2007 JASN IMPACT FACTOR 7.111	HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP
advanced	CURRENT ISSUE	ARCHIVES	JASN Express	ONLINE SUBMISSION

This Article Abstract Full Text (PDF) All Versions of this Article: ASN.2005080891v1 17/3/881    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roos-van Groningen, M. C. Articles by Eikmans, M. Search for Related Content PubMed PubMed Citation Articles by Roos-van Groningen, M. C. Articles by Eikmans, M.

Molecular Comparison of Calcineurin Inhibitor–Induced Fibrogenic Responses in Protocol Renal Transplant Biopsies

Marian C. Roos-van Groningen^*, Eduard M. Scholten, Patrick M. Lelieveld^*, Ajda T. Rowshani, Hans J. Baelde^*, Ingeborg M. Bajema^*, Sandrine Florquin, Frederike J. Bemelman, Emile de Heer^*, Johan W. de Fijter, Jan A. Bruijn^* and Michael Eikmans^*

Departments of ^* Pathology and Nephrology, Leiden University Medical Center, Leiden, and Departments of Internal Medicine, Renal Transplant Unit, and Pathology, Academic Medical Center, Amsterdam, The Netherlands

Address correspondence to: Dr. Marian C. Roos-van Groningen, Leiden University Medical Center, Department of Pathology, Building 1, L1-Q, PO Box 9600, 2300 RC Leiden, The Netherlands. Phone: +31-71-5266574; Fax: +31-71-5248158; E-mail: m.c.roos_van_groningen{at}lumc.nl

Received for publication August 30, 2005. Accepted for publication December 27, 2005.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The calcineurin inhibitor cyclosporine (CsA) induces a fibrogenic response that may lead to scarring of the renal allograft. This study investigated whether tacrolimus, a novel calcineurin inhibitor, exerts fibrogenic effects to a similar extent. Sixty patients were enrolled in a randomized study: 29 received CsA, and 31 received tacrolimus. Patients were subjected to tailored exposure-controlled calcineurin inhibitor regimens. Protocol biopsies were obtained at the time of transplantation and 6 and 12 mo after transplantation. Cortical TGF- and collagens 1(I) and 1(III) mRNA steady-state levels were determined with real-time PCR. The extent of protein deposition of TGF-, -smooth muscle actin, and interstitial collagens in the renal cortex was quantified with computer-assisted image analysis. The extent of interstitial collagen deposition measured with Sirius red and the accumulation of -smooth muscle actin and TGF- protein after 6 and 12 mo were similar for both immunosuppressive regimens. mRNA levels of TGF- and collagens 1(I) and 1(III) were not significantly different in the treatment groups either. It is concluded that the fibrogenic response in renal allografts is similar in patients who receive CsA-based regimens and patients who receive tacrolimus-based regimens.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Allograft survival during the first year after kidney transplantation has improved significantly (1, 2). This improvement is largely due to the introduction of potent immunosuppressive agents such as the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (3, 4). Unfortunately, long-term graft survival has not increased commensurately. The main cause of late graft loss is chronic allograft nephropathy (CAN) (5, 6), which is clinically characterized by a slow progressive decrease in renal function. Morphologically, interstitial fibrosis is a prominent feature. The histologic alterations in CAN result from a complex interaction between immunologic and nonimmunologic causes of injury, including (sub)clinical acute rejections and nephrotoxicity as a result of chronic exposure to immunosuppressive agents (7).

CsA is known to have a fibrogenic effect in the renal allograft (8). There is no conclusive evidence that this effect is similar in patients who are treated with tacrolimus (9, 10). A large, randomized study that compared the efficacy and safety of tacrolimus- and CsA-based regimens showed that the adverse effect profile of CsA is less favorable than that of tacrolimus (11). However, studies in rats have demonstrated that administration of both CsA (12) and tacrolimus (13, 14) cause nephrotoxicity and induce intrarenal TGF- expression. Application of anti–TGF- antibodies in CsA-treated rats reversed the majority of CsA-associated renal lesions (12). This shows that TGF- mediates the nephrotoxic effects of CsA, which is in accordance with its capacity to induce extracellular matrix (ECM) expression (15–17).

Studies of diagnostic renal biopsies have compared the fibrogenic effects of CsA and tacrolimus (18, 19). However, a disadvantage of these studies may be that differences in the extent of morphologic damage between groups, rather than differences in the effects of the various types of medication themselves, account for any change in the level of expression of TGF- and ECM-related components (20). Studies in protocol renal biopsies that have compared the effects of CsA and tacrolimus on intragraft TGF- expression have shown discordant results (21–24). Discrepancies between findings in these studies may be explained by the fact that patients with acute rejection episodes before or during time of biopsy were included in some of the experiments, which may have resulted in biased TGF- expression levels. Different compartments were studied in the various studies, either glomeruli or the whole cortex, and this may have led to different results (22). Furthermore, most studies have compared immunosuppressive regimens through a mere analysis of mRNA levels, which provides no answer to the question of whether differences in mRNA expression in different types of medication would be accompanied by differences in expression of the corresponding proteins (22, 23).

It was the objective of this study to compare the fibrogenic effect of CsA and tacrolimus at the mRNA and protein levels. Randomized patient groups were used in a prospective manner. Protocol biopsies were taken before transplantation and 6 and 12 mo after transplantation. Controlled target area-under-the-concentration-over-time-curve guided dosing of both calcineurin inhibitors was used to maintain efficacy and minimize toxicity. This was accomplished using a population-based pharmacokinetic model together with limited sampling combined with Bayesian estimation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patient Groups, Drug Monitoring, and Study Design
It has been estimated that a controlled trial regarding the effects of two different immunosuppressive regimens powered to demonstrate a 10% difference in renal allograft fibrosis at 6 mo posttransplantation would require 23 patients to be randomly assigned to each treatment arm (25). Therefore, 65 patients were enrolled in an open, prospective, randomized, clinical trial at the Leiden University Medical Center in The Netherlands. Before transplantation, patients were randomly assigned 1:1 to receive either a CsA microemulsion–based (Neoral; n = 33) or tacrolimus-based (Prograft; n = 32) immunosuppressive regimen. CsA and tacrolimus were started orally 3 h before surgery (initial dose 4 mg/kg twice per day for CsA and 0.1 mg/kg for tacrolimus). In the first week, target 12-h trough levels were aimed at 225 ng/ml (range 200 to 250 ng/ml) and 12.5 ng/ml (range 10 to 15 ng/ml) for CsA and tacrolimus, respectively. CsA and tacrolimus AUC_{0 to 12h} were estimated at weeks 2, 4, 6, 8, 12, 17, 21, 26, 39, and 52 with a population-based two-compartmental pharmacokinetic model that was combined with Bayesian fitting and limited sampling. After each AUC assessment, doses were adjusted to comply with predefined target AUC: CsA AUC_{0 to 12h} was 5400 ng/h per ml within the first 6 wk (corresponding with a mean trough level of 225 ng/ml) and 3250 ng/h per ml after the initial 6 wk (corresponding with a mean trough level of 125 ng/ml). Tacrolimus AUC_{0 to 12h} was 210 ng/h per ml within the first 6 wk (corresponding with a mean trough level of 12.5 ng/ml) and 125 ng/h per ml after 6 wk (corresponding with a mean trough level of 7.5 ng/ml). Immunosuppressive co-medication consisted of prednisolone (in both groups 100 mg at days 1 to 3, 50 mg at day 4, 20 mg at days 5 to 14, 15 mg at days 15 to 21, and 10 mg after day 22), mycophenolate mofetil (1000 mg twice per day in the CsA group and 500 mg twice per day in the tacrolimus group), and basiliximab prophylaxis (20 mg intravenously at day 0 and day 4). Drugs that are known to alter concentrations of the calcineurin inhibitors were prohibited.

Protocol biopsies were taken before transplantation (t0) and 6 and 12 mo after transplantation. Biopsies were scored according to the Banff 97 classification scheme (26). Of all protocol biopsies obtained, two 6-mo biopsies from patients who received CsA were not adequate for evaluation according to the Banff criteria. These were excluded from further study. Four patients from the CsA cohort and one patient from the tacrolimus cohort showed morphologic signs of acute rejection in the protocol biopsies and were excluded. Thus, in our study, 29 patients who received CsA and 31 patients who received tacrolimus were included.

Control Groups
mRNA transcripts from 16 kidneys with normal histology (12 samples from the unaffected part of a tumor nephrectomy kidney and four cadaver donor kidneys that initially were intended for transplantation purposes) and 28 acute rejection biopsies (20 Banff type Ia and 8 Banff type Ib) were studied.

RNA Extraction
Frozen biopsy tissue was available for 34 t0 biopsies, 53 6-mo biopsies, and 48 12-mo biopsies. A 2-µm section from each biopsy was analyzed with light microscopy to demonstrate the presence of the renal cortex according to a procedure that was described in a previous study (27). Ten to fifteen 10-µm sections of renal cortex were cut in a Leica CM3050 S cryostat, collected in an Eppendorf tube, and stored at –70°C until usage. RNA extraction was performed with RNeasy spin columns (Qiagen, Westburg, The Netherlands). RNA concentration was determined with photospectometry. The mean A260 to A280 ratio of the samples was between 1.8 and 2.0. A maximum amount of 1 µg of RNA was used as input for cDNA synthesis (mean input 0.75 ± 0.30 µg of RNA). The cDNA reactions (24 µl total volume) consisted of the following reagents: 0.5 mM dNTP, 100 ng oligo dT (Roche, Mannheim, Germany), 500 ng random hexamer primers (Invitrogen, Breda, The Netherlands), 5 U of avian myeloblastosis virus–reverse transcriptase (RT-AMV) (Roche), 20 U of rRNasin (Promega Benelux BV, Leiden, The Netherlands), and 1x reverse transcriptase buffer (Roche).

Real-Time PCR
A real-time PCR with the ABI Prism 7700 Sequence Detector System (Perkin Elmer Biosystems, Foster City, CA) was performed. This procedure was described in detail previously (28). cDNA samples were diluted at a ratio of 1:50, and 5 µl of the dilution was used for PCR. mRNA levels of TGF- and collagens 1(I) and 1(III) were quantified and normalized for the mean of mRNA levels of the household genes glyceraldehyde-3-phosphate dehydrogenase and hypoxanthine guanine phosphoribosyltransferase-1. A previous study showed that there is a significant correlation between these household genes (29). In our study, levels of glyceraldehyde-3-phosphate dehydrogenase significantly correlated with those of hypoxanthine guanine phosphoribosyltransferase-1 (r = 0.61, P < 0.001). Primer and probe sequences and PCR conditions were described previously (29). To allow comparison of samples from different PCR plates, we included in each PCR run a 1:5 dilution range of a reference sample.

Immunohistochemistry and Quantification of Stainings
Paraffin-embedded tissue was available for 54 6-mo biopsies and 54 12-mo biopsies. Four-micrometer sections were cut and used for immunohistochemistry. For detection of deposition of TGF-, slides were heated in a 0.01-M citrate buffer solution for 10 min and subsequently cooled for 20 min. Slides then were incubated for 1 h at room temperature with polyclonal antibodies against TGF- before 30 min of incubation with anti-rabbit EnVision (Dako, Glostrup, Denmark) as the secondary antibody. The rabbit polyclonal anti-human TGF- antibody that was raised against a synthetic peptide that corresponded to the C-terminal amino acids 371 to 390 of human TGF-₁ had been synthesized in our laboratory (30, 31). Antibody specificity was confirmed further by Western blotting and immunoabsorption assays. To detect -smooth muscle actin (-SMA)-positive cells, slides were stained for 1 h with mAb against -SMA (Promega Benelux BV) and thereafter incubated for 30 min with anti-mouse EnVision (Dako). NovaRed (Vector, Burlingame, CA) was used to visualize the immunohistochemical signal in both stainings. Further details on staining protocols were described in a previous report (32). For each immunohistochemical assessment, all sections were stained simultaneously in one session. The extent of interstitial collagen accumulation was visualized in both paraffin sections of 6-mo and 12-mo biopsies and frozen sections of t0 biopsies with Sirius red staining, a process that was described previously (33). Slides from frozen biopsies first were fixed in formalin for 10 min before Sirius red staining.

Digital analysis was performed on each biopsy with a Zeiss microscope that was equipped with a full-color 3CCD camera (Sony DXC 950p, Sony Corp., Tokyo, Japan) and KS-400 image analysis software version 3.0 (Zeiss-Kontron, Eching, Germany) to quantify the extent of staining. An average of 10 microscopic sections were examined from each slide, commencing at the capsular side and following in a linear manner. Using the x20 objective, an average of 63.2 ± 19.5% of the total cortex was analyzed in each biopsy. For both Sirius red and the -SMA staining, blood vessels larger than adjacent tubules were excluded. The procedure was described previously (28).

Statistical Analyses
Differences between groups were assessed with independent-samples t test. Correlations between expression levels were analyzed with Pearson correlation tests. Analyses were performed with SPSS software (version 10.0; SPSS, Inc., Chicago, IL). P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patient Population
Twenty-nine participants received CsA, and 31 received tacrolimus. Demographic characteristics in the treatment groups were similar, as depicted in Table 1. There were no significant differences between treatment groups regarding the donor’s and the recipient’s age and gender, cold ischemia time, occurrence of delayed graft function, donor source, BP, antihypertensive medication use, total HLA mismatches, incidence of cytomegalovirus infection, and GFR 6 and 12 mo after transplantation. There was no significant difference in the number of patients who showed borderline subclinical rejection after 6 and 12 mo between treatment groups. In these groups, GFR was not different for the patients with borderline subclinical rejection and for those without. During the first 12 mo after transplantation, 10 biopsies were taken on clinical indication in addition to the protocolized biopsies (five in each treatment group). These were taken within the first 2.5 mo after transplantation. In the CsA group, four biopsies showed signs of acute rejection and one was taken because of delayed graft function (no morphologic abnormalities). In the tacrolimus group, three biopsies were taken because of delayed graft function and one because of a slight increase in creatinine (all demonstrating no morphologic abnormalities). The fifth biopsy taken showed signs of acute transplant glomerulopathy.

mRNA Assessments
The mRNA levels of TGF- and collagens 1(I) and 1(III) at t0, after 6 mo, and after 12 mo for the study groups are summarized in Table 2. In both treatment groups, the mean TGF- mRNA levels 6 and 12 mo after transplantation were significantly higher than those at the time of transplantation (P < 0.005). No significant difference was found in TGF- mRNA levels between treatment regimens at t0 (CsA 0.14 ± 0.09 versus tacrolimus 0.11 ± 0.08), at 6 mo (CsA 0.54 ± 0.30 versus tacrolimus 0.53 ± 0.37), or at 12 mo (CsA 1.03 ± 0.60 versus tacrolimus 0.91 ± 0.62; Figure 1A). There were no significant differences in collagens 1(I) and 1(III) mRNA expression levels at t0, after 6 mo, or after 12 mo in the treatment groups either (Figure 1, C and D).

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Fibrogenic mRNA molecules and TGF- protein expression in protocol biopsies of cyclosporine (CsA)- and tacrolimus-treated patients. mRNA expression and protein deposition were determined using real-time PCR and immunohistochemistry, respectively. (A) TGF- mRNA levels in protocol biopsies at t0, after 6 mo, and after 12 mo did not significantly differ between treatment regimens. As a comparison, expression was measured in 28 biopsy samples with acute rejection (AR); TGF- mRNA levels were significantly higher in clinical rejection biopsies than in protocol biopsies. (B) TGF- protein staining in protocol biopsies after both 6 and 12 mo was not significantly different in the immunosuppressive medication groups either. (C and D) No significant difference was found between medication regimens for both collagen 1(I) and collagen 1(III) mRNA levels at any time.

Interstitial Protein Deposition
The extent of protein deposition of TGF- and -SMA and the extent of Sirius red staining for both treatment groups are summarized in Table 2. Consistent with the findings for TGF- mRNA expression, no significant difference in TGF- protein expression levels was seen between treatment groups after 6 mo (CsA 5.61 ± 6.12% versus tacrolimus 5.48 ± 4.04%) and after 12 mo (CsA 4.48 ± 3.08% versus tacrolimus 4.64 ± 4.46%; Figure 1B).

The Sirius red staining was performed in duplicate on all sections. A significant correlation was found between duplicate measurements (6 mo r = 0.75, 12 mo r = 0.80; P < 0.001). For each patient, the mean of the duplicate measurements was calculated. There was no significant difference in the extent of Sirius red staining between CsA and tacrolimus treatment regimens (t0: CsA 14.2 ± 5.8% versus tacrolimus 14.8 ± 6.4%; 6 mo: CsA 24.3 ± 5.0% versus tacrolimus 23.4 ± 4.0%; 12 mo: CsA 23.4 ± 5.1% versus tacrolimus 23.5 ± 4.5%; Figure 2A). In accordance with the results found for Sirius red, the extent of -SMA staining after 6 mo (CsA 7.08 ± 2.60% versus tacrolimus 6.88 ± 2.39%) and after 12 mo (CsA 7.75 ± 2.78% versus tacrolimus 7.01 ± 2.49%) was not significantly different between the treatment groups (Figure 2B).

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. The extent of interstitial collagen deposition and the accumulation of -smooth muscle actin (-SMA) protein. (A) Sirius red staining was performed to assess the extent of interstitial collagen accumulation. No difference in interstitial staining was found between CsA and tacrolimus treatment regimens. (B) -SMA staining was performed as a marker for interstitial myofibroblasts. There was no significant difference in the extent of -SMA staining between treatment groups after 6 and 12 mo.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chronic graft damage can be attributed partly to the toxic effect of immunosuppressive agents (7). CsA has been shown to cause fibrosis (8), but it is controversial whether tacrolimus induces a similar effect on renal allografts (9, 10). The objective of our study was to compare the fibrogenic effect of CsA and tacrolimus at the mRNA and the protein level in whole cortical tissue of protocol transplant biopsies from rejection-free patients who were exposed to tailored regimens of these drugs. With both CsA and tacrolimus, a progressive increase in expression of fibrogenic molecules was observed over time. We found no significant difference in expression of fibrosis-related components between treatment groups.

Opposing results have been found in recently published studies of renal protocol transplant biopsies with respect to the influence of CsA and tacrolimus on fibrogenesis and intragraft expression levels of TGF- and ECM molecules (22–24). Most of these studies included biopsies that were taken within 1 yr after transplantation and demonstrated lower TGF- expression levels in tacrolimus-treated recipients (18, 19, 22–24). This may suggest that fibrosis develops slower in tacrolimus-treated patients than in patients who were treated with CsA. However, several of these studies included renal biopsies that were taken for diagnostic purposes. Thus, acquired results may be distorted through the presence of infiltrating cells as a result of acute rejection or through the presence of chronic damage that resulted from long-term medication use. Furthermore, previous studies in protocol biopsies did not examine mRNA and protein levels simultaneously and did not answer conclusively the question of whether increments in mRNA levels of fibrogenic molecules result in increased deposition of the corresponding proteins.

Previous studies showed that the extent of fibrosis, demonstrated by Sirius red staining, in 6-mo protocol biopsies predicts renal function deterioration (34, 35). We found that the extent of fibrosis, measured with digital analysis of Sirius red staining, did not significantly differ between CsA and tacrolimus in our cohort at any moment. The development of interstitial fibrosis is for a large part mediated by the action of interstitial myofibroblasts, which are positive for -SMA (36). We performed immunohistochemical staining for -SMA in our patient cohorts and did not find a significant difference between medication groups.

We found higher mRNA expression levels of TGF- and collagen 1(I) in acute rejection biopsies than in the protocol biopsies without rejection (Figure 1). This is another indication that the expression of fibrogenic molecules can be affected by the presence of such rejection episodes. Recipients who showed clinical or morphologic signs of acute rejection in their protocol biopsies therefore were excluded from this study. No significant differences were found for TGF- mRNA expression levels and TGF- protein deposition between patients who were treated with either CsA or tacrolimus. This finding is in accordance with previous findings for TGF- mRNA expression in glomeruli (22). No significant correlation between TGF- mRNA levels and TGF- protein levels was found, which might be because the antibodies against TGF- visualize both its active and its latent and inactivated forms. Because TGF- mRNA levels significantly correlated with collagens 1(I) and 1(III) mRNA levels in this study, we suppose that mRNA assessment gives a good impression of the extent of TGF- activity in this case.

In both tacrolimus and CsA treatment groups, TGF- mRNA levels progressively rose from their time of transplantation until 12 mo later. Because the patients who had been treated with tacrolimus and CsA showed no differences with respect to TGF- mRNA expression levels at any moment, these findings suggest that the two drugs have a similar effect on TGF- mRNA synthesis. Similarly, mRNA levels of interstitial collagens 1(I) and 1(III) rose over time in both treatment groups. CsA has been known to target the promoter fragment of collagen III (37) and in the process specifically affects the rate of synthesis of the mRNA transcript. A similar mechanism may be involved in the case of collagen 1(I). It is not clear whether tacrolimus also has the capacity to interact with response elements in the interstitial collagen genes.

Our study puts forward results that seem to be at variance with the results in previous studies in the literature, because no significant difference in fibrogenic response of the kidney graft between tacrolimus and CsA was observed in this study. This difference may be because, in this study, patients received tailored calcineurin-inhibitor regimens. The calcineurin inhibitor regimens follow a population-based two-compartmental pharmacokinetic model, which requires only limited sampling and is combined with Bayesian estimation. As previous studies have shown, this method gives an accurate and precise estimation of systemic exposure while being very flexible in allowing nonrigid sampling times as long as dosing and sampling times are recorded accurately (38, 39).

It seems that this study is the first to have compared predefined area-under-the-concentration-over-time-curve guided dosing regimens of tacrolimus and CsA through an assessment of the expression of TGF- and ECM molecules in protocol renal allograft biopsies that are free of rejection. CsA and tacrolimus have a similar inducing effect on intragraft TGF- and collagens 1(I) and 1(III) mRNA levels within the first 12 mo after transplantation. The extent of deposition of TGF- protein and interstitial collagens did not significantly differ between recipients of either CsA or tacrolimus, which shows that the fibrogenic response of renal allografts is similar for either calcineurin inhibitor.

	Acknowledgments

 
Part of this work was presented in abstract form at the following meetings: the American Transplant Congress, May 15 to 19, 2004, Boston, MA; the American Society of Nephrology, Renal Week 2004, St. Louis, MO, October 29 to November 1, 2004; and the American Society of Nephrology, Renal Week 2005, Philadelphia, PA, November 8 to 13, 2005.

We gratefully acknowledge Annemieke van der Wal for expert technical assistance.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Burke JF Jr, Pirsch JD, Ramos EL, Salomon DR, Stablein DM, Van Buren DH, West JC: Long-term efficacy and safety of cyclosporine in renal-transplant recipients. N Engl J Med 331 : 358 –363, 1994[Abstract/Free Full Text]
2. Hariharan S, Johnson CP, Bresnahan BA, Taranto SE, McIntosh MJ, Stablein D: Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med 342 : 605 –612, 2000[Abstract/Free Full Text]
3. Margreiter R: Efficacy and safety of tacrolimus compared with ciclosporin microemulsion in renal transplantation: A randomised multicentre study. Lancet 359 : 741 –746, 2002[CrossRef][Medline]
4. Vincenti F, Jensik SC, Filo RS, Miller J, Pirsch J: A long-term comparison of tacrolimus (FK506) and cyclosporine in kidney transplantation: Evidence for improved allograft survival at five years. Transplantation 73 : 775 –782, 2002[CrossRef][Medline]
5. Paul LC: Chronic renal transplant loss. Kidney Int 47 : 1491 –1499, 1995[Medline]
6. Schweitzer EJ, Matas AJ, Gillingham KJ, Payne WD, Gores PF, Dunn DL, Sutherland DE, Najarian JS: Causes of renal allograft loss. Progress in the 1980s, challenges for the 1990s. Ann Surg 214 : 679 –688, 1991[Medline]
7. Nankivell BJ, Borrows RJ, Fung CL, O’Connell PJ, Allen RD, Chapman JR: The natural history of chronic allograft nephropathy. N Engl J Med 349 : 2326 –2333, 2003[Abstract/Free Full Text]
8. Myers BD, Ross J, Newton L, Luetscher J, Perlroth M: Cyclosporine-associated chronic nephropathy. N Engl J Med 311 : 699 –705, 1984[Abstract]
9. Randhawa PS, Tsamandas AC, Magnone M, Jordan M, Shapiro R, Starzl TE, Demetris AJ: Microvascular changes in renal allografts associated with FK506 (tacrolimus) therapy. Am J Surg Pathol 20 : 306 –312, 1996[CrossRef][Medline]
10. Paul LC: Chronic allograft nephropathy: An update. Kidney Int 56 : 783 –793, 1999[CrossRef][Medline]
11. Pirsch JD, Miller J, Deierhoi MH, Vincenti F, Filo RS: A comparison of tacrolimus (FK506) and cyclosporine for immunosuppression after cadaveric renal transplantation. FK506 Kidney Transplant Study Group. Transplantation 63 : 977 –983, 1997[CrossRef][Medline]
12. Islam M, Burke JF Jr, McGowan TA, Zhu Y, Dunn SR, McCue P, Kanalas J, Sharma K: Effect of anti-transforming growth factor-beta antibodies in cyclosporine-induced renal dysfunction. Kidney Int 59 : 498 –506, 2001[CrossRef][Medline]
13. Shihab FS, Bennett WM, Tanner AM, Andoh TF: Mechanism of fibrosis in experimental tacrolimus nephrotoxicity. Transplantation 64 : 1829 –1837, 1997[CrossRef][Medline]
14. Ninova D, Covarrubias M, Rea DJ, Park WD, Grande JP, Stegall MD: Acute nephrotoxicity of tacrolimus and sirolimus in renal isografts: Differential intragraft expression of transforming growth factor-beta1 and alpha-smooth muscle actin. Transplantation 78 : 338 –344, 2004[CrossRef][Medline]
15. Border WA, Noble NA, Yamamoto T, Harper JR, Yamaguchi Y, Pierschbacher MD, Ruoslahti E: Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease. Nature 360 : 361 –364, 1992[CrossRef][Medline]
16. Jain S, Furness PN, Nicholson ML: The role of transforming growth factor beta in chronic renal allograft nephropathy. Transplantation 69 : 1759 –1766, 2000[CrossRef][Medline]
17. Ignotz RA, Massague J: Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J Biol Chem 261 : 4337 –4345, 1986[Abstract/Free Full Text]
18. Mohamed MA, Robertson H, Booth TA, Balupuri S, Kirby JA, Talbot D: TGF-beta expression in renal transplant biopsies: A comparative study between cyclosporin-A and tacrolimus. Transplantation 69 : 1002 –1005, 2000[CrossRef][Medline]
19. Khanna A, Plummer M, Bromberek C, Bresnahan B, Hariharan S: Expression of TGF-beta and fibrogenic genes in transplant recipients with tacrolimus and cyclosporine nephrotoxicity. Kidney Int 62 : 2257 –2263, 2002[CrossRef][Medline]
20. Hetzel GR, Plum J, Ozcan F, Heering P, Grabensee B: Transforming growth factor-beta1 plasma levels in stable renal allograft recipients under different immunosuppression. Transplantation 71 : 586 –587, 2001[CrossRef][Medline]
21. Matl I, Viklicky O, Voska L, Lodererova A, Vitko S: The effect of different immunosuppressive regimens on TGF-beta 1 expression in kidney transplant patients. Transpl Int 18 : 668 –671, 2005[CrossRef][Medline]
22. Bicknell GR, Williams ST, Shaw JA, Pringle JH, Furness PN, Nicholson ML: Differential effects of cyclosporin and tacrolimus on the expression of fibrosis-associated genes in isolated glomeruli from renal transplants. Br J Surg 87 : 1569 –1575, 2000[CrossRef][Medline]
23. Baboolal K, Jones GA, Janezic A, Griffiths DR, Jurewicz WA: Molecular and structural consequences of early renal allograft injury. Kidney Int 61 : 686 –696, 2002[CrossRef][Medline]
24. Miyagi M, Muramatsu M, Ishikawa Y, Ishii T, Sakai K, Arai K, Aikawa A, Ohara T, Mizuiri M, Hirayama N, Hasegawa A: Comparison of the therapeutic effects between CsA and FK506 on chronic renal allograft injury and TGF-beta expression. Transplant Proc 34 : 1589 –1590, 2002[CrossRef][Medline]
25. Nicholson ML, Bailey E, Williams S, Harris KP, Furness PN: Computerized histomorphometric assessment of protocol renal transplant biopsy specimens for surrogate markers of chronic rejection. Transplantation 68 : 236 –241, 1999[CrossRef][Medline]
26. Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, Croker BP, Demetris AJ, Drachenberg CB, Fogo AB, Furness P, Gaber LW, Gibson IW, Glotz D, Goldberg JC, Grande J, Halloran PF, Hansen HE, Hartley B, Hayry PJ, Hill CM, Hoffman EO, Hunsicker LG, Lindblad AS, Yamaguchi Y: The Banff 97 working classification of renal allograft pathology. Kidney Int 55 : 713 –723, 1999[CrossRef][Medline]
27. Eikmans M, Baelde HJ, de Heer E, Bruijn JA: Processing renal biopsies for diagnostic mRNA quantification: Improvement of RNA extraction and storage conditions. J Am Soc Nephrol 11 : 868 –873, 2000[Abstract/Free Full Text]
28. Eikmans M, Baelde HJ, de Heer E, Bruijn JA: Effect of age and biopsy site on extracellular matrix mRNA and protein levels in human kidney biopsies. Kidney Int 60 : 974 –981, 2001[CrossRef][Medline]
29. Koop K, Bakker RC, Eikmans M, Baelde HJ, de Heer E, Paul LC, Bruijn JA: Differentiation between chronic rejection and chronic cyclosporine toxicity by analysis of renal cortical mRNA. Kidney Int 66 : 2038 –2046, 2004[CrossRef][Medline]
30. de Boer WI, Vermeij M, Diez de Medina SG, Bindels E, Radvanyi F, van der KT, Chopin D: Functions of fibroblast and transforming growth factors in primary organoid-like cultures of normal human urothelium. Lab Invest 75 : 147 –156, 1996[Medline]
31. de Boer WI, Schuller AG, Vermey M, van der Kwast TH: Expression of growth factors and receptors during specific phases in regenerating urothelium after acute injury in vivo. Am J Pathol 145 : 1199 –1207, 1994[Abstract]
32. Bakker RC, Koop K, Sijpkens YW, Eikmans M, Bajema IM, de Heer E, Bruijn JA, Paul LC: Early interstitial accumulation of collagen type I discriminates chronic rejection from chronic cyclosporine nephrotoxicity. J Am Soc Nephrol 14 : 2142 –2149, 2003[Abstract/Free Full Text]
33. Masseroli M, O’Valle F, Andujar M, Ramirez C, Gomez-Morales M, de Dios LJ, Aguilar M, Aguilar D, Rodriguez-Puyol M, Del Moral RG: Design and validation of a new image analysis method for automatic quantification of interstitial fibrosis and glomerular morphometry. Lab Invest 78 : 511 –522, 1998[Medline]
34. Nicholson ML, McCulloch TA, Harper SJ, Wheatley TJ, Edwards CM, Feehally J, Furness PN: Early measurement of interstitial fibrosis predicts long-term renal function and graft survival in renal transplantation. Br J Surg 83 : 1082 –1085, 1996[Medline]
35. Grimm PC, Nickerson P, Gough J, McKenna R, Stern E, Jeffery J, Rush DN: Computerized image analysis of Sirius Red-stained renal allograft biopsies as a surrogate marker to predict long-term allograft function. J Am Soc Nephrol 14 : 1662 –1668, 2003[Abstract/Free Full Text]
36. Strutz F: The fibroblast—A (trans-)differentiated cell? Nephrol Dial Transplant 10 : 1504 –1506, 1995[Free Full Text]
37. Oleggini R, Musante L, Menoni S, Botti G, Duca MD, Prudenziati M, Carrea A, Ravazzolo R, Ghiggeri GM: Characterization of a DNA binding site that mediates the stimulatory effect of cyclosporin-A on type III collagen expression in renal cells. Nephrol Dial Transplant 15 : 778 –785, 2000[Abstract/Free Full Text]
38. Scholten EM, Cremers SC, Schoemaker RC, Rowshani AT, van Kan EJ, den Hartigh J, Paul LC, de Fijter JW: AUC-guided dosing of tacrolimus prevents progressive systemic overexposure in renal transplant recipients. Kidney Int 67 : 2440 –2447, 2005[CrossRef][Medline]
39. Cremers SC, Scholten EM, Schoemaker RC, Lentjes EG, Vermeij P, Paul LC, den Hartigh J, de Fijter JW: A compartmental pharmacokinetic model of cyclosporin and its predictive performance after Bayesian estimation in kidney and simultaneous pancreas-kidney transplant recipients. Nephrol Dial Transplant 18 : 1201 –1208, 2003[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Hertig, D. Anglicheau, J. Verine, N. Pallet, M. Touzot, P.-Y. Ancel, L. Mesnard, N. Brousse, E. Baugey, D. Glotz, et al. Early Epithelial Phenotypic Changes Predict Graft Fibrosis J. Am. Soc. Nephrol., August 1, 2008; 19(8): 1584 - 1591. [Abstract] [Full Text] [PDF]

				T. R. Srinivas and H.-U. Meier-Kriesche Minimizing Immunosuppression, an Alternative Approach to Reducing Side Effects: Objectives and Interim Result Clin. J. Am. Soc. Nephrol., March 1, 2008; 3(Supplement_2): S101 - S116. [Abstract] [Full Text] [PDF]

				J. Schiff, E. Cole, and M. Cantarovich Therapeutic Monitoring of Calcineurin Inhibitors for the Nephrologist Clin. J. Am. Soc. Nephrol., March 1, 2007; 2(2): 374 - 384. [Abstract] [Full Text] [PDF]

				E. M. Scholten, A. T. Rowshani, S. Cremers, F. J. Bemelman, M. Eikmans, E. van Kan, M. J. Mallat, S. Florquin, J. Surachno, I. J. ten Berge, et al. Untreated Rejection in 6-Month Protocol Biopsies Is Not Associated with Fibrosis in Serial Biopsies or with Loss of Graft Function J. Am. Soc. Nephrol., September 1, 2006; 17(9): 2622 - 2632. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: ASN.2005080891v1 17/3/881    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Roos-van Groningen, M. C. Articles by Eikmans, M. Search for Related Content PubMed PubMed Citation Articles by Roos-van Groningen, M. C. Articles by Eikmans, M.