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J Am Soc Nephrol 15: 2404-2413, 2004
© 2004 American Society of Nephrology
doi: 10.1097/01.ASN.0000136132.20189.95
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J Am Soc Nephrol 15:2404-2413, 2004
© 2004 American Society of Nephrology

BASIC SCIENCE

Bradykinin Decreases Plasminogen Activator Inhibitor-1 Expression and Facilitates Matrix Degradation in the Renal Tubulointerstitium under Angiotensin-Converting Enzyme Blockade

Hirokazu Okada*, Yusuke Watanabe*, Tomohiro Kikuta*, Tatsuya Kobayashi*, Yoshihiko Kanno*, Takeshi Sugaya{dagger} and Hiromichi Suzuki*

*Department of Nephrology, Saitama Medical College, Saitama, Japan; and {dagger}Center of Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan

Correspondence to Dr. Hiromichi Suzuki, Department of Nephrology, Saitama Medical College, 38 Morohongo, Moroyama-machi, Irumagun, Saitama 350-0495, Japan. Phone: 81-49-276-1611; Fax: 81-49-295-7338;


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
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ABSTRACT. A number of experimental and clinical investigations support the notion that angiotensin-converting enzyme inhibitor (ACEi) and angiotensin II type 1 receptor blocker (ARB) compounds attenuate renal fibrosis. Fibrosis can be attenuated by either suppressing matrix formation or facilitating matrix degradation. In this study, drugs of ACEi and ARB classes were tested for their ability to facilitate matrix degradation in the kidney. A murine model system in which cyclosporin A (CsA) treatment for a specified period caused interstitial matrix deposition in the kidney was used. CsA was then discontinued, and experimental procedures were initiated to investigate matrix degradation. Benazepril, an ACEi, facilitated matrix degradation via the bradykinin (BK) B2 receptor on tubular epithelial cells in the kidney, whereas CGP-48933, an ARB, did not. In this murine model of CsA nephropathy under ACE blockade, plasminogen activator inhibitor-1 (PAI-1) expression was decreased in tubular epithelial cells, possibly leading to conversion of plasminogen to plasmin by plasminogen activator and subsequent activation of matrix metalloproteinases. These findings were confirmed in this study by measurements of plasmin activity, collagenolytic activity, and matrix metalloproteinase activities in the kidneys. In tubular epithelial cells stimulated in vitro, BK suppressed PAI-1 gene expression. All of these results suggest that ACEi can decrease PAI-1 expression via BK, thereby facilitating matrix degradation via activation of degradative enzymes to reduce interstitial matrix deposition. E-mail: iromichi@saitama-med.ac.jp


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A number of clinical trials have confirmed that angiotensin-converting enzyme inhibitors (ACEi) can confer renoprotection to a variety of renal diseases such as type I diabetic nephropathy (1); nondiabetic, proteinuric nephropathy (2,3); and especially IgA nephropathy (4). Angiotensin II (Ang II) is now considered to be a very active molecule that plays a role not only as a vasoactive hormone but also as a growth and a proinflammatory/profibrotic factor (5). The DIABIOPSIES group reported that expansion of the interstitium was limited by ACEi in type 2 diabetic nephropathy (6). We recently found that ACEi significantly slowed the decline in renal function of patients with IgA nephropathy even with renal fibrosis (manuscript submitted).

Effective prevention of organ fibrogenesis by ACEi has been attributed mainly to blockade of Ang II actions through a decrease in circulating and tissue levels of Ang II under ACE blockade (7). However, in humans who underwent ACE blockade by ACEi, tissue levels of Ang II were likely to equal or exceed the basal levels of Ang II because of "the escape phenomenon" through non-ACE pathways of Ang II generation, such as the chymase pathway (8,9). In addition, treatment with an Ang II type 1 receptor blocker (ARB) as well as Ang II type 1A receptor gene knockout significantly attenuated the progression of antiglomerular basement membrane (anti-GBM) nephritis in rodents (10,11). However, treatment with an ACEi was less effective to slow the progression of anti-GBM nephritis in rodents (personal observation), which suggests that ACEi affects kidney disease in another way. ACE degrades bradykinin (BK) so that treatment with ACEi increases BK concentration (12). In addition, in proximal tubular epithelial cells, where ACE and BK B2 receptor molecules are in close proximity, possibly forming a heterodimer, ACEi is believed to augment indirectly the effect of BK on BK B2 receptor (13). Recently, Bascands et al. (14) reported that BK B2 receptor activation reduces renal fibrosis in unilateral ureteral obstruction (UUO) model by increasing extracellular matrix (ECM) degradation through activation of plasminogen activator (PA). In the present study, we demonstrated that interstitial ECM deposition could be induced by cyclosporin A (CsA) in mice, which could be significantly removed by ACEi by facilitating ECM degradation through a decrease in plasminogen activator inhibitor-1 (PAI-1) expression by enhancement of BK activity in the kidney.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CsA Nephropathy Model in Mice
SJL mice that weighed 30 g each were fed a low-salt diet (0.02%), and seven groups of six mice were used in the study. The experimental protocol is shown in Figure 1a. The normal control (NCt) group of animals received subcutaneous injections of 0.1 ml of olive oil (vehicle only) every 24 h for 28 d. A daily dose of CsA (30 mg/kg; Novartis Pharmaceutical Inc., Tokyo, Japan) in olive oil was injected subcutaneously to the other six groups of mice for 28 d and then discontinued. One group of CsA-treated mice was killed on day 28 as the D28 positive control (D28PCt), whereas the other groups then started receiving experimental procedures aimed at ECM degradation. From day 29 and for the next 28 d, the NCt group and another group of CsA-treated mice received intraperitoneal injections of 1 ml of saline (D56NCt and D56PCt, respectively). Similarly, from day 29 through day 56, four other groups of CsA-treated mice received intraperitoneal injections of the following: (1) an ACEi, benazepril hydrochloride (10 mg/kg per d; Novartis Pharmaceutical; D56ACEi group); (2) ACEi and a B2 receptor blocker FR173657 (60 mg/kg per d; Fujisawa Pharmaceutical Co., Osaka, Japan; D56ACEi/BB group) (15); (3) an ARB CGP-48933 (30 mg/kg per d; Novartis Pharma AG, Basel, Switzerland; D56AT1B group); and (4) CGP-48933 and an ARB PD-123319 (20 mg/kg per d; Sigma, St. Louis, MO; D56AT1B/AT2B group). Mice of these groups were killed on day 56. FR173657 is an orally active, nonpeptide BK B2 receptor antagonist that selectively inhibits BK binding to B2 receptors in rodents and humans (16). The dose chosen for each drug was as determined in previous studies (11,15,17). In cases of benazepril and FR173657, the doses previously determined for rats were used in mice in this study after adjustment of each mouse weight because the general pharmacology of both drugs was mostly identical in rats and mice (18–20). When the rats were killed, one and a half kidneys were obtained from each mouse for protein/RNA extractions as described below. The remaining kidney tissue was fixed in 4% PBS paraformaldehyde overnight and processed into paraffin blocks for histopathologic analysis.



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  		Figure 1. Angiotensin-converting enzyme inhibitor (ACEi) decreased interstitial extracellular matrix (ECM) deposition. (a) In vivo experimental protocol. (b) Normal renal histology in the D56NCt group. (c) Increased interstitial ECM deposition surrounding well-preserved tubules in the D28PCt group. (d) Sustained interstitial ECM deposition in the D56PCt group. (e) ECM deposition was reduced in the D56ACEi group. (f) BB attenuated the ACEi effect in the D56ACEi/BB group. Interstitial ECM deposition substantially remained in the D56AT1B group (g) and the D56AT1B/AT2B group (h). (i) The percentage of interstitial area occupied by ECM was quantified as described in Materials and Methods. ACEi significantly decreased interstitial ECM deposition in the D56ACEi group. Magnification, x200 in b through h, Masson’s trichrome stain.

 
Morphologic Examination and Immunohistochemistry
Four-micrometer-thick sections were cut from the paraffin blocks and stained with hematoxylin-eosin and Masson’s trichrome (MT). The interstitial fibrosis in the MT-stained section was quantitatively assessed at x200 magnification using a Mac SCOPE (Ver. 2.5, Mitani Corp., Fukui, Japan). At first, all glomeruli and vessels were subtracted from a given field, yielding a target area of tubulointerstitium. The collagenous fibrotic area in blue was then quantitatively determined; each was expressed as a mean percentage area per field of 10 randomly chosen cortical fields. Renal biopsy specimens from renal transplant patients who were being treated with CsA were used after written consents were obtained for detection of BK B2 receptor. The indirect immunoperoxidase method was described previously (21). Rabbit polyclonal anti–PAI-1 (1:200; American Diagnostica, Greenwich, CT) and mouse monoclonal anti-BK B2 receptor (1:100; Transduction Laboratories, Lexington, KY) were applied to sections as primary antibodies. At the final color development, an AEC Standard Kit (Dako, Carpinteria, CA) was used for BK B2 receptor detection, and a Catalyzed Signal Amplification System Peroxidase (K1500; Dako) was used for PAI-1 detection, according to the manufacturer’s instructions. The negative control was performed by omitting the primary antibody.

Cell Culture
Cultured murine proximal tubular epithelial cells (mProx24) were maintained in DMEM that contained 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (22). Expression of BK B2 receptor in mProx24 was confirmed by reverse transcription–PCR (RT-PCR) and immunoblotting as described below. The cells were seeded in six-well plates (1 x 105 cells/well) and incubated overnight in growth medium. Medium was then changed to K-1 medium (50:50 Ham’s F-12/DMEM with 5 µg/ml transferrin, 5 µg/ml insulin, and 5 x 10–8 M hydrocortisone) (23). After 48 h, BK (Sigma), TGF-{beta}1 (R&D Systems, Minneapolis, MN), or TNF-{alpha} (R&D Systems) was added to the medium at a final concentration of 0.1 µM, 3.0 ng/ml, and 10.0 ng/ml, respectively. In additional experiments, either TGF-{beta}1 (3.0 ng/ml) or TNF-{alpha} (10.0 ng/ml) was co-administered with BK (0.1 µM). For blocking BK B2 receptor, FR173657 (1.0 µM) was added to the medium 30 min before the treatment. After another 12 h of incubation, cells in the six-well plates were harvested for mRNA extraction as described below.

Real-Time RT-PCR
Total RNA was extracted from the homogenates of kidney tissues and cultured cells with TRIzol (Life Technologies BRL, Grand Island, NY) according to the manufacturer’s instructions. All RNA samples were treated with the RNase-free DNase I (Qiagen, Basel, Switzerland) before the RT-PCR. Real-time quantitative one-step RT-PCR assay was performed to quantify mRNA of {alpha}1(I) procollagen ({alpha}1COLI), TGF-{beta}1, tissue-type plasminogen activator (t-PA), PAI-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), BK B2 receptor, and glyceraldehyde-3-phosphate dehydrogenase using QuantiTect SYBR Green RT-PCR (Qiagen) and an ABI PRISM 7700 sequence detection system (Applied Biosystems, Tokyo, Japan). The primers used for real-time RT-PCR were as follows: {alpha}1COLI primer, forward 5'-TGTAAACTCCCTCCACCCCA-3', reverse 5'-TCGTCTGTTTCCAGGGTTGG-3'; TGF-{beta}1 primer, forward 5'-CAGTGGCTGAACCAAGGAGAC-3', reverse 5'-ATCCCGTTGATTTCCACGTG-3'; t-PA primer, forward 5'-GTGGAATATTGCCGGTGCA-3', reverse 5'-CCATTGAAGCATCTTGGTTCG-3'; PAI-1 primer, forward 5'-TCGTGGAACTGCCCTACCAG-3', reverse 5'-ATGTTGGTGAGGGCGGAGAG-3'; TIMP-1 primer, forward 5'-TGTGGGAAATGCCGCAGATA-3', reverse 5'-TTCACTGCGGTTCTGGGACT-3'; BK B2R primer, forward 5'-AGAGGAAGGCCACCGTGCTA-3', reverse 5'-AGCGTGTCCAGGAAGGTGCT-3'; and glyceraldehyde-3-phosphate dehydrogenase primer, forward 5'-TGCAGTGGCAAAGTGGAGATT-3', reverse 5'-TTGAATTTGCCGTGA GTGGA-3'. All of these oligonucleotides were designed by using Primer Express software (Perkin Elmer, Foster City, CA). Preliminary RT-PCR experiments with these primer sets yielded single products of appropriate size.

Indirect ELISA
Kidney tissues were homogenized/sonicated extensively in saline. The samples were then centrifuged for 5 min (14,000 x g), and protein concentration in the supernatant was measured using the Bradford assay (Bio-Rad, Hercules, CA). The concentration of collagen type 1 (COLI) in the supernatant was measured by indirect ELISA, as described previously (24).

Immunoblotting
Immunoblotting was performed as described previously (25) using rabbit polyclonal anti–PAI-1 (1:1000) and mouse monoclonal anti-B2 receptor (1:200). The immunoreactive proteins were detected by enhanced chemiluminescence and captured on x-ray film. Biotinylated molecular weight standards were run with each blot. The intensity of each band was measured using a Mac SCOPE.

Plasmin Activity and Collagenolytic Activity
Total renal tissue plasmin activity was measured using a plasmin-specific chromogenic substrate, Chromozym PL (Roche Molecular Biochemicals, Indianapolis, IN) according to the method described by Huang et al. (26). This substance is specifically cleaved by plasmin into a residual peptide and 4-nitroaniline, which can be detected spectrophotometrically. Renal tissue was suspended at 50 mg/ml 50 mM Tris (pH 8.2) with 0.1% Triton X-100 and homogenized. After centrifugation at 200 x g for 10 min at 4°C, 80 µl of supernatant and 20 µl of 3 mM Chromozym were added per well in a 96-well plate. Absorbance was measured at 405 nm. The standard linear curve was generated with serial dilutions of porcine plasmin (Roche). Results were expressed as 10–4 U/ml. The rest of the supernatant prepared for the plasmin activity measurement (100 µl) was used to measure collagenolytic activity as described previously (27).

Gelatin Zymography
For detecting matrix metalloproteinase (MMP) activities, gelatin zymography was performed with a Gelatin Zymography Kit (YU-68001; Yagai, Yamagata, Japan). Briefly, kidney tissues were homogenized in the extraction buffer (0.05M Tris-HCl [pH 6.8], 2% SDS, 10% glycerol). The samples were then centrifuged for 5 min (14,000 x g), and the concentration of protein was measured in the supernatant using the Bradford assay. Fifteen micrograms of each sample was loaded into wells of the gel. Human ProMMP-2, MMP-2, ProMMP-9, and MMP-9 were also loaded into the outer wells as standards. After electrophoresis, the gel was incubated overnight at 37°C in a solution that contained 50 mM Tris (pH 7.8) and 10 mM CaCl2 and subsequently stained with 0.002% Coomassie blue. The size of each lytic band was measured using a Mac SCOPE.

Statistical Analyses
All values were expressed as mean ± SD. ANOVA with subsequent Bonferroni/Dunnett test was used to determine the significance of difference in multiple comparisons. Mann-Whitney U test was used to compare means of two groups. Values of P < 0.05 were considered to be statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ACEi Decreased Interstitial ECM Deposition in CsA Nephropathy
The summary of in vivo data are shown in Table 1. For determining whether renin angiotensin system (RAS) blockade removes accumulated ECM in the renal interstitium after CsA treatment was stopped at day 28, mice were treated for the next 28 d and evaluated at day 56. By day 28, mice in the D28PCt group showed significantly widened interstitium/increased COLI protein deposition in the kidney (Figure 1b, c and i, and 2aGo). Despite discontinuation of CsA administration and a subsequent decrease in {alpha}1COLI mRNA expression (Figure 2b), high levels of collagenous ECM deposition remained in the interstitium of the D56PCt group at day 56 (Figure 1, d and i, and 2a Go). This may have been due to insufficient ECM degradation. Treatment with benazepril significantly decreased the interstitial ECM deposition from day 29 to Day 56 in the D56ACEi group, and FR173657 abolished its antifibrotic effect in the D56ACEi/BB group (Figure 1, e, f, and i, and 2aGo). Although BK activity could not be measured directly in the kidneys, these findings suggested that enhanced BK activity under ACE blockade facilitated the ECM degradation pathway in the D56ACEi group. In contrast, treatment with CGP-48933 regardless of co-treatment with PD-123319 did not significantly affect interstitial ECM deposition from day 29 to day 56 in the D56AT1B and D56AT1B/AT2B groups, compared with the D28PCt (Figure 1, g through i, and 2a HREF="#FIG2">Go). The expression of TGF-{beta}1 was significantly increased in the kidney by day 28 in the D28PCt group but then spontaneously returned to the basal level by day 56 in the D56PCt group and all the other groups that were treated with relevant drugs (Table 1).


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  		Table 1. Relative changes in the matrix turnover parameters in the resting cyclosporin A nephropathy model compared with D28PCta
 


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  		Figure 2. Quantitative changes in type I collagen. (a) Collagen type 1 (COLI) protein deposition. COLI protein deposition in the kidney was significantly decreased in the D56ACEi group, which is consistent with the result in Figure 1i. (b) {alpha}1(I) procollagen ({alpha}1COLI) gene expression. Renal {alpha}1COLI gene expression was equally decreased in each group at day 56 regardless of any treatment. Such an ACEi effect was totally abolished by co-treatment with BB in the D56ACEi/BB group. The quantitative data were obtained from three mice in each group.

 
ACEi Facilitated ECM Degradation in CsA Nephropathy
The ECM degradation pathway in the kidney consists of a variety of degradative enzymes and related proteins, such as MMP, collagenases and gelatinases, TIMP, plasminogen/plasmin, t-PA, and PAI-1. In the present study, we investigated the gene/protein expression of t-PA, PAI-1, and TIMP-1 and plasmin activity, collagenolytic activity, and MMP activities in kidneys with CsA nephropathy. The expression of the t-PA gene was significantly increased by day 28 in the D28PCt group and then returned to the basal level by day 56 in the D56PCt group and in all of the other groups that were treated with relevant drugs (Table 1). In contrast, treatment with benazepril significantly decreased expression of PAI-1 mRNA/protein from day 29 to day 56 in the D56ACEi group, and FR173657 reversed this effect in the D56ACEi/BB group (Figure 3, a and b). Treatment with benazepril hydrochloride for 4 wk was also shown to decrease PAI-1 mRNA expression in the kidney of the normal mice (NCt-ACEi; Figure 3a). It is unlikely that FR173657 blocked profibrotic actions of TGF-{beta}1 in the D56ACEi/BB group, because FR173657 did not affect TGF-{beta}1 induction of PAI-1 mRNA in mProx24 in vitro as described below (Figure 4f). It is possible that enhanced BK activity under ACE blockade resulted in the decreased expression of PAI-1 mRNA/protein in the D56ACEi group. The level of TIMP-1 gene expression was not significantly altered in any of the experimental groups (Table 1). Therefore, overall plasmin activity was significantly increased in the D56ACEi group at day 56, and such an increase was erased by FR173657 in the D56ACEi/BB group (Figure 3c). All of these findings are consistent with the significant enhancement of activities of MMP-9 and overall collagenolysis induced by treatment with benazepril from day 29 to day 56 in the D56ACEi group (Figure 3, d and e). MMP-2 activity was not significantly activated in any of the experimental groups (data not shown).



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  		Figure 3. ACEi facilitated the ECM degradation pathway. (a) Plasminogen activator inhibitor-1 (PAI-1) mRNA expression. (b) PAI-1 protein expression. ACEi did not affect expression of the tissue-type plasminogen activator (t-PA) gene but significantly decreased PAI-1 gene/protein expression in the kidney of the D56ACEi group. (c) Plasmin activity. The plasmin activity was significantly increased in the kidney of the D56ACEi group. (d) Matrix metalloproteinase (MMP) activities. The MMP-9 activity was significantly increased in the kidney of the D56ACEi group. (e) Collagenolytic activity. The whole collagenolytic activity was also significantly increased in the kidney of D56ACEi groups, which is consistent with the data in a through d. Such ACEi effects were totally abolished by co-treatment with BB in the D56ACEi/BB group in a through e. These quantitative data were obtained from three independent experiments.

 


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  		Figure 4. Bradykinin (BK) decreased PAI-1 gene expression in murine proximal tubular epithelial cells (mProx24). (a) BK B2 receptor localization in human cyclosporine A (CsA) nephropathy. In the fibrosing kidney, B2 receptor was observed in a cytoplasmic granular pattern in the tubular epithelial cells (arrows). (b) Negative control for a. (c) BK B2 receptor protein expression in murine CsA nephropathy at day 28. (d) PAI-1 protein localization in murine CsA nephropathy at day 28. PAI-1 protein was observed exclusively in the tubular epithelial cells (arrows) and arterioles (arrowheads). (e) Negative control for d. (f) PAI-1 gene expression in mProx24. Co-treatment with BK significantly decreased PAI-1 gene expression in mProx24 treated with TGF-{beta}1. FR173657 significantly blocked such a BK action but did not affect TGF-{beta}1 induction of PAI-1 mRNA expression in mProx24. The quantitative data in f were obtained from three independent experiments. Magnification, x200 in a, b (AEC), d, and e (diaminobenzidine).

 
BK Decreased PAI-1 Expression in Stimulated mProx24
The distribution of BK B2 receptor in the fibrosing kidney was largely unknown. However, we have found these receptors by immunohistochemistry with murine monoclonal anti-B2 receptor antibody in vesicles (endosome-like structures) of the tubular epithelial cells of renal biopsies from patients who were suspected of having CsA nephropathy (Figure 4a and b). Immunoblotting revealed that the BK B2 receptor protein is expressed in murine kidneys with or without CsA nephropathy (Figure 4c). In addition, we identified PAI-1 protein expression in the tubular epithelial cells in the day 28 CsA nephropathic kidney in the D28PCt group (Figure 4d and e). Therefore, BK seems to suppress PAI-1 gene expression in the tubular epithelial cells of the kidney via the B2 receptor. To test this idea, we performed in vitro experiments using mProx24 cells that were positive for B2 receptor mRNA/protein (data not shown). Treatment with TGF-{beta}1 stimulated mProx24 cells to express the PAI-1 gene, and treatment of the cells at the same time with BK significantly reduced the induction of the PAI-1 gene (Figure 4f). This was unlikely to be a nonspecific event because co-treatment with BK did not affect TNF-{alpha} induction of t-PA gene expression in mProx24 cells (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we submit that ACEi facilitated ECM degradation in renal interstitial fibrosis through the reduction of PAI-1 expression in tubular epithelial cells by BK and that ACEi was more effective than ARB in degrading the ECM. PAI-1, a member of the SERPIN family, is the major inhibitor of t-PA in vivo that indirectly blocks the activation of plasminogen/MMP and thus contributes to ECM deposition (28). This effect has been demonstrated in sclerotic glomeruli and fibrosing tubulointerstitium in the kidney (29). The antifibrotic action of BK through facilitation of ECM degradation found in our study is consistent with the study of Bascands et al. (14). They demonstrated that renal fibrogenesis in the UUO model using BK B2 receptor gene knockout mice was worse than in UUO wild-type mice. Renal PA activity was significantly lower in UUO B2 receptor gene knockout mice than in UUO wild-type mice despite similar gene expression levels of t-PA and urokinase-type PA. Because of similar levels of renal PAI-1 gene expression in both groups of UUO mice, the authors were unable to account for how BK increased renal PA activity in wild-type mice. In contrast, we demonstrated that BK did not affect t-PA gene expression but decreased PAI-1 expression and increased activities of plasmin, MMP-9, and overall collagenolysis in the renal tubulointerstitium under ACE blockade, which likely facilitated the ECM degradation pathway. MMP-9 plays an important role in tissue remodeling and inflammatory processes (30,31). MMP-9 is secreted as an inactive enzyme. It is then activated by other proteases, such as collagenases, trypsin, and plasmin, to cleave ECM proteins, such as collagens I, III, and IV and entacin and elastin. The result of an increase in MMP-9 activity obtained in this study does not simply suggest that BK enhanced ECM degradation via active MMP-9, which may have broken basement membranes to yield cellular translocations such as immune cell infiltrations and worsen interstitial changes (30,31). PAI-1 was also demonstrated to be involved in an increase in the number of monocytes/macrophages and fibroblasts in the UUO model (32). However, these were considered unlikely factors because the tubulointerstitial alterations found in the present study were mild and there were no significant differences in the number of interstitial cells in the kidney among all of the groups at day 56.

The molecular mechanism of a decrease in PAI-1 gene expression in tubular epithelial cells by BK was not identified in this study. BK induces arachidonic acid release from tubular epithelial cells via phospholipase A2 phosphorylation by protein kinase C (33). Therefore, it is possible that some prostaglandins derived from arachidonic acid increase intracellular cAMP, which has been demonstrated to decrease PAI-1 expression in tubular epithelial cells (34). BK also increases nitric oxide (NO) synthesis in the kidney, and NO decreases TGF-{beta}1 expression, which may suppress PAI-1 expression (35,36). In progressive CsA nephropathy undergoing continuous CsA treatment, an increase in NO synthesis by any means decreases TGF-{beta}1 and PAI-1 expression, attenuating renal fibrogenesis (36). However, in this study, it seemed less involved because TGF-{beta}1 mRNA level in the kidney of the D56PCt group was not significantly different from the one in the D56ACEi group (Table 1).

Ang II and its hexapeptide metabolite, Ang IV, and aldosterone increase PAI-1 expression in mesangial cells, vascular smooth muscle cells, and tubular epithelial cells in the kidney (28). The positive effect of Ang II on PAI-1 induction in tubular epithelial cells can be mediated through a putative Ang IV receptor recently identified in tubular epithelial cells even under ARB treatment (37). In addition to BK action described in the present study, this may partially explain why ACEi inhibited PAI-1 expression in tubular epithelial cells more effectively than ARB.

ACEi and ARB are almost equally effective in preventing renal fibrogenesis in a variety of renal disease models, such as subtotal nephrectomy, UUO, and progressive CsA nephropathy undergoing continuous CsA treatment (7). The importance of low-salt diet suggested the role of RAS in the progressive CsA nephropathy model, which was supported by antifibrotic action of ACEi, ARB, and aldosterone receptor blockers (38,39). Previously, Bennett et al. (38) showed that both an ARB, losartan, and an ACEi, enalapril, prevented interstitial fibrosis in progressive CsA nephropathy in rats. In their study, the {alpha}1COLI mRNA level in the enalapril-treated kidney was significantly higher than that in the losartan-treated kidney, but overall ECM deposition was similarly reduced in both losartan- and enalapril-treated kidneys, which was not explained by the authors. Our finding that ACEi facilitates ECM degradation more effectively than ARB may account for their results. Although ARB has been reported to resolve tissue fibrosis in a few studies (29,40), in the present study, we were unable to demonstrate that ARB facilitated the regression of interstitial ECM deposition in the CsA nephropathy model. The dose of CGP-48933 used in this study (30 mg/kg per d) seemed to be appropriate because it was enough to lower BP and attenuate nephritic changes in mice with anti-GBM nephritis (11). It is conceivable that Ang II was less involved in matrix turnover in the CsA nephropathy model than once presumed. Nevertheless, we do not deny an antifibrotic action of ARB in other pathologic situations. As described above, ARB lessens renal fibrogenesis where it is RAS dependent, such as in the progressive CsA nephropathy model (38). It also attenuates the inflammatory reactions in the anti-GBM nephritis model more significantly than ACEi, resulting in less renal fibrogenesis (11). Taken together, these observations suggest that ACEi seems to be more antifibrotic than ARB, and ARB seems to be more anti-inflammatory than ACEi, which likely accounts for the overall benefit with the combined therapy of ACEi and ARB on a mixed population of patients with nondiabetic, proteinuric nephropathy (41). These different drugs may be useful in specific ways on a case-by-case basis in patients with progressive renal disease to control inflammatory and fibrotic processes.

See related editorial, "Bradykinin and Renal Fibrosis: Have we ACE’d it?," on pages 2504–2506.


   Acknowledgments
 
We are grateful to M. Otobe for technical assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 27, 2004. Accepted for publication June 2, 2004.




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