Identification of potential candidate genes for hypertensive nephropathy based on gene expression profile

Hypertensive nephropathy (HTN) is a kind of the kidney injury due to chronic high blood pressure [1]. Hypertension-induced renal damage is an increasingly common disease recently, and approximately 25 % of patients currently treated with dialysis are hypertensive before renal replacement therapy started [2]. Although the antihypertensive drugs like cilnidipine (2-methoxyethyl cinnamyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate) and avosentan (N-[6-methoxy-5-(2-methoxyphenoxy)-2-(pyridin-4-yl)pyrimidin-4-yl]-5-methylpyridine-2-sulfonamide) are commonly used for the treatment of HTN [3, 4], the effect of clinical treatment for HTN is still not ideal [5]. Due to the increasing morbidity and mortality of renal disease, molecular mechanisms of HTN are urgently required to be revealed, which contributes to the improvement of therapeutic strategies to control blood pressure and delay progression of HTN [6].

Recently, the studies based on gene or protein investigation are successfully used to reveal the potential mechanisms of HTN. For instance, using distinct lines of the spontaneously hypertensive rat, Dmitrieva et al. have shown a major change in transcriptional control by hepatocyte nuclear factor 1 that affects pathways like redox and other genes, which further lead to the hypertensive renal injury [7]. Periostin, also called osteoblast-specific factor 2, strongly associated with plasma creatinine, proteinuria and renal blood flow, has been identified as a critical marker of progression and regression in HTN [8]. Moreover, SMAD family member 7 has also been discovered to inhibit AngII-mediated HTN through the Sp1/SMAD family member 3/nuclear factor kappa B (NF-κB)/miR-29b regulatory network, and it is identified as a therapeutic biomarker for AngII-mediated HTN [9]. Furthermore, the role of microRNAs (miRNAs) in HTN has also been investigated in recent years. A set of miRNAs (e.g. miR-429, miR-200a, miR-205, miR-200b, miR-141, and miR-192) have been found to be highly expressed in hypertensive nephrosclerosis, and the degree of upregulation is closely related to disease severity [10]. Hsa-miR-181a has confirmed to regulate REN (renin) and apoptosis-inducing factor, mitochondrion-associated, 1 mRNA, and modulate REN expression in HTN [11]. Using a mRNA expression profiling dataset GSE37460, Berthier et al. have discovered a series of pathways, such as endothelial cell activation/injury, immune cell infiltration/activation, and tissue remodeling/fibrosis, with macrophage/dendritic cell activation in both murine models and human lupus nephritis, and they have also found that nuclear factor κB1 and peroxisome proliferator-activated receptor γare major regulatory nodes in the tubulointerstitial and glomerular networks [12]. However, the differences between human HTN and healthy controls remain unclear, and more genes and pathways associated with HTN have not been found.

In the present study, based on the expression profile data of healthy living donor samples and HTN samples deposited by Berthier et al. [12], a bioinformatics analysis was performed. After identification of differentially expressed genes (DEGs) and functional enrichment analysis of the DEGs, protein-protein interactions (PPIs) of the DEGs were analyzed. Furthermore, miRNAs that regulate DEGs were further investigated. These results may contribute to a better understanding of the molecular mechanisms of HTN pathogenesis, and provide valid biological information for further investigation of HTN.

As the study did not involve any human or animals, the ethical approval was not required.

HTN is an increasingly common kidney disease in patients with hypertension recently [6]. However, the potential mechanisms of the progress of HTN is still unclear. In this study, a bioinformatics analysis of gene expression profile for healthy living donor samples and HTN samples was performed to explore the mechanisms of HTN. In total, 51 up-regulated DEGs and 140 down-regulated DEGs were identified in the HTN samples compared with the healthy controls. The DEGs were significantly enriched in pathways like drug metabolism, focal adhesion and arachidonic acid metabolism. Furthermore, in the miRNA-DEG regulatory network, hsa-miR-335-5p and hsa-miR-26b-5p were the two most outstanding miRNAs.

In the present study, CYP3A4 and AGTR1 were the outstanding down- and up-regulated DEGs with the highest degree in the PPI network, respectively. CYP3A4 encodes an enzyme belonging to the cytochrome P450 (CYP P450) superfamily, which is a group of heme-thiolate monooxygenases and participate in a variety of oxidation reactions [21]. CYP P450 expression can be altered by inflammation [22], which is involved in renal injury [23]. The expression of CYP3A4 is induced by glucocorticoids and involved in the metabolism of multiple drugs [24]. A previous study has reported that CYP3A4 T16090C SNP responses to amlodipine among African-Americans with early HTN [25]. Moreover, the expression of CYP3A4 is elevated in patients with end-stage renal disease [26]. CYP4A11, a homologue of CYP3A4, had a higher degree in the PPI network and interacted with CYP4A11. In this study, CYP4A11 was significantly enriched in the pathway of arachidonic acid metabolism. CYP P450 metabolites of arachidonic acid play an important role in the control of blood pressure, chronic kidney disease through the maintenance of the glomerular permeability barrier to albumin [27, 28]. Furthermore, 20-hydroxyeicosatetraenoic acid (20-HETE) has renoprotective actions in hypertension, and mutations in CYP4A11 that produces 20-HETE have been linked to elevated blood pressure in humans [29, 30], indicating the important role of CYP4A11 in HTN. In the present study, CYP4A11 was predicted to be regulated by hsa-miR-26b-5p. An recent study has demonstrated that expression of miR-26b-5p is significantly decreased in basal serum samples from those patients with acute kidney injury, and it is a diagnostic biomarker of acute kidney injury [31]. Currently, there is no any other evidence to prove the associations of CYP3A4, CYP4A11 and hsa-miR-26b-5p with HTN. Given the above studies, we speculated that CYP3A4 and CYP4A11, as well as hsa-miR-26b-5p may play pivotal roles in the progress of HTN.

AGTR1 also had a higher degree in the PPI network, and it was modulated by hsa-miR-26b-5p. AGTR1 encodes of angiotensin II type 1 receptor, which is an important effector in the control of blood pressure [32]. Previous studies have shown that variants on genes including AGTR1 are associated with hypertension [33, 34]. The (−535) T allele of AGTR1 is believed to increase hypertension risk among African Americans [35]. Moreover, AGTR1 polymorphisms are believed to be associated with the renal function [36, 37]. Durvasula et al. have reported that intrarenal production of angiotensin II plays an important role in mediating HTN through inducing podocyte injury and promoting the development of glomerulosclerosis [38]. Furthermore, a previous study has found that angiotensin II-induced arterial hypertension and vascular dysfunction are mediated by lysozyme M–positive monocytes [39], which participate in renal injury [40]. Although there is no direct evidence to prove the association of AGTR1 and HTN, we speculate that AGTR1 may exert critical functions in the progress of HTN.

However, this study has several limitations. The major limitation is that the aforementioned results should be validated by other microarray data or experimental studies, which will be conducted and reported later. Furthermore, more patients with HTN should be included for the analysis. Additionally, the clinical data of the patients are not available, thus the patients may be heterogenous. In the further study, more samples from patients with HTN will be used for the verification experiments to confirm our results.

In conclusion, 51 up-regulated DEGs and 140 down-regulated DEGs were identified in the HTN samples compared with the healthy controls. The DEGs such as CYP3A4, CYP4A11 and AGTR1, may be crucial in the progress of HTN, via the regulation by miRNAs (e.g. hsa-miR-26b-5p) and participation in the biological pathways (e.g. arachidonic acid metabolism). Notably, the above discussed genes and miRNA are new-found to be correlated with HTN in this study, and they are worth further investigation. These findings provide new information for further experimental studies. If these genes and miRNAs are confirmed by experiments, they will be promising to be used in the diagnosis or clinical therapy of HTN.