Interstitial expression of
-SMA: an early marker of chronic renal allograft dysfunction
Chérif Badid1,
Alexis Desmouliere2,
Daniela Babici1,
Aoumeur Hadj-Aissa1,
Brigitte McGregor1,
Nicole Lefrancois3,
Jean Louis Touraine3 and
Maurice Laville1,
1 Département de Néphrologie, Hôpital Edouard Herriot et EA 645, Laboratoire de Physiologie de l'Environnement, Faculté de Médecine Grange Blanche, Lyon,
2 GREF, INSERM 9917, Université Victor Segalen Bordeaux 2, Bordeaux and
3 Département de Transplantation et d'Immunologie Clinique, Hôpital Edouard Herriot, Lyon, France
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Abstract
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Background. Renal myofibroblast infiltration has been shown to be strongly associated with renal function decline in several chronic renal diseases. The purpose of the present study was to investigate whether early detection of myofibroblast infiltration using
-smooth-muscle actin (
-SMA) expression in time-zero biopsies predicts renal allograft dysfunction.
Methods. We studied renal tissue from 38 renal transplant patients from whom biopsies had been taken after vascular anastomosis during transplantation to ascertain whether myofibroblasts infiltration predicts renal graft survival. Immunohistochemistry was performed on time-zero biopsies to determine
-SMA expression, and this was compared to annual glomerular filtration rate (GFR) variation and other parameters including cold ischaemic time (CIT), donor and recipient age, number of acute rejections, and delayed graft function (DGF). GFR was measured by inulin clearance during of 3 years of follow-up after the transplantation. Progressors were defined as patients with an annual GFR decline >5 ml/min/year.
Results. We found a significant correlation between interstitial
-SMA expression in time-zero biopsies and GFR evolution during the post-transplantation course (r=0.60, P<0.001). Although progressors had greater interstitial
-SMA expression than non progressors (7.9±0.7 vs 4.3±0.4%), they showed only a tendency towards higher glomerular
-SMA expression. In addition, progressors had more interstitial fibrosis in time-zero biopsies than non-progressors. There was no relationship between
-SMA expression and CIT, donor and recipient ages, number of acute rejections, and occurrence of DGF.
Conclusion. This study suggests that
-SMA evaluation in time-zero biopsies, especially the combination of
-SMA expression and interstitial fibrosis, can strongly predict chronic renal allograft dysfunctions.
Keywords: chronic renal allograft dysfunction; cold ischaemic time; glomerular filtration rate; myofibroblast;
-smooth muscle actin
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Introduction
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While short-term renal graft survival rates have significantly improved during the last decade, chronic renal allograft rejection still remains an important cause of late kidney transplant loss. Chronic rejection is clinically characterized by a progressive deterioration in glomerular filtration rate, is associated with proteinuria and hypertension, and usually commences 36 months after transplantation with histological findings of fibro-obliterative vasculopathy, tubular atrophy, and progressive interstitial fibrosis. Some attention has been given to the role of myofibroblasts in the progression of renal diseases and to correlations with renal function decline [13]. The differentiation into myofibroblasts is one of the key early cellular events that initiates the development of organ fibrosis [4,5]. These cells appear to participate actively in tissue repair and in production of extracellular matrix that reduces the filtration surface area in the kidney. In addition, these contractile cells are characterized by the presence of cytoplasmic microfilament bundles expressing
-smooth-muscle actin (
-SMA), the actin isoform characteristic of vascular smooth-muscle cells [6,7]. Interstitial myofibroblast infiltration has been shown to correlate strongly with prognosis in many glomerular diseases, and we have recently demonstrated that interstitial
-SMA expression is an early prognostic marker in membranous nephropathy [3]. However, little attention has been given to the possible role of myofibroblast differentiation in the development of chronic allograft dysfunction. Many important elements involved in renal allograft dysfunction have been determined during the last two decades, including non-immunological factors such as donor age, cold ischaemic time (CIT) and delayed graft function (DGF) [810]. The aim of the present study was to investigate the prognostic value of
-SMA expression retrospectively in biopsies sampled immediately after vascular anastomosis during renal transplantation. We examined the relationship between
-SMA expression at the time of biopsy and renal function outcome evaluated by inulin clearance.
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Subjects and methods
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Patients
We studied time-zero graft biopsy specimens obtained from recipients undergoing renal transplantation at our institution between May 1993 and June 1995. Selection criteria were the availability of time-zero-biopsy specimens and the availability of at least two successive inulin clearances within the first 2 post-transplant years. Excluded from the study were patients without at least two inulin clearance measurements, patients not followed in our institution after transplantation, or patients receiving a double transplantation (kidney and pancreas or kidney and liver). All biopsies were taken during the transplantation procedure within 30 min after the reperfusion. Of more than 150 transplantations performed during the study period, 38 time-zero biopsies were evaluated. The age of the patients (25 males and 13 females) ranged from 26 to 56 years (mean, 39±2.5 years). The age of the donors ranged from 16 to 57 years (mean, 34.8±2 years). All kidneys, harvested from heart-beating cadaveric donors, were perfused with a preservative solution (University of Wisconsin solution (UW, n=20) or Collins solution (n=18) and were preserved in cold storage at 4°C [11]. All patients were followed with inulin clearance measurements for 36 months after the time-zero biopsy, and all received the same combination of cyclosporin (CsA), prednisolone (P), and azathioprine (Az) associated with anti-lymphocyte serum for the first 10 days. CsA was started at 2 mg/kg intravenously until the oral route could be started at 6 mg/kg/day to maintain whole-blood concentration between 100 and 200 ng/ml. Az was given at a dose of 12 mg/kg/day and adjusted according to the white-cell count, and P was given at a dose of 2/mg/kg/day, tapered to 10 mg by 6 months. None of the patients had received recent immunosuppressant therapy such as mycophenolate mofetil or tacrolimus.
Evaluation of disease progression
Glomerular filtration rate (GFR) was measured by inulin clearance in the same laboratory. We used the continuous infusion technique urine collection from spontaneous voiding, as previously described [3]. Briefly, a priming infusion of 40 mg/kg body weight inulin (polyfructosan, Inutest®, Laevosan, Linz, Austria) was given, followed by infusion at a rate of 0.33 mg/kg/min. Three to four 30-min urine collections were performed. Blood samples were drawn at the mid-point of each urine collection period. Inulin was measured in plasma and urine using standard colorimetric methods on an autoanalyser Technicon® after acid hydrolysis of inulin into fructose. These clearance measurements were normalized to 1.73 m2 of body surface area. The first GFR measurement was performed between 3 and 6 months after the transplantation and considered as GFR0. Evolution of graft function was assessed by annual change in GFR (GFR1-GFR0/GFR0/year) at 1, 2 and 3 years after transplantation. Patients who showed an annual loss of GFR>ml/min/year were defined as progressors.
Histology and immunohistochemistry
All tissue samples were processed in the same light microscopy laboratory using standard methods. Histology and immunohistochemistry were performed on paraffin-embedded sections (3 µm). Interstitial fibrosis was independently evaluated by two observers (CB and BMcG) who were blinded to other clinical information. The fibrosis was assessed using Masson's trichrome staining at low power view (x100). A semi-quantitative scale from 0 to 3 was used as follows: 0, absent; 1, <25% of specimen area; 2, 2550% of specimen area; 3, >50% of specimen area.
For immunohistochemistry, endogenous peroxidase activity was blocked by treatment with hydrogen peroxide (H2O2, 0.3%) in 100% methanol for 20 min. To block non-specific antibody sites, the sections were incubated with normal goat serum (Dako, Trappes, France) for 30 min. Sections were then incubated with a mouse monoclonal antibody against
-SMA (Dako) for 45 min [6]. Sections were then consecutively incubated with the biotinylated secondary antibody for 30 min and streptavidin biotinylated peroxidase complex (streptABC/HRP, Dako) for 30 min. Peroxidase activity was revealed using a 33' diaminobenzidine tetrahydrochloride substrate solution in phosphate-buffered saline containing H2O2 for 57 min. Sections were then counterstained with Harris haematoxylin, washed in water, and coverslipped. The percentage of surface area labelled by
-SMA antibody was evaluated by image analysis using Quantimet 600 (Leica, Cambridge, UK) as previously described [12]. The percentage of stained area was calculated as the ratio of suitable binary thresholded image to the total field area. Sections were analysed twice using 10 of these interstitial fields (x400), and at least 5 glomeruli were then averaged; blood vessels that expressed
-SMA in normal tissue were excluded.
Acute allograft rejection was diagnosed in all cases by renal biopsy. The severity of acute rejections were according to the Banff classification [13]. We graded tubulitis (t), glomerulitis (g), arteritis (v), and interstitial inflammation (i) from 0 to 3. Rejection episodes were treated with 4 mg/kg methylprednisolone that was tapered to 1 mg/kg at one week and to 20 mg/day over 2 more weeks. In four cases, OKT3 was administered at 5 mg/day for 10 days because of cortico-resistance.
Statistical analysis
Data are expressed as means±SEM. Correlations between annual GFR changes and
-SMA labelling in time-zero biopsies were evaluated by linear regression analysis. Clinical parameters as well as
-SMA expression were compared among groups by one-way analysis of variance (ANOVA), followed by Fischer's PLSD test. A P value <0.05 was considered statistically significant.
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Results
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Twenty-four patients had stable allograft function and 14 patients had a sustained and marked decline in GFR. Twenty of 38 patients (53%) had acute rejections during the first 3 post-transplant months and were treated by corticosteroid pulses, and 11/20 (55%) had a second rejection during the 3 years of follow-up. All patients responded to corticosteroid pulses except for four patients who received OKT3 boluses. Two patients had DGF, defined as reinstitution of dialysis in the 6 days after transplantation. A comparison of progressors and non-progressors showed a tendency towards a higher number of acute rejection episodes in the progressors, but differences in incidence and severity of acute rejections were not significant (Table 1
). OKT3 was administered to one patient in the progressor group and to three patients in the non-progressor group. There were no differences between the two groups in donor serum creatinine concentrations, in CIT, or in DGF. There was no difference in donor age. However, progressors had a greater expression of interstitial
-SMA than non-progressors (Table 1
). There was a non-significant tendency towards a higher glomerular
-SMA expression in progressors. In addition, progressors had higher interstitial fibrosis scores than non progressors (Table 1
). Comparisons of clinical parameters in patients having no rejection and those having rejection in the first 3 post-transplant months showed no differences in intensity of interstitial and glomerular
-SMA staining on time-zero biopsies, GFR variation, the CIT, the donor serum creatinine, or in donor or recipient age. In the entire group of patients,
-SMA staining ranged from 0.8 to 19.2% (mean, 5.5±1.2%) for the interstitium and from 1.3 to 25.5% (mean, 10.7±0.8%) for glomeruli. Comparison of clinical parameters of patients having >5.5% interstitial
-SMA staining with patients having <5.5% (5.5% representing the mean value for
-SMA staining in our population) revealed no differences in incidence of acute rejection, concentration of serum creatinine at the time of biopsy, CIT, or donor age. However, patients having >5.5% interstitial
-SMA staining demonstrated a significantly accelerated annual loss of GFR compared with patients having <5.5% (Table 2
). The subdivision of our population into four groups (A, <3.5%; B, 3.55.5%; C, 5.57.5%; D, >7.5% for interstitial
-SMA staining) revealed a strong relationship between GFR variations and interstitial
-SMA staining (Figure 1
). In addition, patients having >5.5% interstitial
-SMA staining had higher interstitial fibrosis scores than those having <5.5% (Table 2
).
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Table 1. Clinical and immunohistochemical parameters in patients who subsequently progressed and those with stable renal function
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Table 2. Comparison of clinical parameters in patients who had <5.5% of interstitial -SMA staining and those who had >5.5%
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There was a marked upregulation of interstitial
-SMA in time-zero biopsies from progressors (Figure 2
). Patients having progression showed significant numbers of
-SMA-positive cells surrounding the tubules. In contrast, patients with stable allograft function showed no or few interstitial myofibroblasts surrounding tubules; these were often located next to the peritubular capillaries. There was strong correlation between interstitial
-SMA staining and the annual GFR change (r=0.60, P<0.001) during the 3 years of post-transplantation follow-up (Figure 3
). However, no significant correlation was found between glomerular
-SMA expression and allograft function outcome (r=0.2, P=NS), although there was a tendency towards higher glomerular
-SMA expression in progressors (Table 1
). There were no correlations between
-SMA expression in time-zero biopsies and donor age, donor creatinine, or CIT.
Interstitial fibrosis was variable, showing scores from 0 to 3. Progressors had more interstitial fibrosis in time-zero biopsies than non-progressors (1.7±0.19 vs 1.2±0.14, P=0.049) (Table 1
). Among the 14 patients having progression, seven showed significant interstitial fibrosis ranging from 2 to 3, while the other progressors had a low score of interstitial fibrosis (score <2). Among these seven progressors not having significant fibrosis, five (71%) had a significant expression of interstitial
-SMA (>5.5%). However, there was no significant difference in interstitial fibrosis between patients having >5.5% (mean score, 1.6±0.2) and those having <5.5% (mean score, 1.3±0.2) interstitial
-SMA staining. In addition, among 25 patients having no significant fibrosis (score <2), nine (36%) showed a significant expression of interstitial
-SMA (>5.5%) and five of these nine (55.5%) were progressors. Among the 14 patients who progressed, 12 (85.7%) showed some significant fibrosis (score >2) or significant interstitial
-SMA staining (>5.5%).
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Discussion
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This study is the first to demonstrate that
-SMA is expressed early in time-zero biopsies from renal grafts and is a predictive factor for renal function. There is a strong relationship between tubulointerstitial changes and long-term renal function in many glomerular diseases [14,15]. The degree of interstitial remodelling, especially interstitial fibrosis, in post-transplant renal biopsies has been shown to be associated with poor post-transplant graft performance [16]. In the present study, we confirmed the importance of pre-existing fibrosis in renal-graft outcome. However, in our population, only 50% of patients who progressed had significant fibrosis, whereas among progressors not having significant fibrosis, 71% had significant
-SMA expression. These results suggest that the presence of
-SMA associated with interstitial fibrosis may provide early identification for the majority of patients at risk of chronic graft dysfunction. Myofibroblasts are well known to be the major cell type responsible for extracellular matrix synthesis and fibrosis development [4,17]. The prognostic value of these cells in renal-function decline has been demonstrated in many glomerular diseases [1,3]. Myofibroblast differentiation precedes histological evidence of tubulointerstitial fibrosis and is an early event during fibrosis, as was demonstrated in the remnant kidney model of glomerulosclerosis [2]. Interstitial cells expressing
-SMA were observed within 7 days after subtotal nephrectomy. Myofibroblasts are significantly increased in chronic graft dysfunction and were the major component of the interstitial infiltrate [18,19]. A relationship between an early differentiation process and the late development of chronic dysfunction has been recently suggested [20]. In addition, an early expression of myofibroblasts was observed in renal-transplant biopsies taken 3 weeks after transplantation, and this expression correlated with allograft outcome [20]. Our study is consistent with these results in showing an earlier expression of
-SMA. In a study by Nicholson et al. [21], staining for
-SMA in 6-month biopsy specimens did not correlate with the subsequent course of renal function, whereas the density of collagen III was predictive of renal dysfunction. Nicholson et al. [21] analysed late biopsies and found that interstitial fibrosis was more developed, resulting in a reduction in interstitial cells, especially of myofibroblasts. As fibrosis worsened, cellularity diminished because cells and particularly myofibroblasts were replaced by extracellular matrix. In addition, some authors have reported that glomerular
-SMA was associated with chronic dysfunction while interstitial
-SMA was not [19]. In fact, interstitial fibrosis in late biopsies was already well developed, resulting in a reduction in interstitial cells. In the present study, despite a tendency towards higher glomerular
-SMA expression in progressors, interstitial
-SMA was better correlated with renal function outcome. Taken together, these observations indicate that further studies are needed to examine the role of
-SMA in early fibrosis development.
We were unable to determine whether
-SMA expression occurred pre-transplantation or post-transplantation during acute ischaemia or after reperfusion.
-SMA expression may reflect injury that occurred before transplantation in kidney donors. However, there was no relationship between intensity of staining and donor age or serum creatinine at the time of transplantation. Another possibility is that
-SMA expression reflects injury appearing during ischaemia or during the reperfusion period. This hypothesis is supported by observations of acute and transient expression of
-SMA in hepatic stellate cells after anoxia and hypoxia or both that occurred during ischaemiareperfusion at the time of liver transplantation as well as during ex vivo pig liver perfusion [22]. In a canine model of coronary occlusion and reperfusion, myofibroblastic differentiation has also been observed [23]. The nature of the stimuli enhancing
-SMA expression during transplantation remains to be elucidated. Studies with pig liver perfusion showing an early expression of
-SMA suggested that this phenotypic differentiation can occur without the involvement of inflammatory processes [22]. Cell infiltration and matrix accumulation accompanying acute renal ischaemia and reperfusion have frequently been noted and their importance in long-term histology and matrix change has been well demonstrated [24]. Our study included a relatively small number of biopsies, and it was not possible to perform a meaningful analysis of the relationship between
-SMA and chronic allograft dysfunction. However, we observed a variable expression of
-SMA in time zero biopsies. This expression was not associated with CIT or with donor age but was strongly associated with renal function outcome. We believe that
-SMA evaluation in graft biopsies will provide superior monitoring of immunosuppressive drug effects, in particular with antiproliferative immunosuppressants such as mycophenolate mofetil, which has been shown to reduce fibrosis development after renal mass reduction in the rat [25].
In conclusion, this study suggests that early detection of
-SMA-positive cells in time zero biopsies, especially in combination with interstitial fibrosis, is a valuable marker of renal transplant quality. It may also be of potential benefit for the management of patients at risk and for adaptation to immunosuppressive drugs.
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Acknowledgments
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We are very grateful to Pr Bernengo for his expert assistance in image analysis (Service Commun de Quantimétrie et de Microscopie Confocale, Domaine Rockefeller, Lyon, France).
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Notes
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Correspondence and offprint requests to: Dr M. Laville, Département de Néphrologie, Hôpital Edouard Herriot, 5 place d'Arsonval 69437, Lyon Cedex 03, France. Email: maurice.laville{at}chu\|[hyphen]\|lyon.fr 
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Received for publication: 23. 7.01
Accepted in revised form: 18. 5.02