Background

Angiotensin-converting enzyme inhibitors (ACE-I) protect against the development of glomerulosclerosis using mechanisms partly dissociated from their systemic antihypertensive action. The aim of the current study was to delineate the mechanism of action underlying the antifibrotic effects of the ACE-I perindoprilat in the context of macrophage-mediated scarring in human mesangial cells.

Methods

Mesangial cells were treated with macrophage-conditioned medium (MPCM) in the presence or absence of the ACE-I perindoprilat.

Results

Forty mol/L perindoprilat reduced MPCM-induced mesangial cell fibronectin levels by 19.4 0.6% (P < 0.001). Immunoprecipitation of ³⁵S-methionine biosynthetically labeled fibronectin and Northern analysis suggested that the decrease in fibronectin levels was not caused by reduced synthesis. MPCM stimulated the production of matrix metalloproteinases (MMP) 2, 3, and 9 in mesangial cells; however, these were not significantly altered by ACE-I treatment, and neither was production of their tissue inhibitor of metalloproteinases (TIMP-1). Addition of exogenous bradykinin to MPCM-treated mesangial cells resulted in a 22.5 1.4% (P < 0.02) reduction in secreted fibronectin levels, while semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting demonstrated that bradykinin B2 receptor expression was up regulated by 71 30% in MPCM-stimulated mesangial cells in response to ACE-I treatment (P = 0.032). Moreover, the bradykinin B2 receptor antagonist HOE 140 attenuated the beneficial effects of perindoprilat. MPCM-stimulated mesangial cell protein expression levels of plasminogen activator system components tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were altered after treatment with ACE-I.

Conclusion

These results suggest that ACE-I-induced renoprotection, in the context of macrophage-stimulated mesangial cell scarring, is mediated, at least in part, via the actions of bradykinin.

METHODS

Unless otherwise stated, all reagents were purchased from Sigma-Aldrich Chemical Co. (Dorset, UK).

Cell culture

Human mesangial cells were cultured, using standard serial sieving techniques, from the glomerular explants of human kidney specimens derived from the normal poles of nephrectomized kidneys from patients with renal carcinoma. The cells were cultured in RPMI 1640 (Invitrogen, Paisley, UK), supplemented with 20% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin (Invitrogen), 100 g/mL streptomycin (Invitrogen), 5 g/mL bovine insulin, and 2 mmol/L glutamine (Invitrogen). Cultured cells were characterized by their stellate fusiform morphology, their positive staining for actin, myosin, vimentin, and desmin, and negative staining for factor VIII-related antigen and cytokeratin.

Mesangial cells of passages 2 through 8 were cultured in 24-well plates (Costar-Corning, Buckinghamshire, UK) or 25 cm² flasks (Costar-Corning), allowed to grow to confluence, and then rendered quiescent in RPMI medium containing 0.5% FCS for 48 hours before use.

Cells of the human monocyte/macrophage cell line U937 (ECCAC no. 85011440) were grown in RPMI 1640 supplemented with 10% FCS, 100 U/mL penicillin, 100 g/mL streptomycin, and 2 mmol/L glutamine.

Preparation of U937 cell-conditioned medium

U937 cells were seeded into 75 cm² at a cell density of 10⁶ cells per mL in serum-free RPMI 1640 (100 U/mL penicillin, 100 g/mL streptomycin, 2 mmol/L glutamine) and stimulated with 10^-8 mol/L phorbol 12-myristate 13-acetate (PMA) to allow them to adhere to the plastic substratum. Adherent cells were then stimulated with lipopolysaccharide (LPS) (from Escherichia coli 026 B6) at a final concentration of 1 g/mL in serum-free RPMI 1640 for 16 hours. After washing 3 times with Hank's balanced salt solution (HBSS; Invitrogen), the cells were cultured for another 30 hours in serum-free RPMI 1640. The conditioned medium (MPCM) was harvested and centrifuged for 5 minutes at 1000 rpm and frozen at -20°C until required.

Culture of mesangial cells in the presence of MPCM

Confluent, quiescent mesangial cells were exposed to a 50% solution of MPCM in the presence or absence of the ACE inhibitor perindoprilat (a gift from Servier, France). The cultures were maintained in this medium for 1 or 3 days. The tissue culture supernatants were harvested and stored at -20°C for subsequent analysis.

Mesangial cells were additionally treated with MPCM 0.1 mol/L bradykinin or MPCM ACE-I 1 mol/L bradykinin B2 receptor antagonist HOE 140.

For Northern and Southern analyses, mesangial cells were exposed to 50% MPCM additions for 18 hours before RNA processing.

Preparation of cell lysates

After removal of tissue culture supernatants, cell monolayers were washed with phosphate-buffered saline solution (PBS), scraped into 250 L 1% Nonidet P40 in wash buffer (PBS containing 0.3 mol/L NaCl and 1% Tween 20), and then incubated at room temperature (RT) for approximately 30 minutes. The cell scrapings were transferred to 2 mL tubes, sonicated with a 5-second burst, and centrifuged for 1 minute at 11,600g. Sonication and centrifugation were repeated, and the lysate supernatants were assayed for fibronectin and total cell protein.

ELISAs

Culture supernatants and cell lysates were assayed for fibronectin using a modified version of an "in house" enzyme-linked immunosorbent assay (ELISA)¹⁵. Briefly, 96-well microtiter plates (Costar-Corning) were coated with 100 L/well rabbit antihuman fibronectin antibody (in 0.05 mol/L carbonate buffer, pH 9.6) at 4°C overnight. The plates were blocked with 2% bovine serum albumin (BSA) (in wash buffer) for 1 hour at RT. After washing the plates 4 times, 50 L/well of human fibronectin standard (2000–31.3 ng/mL in doubling dilutions), supernatant, or cell lysate were added to the wells and incubated for at least 2 hours at RT. After another 4 washes, 50 L of monoclonal antihuman fibronectin antibody was added and incubated for 1 hour at RT. After washing, 50 L of rabbit antimouse immunoglobulin G (IgG) conjugated to horseradish peroxidase (HRP) (Dako, Ltd., Ely, UK) was added to the wells and incubated at RT for 1 hour. After 4 more washes, 50 L of 0.67 mg/mL 1,2 phenylenediamine dihydrochloride was added in 0.03 mol/L citrate buffer, pH 5.0, containing 0.012% H₂O₂. Following color development, 75 L of 1 mol/L H₂SO₄ were added to stop the reaction, and absorbance was read at 492 nm on a Titertek MultiskanPlus microtiter plate reader (Flow Laboratories, Oxfordshire, UK).

The following ELISAs were carried out according to manufacturer's instructions: (1) bradykinin enzyme immunoassay (EIA) (Peninsula Laboratories, Inc., St. Helens, Merseyside, UK); (2) BIOTRAK matrix metalloproteinase (MMP)3, MMP2, and tissue inhibitor of metalloproteinase-1 (TIMP-1) (Amersham Pharmacia Biotech, Hertfordshire, UK); (3) Quantikine MMP 9 (R&D Systems, Abingdon, Oxon, UK); (4) Quantimatrix human laminin (Chemicon International, Temecula, CA, USA); (5) Immunolyse tissue plasminogen activator (tPA) and Immunolyse plasminogen activator inhibitor-1 (PAI-1) (Biopool International, CA, USA); (6) urokinase plasminogen activator (uPA) (Oncogene Science, Bayer Diagnostics, East Walpole, MA, USA).

Zymography

MMP2 and MMP9 activities were determined using gelatin zymography. Cell supernatants were diluted 1:5 in PBS, 30 L of the dilution was mixed with an equal volume of nonreducing sample buffer and loaded on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel containing 1 mg/mL gelatin. After electrophoresis at 4°C, the gels were washed in 2 changes of 2.5% (v/v) Triton X-10 at RT, after which they were incubated overnight at 37°C in activation buffer (50 mmol/L Tris-HCl, 100 mmol/L NaCl, 10 mmol/L CaCl₂, 0.05% Brij 721). The gels were then stained for at least 2 hours with Brilliant Blue R, and destained in an aqueous solution methanol:acetic acid:water (40:10).

Protein determination

The protein content of cell lysates dissolved in 1% Nonidet NP40 was determined using a commercial BioRad detergent compatible (DC) protein assay, using BSA standards according to the manufacturer's instructions.

Northern blotting

Northern analysis was carried out using a method previously described¹⁴. Briefly, total RNA was extracted from mesangial cells using TRIzol reagent (Invitrogen). Thirty-g aliquots of RNA were resolved on a 1% agarose-formaldehyde gel [in 3-[N-morpholino]propane sulfonic acid (MOPS)], blotted onto a Hybond-N nylon membrane (Amersham Pharmacia), and hybridized with a [³²P]-dCTP cDNA probe that had been labeled using a random primer labeling system (Prime-a-Gene; Promega, Southampton, UK) in a solution containing 50% formamide, 1% SDS, 5 Denhardt's, 5 SSPE, and 200 g/mL denatured salmon sperm DNA at 42°C. Membranes were exposed to X-Omat AR (Kodak, Hemel, Hempstead, UK) or Biomax MS (Kodak, UK) films with X-Omatic or Biomax transcreen HE intensifying screens (Kodak, UK), respectively, at -70°C. The resulting autoradiographs were scanned using a BioRad GS 700 imaging densitometer. RNA loading was normalized using a cDNA probe for the nonstructural protein cyclophilin. Cyclophilin expression was not affected by treatment with MPCM.

RT-PCR

Aliquots of total RNA (0.5 g) were reverse-transcribed using avian myeloblastosis virus (AMV) reverse transcription system (Promega) according to the manufacturer's instructions. The resulting cDNA was amplified using ReddyMix™ PCR Mastermix (ABgene, Surrey, UK) and 50 pmol of specific sense and antisense primers.

Thermocycling conditions were: 1 cycle at 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 55°C for 1 minute, 68°C for 2 minutes, 1 cycle at 68°C for 7 minutes, and 10-minute soak at 4°C.

Southern blotting

Aliquots of reverse transcription-polymerase chain reaction (RT-PCR) cDNA (20 L) were resolved on 1% Tris-acetate EDTA (TAE)-agarose gels. The gels were denatured for 45 minutes in a solution containing 1.5 mol/L NaCl and 0.5 mol/L NaOH. The gels were rinsed in water, and then neutralized in a solution containing 1.5 mol/L NaCl and 0.5 mol/L Tris-HCl, pH 7.5. Thereafter, the gels were blotted and hybridized as described for Northern blotting.

Oligonucleotide primers

Probes were created by RT-PCR of RNA extracted from mesangial cells stimulated with MPCM followed by amplification using the following primers:

Bradykinin B2 sense 5'-ATG CTC AAT GTC ACC TTG CAA-3', antisense 5'-CTG ATG ACA CAA GCG GTG ACG-3'. tPA sense 5'-GAC TGG ACG GAG TGT GAG CTC TCC-3', antisense 5'-GTC TAC ACC AAG GTT ACC AAC TAC-3'¹⁶. PAI-1 sense 5'-GTG CAC CAT CCC CCA TCC TAC GTG-3', antisense 5'-GGG TTC CAT CAC TTG GCC CAT GAA-3'¹⁷. Kininogen sense 5'-CTA GAT TGC AAC GCT GAA G-3'¹⁸. High kininogen antisense 5'-GTC ATG TCT ACG AGT ATG C-3'¹⁸. Low kininogen sense 5'-GTC ACC TAA GGT CCT GCG AG-3' antisense 5AGG CAG AGT GAT AAA ATG GC-3'¹⁹. Tissue kallikrein sense 5'-AAC ACA GCC CAG TTT GT-3', antisense 5'-CTT CAC ATA AGA CAG CAC-3'²⁰.

The PCR products were validated by sequencing using the ABI PRISM™ dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Perkin-Elmer Corporation, Warrington, UK) according to manufacturer's instructions.

Metabolic labeling/immunoprecipitation

Confluent, quiescent mesangial cells were stimulated with MPCM in the presence or absence of perindoprilat or medium alone diluted 1:1 in methionine-free RPMI. Each well was pulsed with 20 Ci ³⁵S-methionine (ICN Flow). After 3 days, the culture supernatants were retained, and the cell monolayers were washed twice in PBS. Cell lysates were prepared as described above. To immunoprecipitate the newly synthesized fibronectin, 20 L of rabbit antihuman fibronectin antibody was added to 400 L culture medium for approximately 4 hours. Antigen:antibody complexes were precipitated with 50 L of insoluble protein A cell suspension (10% wet vol/vol of nonviable Staphyloccocal aureus cells (Cowan strain) by overnight incubation at 4°C. The samples were centrifuged for 10 minutes at 3000g, and the pellets were washed 3 times with ice-cold immunoprecipitation buffer (PBS containing 0.5 mol/L NaCl, 0.1% SDS, and 1% Triton X-100, pH 7.4), vortexing the pellet thoroughly between each wash. The pellets were dissolved in 70 L sample buffer and boiled for 7 minutes. The dissolved pellets were resolved by electrophoresis on 5% SDS-polyacrylamide gels. Newly synthesized fibronectin was detected by autoradiography of dried gels. Bands were quantified by scanning densitometry on a BioRad densitometer.

Bradykinin receptor radioligand-binding assays

Mesangial cells in 24-well culture plates were exposed to MPCM, MPCM perindoprilat, or medium alone for 18 hours. The cells were then placed on ice, and 500 L aliquots of the culture supernatant were removed and retained. Prolyl^2,4-3,4(n)-³H-bradykinin [2 L (0.04 Ci)] (Amersham Biosciences) was then added to the remaining 500 L of culture medium in each well and incubated on ice in a refrigerator at 4°C for 2 hours. To determine nonspecific binding (NS), 10 mol/L unlabeled bradykinin was added to some of the wells that had been treated with ³H-bradykinin. After the 4°C incubation, the culture supernatants were removed, and the cells were washed with cold Tris-buffered saline (TBS), pH 8.0. The cell monolayers in each well were scraped into 210 L of 0.5 mol/L NaOH. The plates were then incubated at 65°C for 15 minutes to solubilize the protein. A portion of each cell lysate (180 L) or 200 L of retained radioactive culture supernatant was placed into scintillation vials containing 4 mL Ecoscint scintillation fluid (National Diagnostics, Hull, UK) and 50 L concentrated HCl. Counts per minute (cpm) were determined on a liquid scintillation counter (LKB Wallac; Beckman Coulter, Fullerton, CA, USA). Bradykinin binding in cell lysates (cpm) as a percentage of total bradykinin input and correcting for nonspecific binding is expressed as a function of cell protein.

STATISTICS

Mesangial cell fibronectin levels were corrected for cell protein. Fibronectin levels have been expressed as fold-increase over control (medium) levels. Where appropriate, data in other experiments were expressed as percent of MPCM-stimulated levels. Representative autoradiographs of Northern or Southern blots are shown, but densitometric analysis incorporates data from all experiments after normalization for the appropriate housekeeping gene. Results are expressed as mean SEM. For comparison of the mean between two groups, an unpaired t test was employed. Statistical significance was defined as P < 0.05.

RESULTS

Effect of perindoprilat on mesangial cell fibronectin levels

Stimulation of confluent, quiescent mesangial cells with MPCM for 3 days resulted in a 79 15% increase in secreted, and a 77 17% increase in cell-associated, fibronectin levels (P < 0.001) over those of control cells (medium alone) Figure 1. Addition of 40 mol/L perinoprilat significantly attenuated this response, resulting in decreases in MPCM-stimulated fibronectin levels of 19.4 0.6% (P < 0.001) and 21.7 1.0% (P < 0.001) for secreted and cell-associated fibronectin levels, respectively Figure 1. The dose of 40 mol/L had previously been determined as optimal from a dose-response curve carried out in preliminary experiments (data not shown). Addition of 1 mol/L LPS to mesangial cells had no effect on fibronectin production (data not shown).

Figure 1.

Effect of perindoprilat on mesangial cell fibronectin levels. Confluent, quiescent cultures of human mesangial cells were treated with MPCM 40 mol/L perindoprilat for 3 days. Culture supernatants and cell lysates were assayed for fibronectin. Results are expressed as mean SEM (fold-increase over medium alone and corrected for cell protein). *P < 0.001 vs. medium alone; **P < 0.001 vs. MPCM, N = 14. MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (63K)

Similarly, perindoprilat reduced MPCM-stimulated supernatant laminin protein levels by 23.2 1.72% (P = 0.001) (data not shown).

Effect of perindoprilat on fibronectin synthesis

In order to assess whether the fibronectin levels had decreased because of a reduction in synthesis, immunoprecipitation of metabolically labeled ³⁵S-methionine-fibronectin in the presence or absence of 40-mol/L perindoprilat was carried out. The resulting autoradiographs demonstrated that perindoprilat did not appear to decrease fibronectin protein synthesis Figure 2. Moreover, this was supported by Northern blot analysis, in which fibronectin mRNA levels were unchanged in the presence of perindoprilat Figure 3.

Figure 2.

Effect of perindoprilat on fibronectin synthesis. Representative autoradiograph of immunoprecipitated ³⁵S-methionine-fibronectin obtained from the culture supernatants of mesangial cells incubated in the presence of MPCM 40 mol/L perinoprilat. A representative blot from 3 experiments is shown. MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (28K)

Figure 3.

Effect of perindoprilat on fibronectin mRNA levels. Northern analysis probing for fibronectin was carried out on RNA extracted from mesangial cells incubated with MPCM 40-mol/L perindoprilat for 18 hours. A representative blot from 4 experiments is shown. MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin converting enzyme inhibitor.

Full figure and legend (88K)

Effect of perindoprilat on fibronectin degradation

Because a decrease in fibronectin levels can also occur as a result of matrix degradation we investigated parameters which may be involved in matrix degradation. Tissue culture supernatant levels of the MMP 2, 3, and 9 were measured by ELISA. While MPCM up-regulated MMP levels over control (medium alone), total MMP protein levels were not significantly changed by treatment with perindoprilat, and neither was expression of TIMP-1 Table 1. Gelatin zymography confirmed these data, demonstrating that there was no difference in MMP2 or 9 activities following ACE treatment of MPCM-stimulated mesangial cells (data not shown). Together these data suggest that if ACE-I induced degradation was occurring, it was being mediated by proteinases other than MMP 2, 3, and 9.

Table 1 - Effect of perindoprilat on supernatant levels of matrix metalloproteinases (MMPs 2, 3, 9) and tissue inhibitor of metalloproteinases (TIMP-1).

Full table

Renin-angiotensin and kallikrein-kinin systems

ACE activity could not be detected in our supernatants or MPCM at the sensitivity levels of an automated Sigma ACE detection assay. However, ACE gene expression was detected by semiquantitative RT-PCR, and was found not to change in response to ACE-I treatment (data not shown). ACE provides a common link between the renin-angiotensin and the kallikrein-kinin systems—two interactive, but often antagonistic, systems involved in renal homeostasis. Inhibition of ACE activity results not only in a decrease in Ang II levels, but is accompanied by an increase in local levels of bradykinin. Semiquantitative RT-PCR demonstrated that mesangial cells not only expressed all the components of the RAS (renin, ACE and angiotensinogen) but they also expressed message for high and low kininogens and the serine protease tissue kallikrein Figure 4a, confirming that mesangial cells possess the biochemical machinery necessary to generate vasoactive kinins from an endogenous source.

Figure 4.

Expression of renin angiotensin and kallikrein-kinin systems in human mesangial cells. (A) RNA from control mesangial cells was reverse transcribed and amplified by polymerase chain reaction (PCR) using the appropriate primers. PCR products were resolved by electrophoresis on Tris-acetate EDTA (TAE) agarose gels. Gels reveal the presence of the components of kallikrein-kinin and renin-angiotensin systems in human mesangial cells. (B) RNA from mesangial cells treated with MPCM perinoprilat or medium alone was reverse transcribed and the gene for low kininogen amplified by PCR and Southern blotted. A representative autoradiograph is presented. Densitometry includes data from 4 independent experiments. Abbreviations are: LK, low kininogen; HK, high kininogen; KALL, kallikrein; A'GEN, angiotensinogen; MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor; TAE, Tris-acetate EDTA.

Full figure and legend (137K)

In addition, semiquantitative RT-PCR demonstrated that MPCM stimulation reduced mesangial cell expression levels of low kininogen, the preferred bradykinin precursor in the kidney, below basal levels. Treatment with perindoprilat partially attenuated this reduction, returning levels closer to those of the control Figure 4b.

The role of bradykinin

We investigated the possibility that raised bradykinin levels may play a role in the observed antifibrotic effects. Bradykinin (0.1 mol/L) was added directly to MPCM-stimulated and control mesangial cells. This resulted in a 21.5 1.4% (P < 0.02 vs. MPCM) reduction in secreted fibronectin levels compared with those induced by MPCM alone Figure 5. Bradykinin also significantly decreased basal fibronectin levels by 14.1 0.4% (P < 0.001) Figure 5.

Figure 5.

Effect of exogenous bradykinin on MPCM-stimulated mesangial cell fibronectin levels. Mesangial cells were stimulated with MPCM 0.1 mol/L bradykinin for 3 days. Supernatants were assayed for fibronectin by enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean SEM (Fibronectin levels are expressed as a fold-increase over medium alone and corrected for cell protein). **P < 0.02 vs. MPCM alone; *P > 0.001 vs. medium alone, N = 5. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; Bk, bradykinin.

Full figure and legend (22K)

Paradoxically, however, mesangial cell culture supernatant levels of bradykinin at day 3, as measured by EIA, were found to be significantly reduced following treatment with perindoprilat, and not increased as might have been expected (2.75 0.64, *1.46 0.23, 0.56 0.08 pg bradykinin/g cell protein for MPCM, MPCM + perindoprilat, medium alone, respectively, *P = 0.036 vs. MPCM) Figure 6.

Figure 6.

Effect of perindoprilat on supernatant bradykinin levels. Mesangial cell culture supernatants from cells stimulated with MPCM 40 mol/L perindoprilat were assayed for bradykinin by enzyme immunoassay (EIA). Results are expressed as mean SEM (pg bradykinin/g cell protein). *P = 0.002 vs. MPCM alone, N = 6. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (29K)

Effect of perindoprilat on bradykinin receptor expression

One possible explanation for the observed decrease in bradykinin levels could be that ACE-I treatment up-regulates the expression of the bradykinin B2 receptor, with the result that more bradykinin is sequestered from the local surroundings via ligation to receptors on the cell membrane.

Semiquantitative RT-PCR and Southern blotting of RNA from mesangial cells stimulated with MPCM demonstrated that bradykinin B2 receptor expression was indeed significantly up-regulated 71 30% (P = 0.032) in the presence of perindoprilat Figure 7.

Figure 7.

Effect of perindoprilat on expression of bradykinin B2 receptor. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out on RNA from mesangial cells exposed to MPCM perindoprilat for 18 hours. Southern blots were probed for bradykinin B2 receptor. A representative blot from 10 experiments is shown. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (46K)

Moreover, bradykinin B2 receptor binding using ³H-bradykinin supported these observations at protein level. More ³H-bradykinin (71.0 15.0%) bound to the surface of ACE-I–treated mesangial cells than nontreated cells (*P = 0.002 vs. MPCM alone) Figure 8.

Figure 8.

Effect of perindoprilat on the binding of H³-bradykinin to MPCM-treated mesangial cells. Mesangial cells were treated with MPCM perindoprilat for 18 hours. H³-bradykinin was added to the mesangial cells to determine the level of surface bradykinin B2 receptor expression. Results are expressed as mean SEM (bradykinin binding/g cell protein). P = 0.002 vs. MPCM, N = 5. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (19K)

Effect of bradykinin-B2 receptor inhibitor HOE 140

If the antifibrotic effects of perindoprilat were mediated by ligation of bradykinin to the B2 receptor, treatment with a selective bradykinin B2 receptor antagonist would be expected to attenuate the beneficial effects of the ACE-I. Incubation of mesangial cells with the bradykinin B2 receptor antagonist HOE 140 (1 mol/L) resulted in a reversal of the perindoprilat-mediated reduction in mesangial cell fibronectin levels Figure 9.

Figure 9.

Effect of bradykinin B2 receptor antagonist HOE 140 on the antifibrotic effects of perindoprilat. Mesangial cells were treated with MPCM + 40 mol/L perindoprilat 1 mol/L HOE 140 for 3 days. Culture superntatants were assayed for fibronectin by enzyme-linked immunosorbent assay (ELISA). Results are expressed as mean SEM (fibronectin levels in treated cell supernatants are expressed as a fold-increase over medium alone and corrected for cell protein). *P < 0.001 vs. MPCM, N = 6. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (31K)

Effect of perindoprilat on the tissue plasminogen activator system

The plasminogen activator system is also known to be involved in extracellular matrix degradation. Bradykinin has previously been shown to modulate levels of the fibrinolytic enzyme tPA²², while ACE-I has been shown to modulate levels of the tPA inhibitor PAI-1²². Little information is available in the literature about the effects of bradykinin or ACE-Is on urokinase plasminogen activator (uPA) activity. In order to investigate a possible link between the observed antifibrotic effects induced by perindoprilat and the expression of these agents, mRNA levels of tPA, uPA, and PAI-1 in MPCM-stimulated mesangial cells in the presence or absence of perindoprilat were examined. Northern analysis revealed a 40 8.0% (P = 0.006) increase in tPA mRNA levels following ACE inhibitor treatment. uPA and PAI-1 mRNA levels were not significantly affected Figure 10.

Figure 10.

Effect of 40 mol/L perindoprilat on plasminogen activator system (mRNA levels). Northern analyses probing for tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) were carried out on RNA extracted from mesangial cells treated with MPCM 40 mol/L perindoprilat for 18 hours. Representative blots from 3 experiments are shown. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (138K)

However, measuring total tPA protein levels by ELISA demonstrated that tPA levels in the culture supernatants of ACE-I treated cells were unexpectedly, significantly, reduced by 14.0 0.5% (P < 0.001) compared with non-ACE-I–treated cells. uPA levels were not significantly affected, while PAI-1 protein levels were reduced by 9.0 0.25% (P < 0.001) Figure 11. Exogenous bradykinin treatment of MPCM-stimulated mesangial cells followed a similar pattern. Exogenous bradykinin reduced tPA levels by 25.13 2.37% (P < 0.001), had no significant effect on uPA levels, and reduced PAI-1 levels by 18.75 1.38% (P = 0.02) compared with nonbradykinin-treated cells Figure 12.

Figure 11.

Effect of 40 mol/L perindoprilat on tissue plasminogen activator (tPA) system (protein levels). Supernatants from mesangial cells treated ACE-I were analyzed by enzyme-linked immunosorbent assay (ELISA) for expression of total protein levels of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). Results are mean SEM. tPA, uPA, PAI-1 levels in culture supernatants are expressed as % of those in untreated MPCM-stimulated mesangial cells. *P < 0.001, N = 39, 18, 20, respectively. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (68K)

Figure 12.

Effect of exogenous bradykinin on tissue plasminogen activator (tPA) system. Supernatants from mesangial cells treated with exogenous 0.1 mol/L bradykinin were analyzed by enzyme-linked immunosorbent assay (ELISA) for the components of the tissue plasminogen activator system (tPA), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). Results are mean SEM and expressed as a % of those in untreated MPCM-stimulated mesangial cells. *P = 0.007; **P = 0.02, N = 4. Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; Bk, bradykinin.

Full figure and legend (63K)

Effect of the bradykinin B2 receptor antagonist HOE-140 on plasminogen activator system

In order to observe the effect of bradykinin B2 receptor antagonism on components of the plasminogen activator system, supernatants from MPCM-stimulated mesangial cells treated with perinoprilat HOE 140 were analyzed for tPA, uPA, and PAI-1 levels by ELISA. HOE 140 treatment reversed the effects of perinoprilat on tPA and PAI-1 levels Figure 13, supporting the concept of a direct role of the bradykinin B2 receptor in modulating the components of the plasminogen activator system.

Figure 13.

Effect of bradykinin B2 receptor antagonist HOE 140 on tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) levels in perindoprilat-treated MPCM-stimulated mesangial cells. Supernatants from mesangial cells treated ACE-I bradykinin B2 receptor antagonist HOE-140 were analyzed by enzyme-linked immunosorbent assay (ELISA) for the expression of total protein levels of tPA, uPA, and PAI-1. Results are mean SEM. tPA, uPA, and PAI-1 levels in culture supernatants are expressed as a % of those in untreated MPCM-stimulated mesangial cells. P < 0.001, N = 6 to 8). Abbreviations are: MPCM, macrophage-conditioned medium; Med, medium; ACE-I, angiotensin-converting enzyme inhibitor.

Full figure and legend (45K)

DISCUSSION

ACE-I are valuable therapeutic agents for the management of hypertension, and have specific benefits in delaying the progression of chronic renal failure. While the antihypertensive action of ACE-I may account for their renoprotective effects, other mechanisms may also be important. It is, therefore, of note that in studies in both experimental animals and humans, other classes of antihypertensive agents exhibited less renoprotection than ACE-I, despite equivalent blood pressure control. Thus, it would appear that the beneficial effects of ACE-I are mediated by mechanisms in addition to, or independent of, their systemic hemodynamic actions. These may involve specific actions on intraglomerular hemodynamics, but also include direct effects on the cellular production of profibrotic cytokines and extracellular matrix. A large body of evidence supports the hypothesis that the effects of ACE inhibitors result from the inhibition of Ang II formation. However, accumulating data also suggest that some of their actions may be caused by reduced bradykinin catabolism, with resultant accumulation of endogenous bradykinin levels²³.

The current study has demonstrated that the antifibrotic effects of the ACE-I perindoprilat, in the context of macrophage-induced mesangial cell injury, are mediated, at least in part, by effects on the bradykinin axis. RT-PCR demonstrated that mesangial cells have the necessary biochemical machinery to generate endogenous kinins. Moreover, we demonstrated that MPCM treatment reduced basal mRNA levels of the bradykinin precursor low kininogen, which were partially restored following treatment with ACE-I. Addition of exogenous bradykinin reduced MPCM-induced fibronectin levels in mesangial cells, bradykinin B2 receptor mRNA levels were up-regulated in the presence of perindoprilat, as was binding of [H]³-bradykinin. Furthermore, inhibition of B2 receptor activity with HOE 140 attenuated the beneficial effects of perindoprilat treatment. Bradykinin exerts most of its physiologic effects via the B2 receptor, which is constitutively expressed in many cell types²⁴. There is increasing experimental evidence to suggest that ACE-I may potentiate the effects of bradykinin using mechanisms that are independent of their ability to inhibit ACE activity, per se^{25,26,27,28,29}. For example, ACE inhibitors can potentiate bradykinin activity in the presence of ACE-resistant bradykinin B2 receptor agonists²⁶. Furthermore, the bradykinin-potentiating effects of ACE-inhibitors are not mimicked by the synthetic ACE substrate hippuryl-L-histidyl-L-leucine, which is as equally effective at blocking bradykinin catabolism as are ACE-I^26,27. Moreover, when B2 receptors are desensitized and no longer responsive to extra agonist, ACE inhibitors can reactivate B2 receptor-mediated signaling³⁰. Several possibilities may explain these phenomena. There is evidence to suggest that ACE -I may exert their effect directly on the bradykinin-B2 receptor^26,27, although ACE-I binding to receptor is yet to be demonstrated³¹. It has also been suggested that binding of ACE-I to ACE results in a conformational change, which is transduced directly to the B2 receptor in a sort of ACE:B2 receptor "cross-talk"^30,32. It has been suggested that the variability in ACE-I efficacy, as seen with different molecules of the same class, may be dependent on the ACE-I's unique structural properties, which are able to facilitate bradykinin B2 receptor signaling²⁵.

In the current study, bradykinin levels were significantly reduced in day 3 culture supernatants of mesangial cells treated with both MPCM and ACE-I compared with MPCM alone, results which were contrary to what might have been expected following inhibition of bradykinin catabolism. However, these observations may be explained when increased ligation to an increased number of receptors is taken into account.

The small increase in endogenous bradykinin levels over control observed in response to MPCM alone was probably insufficient to have a beneficial effect on mesangial cells comparable to that of adding exogenous bradykinin. Moreover, this slight increase is likely to be under post-transcriptional control because mRNA levels of the bradykinin precursor low kininogen in response to MPCM were lower than basal mRNA levels.

Bradykinin has been shown to be a potent stimulator of tPA in endothelial cells³³, perfused tissue preparations³⁴, and in whole animals³⁵. A study by Brown et al³⁵ demonstrated that intravenous infusion of bradykinin caused a significant increase in plasma levels of tPA, which was further potentiated by treatment with ACE-I. Bradykinin appeared to have no effect on PAI-1 levels³⁵. Schanstra et al demonstrated that transgenic rats overexpressing increased endogenous bradykinin exhibited reduced interstitial fibrosis in the unilateral ureteral obstruction model of renal injury. Moreover, they demonstrated that genetic manipulation or pharmacologic blockade of the bradykinin B2 receptor increased interstitial fibrosis, and was accompanied by reduced activity of extracellular matrix degrading enzymes³⁶.

In the present study, no measurable difference in the expression of the matrix metalloproteinases 2, 3, or 9 following ACE-I treatment could be detected. However, this was not the case for components of the plasminogen activator system. Protein expression profiles of tPA and PAI-1 were altered in response to ACE-I treatment. The protein profiles differed from mRNA expression levels, suggesting that the cells' plasminogen activator phenotype was modulated post-transcriptionally. This pattern of plasminogen activator system expression following ACE treatment was echoed by addition of exogenous bradykinin. Moreover, antagonism of the bradykinin receptor resulted in a reversal of the effects of perindoprilat on plasminogen activator components. Together, these data lend support to the hypothesis that ACE inhibition, with its resulting reduction in extracellular matrix expression, involves bradykinin, its receptor, and the plasminogen activator system acting in a coordinated manner.

The apparent paradoxic reduction in tPA protein levels, though unexpected, may be explained by an increased turnover of the protease via the ubiquitous low-density lipoprotein receptor-related protein (LRP)³⁷. LRP is a multifunctional endocytic receptor implicated in the modulation of a number of cellular processes, including extracellular matrix accumulation and catabolism, and the turnover of proteases, and protease/inhibitor complexes. Noguchi et al have previously demonstrated that in mouse hepatocytes interleukin-1 (IL-1) was able to up-regulate tPA mRNA levels while concomitantly down-regulating tPA activity and protein levels by up-regulating the expression of LRP³⁷. A similar scenario may account for our observations. However, investigations into this phenomenon are beyond the scope of the current study.

CONCLUSION

This study provides further insight into the potential mechanisms underlying the unique renoprotective actions of ACE-I. It demonstrates the importance of bradykinin, its B2 receptor, and the plasminogen activator system in modulating the renoprotective effects of ACE-I.

References

References

1.	ANDERSON S, RENNKE HG & BRENNER BM. Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat. J Clin Invest 1985; 77: 1993–2000.
2.	PARVING HH, ANDERSEN AR, SMIDT UM & SVENDSEN PA. Early aggressive antihypertensive treatment reduces rate of decline in kidney function in diabetic nephropathy. Lancet 1983; 1: 1175–1179. | PubMed | ISI | ChemPort |
3.	ALVESTRAND A, GUTIERREZ A, BUCHT H & BERGSTROM J. Reduction of blood pressure retards the progression of chronic renal failure in man. Nephrol Dial Transplant 1988; 3: 624–631. | PubMed |
4.	BRAZY PC, STEAD WW & FITZWILLIAM JF. Progression of renal insufficiency: Role of blood pressure. Kidney Int 1989; 35: 670–674. | PubMed | ISI | ChemPort |
5.	CAMPESE VM & BIGAZZI R. The role of hypertension in the progression of renal disease. Am J Kidney Dis 1991; 17: 43–47. | PubMed | ISI | ChemPort |
6.	MATHIESON ER, HOMMEL E & GIESE E et al. Efficacy of captopril in postponing nephropathy in normotensive insulin dependent diabetic patients with microalbuminuria. BMJ 1991; 303: 81–87. | PubMed |
7.	JACKSON B & JOHNSTON CI. The contribution of systemic hypertension to progression of chronic renal failure in the rat remnant kidney. Effect of treatment with angiotensin converting enzyme inhibitor or a calcium inhibitor. J Hypertens 1988; 6: 495–501. | PubMed | ChemPort |
8.	RUILPE LM, MIRANDA M & MORALES JM. Converting enzyme inhibition in chronic renal failure. Am J Kidney Dis 1989; 13: 120–126. | PubMed |
9.	DZAU VJ & INGELFINGER JR. Molecular biology and pathophysiology of the intrarenal renin and angiotensin system. J Hypertens 1989; 7: S3–S8. | ChemPort |
10.	ARAI M, WADA A & ISAKA Y et al. In vivo transfection of genes for renin and angiotensinogen into the glomerular cells induced phenotypic change of the mesangial cells and glomerular sclerosis. Biochem Biophys Res Comm 1995; 206: 525–532. | PubMed |
11.	KAGAMI S, BORDER WA, MILLER DE & NOBLE NA. Angiotensin II stimulates extracellular matrix protein synthesis through induction of transforming growth factor- expression in rat glomerular mesangial cells. J Clin Invest 1994; 93: 2431–2437. | PubMed | ISI | ChemPort |
12.	REGIOLI D & BARABE J. Pharmacology of bradykinin and related kinins. Pharmacol Rev 1980; 32: 1–46. | PubMed | ISI | ChemPort |
13.	BAUMGARTEN CR, LINZ W & KUNKEL G et al. Ramiprilat increases bradykinin out-flow from the hearts of rat. Br J Pharmacol 1993; 108: 293–295. | PubMed | ISI | ChemPort |
14.	PAWLUCZYK IZA & HARRIS KPG. Macrophages promote pro-sclerotic responses in cultured rat mesangial cells. A mechanism for the initiation of glomerulosclerosis. J Am Soc Nephrol 1997; 8: 1525–1536. | PubMed | ISI | ChemPort |
15.	BURTON CJ, COMBE C, WALLS J & HARRIS KPG. Fibronectin production by human tubular cells: The effect of apical protein. Kidney Int 1996; 50: 760–767. | PubMed | ISI | ChemPort |
16.	WILSON HM, HAITES NE & BOOTH NA. Opposing effects of interleukin-1 and transforming growth factor beta on the regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 expression by human mesangial cells. Exp Nephrol 1997; 5: 233–238. | PubMed |
17.	WUN T-C & KRETZMER KK. cDNA cloning and expression in E. coli of a plasminogen activator inhibitor (PAI) related to a PAI produced by Hep G2 hepatoma cell. FEBS Lett 1987; 210: 11–16. | Article | PubMed | ISI | ChemPort |
18.	HERMANN A, BRAUN A & FIGUEROA CD et al. Expression and cellular localisation of kininogens in the human kidney. Kidney Int 1996; 50: 79–84. | PubMed |
19.	SONG Q, WANG D-Z & RUSSELL A et al. Cellular localisation of low-molecular-weight kininogen and bradykinin B2 receptor mRNAs in human kidney. Am J Physiol 1996; 270: F919–F926. | PubMed |
20.	WOL WC, HARLEY RA & SLUCE D et al. Localisation and expression of tissue kallikrein and kallistatin in human blood vessels. J Histochem Cytochem 1999; 47: 221–228. | PubMed | ISI | ChemPort |
21.	SMITH D, GILBERT M & OWEN WG. Tissue plasminogen activator release in vivo in response to vasoactive agents. Blood 1985; 66: 835–839. | PubMed |
22.	OIKAWA T, FREEMAN M & LO W et al. Modulation of plasminogen activator inhibitor-1 in vivo: A new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition. Kidney Int 1997; 51: 164–172. | PubMed | ISI | ChemPort |
23.	WIEMER G, SCHOLKENS BA, BECKER RHA & BUSSE R. Ramiprilat enhances endothelial autacoid formation by inhibiting breakdown of endothelium derived bradykinin. Hypertension 1997; 18: 558–563.
24.	MARGOLIUS HS. Kallikreins and kinins: Some unanswered questions about system characteristics and roles in human disease. Hypertension 1995; 26: 221–229. | PubMed | ChemPort |
25.	HECKER M, BLAUKAT A & BARA A et al. ACE inhibitor potentiation of bradykinin-induced venoconstriction. Br J Pharmacol 1997; 121: 1475–1481. | PubMed |
26.	HECKER M, PORSTI I, BARA AT & BUSSE R. Potentiation by ACE inhibitors of the dilator response to bradykinin in the coronary microcirculation: interaction at the receptor level. Br J Pharmacol 1994; 111: 238–244. | PubMed |
27.	BOSSALLER C, AUCH-SCHWELK W & GRAFE M et al. Effects of converting enzyme inhibition on endothelial bradykinin metabolism and endothelium vascular relaxation. Agents Actions 1992; 38 Suppl: 171–177.
28.	CAMPBELL DJ, KLADIS A & DUNCAN AM. Effects of converting enzyme inhibitors on angiotensin and bradykinin peptides. Hypertension 1994; 23: 439–449. | PubMed | ISI | ChemPort |
29.	MINSHALL RD, TAN F & NAKAMURA F et al. Potentiation of the actions of bradykinin by angiotensin converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I converting enzyme in CHO cells. Circ Res 1997; 81: 848–856. | PubMed | ISI | ChemPort |
30.	BENZING T, FLEMING I & BLAUKAT A et al. Angiotensin-converting enzyme inhibitor Ramiprilat interferes with the sequestration of the B2 kinin receptor within the plasma membrane of native endothelial cells. Circ 1999; 99: 2034–2040.
31.	BUSSE R & FLEMMING I. Molecular responses of endothelial tissue to kinins. Diabetes 1996; 45: S8–S13. | PubMed | ISI |
32.	BUSSE R & LAMONTAGNE D. Endothelium-derived bradykinin is responsible for the increase in calcium produced by angiotensin-converting enzyme inhibitors in human endothelial cells. Naunyn Schmiedebergs Arch Pharmacol 1991; 344: 126–129. | PubMed | ChemPort |
33.	EMEIS JJ & TRANQUILLE N. The role of bradykinin in secretion from vascular endothelial cells. Agents Actions 1992; 38 Suppl: 285–291.
34.	MARKWARDT F & KLOCKING HP. Studies on the release of plasminogen activator. Throm Res 1983; 30: 195–203.
35.	BROWN NJ, NADEAU JH & VAUGHAN DE. Selective stimulation of tissue type plasminogen activator (tPA) in vivo by infusion of bradykinin. Thromb Haemost 1997; 77: 522–525. | PubMed | ChemPort |
36.	SCHANSTRA JP, NEAU E & DROGOZ P et al. Bradykinin reduces renal interstitial fibrosis by increasing extracellular matrix degradation. J Clin Invest 2002; 110: 371–379. | Article | PubMed | ISI | ChemPort |
37.	NOGUCHI T, NOGUCHI M & MASUBUCHI H et al. IL-1 down regulates tissue-type plasminogen activator by up-regulating low density lipoprotein receptor-related protein in AML 12 cells. Biochem Biophys Res Com 2001; 288: 42–48. | PubMed |

Acknowledgments

This study was funded by the National Kidney Research Fund of Great Britain, grant no. R19/2/98. Parts of this study were presented in abstract form at the ASN/ISN World Congress of Nephrology, San Francisco, California, 2001.