Acute kidney injury is a common and vital diagnostic and therapeutic challenge for clinicians and is typically diagnosed by the accumulation of end products of nitrogen metabolism (urea and creatinine) or decreased urine output, or both[1, 22]. According to the different anatomical structures, AKI was divided into three types of prerenal, renal, postrenal impairment. The pathophysiology of AKI shares common pathogenic denominators including cell death, cell injury, inflammation, and fibrosis, regardless of the initiating insults. AKI can be converted to CKD easily in case of no early diagnosis and effective treatment measures. At present, owing to the lack of sensitive and specific means of AKI prevention and therapy, it is crucial to study the diagnostic biomarkers, novel therapeutic targets and potential pathophysiological mechanism in AKI. Therefore, microarray and high throughout sequencing analysis were used in present study to investigate the function of genes at the whole genome level. WGCNA, a systematic biology method, was used to investigate co-expression in AKI and non-AKI tissues. In addition, the current study explored potential molecular mechanisms, miRNAs, transcription factors and immune cell infiltration and constructed a diagnostic model by LASSO logistic regression.
With adjusted P value < 0.05 and log(2)(|Foldchange|) > 1 as the cutoff, 2202 DEGs (572 upregulated and 1630 downregulated genes) were identified, which have potential to be novel drivers and may play a role in the pathophysiological mechanism underlying AKI development. Fifteen hub genes (KMT2B, NOC2L, COL1A1, BAZ1A, PABPN1, HNRNPD, H6PD, SYNE1, DST, RANGAP1, DEK, MACF1, CHD3, CXXC1 and UBTF) of brown module were selected using comprehensive analytical method of WGCNA, which were further successfully validated using another dataset of GEO database. To further understand the molecular mechanism, GO and KEGG pathways enrichment analysis of the DEGs were performed. As for GO analysis, it was identified that DEGs were enriched in oxidation-reduction process, cell adhesion, proliferation, migration, metabolic process, mitochondria, iron ion binding, heparin binding, oxygen binding, and so on. As for KEGG pathways enrichment analysis, it was identified that DEGs were enriched in metabolic pathways, biosynthesis of antibiotics, Rap1 signaling pathways, carbon metabolism, drug metabolism, and so on. Recent studies have shown that the primary site of damage during AKI, proximal tubular epithelial cells, are highly metabolically active, relying on fatty acids to meet energy demands, which are rich in mitochondria and peroxisomes. The two organelles mediate fatty acid oxidation. Mitochondria are cytoplasmic organelles with a double phospholipid membrane that generate energy via oxidative phosphorylation. Mitochondria are also associated with calcium homeostasis, intracellular reactive oxygen species (ROS) generation and cell signaling functions[28, 29]. Mitochondrial fatty acid β-oxidation serves as the preferred source of ATP in the kidney and its dysfunction results in ATP depletion and lipotoxicity to elicit tubular injury and inflammation and subsequent fibrosis progression. The kidney is a highly metabolic organ with high levels of oxidation within cellular mitochondria. Metabolic process includes glucose metabolism, lipid metabolism, drug metabolism and so on which have been discovered in enrichment analysis of DEGs. Lipid metabolism plays a basic role in renal physiology, especially in tubules. Some studies have revealed the emerging association between increased metabolites and AKI pathogenesis and progression from different perspectives, which were consistent with our study[33, 34].
The present study identified that fifteen genes were significantly associated with AKI development as hub genes of brown module. The Mixed Lineage Leukemia 2 (MLL2) protein, also known as KMT2B, belongs to the family of mammalian histone H3 lysine 4 (H3K4) methyltransferases. Moreover, KMT2B plays a key role in development and germ line deletions of MLL2 have been associated with early growth retardation, neural tube defects and apoptosis that leads to embryonic death. The research has revealed that KMT2B acts as a chromatin modifier gene harbors mutations in Renal cell carcinomas through high-throughput sequencing efforts. However, to our knowledge, no experimental studies of KMT2B in acute kidney injury have been reported to date, which is worthy of further study. NOC2L, acts as an inhibitor of histone acetyltransferase activity, prevents acetylation of all core histones by the EP300/p300 histone acetyltransferase at p53/TP53-regulated target promoters in a histone deacetylases-independent manner with chronic kidney disease. COL1A1, acts as type I collagen, is a member of group I collagen (fibrillary forming collagen). The mutations of COL1A1 can cause Osteogenesis imperfecta has been reported extensively. Recent researches have revealed that COL1A1 is highly associated with chronic kidney disease, cardiovascular diseases and bone metabolism disorders. Moreover, the experimental and theory studies of COL1A1 in acute kidney injury are required in further study. Heterogeneous nuclear ribonucleoprotein D (HNRNPD), has been shown to regulate gene expression at the translational and even the transcriptional level and regulate AU rich elements (ARE)-mRNA turnover, primarily functioning to promote rapid ARE-mRNA degradation and various kidney cells express multiple isoforms of HNRNPD. H6PD is a steroid conversion and receptor gene which plays a crucial role in steroid conversion and response in kidney transplantation. The research of SYNE1 revealed that the essential roles in mediating sunitinib cytotoxicity and the loss of function rendered renal cell carcinoma cell resistant to sunitinib in vitro and in vivo. The rest of the hub genes were rarely identified and verified in various kidney disease, especially in acute kidney injury, which demand us to move one step further uncovering the pathophysiologic mechanisms and biological principles. These hub genes will be potential biomarkers and therapeutic targets of acute kidney injury.
The present study identified that BRD2, EP300, ETS1, MYC, SPI1, ZNF263 were significantly enriched transcriptional factors for fifteen hub genes. BRD2 can specially bind acetylated histone H4 and mediate transcription, which belongs to the bromodomain and extraterminal domain (BET) family regulating the expression of many immunity-associated genes and pathways. EP300, a protein with an essential role in controlling cell growth, cell division and prompting cells to differentiate to take on specialized functions, can mediate epigenetic variation of kidney disease as a transcriptional factor[44, 45]. ETS1 is a member of the ETS family and regulates the expression of a variety of genes including growth factors, chemokines and adhesion molecules and plays a crucial role in the cell cycle progression of renal tubules in acute renal failure (ARF). The ETS1 pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF. MYC acts as a transcriptional factor becomes activated in resident kidney stromal cells early after kidney injury and can regulate metabolic switch in fibrosis initiation and progression. Another study revealed MYC acts as a transcriptional factor participating in the positive feedback loop of MEG3/miR-145-5p/RTKN/ Wnt/β-catenin/c-MYC to promote renal ischemia-reperfusion injury by activating mitophagy and inducing apoptosis. SPI1 can activate H19 which overexpression confers protection against renal injury by stimulating proangiogenic signaling in endothelial cells and tubular epithelial cells of ischemic kidney tissue. ZNF263 acts as a transcriptional factor can regulate a crucial enzyme involved in imparting anticoagulant activity to heparin and also can influence the gene expression of ZRANB2 in human kidney cells. Our study demonstrated that miR-181c-5p, miR-218-5p, miR-485-5p, miR-532-5p and miR-6884-5p were significantly enriched target miRNAs for fifteen hub genes. miR-181c-5p is a member of miR-181c family, and plays a crucial role in regulating extracellular matrix proteins during AKI occurrence and progression. miR-218-5p participates in regulating sepsis-induced acute kidney injury by miR-218-5p/ hemeoxygenase-1 signaling pathway. Wang et al reported that miR-218-5p expressed in endothelial progenitor cells contributes to the development and repair of the kidney microvasculature. miR-485-5p regulates the function of TP53 signaling pathway with signal transduction in response to DNA damage and cell cycle regulation of kidney tissues. The research of miR-532-5p uncovered that LINC00052 ameliorates acute kidney injury by sponging miR-532-5p and activating the Wnt signaling pathway. miR-6884-5p regulates the functions of proliferation, invasion, Epithelial-Mesenchymal Transformation in tumors, however, there are rarely researches to reveal the function and mechanism of kidney diseases. Our study results revealed that the expression of Macrophages M2 in AKI cohort was decreasing significantly compared with non-AKI cohort, which meant M2 macrophages were a protective factor for AKI development. The research showed that M2 macrophages can effectively alleviate acute kidney injury by decreasing inflammatory response and promoting primary proximal tubular epithelial cells proliferation, which was consistent with our findings.
In conclusion, the present study identified fifteen hub genes in acute kidney injury using WGCNA and constructed a diagnostic model by LASSO logistic regression. Various pathways, Transcription factors, miRNAs and 22 immune cell subtypes which were associated with AKI were analyzed and provided some basis for future experimental studies.