Table 1.
Basic clinical parameters of the patients included from the public domain (GEO database) for analysis of lymphotoxin expression in the transcriptome of graft biopsies.
(n = number, m = male, f = female, nd = not determined).
Table 2.
Basic clinical parameters of the patients with transplant glomerulopathy included in the microarray analysis.
(n = number, m = male, f = female, nd = not determined, TG = transplant glomerulopathy).
Table 3.
Basic clinical parameters of the patients included from the ERCB-KFB cohort for RTPCR expression analysis.
(n = number, m = male, f = female, nd = not determined, AR = acute rejection, IFTA = interstitial fibrosis and tubular atrophy, Pre-Tx = pretransplant).
Table 4.
Basic clinical parameters of patients from which allograft biopsies were stained for LTβ.
(n = number, m = male, f = female, nd = not determined, AR = acute rejection, IFTA = interstitial fibrosis and tubular atrophy).
Fig 1.
Upregulation of LT and non-canonical NF-κB pathway target transcripts in human renal allograft biopsies with AR and IFTA.
LTβ, TNFRs1 and 2, LIGHT and HVEM mRNA expression in human renal biopsies with AR (n = 8; A) or chronic allograft nephropathy (n = 21; B) was significantly increased when compared to controls, which were obtained from nephrectomy specimens after explantation (n = 12). Also, CXCL12, CXCL13, CCL 19 and BAFF showed significant upregulation in AR (C) and IFTA (D). Displayed are fold changes compared to controls (nonaffected renal cortex from renal cancer nephrectomy samples). All fold changes shown were statistically significant compared to controls. (* q-value < 0.05, ** q < 0.01, *** q < 0.001).
Fig 2.
Clustered heatmap of the gene set influenced by lymphotoxin from array data.
Samples (x-axis) show transplant glomerulopathy clusters 1 and 2 in comparison to control kidneys. Genes (y-axis) are clustered and colored based on their relative expression levels. The color scale is a double gradient, green is very low expression, average expression is dark and higher expressions are red.
Fig 3.
Upregulation of LT mRNAs in human renal allograft biopsies.
(A) Detailed pathological changes of ERCB renal biopsy specimens in the different groups: BR (n = 10), AR (n = 22) and chronic injury (IFTA) (n = 7); atii acute tubulointerstitial inflammation, ctii chronic tubulointerstitial inflammation/interstitial fibrosis tubular atrophy, ag acute glomerulitis, cg chronic glomerulitis/transplant glomerulopathy, av acute intimal arteritis, cv chronic intimal arteritis/ arterial fibrous intimal thickening, mm mesangial matrix increase. BR borderline rejection, AR acute rejection, IFTA interstitial fibrosis/tubular atrophy. Displayed are mean score + SEM. (B) Displays significant differences between groups. (C) LTα, LTβ, LTβ receptor, DcR3 and LIGHT mRNA expression was quantified by quantitative RT-PCR (). Control biopsies were taken from living donors (LD) before implantation (n = 10). Significant upregulation is shown for Ltα, LTβ, DcR3 and LIGHT in borderline and AR. No differential regulation was observed for HVEM and the LTβR. There was a tendency towards higher expression of Ltα, LTβ, DcR3 and LIGHT mRNA in IFTA compared to controls, however results did not reach significance. The graphs show expression ratios of each gene normalized to 18 srRNA (* p< 0.05; **p < 0.01, *** p<0.001.
Fig 4.
Semiquantitative expression of LTβ in human allograft biopsies.
LTβ immunohistochemistry was performed in biopsies with acute rejection (AR), IFTA (IFTA) and controls (ctrl). Upper panel shows semiquantitative analysis of LTβ positivity in tubular epithelial cells (TECs; left), LTβ positivity in infiltrating inflammatory cells in follicular infiltrates (middle) and LTβ positivity in diffusely infiltrating inflammatory cells (right). The lower panel shows examples of LTβ positivity in TECs (left; arrow depicts positively stained tubular epithelial cell), follicular infiltrates (middle; arrow depicts follicular infiltrate) and diffusely infiltrating cells (right) (scale bar = 50 μM; * p < 0.05; ** p < 0.01, *** p < 0.001).
Fig 5.
Quantification of lymphotoxin mRNAs in mouse renal allografts.
Mean fold-induction of LTα, LTβ, LTβ receptor (all upper panel), HVEM and LIGHT mRNAs (lower panel) quantified by quantitative RT-PCR, demonstrated for full mismatched allografts (AG) compared to isografts (IG) (control) seven days post transplantation. A very significant induction was found for LTα, LTβ, HVEM, LIGHT, but no regulation was found for the LTβ receptor. (* p< 0.05, ** p < 0.01, *** p<0.001, ns = non significant.)