Characterization of the macrophage transcriptome in glomerulonephritis-susceptible and -resistant rat strains

Abstract

Crescentic glomerulonephritis (CRGN) is a major cause of rapidly progressive renal failure for which the underlying genetic basis is unknown. Wistar-Kyoto (WKY) rats show marked susceptibility to CRGN, whereas Lewis rats are resistant. Glomerular injury and crescent formation are macrophage dependent and mainly explained by seven quantitative trait loci (Crgn1-7). Here, we used microarray analysis in basal and lipopolysaccharide (LPS)-stimulated macrophages to identify genes that reside on pathways predisposing WKY rats to CRGN. We detected 97 novel positional candidates for the uncharacterized Crgn3-7. We identified 10 additional secondary effector genes with profound differences in expression between the two strains (>5-fold change, <1% false discovery rate) for basal and LPS-stimulated macrophages. Moreover, we identified eight genes with differentially expressed alternatively spliced isoforms, by using an in-depth analysis at the probe level that allowed us to discard false positives owing to polymorphisms between the two rat strains. Pathway analysis identified several common linked pathways, enriched for differentially expressed genes, which affect macrophage activation. In summary, our results identify distinct macrophage transcriptome profiles between two rat strains that differ in susceptibility to glomerulonephritis, provide novel positional candidates for Crgn3-7 and define groups of genes that play a significant role in differential regulation of macrophage activity.

Figure 1. Validation of microarray data with quantitative PCR of selected differentially expressed genes

A) positional differentially expressed QTL candidate genes; B and C) secondary effector genes. Relative gene expression was compared to Hprt for WKY and LEW basal macrophages. The secondary effector genes are presented in two separate graphs (B, C) according to scale of relative gene expression. All samples were amplified using an independent set of biological duplicates with three technical replicates per sample. * = p < 0.05 using a Mann-Whitney non-parametric test (one-tailed). Error bars represent standard deviation.

Figure 2. Selected gene expression in a panel of congenic strains

Relative gene expression compared to Hprt for WKY, LEW, WKY.LCrgn1, LEW.WCrgn1, WKY.LCrgn2, and WKY.LCrgn1,2 in basal macrophages. The sample order shown in the top row of graphs is maintained for all subsequent graphs. All samples were amplified using an independent set of biological duplicates with three technical replicates per sample. * = p < 0.05 **P < 0.01, ***P < 0.001 statistically significantly different to WKY, using a one-way ANOVA with Bonferonni’s correction. Error bars represent standard deviation.

Figure 3. Comparison of alternative splicing microarray data predictions with quantitative PCR of selected transcript isoforms

A) Microarray data showing increased expression of probe-sets that correspond to Dnm3 exons 17-21, in WKY basal macrophages and increased expression of probe-sets that correspond to Sgms1 exons 8-10, for WKY LPS-stimulated macrophages. Data is illustrated as a gene view plot showing mean log₂ signal probe-set intensities by strain. Error bars represent standard errors. B) qPCR data confirming the microarray predictions. Relative expression, compared to Hprt, for WKY and LEW basal macrophages of Dnm3 exon 20 and control exons 15-16, together with WKY and LEW LPS-stimulated macrophages of Sgms1 exon 10 and control exons 3-4. All samples were amplified using an independent set of biological duplicates with three technical replicates per sample. ***P < 0.001, using a one-way ANOVA with Bonferonni’s correction. Error bars represent standard deviation.