NDT Advance Access originally published online on May 16, 2006
Nephrology Dialysis Transplantation 2006 21(9):2417-2424; doi:10.1093/ndt/gfl230
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Experimental Nephrology
The role of the 
-1 adrenoceptor in modulating human mesangial cell matrix production
John Walls Renal Unit, Leicester General Hospital and Department of Infection, Immunity and Inflammation, University of Leicester, UK
Correspondence and offprint requests to: Dr I. Z. A. Pawluczyk, John Walls Renal Unit, Leicester General Hospital, Gwendolen Road, Leicester LE5 4PW, UK. Email: izap1{at}le.ac.uk
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Abstract |
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Background. The sympathetic nervous system is frequently activated in hypertension and may modify various aspects of renal function. Whether modulation of the sympathetic nervous system directly influences the development of renal fibrosis is yet to be established. The current study investigates the role of the
-1 adrenoceptor on human mesangial cell scarring. Methods. Human mesangial cells were injured with macrophage-conditioned medium (MPCM) and treated with doxazosin for 1 or 3 days.
Results. -1 Adrenoceptor antagonist doxazosin of 2 µmol/l reduced fibronectin protein in MPCM-injured female mesangial cells by 31 ± 1.03% (P < 0.001) and by 9.5 ± 0.3% (P = 0.01) in male mesangial cells. The differential response between sexes was significant (P = 0.004). -1B Adrenoceptors were detected in human mesangial cells by reverse transcription-polymerase chain reaction with expression in female cells being 87% higher than in males (P = 0.04). Injury with MPCM reduced -1B adrenoceptor mRNA expression in both cell types. Doxazosin had no effect on the protein levels of transforming growth factor-ß (TGF-ß) or interleukin-1ß (IL-1ß), however, a small reduction in tumour necrosis factor- (TNF-) levels was observed. Doxazosin had no effect on the modulators of matrix turnover matrix metalloproteinases MMP3, MMP9 and tissue inhibitor of matrix metalloproteinases (TIMP-1), although a significant reduction in tissue plasminogen activator (tPA); (36.5 ± 2.6%, P < 0.001) was observed. Doxazosin caused an up-regulation of kallikrein expression, both at mRNA and protein levels. Co-treatment with the bradykinin B2 receptor antagonist HOE140 was able to attenuate the effects of doxazosin treatment on fibronectin levels.
Conclusion. These data suggest that inhibition of -1B adrenoceptors in mesangial cells exerts an anti-fibrotic effect in a sex-specific manner via modulation of the kallikrein–kinin/plasminogen activator system.
Keywords: -1 adrenoceptor; fibronectin; kallikrein; mesangial cells
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Introduction |
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Progressive chronic renal failure is associated with a high morbidity and mortality. In addition to the loss of renal function, patients suffer from accelerated atherosclerosis and most will eventually die from cardiovascular disease. It is therefore vital that the pathophysiology of this condition is understood, so appropriate therapies can be developed to prevent or delay its onset.
Systemic and glomerular hypertension is known to contribute to the initiation and perpetuation of chronic renal disease and it is well established that treatment of systemic hypertension significantly delays the development of glomerulosclerosis, the histological hallmark of chronic renal failure, in all renal diseases. Evidence from in vivo and in vitro studies has shown that drugs which inhibit the renin–angiotensin system (Angiotensin converting enzyme (ACE) inhibitors and Angiotensin II (AII) receptor antagonists) retard the development of glomerulosclerosis to a greater degree than might be expected from their effect on blood pressure alone. However, to date, few studies have investigated potential blood pressure-independent effects of other anti-hypertensive drugs.
-1 Adrenoceptors play a pivotal role in modulating vascular tone and systemic haemodynamics and blockers of these receptors have been extensively utilized to treat hypertension. However, to date, there have been few studies to determine whether such drugs are able to convey a biological effect on the kidney over and above that observed with their blood pressure-lowering effects per se. The sympathetic nervous system is frequently activated in hypertension and renal disease, and may modify various aspects of renal function in general. However, the precise role of sympathetic nervous system activation in the pathogenesis of glomerulosclerosis remains poorly defined. In type 1 diabetes with microalbuminuria, the -1 antagonist doxazosin has been shown to reduce norepinephrine-induced hand vein vasoconstriction and albumin excretion suggesting that exaggerated vascular and glomerular reactivity to adrenergic stimuli may play a role in the development of diabetic nephropathy [1]. Doxazosin has also been shown to inhibit growth factor-induced cellular proliferation in mesangial cells [2] suggesting a potential mode of action beyond the simple inhibition of the effects of circulating catecholamines.
Mesangial cells are thought to express -1 adrenoceptors since they respond to alpha agonists with a contractile response. The mesangial cell is the glomerular homologue of the smooth muscle cell in which adrenergic stimulation with norepinephrine has been shown in vitro to directly modulate TGF-ß expression and extracellular matrix protein synthesis—an effect, which was inhibited by the selective -1 blocker prazosin [3].
We have previously shown that the modulation of the bradykinin/tissue plasminogen activator (tPA) axis is involved in the anti-fibrotic effects of ACE inhibitor action [4]. Although the adrenergic system has not classically been associated with the kallikrein–kinin system, of which bradykinin is the major effector molecule; there is accumulating evidence for interactions between the two systems. Doxazosin has previously been shown to increase urinary kallikrein levels in patients with mild-to-moderate uncomplicated essential hypertension [5]. Conversely, normal Sprague–Dawley rats treated with the -adrenergic agonist clonidine showed reduced urinary kallikrein levels [6]. Furthermore, treatment of hypertensive patients with doxazosin has been shown to improve the activity of the fibrinolytic system by increasing their fibrinolytic indices [tPA/plasminogen activator inhibitor-1 (PAI-1) ratio] [7].
The purpose of the proposed study is to delineate the potential role of the 1-adrenoceptor in the production of a profibrotic response in human mesangial cells. The effects of doxazosin, a quinazoline-derivative and specific post-synaptic 1-adrenoceptor antagonist [8], on mesangial cell fibronectin production were investigated in the context of adrenergic stimulation and injury by macrophages. In addition, the functional role of the kallikrein–kinin system in this response was examined.
While in vivo studies have clearly demonstrated the beneficial effects of anti-hypertensive treatment, the mechanisms underlying reno-protective effects are more difficult to define since direct cellular effects cannot be readily dissected from the effects on the modulation of haemodynamics. In order to investigate mechanisms of action independent of the influence of any haemodynamic effects, an experimental cell culture model of mesangial cell injury was employed. Such observations may thus provide a scientific basis for the rational choice of anti-hypertensive medication in progressive renal disease.
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Materials and methods |
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Unless otherwise stated, all reagents were purchased from Sigma-Aldrich Chemical Co. (Dorset, UK).
Cell culture
Human mesangial cells were cultured from the glomerular explants of human kidney specimens derived from the normal poles of nephrectomized kidneys from patients with renal carcinoma using standard serial sieving techniques. The cells were cultured in RPMI 1640 (Invitrogen, Paisely, UK) supplemented with 20% heat-inactivated fetal calf serum (FCS), 100 U/ml penicillin (Invitrogen), 10 µg/ml streptomycin (Invitrogen) 5 µg/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 2–10 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 h prior to 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, 10 µg/ml streptomycin and 2 mmol/l glutamine.
Preparation of U937 cell-conditioned medium
U937 cell MPCM was prepared as previously described [5].
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 -1 adrenoceptor antagonist doxazosin. 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 ±1 µmol/l bradykinin B2 receptor antagonist HOE 140.
For northern and Southern analyses, mesangial cells were exposed to 50% MPCM ± additions for 18 h prior to RNA processing.
Preparation of cell lysates
Cell lysates were prepared as previously described [5].
ELISAs
Culture supernatants and cell lysates were assayed for fibronectin as previously described [5]. The following enzyme-linked immunosorbent assays (ELISAs) were carried out according to the manufacturer's instructions:
TGF-ß, IL-1ß, TNF-, MMP3, MMP9 and TIMP-1 (R&D Systems, Abingdon, Oxon, UK); Imulyse tPA and PAI-1 (Biomed).
Catecholamine analysis
Analysis of catecholamines was carried out by the Biochemistry Department, Leicester Royal Infirmary using a standard HPLC technique on MPCM and mesangial cell culture supernatants that had been extracted on to alumina.
Protein determination
The protein content of cell lysates dissolved in 1% Nonidet NP40 was determined using a commercial BioRad DC protein assay, using BSA standards according to the manufacturer's instructions.
Northern blotting
Northern analysis was carried out using a method previously described [4].
Reverse transcription–polymerase chain reaction (RT–PCR)
Aliquots of 0.5 µg of total RNA were reverse transcribed using AMV reverse transcription system (Promega) according to the manufacturer's instructions. The resulting cDNA was amplified using ReddyMixTM PCR Mastermix (ABgene, Surrey, UK) and 50 pmol of specific sense and anti-sense primers.
Thermocycling conditions were optimized for each primer set.
-1A Adrenoceptor sense 5' GTG GCC AAG AGG GAG CGG GGC 3', anti-sense 5' CGA AGC AGC CGA CCA CGA TGC CCA G 3' (bases 6–29 and 1380–1404: accession no. AF395806
[GenBank]
).
-1B Adrenoceptor sense 5' ATG AAT CCC GAC CTG GAC ACC GG 3', anti-sense 5' GAT GGC CTT CCT CCG GGT GAC CA 3' (bases 172–194 and 638–660: accession no. NM000679).
Tissue kallikrein sense 5'-AAC ACA GCC CAG TTT GT-3', anti-sense 5'-CTT CAC ATA AGA CAG CAC-3' (bases 292–308 and 778–795: accession no. NM 002257.2).
Southern blotting
Aliquots of 20 µl of RT–PCR cDNA were resolved on 1% TAE-agarose gels. The gels were denatured for 45 min 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.
Statistics
Mesangial cell fibronectin levels were corrected for cell protein and are expressed as fold increase over control levels (medium alone). 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 means ± SEM. For comparison of means between the two groups, an unpaired t-test was employed. To compare values between multiple groups, an analysis of variance (ANOVA) with Bonferroni correction was applied. Statistical significance was defined as P < 0.05.
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Results |
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Effect of
-1 adrenoceptor agonist phenylephrine on fibronectin levelsIn order to assess the effect of catecholamine exposure on mesangial cells with respect to extracellular matrix protein production, human female mesangial cells were exposed to 10 µmol/l of the selective
-1 agonist phenylephrine (PE). PE increased supernatant fibronectin protein levels by 19.8 ± 6.8% (P = 0.03) over basal levels (Figure 1). This effect on fibronectin was abrogated by the -1 adrenoceptor antagonist doxazosin suggesting it was mediated via the -1 adrenoceptor (Figure 1).
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Effect of
-1 adrenoceptor blockade on MPCM-stimulated human mesangial cell fibronectin protein and mRNA levelsWe have previously shown that exposure of human mesangial cells to MPCM increases supernatant fibronectin levels [4]. Although the fold increase in fibronectin in response to MPCM was greater in female than in male cells, absolute fibronectin levels in males were higher (e.g. basal levels 124.8 ± 16.8 ng/µg cell protein in males and 52.4 ± 6.2 µg/ml in females).
Co-incubation of mesangial cells with MPCM in the presence of 2 µmol/l doxazosin resulted in an attenuation of the increase in supernatant fibronectin levels seen following exposure to MPCM (Figure 2A). Although we did not set out to look for a comparison of the effects of gender, we found that the observed reduction in fibronectin levels in response to doxazosin was significantly greater in female than in male cells [reduced by 30.9 ± 1.3% (P < 0.001 vs MPCM alone) in females and by 9.5 ± 0.3% (P = 0.01 vs MPCM alone) in males] (P = 0.004 for males vs females) (Figure 2A). Northern blotting suggested that observed reduction in supernatant fibronectin was not due to reduced synthesis since treatment with doxazosin appeared to have no effect on MPCM-induced fibronectin mRNA levels (Figure 2B). (The catecholamines adrenaline and noradrenaline could not be detected in MPCM and mesangial cell culture supernatants at the limits of detection (10 nmol/l) of the analytical method used).
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Expression of
-1 adrenoceptors in mesangial cellsTo further explore the apparent sexual dimorphism of the response to
-1 adrenoceptor blockade, the expression levels of -1 adrenoceptors in male and female mesangial cells was examined. Semi-quantitative RT–PCR demonstrated that -1 type A adrenoceptor mRNA was not readily detected in human mesangial cells (although it was detected in whole kidney) (Figure 3). -1B Adrenoceptors, on the other hand, were detected in human mesangial cells. Furthermore, receptor expression in female mesangial cells was 87% greater than in males (P = 0.04). Exposure of mesangial cells to MPCM reduced -1B adrenoceptor mRNA expression in cells from either sex. Doxazosin treatment partially restored MPCM-reduced -1B receptor expression (Figure 3).
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As female cells exhibited a greater response to
-1 adrenoceptor antagonism than male cells, all subsequent analyses to delineate a mechanism of action were carried out on female mesangial cells.
Effect of -1 adrenoceptor blockade on the levels of fibrogenic cytokines
To determine possible mechanisms underlying the observed anti-fibrotic effects of -1 adrenoceptor blockade, we examined supernatants from MPCM-stimulated female mesangial cell culture supernatants for the presence of various pro-fibrogenic cytokines. Doxazosin had no effect on the supernatant levels of the growth factor TGF-ß or the pro-inflammatory cytokine IL-1ß. However, a small, but significant reduction in TNF- levels (reduced by 9.9 ± 0.3%, P = 0.008) was observed following doxazosin treatment (data not shown). TNF- could not be detected in MPCM alone at the limits of detection of the assay.
Effect of -1 adrenoceptor blockade on modulators of matrix turnover
The lack of effect of doxazosin on fibronecin synthesis raised the possibility that the effect on supernanant fibronectin might be a result of alteration in fibronectin turnover. The expression of the modulators of extracellular matrix turnover, the matrix metalloproteinases (MMP) 3, 9 and their inhibitor TIMP-1 was examined. No difference in expression in any of these agents was observed in response to doxazosin as assessed by ELISA (data not shown). Similarly, gelatin zymography showed no difference in MMP9 and MMP2 activity with doxazosin treatment. However, when the expression of components of the plasminogen activator system was examined, doxazosin treatment appeared to cause a significant reduction in tPA (reduced by 36.5 ± 2.6%, P < 0.001; Figure 4A). PAI-1 protein levels in MPCM-exposed cells were also slightly reduced, but the reduction did not reach statistical significance (Figure 4B). Northern blotting demonstrated that doxazosin had no effect on tPA or PAI-1 mRNA levels indicating that the effects of doxazosin treatment on tPA (and PAI-1) expression occur post-transcriptionally (data not shown).
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Interaction of
-1 adrenoceptor blockade and the kallikrein–kinin systemThe expression of kallikrein by mesangial cells and the effect of treatment with doxazosin were examined. Semi-quantitative RT–PCR demonstrated that doxazosin dramatically up-regulated expression of kallikrein mRNA levels in MPCM-exposed mesangial cells (Figure 5A). Moreover, western blotting demonstrated that doxazosin was able to increase kallikrein protein levels in MPCM-exposed mesangial cells, thereby supporting the mRNA data (Figure 5B).
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The bradykinin B2 receptor (BkB2-R) mediates the physiological effects of the kallikrein–kinin system. The relevance of doxazosin-mediated up-regulation of kallikrein levels to the observed anti-fibrotic effects of doxazosin on mesangial cells was assessed by treating MPCM-exposed mesangial cells with doxazosin in the presence of the BkB2-R antagonist HOE140. HOE140 attenuated the reduction in supernatant fibronectin seen with doxazosin (Figure 6). This suggests that the BkB2-R is implicated in the observed anti-fibrotic actions of doxazosin.
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Discussion |
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The current study demonstrates that blockade of the
-1 adrenoceptor is able to exert anti-fibrotic effects in human mesangial cells. The ability for catecholamines to induce extracellular matrix production and proliferation in cultured smooth muscle cells and the capacity for -adrenoceptor antagonists to prevent these events has previously been reported [3]. However, the ability of -1 adrenoceptor blockade to attenuate non-catecholamine induced effects has not previously been reported. Although, macrophages are known to produce small levels of catecholamines, neither adrenaline nor noradrenaline could be detected in our conditioned media at the sensitivity levels of the analytical method used.
The response of mesangial cells to -1 adrenoceptor blockade, with respect to reducing fibronectin levels, was sex sensitive; with female cells responding significantly better than male cells. Furthermore, female mesangial cells expressed higher levels of -1B receptor than males. Injury with MPCM reduced mesangial cell receptor expression and -1 adrenoceptor blockade with doxazosin was able to restore it, at least partially. Previously, it has been shown that treatment of rat aortas with medium conditioned by endotoxin-stimulated peritoneal macrophages reduces the ability of the aorta to respond to epinephrine suggesting a reduction in receptor density, an effect thought to be mediated by IL-1ß [9]. A similar mechanism may be responsible for the MPCM-induced decrease in expression of -1B receptors observed in the current experiments, and may contribute to the increase in fibronectin levels observed in response to MPCM. -Adrenoceptor expression is known to be under hormonal influence with oestrogen increasing receptor density [10] and testosterone and progesterone reducing levels [11]. Similarly oestrogen has been shown to suppress stress-induced rises in plasma epinephrine [12]. Hormonally increased adrenoceptor expression has already been associated with cardio- and neuroprotective properties and a similar mechanism may also hold true for renoprotection, since it is known that renal disease progresses less quickly in females than in males.
Of the commonly encountered pro-fibrotic growth factors (TGF-ß and platelet derived growth factor (PDGF)) and proinflammatory cytokines (TNF- and IL-1ß), known to be associated with mesangial cell scarring, only TNF- protein expression was shown to be decreased following blockade of the -1 adrenoceptor with doxazosin. This is consistent with observations of other investigators who have shown that TNF expression can be modulated with anti-hypertensive drugs [13].
The degree of matrix accumulation depends on the balance between matrix synthesis and degradation. Although mesangial cell MMP-2, 3 and 9 and TIMP-1 levels were apparently unaffected by blockade of the -1 adrenoceptor with doxazosin, changes were observed in the expression levels of the plasminogen activator system. The protein levels of tPA (and PAI-1) were reduced by doxazosin, while tPA mRNA levels remained unchanged. The post-transcriptional decrease in tPA levels is most probably due to increased turnover/clearance as a result of endocytosis by one of two major receptors, the low density lipoprotein receptor-related protein (LRP) and/or the mannose receptor [14]. These observations are consistent with the in vivo observations that hypertensive patients are hypo-fibrinolytic [15], and that treatment of hypertensive patients with doxazosin improves the activity of the fibrinolytic system, increasing their fibrinolytic indices (tPA/PAI-1 ratio) [7].
Although the -1 adrenoceptor antagonist doxazosin exerts its anti-hypertensive actions via mechanisms classically unrelated to the renin–angiotensin and kallikrein–kinin systems, physiologically important interactions between the systems at various levels have previously been described [16–19]. Angiotensin is able to reduce -1A type adrenoceptors in cardiac myocytes in a dose-dependent manner [20], while in rat vascular smooth muscle cells angiotensin induces transcription and expression of both -1B and -1D subtypes of the adrenoreceptor [21]. These effects appear to be the result of a direct action of AII on the -1 adrenoceptor. In contrast, AII has also been shown to raise circulating norepinephrine levels in intact animals, indirectly stimulating adrenoceptor expression [16–19,22]. Treatment of hypertensive patients with doxazosin has previously been shown to increase urinary kallikrein levels [5]. Conversely, treatment of rats with the -1 agonist clonidine was able to reduce urinary kallikrein in Sprague–Dawley rats [6]. The current study confirmed that doxazosin up-regulated both kallikrein mRNA and protein levels in MPCM-stimulated mesangial cells. Moreover, co-treatment of MPCM-stimulated mesangial cells with the BkB2-R antagonist HOE140 attenuated the fibronectin-reducing effects of doxazosin. Previous reports had suggested that activation of BkB2-R could induce phosphorylation of -1B adrenoceptors [23] resulting in receptor desensitization. Our current data are consistent with the view that blockade of the -1 adrenoceptor with doxazosin, like ACE-inhibitors, can exert effects downstream of -1 adrenoceptor antagonism resulting in the modulation of the kallikrein–kinin system with the subsequent induction of anti-fibrotic effects. In the current study, a small decrease in fibronetin levels was observed with HOE140 alone. This may be due to the fact that under conditions of low bradykinin receptor activity, HOE140 can function as a partial agonist [24].
In conclusion, we have shown for the first time that blockade of the -1 adrenoceptor with doxazosin is able to exert anti-fibrotic effects on mesangial cells in a sex-specific manner via the kallikein–kinin system and its ability to modulate the plasminogen activator system. Such observations may have implications for the rational choice of anti-hypertensive agents in the treatment of hypertension in patients with chronic kidney disease.
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Acknowledgments |
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The study was funded in part by an unrestricted grant from Pfizer who also provided the doxazosin.
Conflict of interest statement. None declared.
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Notes |
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Parts of this article were presented in abstract form at the annual American Society of Nephrology Meeting in St Louis, 2004.
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[Abstract/Free Full Text]
Accepted in revised form: 3. 4.06
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