Transcriptomic Analysis Revealed an Important Role of Peroxisome-Proliferator-Activated Receptor Alpha Signaling in Src Homology Region 2 Domain-Containing Phosphatase-1 Insufficiency Leading to the Development of Renal Ischemia-Reperfusion Injury

Abstract

In kidney transplantation, the donor kidney inevitably undergoes ischemia-reperfusion injury (IRI). It is of great importance to study the pathogenesis of IRI and find effective measures to attenuate acute injury of renal tubules after ischemia-reperfusion. Our previous study found that Src homology region 2 domain-containing phosphatase-1 (SHP-1) insufficiency aggravates renal IRI. In this study, we systematically analyzed differences in the expression profiles of SHP-1 (encoded by Ptpn6)-insufficient mice and wild-type mice by RNA-seq. We found that a total of 161 genes showed at least a twofold change, with a false discovery rate <0.05 in Ptpn6 ^+/mev mice after IRI and 42 genes showing more than a fourfold change. Of the eight genes encoding proteins with immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that bind to Ptpn6, three were upregulated, and five were downregulated. We found that for the differentially expressed genes (DEGs) with a fold change >2, the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were the cell division pathway and peroxisome-proliferator activated receptor PPARα signaling pathways. Furthermore, the downregulated genes of the PPARα signaling pathway were mainly related to fatty acid absorption and degradation. Using an agonist of the PPARα signaling pathway, fenofibrate, we found that renal IRI was significantly attenuated in Ptpn6 ^+/mev mice. In summary, our results show that insufficiency of SHP-1 inhibits the expression of genes in the PPARα signaling pathway, thereby leading to increased reactive oxygen species (ROS) and exacerbating the renal IRI. The PPARα signaling agonist fenofibrate partially attenuates renal IRI induced by SHP-1 insufficiency.

Keywords: PPARα signaling; SHP-1; bioinformatics; kidney transplantation; renal ischemia-reperfusion injury.

FIGURE 1

SHP-1 insufficiency in vivo aggravated renal IRI and increased apoptosis. (A) The steps involved in the establishment of the mouse renal IRI model and analysis process. Briefly, the control group and the heterozygous mice underwent unilateral nephrectomy. After right nephrectomy, the left kidney was clipped for 34 min, and 24 h later, the left kidney was removed for subsequent experiments. (B) Creatinine levels of wild-type and Ptpn6^+/mev mice after renal IRI (n = 3 in each group). (C) Blood urea nitrogen levels of wild-type and Ptpn6 + ⁣/^mev mice after renal IRI (n = 3 in each group). (D) H&E staining of wild-type and Ptpn6^+/mev mice after renal IRI; the quantitative results are shown. Scale bars = 50 μm. The pathological scoring is as follows: score 1, less than 10%; score 2, 10% to less than 25%; score 3, 25–75%; and score 4, more than 75% of cortex with tubular damage. (E) TUNEL in the wild-type and Ptpn6^+/mev mouse groups after renal I/R and their quantitative results. Scale bars = 50 μm. (F) Accumulation of ROS levels in the kidneys of ROS in the wild-type and Ptpn6^+/mev mouse groups after renal I/R. Scale bars = 50 μm. (G,H) Quantitative results of H&E staining and TUNEL staining. *p < 0.05 and **p < 0.01.

FIGURE 2

Differentially expressed genes (DEGs) in the kidneys of wild-type and Ptpn6^+/mev mice after renal IRI. (A) The PCA plot of the Ptpn6^+/mev samples (circles) shows distinct differences between samples from wild-type mice (triangles): each graph represents all DEGs of one animal, and the most similar Ptpn6^+/mev samples (circles) are located the closest. (B) A total of 336 genes out of 19,529 genes detected by RNA-seq were differentially expressed (FDR < 0.05). (C) A total of 161 genes showed at least twofold up-or down-regulation at FDR < 0.05. (D) Fourteen highly DEGs (FDR < 0.0001) in homozygous Ptpn6^+/mev kidneys compared to wild-type littermates. (E) Expression changes of 14 DEGs in different types of renal cells.

FIGURE 3

Kyoto encyclopedia of genes and genomes (KEGG) analysis of all DEGs and the expression of PPARα signaling pathway related genes. (A) KEGG pathway annotation of all DEGs. (B) The top 20 enriched KEGG pathways of all DEGs. A larger rich factor indicates a higher degree of enrichment. The plot is plotted with the pathways ranked by P-value from smallest to largest for the top 20. (C) KEGG enrichment network diagram of all DEGs. (D) The expression of PPARα signaling pathway related genes were obtained by RNA-seq analysis. *p < 0.05.

FIGURE 4

Fenofibrate reverses renal tubular injury caused by a decrease in lipid metabolism gene expression and the accumulation of reactive oxygen species mediated by SHP-1 insufficiency in mice with ischemia-reperfusion injury. (A) Schematic diagrams of the experimental procedure. (B,C) Levels of creatinine and urea nitrogen in Ptpn6^+/mev mice given fenofibrate and vehicle control (corn oil). (D,E) H and E staining in the kidneys of Ptpn6^+/mev mice given fenofibrate and in those given vehicle control after IRI; the quantitative results are shown. Scale bars = 50 μm. (F,G) Accumulation of ROS levels in the kidneys of Ptpn6^+/mev mice given fenofibrate and in those given vehicle after IRI; the quantitative results are shown. Scale bars = 50 μm. (H) Expression of Acaa1b, Ehhadh, Cyp4a10, and Cyp4a14 in Ptpn6^+/mev mice given fenofibrate and in those given vehicle control after IRI. *p < 0.05, **p < 0.01.

FIGURE 5

Schematic of the mechanisms that aggravate IRI after SHP-1 knockdown, possibly through downregulation of the PPARα signaling pathway.