Expression of complement components differs between kidney allografts from living and deceased donors

Abstract

A disparity remains between graft survival of renal allografts from deceased donors and from living donors. A better understanding of the molecular mechanisms that underlie this disparity may allow the development of targeted therapies to enhance graft survival. Here, we used microarrays to examine whole genome expression profiles using tissue from 53 human renal allograft protocol biopsies obtained both at implantation and after transplantation. The gene expression profiles of living-donor kidneys and pristine deceased-donor kidneys (normal histology, young age) were significantly different before reperfusion at implantation. Deceased-donor kidneys exhibited a significant increase in renal expression of complement genes; posttransplantation biopsies from well-functioning, nonrejecting kidneys, regardless of donor source, also demonstrated a significant increase in complement expression. Peritransplantation phenomena, such as donor death and possibly cold ischemia time, contributed to differences in complement pathway gene expression. In addition, complement gene expression at the time of implantation was associated with both early and late graft function. These data suggest that complement-modulating therapy may improve graft outcomes in renal transplantation.

Figures

Figure 1.

(A) Gene ontology analysis of the biologic processes of the genes differently expressed between living- and deceased-donor implantation kidneys. Of the 932 significant probe sets, 648 mapped to different genes, which were assigned to 850 biologic processes. Genes for which no biologic process could be assigned were omitted from this display; only categories with more than 15 assigned genes are shown. The numbers represent the number of genes associated with a biologic process. Gene ontology analysis was done with the PANTHER program. (B) Subdivisions of the category immunity and defense responses in A. The 70 genes in this category in A were assigned with more detailed biologic processes. Categories with more than four assigned genes are shown. (C) Canonical pathways significantly overrepresented in this gene set consisting of 932 probe sets (648 unique identified genes). Significance values refer to the −log [P value], which is obtained by the Ingenuity pathway program, which is based on the Ingenuity Pathways Knowledge Base. (D) Significant overrepresentation of complement genes of both the classical and the alternative complement pathway in the difference between living- and deceased-donor kidneys at implantation. Complement genes expressed significantly higher in deceased- compared with living-donor kidneys are indicated in red. Complement activation inhibitor CR1 is expressed significantly lower in deceased-donor kidneys at implantation and is indicated in green.

Figure 2.

(A) Complement gene expression in microarrays on the training set biopsies from deceased-donor baseline biopsies (n = 9), relative to gene expression in living-donor baseline biopsies (n = 9). P values were obtained using Wilcoxon-Mann-Whitney U nonparametric ANOVA. *P < 0.05. (B) Complement gene expression in microarrays on the test set 1 biopsies from deceased-donor baseline biopsies (n = 5), relative to gene expression in living-donor baseline biopsies (n = 5). P values were obtained using Wilcoxon-Mann-Whitney U nonparametric ANOVA. *P < 0.05. (C) qRT-PCR validation of the microarray results for different complement cascade genes in the training set (n = 9 living-donor [LD] and 9 diseased-donor [DD] samples). P values were obtained using Wilcoxon-Mann-Whitney U nonparametric ANOVA.

Figure 3.

(A) Evolution of complement gene expression in implantation and posttransplantation biopsies according to donor source, expressed as fold difference from the expression level in implantation biopsies from living-donor kidneys. *ANOVA P value compared with baseline living-donor biopsy expression <0.05, after Bonferroni correction. (B) Paired evolution of complement gene expression in implantation and posttransplantation biopsies of living-donor kidneys (n = 8, obtained from 3 up to 24 mo after transplantation). P values were obtained using paired t test. This figure demonstrates that enhanced local complement gene expression in renal allografts not only is an immediate peritransplantation phenomenon but also is observed in well-functioning, nonrejecting kidneys from living donors. (C, top) C3d positivity apically in the tubular epithelium. Absence of C3d staining apically in the tubular epithelium is shown in the inset. Positivity of the tubular basement membrane is used as internal control. (Bottom) C4d positivity in the cytoplasm of the tubular epithelium. Absence of C4d staining in the cytoplasm of the tubular epithelium is shown in the inset. Mesangial positivity is used as internal control. Magnification, ×400.

Figure 4.

Graft function evolution in living-donor (n = 14) and deceased-donor (n = 14) kidneys included in this study. *Wilcoxon-Mann-Whitney U P < 0.05; **Wilcoxon-Mann-Whitney U P < 0.01. Overall mixed model repeated measures P value for Schwartz GFR according to donor origin, P = 0.04. Data are means ± SEM.