miR-769-5p has diagnostic value in acute kidney injury in intensive care unit patients and mediates disease development by targeting SIRT6

Abstract

Background

This investigation was designed to assess the diagnostic value of miR-769-5p in acute renal injury (AKI) among intensive care unit (ICU) patients and explore its mechanism by targeting silent information regulator 6 (SIRT6).

Methods

80 sepsis patients without AKI and 82 with sepsis-induced AKI (S-AKI) were enrolled. HK-2 cells were induced with lipopolysaccharide (LPS) to construct an in vitro cell model. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to quantify mRNA levels of miR-769-5p, SIRT6, and renal tubular injury markers kidney injury molecule-1 (KIM-1) and Neutrophil gelatinase-associated lipocalin (NGAL). The receiver operating characteristic (ROC) curve was applied to assess miR-769-5p’s diagnostic ability for S-AKI. Cell proliferation, apoptosis, inflammatory cytokines, and oxidative stress markers were assessed using CCK-8, flow cytometry, ELISA, and commercial kits, respectively. Finally, RNA immunoprecipitation assay and Dual-luciferase reporter assay confirmed miR-769-5p’s direct targeting of SIRT6.

Results

miR-769-5p expression was higher in S-AKI patients compared to Sepsis patients, while SIRT6 was downregulated. miR-769-5p with 87.8% sensitivity and 83.8% specificity, could identify S-AKI patients from Sepsis patients. In HK-2 cells, LPS increased miR-769-5p level and decreased cell viability. Additionally, inhibiting miR-769-5p alleviated LPS-induced cell growth restraint and apoptosis promotion, and the LPS-promoted expression of inflammatory factors, oxidative stress indicators, and tubular injury markers were also weakened by low miR-769-5p expression. miR-769-5p targeted SIRT6, which was downregulated in AKI.

Conclusions

miR-769-5p has diagnostic value in identifying the occurrence of AKI in sepsis patients. Targeted regulation of miR-769-5p may offer a new treatment strategy for AKI.

Background

Acute renal injury (AKI) represents a prevalent and severe complication among patients in the intensive care unit (ICU), characterized by high morbidity and mortality rates [1, 2]. Especially in patients admitted to ICU due to sepsis, AKI caused by sepsis poses a great threat to the life and health of patients [3]. Sepsis, as a systemic inflammatory response syndrome induced by infection, is often accompanied by complex pathophysiological changes [4, 5]. Given its marked perfusion and rich metabolic activity, the kidney is extremely vulnerable to sepsis-related inflammatory mediators, which in consequence causes AKI [6, 7]. Thus, it is crucial to offer a novel perspective on the pathogenic mechanism of AKI and to explore possible therapeutic strategies for AKI.

In the past few years, the regulatory role of microRNA (miRNA) in the occurrence and development of diseases has attracted extensive attention [8]. MiRNAs are a set of endogenous non-coding single-stranded RNA species, these regulate gene expression during the post-transcriptional stage by binding to complementary segments of target messenger RNAs and are involved in inflammation, immunity, and tumorigenesis [9]. Research has indicated that miRNAs are capable of modulating imbalanced inflammatory reactions [10]. They achieve the regulation of inflammatory responses by influencing inflammatory factors and the signaling pathways related to these factors at the post-transcriptional stage, as seen in the role of exosomes in inflammatory diseases and tumor-related inflammation [11]. In the field of kidney diseases, more and more studies have shown that miRNAs, including miR-16-5p [12], let-7a [13], and miR-17-5p [14], are involved in kidney development, physiological function maintenance, and pathological process of diseases [15]. As a component of miRNAs, miR-769-5p has been demonstrated to be related to glioma [16], bladder cancer [17], and non-small cell lung cancer [18], showing differential expression in these malignancies. Sepsis is an inflammation-related disease, and miR-769-5p promotes the secretion of inflammatory factors in periodontal ligament cells, thereby participating in the advancement of gum inflammation [19]. Meanwhile, miR-769-5p is also associated with granulomatosis with polyangiitis [20]. Notably, Ma et al. analyzed and screened differentially detected microRNAs in sepsis-caused renal impairment through sequencing, including miR-769-5p [21]. Nonetheless, the role of miR-769-5p in sepsis AKI continues to be indistinct.

At the same time, silent information regulator 6 (SIRT6), as a NAD + -dependent histone deacetylase, plays a key role in various biological processes such as cell metabolism, aging, inflammation, and stress response [22, 23]. Previous research findings have indicated that the expression of SIRT6 is down-regulated in kidney-related diseases [24]. Our preliminary database prediction indicates a potential targeted binding relationship between miR-769-5p and SIRT6. Thus, we speculate that miR-769-5p may affect the progression of acute kidney injury (AKI) by targeting SIRT6. Given that miRNAs usually function by targeting specific genes, whether there is a targeted regulatory relationship between miR-769-5p and SIRT6 to affect the continuation of AKI becomes a key scientific question in this study.

This research endeavors to systematically judge the diagnostic merit of miR-769-5p in AKI in ICU patients, and to delve into the potential molecular principle of miR-769-5p mediating disease development by targeting SIRT6, to provide a new theoretical basis and potential targets with the intention of early recognition and cure of AKI.

Methods

Participants

Firstly, 162 subjects met the WHO diagnostic criteria were collected from the ICU department of Beijing Rehabilitation Hospital, Capital Medical School from January 2021 to June 2024, and they were divided into sepsis group (n = 80) and sepsis-induced acute kidney injury group (n = 82). The sepsis group was defined as patients admitted to the ICU as a result of sepsis and without the presence of AKI, and the sepsis-induced AKI group was defined as patients with acute kidney injury caused by sepsis. The criteria for exclusion were as follows: (1) patients with significant chronic maladies aside from sepsis (such as malignant tumors, autoimmune diseases, etc.); (2) patients with recent use of drugs that may interfere with renal function or inflammatory markers. Each subject signed a consent with full knowledge, and the study protocol was ratified by the Ethics Committee of Beijing Rehabilitation Hospital, Capital Medical School (Ethics Approval Number: 2020bkky-098).

Clinical sample collection

Clinical baseline data such as age, body mass index (BMI), gender, history of hypertension and diabetes were amassed. Blood urea nitrogen (BUN), serum creatinine (Scr), Lactate, procalcitonin (PCT), and other clinical indicators were measured by an automatic biochemical analyzer, immunoluminescence method, and other detection methods. Patients’ conditions were scored according to acute physiology and chronic health evaluation II (APACHE II) and sequential organ failure assessment (SOFA) scores, they were evaluated, and the duration of hospitalization was meticulously documented.

ROC curve power analysis for sample size calculation

In this study, we employed the sample size calculation method for receiver operating characteristic (ROC) curves proposed by Obuchowski (2005), a widely accepted approach in diagnostic trial design. The formula used was: N = (Z_1−α/2+Z_1−β)²⋅(0.5⋅AUC₀ ^− 1+1)/(AUC₁ − AUC₀)². Here, AUC₀ represents the area under the ROC curve (AUC) under the null hypothesis, set at 0.5, and AUC₁ denotes the anticipated AUC before the study, set at 0.944 based on prior research [25]. α = 0.05, and β = 0.20, corresponding to a statistical power of 80%, Z_1−α/2 = 1.96 and Z_1−β = 0.84. Validation calculations using the R language’s powerROC package confirmed a minimum required sample size of 61 cases. However, we included an actual sample size of 162 cases to enhance statistical robustness.

Cell culture

HK-2 cells (ATCC, USA) were seeded in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) medium fortified with 10% fetal bovine serum (Ausbian, Australia) along with 1% penicillin-streptomycin (Gibco, USA). Subsequently, incubation of the cells took place at 37 °C in an atmosphere composed of 5% CO₂. During the culture period, the growth status of the cells, including cell morphology, density, and adherence, was regularly observed using an inverted microscope. HK-2 cells induced by 0, 1, 5, and 10 µg/mL lipopolysaccharide (LPS) for 24 h. Based on previous studies, 10 µg/mL LPS was selected to induce HK-2 for 24 to an in vitro cell model [26, 27].

Cell transfection

HK-2 cells were injected into 6-well culture plates and cultivated according to the above-mentioned culture conditions. At the point when the cells occupied 50-60% of the culture space, the transfection operation was started. First, to investigate the role of miR-769-5p, miR-769-5p mimic, miR-769-5p inhibitor, and their respective negative controls (mimic NC, inhibitor NC) were introduced into the cells. In the specific transfection process, we mixed diluted Lipofectamine™2000 (Invitrogen, USA) with miR-769-5p mimic, inhibitor, and their negative controls (synthesized by Genepharma, China) to form transfection complexes. Then, the transfection assemblage was dropwise administered to the cells in the culture plate. 6–8 h after transfection, the medium was substituted by fresh medium. After continued culture for a proper time, cells were harvested for subsequent analysis.

RNA extraction and Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)

In the RT-qPCR experiment of acute kidney injury study, total RNA was extracted from HK-2 cells after LPS induction and transfection with TRIzol™ Reagent (Invitrogen, USA). The concentration and purity of total RNA were gauged after chloroform extraction and isopropanol precipitation purification. For miRNAs such as miR-769-5p, cDNA was generated with the use of miScript II RT Kit (Qiagen, Germany). Reverse transcription was performed for mRNA of SIRT6, renal tubular injury markers kidney injury molecule-1 (KIM-1), and Neutrophil gelatinase-associated lipocalin (NGAL) using SuperScript™ IV Reverse Transcriptase Kit (Invitrogen, USA). miR-769-5p were amplified using miScript SYBR Green PCR Kit (Qiagen, Germany). The SYBR Green Quantitative RT-qPCR kit (Invitrogen, USA) was used to amplify SIRT6 mRNA and detect KIM-1 and NGAL. U6 was utilized as the reference gene for miR-769-5p, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was applied as the reference gene for SIRT6, KIM-1, and NGAL. The 2 ^−ΔΔCT method was applied to compute the relative expression levels of target genes relative to their respective reference genes to explore the pathogenesis of acute kidney injury.

Cell viability assay

HK-2 cells were digested and counted by trypsin (Gibco, USA), and seeded in a 96-well plate at appropriate density for 24 h to make them adherent. After that, the medium was absorbed and discarded, and 10 µl cell counting kit (CCK)-8, reagent (Dojindo, Japan), and 100 µl serum-free medium were introduced into each well. The mixture was maintained in the incubator for 1 h, and then the optical density (OD) value was gauged at 450 nm by a microplate reader (Bio-Tek, USA). Finally, the cell survival ability of each treatment group was calculated based on the control group, and the significance of the difference was analyzed by GraphPad Prism software.

Cell apoptosis assay

Apoptosis was detected using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (cwbio, China). First, after treatment, the cell concentration was adjusted to approximately 1 × 10⁶ cells/ml. Then, in strict accordance with the kit instructions, Annexin V binding buffer, Annexin V-FITC, and PI were added to the cell suspension in sequence. During this process, the cells were kept in a dark environment for 15 min and then gently shaken to ensure uniform mixing. Finally, the samples were loaded into a flow cytometer to measure the proportion of apoptotic cells accurately.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of cytokines (tumor necrosis factor α [TNF-α], interleukin 6 [IL-6], interleukin 1 β [IL-1β]) in the culture supernatant were quantified with an Invitrogen ELISA kit. First, prepare reagents and samples as per the kit’s instructions. Add samples and standards (with three replicates each) to pre-coated ELISA plates. Incubate at the specified temperature and time, then wash to remove unbound substances. Put in the detection antibody, incubate the sample once more, and rinse it. After that, add the substrate solution and allow the reaction to take place in the absence of light. Finally, add the stop solution and measure absorbance with a microplate reader to calculate cytokine concentrations from the standard curve.

Oxidative stress markers determination

The supernatant from HK-2 cells was collected. Subsequently, the expression levels of oxidative stress markers malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were measured using the commercially available test kits from Nanjing Jiancheng Bioengineering Institute, following the manufacturer’s protocols.

Reactive oxygen species (ROS) level detection

Cellular ROS levels were quantified using the 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA)-Cellular ROS assay kit (Abcam, USA). Briefly, cells were seeded in 24-well plates, washed once with buffer solution, and incubated with diluted DCFDA solution for 45 min in the dark. After two additional buffer washes, fluorescence signals were measured using Image J software.

RNA Immunoprecipitation (RIP) assay

The RIP assay was carried out in accordance with the guidelines of the Magna RIP kit. (Millipore, USA). HK-2 cells were collected and lysed to obtain cell lysates. Cell lysates underwent incubation with the anti-Ago2 antibody or anti-igG antibody to form immunoprecipitation complexes. Finally, the degrees of miR-769-5p and SIRT6 in the co-precipitated complex were determined by RT-PCR.

Dual-luciferase reporter (DLR) assay

The dual luciferase reporter plasmids, with wild-type (WT) and mutant (MUT) SIRT6 sequences, were commissioned from GenePharma (China). When the growth density of HK-2 cells reached 70-80%, the reporter plasmid and miR-769-5p mimics (GenePharma, China) were jointly transfected into cells by Lipofectamine™3000 reagent (Invitrogen, USA). After 48 h, the activity of dual-luciferase in the samples was measured according to the dual-luciferase reporter gene detection kit (Beyotime, China).

Statistical analysis

After data collection was completed in all the above experiments, Statistical analysis was conducted using SPSS 23.0. Categorical variables were analyzed by Chi-square test, continuous variables by independent-sample T-test, and correlation analysis by Pearson correlation analysis. Multivariate logistic regression analysis was used to determine the independent influencing factors of acute kidney injury in patients with sepsis. To evaluate the data correlation, the Pearson correlation coefficient was determined (when 0.3 < r < 0.4, weak correlation; when 0.5 < r < 0.7, medium strength correlation; when 0.7 < r < 0.9, strong correlation; and when 0.9 < r ≤ 1, very strong correlation). P < 0.05 was considered statistically significant. Experimental data are presented as mean ± standard deviation.

Results

The clinical baseline data of all subjects were statistically analyzed

As shown in Table 1, regarding the baseline characteristics of patients, there were no statistically notable disparities in age, BMI, and gender distribution between the Sepsis group and the sepsis-induced acute kidney injury (S-AKI) group (P > 0.05). Secondly, in terms of medical history, there was no notable disparity in the history of hypertension and diabetes between the two groups (P > 0.05). In terms of clinical indicators, BUN, Scr, lactic acid, PCT, APACHE II score, SOFA score, and duration of hospitalization in the S-AKI group considerably surpassed those within the sepsis group (P < 0.05). Patients with S-AKI are more severe, with more prominent renal damage and inflammatory response.

Table 1.

Comparison of baseline levels and clinical features between the two groups

Parameters	Sepsis(n = 80)	S-AKI(n = 82)	P value
Age (years)	47.19 ± 15.51	44.15 ± 14.38	0.197
BMI (kg/m2)	24.77 ± 3.07	25.17 ± 2.76	0.386
Sex ( n/%)			0.750
Male	38 /47.5%)	41 /50.0%
Female	42 /52.5%)	41 /50.0%
History of disease
Hypertension (n/%)	39/48.48	42/51.2	0.753
diabetes	36/45.0%	43/52.4	0.344
Heart rate (min)	118.23 ± 27.36	118.37 ± 27.91	0.947
BUN (mmol/L)	15.67 ± 11.31	24.17 ± 15.11	0.001
Scr (µmol/L)	68.41 ± 52.24	321.09 ± 177.04	< 0.001
Lactate(mmol/l)	2.35 ± 1.66	3.69 ± 1.84	< 0.001
PCT (ng/ml)	4.05 ± 1.81	4.96 ± 2.52	0.009
APACHEII	7.13 ± 3.61	15.06 ± 7.92	<0.001
SOFA	6.75 ± 2.75	8.82 ± 5.80	0.004
Length of hospital stay (days)	14.94 ± 3.85	17.59 ± 5.79	0.001

Note: S-AKI, Acute kidney injury induced by sepsis; BMI, body mass index; BUN, blood urea nitrogen; SCr, serum creatinine; PCT, procalcitonin; APACHE II, acute physiology and chronic health evaluation II; SOFA, sequential organ failure assessment. P < 0.05 means a significant difference. All data in the table are expressed as mean ± standard deviations

Analysis of miR-769-5p expression levels in patients

To conduct a deeper exploration of the potential part that miR-769-5p plays in S-AKI, we first analyzed the expression quantity of miR-769-5p in those patients who have sepsis and S-AKI. The outcomes demonstrated that the change fold of miR-769-5p manifestation within the S-AKI group was substantially higher in contrast to that in the Sepsis group (P < 0.001, Fig. 1A).

Fig. 1.

Expression characteristics and diagnostic value of miR-769-5p in sepsis and sepsis-induced Acute kidney injury (AKI). (A) Fold change expression of miR-769-5p in Sepsis and S-AKI groups. (B) ROC curve of miR-769-5p in diagnosing sepsis-induced AKI. ***P < 0.001

Correlation between miR-769-5p expression levels and clinical characteristics

Subsequently, we performed a Pearson coefficient correlation analysis on the miR-769-5p expression level and patients’ clinical measures. We found that miR-769-5p was notably positively related to BUN, Scr, Lactate, and PCT. Moreover, as APACHE II score and SOFA are indicators of sepsis severity, miR-769-5p was also remarkably positively related to them. Notably, patients having a high level of miR-769-5p expression endured prolonged hospital confinement (P < 0.001, Table 2).

Table 2.

The correlation between miR-769-5p expression levels and clinical parameters in S-AKI group

Items	r	P-value
BUN (mmol/L)	0.649	< 0.001
Scr (µmol/L)	0.745	< 0.001
Lactate(mmol/l)	0.771	< 0.001
PCT (ng/ml)	0.721	< 0.001
APACHEII	0.801	< 0.001
SOFA	0.673	< 0.001
Length of hospital stay (days)	0.566	< 0.001

Notes: S-AKI, Acute kidney injury induced by sepsis; BUN, blood urea nitrogen; SCr, serum creatinine; PCT, procalcitonin; APACHE II, acute physiology and chronic health evaluation II; SOFA, sequential organ failure assessment

The independent influencing factors of acute kidney injury were analyzed

Results from multivariate logistic regression analysis demonstrated that miR-769-5p, BUN, and Scr were independent risk factors for AKI in patients with sepsis (P < 0.05, Table 3). The hazard ratio (HR) proved that miR-769-5p had the highest HR (4.432). Although factors such as lactate, PCT, APACHE II, SOFA score, and length of hospital stay may differ in univariate analysis and potentially influence the occurrence of kidney injury, multivariate regression analysis revealed that they were not independent risk factors for acute kidney injury in patients with sepsis (P > 0.05, Table 3). Finally, we evaluated the diagnostic value of miR-769-5p using the ROC curve. The ROC curve in Fig. 1B showed that the AUC of miR-769-5p in the diagnosis of acute kidney injury was 0.928 (95%CI: 0.891–0.964), with a sensitivity of 87.8% and a specificity of 83.8%.

Table 3.

Multivariable logistic regression analysis of risk factors for sepsis-induced acute kidney injury

Parameters	HR	95% CI	P value
MiR-769-5p	4.432	1.703–11.533	0.002
BUN (mmol/L)	2.798	1.086–7.210	0.033
Scr (µmol/L)	3.735	1.529–9.125	0.004
Lactate(mmol/l)	2.904	0.751–11.230	0.122
PCT (ng/ml)	2.484	0.542–11.381	0.241
APACHEII	3.216	0.782–13.230	0.105
SOFA	1.627	0.505–5.241	0.415
Length of hospital stay (days)	1.393	0.561–3.457	0.475

Note: BUN, blood urea nitrogen; SCr, serum creatinine; PCT, procalcitonin; APACHE II, acute physiology and chronic health evaluation II; SOFA, sequential organ failure assessment. P < 0.05 means a significant difference

Effects of LPS on miR-769-5p expression and cell viability

Under the action of varying concentrations of LPS (0, 1, 5, 10 µg/ml), the expression of miR-769-5p increased gradually with the increase of LPS concentration. Compared with the 0 µg/ml group, the 5 µg/ml group had an appreciable difference. The difference was extremely pronounced in the 10 µg per milliliter group (P < 0.01, P < 0.001, Fig. 2A). Therefore, we selected 10 µg for subsequent studies. When treated having different levels of LPS concentration, the cell viability slowly decreased as the concentration of LPS went up. Compared with the 0 µg/ml group, the difference in the 5 µg/ml group was significant, and the difference in the 10 µg/ml group was highly significant (P < 0.01, P < 0.001, Fig. 2B).

Fig. 2.

Impact of LPS on miR-769-5p expression and cell viability. (A) Fold change expression of miR-769-5p under different LPS concentrations. (B) Cell viability under different LPS concentrations. **P < 0.01, ***P < 0.001

Effect of miR-769-5p inhibition on LPS-treated cells

We transfected miR-769-5p mimic and inhibitor in HK-2 cells, and both of them effectively elevated and inhibited miR-769-5p expression (P < 0.001, Fig. 3A). In comparison to the control group, the expression of miR-769-5p was markedly elevated in the LPS treatment group and significantly decreased in the miR-769-5p inhibitor treatment group (P < 0.001, Fig. 3B). At diverse time intervals (0, 24, 48, 72 h), the cell growth in the LPS-treated group was repressed in contrast to the control group, while the OD value of the miR-769-5p inhibitor treatment group at every time point far exceeded that of the LPS-treated group (P < 0.001, Fig. 3C). Figure 3D showed that the apoptosis rate of the LPS treatment group was strikingly higher than that of the control group (P < 0.001), and the apoptosis rate of the miR-769-5p inhibitor treatment group was lower than that of the LPS treatment group (P < 0.01).

Fig. 3.

Impacts of miR-769-5p inhibition on LPS-treated cells. (A) Effect of transfection of miR-769-5p mimic and inhibitor on miR-769-5p levels in HK-2 cells. (B) Fold change embodiment of miR-769-5p in different treatment groups. (C) OD values representing cell proliferation at different time points in different treatment groups. (D) Apoptotic cell percentages in different treatment groups. ***P < 0.001 vs. control. ## P < 0.01, ### P < 0.001 vs. inhibitor NC

Effect of miR-769-5p inhibition on the expression of markers of inflammation and renal tubular injury induced by LPS

In terms of inflammatory factors, the abundances of IL-6, IL-1β, and TNF-α were significantly elevated in the culture supernatant of the LPS-treated group compared with the controls, however, this elevation was all typically attenuated by miR-769-5p inhibitor(P < 0.001, Fig. 4A). The impact of miR-769-5p on oxidative stress was further evaluated. As demonstrated in Fig. 4B, LPS induced ROS production, which was significantly suppressed by miR-769-5p downregulation. Moreover, the LPS-mediated elevation of MDA levels and the reduction of SOD and CAT activities were effectively reversed by miR-769-5p inhibition (P < 0.05, Fig. 4C-E). In terms of the manifestation of kidney tubule damage indicator, compared with the control group, the relative mRNA presentation of KIM-1, a marker related to renal tubular injury and NAGL in the LPS treatment group were notably augmented, the miR-769-5p inhibitor treatment group had a significantly lower mRNA expression of KIM-1 and NGAL (P < 0.001, Fig. 4F-G).

Fig. 4.

Effects of miR-769-5p inhibition on inflammatory factors and renal tubular injury markers. A. Cytokine (IL-6, IL-1β, and TNF-α) concentrations in cultured supernatant. B-E. Commercially available assay kits to analyze the activity of oxidative stress indicators (ROS, MDA, SOD, and CAT). F. Relative mRNA index of Renal Tubular Injury KIM-1. G. Relative mRNA transcription of the renal tubular injury biomarker NGAL. ***P < 0.001 vs. control. ## P < 0.01, ### P < 0.001 vs. inhibitor NC

The targeting relationship between miR-769-5p and SIRT6 and its expression characteristics in sepsis-related kidney injury

As shown in Fig. 5A, the TargetScan database predicted that hsa-miR-769-5p has complementary paired sequences with the 3’UTR region (positions 341–347) of SIRT6, suggesting a potential targeting relationship. miR-769-5p mimic appreciably reduced the luciferase activity of WT-SIRT6 (P < 0.001, Fig. 5B), and miR-769-5p inhibitor significantly increased it. There was no variance between the groups undergoing treatment of the MUT-SIRT6 group, confirming that miR-769-5p targeted the 3’ UTR region of SIRT6 to regulate luciferase activity. RIP experiment suggested that in contrast to the IgG group, the enrichment fold of miR-769-5p and SIRT6 in the Ago2 group was significantly increased (P < 0.001, Fig. 5C), further confirming the intracellular interaction between them. The mRNA expression ratio of SIRT6 in the S-AKI group was significantly lower than that in the Sepsis group (P < 0.001, Fig. 5D). Figure 5E scatter plot revealed that a marked negative association between them (r = -0.8961, P < 0.001). When contrasted with the control group, the change fold of SIRT6 expression in the LPS treatment group was significantly decreased, while the group that received treatment with the miR-769-5p inhibitor showed a significant elevation (P < 0.001, Fig. 5F).

Fig. 5.

Aimed-at relationship between miR-769-5p and SIRT6 and its expression characteristics in sepsis-related kidney injury. (A) Complementary paired sequences of hsa-miR-769-5p and SIRT6 3’UTR region. (B) Luciferase activity in different treatment groups for WT-SIRT6 and MUT-SIRT6. (C) Fold enrichment of miR-769-5p and SIRT6 in Ago2 and IgG groups. (D) Relative mRNA expression of SIRT6 in Sepsis and S-AKI groups. (E) Correlation between miR-769-5p and SIRT6 expression. (F) Fold change expression of SIRT6 in different treatment groups. ***P < 0.001 vs. control. ### P < 0.001 vs. inhibitor NC

Discussion

AKI is very widespread and seriously harmful in ICU patients, having a high prevalence and a high mortality level, especially AKI caused by sepsis, which seriously threatens the life and health of patients [28]. Timely diagnosis of AKI is essential to improve the prognosis and reduce mortality [29]. In recent years, the application of non-invasive biomarkers such as miRNA in clinical diagnosis has attracted much attention [30]. A group of endogenous non-coding single-stranded RNA molecules, miRNA has the advantage of high stability and can be detected in body fluids, which can provide a new way for disease diagnosis [8]. In the field of kidney diseases, many miRNAs have been explored for diagnosis [30]. During this investigation, we began by examining the diagnostic value of miR-769-5p in acute kidney injury in ICU patients. The outcomes demonstrated that the expression of miR-769-5p was significantly mounted in patients with S-AKI. What’s more, miR-769-5p has the potential to function as a biomarker for non-invasive identification of AKI with high sensitivity and specificity that can differentiate between patients with S-AKI and sepsis, which is expected to provide help for early clinical diagnosis.

A class of small non-coding RNAs called miRNAs undertake essential regulatory tasks in diverse biological processes. miR-769-5p, a part of the miRNA family, has been reported to participate in the pathogenesis of several diseases. In the context of AKI, its role has drawn increasing attention. In this study, to further examine the association between miR-769-5p and the occurrence and development of AKI, we analyzed its relationship with related factors. Scr is a well-established marker of glomerular filtration function. Lactate levels reflect tissue perfusion status and anaerobic metabolism [31]. PCT serves as a specific inflammatory biomarker for sepsis, while BUN is a recognized indicator of renal function, with elevated levels suggesting impaired renal excretory capacity. The APACHE II score and the SOFA score are used to assess disease severity [32]. Consistent with prior research, we observed aberrant expression of these parameters in S-AKT. Notably, for the first time, our study revealed a strong correlation between miR-769-5p and Scr, lactate, PCT, and APACHE II scores, as well as a moderately strong correlation with BUN and SOFA scores. These findings suggest that miR-769-5p may be intricately involved in the pathophysiological mechanisms of S-AKI. It could potentially influence the extent of renal injury and systemic inflammation by modulating pathways related to inflammation, oxidative stress, and renal tubular cell apoptosis. Consequently, miR-769-5p may play a regulatory role in renal dysfunction, inflammatory responses, and disease progression during AKI, underscoring its potential clinical utility as a diagnostic marker for S-AKI.

LPS represents a primary element within the cell wall of Gram-negative microbes and is emitted into the host when bacterial infection occurs, and its induced cells are widely used in sepsis and kidney injury [33]. In the following experiments, we explored the effectuation of miR-769-5p on cell functions through cell experiments. The data we obtained showed that as the concentration of LPS augmented, the expression of miR-769-5p increased, and the cell viability decreased. Inhibition of miR-769-5p could alleviate the repressive action of LPS on cell proliferation and reduce cell apoptosis. This indicated that we successfully simulated the LPS-induced cell injury model and that the downregulation of miR-769-5p had a positive role in alleviating cell damage. Therefore, miR-769-5p may be a key factor regulating cell survival and function in AKI-related injury, and inhibiting its expression is expected to be a potential strategy to improve cell injury. The inflammatory process can lead to the ejection of a significant number of cytokines and damage the kidney tissue. Previous studies have shown that miR-769-5p has a potential regulatory role in the inflammatory process. Previous studies have reported miR-767-5p’s involvement in periodontitis progression by modulating inflammatory responses in periodontal membrane cells [19] and its association with nodular polyangiitis [34]. Consistent with these findings, our study confirms miR-767-5p’s role in inflammation. Notably, we demonstrate for the first time that miR-767-5p silencing mitigates LPS-induced inflammatory response in HK-2 cells. Additionally, previous studies have demonstrated that oxidative stress contributes to sepsis-associated AKI pathogenesis, as renal tissues—with high vascular density and metabolic demands—are susceptible to ROS-mediated damage [35]. miR-769-5p has also been implicated in myocardial ischemia-reperfusion injury by modulating hypoxia/reoxygenation-induced oxidative stress [36, 37]. Consistent with these findings, our LPS-induced cell models exhibited dysregulated oxidative stress markers. Notably, we demonstrate for the first time that miR-769-5p downregulation significantly attenuates all LPS-driven oxidative stress responses in HK-2 cells. KIM-1 and NGAL are markers of AKI injury that predict the onset [38], severity, and incidence of poor prognosis of AKI, and also indicate cellular damage to renal tubular structural pinches [39]. In addition, Li et al. found that exposure of kidney tissues and HK-2 cells to LPS resulted in the upregulation of KIM-1 and NGAL [12]. Consistent with the recent document, our results illuminate the KIM-1 and NGAL are abnormally elevated in LPS-induced HK-2. Intriguingly, we identified miR-768-5p for the first time as a functional mediator regulating the levels of KIM-1 and NGAL in the S-AKI. Furthermore, our findings indicate that miR-769-5p downregulation enhances renal tubular epithelial cell viability and reduces apoptosis, suggesting its regulatory role in preserving renal tubular cell function and integrity. These cells are vital for renal reabsorption and secretion. In S-AKI, damage to them disrupts renal function, increasing BUN and Scr levels. Modulating miR-769-5p expression may shield renal tubular cells from injury, thereby safeguarding renal function. Flammarion and oxidative stress are primary causes of renal injury in S-AKI, with ROS and inflammatory mediators directly harming renal tubular epithelial cells, causing cell death and tissue damage. Conversely, miR-769-5p downregulation could mitigate renal tubular injury by suppressing oxidative stress and inflammatory response. Therefore, miR-769-5p may serve as a target miRNA, and inhibiting its level may alleviate S-AKT progression.

SIRT6, a NAD-dependent deacetylase in the sirtuin family, is crucial for DNA repair, gene regulation, and metabolism [40]. In diseases like cancer (as a tumor suppressor) and neurodegenerative conditions (protecting neurons), it has well-known functions. In S-AKI, SIRT6 safeguards renal cells by modulating inflammation and oxidative stress [41]. Our database prediction suggested miR-769-5p might target SIRT6 [28]. Our dual luciferase reporter and RIP assays confirmed this targeting. In AKI patients, SIRT6 expression was down-regulated and strongly negatively correlated with miR-769-5p. It is suggested that miR-769-5p may act directly with SIRT6 mRNA to reduce SIRT6 expression by binding to specific sequences, promoting its degradation, or inhibiting its translation process. As a cytoprotective protein, SIRT6 downregulation may reduce cell resistance to injury [23]. miR-769-5p, by targeting SIRT6, could disrupt cell signal pathways, promoting AKI. Previous research indicates that SIRT6 may mitigate sepsis-associated S-AKI via the autophagy pathway [42]. Additionally, USP10 has been implicated in sepsis-induced renal dysfunction, acting through SIRT6 to activate the NRF2/ARE signaling pathway, thereby reducing apoptosis and oxidative stress in renal tubular epithelial cells [43]. While our study demonstrates that inhibiting miR-769-5p alleviates LPS-induced apoptosis and oxidative stress injury in HK-2 cells and that miR-769-5p targets SIRT6, further investigation is required to confirm whether miR-769-5p participates in S-AKI progression through the NRF2/ARE signaling pathway or autophagy pathway. This discovery offers new insights into AKI pathogenesis and a potential therapeutic target direction.

Although we propose that targeted regulation of miR-769-5p-SIRT6 could serve as a therapeutic approach, it is essential to acknowledge the associated limitations. Firstly, the small sample size may compromise the generalizability of our conclusions. Additionally, to ensure the robustness of our findings, we employed stringent inclusion and exclusion criteria to minimize confounding factors; however, these criteria may also limit the applicability of our results to real-world clinical scenarios. Consequently, we plan to expand the sample size and select more representative samples for validation. Second, the dynamic of miR-769-5p in disease progression and whether it can be used as a monitor of therapeutic efficacy need to be further validated. Third, the specific mechanisms and pathways of the miR-769-5p/SIRT6 axis involved in S-AKI need to be further analyzed and validated. In addition, challenges to the development of miRNAs in therapy remain to be addressed; these include tissue-specific delivery, potential off-target effects, and safety.

Conclusions

In summary, the current research verified the crucial role of miR-769-5p in AKI diagnosis and pathogenesis. miR-769-5p might be employed as a potential biomarker for S-AKI, and targeted regulation of miR-769-5p might offer a novel approach to AKI treatment. In times ahead, we will further study the specific molecular mechanism of miR-769-5p regulating AKI, and carry out more clinical studies to verify its diagnostic and therapeutic value, to offer a more solid theoretical and practical groundwork for the prevention and treatment of AKI.

Abbreviations

AKI

Acute renal injury

S-AKI

Sepsis-induced acute kidney injury

ICU

Intensive care unit

miRNA

MicroRNA

SIRT6

Silent information regulator 6

BMI

Body mass index

BUN

Blood urea nitrogen

Scr

Serum creatinine

PCT

Procalcitonin

SOFA

Sequential organ failure assessment

ELISA

Enzyme-linked immunosorbent assay

RIP

RNA immunoprecipitation

DLR

Dual-luciferase reporter

RT-qPCR

Real-time Quantitative Reverse Transcription Polymerase Chain Reaction

KIM-1

Kidney injury molecule-1

NGAL

Neutrophil gelatinase-associated lipocalin

ROC

Receiver operating characteristic

CCK-8

Cell counting kit

ELISA

Enzyme-linked immunosorbent assay

LPS

Lipopolysaccharide

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

OD

Optical density

FITC

Fluorescein isothiocyanate

PI

Propidium iodide

TNF-α

Tumor necrosis factor α

IL-6

Interleukin 6

IL-1β

Interleukin 1 β

HR

Hazard ratio

DCFDA

2’,7’-dichlorodihydrofluorescein diacetate

MDA

Malondialdehyde

SOD

Superoxide dismutase

CAT

Catalase

Author contributions

Yanping Peng, Yao Ma, Xusheng Yang, Yang Zhao, Yunxiao Jia, Yunxing Guo made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, and draft of the manuscript. Xiaodan Sun revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.

Funding

No funding.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

Subjects signed an informed consent form before enrollment. The study protocol was approved by Beijing Rehabilitation Hospital, Capital Medical School and strictly followed the principles of the Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.