Identification of fibroblast activation-related genes in two acute kidney injury models

Introduction

Acute kidney injury (AKI) is a renal complication that occurs in up to 20% of hospital admissions (Heung & Chawla, 2012), and part of it can eventually progress to chronic kidney disease (CKD) through interstitial fibrosis (Rodriguez-Romo et al., 2016). Both AKI and CKD represent significant health burdens and devastating conditions with high morbidity and mortality (Basile et al., 2016; Rewa & Bagshaw, 2014). Myofibroblasts (MFs) are the principal cells responsible for extracellular matrix (ECM) production. Generally, MFs do not exist in normal kidneys, while 50% of MFs in fibrotic kidneys are derive from renal resident fibroblasts (LeBleu et al., 2013). The activation of fibroblasts and excessive deposition of the ECM leads to damage of the renal parenchyma and progressive loss of renal function, which eventually progresses to end-stage renal disease (ESRD) (Eddy, 2005). Thus, renal fibrosis is the hallmark of CKD, and it has been demonstrated that the activation of fibroblasts leads to fibrosis (Grgic et al., 2012). However, the molecular mechanisms underlying fibroblast activation are not entirely understood.

Recently, some scholars have found that the activation of Sirtuin 2(SIRT2) promotes fibroblast activation and aggravates renal fibrogenesis (Ponnusamy et al., 2015). As expected, its inhibitor suppresses fibroblast activation through murine double minute2(MDM2), suggesting the SIRT2-MDM2 signaling pathway may be a therapeutic target for renal tubulointerstitial fibrosis (He et al., 2018). In the study of ischemic renal fibrosis, Cyr61 may not only inhibit the fibrotic phenotype of fibroblasts, but also inhibit the proliferation of fibroblasts through the p53/P21/Rb pathway (Liu et al., 2019). Glycogen synthase kinase 3 (GSK3) could promote renal fibrosis through TGF-β signaling, and the pharmacological inhibition of GSK3 could suppress fibroblast activation, even after the detection of AKI in patients (Singh et al., 2015). At present, most studies have proved a detrimental role of the fibroblast activation of renal fibrosis on CKD. Furthermore, the role of fibroblast activation of AKI has been largely ignored. It was reported that fibroblast activation is an extremely early event in AKI, and the blockade of early activation of fibroblasts by genetic and pharmacologic approaches aggravate renal damage and dysfunction after AKI, indicating that renal fibrosis is a complex and multistep pathological process (Zhou et al., 2019). Thus, there is an urgent need to identify novel and predictive biomarkers as therapeutic targets to interfere the progression of AKI to CKD.

The present study identified differentially expressed genes (DEGs) in kidney ischemia-reperfusion injury (IRI) and folic acid (FA)-induced injury models of C57BL/6 mice using the bioinformatics analysis of Gene Expression Omnibus (GEO) database. Then, functional enrichment analysis including Gene Ontology (GO) and KEGG, as well as protein-protein interaction (PPI) network analysis were carried out to explore the potential pathways and hub genes for further research.

Materials and Methods

Results

DEGs screening

After integrated analysis of the two datasets (GSE62732 and GSE121190) between acute injured kidney and normal samples, 457 DEGs (—log2 FC— >1 and FDR <0.05, Figures1) were screened, which included 215 upregulated genes and 242 downregulated genes. Venn diagram and volcano plots (Figs. 1A and 1B) were generated to present the correlation between DEGs. Principal component analysis (PCA) was performed to identify similar gene expression patterns in samples between two subgroups. The results showed that there were significant distinctions between the two subgroups Fig. 1C). A heatmap was constructed to visually present the expression change of the upregulated and downregulated DEGs in the two datasets, and reveal the distribution of the gene expression data of each subset (Fig. 1D).

The GO and KEGG pathway enrichment analysis

The DAVID database was used to determine potential function of DEGs and identify the overrepresented GO categories in the biological process of these 457 DEGs. The top 10 GO terms from the biological process (BP), cellular component (CC) and molecular function (MF) domains in the co-expressed DEGs were ranked according to enriched gene count and P-value (Fig. 2A). In the BP domain, the most meaningful enriched GO terms primarily focused on transport (GO:0006810), the oxidation–reduction process (GO:0055114), and the metabolic process (GO:0008152). The most enriched GO CC terms were correlated to the membrane (GO:0016020), extracellular exosome (GO:0070062), and extracellular region (GO:0005576). For the MF terms, the represented terms were metal ion binding (GO:0046872), hydrolase activity (GO:0016787), and oxidoreductase activity (GO:0016491).

The KEGG pathway enrichment analysis results were listed and ranked by enriched gene count and P-value. Overall, the top seven pathways were selected from the 269 pathways correlated to fibroblast activation in the IRI and FA-induced kidney injury models (Fig. 2B). The common pathways in both sets were the PI3K-Akt signaling pathway (mmu04151), protein digestion and absorption (mmu04974), and focal adhesion (mmu04510).

Observation of cell morphology

Under an inverted phase contrast microscope (X100), we observed the cellular morphology of NRK-49F cells exposed to normoxia and H/R conditions (Fig. 4A). The cells grown under normoxic conditions showed normal fusiform or spindle-shaped, while the cells induced by H/R were longer and the number of cells decreased. Compared with the control group, the growth rate of cells reduced slightly after H/R injury, the fibroblasts were then activated and showed the characteristics of muscle fiber cells.

Discussion

Tubulointerstitial fibrosis is the common endpoint of virtually all progressive renal diseases, regardless of the primary cause (Boor, Ostendorf & Floege, 2010). The transformation of fibroblasts to myofibroblasts is the core event in the development of renal fibrosis (Zeisberg & Neilson, 2010). Early diagnosis and intervention are particularly essential to delay the further deterioration of renal function. Therefore, it is necessary to detect the biomarkers for the early activation of fibroblasts. To our knowledge, little attention has been given to the mechanism of fibroblast activation in AKI. In order to explore the related genes that may play an important role in this process, the gene expression profiles of fibroblasts in two different AKI models of IRI and FA-induced injury were detected by bioinformatics analysis. A total of 454 differentially expressed mRNAs were identified, which included 215 upregulated mRNAs and 242 downregulated mRNAs.

In the GO analysis, it was found that the most enriched BP and MF terms were correlated to intracellular energy metabolism and redox reactions. Recently, emerging evidence has confirmed that the energy metabolism in the progression of renal fibrosis changes from mitochondrial oxidative phosphorylation to glycolysis (Lan et al., 2016; Liu et al., 2017). The results of the KEGG pathway enrichment indicated that the PI3K-Akt signaling pathway was the most significantly enriched pathway between acute injured kidney and non-injured kidney samples. A previous study demonstrated that LPS activates the PI3K-Akt-mTOR/PFKFB3 pathway, and promotes the collagen synthesis of lung fibroblasts through aerobic glycolysis to aggravate pulmonary fibrosis (Hu et al., 2020). Furthermore, in vivo and in vitro evidences have suggested that DHA and Nrf2 may attenuate renal fibrosis through the regulation of fibroblast proliferation and differentiation by inhibiting the PI3K/AKT pathway, and both of these have been used as therapeutic antifibrotic targets for the treatment of renal fibrosis (Wang et al., 2019; Zhang et al., 2019). The PPI network was constructed using the STRING online database and Cytoscape software. A total of 10 hub genes were identified according to the degree of connectivity. Then, qRT-PCR assay was performed for the H/R model and normal cultured primary fibroblasts. Then, three hub genes (Hnf4α, Pck1 and Timp-1), which were closely correlated to fibroblast activation, were filtered out in AKI.

Hnf4α is a cell-specific transcription factor in the nuclear hormone receptor superfamily (Grigo et al., 2008). This is required for homeostasis and metabolism. Furthermore, this is highly expressed in the liver, kidneys and intestines, but is downregulated in the pancreas, stomach and epididymis in adult vertebrates (Odom et al., 2004; Tanaka et al., 2006).In the study of hepatic fibrosis, it was found that the progression of hepatic fibrosis was correlated to the decreased expression of Hnf4α. The overexpression of Hnf4α could promote the expression of E-cadherin, and significantly inhibit the expression of some profibrogenic markers, such as α-SMA, Timp1 and other interstitial markers. Furthermore, inducing the mesenchymal-epithelial transition (MET) process deactivates myofibroblasts and ameliorates the ECM deposition (Yue et al., 2010). At present, no direct evidence has confirmed whether Hnf4α can block renal fibrosis. However, some scholars have proven that the combined expression of four transcription factors, including Hnf4α, has the ability to convert mouse and human fibroblasts to induced-renal tubular epithelial cells (Kaminski et al., 2016). During kidney development, it has been confirmed that Hnf4α is involved in the development of metanephric mesenchyme (MM) into nephrons through MET. In fibroblasts with a forced expression of Hnf4α protein, it was observed that Hnf4α induced the increased expression of epithelial cells (Krt8 and CdH1), and decreased the interstitial gene expression (Acta1 and Vim) (Kanazawa et al., 2011). Meanwhile, the gene expression profiling of unilateral ureteral obstruction (UUO) revealed that Hnf4α might play an important role in early gene expression events related to kidney injury and fibrosis caused by an obstruction (Wu & Brooks, 2012). Therefore, the decreased expression of Hnf4α after the early activation of fibroblasts in AKI may aggravate renal fibrosis by promoting EMT.

Pck1 is a member of the phosphoenolpyruvate carboxykinase family, which is located in the cytoplasm, and is also known as PEPCK-C. This catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, which is the rate-limiting enzyme of gluconeogenesis (Tontonoz et al., 1995). The kidney is one of the most important sites of gluconeogenesis in humans, and the downregulation of key gluconeogenic enzymes, including Pck1, would deleteriously affect RCC cancer cell proliferation and survival (Khan et al., 2015). From the mechanism of cellular energy metabolism, it was found that the main feature of fibroblast activation in the process of renal fibrosis was the transition from oxidative phosphorylation to glycolysis, and that inhibiting glycolysis could suppress fibroblast activation and reduce renal fibrosis in the UUO model (Ding et al., 2017; Wei et al., 2019). Gluconeogenesis and glycolysis are two opposite pathways. When the level of intracellular energy decreases, this is conducive to glycolysis, but not to gluconeogenesis, and the factors that can reduce the level of gluconeogenesis would in turn improve the energy supply of glycolysis. Aerobic glycolysis has been considered as the dominating metabolic pathway of renal tubular epithelial cells in the progression of AKI. Glycolysis inhibitors can suppress fibroblast activation after FA-induced injury through the early inhibition of lactate production in renal tubules (Shen et al., 2020). Recent studies have found that the transcription level of Pck1 is downregulated in the acute renal injury stage of FA-induced fibrosis, suggesting that this participates in mitochondrial energy metabolism, and promotes the progression of renal interstitial fibrosis (Shen et al., 2018). Compared with the detection of total RNA in mouse kidneys, the transcript level of Pck1 in fibroblast mRNA alone would be more meaningful when determining whether this can be used as a precise marker of renal fibrosis.

Timp1 is a member of the tissue inhibitors of metalloproteinases (TIMPS), which are endogenous protein regulators of the family of matrix metalloproteinases (MMPs) (Murphy, 2011). Except for inhibiting matrix degradation, Timp1 is involved in inflammation, and cell proliferation and apoptosis (Peng et al., 2011). In the study of sepsis-associated acute kidney injury (SA-AKI), septic patients had higher serum TIMP-1 levels which could serve as a potential diagnostic biomarker of SA-AKI (Bojic et al., 2015). As a recognized profibrogenic-marker, Timp1 plays a variety of roles in the progression of fibrotic diseases. In the UUO model, the overexpression of Timp1 deteriorates renal interstitial fibrosis through the inflammatory pathway induced by upregulating ICAM-1l (Cai et al., 2008). It has been identified that Timp1 stimulates fibroblasts activation and proliferation via the interaction of CD63/integrin β1, and the activation of the intracellular ERK pathway in the development of lung fibrosis (Dong & Ma, 2017). In addition, hypoxia also increased the expression of Timp1 in mouse fibroblast AKR-2B cells, and induced a phenotypic switch of fibroblasts to myofibroblasts through the MMP-2/TIMP mediated pathway (Misra et al., 2010). The expression level of Timp1 varies in different stages and cells of fibrosis. It has been found that Timp1 is active in the early stage of fibrosis in the UUO model, and initial Timp1 mRNA was transcribed by unidentified interstitial cells and few ED1+ macrophages, but this was mainly transcribed by α-SMA+ myofibroblasts until the later stage of post-ligation (Duymelinck et al., 2000). Nevertheless it is noteworthy that the elimination of Timp1 alone did not block the progression of renal interstitial fibrosis, which might be due to the compensatory effect of other protease inhibitors, such as TIMP-2, TIMP-3, or PAI-1 (Kim et al., 2001). Different from previous studies that focused on chronic fibrosis models, it was found that the expression of Timp1 increased in fibroblasts in two different AKI models, suggesting that the early fibroblast activation that led to renal fibrosis is an early event in the process of AKI transition to CKD.

Conclusions

In summary, a comprehensive bioinformatics analysis of the possible DEGs and pathways involved in two different AKI models was conducted. DEGs, such as Hnf4α, Pck1 and Timp-1, as well as the PI3K-Akt signaling pathway, protein digestion and absorption pathway, focal adhesion pathway and gluconeogenesis/glycolysis pathway, may play a pivotal role in the early activation of fibroblasts. The findings of the present study may facilitate our understanding the different roles of fibroblast activation in AKI.

However, the present study also has some limitations. First, the sample size of the microarray analysis was small, which may cause a potential false positive rate. Second, in vivo experimental verifications were absent from the present study. Therefore, in-depth experiments with a large sample size should be considered to corroborate these discoveries.

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