Comparative Transcriptional Profiling of 3 Murine Models of SLE Nephritis Reveals Both Unique and Shared Regulatory Networks

<div><p>Objective</p><p>To define shared and unique features of SLE nephritis in mouse models of proliferative and glomerulosclerotic renal disease.</p><p>Methods</p><p>Perfused kidneys from NZB/W F1, NZW/BXSB and NZM2410 mice were harvested before and after nephritis onset. Affymetrix based gene expression profiles of kidney RNA were analyzed using Genomatix Pathway Systems and Ingenuity Pathway Analysis software. Gene expression patterns were confirmed using real-time PCR.</p><p>Results</p><p>955, 1168 and 755 genes were regulated in the kidneys of nephritic NZB/W F1, NZM2410 and NZW/BXSB mice respectively. 263 genes were regulated concordantly in all three strains reflecting immune cell infiltration, endothelial cell activation, complement activation, cytokine signaling, tissue remodeling and hypoxia. STAT3 was the top associated transcription factor, having a binding site in the gene promoter of 60/263 regulated genes. The two strains with proliferative nephritis shared a macrophage/DC infiltration and activation signature. NZB/W and NZM2410 mice shared a mitochondrial dysfunction signature. Dominant T cell and plasma cell signatures in NZB/W mice reflected lymphoid aggregates; this was the only strain with regulatory T cell infiltrates. NZW/BXSB mice manifested tubular regeneration and NZM2410 mice had the most metabolic stress and manifested loss of nephrin, indicating podocyte loss.</p><p>Conclusions</p><p>These findings identify shared inflammatory mechanisms of SLE nephritis that can be therapeutically targeted. Nevertheless, the heterogeneity of effector mechanisms suggests that individualized therapy might need to be based on biopsy findings. Some common mechanisms are shared with non-immune–mediated renal diseases, suggesting that strategies to prevent tissue hypoxia and remodeling may be useful in SLE nephritis.</p></div

Literature-based analysis of genes shared among all three strains using Genomatix Pathway System (GePS) software.

<p>263 human gene orthologs were regulated in the same direction in the nephritic vs. prenephritic kidneys in NZB/W, NZM2410 and NZW/BXSB. The picture shows the 204 genes that were co-cited in PubMed abstracts in the same sentence. Orange represents the genes that are upregulated and green represents the genes that are downregulated in nephritic compared to prenephritic mice.</p

Top 10 canonical pathways significantly regulated sorted by Benjamini-Hochberg Multiple Testing corrected p-value (p-value<0.05), as assessed by IPA (Ingenuity Pathway Analysis).

<p>Genes tested by RT-PCR are highlighted in bold and underlined.</p>*<p>(number of genes regulated in the same direction).</p>†<p>top transcription factors for each analysis as assessed by GePS.</p

Literature-based analysis of limited gene expression patterns using Genomatix Pathway System (GePS) software.

<p><b>A</b>. 103 shared genes were regulated in the same direction in the nephritic vs. prenephritic kidneys in NZW/BXSB and NZB/W mouse models. The picture shows the 72 that were co-cited in the same sentence of PubMed abstracts. <b>B</b>. 240 genes were regulated in the same direction in the nephritic vs. prenephritic kidneys in NZB/W and NZM2410 mouse models. The picture shows the 124 genes that were co-cited in PubMed abstracts in the same sentence. Orange represents the genes that are upregulated and green represents the genes that are downregulated in nephritic compared to prenephritic mice.</p

A. Glomerular and interstitial damage scores in pre-nephritic (Pre) and nephritic (N) mice (mean + SD) of NZB/W (B/W), NZW/BXSB (W/B) and NZM2410 NZM) strains (* p<0.001; ** p<0.01). The high tubulointerstitial score in the NZM2410 strain reflects severe tubular atrophy. B. Shared and unique gene expression profiles of each of the three strains. Parentheses indicate the number of genes with a human ortholog. C. 3D principal component analysis from the 14780 genes passing the cutoff value (see Materials and Methods) after normalization and batch correction of the arrays from the 3 mouse strains together.

<p>A. Glomerular and interstitial damage scores in pre-nephritic (Pre) and nephritic (N) mice (mean + SD) of NZB/W (B/W), NZW/BXSB (W/B) and NZM2410 NZM) strains (* p<0.001; ** p<0.01). The high tubulointerstitial score in the NZM2410 strain reflects severe tubular atrophy. B. Shared and unique gene expression profiles of each of the three strains. Parentheses indicate the number of genes with a human ortholog. C. 3D principal component analysis from the 14780 genes passing the cutoff value (see Materials and Methods) after normalization and batch correction of the arrays from the 3 mouse strains together.</p

Expression and significance of selected genes.

<p>The genes passing the defined filter criteria are highlighted in bold. np: genes not passing the Affymetrix negative controls cut-off. In italic are the genes not significantly regulated (q-value>0.05).</p

A. One way cluster analysis of genes with significantly altered expression in the PCR validation set (See Table S3). Gene expression was scaled to the mean of pre-nephritic controls for each strain. Significantly up or downregulated (>2 fold) genes by SAM with q value <0.05 are shown (corresponding to 137 genes). B. Summary of the unique and shared pathogenic pathways identified in the kidneys of the three mouse strains.

<p>A. One way cluster analysis of genes with significantly altered expression in the PCR validation set (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077489#pone.0077489.s003" target="_blank">Table S3</a>). Gene expression was scaled to the mean of pre-nephritic controls for each strain. Significantly up or downregulated (>2 fold) genes by SAM with q value <0.05 are shown (corresponding to 137 genes). B. Summary of the unique and shared pathogenic pathways identified in the kidneys of the three mouse strains.</p

A–L. Flow cytometry analysis of proliferating cells. After gating for singlets (A), whole kidney cells from prenephritic (Pre - B, F) and nephritic (Neph - C, G, I, J) NZW/BXSB and nephritic NZB/W (D, H, K, L) mice were analyzed for BrDU incorporation. A BrDU⁺/F4/80⁻ population is seen only in nephritic NZW/BXSB mice (F–H). Proliferating CD11b⁺/F4/80⁺ macrophages are observed only in nephritic NZB/W mice (I–L). A non-BrDU treated control is shown in E. M–P. Immunohistochemistry of kidneys from nephritic NZW/BXSB (M, N), 8 week NZW/BXSB (O), and nephritic NZB/W (P) mice stained with antibodies to Ki67 (M) and PCNA (N–P). 40× magnification. Data are representative of 3 mice per stain.

<p>A–L. Flow cytometry analysis of proliferating cells. After gating for singlets (A), whole kidney cells from prenephritic (Pre - B, F) and nephritic (Neph - C, G, I, J) NZW/BXSB and nephritic NZB/W (D, H, K, L) mice were analyzed for BrDU incorporation. A BrDU⁺/F4/80⁻ population is seen only in nephritic NZW/BXSB mice (F–H). Proliferating CD11b⁺/F4/80⁺ macrophages are observed only in nephritic NZB/W mice (I–L). A non-BrDU treated control is shown in E. M–P. Immunohistochemistry of kidneys from nephritic NZW/BXSB (M, N), 8 week NZW/BXSB (O), and nephritic NZB/W (P) mice stained with antibodies to Ki67 (M) and PCNA (N–P). 40× magnification. Data are representative of 3 mice per stain.</p

Distribution of GIMS score quantiles across gene sets predicted to be enriched in podocyte or mesangial cells.

<p>** “LoF-Tolerant” and “All” genes are included as reference sets. *Note; lower GIMS score quantile=stronger negative selection.</p

Overview of framework to generate GIMS score.

<p>Comparative genomic metrics (GERP++), functional genomic metrics (Polyphen2), and population genetic metrics (SNPs/kb and %RARE) from the 1000 Genomes Project were combined using meta-analysis into a single GIMS scores for 20,079 genes. Gene set enrichment analyses were then performed to evaluate the performance of GIMS scores and test for enrichment of selection in nephrotic syndrome relevant gene sets. 1000<i>G</i>=1000 Genomes Project; SNP/kb= Single Nucleotide polymorphisms/kilobase; FSGS=focal segmental glomerulosclerosis.</p