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Expression of Surfactant Protein-C, S100A8, S100A9, and B Cell Markers in Renal Allografts: Investigation of the Prognostic Value

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Expression of Surfactant Protein-C, S100A8, S100A9, and B Cell Markers in Renal Allografts: Investigation of the Prognostic Value Expression of Surfactant Protein-C, S100A8, S100A9, and B Cell Markers in Renal Allografts: Investigation of the Prognostic Value Michael Eikmans,* Marian C. Roos-van Groningen,* Yvo W.J. Sijpkens,† Jan Ehrchen,‡ Johannes Roth,‡§ Hans J. Baelde,* Ingeborg M. Bajema,* Johan W. de Fijter,† Emile de Heer,* and Jan A. Bruijn* *Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; †Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands; and ‡Institute of Experimental Dermatology and §Department of Pediatrics, University of Mu¨nster, Munich, Germany The intent of this study was to identify genes of which expression during acute rejection is associated with progression to chronic allograft nephropathy using gene expression profiling. Ten patients who had graft loss through chronic allograft nephropathy (progression [PR] group) and 18 patients who had stable graft function over time (nonprogression [NP] group) were studied. Rejection severity and extent of infiltrating leukocytes in acute rejection biopsies were similar for both groups. Microarray analysis and real-time PCR validation showed that surfactant protein-C (SP-C), S100 calcium-binding protein A8 (S100A8), S100A9, and ␤-globin levels distinguished the two groups. Relationship between expression of B cell markers and prognosis was also examined. Location in the graft of the protein and mRNA expression of candidate genes was investigated. The prognostic value of mRNA transcripts was tested in an independent cohort of 43 rejection biopsies. mRNA and protein expression of S100A8 and S100A9 in infiltrating cells was significantly higher in the NP group compared with the PR group. Expression of SP-C was four-fold higher in the PR group and was detected in glomeruli. No association between B cell clusters and outcome was found. In the second group of acute rejection biopsies, SP-C mRNA levels predicted renal function course beyond 6 mo in multivariate analysis. Relatively high expression of S100A8 and S100A9 during acute rejection is associated with a favorable prognosis, and high SP-C expression is associated with an unfavorable prognosis. Messenger RNA transcripts complement the biopsy in the prediction of graft function deterioration. J Am Soc Nephrol 16: 3771–3786, 2005. doi: 10.1681/ASN.2005040412 hanges in mRNA levels of inflammatory components occurrence of acute interstitial rejection (15). Second, the nature and extracellular matrix (ECM)-regulating molecules of the inflammatory infiltrate and the restorative effect of the C within the first 12 mo after transplantation are associ- renal tissue upon acute rejection may partly account for pa- ated with progressive allograft dysfunction (1–4). Various re- tients’ predisposition to develop CAN (16). Recently, Sarwal et ports have emphasized the capacity of microarray technology al. (4) identified three distinct subtypes of acute rejection with to find genes and elucidate molecular pathways that are in- cDNA microarray profiling and showed that the composition volved in the progression of renal allograft damage (4–10). of the infiltrate is related to the prognosis: A high expression of Identification of prognostic factors in the transplantation set- B cell–specific genes during acute rejection predicts an adverse ting may lead to the development of improved intervention outcome. strategies and may contribute to a better understanding of the We intended to identify genes whose early expression after pathophysiology of chronic allograft nephropathy (CAN). transplantation is related to the occurrence of CAN. Such genes In kidney transplantation, CAN is the major cause of late may be involved in the pathogenesis of CAN. Unlike Sarwal’s graft failure (11). The number of acute rejection episodes and approach, we selected two groups of patients who expressed their timing and severity are the strongest predictors of CAN acute rejection within the setting of a case-control study: Those and graft failure (12–14). However, not all acute rejection epi- who had an unfavorable prognosis and lost their grafts as a sodes lead to CAN. First, the occurrence of acute vascular result of CAN within 5 yr after transplantation and those who rejection is associated with a more adverse prognosis than the retained stable graft function after acute rejection for at least 6 yr. In a previous study, TGF-␤ mRNA levels in early acute Received April 19, 2005. Accepted September 15, 2005. rejection biopsies were associated with graft prognosis (2). In this study, we performed gene expression profiling to identify Published online ahead of print. Publication date available at www.jasn.org. molecules for which the expression during acute rejection is Address correspondence to: Dr. Michael Eikmans, Leiden University Medical Center, Department of Pathology, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden, The Nether- associated with graft outcome. Several candidate genes were lands. Phone: ϩ31-71-526-6574; Fax: ϩ31-71-524-8158; E-mail: [email protected] studied to investigate location of the mRNA and the protein in Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1612-3771 3772 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 3771–3786, 2005 the graft tissue and to test whether protein levels also could Control Groups discriminate patient groups with different outcomes. The pre- In all molecular analyses, two control groups were analyzed. One dictive value of the candidate genes was investigated further in group consisted of 6-mo protocol transplant biopsies. The patients Ϯ an independent cohort of acute rejection biopsies. (45.0 6.0 yr) showed stable graft function at time of biopsy, and they received cyclosporine as maintenance therapy (initial dosage of 4 mg/kg twice daily). The biopsies showed no evidence of CAN, acute Materials and Methods rejection, or drug toxicity. The other control group consisted of three Selection of Patient Groups cadaveric donor kidneys that initially were intended for transplanta- To establish two patient groups that represent extremes with respect tion purposes: One sample from the unaffected part of a tumor ne- to outcome, we constructed from 654 consecutive kidney transplants phrectomy kidney and two autopsy kidneys. Mean age was 55.7 Ϯ 18.7 regression lines through reciprocal serum creatinine values beyond 6 yr. The kidneys showed normal histology, and the patients had normal mo after transplantation. These had been performed between 1986 and renal function and no proteinuria. For the immunohistochemical stain- 1995 at the Leiden University Medical Center (17,18). From this cohort, ings, follow-up biopsies from the PR group (patients 1, 3 through 5, and patients who met all of the following criteria were selected: (1) Recip- 7 through 9; see Table 4), showing signs of CAN, were analyzed. ient of a cadaveric donor kidney, (2) treated with cyclosporine (Sand- immune formula; initial dosage of 4 mg/kg twice daily) as maintenance Assessment of Morphologic Alterations immunosuppressive regimen, (3) a graft survival of at least 6 mo, and The extent of infiltrate, chronic damage, and C4d staining during (4) at least one biopsy-supported rejection episode within the first 6 mo acute rejection in individual patients was determined. The extent of after transplantation (n ϭ 200). Two patient groups were defined: One interstitial fibrosis was assessed in Sirius red–stained paraffin sections, group consisted of patients who lost their graft as a result of biopsy- according to methods that have been described elsewhere (19). The supported CAN within 5 yr (progression [PR] group; n ϭ 29). From this percentage of the Sirius red–positive area of the total tubulointerstitial group, 10 patients (11 samples) were included. The other group re- surface was quantified by digital image analysis (20). Histology of all tained stable graft function for at least 6 yr (nonprogression [NP] group; biopsies was evaluated according to Banff 1997 criteria (21). Differences n ϭ 160). From this group, 18 patients (20 samples) were included. in renal gene expression levels between patient groups may be the Slope values of the regression lines and graft survival plots are shown result of differences in the number of inflammatory cells. Therefore, the in Figure 1. total extent of infiltrate, the amount of granulocytes, and the amount of From the 28 patients who were included in the selection, either the plasma cells between groups were compared. These were scored semi- only or the last biopsy with rejection occurring within the first 6 mo quantitatively on a scale from 0 to 3 in silver stainings. The total amount after transplantation was used for molecular analyses. Of three pa- of leukocytes and monocytes was assessed by stainings on paraffin tients, two biopsy samples were included. Table 4 shows the rejection sections for CD45 (Dakocytomation, Leuven, Belgium) and CD68 episodes during which the biopsies were obtained. Of the 31 biopsies (Dako, Glostrup, Denmark), respectively. The extent of staining in the studied, 15 were taken during a first-rejection episode and 16 were renal cortices was quantified by image analysis. Immunofluorescence taken during a follow-on rejection episode. Treatment of rejections staining for C4d was performed on frozen slides using mouse anti-C4d consisted of methylprednisolone, anti-thymocyte globulin, and meth- antibody (Quidel, San Diego, CA). The extent of staining in the peritu- ylprednisolone for first, second, and third rejection
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