Besides its pivotal role in the regulation of extracellular volume and systemic blood pressure, the renin-angiotensin system (RAS) also functions at the intracrine, autocrine, and paracrine levels1,2,3,4,5,6,7,8. Its components [angiotensinogen, renin, angiotensin converting enzyme (ACE), and aminopeptidases (APs)] have been found in many tissues, and there is overwhelming evidence of a local angiotensin II (Ang II) biosynthesis [reviewed in 9]. The kidney and the blood vessels represent two examples of target tissues as well as sites of production and degradation of Ang II10,11. This is particularly evident in the proximal tubule, where the Ang II concentration is 1000 times higher than in plasma12,13.
Several studies have clearly shown that intrarenal RAS plays an important role not only in the regulation of renal hemodynamics but also in the pathogenesis of renal fibrosis1,2,14,15,16. Recent data suggest that the Ang II blockade may also act through pressure-independent mechanisms to induce the repair of tissue injury. Indeed, it has been demonstrated that ACE inhibitors and Ang II receptor antagonists attenuate progressive renal fibrosis in several disease models, and slow disease progression in several forms of human glomerulonephritides14,17,18,19,20,21,22,23. The precise mechanisms by which Ang II induces renal fibrosis are still unclear.
Until recently, Ang II and its metabolite Ang III were considered to be the only bioactive agents24. Shorter fragments, such as Ang IV, were denied any physiological interest, as they lacked the effects of Ang II and Ang III24. However, new findings indicate that the actions of the RAS are mediated, in addition to Ang II and Ang III, by a variety of angiotensin peptides binding to multiple receptors25,26,27,28,29,30. Locally synthesized Ang II therefore has the potential to directly activate its specific neighboring receptors, but it is also hydrolyzed by several cell-associated brush border enzymes into shorter fragments, such as Ang IV, which may activate their own receptors.
In endothelial cells, it has been reported that the induction of plasminogen activator inhibitor-1 (PAI-1) expression by Ang II is mediated by Ang IV via a receptor that is different than AT1 and AT231. PAI-1 is a major physiological inhibitor of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and, therefore, plays an important role in the regulation of the PA/plasmin system32. This system is not only a key regulator of fibrinolysis, but also participates in extracellular matrix (ECM) degradation. Indeed, PAI-1 prevents the transformation of the metalloproteinases, which are potent ECM degradation enzymes, from the latent to the active form by blocking the activation of plasminogen in plasmin32.
Tubulointerstitial fibrosis is the net result of increased ECM deposition and/or diminished ECM degradation. Renal tubular cells, previously considered to be passive targets of the inflammatory process, seem to be implicated in the pathogenesis of renal tubulointerstitial fibrosis [reviewed in 33]. Indeed, in response to different stimuli, they are able to produce an array of inflammatory and profibrogenic molecules. Moreover, they express angiotensinogen, renin, ACE, and AP and possess specific receptors for Ang II and its metabolites9,26. Interestingly, an increase of ACE and PAI-1 has been reported in pathological conditions involving the renal tubule34,35,36.
Therefore, we evaluated the effects of Ang II and Ang IV on the expression of PAI-1 mRNA in a well-characterized and immortalized human proximal tubular cell line37. Moreover, we addressed the hypothesis that PAI-1 mRNA expression by human proximal tubular cell may be mediated by a specific receptor for Ang IV, and we speculated on the role played by the Ang II metabolites in the pathogenesis of tubulointerstitial fibrosis.
METHODS
Reagents
Dulbecco's modified Eagle's medium (DMEM)/F12, fetal bovine serum (FBS), L-glutamine, insulin, transferrin, and sodium selenite were obtained from Sigma Cell Colture (Milan, Italy). Trypsin, penicillin, and streptomycin were obtained from Mascia Brunelli (Milan, Italy). Epidermal growth factor was obtained from Calbiochem (La Jolla, CA, USA). PGE1, hydrocortisone, and T3 were purchased from Sigma Chemical Co. (Milan, Italy). Ang II and Ang IV were obtained from Peninsula Laboratories (Belmont, CA, USA). Amastatin was obtained from Sigma Chemical Co. (Milan, Italy). Losartan was kindly donated by DuPont Merck Pharmaceutical Company (Wilmington, DE, USA). [32P]dCTP and [methyl-3H]-thymidine were purchased from Amersham (Little Chalfont, Buckinghamshire, UK). All other chemicals were reagent grade.
Cell culture
HK2 cells, an immortalized proximal tubular epithelial cell (PTEC) line from normal adult human kidney37, were obtained from ATCC (Rockville, MD, USA). Cells were grown to confluence in DMEM/F12 medium supplemented with 5% heat-inactivated FBS, 100 U/ml penicillin, 100 
g/ml streptomycin, 2 mM L-glutamine, 5 g/ml insulin, 5 g/ml transferrin, and 5 ng/ml sodium selenite, 5 pg/ml T3, 5 ng/ml hydrocortisone, 5 pg/ml PGE1, and 10 ng/ml epidermal growth factor. For passage, confluent cells were washed with phosphate-buffered saline (PBS), removed with trypsin 0.05%/ethylenediaminetetraacetic acid (EDTA) 0.2% in PBS, and plated in DMEM/F12. Following these culture conditions, the cells retained a phenotype indicative of well-differentiated PTECs as previously demonstrated37,38 and as confirmed by our studies. Cells were positive for cytokeratin, antiadenosine deaminase-binding protein antibody (CD26), alkaline phosphatase, and CHIP28 (a proximal tubule water channel), and were negative for Tamm-Horsfall protein (data not shown). The cells were washed with fresh serum-free DMEM/F12 medium and starved in serum-free medium for 24 hours, and then the indicated concentrations of Ang II or Ang IV were added in the presence or absence of the AT1 receptor antagonist (DuP 753/MK 954, losartan), AT2 receptor antagonist [N-Nicotinoyl-Tyr-N3- (N
-CBZ-Arg)-Lys-His-Pro-Ile], or Amastatin. Primary PTECs were obtained according to the method described by Detrisac et al from apparently normal portions of nephrectomy specimens39. PTECs were cultured following the protocol used for HK2 cells.
DNA synthesis
DNA synthesis was measured as the amount of [methyl-3H]-thymidine incorporated into trichloroacetic (TCA)-precipitable material. Cells were plated in 24-well dishes at a density of 4 
104 per well, were grown to confluence, and were made quiescent by placing them in serum-free medium for 24 hours. Then cells were incubated with Ang II and Ang IV at the indicated concentrations for 24 hours at 37°C. At the end of the incubation period, cells were pulsed for four hours with 1.0 Ci/ml of3H-thymidine. The medium was then removed, and cells were washed twice in ice-cold 5% TCA and incubated in 5% TCA for five minutes. Cells were solubilized by adding 0.75 ml of 0.25 N NaOH in 0.1% sodium dodecyl sulfate (SDS). One-half milliliter aliquots were then neutralized and counted in scintillation fluid by using a 
counter.
RNA isolation and Northern blot analysis
Proximal tubular epithelial cells were grown to confluence, rested for 24 hours in serum-free medium, and then incubated with Ang II or Ang IV with or without the antagonists at the indicated concentrations for 0, 3, 6, 12, and 24 hours at 37°C. At the end of the incubation period, cells were lyzed with 4 M guanidium isothiocyanate containing 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, and 0.1 mM 2--mercaptoethanol. Total RNA was isolated by the single-step method, using phenol and chloroform/isoamylalcohol40.
Plasminogen activator inhibitor-1, PAI-2, u-PA, t-PA, and transforming growth factor-1 (TGF-1) gene expression was studied by Northern blotting as previously described41. The cDNA probes used for Northern blotting analysis were a 790 bp fragment encoding the human PAI-2 cDNA, a 600 bp fragment of the human u-PA cDNA (kindly provided by Dr. S. Moll and Dr. D. Belin, Laboratoire de Nefrologie, Geneve, France), a 1200 bp fragment of the human PAI-1 cDNA, a 770 bp fragment of the human t-PA cDNA (kindly provided by Dr. R. Lorenzet, M. Negri Sud, S. Maria Imbaro; originally from Dr. D. Loskutoff, Scripps Clinic, La Jolla, CA, USA), and a 2140 bp fragment encoding the human TGF-1 cDNA (kindly provided by Dr. S. Milani, Department of Gastroenterology, University of Florence, Florence, Italy). Briefly, electrophoresis of 20 g total RNA from each experimental condition was carried out in 1% agarose gel with 2.2 M formaldehyde. The gel was stained with ethidium bromide to evaluate the 28S and 18S ribosomal bands and was transferred overnight to a nylon membrane (Schleicher & Schuell, Dassel, Germany). The membrane was prehybridized at 42°C for two hours in 50% formamide, 0.5% SDS, 5 standard saline citrate (SSC), and 0.1 mg/ml salmon sperm DNA. The cDNA probes were labeled by random priming using a commercially available kit (Amersham) and [32P]dCTP (specific activity, 3000 Ci/mmol). The probe (106 cpm/ml) was added to 10 ml of prehybridization solution, and the blots were hybridized for 16 hours at 42°C. The membranes were then washed once in 2 SSC, 0.1% SDS at room temperature for five minutes, once in the same buffer at 55°C for 30 minutes, and in 1 SSC, 0.1% SDS at 55°C for an additional 30 minutes. After drying, membranes were exposed to a Kodak X-OMAT film with intensifying screens at –70°C.
Measurement of plasminogen activator inhibitor-1 in conditioned medium and extracellular matrix
Proximal tubular epithelial cells were grown to confluence, rested for 24 hours in serum-free medium, and then incubated with Ang II or Ang IV at the indicated concentrations for 24 hours at 37°C. At the end of incubation, conditioned media were collected, centrifuged, and stored at -80°C until assayed. The cell monolayer was washed twice with culture medium and then treated with 0.5 ml of 0.02 M Tris, 0.15 M NaCl, pH 7.4, containing 0.5% Triton X-100. After incubation for 45 minutes at room temperature on a rotating plate, the resulting cell extract was removed by aspiration, and the Triton X-100–insoluble ECM was washed twice with Tris buffer. ECM-associated PAI-1 was then brought into solution by the addition of 0.3 ml of 1 g/ml of t-PA and incubated for two hours at 37°C. This treatment resulted in more than an 80% release of PAI-1 as PAI-1/t-PA complex, as previously reported42. PAI-1 antigen was measured by an enzyme-linked immunosorbent assay (ELISA) method (Biopool, Umea, Sweden) and expressed as ng per 100,000 cells (conditioned media) or ng/ml (ECM associated).
Statistical analysis
Results are presented as mean 
SD. Statistical significance was determined using one-way analysis of variance and the unpaired t-test. A P value of less than 0.05 was considered statistically significant. Triplicate wells were analyzed for each experiment, and each experiment was performed independently at least three times.
RESULTS
Effect of Ang II and IV on PTEC proliferation
As previously reported1,2, the treatment of PTEC with Ang II did not cause an increase of either [3H]-thymidine incorporation or cell number (P = NS; Figure 1). Similar effects were observed with Ang IV (P = NS; Figure 1).
Figure 1.
(A) Effect of angiotensin (Ang) II (
) and Ang IV (
) on proximal tubular epithelial cell (PTEC) DNA synthesis. Tritiated thymidine incorporation is expressed as count per min/well. (B) Effect of Ang II and IV on PTEC proliferation. Ang II and IV did not cause a significant and dose-dependent increase in DNA synthesis, as well as in cell number. Data represent means SD (N = 3, each point in quadruplicate).
Effect of Ang II and IV on PAI-1 mRNA expression
HK2 cells were exposed to Ang II or Ang IV for three hours over the concentration range of 0 to 20 nM. At the end of incubation, cells were lyzed. RNA was extracted, and PAI-1 gene expression was evaluated by Northern blotting. In basal conditions, PAI-1 mRNA was expressed at low but detectable levels Figure 2. Ang II and Ang IV induced a dose-dependent increase of PAI-1 expression Figure 2, which reached its plateau at a concentration of 10 nM of either agent. Exposure of the cells to 10 nM Ang II or Ang IV resulted in a 4.2 2.1-fold and 7.8 3.3-fold increase in the expression of PAI-1, respectively Figure 2. A time-dependent increase of Ang II- and Ang IV-induced PAI-1 expression was also demonstrated, which seemingly peaked at three hours Figure 3 a, c). The same results were obtained with primary PTEC (data not shown).
Figure 2.
Effect of different angiotensin (Ang) II () and Ang IV () concentrations on plasminogen activator inhibitor-1 (PAI-1) expression by proximal tubular epithelial cells (PTECs). Confluent cultures of PTEC were washed, serum starved, and then incubated with vehicle, Ang II, or Ang IV for three hours at the indicated concentrations. The expression of PAI-1 mRNA after exposure to Ang II and Ang IV was determined by densitometry and was normalized using the 28S band. Data represent means SD (N = 3, each point in triplicate).
Figure 3.
(A and B) Time course of Ang II and Ang IV-induced plasminogen activator inhibitor-1 (PAI-1) expression by proximal tubular epithelial cells (PTECs). Confluent cultures of PTECs were washed, serum starved, and incubated with vehicle, Ang II (10 nM), and Ang IV (10 nM) for the indicated time periods. The expression of the PAI-1 mRNA is demonstrated in the upper panel. To control for variation in gel loading, the relative amounts of RNA in each lane were visualized by staining with ethidium bromide. (C) Time course of Ang II () and Ang IV (
) induced increase of PAI-1 expression by PTECs. The expression of PAI-1 mRNA after exposure to Ang II and Ang IV was determined by densitometry and normalized using the 28S band.
Effects of amastatin, AT1 and AT2 receptor antagonists on the response to Ang II and IV
Amastatin is a potent inhibitor of aminopeptidases P and APN, which prevent the conversion of Ang II to smaller fragments, including Ang IV9. Figure 4 shows that amastatin strongly reduced Ang II-induced PAI-1 mRNA expression in a dose-dependent manner, and at a concentration of 1 mM, it completely abrogated the effect of 10 nM Ang II. In contrast, amastatin was unable to prevent the expression of PAI-1 mRNA induced by 10 nM Ang IV Figure 5. Furthermore, Ang IV-dependent induction of PAI-1 mRNA was supported by an experiment in which cells stimulated by Ang II, pretreated with losartan and N-Nicotinoyl-Tyr-n3-(n-CBZ-Arg)-Lys-His-Pro-Ile, specific antagonists of AT1 and AT2 receptors, did not show any variation of PAI-1 mRNA expression when compared with cells treated with Ang II alone Figure 6.
Figure 4.
Effect of the aminopeptidase inhibitor amastatin on Ang II-induced PAI-1 expression in proximal tubular epithelial cells (PTECs). Confluent cultures of PTECs were washed, serum starved, and incubated with vehicle or Ang II (10 nM) with or without increasing concentration of amastatin (0 to 1000 nM). Data represent the means SD (N = 3, each point in triplicate).
Figure 5.
Effect of the aminopeptidase inhibitor amastatin on angiotensin (Ang) II/IV-induced plasminogen activator inhibitor-1 (PAI-1) expression in proximal tubular epithelial cells (PTECs). Confluent cultures of PTECs were washed, serum starved, and incubated with vehicle (A and D), Ang II (10 nM; B), or Ang IV (10 nM; E). In (C and F), cells were incubated with Ang II and Ang IV plus amastatin (1000 nM), respectively.
Full figure and legend (77K)Figure 6.
(A) Effect of losartan on angiotensin (Ang) II-induced plasminogen activator inhibitor-1 (PAI-1) expression by proximal tubular epithelial cells (PTECs). Confluent cultures of PTECs were washed, serum starved, and incubated with vehicle (lane 1) or Ang II (10 nM; lane 2). In lanes 3 and 4, cells were preincubated with an AT1 receptor antagonist (1 M; losartan; lane 3) or with amastatin (1 M; lane 4). The expression of PAI-1 mRNA after exposure to Ang II was normalized using the 28S band. (B) Effect of N-Nicotinoyl-Tyr-N3-(N-CBZ-Arg)-Lys-His-Pro-Ile on Ang II-induced PAI-1 expression by PTECs. Confluent cultures of PTECs were washed, serum starved, and incubated with vehicle (lane 1) or Ang II (10 nM; lane 2). In lanes 3 and 4, cells were preincubated with an AT2 receptor antagonist [1 M; N-Nicotinoyl-Tyr-N3-(N-CBZ-Arg)-Lys-His-Pro-Ile; lane 3] or with amastatin (1 M; lane 4). Expression of PAI-1 mRNA after exposure to Ang II was normalized using the 28S band.
Effect of angiotensin II and IV on u-PA, t-PA, and PAI-2 expression
Angiotensin II and Ang IV did not induce any variation in u-PA mRNA expression Figure 7, whereas t-PA and PAI-2 mRNA expression were undetectable (data not shown).
Figure 7.
Effect of angiotensin (Ang) II and Ang IV on u-PA and transforming growth factor-
(TGF-) expression by proximal tubular epithelial cells (PTECs). Confluent cultures of PTECs were washed, serum starved and incubated with vehicle, Ang II (10 nm), or Ang IV (10 nm) for 0 to 24 hours. No differences were observed in Ang II and Ang IV induced u-PA expression at any time point. Ang II induced an increase of TGF- mRNA expression, with a maximal effect at 12 hours. Ang IV was devoid of any effect on TGF- mRNA expression. Expression of u-PA and TGF-1 mRNA after exposure to Ang II and Ang IV was normalized to GAPDH expression.
Effects of Ang II and IV on TGF- expression
It has been previously demonstrated that Ang II is able to induce a profibrotic factor, that is, TGF-, in PETCs via the AT1 receptor. Thus, to demonstrate that HK2 cells were responsive to Ang II and that the lack of a direct effect of Ang II on PAI-1 expression was not due to the absence of AT1 receptors, the same total RNA shown in Figure 3 was hybridized with TGF- cDNA. As shown in Figure 7, although Ang II induced a 2.8 0.6-fold increase of TGF-1 mRNA expression, with a maximal effect at 12 hours, Ang IV was devoid of any effect on TGF- mRNA expression. Finally, losartan was able to prevent TGF- mRNA expression induced by Ang II Figure 8.
Figure 8.
Effect of losartan on angiotensin (Ang) II-induced TGF- expression by proximal tubular epithelial cells (PTECs). Confluent cultures of PTECs were washed, serum starved, and incubated with vehicle (lane 1) or Ang II (10 nM; lanes 2 through 5). In lanes 3 through 5, cells were preincubated with amastatin (1 M; lane 3), with an AT2 receptor antagonist [1 M; N-Nicotinoyl-Tyr-N3-(N-CBZ-Arg)-Lys-His-Pro-Ile; lane 4] and with an AT1 receptor antagonist (1 M; losartan; lane 5). Expression of TGF- mRNA after exposure to Ang II was normalized using the 28S band.
Effects of Ang II and IV on PAI-1 protein synthesis
To demonstrate that the rise in PAI-1 mRNA in PTEC culture was accompanied by the synthesis of PAI-1 protein, PAI-1 antigen was measured by a commercially available ELISA assay kit. PAI-1 antigen was significantly increased in both Ang II- and Ang IV-stimulated PTECs (basal, 9.3 0.6 ng/100,000 cells; Ang II, 12.3 1.9 ng/100,000 cells; and Ang IV, 13.9 1.7 ng/100,000 cells, P < 0.02). Because this difference was less pronounced than that observed at mRNA level, we determined the amount of PAI-1 associated with the ECM and found that in both Ang II- and Ang IV-treated cells, the amount of ECM-associated inhibitor was higher than in control cells (basal, 35 0.6 ng/ml; Ang II, 51.9 8.5 ng/ml; and Ang IV, 40.8 2.16 ng/ml, P < 0.02). It is conceivable that treatment with Ang II and Ang IV enhances PAI-1 production as well as PAI-1 accumulation within the ECM. It should be noted that ECM-associated PAI-1 is active and accessible to soluble plasminogen activators even in the presence of adherent cells42.
DISCUSSION
In this study, for the first time to our knowledge, we demonstrated that the exapeptide Ang IV induces PAI-1 mRNA expression in human PTECs in a time- and dose-dependent manner. Moreover, our results suggest that PAI-1 mRNA expression induced by Ang II in PTECs is mediated by Ang IV, supporting the data reported by Kerins, Hao, and Vaughan in endothelial cells31. This conclusion was drawn from the demonstration that the use of amastatin, a potent inhibitor of APs, abrogated PAI-1 mRNA expression induced by Ang II but not by Ang IV.
Aminopeptidases (APs) are enzymes that degrade Ang II into Ang III and Ang IV9. At least two APs, A and N, are involved in this process. APA, also known as angiotensinase, converts Ang II into Ang III, whereas APN converts Ang III into the hexapeptide Ang IV9. Both enzymes, APA and APN, have been found on the brush border of the proximal tubule43,44. Amastatin is a potent inhibitor of both aminopeptidases with an IC50 of 0.2 M45. Moreover, the kidney expresses a specific receptor for Ang IV, named AT4. Harding et al have shown that it is preferentially concentrated in the outer stripe of medulla46. Finally, it has been reported that PTECs may express angiotensinogen, renin, and ACE10,11,12,13,47.
It is conceivable that in the setting of an inflammatory process, such as tubulointerstitial nephritis, angiotensinogen gene transcription may be activated by proinflammatory cytokines, that is, interleukin-1 and tumor necrosis factor-, produced by macrophages or intrinsic renal cells33. Indeed, both of these cytokines induce two transcription factors, nuclear factor-
B (NF-B) and a member of the CCAT/enhancer binding protein family, both of which are able to bind and to induce a single regulatory site in the angiotensinogen promoter48. Moreover, Ang II per se may activate NF-B. This was indirectly demonstrated by Morrissey and Klahr, who were able to obtain a significant reduction of NF-B activity rat kidneys with unilateral obstruction by using with enalapril49. Thus, it can be hypothesized that at the tubular level the activation of the intrinsic RAS system by inflammatory stimuli may create a positive loop perpetuating the renal damage. The finding that tubular cells express APA and APN is of significant interest43,44. Therefore, in an inflammatory process, the presence of higher amounts of intratubular Ang II may be accompanied by an increase of its metabolites, including Ang IV. This Ang II metabolite inducing PAI-1 may in turn be responsible for the increased accumulation of ECM, by inhibiting its degradation32. The proinflammatory and profibrotic role of the intrinsic RAS system is indirectly supported by both animal and human studies34,50,51,52,53,54,55,56,57,58. In different models of tubulointerstitial injury, the administration of ACE inhibitors blunted the progression of tissue damage55,56,57,58. Recently, Oikawa et al reported that inhibition of the RAS system in irradiated rats may anchor the degree of injury by accelerating fibrinolysis and degradation of ECM34. The authors demonstrated that the inhibition of the RAS system reduced the PAI-1 expression in the irradiated kidney without affecting either t-PA or u-PA mRNA expression. These data suggest that RAS inhibition may diminish renal fibrosis via the inhibition of PAI-1 expression. Moreover, several studies have clearly shown that ACE inhibitor treatment reduces the progression of renal damage in both primary and secondary human glomerulonephritides17,20,21,59. Although prior studies have demonstrated that Ang II stimulates the expression of PAI-1, the specific angiotensin receptor subtype mediating this response was not defined15,34,51,60,61,62,63. Kagami et al demonstrated that the addition of Ang II to mesangial cells in culture increased the expression of PAI-1 mRNA15. No aminopeptidase inhibitors were used in this study. However, they excluded that the increased expression of PAI-1 mRNA induced by Ang II was mediated by TGF-. Indeed, coincubation of mesangial cells with Ang II and a neutralizing antibody to TGF1 did not alter the PAI-1 mRNA expression. In our study, we demonstrated that Ang II is capable of stimulating the expression of PAI-1 and that this effect is Ang IV mediated. Moreover, Ang II was able to induce TGF- expression directly, whereas Ang IV failed to evoke such an effect. Our findings appear particularly interesting because they may support the hypothesis that the activation of RAS system induces fibrosis by following two separate pathways: through TGF- that is induced by Ang II, and through PAI-1 that is mediated by Ang IV. This hypothesis may have important clinical implications. Specific AT1 or AT2 receptor antagonists, although preventing NF-B induction by Ang II49, may lack any favorable effect on the fibrinolytic system. On the other hand, captopril has been shown to decrease PAI-1 activity in patients with recent uncomplicated myocardial infarction64,65,66,67. Obviously, clinical prospective studies comparing these two classes of drugs interfering with the RAS system at different sites are now warranted to support the previously mentioned hypothesis.
In conclusion, our study demonstrates that (a) Ang IV is the form of angiotensin that stimulates PAI-1 mRNA expression in human proximal tubular cells. (b) The effects of Ang IV seem to be mediated by a receptor that is specific for Ang IV. (c) Ang IV, inducing PAI-1 mRNA expression, may be implicated in the pathogenesis of renal interstitial fibrosis.
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Acknowledgments
This study was partly supported by: the Associazione per il Progresso Scientifico in Nefrologia e Trapianto, Bari, Italy; the Extramural Grant Program Baxter, Chicago, IL, USA (PI: L. Gesualdo, round 1997-2000); the Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Roma, Italy (grants 60%: 98/2090108/1; 97-7703 and 40%: 96-7404); and the Consiglio Nazionale delle Ricerche (grant 98.513.04 and the target Project on Biotechnology). The authors are grateful to Dr. Salvatore Di Paolo and Miss Francesca Deleo for the critical review of the manuscript. Portions of this work were presented at the American Society of Nephrology meeting, San Antonio, TX, November 1–5, 1997. The abstract has been published in the J Am Soc Nephrol 8(Suppl 9):515A, 1997.
