, Introduction
Scrub typhus, caused by the bites of Orientia tsutsugamushi-infected chiggers, is an acute febrile illness that can involve multiple organs, including the lungs, liver, kidneys, and central nervous system [1]. Most patients with scrub typhus recover after antibiotic therapy. However, complications, such as pneumonia, meningitis, and acute kidney injury (AKI) occasionally result in fatalities [2]. Reportedly, the incidence of AKI in scrub typhus varies in the range of 12% to 41% [3,4], and old age and hypoalbuminemia are known to be clinical parameters for predicting the occurrence of AKI in patients with scrub typhus [4,5]. Recently, biomarkers, such as serum neutrophil gelatinase-associated lipocalin have been helpful in predicting scrub typhus-associated AKI [6].
Further, it has been observed that several microRNAs (miRNAs) are differentially expressed in different organs and that distinct subsets of miRNAs are expressed in various kidney diseases. Therefore, several miRNAs can function as potential biomarkers for various kidney diseases [7]. Previous studies on kidney diseases have been primarily focused on urinary miRNAs [8], whereas some recent reports have indicated a unique profile of circulating miRNAs in kidney diseases, including AKI [9], suggesting that circulating miRNAs are biomarkers for kidney diseases. Although AKI can occur in patients with scrub typhus, studies with a focus on the role of miRNAs in scrub typhus-associated AKI are limited. Yun et al. [10] recently proposed that urinary exosomal miRNA-21 can be used as a biomarker for predicting AKI in patients with scrub typhus. However, data showing the potential role of circulating miRNAs as diagnostic, prognostic, and therapeutic biomarkers for scrub typhus-associated AKI are limited.
In this study, we aimed to identify AKI-specific miRNAs in patients with scrub typhus by comparing the circulating miRNA profiles of patients with scrub typhus and healthy volunteers (HVs) using miRNA microarrays.
Methods
Ethics approval and consent to participate
All the participants provided informed consent for participation in the study and for the use of human-derived materials. Further, the study was conducted in compliance with the Declaration of Helsinki and Istanbul and was approved by the Institutional Review Board of the Presbyterian Medical Center in Jeonju, Republic of Korea (No. 2020-09-041).
Study population
Patients diagnosed with scrub typhus, confirmed via a positive immunoglobulin M enzyme-linked immunosorbent assay (ELISA) for scrub typhus at the Presbyterian Medical Center (Jeonju, Korea) between January 2016 and December 2021, were recruited. Patients who were transferred to another hospital for a higher level of care, treated closer to their own homes during the course of treatment, or had concomitant infections, such as leptospirosis, malaria, or dengue fever, were excluded from the study. Patients who were not admitted to our hospital as well as those who were not followed up during the complete recovery of their renal function or for at least 3 months after discharge were excluded. Thus, we prospectively enrolled 40 patients with scrub typhus and divided them into two groups: those with AKI (AKI group) and those without AKI (non-AKI group). Each group consisted of 20 age- and sex-matched patients. In addition, we recruited 20 HVs from individuals who visited our center for routine health checks. All the volunteers provided informed consent and confirmed that they had no history of any medical conditions or long-term medication use. Thus, the study comprised three groups, namely, the AKI, non-AKI, and HV groups (n = 20 per group).
Clinical and laboratory information
The baseline demographics and clinical and laboratory data of the patients were reviewed at hospitalization and during the follow-up period. Further, the detailed clinical histories of all the patients were obtained. All the participants also underwent thorough physical and biochemical examinations. A standard set of investigations, including the following, complete blood count analysis, liver and renal function tests, chest radiography, three peripheral blood smears for malaria, urinalysis (including urea and electrolytes), two blood culture analyses, and a standard set of febrile serological investigations (including an ELISA for the detection of scrub typhus; InBios International Inc.), were performed. The definition of AKI was based on the Risk, Injury, Failure, Loss of kidney function, and End-stage kidney disease (RIFLE) criteria [11]. The patients were categorized into the R, I, or F groups. The estimated glomerular filtration rate (eGFR) was calculated using the abbreviated Modification of Diet in Renal Disease (MDRD) equation [12]. When data on baseline serum creatinine level was not available, eGFR was calculated using the standard four-variable MDRD formula assuming 75 mL/min/1.73 m2 as the cut-off eGFR. The RIFLE class was determined based on the worst class considering the serum creatinine levels, eGFR, and urine output criteria. Renal replacement therapy was initiated using the standard indications.
Serological workups were also performed to differentiate between endemic zoonoses, including leptospirosis and hemorrhagic fever with renal syndrome (HFRS). Leptospirosis was diagnosed based on a ≥4-fold increase in indirect immunofluorescent assay (IFA) titer in paired serum samples, or an antibody titer ≥1:800 in one serum sample using microscopic agglutination tests. Further, HFRS was diagnosed based on a single titer ≥1:80 or ≥4-fold rise in IFA titer in paired serum samples.
MicroRNA microarray analysis
Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Thereafter, RNA quality and quantity were assessed using an Agilent 2100 bioanalyzer (Agilent Technologies). Starting with 250 ng of total RNA, the labeling process began with poly A tailing for each RNA strand using poly A polymerase, followed by the ligation of the biotin-labeled 3DNA dendrimer. Next, the biotinylated RNA strands were hybridized at 48 °C for 18 hours using GeneChip miRNA 4.0 Array (Affymetrix), which was washed and stained using Fluidics Station 450 (Affymetrix). The amplified fluorescence signals from the branched structure of the 3DNA dendrimer were scanned using a GeneChip Scanner 3000 7G (Affymetrix). Further, the arrays were analyzed using a GeneChip Scanner (Affymetrix) with the associated software. MiRNA expression levels were determined using Transcriptome Analysis Console (Affymetrix). The relative signal intensities of the different miRNAs were generated using the robust multi-array average algorithm. Further, target predictions were performed using miRBase, miRDB, TargetScan, miRTarBase, and microRNA.org. Hierarchical clustering and Venn diagrams for the differentially expressed miRNAs were generated using GeneSpring GX 7.3 software (Agilent Technologies).
Identification of microRNA target genes and their molecular pathways
For the analysis of miRNA gene interactions, we uploaded the differentially expressed miRNAs in the patients with scrub typhus with and without AKI relative to the HVs into commonly used analysis programs, such as DIANA-miRPath and miRSystem. For the analysis using DIANA-miRPath v.3.0, we used DIANA-microT-CDS and TargetScan 6.2 [13].
Statistical analysis
All statistical analyses were performed using IBM SPSS version 22.0 (IBM Corp.). Normally distributed continuous variables are expressed as the mean ± standard deviation, while nonnormally distributed variables were expressed as medians with interquartile ranges. To determine statistically significant differences between continuous variables, we performed the t test, and for categorical variables, we performed the chi-square test. Patients with scrub typhus were further divided into two groups, and continuous variables were compared among the three subgroups (HVs vs. AKI vs. non-AKI) using the Kruskal-Wallis multiple comparison test. Receiver operating characteristic (ROC) curve analysis was performed to calculate the area under the curve for each miRNA in the diagnosis of AKI in patients with scrub typhus. Further, statistical significance was set at a p-value of <0.05 (two-sided).
Results
Baseline clinical characteristics
Among the patients with scrub typhus, 20 (50.0%) were male and their mean age was 71 years (range, 55–90 years). The baseline characteristics of the study participants are shown in Table 1. The HVs had no history of hypertension, diabetes, or medication use. A comparison of clinical characteristics between the AKI and non-AKI groups showed no sex-related differences. Further, no differences in comorbidities, such as diabetes mellitus and hypertension, were also observed. The patients with AKI showed a poorer renal function (eGFR, 41 ± 16 mL/min/1.73 m2 vs. 83 ± 19 mL/min/1.73 m2, p < 0.001) at admission as well as lower serum albumin levels than those in the non-AKI group (3.1 ± 0.6 mg/dL vs. 3.6 ± 0.9 mg/dL, p = 0.005), whereas plasma alanine aminotransferase concentrations and total bilirubin levels did not differ between these two groups. We also observed a longer duration of hospital stay for patients in the AKI group than for those in the non-AKI group (10.3 ± 10.0 days vs. 5.7 ± 2.3 days, p = 0.06).
Characterization of small RNA composition changes
RNA quality was analyzed using a bioanalyzer for microarray analysis. To examine small RNA composition, we conducted microarray analysis, which showed small RNAs, including miRNAs, small nucleolar RNAs, and small Cajal body-specific RNAs (scaRNAs). The proportions of these small RNAs were higher in patients with scrub typhus than in the HVs, whereas the proportion of stem loops was lower in the patients than in the HVs (Supplementary Fig. 1, available online).
Comparison of microRNAs among patients with and without acute kidney injury and healthy volunteers
RNA sequencing showed that relative to the HV group, 120 and 449 miRNAs were upregulated and downregulated, respectively, in the non-AKI group (Fig. 1A), and 101 and 468 miRNAs were upregulated and downregulated, respectively, in the AKI group (Fig. 1B). Furthermore, 11 and 110 miRNAs were upregulated and downregulated, respectively, in the AKI group relative to the non-AKI group (Fig. 2). Among these miRNAs, 14 were significantly upregulated or downregulated in the AKI group relative to their levels in the HV and non-AKI groups (Fig. 3). The areas under the ROC curve for each of these 14 miRNAs are shown in Supplementary Fig. 2 (available online). Notably, the area under the ROC curve was 0.784 for miR-4443. We also evaluated the biological pathways associated with these miRNAs using miRSystem. The possible pathways associated with scrub typhus-associated AKI thus obtained are shown in Table 2. Further, the miRNAs associated with tumor necrosis factor alpha (TNF-α) are listed in Table 3. Several genes that were upregulated or downregulated by the 14 differentially expressed miRNAs showed association with the nuclear factor kappa B (NF-κB) pathway (Supplementary Fig. 3, available online).
Correlation between microRNAs and clinical parameters
Among the differentially expressed miRNAs in patients with AKI, the expression levels of miRNA-18a-5p, miRNA-30e-5p, miRNA-15a-5p, miRNA-1224-5p, miRNA-660-5p, miRNA-27b-3p, miRNA-1587, miRNA-4443, and miRNA-6826-5p showed correlation with serum albumin level at admission (Table 4). In the case of renal function, eGFR showed a correlation with the expression levels of miRNA-30e-5p, miRNA-6808-5p, miRNA-6785-5p, miRNA-1224-5p, miRNA-3187-3p, miRNA-1587, miRNA-4443, and miRNA-6826-5p. Additionally, of these 14 miRNAs, miRNA-1224-5p, miRNA-1587, miRNA-4443, and miRNA-6826-5p showed significant associations with eGFR, serum albumin, and age.
Discussion
In this study, we conducted microarray analysis to examine the circulating miRNA profiles of patients with scrub typhus with AKI and compared them with those of patients without AKI or HVs. Thus, we identified AKI-specific miRNAs in patients with scrub typhus relative to those in HVs or patients without AKI. Further, these AKI-specific miRNAs showed a correlation with prognostic clinical parameters in scrub typhus-associated AKI.
Reportedly, patients with various kidney diseases, including AKI show differentially expressed circulating miRNAs [7]. Berillo et al. [14] suggested circulating let-7g-5p and miR-191-5p as biomarkers in patients with chronic kidney disease with hypertension. Lorenzen et al. [15] reported 13 deregulated miRNAs that are expressed in the serum of patients with AKI, and among these, miRNA-210 was identified as a strong predictor of survival in critically ill patients. It has also been shown that miRNAs are helpful in explaining the pathogenesis of AKI [16,17], in which the PTEN/AKT/mTOR pathway is associated with apoptosis and the inflammatory response of tubular epithelial cells. Although tropical acute febrile illnesses, such as scrub typhus, malaria, and leptospirosis can also lead to AKI development [3], only a few studies have reported the role of miRNAs in scrub typhus. Jiang et al. [18] revealed the dynamic regulation of serum exosomal miRNA in mice infected with O. tsutsugamushi, and Yun et al. [10] suggested urinary exosomal miRNA-21 as a marker for scrub typhus-associated AKI. However, the role of circulating miRNAs in scrub typhus-associated AKI has not been reported in any previous study. Therefore, to the best of our knowledge, this study is the first to demonstrate the clinical implications of circulating miRNAs in patients with scrub typhus and kidney dysfunction.
Specifically, in the present study, we identified 14 miRNAs that were differentially expressed in patients scrub typhus with AKI relative to patients without AKI and HVs. Using these 14 miRNAs, we identified 10 pathways associated with AKI in patients with scrub typhus. Mitogen-activated protein kinases (MAPKs) are an important regulatory factor in the human body. Reportedly, it can regulate multiple downstream pathways, energy metabolism, and cell death; thus, it has an effect on kidney injury [19,20]. In case of scrub typhus, O. tsutsugamushi activates MAPK in macrophage cells, and thus, induces TNF-α production [21]. It has also been observed that several intracellular bacteria use the clathrin-mediated endocytic pathway [22]. Additionally, O. tsutsugamushi can invade host cells via the clathrin-dependent endocytic pathway [23], which is enhanced by the TNF receptor ligand [24]. Ras signaling activation is also required for TNF-α production [25,26] and is also associated with AKI via the promotion of oxidative stress [27]. Thus, TNF-α possibly plays an important role in the development of AKI in patients with scrub typhus. Meanwhile, studies have shown the existence of a close association between the TNF and NF-κB pathways, reflecting the biological relationship between the two pathways [28,29]. Specifically, Jan et al. [28] reported that in patients with scrub typhus, a surface antigen of O. tsutsugamushi activates human monocyte-derived dendritic cells via the NF-κB pathway. In this study, after analyzing AKI-specific miRNAs among the 14 differentially expressed miRNAs in patients with scrub typhus, we also observed an association between several genes and the NF-κB pathway.
TNF-α is an important cytokine that mediates diverse cellular immune responses with respect to infection and inflammation and is particularly known to play an important role in the host’s defense against a variety of intracellular pathogens [30]. Circulating cytokines, including TNF-α repress klotho expression in the kidneys, lead to renin-angiotensin-aldosterone system activation and kidney injury [31,32]. Compared to healthy controls, serum TNF-α level is higher in patients with scrub typhus [33] and is reported to be correlated with disease severity [34]. Further, Kim et al. [35] reported that increased TNF-α levels can be useful for predicting the poor prognosis of patients with scrub typhus. In a previous study, we identified hypoalbuminemia as a marker of scrub typhus-associated AKI development [5]. Studies in both experimental and human settings have also shown that TNF-α is associated with a decrease in serum albumin levels [36,37]. Therefore, our results provide deeper insight into the association between hypoalbuminemia and scrub typhus-associated AKI as it clarifies the key role of TNF-α in the pathogenesis of scrub typhus.
Of the 14 differentially expressed miRNAs in patients with AKI identified in this study, miRNA-1224, miRNA-1587, miRNA-4443, and miRNA-6826, which reportedly, are associated with scrub typhus-associated AKI, showed association with renal function, serum albumin, and age [4,5]. MiRNA-4443 is an important regulatory molecule that regulates four signaling pathways, namely, the janus kinase 2/signal transducer and activator of transcription 3, transforming growth factor beta 1, NF-κB, and Ras pathways, by inhibiting their target genes. Thus, it affects the development of human diseases, especially cancer [37,38]. Additionally, miRNA-4443 is associated with T cell-mediated inflammatory processes, including CD4+ T cell dysfunction [39], which has been observed in scrub typhus [40]. In this study, miRNA-4443 was not only associated with prognostic clinical variables but also showed good sensitivity and specificity for the detection of AKI in patients with scrub typhus. Therefore, miRNA-4443 may be useful as a biomarker for predicting AKI in patients with scrub typhus.
This study had some limitations. First, the number of enrolled participants was relatively small; therefore, larger prospective randomized controlled trials are required to validate our results. Second, we could not validate our findings in different cohorts; this would be considered in future studies. Lastly, we did not assess blood TNF-α levels in patients with scrub typhus and HVs; in future studies, we plan to measure these levels to evaluate their clinical relevance and utility.
In conclusion, we identified circulating miRNAs associated with AKI in patients with scrub typhus that may be used as surrogate markers for predicting AKI. The findings of this study enhance understanding regarding the pathogenesis of scrub typhus-associated AKI and the clinical implications of the associated circulating miRNAs. It also highlights potential targets for further research and clinical applications.
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