in proteinuria over weeks 3 to 9 after ACGN induction.In contrast, mice that were treated with Smad7 exhibited a significantreduction of proteinuria over weeks 6 to 9 (Figure 2A). Thiswas associated with a significant reduction in both serum levelsof serum urea nitrogen (Figure 2B) and creatinine (Figure 2C).Histologically, as shown in Figure 3, diseased mice that weretreated with the empty vectors revealed extensive crescent formation,glomerulosclerosis, and remarkable interstitial mononuclearcell infiltration (mainly periglomerular; Figure 3B). In contrast,as shown in Figure 3, C and J, ACGN mice that were treated withSmad7 substantially inhibited crescent formation, glomerulosclerosis,and interstitial mononuclear cell infiltration (all P < 0.01,respectively). It is interesting that there was no significantdifference in the intensity of IgG and C3 deposition in theglomerulus between the empty vector and the Smad7-treated ACGNmice (Figure 3, D through I and K), indicating that treatmentwith Smad7 had no effect on immune complex deposition.
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.
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, x400.
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 treatmentresulted in a significant increase in the level of renal Smad7mRNA. All fibrogenic markers including mRNA were upregulatedin ACGN that were mice treated with empty vector. In contrast,this was associated with the marked inhibition of -SMA (Figure 4B),collagen I (Figure 4C), collagen III (Figure 4D), and collagenIV (Figure 4E) mRNA expression. Similarly, IHC demonstratedthat increased expression of -SMA and collagens I, III, andIV within the diseased kidney of ACGN mice that were treatedwith empty vectors was inhibited by gene transfer with Smad7(Figure 5).
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) -Smooth muscle actin (-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.
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–-SMA. (D through F) Anti–collagen I. (G through I) Anti–collagen III. (J through L) Anti–collagen IV. Magnification, x400.
Blockade of the TGF-/Smad2/3 Signaling Is a Mechanism by Which Smad7 Inhibits Renal Fibrosis
We next investigated the mechanisms by which Smad7 inhibitsrenal fibrosis in ACGN. As shown in Figure 6, A and B, real-timePCR and ELISA analyses showed that compared with normal controlmice, renal TGF-1 mRNA and protein were significantly upregulatedin ACGN mice that were treated with empty vectors (P < 0.05),which resulted in a significant increase in phosphorylationof Smad3 (Figure 6, C and D) and Smad2. However, overexpressionof Smad7 abrogated an increase in both TGF-1 mRNA (Figure 6A)and protein (Figure 6B), thereby blocking Smad3 (Figure 6, Cand D) and Smad2 activations. To confirm the expression of theseproteins in renal tissues, we performed IHC. As shown in Figure 7,A through C, both normal control and empty vector–treatedACGN mice exhibited a weak Smad7 staining in both glomeruliand tubulointerstitium (Figure 7, A and B). In contrast, ACGNmice that were treated with Smad7 showed a moderate to strongSmad7 protein expression within kidney, particularly in theglomeruli and tubulointerstitial tissues (Figure 7C). For detectionof the transfected Smad7 gene expression within the kidney,a monoclonal anti–flag m2 antibody was applied. As shownin Figure 7, D through F, whereas mice that were treated withSmad7 revealed a moderate to strong positivity for the flagm2 in both the glomerulus and tubulointerstitial tissues (Figure 7F),both normal control and diseased mice that were treated withempty vectors showed negative signals for the flag m2 in theglomerulus, although the tubular staining remained strong (Figure 7,D and E). The latter may be associated with the cross-reactivityof the anti–flag m2 antibody to an isoform of Mg2+-dependentprotein phosphatase as previously reported (13).
Figure 6. Effects of Smad7 gene therapy on renal TGF-1 expression and Smad2/3 activation. Mice were killed at week 9 after disease induction. (A) TGF-1 mRNA levels of kidney as detected by real-time PCR. (B) TGF-1 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.
Figure 7. Effects of Smad7 gene therapy on renal TGF-/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-1. Magnification, x400.
By IHC, low levels of TGF-1 and phosphorylated Smad2/3 withinthe nuclei were found in the normal control kidney with moderateSmad7 expression (Figure 7, A, D, G, and J). However, TGF-1was upregulated in the diseased mice that were treated withempty vectors (Figure 7K), resulting in a marked activationof Smad2/3 (Figure 7H), whereas renal Smad7 remained lower (Figure 7,B and E). In contrast, gene transfer with Smad7 resulted ina marked upregulation of Smad7, which was associated with inhibitionof renal TGF-1 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 progressionof crescentic glomerulonephritis. In the context of renal immuneinjury, we first tested whether overexpression of Smad7 inhibitsrenal inflammation in mouse with ACGN. IHC showed that a profoundT cell and macrophage infiltration was noted in the kidney ofACGN 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 overexpressionwas dominant in blocking monocyte/macrophage over T cell infiltrates.Further studies with real-time PCR showed that inhibition ofT cell and macrophage infiltration was associated with suppressionof inflammatory cytokines (IL-1 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 Smad7treatment resulted in a great suppression of inflammatory cytokinesin ACGN mice, MCP-1 levels remained high when compared withnormal control mice (Figure 9C). Similarly, Smad7 treatmentinhibited the protein levels of IL-1, IL-6, MCP-1, iNOS, andICAM-1 in the glomerulus of the ACGN model as demonstrated byIHC (Figure 10).
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, x400.
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-1 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.
Figure 10. Effects of Smad7 gene therapy on renal inflammatory cytokine (IL-1 and IL-6), MCP-1, iNOS, and NF-B p65 expression. Mice were killed at week 9 after disease induction. (A through C) Anti–IL-1. (D through F) Anti–IL-6. (G through I) Anti–MCP-1. (J through L) Anti-iNOS. (M through O) Anti–NF-B p65. (P) Semiquantitative analysis of NF-B p65 (IHC). (Q) NF-B 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, x400 each.
Blockade of NF-B Activation Is a Key Mechanism by Which Smad7 Inhibits Renal Inflammation
We further investigated whether inhibition of NF-B activationis a central mechanism whereby overexpression of Smad7 inhibitsrenal inflammation in the ACGN model. As shown in Figure 10,NF-B p65 was markedly activated in the diseased mice that weretreated with empty vectors, compared with the normal controlkidney, as evident by its nuclear location in the glomeruliand tubulointerstitium as well as in mononuclear cells thatinfiltrated the periglomerular regions (Figure 10, M, N, andP). In contrast, NF-B p65 activation was virtually blocked bygene transfer of Smad7 (Figure 10, O and P). Consistent withthe IHC, ELISA assay for renal tissue nuclear protein extractsalso demonstrated that the Smad7-treated ACGN mice produceda significant inhibition of NF-B p65 activation, compared withthe empty vector–treated ACGN mice (16.46 ± 2.48versus 40.92 ± 11.10 ng/ml; P < 0.01; Figure 10Q).
This study provides the first demonstration that gene therapywith inducible Smad7 can effectively ameliorate ACGN, a modelof autoimmune kidney disease. This was demonstrated by the inhibitionof renal inflammation and fibrosis with crescentic glomerulonephritis.Blocking of TGF-/Smad signaling and NF-B activation is the centralmechanism by which gene transfer of Smad7 attenuated progressiverenal injury in a mouse model of ACGN.
In this study, Smad7 gene was successfully transfected intothe kidney using the noninvasive ultrasound-microbubble–mediatedsystem after intravenous injection of a mixture of microbubbles(Optison) and pTRE-m2Smad7 plasmids. The Smad7 transgene expressionwas identified in the majority of glomerular cells with theanti–flag m2 mAb, although Smad7 transgene expressionby tubular cells remains unidentified because of the antibodycross-reactivity to the tubule cells as described previously(13). Ultrasound-mediated microbubble cavitation could be akey mechanism by which ultrasound treatment largely enhancesSmad7 gene transfection rate and transgene expression (13).
As expected, overexpression of Smad7 blocked Smad2/3 activationand renal fibrosis in ACGN, which is consistent with previousreports in obstructive kidney disease (16) and remnant kidneydiseases (13). However, inhibition of immune-mediated kidneydisease, particularly crescentic glomerulonephritis by overexpressionof Smad7, is novel, although it is noted that expression ofSmad7 is able to block renal inflammation in nonimmunologicallymediated renal injury in a mouse obstructive kidney model andin a rat model of kidney disease (13–16). It is generallybelieved that TGF- is an anti-inflammatory cytokine and immunemodulator (28). Thus, blockade of TGF- may promote inflammationand immune-mediated injury (29). Lessons learned from the TGF-knockout mice with massive systemic inflammation reveal thecritical role for TGF- in anti-inflammation. The use of neutralizingantibodies to block TGF- is likely to prevent the developmentof fibrosis but may also enhance inflammatory response and renalinjury as reported in a db/db diabetic mouse model and a puromycinaminonucleoside nephropathy in rats (30,31), suggesting thecomplexity of TGF- in the pathophysiologic progression of renaldisease. The findings that blockade of downstream of TGF- signalingwith Smad7 inhibited renal fibrosis and immune-mediated renalinjury indicate an additional role for Smad7 in anti-renal inflammationin ACGN. It is generally accepted that glomerular crescent formationis a severe form of immune-mediated glomerulonephritis as demonstratedby the participation of macrophages, T cells, and neutrophils(27,32,33). In past decades, many studies have shown that deletionof T cells as well as macrophages attenuates crescentic glomerulonephritis(23,25,34), demonstrating an essential role for T cells andmacrophages in the pathogenesis of crescentic glomerulonephritis.In addition, NF-B–dependent proinflammatory cytokines,including IL-1 and TNF-, have been shown to play a criticalrole in the pathogenesis of crescentic glomerulonephritis (35,36).Blockade of NF-B inhibits crescentic glomerulonephritis in arat model of anti–glomerular basement membrane disease,demonstrating a critical role for the NF-B pathway in progressiverenal injury (37). Thus, blockade of NF-B activation in bothglomeruli and interstitial tissues could be a key mechanismby which Smad7 inhibits immune-mediated renal injury in ACGN.It is likely that Smad7 inhibited renal inflammation througha combined suppression of both NF-B activity and mononuclearleukocyte infiltration in the kidney.
Activation of Smad3 has been shown to play a critical role infibrosis. This is further delineated by the findings from Smad3knockout mice. Many studies have shown that mice that are nullfor Smad3 are protected against fibrosis in a number of diseasesettings, including tubulointerstitial fibrosis in obstructivekidney disease (38,39). Beyond the context of fibrosis, micethat lack Smad3 also exhibit reduced inflammatory response,such as macrophage and T cell infiltration in obstructive kidneyand in skin wound healing (39,40), indicating an additionalrole for Smad3 in inflammation. This is further demonstratedby the recent studies that TGF- signals through Smad3 to exertits inhibitory effect on T cell proliferation, MCP-1–drivenleukocyte infiltration, adhesion molecules (41), and Th1-cytokineIL-2 production (42). On the basis of our observations thatoverexpression of Smad7 is able to block Smad2/3 activationand inhibition of NF-B activation, this study demonstrated thatthere are three major mechanisms by which Smad7 inhibits progressiverenal injury in ACGN. First, overexpression of Smad7 may actas a negative regulator of TGF- signaling to inhibit renal fibrosisby blocking the TGF-/Smad-dependent pathway. Second, Smad7 mayblock renal inflammation via inhibition of the NF-B–mediatedinflammatory pathway, although inhibition of Smad3 activationby Smad7 may also have an impact on renal inflammation. Finally,one potential mechanism that should also be considered is thepossible effect of Smad7 gene therapy on the circulating and/ortissue inflammatory cells themselves. Inhibition of T cell andmacrophage infiltration and activation could account for thefact that gene therapy with Smad7 blocks renal fibrosis andimmune-mediated renal injury in ACGN.
This study was supported by grants from the Ministry of Economy(95-EC-17-A-20-S1-028), Tri-Service General Hospital (TSGH-C92-4-S02),Taiwan, Republic of China, and the Research Grant Council ofHong Kong (RGC CERG HKU7592/06M).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
/Smad signaling in autoimmune kidney disease. This study tested the hypothesis that blocking TGF-
B activation (P < 0.01), thereby inhibiting 
-smooth muscle actin; collagen I, III, and IV accumulation; and expression of inflammatory cytokines (IL-1
), empty vector (
), or normal control (
). *P < 0.05; **P < 0.01; ***P < 0.005.
