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Early Interstitial Accumulation of Collagen Type I Discriminates Chronic Rejection from Chronic Cyclosporine Nephrotoxicity

Rene C. Bakker^*, Klaas Koop, Yvo W. Sijpkens^*, Michael Eikmans, Ingeborg M. Bajema, Emile de Heer, Jan A. Bruijn and Leendert C. Paul^*

Departments of *Nephrology and Pathology, Leiden University Medical Center, Leiden, The Netherlands.

Correspondence to Dr. Rene C. Bakker, Department of Nephrology, Leiden University Medical Center, Building 1, C3-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: +31-715262148; Fax: +31-715248118;

	Abstract

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ABSTRACT. Little is known regarding the composition of the interstitial extracellular matrix of kidney allografts with deteriorating function. Collagen I, III, and IV, the collagen IV 3 chain, and the laminin 2 chain were investigated in biopsies of allografted kidneys with chronic cyclosporine A nephrotoxicity (CsAT) (n = 17), chronic rejection (CR) (n = 12), or chronic allograft nephropathy (CAN) (n = 19). -Smooth muscle actin expression was also examined. Normal native kidneys were used as control samples (n = 11). Biopsy samples were studied with routine light microscopy and immunostaining. The mean interstitial fibrosis scores were significantly higher for the CR and CAN groups, compared with the chronic CsAT group. The cortical tubulointerstitial areas of the CR and CAN groups, but not the chronic CsAT group, contained more collagen I than did normal control samples. Differences were noted even in biopsies with mild fibrosis. Accumulation of collagen III, IV, and IV 3 was increased in all patient groups. Collagen III accumulation was greater in the CR and CAN groups than in the chronic CsAT group. Receiver-operating characteristic curve analysis demonstrated that collagen I staining had the best discriminatory value in differentiating CR from chronic CsAT, with a sensitivity of 63% and a specificity of 94% at a cutoff value of 19%. Laminin 2 staining did not differentiate CR from CsAT. Increased -smooth muscle actin staining did not differ among the three groups. It was concluded that, during chronic CsAT, collagen III and IV were preferentially accumulated in the tubulointerstitium. Early increases in the deposition of collagen I, with collagen III and IV, were more specific for CR. CR seems to elicit a more pronounced fibrotic response than does chronic CsAT. E-mail: RNR@euronet.nl

	Introduction

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Late loss of allograft function remains a major problem in renal transplantation (1). Kidney allograft failure is usually preceded by a process of chronic transplant dysfunction, which is characterized by a relatively slow but variable rate of decline in GFR, increasing proteinuria, and increases in BP. The pathologic changes in deteriorating grafts are often less specific. The term chronic allograft nephropathy (CAN) has been adopted to classify these changes, which include chronic obliterative vascular alterations, tubular atrophy, glomerulosclerosis, and interstitial fibrosis (2).

CAN includes both chronic rejection (CR) and chronic calcineurin inhibitor toxicity (2). The nature of vascular, glomerular, and interstitial changes in CAN biopsy specimens can sometimes help to define more specifically the cause of graft dysfunction. Concentric intimal thickening of arteries and arterioles, often accompanied by a moderate degree of mononuclear cell infiltration of the vessel walls, and duplication of the glomerular basement membrane are thought to indicate CR. Smooth muscle cell necrosis and peripheral nodular medial hyalinosis of arterioles are considered hallmarks of cyclosporine A (CsA) nephrotoxicity (CsAT) (3,4).

Extracellular matrix (ECM) accumulates in the cortical interstitium regardless of the cause of chronic graft dysfunction, as observed in native kidneys with chronic diseases (4,5). Surprisingly, the ECM composition of deteriorating allografted kidneys has rarely been studied. Determination of the ECM molecules that accumulate could enhance our understanding of the pathogenesis of graft dysfunction and could potentially help define the cause, if disease-specific changes are observed. In this study, we investigated whether the ECM composition differs between allografts that lose function because of CR and allografts with chronic CsAT. We studied the cortical interstitial ECM composition of kidney allografts of three groups of patients, i.e., patients with chronic CsAT, patients with CR, and patients who were receiving CsA treatment but were most likely to experience CR.

	Materials and Methods

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Patient Selection
All kidney allograft biopsies that were performed in our center for clinical reasons >1 yr after transplantation, in the period between March 1, 1976, and March 1, 2001, were reviewed. Cases that had available frozen-tissue samples and that met the criteria defined below were included. Patients with graft artery stenosis or diabetes mellitus were excluded. In addition, biopsy samples with histologic signs of de novo or recurrent glomerulonephritis or acute rejection were excluded. The group designated the chronic CsAT group (n = 17) consisted of patients who exhibited progressive declines in allograft function only after a switch was made from a CsA formulation with a lower bioavailability (Sandimmune; Sandoz, Basel, Switzerland) to one with a higher bioavailability (Neoral; Novartis, Basel, Switzerland) (6). A brief period of acute rejection in the early posttransplantation period was allowed and occurred in eight cases. Before the switch, the patients used Sandimmune once daily, aiming at a 24-h trough level of 100 µg/liter, and demonstrated stable graft function for a mean of 5.1 ± 3.4 yr. After conversion to twice-daily Neoral therapy, a higher target 12-h trough level of 150 µg/liter was adopted. To reach this level, the mean CsA dose was increased from 3.2 to 3.5 mg/kg (6). Nephrotoxicity was not observed in the first months after conversion but became evident after 12 mo (6). Biopsies were obtained a mean of 2.4 ± 1.2 yr after switching. None of the studied biopsies in this group exhibited signs of chronic allograft glomerulopathy. No positive staining for C4d in the peritubular capillaries (see below) was observed.

The patient group designated the CR group (n = 12) consisted of patients who exhibited progressive declines in renal allograft function with a calcineurin inhibitor-free immunosuppressive regimen. Immunosuppressive therapy consisted of prednisone and azathioprine. All of these patients initially demonstrated good allograft function (Table 1). Thirty-three percent exhibited C4d positivity in their peritubular capillaries.

Clinical and laboratory data were obtained in chart reviews. The collected data included posttransplantation time, allograft age (donor age plus time after transplantation), systolic and diastolic BP, number of antihypertensive medications used, creatinine clearance (as estimated with the Cockroft-Gault formula), loss of allograft function, proteinuria, and CsA trough levels at the time of biopsy. The loss of allograft function was defined as the difference in the creatinine clearance measured before the worsening of allograft function, as assessed by the breakpoint in the regression lines of 1/creatinine clearance, and the creatinine clearance measured at the time of the index biopsy (7).

Light Microscopy
After routine staining, the biopsy specimens were coded and reevaluated with light microscopy. An experienced pathologist who was blinded with respect to the instituted immunosuppressive regimen scored the histopathologic changes. The Banff 97 classification system for histopathologic scoring of allografted kidneys was used (8).

Immunostaining
The ECM components that were studied were detected with an indirect immunoperoxidase technique. Staining for each molecule was performed in one session. The primary antibodies used were polyclonal mouse anti-human collagen I, anti-human collagen III, and anti-human collagen IV (Harlan Sera-Lab, Sussex, UK) and monoclonal mouse antibodies against the collagen IV 3 (Wieslab, Lund, Sweden) and laminin 2 (C4) (Developmental Studies Hybridoma Bank, Iowa City, IA) chains. The secondary antibody consisted of peroxidase-coupled rabbit anti-mouse IgG (Dako, Glostrup, Denmark). Cryostat sections (4 µm) were mounted on glass slides, dried for 1 h, and stored at -20°C until used. The tissue was fixed with 4% formalin for 10 min. After blocking of endogenous peroxidase by incubation for 30 min with 0.1% H₂O₂ in phosphate-buffered saline, the sections were incubated for 30 min with 5% normal rabbit serum. The sections were then incubated with the primary antibody for 1 h, followed by incubation with the secondary antibody for 30 min. The sections were incubated for 10 min in a filtered solution of 0.5 mg/ml diaminobenzidine and 0.02% H₂O₂ and were then incubated for 5 min with 0.05% Cu₂SO₄ in saline solution. Counterstaining with hematoxylin was performed for 15 s. Between all steps, the slides were rinsed with phosphate-buffered saline.

Immunofluorescence staining for C4d was performed on untreated slides, as described previously (9). Mouse anti-C4d (Quidel, San Diego, CA) was used as the primary antibody (9). The secondary antibody was FITC-conjugated goat anti-mouse Ig (Sigma Chemical Co., St. Louis, MO). Staining was independently evaluated by two authors (Drs. Koop and Eikmans), who were blinded with respect to the diagnoses. A previously described scoring method was used (10).

Image Analysis
After immunostaining, the slides were coded and analyzed in a blinded manner. Digital image analysis was performed with a Zeiss microscope equipped with a full-color 3CCD camera (DXD 950p; Sony Corp., Tokyo, Japan) and KS-400 image analysis software, version 3.0 (Zeiss-Kontron, Eching, Germany). This method of digital image analysis has been validated and demonstrated to be highly reproducible (11,12). Images of the renal cortex of each biopsy specimen were obtained and analyzed with the aid of automated script protocols (macros) developed in our department. Recording and analyses of images were performed with fixed settings. The software allowed censoring of regions of noninterest. Larger vessels and glomeruli were omitted from the analyses. The deposition of each ECM component was determined in each biopsy sample by calculation of the percentage of surface area stained by the indicator dye, relative to the total surface area. The degree of deposition was assessed by evaluation of the entire renal cortex, with a minimum of five microscopic fields per biopsy sample (at x200 magnification).

The biopsy specimens that were stained for the laminin 2 and collagen IV 3 chains were also directly evaluated for interstitial or tubular expression of those molecules, by two investigators (Drs. Bakker and Koop) who were blinded with respect to the diagnoses. In the few cases of discordant scoring, decisions were reached by consensus.

Statistical Analyses
Statistical analyses were performed with SPSS for Windows software (SPSS, Inc., Chicago, IL). One-way ANOVA was used for comparisons of continuous variables with normal distributions. Either a Bonferroni or Games-Howel post hoc procedure was used when appropriate. The Mann-Whitney U test was used to compare continuous variables that were not distributed normally. The data are expressed as mean ± SD or mean ± SEM, as indicated. In a general linear model, values were adjusted for differences in the loss of allograft function and creatinine clearance at the time of biopsy. Correlations between the degree of deposition of individual ECM components and the clinical characteristics of the patients were evaluated with the Pearson correlation test. P < 0.05 was considered statistically significant. Using a receiver-operating characteristic curve, we determined the cutoff point of the percentage of staining for the various ECM molecules that predicted the presence of CR with the best combination of sensitivity and specificity.

	Results

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Patient Data
The clinical characteristics of the patients are presented in Table 1. The mean age of the allografted kidney was lower for the patients with CR, compared with the patients with chronic CsAT or CAN. The loss of creatinine clearance was greater for the patients with CR. The time after transplantation, BP values, number of antihypertensive drugs prescribed, and creatinine clearance at the time of biopsy were similar among the groups. No difference in CsA trough levels was observed between the groups receiving CsA.

Light-Microscopic Findings
The chronic histopathologic changes that were observed in the biopsy samples in routine light-microscopic analyses are summarized in Table 2. The mean interstitial fibrosis scores for the CR and CAN groups were significantly higher than that for the chronic CsAT group (CR group versus chronic CsAT group, P = 0.011; CAN group versus chronic CsAT group, P = 0.001). The tubular atrophy scores were higher for the CR and CAN groups, but the differences did not reach statistical significance (CR group versus chronic CsAT group, P = 0.422; CAN group versus chronic CsAT group, P = 0.159). The percentages of global glomerulosclerosis did not differ among the groups. The mean peripheral arteriolar hyaline thickening score for the chronic CsAT group was higher than those for the CR and CAN groups, as expected from the case definitions.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Staining areas (mean ± SEM) for collagen (Col) I (A) and collagen III (B). CsAT, cyclosporine A nephrotoxicity; CAN, chronic allograft nephropathy; CR, chronic rejection. *P < 0.05, **P < 0.01.

View larger version (131K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Immunoperoxidase staining for cortical collagen (Col) I and III. Representative images of a normal control sample (left) and a kidney allograft with CR (right) are shown.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Staining areas (mean ± SEM) for collagen (Col) IV (A) and the collagen IV 3 chain (B). *P < 0.05, **P < 0.01.

Because an earlier report suggested that immunostaining for the 3 chain of collagen IV and the 2 chain of laminin could differentiate CR from chronic CsAT (13), we also evaluated these molecules. The areas stained for the collagen IV 3 chain were increased in all patient groups, compared with the control group, to the same extent (mean area ± SEM for the CAN, CR, chronic CsAT, and control groups, 6.7 ± 1.1, 5.3 ± 1.2, 7.8 ± 0.8, and 1.1 ± 0.3%, respectively) (Figure 3B). The collagen IV 3 chain was expressed in a normal pattern, namely, at the distal tubular basement membrane (TBM), in all three patient groups. No interstitial staining or new expression at the proximal TBM was noted. In none of the groups did the areas staining for the laminin 2 chain change significantly, compared with the control group (Figure 4). However, we occasionally observed some new abnormal tubular expression in each of the groups (CsAT group, nine biopsy samples, 53%; CAN group, eight samples, 42%; CR group, six samples, 50%). The new expression was mostly confined to atrophic tubules or tubules surrounded by infiltrate, the proximal or distal morphologic features of which were difficult to assess. Less than 1% of tubules were affected in these samples, except for one sample in the CsAT group, three samples in the CAN group, and two samples in the CR group, in which 1 to 10% of tubules exhibited staining. No interstitial staining for the laminin 2 chain was observed, except for faint staining in some areas of periglomerular fibrosis.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Box and whisker plots of the median staining areas for the laminin 2 chain. The boxes contain 50% of the values. The upper and lower borders indicate the 25th and 75th percentiles, respectively. The upper and lower whiskers indicate the highest and lowest values, respectively. The black lines in the boxes indicate the medians. No significant differences were observed among the groups (P values from the Mann-Whitney test: CsAT group versus CAN, CR, and control groups, 0.847, 0.925, and 0.056, respectively; CAN group versus CR and control groups, 0.868 and 0.172, respectively; CR group versus control group, 0.463).

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Box and whisker plots of the median staining areas for -smooth muscle actin (SMA). *P < 0.05, **P < 0.01, compared with control group.

Receiver-operating characteristic curve analysis demonstrated that collagen I staining had the best discriminatory value in differentiating CR from chronic CsAT, with a sensitivity of 75% and a specificity of 88% at a cutoff value of 13% cortical interstitial staining and with values of 63% and 94%, respectively, at a cutoff value of 19% staining. The fraction of the area under the curve was 0.92 of the total area (P = 0.001). None of the ratios of the mean staining areas for the studied molecules proved to be useful for discrimination of CR from chronic CsAT.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of this study was to examine the composition of the cortical interstitial ECM of kidney allografts with either CR or chronic CsAT. We wondered whether quantitative and/or qualitative differences in ECM accumulation occur. Therefore, we quantitatively compared the deposition of several ECM molecules. We chose to study the molecules collagen I, III, and IV, because they are known to accumulate in the renal cortical interstitium during native kidney disease (5,14). The laminin 2 and collagen IV 3 chains were investigated because an earlier report suggested that de novo expression of these molecules at the proximal TBM could differentiate CR from chronic CsAT (13).

In this study, we observed quantitative and qualitative differences in the ECM composition of kidney allografts with CR or chronic CsAT. Our results demonstrated that collagen I, III, and IV accumulated in the renal cortical interstitium during CR. The most prominent increases were observed for collagen I and III. A similar pattern of ECM deposition was observed in the kidneys of patients who likely had CR but were receiving CsA. In the kidneys of patients with chronic CsAT, however, only collagen III and IV accumulated significantly.

Regulation of the renal interstitial ECM composition in vivo is complex and encompasses changes in ECM biosynthesis and degradation. In normal kidneys, collagen I and III are observed in the blood vessels and the interstitium (albeit in small amounts) (Figure 2) (15). Collagen IV is a natural component of the glomerular basement membrane and TBM, and its 3 chain is normally observed in the basement membranes of glomeruli and distal tubules (15). Laminin is observed in vessel walls and all basement membranes. The 2 chain, however, is observed only in glomeruli and vessel walls (16).

Quantitative data on the ECM composition of the cortical interstitium of human allografts with chronic CsAT or CR were lacking. A few studies reported increases in collagen III levels in grafts with chronic dysfunction but did not compare changes induced by CR and chronic CsAT (17,18). A number of in vitro studies have addressed the effects of CsA on the production of ECM molecules by renal cells. One study reported that CsA stimulated the production of pro-collagen I and IV in cultures of murine proximal tubular epithelial cells and the production of pro-collagen I in cultures of murine renal fibroblasts (19). Increases in the cortical expression of collagen I and IV were observed in vivo in a rat model of CsAT (20). The deposition of collagen III was not assessed in that model, however. Other data on collagen production were derived from a study on cultured human cells, which demonstrated an increase in collagen III production by renal fibroblasts when they were treated with CsA (21). Studies performed with a monkey renal fibroblast cell line (CV1) demonstrated that CsA stimulated the synthesis of type III collagen by a pathway leading to activation of the COL3A1 promoter and upregulation of COL3A1 mRNA (22). We observed the increased deposition of collagen III and IV in the interstitium of the cortex of human renal allografts as a result of chronic CsAT. In contrast to the results of the study performed in rats, we observed no significant increase in collagen I deposition. Although we did not observe a correlation between the time elapsed since the Neoral switch and the degree of collagen I deposition, we cannot exclude the possibility that, with time, collagen I would also accumulate during long-term chronic CsAT. The difference in collagen I staining between the CsAT and CAN/CR subgroups with mild fibrosis suggests that CR may stimulate collagen I accumulation earlier than does CsAT.

In our study, we observed greater expansion of the interstitial space in the CR group, compared with the chronic CsAT group, as well as more deposition of collagen type III. The difference in interstitial fibrosis could not be explained on the basis of differences in clinical variables, because no correlations with those variables were observed.

We wondered whether the differences in ECM deposition between CR and chronic CsAT could be explained on the basis of a difference in the numbers of myofibroblasts present. SMA is a marker of activated myofibroblasts, which are thought to play a major role in the deposition of scar tissue (23). Two earlier reports described increases in interstitial SMA staining in deteriorating human kidney allografts (17,18). In CR, SMA-positive cell numbers were reported to increase with increases in the interstitial area fraction and collagen III deposition (18). In our study, we did not observe differences in SMA staining among the CR, CAN, and chronic CsAT groups; all samples contained more SMA-positive cells than normal. To explain the greater degree of interstitial ECM deposition during CR, compared with chronic CsAT, a higher rate of collagen production by myofibroblasts or a lower rate of ECM degradation by tissue metalloproteinases could be hypothesized. Data derived from a primate model of chronic cardiac rejection support the latter hypothesis, because it was demonstrated that the progression of myocardial fibrosis was associated with increased expression of tissue inhibitor of metalloproteinases 1 and 2 (24).

We also examined the 3 chain of collagen IV and the 2 chain of laminin. Our results are at variance with a report by Abrass et al. (13), which suggested that a pattern of new expression of those molecules at the proximal TBM was specific for CR. In that study, CsAT resulted in the interstitial deposition of collagen I and III. Our study differs in the definitions of cases, the antibodies used, and the mode of analysis. The study by Abrass et al. (13) examined patterns of distribution and provided no quantitative data on the investigated molecules. According to Abrass et al. (13), collagen I and III are not normally observed in the cortical interstitial area and the collagen IV 3 and laminin 2 chains are not expressed at the TBM. However, we and others observed that collagen I and III are normally present in the renal interstitium, albeit in small amounts (5,15,25–27). We also observed that the collagen IV 3 chain is normally expressed at the distal TBM, as noted earlier (16,28). This discrepancy may be related to the different antibodies used. In the study by Abrass et al. (13), allografted kidneys with rejection exhibited acute rejection; although the authors stated that their results also applied to allografts with CR, they did not systematically examine that idea and no data were presented. The new expression of the collagen IV 3 and laminin 2 chains at the proximal TBM observed by Abrass et al. (13) might thus very well be related to tubulitis during acute rejection. In our study, we observed sporadic new expression of laminin 2 at the TBM in a portion of the biopsy samples in each study group, especially in areas of tubular damage. Staining for this molecule did not differentiate CR from chronic CsAT. We observed increased expression of the collagen IV 3 chain in the CR, CAN, and chronic CsAT groups but no abnormal distribution pattern. We observed a small nonsignificant increase in collagen I staining for the chronic CsAT group, compared with normal control values. It is possible that a small increase in collagen I expression by the patients with chronic CsAT, which was below our detection limit, could have resulted in the expression pattern observed in the study by Abrass et al. (13).

In conclusion, we observed a more pronounced fibrotic response in the tubulointerstitium, involving collagen I and III, in allografts with CR, compared with those with chronic CsAT. Early increases in the cortical interstitial deposition of collagen I were more specific for CR. Staining for the collagen IV 3 and laminin 2 chains was not useful for the differentiation of CR from chronic CsAT.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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