Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule

Abstract

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na⁺-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.

Keywords: Shiga toxin type 2 (Stx2); circadian rhythm; human renal proximal tubular epithelial cell (RPTEC); microarray; mouse; renal proximal tubule.

Figure 1

Stx2 induces the sloughing of proximal tubular cells in the murine kidney. Periodic acid-Schiff (PAS) stains of Stx2-injected murine kidney sections are shown. (A) 0 h (naïve), (B) 8 h, and (C) 48 h after Stx2 injection. Arrows indicate sloughing proximal tubular cells. Arrowheads indicate cell debris. A magnified field within (C) is shown in (D) with sloughing cells pointed with arrows. Bars in (A–C) indicate 50 µm. The scale bar in (D) indicates 10 μm.

Figure 2

Murine proximal tubules express Gb3 in vivo. Confocal microscopic observation of (A) anti-Gb3, (B) anti-AQP1, (C) DAPI, and (D) anti-CD31 immunofluorescence staining. (E) A merged picture of A through D. Closed arrowheads show AQP1 positive proximal tubules which are Gb3 positive. An open arrowhead points to AQP1(−)Gb3(+) tubules. Asterisks indicate tubular lumen. Bars indicate 50 µm.

Figure 3

Human proximal tubules express Gb3 in vivo. Confocal microscopic observation of (A) anti-Gb3, (B) anti-AQP1, (C) DAPI, and (D) anti-CD31 immunofluorescence staining. (E) A merged picture of A through D. Closed arrowheads point to AQP1 positive proximal tubules which are Gb3 positive. Open arrowheads point to AQP1(−)Gb3(+) tubules. Asterisks indicate CD31(+) endothelial cells that are Gb3(+). Bars indicate 50 µm.

Figure 4

Microarray analysis. (A) Among genes analyzed by microarray, 147 genes (green circled), and 4013 genes (red circled) from Stx2-treated mouse kidney and RPTEC, respectively, were detected as having log2-fold change less than −1 or more than 1. Genes that were altered by Stx2 treatment against 0 h in both microarrays (common differentially expressed genes/DEGs) totaled 58 (red and green overlapped area). (B) The heatmap of the common 58 genes is shown in mouse kidney sampling timeline. Z equals 0 denotes an average of log2-fold change values of a gene and reflects a trend of gene expression. Thus, greenish color represents a lower value than an average of a particular gene in a row, whereas reddish color represents above the average change. Log2-fold data of the 58 genes from PBS-injected 72 h dataset compared to 0 h is presented as Table S3 for a reference. (C) The Metascape GO enrichment bar graph is shown. X-axis shows −log10 p values of enriched GO terms across 58 common DEGs that higher number of the enriched GO term is more likely relevant to the system. The top 20 enriched GO terms are shown. (D) STRING analysis of 58 common DEGs is shown in the six most connected/related clusters. Spherical colors denote genes belonging to the same cluster. Relations of actions between genes are shown in color- and end shape-coded manner. ENSG00000279576 has now been coded as ENSP00000485396, which denotes human ID of gene MALAT1.

Figure 5

Differentially expressed genes with mouse sampling timeline. (A) PDK4 and CEBPB mRNA expression patterns are shown. (B) immediate-early genes. (C) Matricellular proteins CYR61 (CCN1) and CTGF (CCN2) expressions. (D) UPR-related genes DDIT3 (CHOP, GADD153), DDIT3 binding partner CEBPB and negative feedback factor PPP1R15A (GADD34) are shown. (E) Transcription factors DDIT3 (CHOP, GADD153) and ATF3 and GDF15, which is transcriptionally regulated by these factors, are shown. (F) UPR- and RSR-associated genes are shown. (G) EDN1 in relation to UPR genes are plotted. (H) Early peaks of EDN1 and EGR1 and following peak of PER1 are shown with later gradual increase in all three genes. (I) Genes from Figure 4D purple cluster are circadian rhythm-related genes. (J) Circadian core genes PER1, Arntl (BMAL1), Cry1 and Clock are shown. (K) PER1 and circadian clock-regulated renal genes are shown. (L) Inflammatory genes with NF-kB pathway-related genes are shown. To see individual log2 data points, please refer to Supplemental Figure S4. Figure S5, showing average log2 with standard deviation bars, is presented as a reference.