Ultrasound-Microbubble-Mediated Gene Transfer of Inducible Smad7 Blocks Transforming Growth Factor-β Signaling and Fibrosis in Rat Remnant Kidney
2005; Elsevier BV; Volume: 166; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)62297-3
ISSN1525-2191
AutoresChun‐Cheng Hou, Wansheng Wang, Xiao Ru Huang, Ping Fu, Tso-Hsiao Chen, David Sheikh‐Hamad, Hui Y. Lan,
Tópico(s)Chronic Kidney Disease and Diabetes
ResumoTransforming growth factor (TGF)-β1 has been shown to play a critical role in hypertensive nephropathy. We hypothesized that blocking TGF-β1 signaling could attenuate renal fibrosis in a rat model of remnant kidney disease. Groups of six rats were subjected to 5/6 nephrectomy and received renal arterial injection of a doxycycline-regulated Smad7 gene or control empty vector using an ultrasound-microbubble-mediated system. Smad7 transgene expression within the kidney was tightly controlled by the addition of doxycycline in the daily drinking water. All animals were euthanized at week 4 for renal functional and histological examination. Hypertension of equivalent magnitude (190 to 200 mmHg) developed in both Smad7- and empty vector-treated rats. However, treatment with Smad7 substantially inhibited Smad2/3 activation and prevented progressive renal injury by inhibiting the rise of 24-hour proteinuria (P < 0.001) and serum creatinine (P < 0.001), preserving creatinine clearance (P < 0.05), and attenuating renal fibrosis and vascular sclerosis such as collagen I and III expression (P < 0.01) and myofibroblast accumulation (P < 0.001). In conclusion, TGF-β/Smad signaling plays a critical role in renal fibrosis in a rat remnant kidney model. The ability of Smad7 to block Smad2/3 activation and attenuate renal and vascular sclerosis demonstrates that ultrasound-mediated Smad7 gene therapy may be a useful therapeutic strategy for the prevention of renal fibrosis in association with hypertension. Transforming growth factor (TGF)-β1 has been shown to play a critical role in hypertensive nephropathy. We hypothesized that blocking TGF-β1 signaling could attenuate renal fibrosis in a rat model of remnant kidney disease. Groups of six rats were subjected to 5/6 nephrectomy and received renal arterial injection of a doxycycline-regulated Smad7 gene or control empty vector using an ultrasound-microbubble-mediated system. Smad7 transgene expression within the kidney was tightly controlled by the addition of doxycycline in the daily drinking water. All animals were euthanized at week 4 for renal functional and histological examination. Hypertension of equivalent magnitude (190 to 200 mmHg) developed in both Smad7- and empty vector-treated rats. However, treatment with Smad7 substantially inhibited Smad2/3 activation and prevented progressive renal injury by inhibiting the rise of 24-hour proteinuria (P < 0.001) and serum creatinine (P < 0.001), preserving creatinine clearance (P < 0.05), and attenuating renal fibrosis and vascular sclerosis such as collagen I and III expression (P < 0.01) and myofibroblast accumulation (P < 0.001). In conclusion, TGF-β/Smad signaling plays a critical role in renal fibrosis in a rat remnant kidney model. The ability of Smad7 to block Smad2/3 activation and attenuate renal and vascular sclerosis demonstrates that ultrasound-mediated Smad7 gene therapy may be a useful therapeutic strategy for the prevention of renal fibrosis in association with hypertension. Renal fibrosis is a final common pathway to end-stage renal disease. Recent studies have shown that hypertensive nephropathy is a major leading cause of end-stage renal disease and the renin-angiotensin system plays a pivotal role in the development of progressive renal injury.1Flack JM Peters R Shafi T Alrefai H Nasser SA Crook E Prevention of hypertension and its complications: theoretical basis and guidelines for treatment.J Am Soc Nephrol. 2003; 14: S92-S98Crossref PubMed Google Scholar, 2Klahr S Morrissey J Progression of chronic renal disease.Am J Kidney Dis. 2003; 41: S3-S7Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar Clinical trials have shown that blocking the effects of angiotensin II (Ang II) with angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers can prevent or slow the progression of kidney damage in patients with diabetes and hypertension.1Flack JM Peters R Shafi T Alrefai H Nasser SA Crook E Prevention of hypertension and its complications: theoretical basis and guidelines for treatment.J Am Soc Nephrol. 2003; 14: S92-S98Crossref PubMed Google Scholar, 2Klahr S Morrissey J Progression of chronic renal disease.Am J Kidney Dis. 2003; 41: S3-S7Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 3Taal MW Brenner BM Renoprotective benefits of RAS inhibition: from ACEI to angiotensin II antagonists.Kidney Int. 2000; 57: 1803-1817Crossref PubMed Scopus (404) Google ScholarThe mechanism of renal fibrosis associated with hypertension involves stimulation of transforming growth factor (TGF)-β by Ang II.4Border WA Noble NA Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar, 5Gaedeke J Peters H Noble NA Border WA Angiotensin II, TGF-beta and renal fibrosis.Contrib Nephrol. 2001; 135: 153-160Crossref PubMed Google Scholar, 6Wolf G Link between angiotensin II and TGF-beta in the kidney.Miner Electrolyte Metab. 1998; 24: 174-180Crossref PubMed Scopus (82) Google Scholar According to this view, Ang II and TGF-β are critical mediators of renal fibrosis.4Border WA Noble NA Interactions of transforming growth factor-beta and angiotensin II in renal fibrosis.Hypertension. 1998; 31: 181-188Crossref PubMed Google Scholar, 5Gaedeke J Peters H Noble NA Border WA Angiotensin II, TGF-beta and renal fibrosis.Contrib Nephrol. 2001; 135: 153-160Crossref PubMed Google Scholar, 6Wolf G Link between angiotensin II and TGF-beta in the kidney.Miner Electrolyte Metab. 1998; 24: 174-180Crossref PubMed Scopus (82) Google Scholar Indeed, Ang II can induce extracellular matrix (ECM) expression by glomerular mesangial cells,7Wolf G Haberstroh U Neilson EG Angiotensin II stimulates the proliferation and biosynthesis of type I collagen in cultured murine mesangial cells.Am J Pathol. 1992; 140: 95-107PubMed Google Scholar, 8Kagami S Border WA Miller DE Noble NA Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor-beta expression in rat glomerular mesangial cells.J Clin Invest. 1994; 93: 2431-2437Crossref PubMed Scopus (998) Google Scholar tubular epithelial cells,9Wolf G Zahner G Schroeder R Stahl RA Transforming growth factor beta mediates the angiotensin-II-induced stimulation of collagen type IV synthesis in cultured murine proximal tubular cells.Nephrol Dial Transplant. 1996; 11: 263-269Crossref PubMed Scopus (58) Google Scholar, 10Wolf G Ziyadeh FN Stahl RA Angiotensin II stimulates expression of transforming growth factor beta receptor type II in cultured mouse proximal tubular cells.J Mol Med. 1999; 77: 556-564Crossref PubMed Scopus (89) Google Scholar and vascular smooth cells11Gibbons GH Pratt RE Dzau VJ Vascular smooth muscle cell hypertrophy vs hyperplasia: autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II.J Clin Invest. 1992; 90: 456-461Crossref PubMed Scopus (629) Google Scholar through a TGF-β-dependent mechanism. 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Once activated, Smad2 and Smad3 associate with the common partner Smad4 and translocate to the nucleus, where Smad protein complexes participate in transcriptional control of target genes. In addition, activation of TGF-β signaling can also result in the expression of inhibitory Smads, which include Smad6 and Smad7. These inhibitory Smads decrease Smad2/3 phosphorylation by blocking their access to TGF-β receptors or causing degradation of TGF-β receptors via a negative feedback mechanism.22Kavsak P Rasmussen RK Causing CG Bonni S Zhu H Thomsen GH Wrana JL Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF-β receptor for degradation.Mol Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar Increasing evidence has shown that TGF-β/Smad signaling plays a critical role in renal fibrosis.23Poncelet AC Schnaper HW Sp1 and Smad proteins cooperate to mediate transforming growth factor-β 1-induced α 2(I) collagen expression in human glomerular mesangial cells.J Biol Chem. 2001; 276: 6983-6992Crossref PubMed Scopus (222) Google Scholar, 24Schnaper HW Hayashida T Poncelet AC It's a Smad world: regulation of TGF-beta signaling in the kidney.J Am Soc Nephrol. 2002; 13: 1126-1128PubMed Google Scholar, 25Bottinger EP Bitzer M TGF-β1 signaling in renal disease.J Am Soc Nephrol. 2002; 13: 2600-2610Crossref PubMed Scopus (656) Google Scholar, 26Schiffer M Schiffer LE Gupta A Shaw AS Roberts IS Mundel P Bottinger EP Inhibitory Smads and TGF-beta signaling in glomerular cells.J Am Soc Nephrol. 2002; 13: 2657-2666Crossref PubMed Scopus (76) Google Scholar This is supported by studies showing that Smad2/3 is activated in diabetic nephropathy27Isono M Chen S Hong SW Iglesias-de la Cruz MC Ziyadeh FN Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-beta-induced fibronectin in mesangial cells.Biochem Biophys Res Commun. 2002; 296: 1356-1365Crossref PubMed Scopus (155) Google Scholar, 28Li JH Huang XR Zhu HJ Oldfield M Cooper M Truong LD Johnson RJ Lan HY Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease.FASEB J. 2004; 18: 176-178Crossref PubMed Scopus (104) Google Scholar and that overexpression of Smad7 by gene transfer can inhibit TGF-β-induced Smad2/3 activation and ECM production in vitro29Li JH Zhu HJ Huang XR Lai KN Johnson RJ Lan HY Smad7 inhibits fibrotic effect of TGF-β on renal tubular epithelial cells by blocking Smad2 activation.J Am Soc Nephrol. 2002; 13: 1464-1472Crossref PubMed Scopus (234) Google Scholar, 30Chen R Huang C Morinelli TA Trojanowska M Paul RV Blockade of the effects of TGF-β1 on mesangial cells by overexpression of Smad7.J Am Soc Nephrol. 2002; 13: 887-893PubMed Google Scholar and in the obstructive rat kidney model.31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar, 32Terada Y Hanada S Nakao A Kuwahara M Sasaki S Marumo F Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney.Kidney Int. 2002; 61: S94-S98Crossref PubMed Scopus (96) Google Scholar We, thus, hypothesized that blockade of the TGF-β signaling pathway by gene transfer of an inducible Smad7 gene may be able to attenuate progressive renal injury in the 5/6 nephrectomized rat model and may represent a specific therapeutic strategy to block renal fibrosis in kidney diseases with hypertension. Gene transfer was achieved with the ultrasound-microbubble technique and Smad7 transgene expression was tightly regulated by the addition of doxycycline in the drinking water. The results showed that overexpression of Smad7 attenuated renal fibrosis and prevented renal function impairment in this hypertensive kidney disease model.Materials and MethodsUltrasound-Mediated Gene Transfer of Inducible Smad7 Gene-Loaded Microbubbles into the KidneyTo control Smad7 transgene expression within the kidney at the therapeutic levels without undesirable effects, a doxycycline-regulated Smad7-expressing plasmid was used.31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar Briefly, a mouse Smad7 cDNA with a flag tag (m2) at its NH2 terminus in pcDNA3 (gift from Dr. P. ten Dijke, The Netherlands Cancer Institute, Amsterdam, The Netherlands) was subcloned into a tetracycline-inducible vector, pTRE (Clontech, Palo Alto, CA), to obtain pTRE-m2Smad7. An improved pTet-on vector (Clontech), pEFpurop-Tet-on, was generously provided by G. Vario (Cerylid, Melbourne, Australia). All plasmids were prepared using the EndoFree plasmid kit (Qiagen Inc., Valencia, CA) as per the manufacturer's instructions. To achieve doxycycline-inducible Smad7 transgene expression, the ultrasound-microbubble-mediated system was applied as previously described.31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar Briefly, after mixing pTRE-m2Smad7 and pEFpurop-Tet-on with albumin perflutren-filled microbubbles (Optison; Mallinckrodt, St. Louis, MO) at a ratio of 1:1 (v/v), the mixed solution containing 15 μg of designated plasmids in 0.3 ml was injected into the left remnant kidney via the renal artery while temporarily clipping off proximal portions of the renal artery and vein (less than 5 minutes). Immediately after injection, the ultrasound transducer (Ultax UX-301; Celcom Medico Inc., Fukuoka, Japan) was directly applied to one side of the kidney and a continuous-wave output of 1 MHz at 5% power output was applied for a total of 60 seconds with a 30-second interval. Ultrasound treatment was applied to the other side of the kidney using the same procedure.Animal Model and ProtocolMale Sprague-Dawley rats at 6 weeks of age, weighing 220 to 250 g, were obtained from Harlan (Indianapolis, IN). Progressive renal disease model with hypertension was induced by 5/6 subtotal nephrectomy as previously described.33Ng YY Huang TP Yang WC Chen ZP Yang AH Mu W Nikolic-Paterson DJ Atkins RC Lan HY Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.Kidney Int. 1998; 54: 864-876Crossref PubMed Scopus (344) Google Scholar Briefly, 12 animals underwent subtotal nephrectomy involving right subcapsular nephrectomy and infarction of approximately two-thirds of the left kidney by ligation of the posterior and one or two anterior extrarenal branches of the renal artery. Immediately after the surgery, rats were randomly assigned into groups of six animals and received either Smad7 or empty vectors as previously described.31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar In the treatment group, six rats were injected with a mixture (0.3 ml) of pTRE-m2Smad7 plus Tet-on plasmids plus Optison into the left kidney at a dose of 15 μg/kidney via the renal artery and treated with ultrasound as described above. Control animals received the same procedure, except for substitution of pTRE-m2Smad7 with empty vector. To induce Smad7 transgene expression, 1 ml of doxycycline (Sigma, St. Louis, MO) at a concentration of 200 μg/ml was administered into the peritoneal cavity immediately after ultrasound-microbubble gene transfer, followed by the addition of doxycycline in the daily drinking water (200 μg/ml) for 28 days as previously described.31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar For normal controls, six rats underwent a sham operation consisting of laparotomy and manipulation of the renal pedicles but without damage to the kidney. All rats were euthanized at day 28 after disease induction. Rat serum was collected at day 0 and weeks 2 and 4 for creatinine determination, and 24-hour urine samples were collected in metabolic cages at day 0 and weekly for the determination of urinary levels of protein and creatinine excretion. All procedures were performed in accordance with Institutional Guidelines for Animal Care.Renal Function Assessment and Blood Pressure MeasurementUrinary protein concentrations were determined by the Bradford method, adapted to a microtiter plate assay. Coomassie reagent (USB, Cleveland, OH) was added to the diluted urine samples. After 10 minutes, the absorbance at 595-nm wavelength was read on ELX800 microplate reader (Bio-Tek Instruments, VT). The protein concentrations were calculated by reference to bovine serum albumin (Sigma) standards. Serum and urine creatinine were analyzed using a creatinine kit (Sigma). Systolic blood pressure was recorded by tail plethysmography using the BP2000 blood pressure analysis system (Visitech Systems, Inc., Apex, NC) in conscious rats at baseline and every 2 weeks throughout the experimental time course.Real-Time Polymerase Chain Reaction (PCR)Total RNA was isolated from snap-frozen, noninfarcted kidney tissues using the RNAeasy isolation kit (Qiagen) according to the manufacturer's instructions. Real-time reverse transcriptase (RT)-PCR was performed as previously described.34Li JH Wang W Huang XR Oldfield M Schmidt AM Cooper ME Lan HY Advanced glycation end products induce tubular epithelial-myofibroblast transition through the RAGE-ERK1/2 MAP kinase signaling pathway.Am J Pathol. 2004; 164: 1389-1397Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar Briefly, a real-time PCR was performed using Bio-Rad (Hercules, CA) iQ SYBR Green supermix with Opticon (MJ Research Inc., Waltham, MA), according to the manufacturer's instructions. One hundred μg of total RNA were reverse-transcribed and subjected to PCR as follows: 94°C for 2 minutes followed by 40 cycles at 94°C for 15 seconds, 58°C for 30 seconds, 72°C for 30 seconds, with final extension at 72°C for 10 minutes. The primers used in this study were: rat Smad7, forward 5′- CGCTGTACCTTCCTCCGAT-3′, reverse 5′-ATGAAAGATGGCTGGAAGAGGGTC-3′; α-smooth muscle actin (SMA), forward 5′-ACTGGGACGACATGGAAAAG-3′, reverse 5′-CATCTCCAGAGTCCAGCACA-3′; collagen I, forward 5′-GAGCGGAGAGTACTGGATCG-3′, reverse 5′-TACTCGAACGGGAATCCATC-3′; and collagen III, forward 5′-TGGTCCTCAGGGTGTAAAGG-3′, reverse 5′-GTCCAGCATCACCTTTTGGT-3′. All samples were subjected to RT-PCR for the housekeeping gene GAPDH, forward 5′-TGCTGAGTATGTCGTGGAGTCTA-3′, reverse 5′-AGTGGGAGTTGCTGTTGAAATC-3′ as an internal standard. Reaction specificity was confirmed by gel electrophoresis of products after real-time PCR and melting curve analysis. Ratios for Smad7/GAPDH, α-SMA/GAPDH, collagen I/GAPDH, and collagen III/GAPDH mRNA were calculated for each sample and expressed as the means ± SEM.Western Blotting and ImmunoprecipitationWestern blot analysis was used for detection of Smad7, α-SMA, and collagen type I and type III expression within the kidney, whereas immunoprecipitation was used for detection of phosphorylated Smad2/3 as previously described.28Li JH Huang XR Zhu HJ Oldfield M Cooper M Truong LD Johnson RJ Lan HY Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease.FASEB J. 2004; 18: 176-178Crossref PubMed Scopus (104) Google Scholar, 31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar Snap-frozen, noninfarcted kidney tissues were lysed in 1 ml of 1% Nonidet P-40, 25 mmol/L Tris-HCl, 150 mmol/L NaCl, 10 mmol/L ethylenediaminetetraacetic acid, pH 8.0, containing protease inhibitor cocktail (P2714, at a final dilution of 1:50; Sigma) for 30 minutes on ice. Samples (20 μg) were centrifuged at 14,000 × g for 5 minutes to pellet cell debris, mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer, boiled for 5 minutes, electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel, and electroblotted onto polyvinylidene difluoride nitrocellulose membrane (Amersham International, Buckinghamshire, UK). The membrane was blocked in Tris-buffered saline containing 5% bovine serum albumin and 0.02% Tween 20 and incubated for 1 hour with one of the following: mouse monoclonal antibodies (mAbs) to α-SMA (Sigma), goat polyclonal antibodies to Smad7 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), goat anti-collagen I or anti-collagen III (Southern Biotech, Birmingham, AL). After washing, the membrane was incubated with peroxidase-conjugated goat anti-mouse IgG or rabbit anti-goat IgG in phosphate-buffered saline containing 1% normal goat serum and 1% fetal calf serum. Phosphorylated Smad2/3 was determined by immunoprecipitation with anti-Smad2/3 antibody followed by immunoblotting and detection with rabbit anti-phosphoserine antibody.28Li JH Huang XR Zhu HJ Oldfield M Cooper M Truong LD Johnson RJ Lan HY Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease.FASEB J. 2004; 18: 176-178Crossref PubMed Scopus (104) Google Scholar, 31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar After washing, bands were detected using the ECL kit (Amersham) and signal was captured on X-ray film.Histology and ImmunohistochemistryRenal tissues for histological examination were fixed in 10% formalin and 4-μm paraffin sections were stained with hematoxylin and eosin and periodic acid-Schiff (PAS). To detect activation of Smad2/3 and expression of Smad7, 4-μm paraffin sections were stained with rabbit polyclonal antibodies to p-Smad2/3 (Santa Cruz) and goat antibody to Smad7 (Santa Cruz), using a microwave-based antigen retrieval technique and a peroxidase anti-peroxidase method as previously described.28Li JH Huang XR Zhu HJ Oldfield M Cooper M Truong LD Johnson RJ Lan HY Advanced glycation end products activate Smad signaling via TGF-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease.FASEB J. 2004; 18: 176-178Crossref PubMed Scopus (104) Google Scholar, 31Lan HY Mu W Tomita N Huang XR Li JH Zhu HJ Morishita R Johnson RJ Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2002; 14: 1535-1548Crossref Scopus (321) Google Scholar An irrelevant isotype rabbit or goat IgG was used for negative control. The degree of interstitial myofibroblast accumulation and collagen I and III expression were determined using microwave-based antigen retrieval method with anti-α-SMA mAb (Sigma) and anti-rat collagen I and III polyclonal antibodies (Southern Biotech). Sections were then developed with diaminobenzidine to produce a brown color and counterstained with hematoxylin.Quantitative AnalysesSmad2/3 activation was determined by the extent of its nuclear localization in stained tissue using anti-phosphorylated Smad2/3 antibody. The number of positive cells for activated Smad2/3 was counted in 20 consecutive glomeruli and expressed as cells/glomerular cross-section (gcs), whereas positive cells for activated Smad2/3 in the tubulointerstitium were counted under high-power fields (×40) by means of a 0.25-mm2 graticule fitted in the eyepiece of the microscope, and expressed as cells per cm2. Because Smad7 was widely expressed in the tubulointerstitium in both normal and diseased animals, semiquantitation of Smad7 expression in the tubulointerstitium was scored as follows: 0.5, weak; 1, mild; 2, moderate; 3, strong. The degree of glomerular Smad7 expression and accumulation of α-SMA and collagens type I and III in glomeruli and entire cortical tubulointerstitium (a cross-section of the kidney) were determined using quantitative Image Analysis System (Optima 6.5; Media Cybernetics, Silver Spring, MD). Briefly, the examined area of the glomerulus or the tubulointerstitium was outlined; positive staining patterns were identified, and the percent positive area in the examined glomerulus or tubulointerstitium was then measured. The spaces in Bowman's capsule and large arteries, including the arterial walls and periarterial areas were excluded from the study. Data were expressed as percent positive area examined. To investigate vascular Smad2/3 activation and vascular sclerosis, a point-counting technique was applied. Smad2/3-positive cells in arterial walls and immediate periarterial areas with or without the development of proliferative and sclerotic vasculopathy in the entire cortex were counted under 0.25-mm2 graticule and expressed as cells per cm2, whereas collagen matrix accumulation was measured using quantitative Image Analysis System as described above. The arterial lumen space was excluded from the study and the periarterial area was defined by the outline of tubular basement membrane surrounding the artery examined. Generally, 7 to 10 arteries in the entire cortex were identified and examined. All analyses were performed in the same paraffin-block of renal tissue cross-sections that were far from the infracted areas (two to three low-power fields from the infracted tissues). All scoring was performed in a blinded manner on coded slides.Statistical AnalysesData obtained from this study are expressed as the means ± SEM. Statistical analyses were performed using GraphPad Prism 3.0 (GraphPad Software, Inc., San Diego, CA). Differences in blood pressure, proteinuria, serum creatinine, and creatinine clearance at different time points (weeks 0 to 4) within and between the groups, and differences of Smad2/3 activation, Smad7 expression, and α-SMA and collagen matrix accumulation in sham, control vector-treated, and Smad7-treated animals were assessed by one-way analysis of variance, followed by t-test.ResultsUltrasound-Mediated Ge
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