N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Ameliorates the Progression of Renal Dysfunction and Fibrosis in WKY Rats with Established Anti–Glomerular Basement Membrane Nephritis
* Biomedical Research Laboratories, Daiichi Asubio Pharma Co., Ltd., Osaka; Department of Endocrinology and Metabolism, Kanazawa Medical University, Ishikawa; Department of Medicine, Shiga University of Medical Science, Shiga; Laboratory of Analytical Pharmacology, Faculty of Pharmacy, Meijo University, Nagoya; and || Second Department of Internal Medicine, Asahikawa Medical College, Asahikawa, Japan
Address correspondence to: Dr. Mitsugu Omata, Biomedical Research Laboratories, Daiichi Asubio Pharma Co., Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8513, Japan. Phone: +81-75-962-8538; Fax: +81-75-962-6448; E-mail: mitsugu_omata{at}asubio.co.jp
Received for publication April 11, 2005.
Accepted for publication December 4, 2005.
N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), which is hydrolyzedby angiotensin-converting enzyme, is a natural regulator ofhematopoiesis. Here it is shown that Ac-SDKP inhibits TGF- actionin mesangial cells. Because TGF- is thought to play a pivotalrole in the development and progression of glomerulonephritis,the therapeutic effects of Ac-SDKP on an established model ofrenal dysfunction and histologic alteration in Wistar-Kyotorats with anti–glomerular basement membrane nephritiswas examined. Fourteen days after the induction of anti–glomerularbasement membrane nephritis, the rats were treated subcutaneouslywith Ac-SDKP at a dose of 1 mg/kg per d for 4 wk. Treatmentwith Ac-SDKP significantly improved proteinuria and renal dysfunction,including increased plasma blood urea nitrogen and creatininelevels and decreased creatinine clearance. Histologic examinationshowed severe glomerulosclerosis and interstitial fibrosis inthe vehicle-treated rats, whereas these histologic injurieswere significantly ameliorated in rats that were treated withAc-SDKP. The histologic improvements were accompanied by thesuppression of gene and protein expression of fibronectin, interstitialcollagen, and TGF-1 in the nephritic kidney. Furthermore, treatmentwith Ac-SDKP resulted in the inhibition of Smad2 phosphorylation,an increase in Smad7 expression in the kidney, and reductionof macrophage accumulation into the glomeruli and tubulointerstitiumin nephritic rats. In conclusion, Ac-SDKP significantly amelioratedthe progression of renal dysfunction and fibrosis even afterthe establishment of nephritis. The inhibitory effect of Ac-SDKPwas mediated in part by the inhibition of TGF-/Smad signal transductionand the inflammatory response. These findings suggest that Ac-SDKPtreatment may be a novel and useful therapeutic strategy forthe treatment of progressive renal diseases.
It has been reported that more than 20 million people worldwidehave lost half of their renal function or have shown overt proteinuria(1). The number of patients who have ESRD or must undergo chronicrenal dialysis therapy is ever increasing (2,3). The end-stagelesions, such as glomerulosclerosis and tubulointerstitial fibrosis,are characterized by the advanced loss of renal cells and excessiveaccumulation of extracellular matrix (ECM), including fibronectinand collagens; these lesions represent a common structure independentof the primary underlying disease (4,5). Although most patientswho have chronic kidney disease (CKD) have been treated withseveral pharmacotherapies before reaching end-stage renal failure,no currently available treatment is effective in halting theloss of residual renal function and the progression of renalfibrosis (6–8). Therefore, the development of novel therapeuticagents that interrupt the uncontrollable fibrotic response torenal injury has been eagerly anticipated.
N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an endogenoustetrapeptide that possesses a regulatory effect on the proliferationof hematopoietic stem cells (9). Ac-SDKP, which is normallypresent in plasma and mononuclear cells, is degraded in thecirculation by angiotensin I–converting enzyme (ACE) (10).In some in vivo studies, Ac-SDKP prevented collagen depositionin the left ventricle and kidneys in a hypertensive rat model(11,12) and reversed cardiac inflammation and fibrosis in ratswith heart failure induced by myocardial infarction (13). Morerecently, we also found that treatment with Ac-SDKP preventsthe renal insufficiency and glomerular mesangial matrix expansionobserved in type 2 diabetic db/db mice (14). In some in vitrostudies, Ac-SDKP has been shown to suppress the proliferationof renal fibroblasts (15) and to inhibit DNA and collagen synthesisin cardiac fibroblasts (16). We have shown that Ac-SDKP is ableto inhibit the TGF-–induced expression of plasminogenactivator inhibitor-1 and 2-type I collagen through the suppressionof Smad signaling in mesangial cells (17). Because profibroticcytokine TGF- is thought to play a pivotal role in the progressionof glomerulonephritis and tubulointerstitial fibrosis (18,19),we investigated the ability of Ac-SDKP to inhibit TGF- actionand improve renal fibrosis and dysfunction in a glomerulosclerosismodel. Most patients who receive a diagnosis of CKD alreadyhave renal dysfunction accompanied by some degree of fibroticlesions in the kidneys. Therefore, we administered Ac-SDKP 2wk after nephritis induction in our rat model, a time pointafter the development of renal dysfunction and pathologic changes.We found that Ac-SDKP significantly ameliorated the progressionof renal dysfunction and fibrosis even after the establishmentof nephritis. The inhibitory effects of Ac-SDKP were mediatedin part by the inhibition of TGF-/Smad signal transduction andinflammatory responses, such as monocyte/macrophage accumulation.
Experimental Protocol
All animal experiments were approved by the Animal Care andEthics Committee of Daiichi Asubio Pharma. Male Wistar-Kyoto(WKY) rats were purchased from Charles River Japan (Kanagawa,Japan). On day 0, 0.025 ml of rabbit anti-rat glomerular basementmembrane (GBM) serum (20) was injected intravenously into 187-wk-old rats. Fourteen days after the induction of nephritis,urine samples were collected for 24 h to measure of urinaryprotein excretion. Four rats were selected randomly and killedon day 14. After examination of the urinary protein excretion,the remaining rats were randomly assigned to two groups: Ratsanti-GBM nephritis, which were given no treatment (n = 7), andrats that had anti-GBM nephritis and were treated with 1 mg/kgper d Ac-SDKP (n = 7). An osmotic minipump (Alzet2 ML4, Alzet,Cupertino, CA) was implanted subcutaneously in the neck to delivereither Ac-SDKP or vehicle, saline plus 0.01 N acetic acid. Thedosage of Ac-SDKP and the route of administration were selectedon the basis of previously described procedures (11,14). Ac-SDKPwas infused subcutaneously for 4 wk. Age-matched male WKY ratswithout nephritis were used as normal controls. Serial bloodsamples were taken via tail-vein puncture under light anesthesiainduced by isoflurane inhalation on days 0, 14, 21, 28, and35. Twenty-four-hour urine samples for the measurement of proteinuriawere collected during the same period. At 42 d after nephritisinduction, the animals received perfusion of sterile salineadministered to the thoracic aorta under anesthesia (50 mg/kgpentobarbital). The renal cortex was dissected from the leftkidney, immediately frozen in liquid nitrogen, and stored at–80°C until further analysis. The right kidney wasexcised and immersed in 4% paraformaldehyde for histologic examination.
Determination of Blood and Urine Parameters
Urinary protein concentrations were determined using the methodof Kingsbury et al. (21). Plasma (Pcr) and urinary (Ucr) creatinineand blood urea nitrogen (BUN) levels were determined using anautoanalyzer (HITACHI clinical analyzer 7070; Hitachi High-TechnologiesCo., Tokyo, Japan). For avoidance of errors from incompleteurine collection, urinary protein excretion was normalized tothe levels of urinary creatinine. Creatinine clearance (Ccr)was calculated using the standard formula Ccr = Ucr x urinevolume/Pcr and was expressed as ml/min per 100 g body wt.
Histologic Examination
The longitudinally bisected kidney was embedded in paraffin,sectioned, and stained with periodic acid-Schiff and Masson’strichrome reagents. The sections were evaluated by two independentobservers who were unaware of the origin of the slides. Fordetermination of glomerulosclerosis, 100 glomeruli were randomlyselected in cross-sections that were obtained from each rat.Glomerulosclerosis, which was defined as glomeruli exhibitingadhesion of the capillary tuft to the Bowman’s capsule,capillary obliteration, mesangial expansion, or fibrotic crescents,was graded semiquantitatively as follows: 0, normal; 1, 0 to25% of the glomerular area affected; 2, 25 to 50% affected;3, 50 to 75% affected; and 4, 75 to 100% affected (22). Thedegree of glomerulosclerosis was expressed as the glomerulosclerosisindex (GSI). The GSI of a whole kidney was obtained by averagingthe scores from 100 glomeruli in one section. Furthermore, scoresthat were obtained by two independent investigators were averaged.For quantification of tubulointerstitial fibrosis, 50 fieldswere randomly selected from the cortical region and were analyzedunder high-power magnification (x400). Fibrotic areas in theinterstitium, which were stained blue, were identified on thedigital images using a computer-aided manipulator (LuminaVision;Mitani Co., Fukui, Japan). The percentage of fibrotic area wascalculated. Glomeruli and large vessels were excluded from analysis.The deposition of interstitial collagen was evaluated by stainingwith picrosirius red. Fifty fields were randomly selected fromthe cortical region and analyzed under high-power magnification(x400). Areas of collagen deposition in the interstitium, whichwere stained red, were identified on digital images using LuminaVision.The percentage of red area was calculated.
Immunohistochemistry
Immunohistochemical staining for rat monocyte/macrophage (ED-1)was performed using an avidin-biotin complex staining method(VECTASTAIN Elite ABC kit; Vector Laboratory, Burlingame, CA).After deparaffinization, slides were placed in 0.01 M citrateand heated in a microwave oven at 2450 MHz and 800 W for 10min. The slides were incubated in normal blocking serum for6 h followed by overnight incubation at 4°C with a monoclonalmouse antibody against rat macrophages (ED-1) (Serotec, Oxford,UK). Endogenous peroxidase was inactivated by incubation for10 min with 3% H2O2, and a biotinylated anti-mouse antibodywas added for 45 min at room temperature. After washing, VECTAINelite ABC/Reagent was added for 30 min at room temperature.The color reaction was developed with 3,3'-diaminobenzidineand counterstained with hematoxylin.
The accumulation of monocytes/macrophages into the renal tissuewas evaluated by assessing the number of ED-1–positivecells. The number of ED-1–positive cells in the renalcortex was determined by observation of 50 randomly selectedglomeruli or cortical interstitial areas in each cross-section.
Western Blot Analysis
The expression levels of fibronectin, Smad2, phosphorylatedSmad2, and Smad7 were examined by Western blotting. Briefly,renal cortex tissue was homogenized in 0.5 ml of ice-cold lysisbuffer (23). Samples were centrifuged at 12,000 x g for 10 min,and the supernatants were used for the assay. After mixing withSDS-PAGE sample buffer and boiling for 5 min, samples (20 µg/lane)were electrophoresed on 7.5% SDS polyacrylamide gels and transferredto polyvinylidene difluoride membranes for 1.5 h at 180 mA.Membranes were blocked for 1 h with Block-Ace (Daihippon, Japan)and incubated with primary antibody overnight at 4°C. Primaryantibodies that were used in this study included anti-rat fibronectinantibody (1:300 dilution; Chemicon International, Temecula,CA), anti-phosphorylated Smad2 antibody (1:1000 dilution; CellSignaling Technology, Danvers, MA), anti-Smad2 antibody (1:1000dilution; Cell Signaling Technology), and anti-Smad7 antibody(1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnightat 4°C. After washing, membranes were incubated with horseradishperoxidase–conjugated anti-rabbit IgG for 1 h. Westernblots were visualized using the enhanced chemiluminescence system(LumiGLO Reagent; Cell signaling Technology), which was capturedon x-ray film. The same membranes were stripped and reprobedwith anti–-actin antibody (1:200 dilution; Santa CruzBiotechnology) to confirm equal loading.
Real-Time Reverse Transcription–PCR
Total RNA was isolated with the use of a commercial kit (RNasemini kit; Qiagen, Valencia, CA). cDNA was generated from 1.5µg of total RNA and oligo(dT) primers using the SuperScriptFirst-Strand Synthesis System (Invitrogen, Carlsbad, CA) forreverse transcription–PCR according to the manufacturer’sprotocol. Quantitative real-time TaqMan PCR was performed ina 40-µl reaction volume that contained 1 µl of cDNAtemplate, 20 µl of Universal Master Mixture (Applied Biosystems,Foster City, CA), 100 nM each of sense and antisense primers,and the TaqMan probe. The PCR amplification profiles includedan initial incubation at 50°C for 2 min, denaturation at95°C for 10 min, and 40 to 45 cycles of 95°C for 15s and 60°C for 1 min. The specific primers are listed inTable 1. Reactions were performed in duplicate. All data arepresented after normalization to the glyceraldehyde-3-phosphatedehydrogenase expression observed in the same sample.
Measurement of TGF-1 Protein
Renal cortex tissue was homogenized in 0.5 ml of ice-cold lysisbuffer. Samples were centrifuged at 12,000 x g for 10 min, andthe supernatants were used for the assay. TGF-1 levels weredetermined using an ELISA kit (R&D Systems, Minneapolis,MN) according to the manufacturer’s methods. As this activityrepresents active TGF-1, acid activation of the samples wasrequired to convert the latent TGF-1 to the active form anddetermine the total level of TGF-1. A portion of the renal tissuehomogenate was assayed for total protein using a BCA proteinassay kit (Pierce, Rockford, IL). The total TGF-1 levels wereexpressed as TGF-1 levels per total protein levels in the tissuehomogenate.
Plasma Level of Ac-SDKP
At the end of the experiment, blood was collected from the abdominalaorta in a tube that contained heparin and captopril, an ACEinhibitor, at a final concentration of 10 µM. Sampleswere centrifuged at 8 000 x g for 10 min at 4°C. The recoveredplasma was stored at –80°C until the Ac-SDKP assaywas performed. Plasma Ac-SDKP was quantified with a competitiveenzyme immunoassay (SPI-BIO, Montigny le Bretonneux, France).
Statistical Analyses
Results were presented as the mean ± SEM. Comparisonsbetween two groups were performed using either the t test, whenthe data were normally distributed, or the Mann-Whitney U test,when the observations showed considerable variability. The comparisonof semiquantitative analyses, such as GSI scores, was also evaluatedby the Mann-Whitney U test. Differences among three or moregroups were evaluated using two-way ANOVA, followed by Dunnetttest. P < 0.05 was considered to be statistically significant.
Renal Dysfunction
Fourteen days after the induction of nephritis, proteinuriaand plasma levels of BUN and creatinine were markedly increasedcompared with the levels observed on day 0. After the observationat day 14, animals were randomly assigned to the vehicle-treatedgroup or the Ac-SDKP–treated group. In the vehicle-treatedgroup, proteinuria remained at high levels on days 21, 28, and35. In addition, the plasma BUN and creatinine levels in thevehicle-treated group showed a tendency to increase in a time-dependentmanner. On day 35, the proteinuria in the Ac-SDKP–treatedgroup (52.1 ± 7.5 mg/mg Cr) was significantly lower thanthat in the vehicle-treated group (74.8 ± 3.8 mg/mg Cr;Figure 1A). The BUN level in the Ac-SDKP–treated groupon day 35 was significantly lower than that in the vehicle-treatedgroup by 33.2% (Figure 1B). The plasma creatinine level wasreduced by Ac-SDKP treatment (32.6% reduction relative to vehicletreatment), consistent with the BUN level (Figure 1C). Furthermore,creatinine clearance on day 35 in the Ac-SDKP–treatedgroup was significantly higher than that in the vehicle group(Figure 1D).
Figure 1. Therapeutic effects of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on urinary protein (A), blood urea nitrogen (B), plasma creatinine (C), and creatinine clearance (D) in rats with anti–glomerular basement membrane (ant-GBM) nephritis. Each symbol represents the mean ± SEM of seven rats that were treated with vehicle () or 1 mg/kg per d Ac-SDKP (•). Statistical significance is based on the t test: *P < 0.05 versus the same time point of the vehicle group; #P < 0.01 for the vehicle group versus day 0; ##P < 0.01 for the Ac-SDKP–treated group versus day 0.
Histologic Injury
In comparison with renal sections from normal rats (Figure 2A),the rats with anti-GBM nephritis showed mild glomerulosclerosis(GSI 0.78 ± 0.06) with cellular crescent formation inalmost 50% of the glomeruli on day 14 (Figure 2B). Glomerularlesions in the vehicle-treated rats progressed thereafter, andon day 42, a marked degree of glomerulosclerosis was exhibited(GSI 1.40 ± 0.15; Figure 2C). The glomerulosclerosiswas significantly ameliorated by the treatment with Ac-SDKP(GSI 0.71 ± 0.07; Figure 2, D and I). In rats with anti-GBMnephritis, slight tubulointerstitial fibrosis was seen on day14 (2.99 ± 0.35%; Figure 2F) and interstitial injuryshowed marked development on day 42 (18.49 ± 1.90%; Figure 2G)compared with normal rats (Figure 2E). Treatment with Ac-SDKPsignificantly blunted the formation of interstitial fibrosison day 42 (9.45 ± 1.07%; Figure 2, H and J).
Figure 2. Effects of Ac-SDKP on glomerular injury and tubulointerstitial fibrosis in rats with anti-GBM nephritis. Representative photomicrographs of periodic acid-Schiff–stained glomerulosclerotic lesions in renal sections from normal rats (A), day 14 nephritic rats (B), day 42 nephritic rats that were treated with vehicle (C), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (D). Bar = 30 µm. Glomerulosclerosis was scored on the sclerosis index (I). Each column shows the data from day 14 nephritic rats that were treated with vehicle (
), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of four rats on day 14 in the vehicle group or seven rats on day 42 in the vehicle and Ac-SDKP groups. Statistical significance is based on the Mann-Whitney U test: P < 0.05 versus day 14 in the nephritic rats; **P < 0.01 versus day 42 in the vehicle group. (E through H) Representative photomicrographs of tubular lesions in renal sections from normal rats (E), day 14 nephritic rats (F), day 42 nephritic rats that were treated with vehicle (G), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (H). (J) The percentage of the entire field that was fibrotic and stained blue was calculated. Each column shows data from day 14 nephritic rats that were treated with vehicle (
), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of four rats on day 14 in the vehicle group or seven rats on day 42 in the vehicle and Ac-SDKP groups. Statistical significance: P < 0.05, P < 0.01 versus day 14 in the nephritic rats by Dunnett test; **P < 0.01 versus day 42 in the vehicle group by the t test.
Expression of ECM
Expression of fibronectin mRNA in nephritic kidneys on days14 and 42 was 10-fold higher than that in normal kidneys; thisupregulation was significantly attenuated (54.7% reduction)by Ac-SDKP treatment (Figure 3A). In the vehicle-group, thetype I and III collagen mRNA levels were increased by 14- and6-fold, respectively, on day 14 and further increased to 21-and 17-fold, respectively, on day 42. The upregulation of thesegenes was also significantly suppressed by Ac-SDKP treatment(55.5 and 58.3% reduction at day 42 compared with the vehiclegroup; Figure 3, B and C). The deposition of interstitial collagenprotein stained with picrosirius red was greater on days 14(3.6%) and 42 (9.8%) in rats with anti-GBM nephritis relativeto normal rats (1.9% at day 42). Ac-SDKP treatment significantlyreduced the amount of collagen deposition (4.8%) on day 42 comparedwith the vehicle group (Figure 4). Furthermore, Western blotanalysis demonstrated that the expression of fibronectin proteinwas remarkably increased on day 42 in rats with nephritis; Ac-SDKPtreatment resulted in a significant inhibition of fibronectinexpression compared with the vehicle group (Figure 5).
Figure 3. Inhibitory effects of Ac-SDKP on gene expression of extracellular matrix in the anti-GBM nephritis rats. Levels of mRNA encoding fibronectin (A), type I collagen (B), and type III collagen (C) in the kidney cortex were determined by real-time reverse transcription–PCR (RT-PCR) analyses. Each column shows data from normal rats (light gray column), day 14 nephritic rats that were treated with vehicle (dark gray column), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of four rats for the normal and day 14 vehicle groups or seven rats for the day 42 vehicle and Ac-SDKP groups. Statistical significance: #P < 0.05, ##P < 0.01 versus the normal group; P < 0.05 versus day 14 in the nephritic rats by Dunnett test; *P < 0.05 versus day 42 in the vehicle group by the t test.
Figure 4. Effects of Ac-SDKP on collagen deposition in the tubulointerstitium of anti-GBM nephritic rats. (A through D) Representative photomicrographs of tubular lesions in renal sections from normal rats (A), day 14 nephritic rats (B), day 42 nephritic rats that were treated with vehicle (C), and day 42 nephritis rats treated with 1 mg/kg per d Ac-SDKP (D). (E) The percentage of the entire field that was stained red was calculated. Each column shows data from normal rats (light gray column), day 14 nephritic rats that were treated with vehicle (dark gray column), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of four rats for the normal and day 14 vehicle groups and seven rats for the day 42 vehicle and Ac-SDKP groups. Statistical significance: ##P < 0.01 versus normal group, P < 0.01 versus day 14 in nephritis rats by Dunnett test; **P < 0.01 versus day 42 in the vehicle group by the t test. Bar = 50 µm.
Figure 5. Effects of Ac-SDKP on fibronectin expression in kidneys from anti-GBM nephritis rats. (A) Representative Western blot shows the expression of fibronectin at 220 kD and -actin as a control in the renal cortex on day 42 in nephritic and normal rats. (B) Densitometric quantification of the corresponding bands was performed using an image analyzer. The data are presented after normalization to -actin expression. Each column shows data for the normal rats (
), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of three to four samples. Statistical significance is based on the Mann-Whitney U test: #P < 0.05 versus the normal group; *P < 0.05 versus the vehicle group.
Levels of TGF-1 Gene and Protein Expression
The levels of TGF-1 mRNA obtained from the kidneys of vehicle-treatedrats on day 42 were elevated relative to normal rats. Ac-SDKPtreatment significantly reduced TGF-1 gene expression on day42 relative to vehicle treatment (Figure 6A). Total levels ofTGF-1 protein were increased by nephritis induction and significantlyreduced by Ac-SDKP treatment (Figure 6B).
Figure 6. Effects of Ac-SDKP on TGF-1 gene and protein expression in the kidney cortex of anti-GBM nephritic rats. TGF-1 gene (A) and protein (B) levels were determined by real-time RT-PCR and ELISA, respectively. Renal cortex tissue was isolated from normal rats and from anti-GBM nephritic rats on day 42. TGF-1 gene expression and protein levels are presented after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or total protein levels in the same sample, respectively. Each column shows data from normal rats (
), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of four rats for the normal group or seven rats for the day 42 vehicle and Ac-SDKP groups. Statistical significance: ##P < 0.01 versus the normal group by Dunnett test; *P < 0.05 versus the vehicle group by the t test.
TGF- Intracellular Signaling
To explore the underlying mechanism of Ac-SDKP activity againstrenal fibrosis, we used Western blotting to examine the effectsof Ac-SDKP treatment on the TGF- signaling-related moleculesSmad2 and Smad7. Compared with the normal rat kidneys, vehicle-treatednephritic kidneys exhibited increased phosphorylation of Smad2.Ac-SDKP treatment resulted in a 44% reduction of Smad2 phosphorylationrelative to the vehicle-treated kidneys (Figure 7, A and C).Smad7 protein was moderately expressed in normal kidneys. Itis interesting that Smad7 protein expression showed a 50% reductionin nephritic kidneys relative to normal kidneys. Ac-SDKP treatmentresulted in significant upregulation of Smad7 expression toa level 3.6-fold higher than that observed in vehicle-treatedrats (Figure 7, B and D).
Figure 7. Effects of Ac-SDKP on the phosphorylation of Smad2 and the expression of Smad7 in kidneys from anti-GBM nephritic rats. Representative Western blotting shows the expression of phosphorylated Smad2 and total Smad2 at 58 kD (A) and the expression of Smad7 at 45 kD and -actin as a control (B). (C and D) Densitometric quantification of the corresponding bands was performed using an image analyzer. The data are presented after normalization to total Smad2 expression or -actin expression. Each column shows data from normal rats (
), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of three to four samples. Statistical significance is based on the Mann-Whitney U test: #P < 0.05 versus the normal group; *P < 0.05 versus the vehicle group.
Monocyte/Macrophage Infiltration
ED-1–positive cells were observed only rarely in the glomeruliand the interstitium of normal rats (Figure 8, A and E). Inrats with anti-GBM nephritis on day 14, ED-1–positivecells had markedly accumulated into the glomeruli and the tubulointerstitium(Figure 8, B and F). On day 42, the number of ED-1–positivecells had further increased in the tubulointerstitium (Figure 8G);however, the number of ED-1–positive cells tendedto be lower in the glomeruli (Figure 8C). In the Ac-SDKP–treatedrats, the number of ED-1–positive cells on day 42 wassignificantly reduced in both the glomeruli (Figure 8, D andI) and tubulointerstitium (Figure 8, H and J) compared withthe vehicle-treated rats.
Figure 8. Effects of Ac-SDKP on ED-1–positive monocyte/macrophage infiltration into the glomerulus and tubulointerstitium. (A through D) Representative photomicrographs of glomerular lesions in renal sections from normal rats (A), day 14 nephritic rats (B), day 42 nephritic rats that were treated with vehicle (C), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (D). (E through H) Representative photomicrographs of tubular lesions in renal sections from normal rats (E), day 14 nephritic rats (F), day 42 nephritic rats that were treated with vehicle (G), and day 42 nephritic rats that were treated with Ac-SDKP (H). Quantification of monocyte/macrophage infiltration into the glomerulus (I) and tubulointerstitium (J) was evaluated by counting the number of ED-1–positive cells. Each column shows data from normal rats (light gray column), day 14 nephritic rats that were treated with vehicle (dark gray column), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of four rats for the normal and day 14 vehicle groups or seven rats for the day 42 vehicle and Ac-SDKP groups. Statistical significance: #P < 0.05, ##P < 0.01 versus the normal group by Dunnett test; *P < 0.05 versus day 42 in the vehicle group by the t test. Bar = 30 µm.
Expression of Inflammation-Related Genes
To elucidate the mechanism by which Ac-SDKP inhibits macrophageinfiltration, we evaluated the effects of Ac-SDKP on inflammation-relatedgenes, including monocyte chemoattractant protein-1 (MCP-1),TNF-, IL-1, and intercellular adhesion molecule-1 (ICAM-1).The levels of MCP-1, TNF-, IL-1 and ICAM-1 mRNA were markedlyupregulated on day 42 in nephritic kidneys relative to normalkidneys. The alterations in the expression of these genes weresignificantly suppressed by Ac-SDKP treatment (Figure 9).
Figure 9. Inhibitory effect of Ac-SDKP on the expression of genes that are involved in the inflammatory response in anti-GBM nephritic rats. The mRNA levels of monocyte chemoattractant protein-1 (MCP-1; A), TNF- (B), IL-1 (C), and intercellular adhesion molecule-1 (ICAM-1; D) were determined by real-time RT-PCR analyses. Renal cortex tissue was isolated from anti-GBM nephritic rats on day 42. All data are presented after normalization to the level of GAPDH in the same sample. Each column shows data from normal rats (
), day 42 nephritic rats that were treated with vehicle (), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (). Each column represents the mean ± SEM of four rats for the normal group or seven rats for the day 42 vehicle and Ac-SDKP groups. Statistical significance: ##P < 0.01 versus the normal group by Dunnett test; *P < 0.05 versus the vehicle group by the t test.
Number of Peripheral Leukocytes and the Plasma Concentration of Ac-SDKP
We examined the effects of Ac-SDKP treatment on the number ofperipheral leukocytes in nephritic rats. There were no differencesin the number of peripheral leukocytes among the three groups(normal group 0.98 ± 0.03 x 107 cells/ml; vehicle group1.08 ± 0.07 x 107 cells/ml; Ac-SDKP group 1.14 ±0.09 x 107 cells/ml).
The plasma concentrations of Ac-SDKP were determined using acommercial EIA kit. The endogenous Ac-SDKP concentration was1.98 ± 0.16 nM in the vehicle-treated rats. After thesubcutaneous administration of 1 mg/kg per d Ac-SDKP for 4 wk,the Ac-SDKP concentration was increased to 36.06 ± 8.35nM.
In this study, we used a rat chronic progressive glomerulonephritismodel to investigate the possible efficacy of Ac-SDKP as a novelmedicine for the treatment of established kidney disease. Althoughtreatment was not started until 2 wk after nephritis induction,Ac-SDKP significantly suppressed proteinuria and inhibited theprogression of renal dysfunction. Histologic analysis showedthat the development of the glomerulosclerosis and tubulointerstitialfibrosis was significantly inhibited by Ac-SDKP treatment.
TGF- is known to play a crucial role in the progression of renalfibrosis in clinical and experimental renal disease (18). Ithas been reported that the blockade of TGF- action by overexpressingsoluble TGF- type II receptor ameliorated renal dysfunctionand fibrosis in an anti-GBM nephritis model in rats (19). Recentstudies have suggested that TGF- transmits biologic signalsfrom the plasma membrane to the nucleus using Smads as intracellulareffector molecules (24,25). After TGF- binds to its receptors,the activated receptors induce phosphorylation of the receptor-regulatedSmad2/3 (R-Smads) proteins, which then associate with a commonpartner, Smad4. The heterodimers translocate to the nucleus,where they modulate the expression of TGF- target genes (26).The inhibitory protein, Smad7, is upregulated by some stimuliand acts by occupying the ligand-activated TGF- type I receptor,interfering with the phosphorylation of R-Smad (27,28). It hasbeen demonstrated that the overexpression of Smad7 by gene transferameliorated renal fibrosis in rat in vivo models (29,30). Inour anti-GBM nephritis model, TGF-1 mRNA and protein expressionwas upregulated markedly in the kidney, Smad7 was downregulated,and Smad2 was inversely activated. Recently, it was reportedthat a mouse model of unilateral ureteral obstruction showedrenal fibrosis with a concomitant decrease of Smad7 proteinand activation of Smad2 (31). It is likely that the resultsobserved in our model are partially caused by activation ofthe TGF- signaling pathway, which can aggravate renal fibrosis,as in the unilateral ureteral obstruction model. To elucidatethe mechanism of Ac-SDKP inhibition of renal dysfunction andfibrosis in this nephritis model, we examined the effects ofAc-SDKP on the TGF- signaling pathway. Pokharel et al. (32)reported that Ac-SDKP inhibited the TGF-–stimulated proliferationof cardiac fibroblasts by inhibiting Smad2 phosphorylation.We also have reported that Ac-SDKP inhibited TGF- signalingby suppressing Smad2 activation via the nuclear export of Smad7in human mesangial cells (17). Furthermore, Ac-SDKP amelioratedglomerulosclerosis by inhibiting Smad3 activation in diabeticmice (14). In the model used in this study, Ac-SDKP not onlyreduced TGF-1 protein expression but also inhibited Smad2 activationand restored Smad7 protein expression. From these results, wespeculate that Ac-SDKP inhibits renal fibrosis via blockadeof the TGF-/Smad signal transduction pathway.
In our model, monocytes/macrophages accumulated remarkably intothe glomeruli and tubulointerstitium. Ac-SDKP treatment significantlyattenuated monocyte/macrophage accumulation and suppressed theexpression of mediators of the inflammatory response in therenal tissue. These results led us to speculate that Ac-SDKPinhibits monocyte/macrophage accumulation via the suppressionof proteins that are involved in the inflammatory response.It has been reported that monocytes/macrophages that infiltrateinto the glomeruli and tubulointerstitium may play a pivotalrole in the progression of renal fibrosis. Indeed, blockadeof monocyte/macrophage accumulation by anti—MCP-1 antibodyhas been shown to improve the renal fibrosis in anti-GBM ratnephritis (33,34). Therefore, the antifibrotic action of Ac-SDKPmay also be attributed partially to the suppression of monocyte/macrophageaccumulation. Furthermore, to assess the possibility that Ac-SDKP–inducedattenuation of monocyte/macrophage accumulation into the renaltissue was due to the inhibition of hematopoietic stem cellproliferation, another function of Ac-SDKP, we evaluated theeffects of Ac-SDKP on the number of peripheral leukocytes. IfAc-SDKP exerted an inhibitory effect on the hematopoietic stemcell proliferation, then we would predict a reduction in thenumber of each type of leukocyte, including monocytes, neutrophils,and lymphocytes (35,36). However, in our study, the number oftotal leukocytes was not decreased by Ac-SDKP treatment. Therefore,we speculate that Ac-SDKP did not affect stem cell proliferationand that the numbers of circulating monocytes in Ac-SDKP–andvehicle-treated rats were similar. It follows that Ac-SDKP mayhave inhibited monocyte infiltration without changing the numberof monocytes in the peripheral blood.
We and others have reported that a 10- to 100-nM concentrationof Ac-SDKP is effective against TGF- signal transduction andexpression of extracellular matrix in vitro (17,32). In thisstudy, the plasma Ac-SDKP concentration in the Ac-SDKP–treatedgroup was 36 nM, 18 times greater than the endogenous levelsand within the range of the effective concentrations in thein vitro experiments.
We have demonstrated for the first time that Ac-SDKP significantlyameliorated the progression of renal dysfunction and fibrosis,after the establishment of glomerulonephritis. Thus, Ac-SDKPtreatment may represent a novel therapeutic strategy for haltingthe progression of renal disease.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Blantz RC: Reflections on the past, transitions to the future: The American Society of nephrology.
J Am Soc Nephrol 14
: 1695
–1703, 2003[Free Full Text]
Burkart JM, Pereira BJ, Parker TF 3rd: Strategies for influencing outcomes in pre-ESRD and ESRD patients.
J Am Soc Nephrol 9
: S2
–S3, 1998[Medline]
Port FK, Fenton SSA, Mazzuchi N: ESRD throughout the world: Morbidity, mortality and quality of life.
Kidney Int 57
: S1
–S2, 2000
Remuzzi G, Bertani T: Pathophysiology of progressive nephropathies.
N Engl J Med 339
: 1448
–1456, 1998[Free Full Text]
Eddy AA: Molecular insights into renal interstitial fibrosis.
J Am Soc Nephrol 7
: 2495
–2508, 1996[Abstract]
Brenner BM: Remission of renal disease: Recounting the challenge, acquiring the goal.
J Clin Invest 110
: 1753
–1758, 2002[CrossRef][Medline]
Hebert LA, Wilmer WA, Falkenhain ME: Ladson-Wofford SE, Nahman NS, Rovin BH: Renoprotection: One or many therapies?
Kidney Int 59
: 1211
–1226, 2001[CrossRef][Medline]
Peters H, Border WA, Noble NA: Targeting TGF-beta overexpression in renal disease: Maximizing the antifibrotic action of angiotensin II blockade.
Kidney Int 54
: 1570
–1580, 1998[CrossRef][Medline]
Wdzieczak-Bakala J, Fache MP, Lenfant M, Frindel E, Sainteny F: Ac-SDKP, an inhibitor of CFU-S proliferation, is synthesized in mice under steady-state conditions and secreted by bone marrow in long-term culture.
Leukemia 4
: 235
–237, 1990[Medline]
Azizi M, Rousseau A, Ezan E, Guyene T-T, Michelet S, Grognet J-M, Lenfant M, Corvol P, Menard J: Acute angiotensin-converting enzyme inhibition increases the plasma level of the natural stem cell regulator N-acetyl-seryl-aspartyl-lysyl-proline.
J Clin Invest 97
: 839
–844, 1996[Medline]
Peng H, Carretero O, Raij L, Yang F, Kapke A, Rhaleb NE: Anti-fibrotic effects of N-acetyl-seryl-aspartyl-lysyl-proline on the heart and kidney in aldosterone-salt hypertensive rats.
Hypertension 34
: 794
–800, 2001
Rhaleb NE, Peng H, Yang XP, Liu YH, Mehta D, Ezan E, Carretero OA: Long-term effect of N-acetyl-seryl-aspartyl-lysyl-proline on left ventricular collagen deposition in rats with 2-kidney, 1-clip hypertension.
Circulation 103
: 3136
–3141, 2001[Abstract/Free Full Text]
Peng F, Yang XP, Liu YH, Xu J, Cingolani O, Rhaleb NE, Carretero OA: Ac-SDKP reverses inflammation and fibrosis in rats with heart failure after myocardial infarction.
Hypertension 43
: 229
–236, 2004[Abstract/Free Full Text]
Shibuya K, Kanasaki K, Isono M, Sato H, Omata M, Sugimoto T, Araki S, Isshiki K, Kashiwagi A, Haneda M, Koya D: N-acetyl-seryl-aspartyl-lysyl-proline prevents renal insufficiency and mesangial matrix expansion in diabetic db/db mice.
Diabetes 54
: 838
–845, 2005[Abstract/Free Full Text]
Iwamoto N, Xano HJ, Yoshioka T, Shiraga H, Hitta K, Muraki T, Ito K: Acetyl-seryl-aspartyl-lysyl-proline is a novel natural cell cycle regulator of renal cells.
Life Sci 66
: PL221
–PL226, 2000[Medline]
Rhaleb N, Peng H, Harding P, Tayeh M, LaPointe M, Carretero O: Effect of N-acetyl-seryl-aspartyl-lysyl-proline on DNA and collagen synthesis in rat cardiac fibroblasts.
Hypertension 37
: 827
–832, 2001[Abstract/Free Full Text]
Kanasaki K, Koya D, Sugimoto T, Isono M, Kashiwagi A, Haneda M: N-Acetyl-seryl-aspartyl-lysyl-proline inhibits TGF-beta-mediated plasminogen activator inhibitor-1 expression via inhibition of Smad pathway in human mesangial cells.
J Am Soc Nephrol 14
: 863
–872, 2003[Abstract/Free Full Text]
Border WA, Noble NA: Mechanisms of disease: Transforming growth factor beta in tissue fibrosis.
N Engl J Med 331
: 1286
–1292, 1994[Free Full Text]
Zhou A, Ueno H, Shimomura M, Tanaka R, Shirakawa T, Nakamura H, Matsuo M, Iijima K: Blockade of TGF-beta action ameliorates renal dysfunction and histologic progression in anti-GBM nephritis.
Kidney Int 64
: 92
–101, 2003[Medline]
Taniguchi H, Nagamatsu T, Kojima R, Ito M, Suzuki Y: Marked antinephritic action and less adverse effects of methylprednisolone suleptanate by intermittent administration in rats.
Jpn J Pharmacol 64
: 79
–88, 1994[CrossRef][Medline]
Kingsbury FB, Clark CP, Williams G, Post AL: The rapid determination of albumin in urine.
J Lab Clin Med 11
: 981
–989, 1926
De Boer E, Navis G, Tiebosch AT, De Jong PE, De Zeeuw D: Systemic factors are involved in the pathogenesis of proteinuria-induced glomerulosclerosis in Adriamycin nephrotic rats.
J Am Soc Nephrol 10
: 2359
–2366, 1999[Abstract/Free Full Text]
Isshiki K, Haneda M, Koya D, Maeda S, Sugimoto T, Kikkawa R: Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats.
Diabetes 49
: 1022
–1032, 2000[Abstract]
Miyazono K: TGF-beta signal by Smad proteins.
Cytokine Growth Factor Rev 11
: 15
–22, 2000[CrossRef][Medline]
Heldin C, Miyazono K, ten Dijke P: TGF-beta signaling from cell membrane to nucleus through SMAD proteins.
Nature 390
: 465
–471, 1997[CrossRef][Medline]
Wrana J, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang X, Massague J: TGF-beta signals through a heteromeric protein kinase receptor complex.
Cell 71
: 1003
–1014, 1992[CrossRef][Medline]
Miyazono K: Positive and negative regulation of TGF-beta signaling.
J Cell Sci 113
: 1101
–1109, 2000[Abstract]
Kavsak 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-beta receptor for degradation.
Mol Cell 6
: 1365
–1375, 2000[CrossRef][Medline]
Terada Y, Hanada S, Nakao A, Kuwahara M, Sakai S, Marumo F: Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney.
Kidney Int 61[Suppl 1]
: S94
–S98, 2002
Lan 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 14
: 1535
–1548, 2003[Abstract/Free Full Text]
Fukasawa H, Yamamoto T, Togawa A, Ohashi N, Fujigaki Y, Oda T, Uchida C, Kitagawa K, Hattori T, Suzuki S, Kitagawa M, Hishida A: Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice.
Proc Natl Acad Sci U S A 101
: 8687
–8692, 2004[Abstract/Free Full Text]
Pokharel S, Rasoul S, Roks AJ, van Leeuwen RE, van Luyn MJ, Deelman LE, Smits JF, Carretero O, van Gilst WH, Pinto YM: N-acetyl-Ser-Asp-Lys-Pro inhibits phosphorylation of Smad2 in cardiac fibroblasts.
Hypertension 40
: 155
–161, 2002[Abstract/Free Full Text]
Wada T, Yokoyama H, Furuichi K, Kobayashi KI, Harada K, Naruto M, Su SB, Akiyama M, Mukaida N, Matsushima K: Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1).
FASEB J 10
: 1418
–1425, 1996[Abstract]
Fujinaka H, Yamamoto T, Takeya M, Feng L, Kawasaki K, Yaoita E, Kondo D, Wilson CB, Uchiyama M, Kihara I: Suppression of anti-glomerular basement membrane nephritis by administration of anti-monocyte chemoattractant protein-1 antibody in WKY rats.
J Am Soc Nephrol 8
: 1174
–1178, 1997[Abstract]
Volkov L, Quere P, Coudert F, Comte L, Antipov Y, Praloran V: The tetrapeptide AcSDKP, a negative regulator of cell cycle entry, inhibits the proliferation of human and chicken lymphocytes.
Cell Immunol 168
: 302
–306, 1996[CrossRef][Medline]
Gaudron S, Grillon C, Thierry J, Riches A, Wierenga PK, Wdzieczak-Bakala J: In vitro effect of acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) analogs resistant to angiotensin I-converting enzyme on hematopoietic stem cell and progenitor cell proliferation.
Stem Cells 17
: 100
–106, 1999[Abstract/Free Full Text]
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Top
Abstract
Introduction
action in mesangial cells. Because TGF-
2-type I collagen through the suppression of Smad signaling in mesangial cells (17). Because profibrotic cytokine TGF-
) or 1 mg/kg per d Ac-SDKP (•). Statistical significance is based on the t test: *P < 0.05 versus the same time point of the vehicle group; #P < 0.01 for the vehicle group versus day 0; ##P < 0.01 for the Ac-SDKP–treated group versus day 0.
), day 42 nephritic rats that were treated with vehicle (
), and day 42 nephritic rats that were treated with 1 mg/kg per d Ac-SDKP (
). Each column represents the mean ± SEM of four rats on day 14 in the vehicle group or seven rats on day 42 in the vehicle and Ac-SDKP groups. Statistical significance is based on the Mann-Whitney U test: 
