Glomerular expansion during glomerulosclerosis resulting from mesangial cell (MC) hypertrophy and mesangial matrix accumulation is believed to be a key event leading to diabetic nephropathy. Insulin-like growth factor-I (IGF-I) and to a lesser extent insulin, both induce MC proliferation and collagen secretion most likely via activation of the IGF-I receptor. Insulin thus may be involved in the early steps of the glomerulosclerotic process1,2,3,4. The renoprotective effect of angiotensin-converting enzyme (ACE) inhibitors on the progression of several renal diseases, including diabetic nephropathy, is supported by a large number of reports5,6,7,8,9,10,11,12. Several mechanisms were proposed to account for the beneficial effects of ACE inhibitors. ACE inhibitors can inhibit the angiotensin II (Ang II)-induced increase in glomerular capillary resistance but also act on the Ang II-stimulated secretion of matrix components by renal cells, independently of any hemodynamic effects. Besides the Ang II-dependent mechanism, ACE inhibitors reduce the enzymatic degradation of bradykinin (BK), thereby favoring the stimulation of the B2 receptor (B2 receptor). This is an additional potential mechanism in the protective role of ACE inhibitor that has been poorly investigated. In order to document the role of Ang II in the development of diabetic glomerulosclerosis, the combined effects of Ang II and insulin on their signaling pathways has been investigated in mesangial cells (MCs)13. It was established that Ang II and insulin are additive in stimulating the expression of transforming growth factor-beta (TGF-
), a major prosclerotic cytokine up-regulated by hyperglycemia. In contrast, possible cross-talk between BK and insulin in the kidney has never been investigated. In the past years, our group has documented in detail the presence of the BK receptors in the kidney and in cultured MCs [reviewed in14]. Initially, we observed that B2 receptor stimulation induced proliferation of quiescent MCs15, whereas our subsequent study demonstrated that BK also was able to reduce MC proliferation induced by fetal calf serum (FCS)16. These opposite effects of BK have been confirmed by other groups. The stimulating effect of BK on mitogen-activated protein (MAP) kinase has been demonstrated in MCs17,18, smooth muscle cells19 and in cell lines overexpressing the B2 receptor20,21. More recently BK-induced inhibition of smooth muscle cell proliferation was demonstrated; however, while this effect was independent of phospholipase C (PLC) and phospholipase A2 (PLA2), the authors were not able to propose a more precise mechanism22,23.
We hypothesized that the effect of BK on cell proliferation may be dependent upon growth factor receptor activation. IGF-I is a peptide growth factor synthesized by many cells and tissues including MCs. An increase in renal IGF-I levels has been implicated during the development of diabetic glomerulosclerosis by promoting cell proliferation and collagen secretion24,25,26. In the present article, we extend the knowledge of the mechanism of BK-induced activation of MAP kinase in MCs in the absence and the presence of IGF-I. We found that BK alone induced a transient activation of Erk 1 and 2, whereas it significantly reduced IGF-I-induced Erk 1 and 2 activation, both in MCs and in IG. We propose a mechanism for this inhibitory cross-talk involving a calcium-dependent tyrosine phosphatase. Finally, we were able to demonstrate that BK reduced MC proliferation induced by IGF-I.
METHODS
Rat MC cultures
Mesangial cells were obtained as outgrowths of decapsulated collagenase-digested glomeruli obtained by graded sieving as described previously27. Glomerular explants were allowed to grow to confluency at 37°C in a humidified atmosphere of 5% CO2 in RPMI 1640 medium containing 15% FCS, 100 U/mL penicillin, 100 
g/mL streptomycin and D-valine substituted for L-valine to prevent fibroblast development. With this method, MCs appear in the culture after two to three weeks. They were characterized as previously described by morphological criteria, that is, the presence of multilayers, resistance to puromycin (10 g/mL), sensitivity to mitomycin (5 g/mL), presence of myosin filaments revealed by specific antibodies, presence of actin filaments revealed by fluorescent NBD phallacidin, positive staining for the MC marker Thy-1.1, negative staining for the endothelial cell marker factor VIII, and functional criteria such as an increase in intracellular calcium induced by Ang II27. Experiments were performed with primary cultures and passaged cells (passages 4 to 10).
Cell extract preparation
Confluent cells were washed three times with serum-free culture medium and exposed to various agents in serum-free culture medium at 37°C. The reactions were stopped by rapid aspiration of the medium and rinsing three times with ice-cold phosphate-buffered saline (PBS) containing 1 mmol/L sodium orthovanadate. Cells were lysed under stirring for 30 minutes in 1 mL of ice-cold lysis buffer [10 mmol/L Tris/HCl (pH 7.4 containing 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L egtazic acid (EGTA), 0.2 mmol/L orthovanadate, 1% NP 40 (vol/vol), 1% Triton X-100 (vol/vol), 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 g/mL leupeptin, 1 g/mL aprotinin]. Insoluble material was removed by centrifugation at 13,000 rpm for 20 minutes. Protein concentration was determined by the Bradford protein assay. Proteins of the soluble extract were boiled in Laemli buffer for five minutes at 100°C and stored frozen until sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Isolated glomerulus preparation
Freshly isolated glomeruli are the appropriate ex vivo preparation to extend the in vitro observations obtained with cultured MCs to a more physiological environment. Isolated glomeruli were prepared as routinely performed in the laboratory by graded sieving. Briefly, male Sprague-Dawley rats (12 weeks of age) were exsanguinated and the kidneys were quickly removed. The cortex was forced through three consecutive steel sieves with decreasing pore sizes (180, 125 and 75 m) and the glomeruli were collected on the 75 m sieve. About 12,000 glomeruli per kidney were obtained. Under light microscopy, more than 90% of the glomeruli appeared to be decapsulated and free of surrounding tubules and arterioles. The glomeruli were resuspended in RPMI culture medium and redistributed in experimental tubes containing about 5000 glomeruli per tube. After the appropriate incubation time at 37°C in the presence of various concentrations of BK and/or IGF-I, the incubation was stopped by adding 1 mL of ice-cold PBS containing 1 mmol/L of the tyrosine phosphatase inhibitor orthovanadate. Then the tubes were centrifuged (15,000 rpm, 4°C, 2 min) and the supernatant was discarded. The pellet containing the glomeruli was resuspended in 100 L of lysis buffer, sonicated for 10 seconds and centrifuged (15,000 rpm, 4°C, 15 min). Insoluble material was removed and the proteins of the soluble extracts were boiled in Laemli buffer for five minutes and stored frozen until SDS-PAGE. Protein concentration was determined by the Bradford protein assay.
SDS-PAGE and Western blotting
Equal amounts of proteins (30 g) were separated by 10% SDS-PAGE in Tris/glycine buffer under 150 volts and 30 mA current in a Bio-Rad miniature gel apparatus (Mini-protean; Bio-Rad Laboratories, Richmond, CA, USA). Proteins were then transferred to a nitrocellulose membrane (Amersham SA, Orsay, France). The membrane was blotted with the appropriate antibody. Proteins were visualized using a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin and an enhanced chemiluminescence (ECL) kit (Amersham SA).
Mitogen-activated protein kinase activity
Mitogen-activated protein kinase activity was assessed by Western blotting using antiphospho-Erk antibodies (Promega, Madison, WI, USA), which recognize the active form of Erk 1 (MAP-kinase 1, molecular wt = 44 kD) and Erk 2 (MAP kinase 2, molecular wt = 42 kD). The amount of total Erk also was visualized as a control using an antibody that recognizes total Erk 1 and 2 proteins (Santa Cruz Biotechnology, Le Perray, France) independently of their level of phosphorylation. In a preliminary study, we established that in our experimental conditions the detection of MAP kinase activity by this method was highly correlated with the incorporation of radioactive phosphorus in the myelin basic protein.
Proliferation studies
Mesangial cell proliferation was studied by counting the number of cells (Coulter cell counter ZM; Flow Laboratory, Paris, France). MCs were seeded in 12-well plates (Nunc; Polylabo, Strasbourg, France) at a density of 40,000 cells per well. After a 48-hour initial period in the presence of culture medium containing 15% FCS to allow adhesion of the cells, they were rendered quiescent by 48 hours of serum deprivation in 0.5% FCS medium. Then the effects of IGF-I in the presence and absence of BK were tested. The number of cells in triplicate wells was determined after 48 or 72 hours as previously described15. The results are expressed as absolute values 
SEM.
Statistical analysis
Results are expressed as mean SEM. The t test for unpaired data was used for comparisons between two groups. For multiple comparisons, results were analyzed using a one-way analysis of variance (ANOVA) with a Tukey-Kramer post-hoc test. Means were considered to be significantly different when P < 0.05. All analyses were performed with Statview 5® software (SAS Institute Inc., Cary, NC, USA).
RESULTS
Effect of BK on Erk 1 and 2 phosphorylation in quiescent mesangial cells
The dose- and time-dependent effects of BK on the phosphorylation of Erk 1 and 2 were studied by Western blot analysis Figure 1. Under our cell culture conditions, BK induced Erk 1 and 2 phosphorylation in a dose-dependent manner from 1 to 100 nmol/L, reaching a maximum stimulation of sixfold Figure 1a. The BK-induced stimulation of Erk 1 and 2 phosphorylation was transient, and the maximum sixfold stimulation was observed five minutes after stimulation with 100 nmol/L BK Figure 1b. After 10 minutes of stimulation with BK, Erk 1 and 2 phosphorylation returned to levels detected in the absence of BK. Total Erk 1 and 2 expression, studied with an antibody against phosphorylated and non-phosphorylated forms of Erk, remained unchanged during stimulation with BK. In order to determine the receptor subtype involved in the BK-induced stimulation of Erk 1 and 2 phosphorylation, MCs were pretreated with B1 and B2 antagonists, as shown in Figure 1c. The B1 and B2 antagonists had no effect on Erk 1 and 2 phosphorylation when given alone (Figure 1c, lanes 15 and 16). Pretreatment of MCs with 1 mol/L of the B2 antagonist HOE-140 (Figure 1c, lane 14) but not with 1 mol/L of the B1 antagonist des-Arg9-Leu8-BK (Figure 1c, lane 17), completely abolished the BK-induced stimulation of Erk 1 and 2 phosphorylation (Figure 1c, lane 14 vs. lane 13).
Figure 1.
Dose- (A) and time-dependent (B) effect of bradykinin (BK) and pharmacological profile (C) of BK activation of Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated: (A) for 5 min in the presence of increasing concentrations of BK (0 to 1000 nmol/L); (B) in the presence of 100 nmol/L BK for different times (from 1 to 10 min); (C) in the presence of 100 nmol/L BK with either 1 mol/L of the B2 antagonist HOE 140 (B2A) or 1 mol/L of the B1 antagonist des-Arg9-Leu8-BK (B1A). Erk 1 and 2 phosphorylation (P-Erk) was measured by Western blotting with an antibody against the phosphorylated forms. Analyses were performed on an equal amount of protein (30 g) as measured with an antibody against total-Erk (phosphorylated and non-phosphorylated forms). Erk 1 (
) and Erk 2 (
) phosphorylation (P-Erk 1 and 2) was expressed by the fold increase of the ratio P-Erk versus total-Erk and is shown as mean SE of the scanning densitometry of five experiments. *P < 0.05; **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lane 13).
Involvement of protein kinase C via a pertussis toxin-dependent signaling pathway in the BK-induced phosphorylation of Erk 1 and 2
When cells were pretreated for 24 hours with pertussis toxin (PTX; 100 ng / mL), the stimulating effect of 100 nmol/L BK on Erk 1 and 2 phosphorylation was inhibited (Figure 2, lane 4 vs. lane 2) whereas PTX alone had no effect (Figure 2, lane 3). In addition, the stimulating effect of 100 nmol/L BK also was completely abolished by 10 mol/L U73122, a phospholipase C (PLC) inhibitor (Figure 2, lane 8 vs. lane 6). Next, we investigated the involvement of protein kinase C (PKC; Figure 3). A five-minute treatment with 0.3 mol/L phorbol 12-myristate 13-acetate (PMA), resulting in translocation of isoforms 
, 
and 
but not 
(data not shown), induced a 35-fold increase in Erk 1 and 2 phosphorylation (Figure 3a, lane 1 vs. lane 3). In these conditions where PKC was highly stimulated, no additional stimulation of Erk 1 and 2 phosphorylation was observed in response to stimulation with 100 nmol/L BK for five minutes (Figure 3a, lane 2 vs. lane1). The stimulating effect observed after a five minute treatment with 100 nmol/L BK was prevented by prior incubation with 5 mol/L PKC inhibitor GF 109203X (Figure 3b, lane 7 vs. lane 5). Moreover, when the MCs were pre-incubated for 18 hours with 0.3 mol/L PMA, a situation associated with complete down-regulation of PKC (data not shown), the stimulating effect of BK on Erk 1 and 2 was no longer observed (Figure 3c, lane 11 vs. lane 9). All these data indicate that Erk 1 and 2 phosphorylation can be directly triggered by activation of PKC and that BK-induced Erk1 and 2 phosphorylation is mediated via PLC and PKC recruitment. In all these experiments the total form of Erk 1 and 2 remained unchanged.
Figure 2.
Effects of pertussis toxin (PTX) and phospholipase C inhibitor (U73122) on BK-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated in the presence or absence of 100 nmol/L BK for 5 min and in the presence of PTX (lanes 3 and 4) or of 10 nmol/L U73122 (lanes 7 and 8). Western blotting was performed as described in the legend of Figure 1. Symbols are: () Erk 1; () Erk 2. N = 5; *P < 0.05, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lanes 2 and 6).
Figure 3.
Effects of protein kinase C stimulation (A) and inhibition (B and C) on BK-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated: (A) in the presence or absence of 100 nmol/L BK for 5 min either in the presence of PMA for 5 min (lanes 1 and 2); (B) in the presence of GF109203, a PKC inhibitor (lanes 6 and 7); and (C) after pretreatment with PMA for 18 hours (lanes 10 and 11). Western blotting was performed as described in the legend of Figure 1. Symbols are: () Erk 1; () Erk 2. N = 5, *P < 0.05, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lanes 5 and 9).
BK-induced phosphorylation of Erk 1 and 2 requires tyrosine kinase activity
To determine whether a tyrosine kinase activity was involved in BK-induced Erk 1 and 2 phosphorylation, we evaluated the effects of two different tyrosine kinase inhibitors: PD098059, an inhibitor of MEK1 (MAP kinase kinase); and PPI, an inhibitor of the src protein kinase family. Figure 4 shows that both 20 mol/L PPI (Figure 4, lanes 3 and 4) and 50 mol/L PD098059 (Figure 4, lanes 7 and 8) abolished the stimulating effect of BK, indicating that a tyrosine kinase activity also is responsible for the action of the B2 receptor on MAP kinase activation. The total forms of Erk 1 and 2 remained unchanged in all experiments.
Figure 4.
Effect of tyrosine kinase and src kinase inhibition on BK-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated in the presence or absence of 100 nmol/L BK for 5 min and PPI, an inhibitor of the src family protein (lanes 3 and 4) or PD090059, an Erk 1 and 2 kinase (MEK) inhibitor (lanes 7 and 8). Western blotting was performed as described in the legend of Figure 1. Symbols are: () Erk 1; () Erk 2. N = 5, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lanes 2 and 6).
Effect of BK on Erk 1 and 2 activation in MCs stimulated with IGF-I
Figure 5a demonstrates that IGF-I induced a dose-dependent increase in Erk 1 and 2 phosphorylation in FCS-free incubation medium. Moreover, when the effects of IGF-I were compared with that of insulin (data not shown), it appeared that both insulin and IGF-I stimulated phosphorylation of Erk 1 and 2. However, IGF-I showed a much higher efficacy (10 nmol/L) than insulin (1 mol/L) confirming previous results indicating that insulin probably acts through the stimulation of the IGF-I receptor in MCs2,3. Interestingly, when MCs were pretreated with 100 nmol/L BK for five minutes prior to stimulation with 10 mol/L IGF-I, the maximum IGF-I–induced stimulation of Erk 1 and 2 phosphorylation (Figure 5b, lane 5) was significantly and dose-dependently reduced to reach a 60 8% (P < 0.05) maximum reduction (Figure 5b, lanes 6 to 8). This effect was prevented by B2 antagonist (Figure 5c, lanes 10 to 12 vs. lanes 7 and 8), which had no effect alone on IGF-I (Figure 5c, lane 9). Moreover, the level of total Erk 1 and 2 remained unaffected.
Figure 5.
Effects of BK on IGF-I-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated for 5 min: (A) in the presence of increasing concentrations of IGF-I from 0.1 to 10 nmol/L (lanes 2 to 4); (B) in the presence of 10 nmol/L IGF-I and increasing concentrations of BK from 0.1 to 10 nmol/L (lanes 6 to 8); (C) in the presence of 10 nmol/L IGF-I and increasing concentrations of BK from 0.1 to 10 nmol/L and 1 mol/L of the B2 antagonist HOE 140 (lanes 9 to 12). Western blotting was performed as described in the legend of Figure 1. Symbols are: () Erk 1; () Erk 2. N = 5, *P < 0.05, **P < 0.01 P < 0.01 when compared to the level of P-Erk in the absence of BK (lane1). °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 10 nmol/L IGF-I (lane 5). ++P < 0.01 when compared to the level of P-Erk in the presence of IGF-I and BK (lanes 7 and 8).
Involvement of a calcium-dependent tyrosine phosphatase
As the decrease in Erk 1 and 2 phosphorylation suggests the activation of tyrosine phosphatase, we tested the effect of both serine-threonine and tyrosine phosphatase inhibitors. The inhibitory effect of 100 nmol/L BK on 10 mol/L IGF-I-induced Erk 1 and 2 phosphorylation (Figure 6; lane 3 vs. lane 2) was not altered by 100 mol/L okadoic acid, a serine-threonine phosphatase inhibitor (Figure 6, lane 5 vs. lane 3), okadoic acid alone having no effect on the stimulation by 10 mol/L IGF-I (Figure 6, lane 4 vs. lane 2). In contrast, incubation in the presence of the tyrosine phosphatase inhibitor orthovanadate significantly increased the levels of phosphorylated Erk 1 and 2 (Figure 6, lane 6 vs. lane 2) and interestingly abolished the inhibitory effect of BK on IGF-I–induced phosphorylation of Erk 1 and 2 (Figure 6, lanes 7 and 8 vs. lane 3). In addition, incubation in the presence of the intracellular calcium chelator BAPTA/AM, increased the level of P-Erk 1 and 2 after stimulation by IGF-I (Figure 6, lane 12 vs. lane 10), but also completely abolished the inhibition by BK of Erk 1 and 2 phosphorylation (Figure 6, lane 13 vs. 11). The specific effect of BK and BAPTA/AM on phosphorylated Erk 1 and 2 is demonstrated by the unchanged levels of total Erk 1 and 2.
Figure 6.
Effects of orthovanadate, okadoic acid and BAPTA/AM on BK-induced reduction of Erk 1 and 2 phosphorylation stimulated with IGF-I. Serum-depleted rat MCs were incubated in the presence or absence of IGF-I for 5 min (lanes 2 to 8 and 10 to 13), 100 nmol/L BK (lanes 3, 5, 7,8, 11 and 13), okadoic acid, an inhibitor of serine phosphatase (lanes 4 and 5), orthovanadate, an inhibitor of tyrosine phosphatase (lanes 6 to 8), and BAPTA/AM, a chelator of intracellular calcium (lanes 11 and 13). Western blotting was performed as described in the legend of Figure 1. N = 5, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 10 nmol/L IGF-I (lanes 2 and 4). ++P < 0.01 when compared to the level of P-Erk in the presence of IGF-I and BK (lanes 3 and 11).
Full figure and legend (67K)Effect of BK on IGF-I induced MC proliferation
The effect of BK on MC proliferation is shown in Figure 7. As expected in quiescent MCs, only a high concentration of BK (1 mol/L) induced a slight increase in MC proliferation Figure 7a. In MCs that were serum-depleted to remove stimuli for cell proliferation, IGF-I induced time- and dose-dependent MC proliferation (not shown). The effect of BK was tested in the presence of the peak IGF-I stimulation (10 nmol/L for 48 h). It can be shown that the IGF-I–induced stimulation of cell proliferation was significantly and dose-dependently reduced in the presence of BK (1 nmol/L to 1 mol/L; Figure 7 b). Moreover, no evidence of cell detachment or change in cell viability could be detected.
Figure 7.
Effects BK on MC proliferation. MCs were plated at a density of 40,000 cells per well and were stimulated with increasing concentrations of BK for 48 hours in two different conditions. (A) MCs cultured in RPMI medium containing 0.5% FCS. (B) MCs cultured in RPMI medium containing 10 nmol/L IGF-I. Cell proliferation is shown as percent of control values obtained in the absence of BK and is expressed as mean SE of five experiments. *P < 0.01, **P < 0.01 when compared to proliferation in the absence of BK.
Effect of BK on IGF-I induced Erk 1 and 2 activation in isolated glomeruli
To extend the physiological significance of the inhibitory effect of BK on Erk phosphorylation, we performed similar experiments with freshly isolated rat glomeruli. As shown in Figure 8, when given alone, BK, IGF-I and insulin induced transient phosphorylation of Erk 1 and 2 that peaked at two minutes for BK (Figure 8a, lane 3) and between two and five minutes for IGF-I (Figure 8b, lanes 8 to 10) and insulin (Figure 8c, lanes 14 to 16). However, insulin concentrations higher than those of IGF-I are required to induce similar Erk 1 and 2 phosphorylation levels. In addition, as observed in cultured MCs, BK lowered IGF-I–induced Erk 1 and 2 phosphorylation (Figure 8d, lane 22 vs. 21) without any change in the level of expression of the total forms of Erk 1 and 2. Finally, the inhibitory effect of BK was absent in the presence of orthovanadate (Figure 8d, lanes 25 and 26). These data confirm that the BK-induced inhibition of Erk phosphorylation promoted by IGF-I operates by a mechanism found both in cultured MCs and in IG, indicating that this inhibitory loop is physiologically relevant.
Figure 8.
Time-dependent effect of bradykinin (BK), IGF-I and insulin on Erk 1 and 2 phosphorylation in isolated glomeruli. Isolated glomeruli were incubated in the presence of: (A) 100 nmol/L BK; (B) 65 nmol/L IGF-I; (C) 1 mol/L insulin for increasing times from 0.5 to 20 min; (D) in the presence of 65 nmol/L IGF-I, 100 nmol/L BK and orthovanadate (O-vanadate). Western blotting was performed as described in the legend of Figure 1. Symbols are: () Erk 1; () Erk 2. N = 5, *P < 0.05; **P < 0.01 when compared to the level of P-Erk in the absence of growth factor. °°P < 0.01 when compared to maximal P-Erk level detected in glomeruli stimulated with 10 nmol/L IGF-I (lane 21). ++P < 0.01 when compared to the level of P-Erk in the presence of IGF-I and BK (lane 22).
DISCUSSION
The rationale for this study was to test the hypothesis of the involvement of BK as a possible negative modulator of IGF-I signaling pathways and IGF-I–stimulated cell proliferation. Erk 1 and 2 activity is stimulated during the early phases of hyperglycemia in diabetic rats27 and in MCs exposed to elevated glucose concentrations28. The contribution of IGF-I–induced Erk 1 and 2 activation and cell proliferation is proposed as an essential initial event in the development of diabetic nephropathy. IGF-I is a growth factor locally released by MCs in the glomerulus during the development of diabetic nephropathy and acts in an autocrine and paracrine fashion29. Therefore, we tested the effect of BK on IGF-I–induced Erk 1 and 2 activation.
Although a BK-induced inhibition of fibroblast division stimulated by a mixture of IGF-I and EGF has been reported30, our study documents a possible mechanism of an inhibitory effect of BK on IGF-I–induced Erk 1 and 2 activation and cell proliferation. Interestingly, the inhibitory effect of BK is not only observed in vitro in cultured MCs, but also is detectable in a physiological renal structure such as the isolated glomerulus. However, the importance of IGF-I in the development of glomerulosclerosis of diabetic nephropathy could still appear controversial. The main argument against a noxious action of IGF-I in diabetes mellitus is the initial observation showing that transgenic mice overexpressing growth hormone (GH) develop glomerulosclerosis whereas transgenic mice overexpressing IGF-I do not31. Since that initial observation, the same group revised the mechanism of GH-induced glomerulosclerosis and suggested the involvement of both GH and IGF-I during low-protein-induced reduction of glomerulosclerosis32. The same authors also reported that transgenic mice for GH exhibited elevated plasmatic IGF-I concentrations associated with glomerulosclerosis in spite of normal glycemia33. Moreover, several studies have pointed out that IGF-I acts as an initiation factor by inducing MC proliferation3,34 and collagen secretion4,29,35, two major contributors to the development of diabetic glomerulosclerosis, whereas similar direct effects of GH on MCs have never been reported. Finally, inhibition of early renal growth in diabetic and uninephrectomized rats is prevented by the IGF-I antagonist36. All these arguments support rather than detract from a contribution of IGF-I in diabetic nephropathies. Since MC hyperplasia is a feature common to several human glomerular diseases, the inhibitory effect of BK on the signaling cascade of IGF-I appears to be a potential protective endogenous mechanism. This hypothesis is consistent with the renoprotective effects of ACE inhibitors observed in experimental models and clinical trials as compared with other antihypertensive drugs5,6,7,8,9,10,11,12.
Moreover, beside the effects on MC proliferation and matrix secretion, acute elevation of IGF-I by exogenous administration reduces renal resistance and raises the glomerular filtration rate at least in normal rats37, whereas the hemodynamic effects of chronic endogenous elevation of IGF-I concentration remain controversial. It also has been suggested that IGF-I–induced hyperfiltration can be corrected by a B2-kinin antagonist38. However, that study was conducted for only 60 minutes with exogenous infusion of high doses of IGF-I reaching 50 nmol/L. It is clear that in this acute situation IGF-I predominantly induced a vasodilatory effect. Blockade of the B2 receptor can reduce this effect whereas the effect of chronic B2 blockade on the renal hemodynamics of diabetic rats has never been reported. The variations of the activity and expression of the kallikrein-kinin system during diabetes mellitus expression have been investigated and show a biphasic pattern. During an acute initial phase, glomerular hyperfiltration is prevented by administration of a B2 antagonist. On the other hand, renal kallikrein expression is suppressed in insulin-untreated streptozotocin (STZ)-diabetic rats and this abnormality is reversed by insulin39.
In order to associate the inhibition of MAP kinase activation with a cellular response, we investigated the effect of BK on IGF-I–induced MC proliferation. BK inhibits IGF-I–induced MC proliferation and this is consistent with the inhibition of Erk phosphorylation. Whereas the proliferative action of BK on quiescent cell systems has been extensively investigated, few studies report an inhibitory effect of BK in smooth muscle cells, fibroblasts or breast stromal cells22,23,40,41, and no mechanisms have been proposed to date. The involvement of a tyrosine phosphatase in the reduction of MAP kinase activation therefore appears as a new finding with interesting therapeutic perspectives. In this respect, the pharmacological inhibition of tyrosine kinase activity has demonstrated therapeutic effectiveness in a well-characterized animal model of polycystic kidney diseases resulting in reduction of cystic lesions42.
In addition to the inhibitory effect of BK on IGF-I signaling, the present article brings complementary data concerning the mechanism of stimulation of MAP kinase by the B2 receptor. The pathways by which the B2 receptor activates MAP kinase confirm a high degree of complexity and cell specificity as previously reported for other G-protein coupled receptors43. Using PC-12 cells transfected with dominant interfering mutants of Pyk2, it was shown that BK-induced MAP kinase activity resulted from an interaction between the proteins Pyk2 and src20. The overexpression strategy used20 is not a favorable experimental condition to investigate alternative signaling pathways as reported in the present study. In rat aortic smooth muscle cells, BK stimulated MAP kinase activity through a different pathway that is insensitive to both pertussis and cholera toxins and involves p60 Src kinase and PKC19. In the human colon carcinoma cell line SW-40, a novel mitogenic signaling pathway of BK has been reported and involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase , and PKC21. Taken together, these studies demonstrate the wide possibility of BK signaling pathways mainly depending on the cell-types used. With respect to MCs, it was first demonstrated by confocal microscopy that BK caused nuclear translocation of MAP kinase18. The pathway was only partly clarified more recently, suggesting that the BK-stimulated MAP kinase pathway required only tyrosine phosphorylation, thus appearing to be PKC independent17. The present study provides a much more complete understanding of the mechanism of MAP kinase activation by the endogenous B2 receptor in rat MC primary culture. We demonstrate that: (1) Erk 1 and 2 phosphorylation in MCs is strongly activated by PKC as revealed by the potent effect of a five-minute exposure to PMA; (2) BK-induced stimulation of Erk 1 and 2 was inhibited by inhibitors of PKC, of PLC or of tyrosine kinase; (3) down-regulation of PKC by an 18-hour exposure to PMA resulted in the absence of BK-induced stimulation of Erk 1 and 2 phosphorylation. From the present data it is now clear that the activation of MAP kinase by the B2 receptor is only transient and is dependent on G-protein coupling requiring both PKC and tyrosine kinase activity. From our study in rat MCs, we can conclude that BK-induced Erk 1 and 2 activation in MC requires both activation of PKC and of proteins from the Src kinase family since blockade of either of these two pathways resulted in the complete inhibition of BK-induced Erk 1 and 2 activation. In addition to this stimulating effect observed in quiescent cells, BK had an opposite antiproliferative effect in serum16 or IGF-I–activated MCs. According to the state of the cellular activation we suggest that cross-talk between the B2 receptor and IGF-I signaling reduces IGF-I–stimulated MAP kinase levels through a mechanism involving a calcium-dependent tyrosine phosphatase. Besides the inhibitory effect of BK on the signaling pathway activated by IGF-I, an additive inhibitory effect of BK on IGF-I mRNA in dermal fibroblasts could participate in the attenuation of IGF-I effects44.
The molecular mechanism of activation by BK is currently under investigation. However, the dual effect of BK (that is, activation or inhibition of Erk phosphorylation) is consistent with our recent report showing that BK can both activate and inhibit tyrosine kinase activity by two independent pathways45. The activation of tyrosine kinase is abolished by PTX whereas the inhibition is PTX resistant. It could have important implications for treatment of diseases associated with hyperproliferation with ACE inhibitors. Consistent with our results, a recent study demonstrates that ACE inhibition attenuates IGF-I–induced cardiac fibroblast proliferation46. Whether this BK-dependent mechanism is recruited during chronic treatment with ACE inhibitor remains to be investigated.
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Acknowledgments
Celine Alric was supported by a grant from MNSR and Eric Cellier is a post-doctoral fellow supported by a grant from the Medical Research Council of Canada. This work received funding from INSERM and from Midi-Pyrenees grant n° 99001254.
