Modular Activation of Nuclear Factor-κB Transcriptional Programs in Human Diabetic Nephropathy
Article Figures & Tables
Figures
- FIG. 1.
Flow diagram of the experimental strategy to define the transcriptional regulation in progressive DN via an integrated systems biology approach.
- FIG. 2.
Hierarchical cluster analysis of gene expression profiles from renal biopsies with early or progressive DN and different controls. Tubulointerstitial compartments of renal biopsies from patients with DN (n = 13) and MCD (n = 4), as well as pretransplant biopsies from related LD (n = 3) and CD (n = 4) kidneys, were analyzed. For clinical and histological patient characteristics, see Table 1. Gene expression profiles were analyzed using Affymetrix oligonucleotide arrays HGU133A. Of the 22,283 probe sets, 10,183 probe sets (45.7%) were expressed above background signal. Each row represents a gene; each column represents a biopsy. Transcript abundance is displayed on a red-green color scale, with red expression above and green below the median. Using the SAM algorithm, a multiple comparison analysis of LDs, CDs, MCD, and early and progressive DN with an FDR of 1% detected differential regulation of 1,349 from the 10,183 expressed probe sets. The cluster dendrogram sorts the patients with the most similar gene expression profiles together with the shortest branches. Hierarchical clustering showed three principal branches of DN, CD, and MCD/LD, with the earliest DN (DN1) as the only outlier in the MCD/LD cluster. Differentially regulated mRNAs are provided in the online appendix.
- FIG. 3.
The canonical NF-κB signaling pathway shows a prominent induction in progressive DN. To identify underlying control mechanisms of stress response genes in progressive DN, differentially regulated molecules were categorized into canonical pathways. Starting from 2,738 probe sets regulated between progressive DN and the combined controls (MCD/LD), 1,725 genes could be annotated to biological pathways using the Ingenuity Pathways Knowledge Base. Evaluating the master switch of stress responses, the canonical NF-κB signaling pathway, a differential regulation of 23 of 85 members (27%) in progressive DN compared with none in early DN could be detected. Upregulated genes are marked in red, downregulated genes in green, and green/red symbols correspond to differential regulation of gene isoforms.
- FIG. 4.
NF-κB–dependant target molecules are significantly regulated in progressive DN. Of 232 NF-κB–dependant target genes, 138 (59.5%) showed mRNA expression above background in the kidney expression arrays. An unbiased cluster dendrogram using these 138 genes segregated progressive DN from the combined controls (MCD/LD) and early DN. Of the 138 expressed NF-κB–dependant mRNAs, 54 genes (39%) were significantly induced compared with the combined controls (LD/MCD). In contrast, only one of the 138 genes was upregulated in the early DN, underlining the role of NF-κB in the progression of DN.
- FIG. 5.
Confirmation of predicted gene regulation by the NFKB_IRFF_01 promoter module. A: Microarray and real-time RT-PCR confirm the induction of B2M, HLA-A, HLA-B, VCAM1, and CXCL10/IP10 in progressive DN. B2M, HLA-A, HLA-B, VCAM1, and CXCL10/IP10 expression levels in progressive DN extracted from the array studies are given as ratios to the combined control group (LD/MCD) in the left panel. For confirmation and validation of the hybridization experiments, mRNA expression for CXCL10/IP10 was studied in a cohort of progressive DN patients (n = 22) and different control subjects (LD, n = 9; CD, n = 1; and MCD, n = 7) using real-time RT-PCR (right panel). mRNA expression ratios were normalized to Cyclophilin A. Comparable with the array data (left panel) CXCL10/IP10 mRNA was induced in the tubulointerstitium of patients with advanced DN (DN vs. control, P = 0.013*). Con, control. B: mRNA expression for CCL5/RANTES, EDN1, and IFNB1 is significantly induced in patients with progressive DN. mRNA expression for CCL5/RANTES, EDN1, and IFNB1 was studied in a cohort of progressive DN patients (n = 22) and different control subjects (n = 17) by real-time RT-PCR. mRNA expression ratios normalized to Cyclophilin A revealed a significant induction in the tubulointerstitium of patients with advanced DN (CCL5/RANTES: DN vs. control, *P < 0.001; EDN1: DN vs. control, *P = 0.012; IFNB1: DN vs. control, *P = 0.010*). Con, control.
Tables
- TABLE 1
Clinical and histological characteristics of reference biopsies analyzed by oligonucleotide array–based gene expression profiling and real-time RT-PCR
Sample name Sex Age (years) Histology major diagnosis Histology score Creatinine (mg/dl) Urine proteinuria (g/day) Living donor Array LD1 F 66 LDx NA <1.1 <0.2 LD2 M 26 LDx w/o prev. damage NA 0.9 <0.2 LD3 M 49 LDx w/o prev. damage NA <1.1 <0.2 Mean ± SEM 47 ± 9.6 <0.2 RT-PCR LD4 F 35 LDx NA <1.1 <0.2 LD5 M 39 LDx NA <1.1 <0.2 LD6 F 55 LDx NA <1.1 <0.2 LD7 M 41 LDx NA <1.1 <0.2 LD8 M 61 LDx NA <1.1 <0.2 LD9 F 58 LDx NA <1.1 <0.2 LD10 M 27 LDx NA <1.1 <0.2 LD11 F 54 LDx NA <1.1 <0.2 LD12 F 61 LDx NA <1.1 <0.2 Mean ± SEM 48 ± 14 <1.1 <0.2 Cadaveric donor Array CD1 M 50 CDx, minor int. fibrosis NA 0.9 <0.2 CD2 M 54 CDx w/o prev. damage NA 0.9 <0.2 CD3 M 61 CDx w/o prev. damage NA 1.2 <0.2 CD4 F 51 CDx, minor int. fibrosis NA 0.7 <0.2 Mean ± SEM 54 ± 2.1 0.9 ± 0.1 <0.2 RT-PCR CD5 NA NA CDx NA <1.1 <0.2 MCD/ no histological changes Array MCD1 M 32 Minimal-change GN 1 1.3 11.0 MCD2 F 32 Minimal-change GN 1 0.7 3.0 MCD3 M 16 Minimal-change GN 0 1.2 5.4 MCD4 M 20 Minimal-change GN in remission 0 0.9 0.2 Mean ± SEM 25 ± 3.6 1.0 ± 0.2 4.9 ± 2.3 RT-PCR MCD5 F 57 Minimal-change GN 0 1.1 10 MCD6 M 33 Minimal-change GN 1 1.4 9.1 MCD7 M 24 No histological changes 0 0.6 0.4 Mean ± SEM 38 ± 8 1.0 ± 0.2 6.5 ± 2.5 - TABLE 2
Clinical and histological characteristics of disease biopsies analyzed by oligonucleotide array–based gene expression profiling and real-time RT-PCR
Sample name Sex Age (years) Histology major diagnosis Histology score Creatinine (mg/dl) Proteinuria (g/day) Diabetes type Diabetes duration (years) HbA1c (%) Retinopathy DN Array DN1 M 45 DN 0 0.9 0.7 2 0.3 7.1 No DN2 F 34 DN 1 1.4 0.3 1 31 NA Yes DN3 M 57 DN 3 1.6 9.7 1 20 8.7 Yes DN4 M 61 DN 2 1.1 3.7 2 3 5.7 Yes DN5 F 46 DN 2 2.4 0.4 2 25 7.5 Yes DN6 F 67 DN 4 4.8 2.4 2 5 NA Yes DN7 M 73 DN 2 1.1 2.4 2 1 7.3 No DN8 M 62 DN 2 1.2 2.5 2 5 5.6 No DN9 M 67 DN 2 4.0 3.0 2 NA NA NA DN10 M 62 DN 3 2.3 5.0 2 4 7.8 No DN11 M 68 DN 3 3.5 2.4 2 4 7.2 No DN14 F 70 DN 4 4.0 5.0 — — — No DN15 M 46 DN 3 0.9 6.0 — — — No Mean ± SEM 58.3 ± 3.4 2.2 ± 0.4 3.3 ± 0.7 10.9 ± 3.4 7.1 ± 0.3 RT-PCR DN3 M 57 DN 3 1.6 9.7 1 20 8.7 Yes DN5 F 46 DN 2 2.4 0.4 2 25 7.5 Yes DN6 F 67 DN 4 4.8 2.4 2 5 NA NA DN10 M 62 DN 3 2.3 5.0 2 4 7.8 Yes DN11 M 68 DN 3 3.5 2.4 2 4 7.2 No DN16 M 58 DN 2 1.7 0.7 2 6.5 7.4 No DN17 M 78 DN 4 3.6 3.1 2 32 NA No DN18 F 59 DN 4 3.2 21.4 2 12 NA NA DN19 M 58 DN 4 2.4 14.2 2 31 9.8 Yes DN20 F 63 DN 4 3.3 7.0 2 18 7.9 No DN21 M 63 DN 4 2.1 8.6 2 10 7.1 No DN22 F 47 DN 4 7.0 NA 2 8 13.8 Yes DN23 M 74 DN 3 2.5 0.3 2 8 4.6 No DN24 F 63 DN 3 2.9 6.5 2 7 6.4 No DN25 M 55 DN 4 2.8 7.5 2 6 7.8 Yes DN26 M 57 DN 3 1.6 1.4 2 20 8.8 NA DN27 M 63 DN 2 3.0 0.6 2 6 6.2 NA DN28 M 66 DN 2 9.8 1.7 2 10 6.0 No DN29 M 60 DN 4 2.2 2.0 2 8 6.9 No DN30 M 75 DN 2 1.7 6.2 2 6 7.3 No DN31 M NA DN NA 1.2 10.0 2 <1 NA NA DN32 F 63 DN 4 2.0 5.5 2 23 6.4 NA Mean ± SEM 62 ± 1.7 3.0 ± 0.4 5.5 ± 1.1 11.3 ± 1.7 7.6 ± 0.4 -
Clinical data are presented for all patients involved in the study, either analyzed by microarrays, real-time RT-PCR, or both. Follow-up of the clinical data revealed in 2 (in boldface type) of the 30 patients with nodular glomerulosclerosis, no evidence for clinically overt diabetes but a light chain deposit disease in DN14 and unclassified nodular glomerulosclerosis with mesangial matrix expansion in DN15.
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- TABLE 3
ModelInspector analysis demonstrates a clear overrepresentation of the NF-κB module NFKB_IRFF_01 in 54 upregulated NF-κB–dependant target genes
Modules Induced NF-κ B genes Non-ind. NF-κ B genes All human promoters Number of matches Probability (per 10,000 bp) Number of matches Probability (per 10,000 bp) Number of matches Probability (per 10,000 bp) AP1F_NFKB_01 2 0.29 1 0.11 53 0.02 AP1F_NFKB_02 4 0.58 1 0.11 41 0.01 AP1F_NFKB_03 0 0.00 1 0.11 145 0.04 AP1F_NFKB_04 1 0.15 1 0.11 19 0.01 AP1F_NFKB_EBOX_01 0 0.00 1 0.11 2 0.00 CEBP_NFKB_01 0 0.00 1 0.11 34 0.01 CEBP_NFKB_02 2 0.29 1 0.11 121 0.03 CEBP_NFKB_04 1 0.15 1 0.11 111 0.03 CEBP_NFKB_05 1 0.15 0 0.00 139 0.04 CEBP_NFKB_06 5 0.73 3 0.34 >1,000 0.43 CEBP_NFKB_NFAT_02 0 0.00 1 0.11 204 0.06 CEBP_NFKB_STAT_01 0 0.00 1 0.11 1 0.00 CREB_NFKB_01 1 0.15 1 0.11 138 0.04 CREB_NFKB_03 1 0.15 0 0.00 127 0.04 GATA_GATA_NFKB_NFKB_01 1 0.15 0 0.00 1 0.00 IRFF_NFKB_01 1 0.15 0 0.00 106 0.03 IRFF_NFKB_03 0 0.00 2 0.23 70 0.02 NFKB_AP1F_01 4 0.58 6 0.68 >1,000 0.58 NFKB_AP1F_SP1F_01 0 0.00 2 0.23 14 0.00 NFKB_CEBP_01 1 0.15 4 0.46 596 0.17 NFKB_CREB_01 6 0.88 4 0.46 >1,000 0.58 NFKB_ETSF_01 0 0.00 1 0.11 54 0.02 NFKB_IRFF_01 5 0.73 0 0.00 62 0.02 NFKB_NFKB_02 0 0.00 1 0.11 5 0.00 NFKB_NFKB_03 3 0.44 1 0.11 40 0.01 NFKB_RBPF_01 3 0.44 6 0.68 344 0.10 NFKB_SORY_01 14 2.04 11 1.25 772 0.22 NFKB_SORY_02 10 1.46 5 0.57 348 0.10 NFKB_STAT_01 0 0.00 2 0.23 26 0.01 NFKB_STAT_02 7 1.02 9 1.02 >1,000 0.76 NFKB_STAT_NFKB_01 2 0.29 3 0.34 265 0.08 SP1F_NFKB_01 0 0.00 1 0.11 32 0.01 STAT_NFKB_02 1 0.15 0 0.00 27 0.01 YY1F_NFKB_01 1 0.15 1 0.11 50 0.01 -
Numbers of matches by ModelInspector (Genomatix) analyses of individual experimentally verified NF-κB containing promoter modules in proximal promoter sequences of 54 DN upregulated NF-κB target genes, 84 DN noninduced NF-κB target genes, and the whole human promoter library, including 50.145 proximal promoter sequences are depicted. Total length of analyzed proximal promoter sequences were 68.468 bp in upregulated NF-κB target genes, 87.856 bp in noninduced NF-κB target genes, and 35.125.826 bp in the whole human promoter library. Probabilities of matches with individual modules were shown as frequency of matches observed in 10.000 bp of analyzed proximal promoter sequences. Only the module NFKB_IRFF_01 (in boldface type) was significantly overrepresented in the proximal promoter sequences of induced NF-κB target genes compared with noninduced target genes (P < 0.05).
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