This study enrolled patients with liver cirrhosis who were admitted to the Department of Hepatology, Ningbo No.2 Hospital (Zhejiang, China) from October 2020 to January 2021. The inclusion criteria were being Han Chinese, aged $\ge$18 years old, and being hospitalized for a diagnosis of liver cirrhosis. The exclusion criteria had organic kidney disease, having received renal replacement therapy, intending to undergo or have undergone kidney transplantation or liver transplantation, having liver cancer and other malignant tumors, having a confirmed pregnancy, or having a life expectancy 3 days. Those meeting the inclusion criteria were divided into an AKI group and a non-AKI group. The research protocol was approved by the Medical Ethics Committee of the hospital, and all of the participants provided signed informed consent. The diagnostic criteria for AKI were based on the 2015 International Ascites Club guidelines: a serum creatinine (SCr) concentration $>$0.3 mg/dL (26.5 µmol/L) from baseline within 48 h of admission, or $>$50% from baseline within 7 days of admission.

The data collected on the general condition of the participants comprised their age, sex, primary disease, comorbidities, medical history, family history, having complications of cirrhosis (infection/spontaneous peritonitis, gastrointestinal (GI) bleeding, ascites, and hepatic encephalopathy), body mass index (BMI), systolic blood pressure, and diastolic blood pressure. The laboratory indicators of the participants that were recorded included SCr concentration (baseline SCr concentration, i.e., at admission, and highest SCr concentration), blood urea nitrogen (BUN) concentration, albumin concentration, total bilirubin concentration, alanine aminotransferase (ALT) concentration, aspartate aminotransferase (AST) concentration, glucose concentration, total cholesterol concentration, low-density lipoprotein concentration, triglyceride concentration, blood sodium concentration, C-reactive protein (CRP) concentration, hemoglobin concentration, platelet count, prothrombin time, and international normalized ratio. The simplified Modification of Diet in Renal Disease formula was used to determine the estimated glomerular filtration rate (eGFR).

A sample (0.5 mL) of peripheral venous blood was collected from the patients in the morning while they were at rest and had an empty stomachs. The sample was treated with an anticoagulant (ethylenediaminetetraacetic acid) and then stored at –80 °C. After all of the blood samples had been collected, their DNA was extracted using a blood genomic DNA extraction kit and then stored at –20 °C for later use.

The SNP locus information of SIRT1 was obtained, revealing that the functional SNP site of SIRT1 was rs4746720. Thus, the primer sequence was designed as shown in Table 1.

The kidney proximal tubular epithelial cell line Human Kidney 2 (HK2, cat. ZB188) was purchased from the Shanghai Zhibei Biotechnology Co., Ltd., which has been validated by short tandem repeat profiling and was negative for mycoplasma contamination. Dulbecco’s modified Eagle’s medium/F-12 modification containing 10% fetal bovine serum was added to recovered human proximal tubular epithelial (HK-2) cells, which were then cultured in an incubator at 37 °C under an atmosphere of 5% CO${}_2$. Cells were passaged at a density of 5 $\times$ 10${}^5$ cells/mL.

LipofectamineTM3000 liposomal transfection reagent was used to co-transfect the pmir-GLO-SIRT1-3${}^{\prime }$-UTR-T and pmir-GLO-SIRT1-3${}^{\prime }$-UTR-C luciferase reporter plasmids with an miR-599 mimic and negative control (NC), respectively, into HK-2 cells. A luciferase kit was used to detect the relative luciferase activity of cells after 72 h.

Overexpressed recombinant plasmids pcDNA3.1-SIRT1-T and pcDNA3.1-SIRT1-C bearing different alleles of SIRT1 rs4746720 were constructed, and HK-2 cells were co-transfected with an miR-599 mimic, an miR-599 inhibitor, or an NC, respectively. Total cellular RNA was extracted using TRIzol reagent, and the concentration and purity of the extracted RNA were determined. RNA was reverse transcribed into complementary DNA (cDNA), which was detected by qPCR using an miR-599 upstream primer (5${}^{\prime }$-GCACGGCAGTGTGTGTCAGTGTTTA-3${}^{\prime }$) and downstream primer (5${}^{\prime }$-TATGGTTCTTCACACGACTCCTTCAC-3${}^{\prime }$). The upstream primer of the internal reference U6 was forward 5${}^{\prime }$-CTCGCTTCGGCAGCACA-3${}^{\prime }$, and the downstream primer was 5${}^{\prime }$-AACGCTTCACGAATTTGCG-3${}^{\prime }$. The PCR reaction system comprised 2$\times$ SYBR Color qPCR Master Mix (10 µL), upstream and downstream primers (0.6 µL), cDNA (8.8 µL), and double-distilled H${}_2$O (20 µL). The reaction conditions were 40 cycles comprising pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, and annealing at 60 °C for 30 s. The relative gene expression was calculated using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{CT}}$ method.

Phenylmethylsulfonyl fluoride was added to lyse and extract total proteins, and the bicinchoninic acid assay was used to determine the total protein concentration. Subsequently, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to a polyvinylidene difluoride membrane. Then the membrane was blocked in blocking solution for 1 h, followed by incubation overnight at 4 °C with primary antibodies against SIRT1, PGC-1$\alpha$, nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM) (1:1000), and glyceraldehyde 3-phosphate dehydrogenase (1:5000). Next, the membrane was washed and then incubated for 1 h at room temperature with secondary antibody (1:5000). Finally, enhanced chemiluminescence was used for protein detection, and ImageJ software (v1.42; National Institutes of Health, Bethesda, MD, USA) was used to quantify the bands.

Statistical Package for Social Sciences (SPSS) version 26.0 (IBM SPSS Statistics, Armonk, NY, USA) was used for the data analysis. The normally distributed measurements are expressed as the means $\pm$ standard deviations, the t-test was used for comparisons between groups, and analysis of variance was used for comparisons between multiple groups. The non-normally distributed measures are expressed as medians (interquartile intervals), and the Mann–Whitney U test was used for comparisons between groups. Counts are expressed as cases and percentages, and comparisons between groups were made using a goodness-of-fit chi-square ($\chi$${}^2$) Hardy–Weinberg equilibrium (HWE) test. Logistic regression was used to analyze the degree of association in terms of an odds ratio (OR) with a 95% confidence interval (CI). p 0.05 was considered statistically significant.

Of the 116 participants, 29 (20 males) were assigned to the AKI group and had an average age of 59.0 $\pm$ 14.8 years, and 87 (58 males) were assigned to the control group and had an average age of 60.1 $\pm$ 11.2 years. There were no significant between-group differences in age, sex, BMI, mean arterial pressure, diabetes mellitus, hypertension, ascites, hepatic encephalopathy, GI bleeding, albumin concentration, blood sodium concentration, coagulation function, or baseline SCr concentration (p $>$ 0.05). The infection rate, total bilirubin concentration, ALT concentration, AST concentration, CRP concentration, and model for end-stage liver disease (MELD) scores of the AKI group were significantly higher than those of the control group (p 0.05). The eGFR of the AKI group was significantly lower than that of the control group (p 0.001; Table 2).

SNaPshot gene sequencing technology was used for SIRT1 genotyping. This revealed that SIRT1 rs4746720 sites exhibited CC, CT, or TT genotypes (Fig. 1).

Fig. 1.

The genotyping results of SIRT1 rs4746720 polymorphism in the AKI group and the control group. (A) CC homozygous. (B) CT heterozygotes. (C) TT homozygous. Note: SIRT1 rs4746720 extends forward, and the product is consistent with polymorphism.

The bioinformatics software programs TargetScan and miRanda were used to predict the target gene of microRNA-599 (miRNA-599). To verify the effect of SIRT1 rs4746720 SNP binding with miR-599, whole-gene SIRT1–3${}^{\prime }$-UTR-T/C sequences were synthesized, and the pmir-GLO-SIRT1–3${}^{\prime }$-UTR-T/C double luciferase reporter gene plasmid was constructed. This was used to co-transfect HK-2 cells with NC and miR-599 mimics, respectively, and the changes in luciferase activity were compared. The results showed that the luciferase activity in the SIRT1-3${}^{\prime }$-UTR-T+miR-599 mimic group was significantly lower than that in the SIRT1-3${}^{\prime }$-UTR-T+NC group and the SIRT1-3${}^{\prime }$-UTR-C+miR-599 mimic group (p 0.001), whereas there was no significant difference in luciferase activity between the SIRT1-3${}^{\prime }$-UTR-C+miR-599 mimic group and the SIRT1-3${}^{\prime }$-UTR-C+NC group (p $>$ 0.05). The above-described results showed that the expression of the T allele of SIRT1 rs4746720 was regulated by miR-599, resulting in significant inhibition of the expression of luciferase by miR-599, whereas the C allele of SIRT1 rs4746720 was not regulated by miR-599, resulting in no inhibition of the expression of luciferase by miR-599 (Fig. 2).

Fig. 2.

Results of dual luciferase reporter genes interaction between SIRT1 rs4746720 and miR-599 (${}^{\mathrm{*}\mathbf{}\mathrm{*}\mathrm{*}}$p 0.001).

To further study the effect of the SIRT1 rs4746720-mediated effect of miR-599 on SIRT1 expression, overexpression plasmids pcDNA3.1-SIRT1-T/C, which contained different alleles (i.e., T or C) of SIRT1 rs4746720, were constructed and verified by gene sequencing. Next, pcDNA3.1-SIRT1-T/C plasmids and pcDNA3.1 (empty plasmid) were used to transfect HK-2 cells with NC, an miR-599 mimic, or an miR-599 inhibitor, respectively. Subsequently, the relative expression of miR-599, SIRT1 mRNA, and protein in cells was determined by qPCR and Western blotting, respectively. The results were as follows: (1) Compared with transfection with NC, transfection with the miR-599 mimic caused the expression of miR-599 in the SIRT1-T overexpression group and the empty plasmid group to increase by 41% and 58%, respectively (p 0.01), whereas transfection with the miR-599 inhibitor caused the expression of miR-599 in the SIRT1-T overexpression group, SIRT1-C overexpression group, and empty plasmid group to decrease by 39%, 34%, and 39%, respectively (p 0.05). These results confirmed the effectiveness of transfection with the miR-599 mimic and miR-599 inhibitor, respectively (Fig. 3A). (2) Compared with the empty plasmid+NC group, in the SIRT1-T overexpression+NC group and the SIRT1-C overexpression+NC group, the mRNA and protein expression of SIRT1 was significantly upregulated (p 0.05). This confirmed the functioning of the SIRT1-T/C overexpression plasmids. (3) Compared with the NC group, in the SIRT1-T overexpression group, co-transfection with the miR-599 mimic significantly reduced the expression of SIRT1 mRNA (p 0.01). Protein expression of SIRT1 decreased while with no significance (p $>$ 0.05). Co-transfection with the miR-599 inhibitor increased the expression of SIRT1 mRNA and SIRT1, but this difference was not statistically significant. (4) In the SIRT1-C overexpression group and empty plasmid groups, SIRT1 mRNA and SIRT1 expression were not significantly changed by transfection with the miR-599 mimic or the miR-599 inhibitor (p $>$ 0.05). Taken together, these results indicate that the T allele of SIRT1 rs4746720 may mediate the interaction of miR-599 with SIRT1, resulting in significant inhibition of the mRNA expression of SIRT1, leading to a downward trend in the expression of SIRT1 (Fig. 3B,C).

Fig. 3.

Expression of miR-599 and SIRT1 in HK-2 cells after co-transfection with overexpression plasmid and miR-599. (A) Relative expression of miR-599. (B) Relative expression of SIRT1 mRNA. (C) Relative expression of SIRT1; *p 0.05, **p 0.01, compared with the NC group; #p 0.05, ##p 0.01, ###p 0.001, compared with the empty plasmid group. (D) SIRT1 protein detected by western blot.

The effect of the SIRT1 rs4746720 SNP-mediated interaction of miR-599 with SIRT1 on the PGC-1$\alpha$/NRF1/TFAM signaling pathway downstream of SIRT1 was further investigated by determining the relative mRNA and protein expression of PGC-1$\alpha$, NRF1, and TFAM with qPCR and Western blot analysis, respectively. The following results were obtained: (1) Compared with the empty plasmid+NC group; there was a significantly higher abundance of PGC-1$\alpha$, NRF1, and TFAM mRNA and protein in the SIRT1-T overexpression+NC group and SIRT1-C overexpression+NC group (p 0.001). (2) Compared with the NC group, in the SIRT1-T overexpression group, transfection with the miR-599 mimic caused a significant reduction in the mRNA and protein expression of PGC-1$\alpha$, NRF1, and TFAM (p 0.01), whereas co-transfection with an miR-599 inhibitor caused a significant increase in their levels (p 0.05). (3) In the SIRT1-C overexpression group, co-transfection with the miR-599 inhibitor caused a significant reduction in the mRNA expression of TFAM (p 0.001) but not in the protein expression of TFAM (p $>$ 0.05), as well as a significant reduction in the mRNA levels of NRF1 and TFAM (p 0.001) but not in the protein expression of NRF1 and TFAM (p $>$ 0.05). (4) In the empty plasmid group, co-transfection with the miR-599 mimic and the miR-599 inhibitor, respectively, did not cause significant changes in the mRNA or protein levels of PGC-1$\alpha$, NRF1, and TFAM (p $\mathrm{>}$ 0.05). Taken together, these results suggest that the T allele of SIRT1 rs4746720 may mediate the ability of miR-599 to significantly inhibit the activity of the PGC-1$\alpha$/NRF1/TFAM pathway (Fig. 4).

Fig. 4.

Expression of PGC-1$\mathrm{\alpha }$/NRF1/TFAM in HK-2 cells after co-transfection with overexpression plasmid and miR-599. (A–C) PGC-1$\alpha$/NRF1/TFAM mRNA relative expression. (D–G) PGC-1$\alpha$/NRF1/TFAM protein relative expression; *p 0.05, **p 0.01, ***p 0.001, compared with NC; ###p 0.001, compared to empty plasmid.

SIRT1 is a highly conserved protein deacetylase that is widely expressed in renal tissue. In ischemia-reperfusion injury, sepsis, and AKI caused by cisplatin, SIRT1 acts on many transcription factors to regulate members of downstream pathways, such as PGC-1$\alpha$, forkhead box O, nuclear factor kappa B, and p53, resulting in nephroprotective effects [15, 16, 17]. Specifically, SIRT1 mediates the deacetylation of PGC-1$\alpha$, activates NRF1 and TFAM, promotes mitochondrial biosynthesis and respiratory recovery, and drives proximal tubule repair, which may be a key pathway for renal protection [15, 29, 30]. In our early research using a rat model of AKI in cirrhosis, we found that the expression of SIRT1 and PGC-1$\alpha$ in the renal tissue of rats decreased in the early stages of AKI, and the SIRT1/PGC-1$\alpha$ signaling pathway may be involved in the mechanism of AKI in cirrhotic patients. Chou et al. [31] showed that knockout of SIRT1 increased tumor necrosis factor alpha-mediated renal insufficiency. Thus, SIRT1 and the downstream pathways that it affects play an important role in protecting the kidney against damage with various etiologies. SIRT1 SNPs are associated with aging, cardiovascular disease, alcoholic fatty liver disease, diabetic nephropathy, cardiorenal syndrome type 1, and many other diseases [32, 33, 34, 35]. The current study represents the first examination of the association between SIRT1 SNPs and AKI in cirrhosis. Our data analysis using Haploview 4.2 software (Broad Institute, Cambridge, MA,USA) and functional annotation screened out one functional SNP locus: the rs4746720 locus in the 3${}^{\prime }$-UTR region of SIRT1.

SIRT1 rs4746720 is associated with aging, type 2 diabetes, diabetic nephropathy, and other diseases [36, 37, 38, 39]. In the current study, we found that the TT genotype and T allele frequencies of the SIRT1 rs4746720 locus in the AKI group were higher than those in the control group (41.4% vs. 34.5%, 60.3% vs. 55.7%), and the risk of AKI in cirrhosis was increased in patients with the T allele; however, the latter difference did not reach statistical significance (p = 0.534). Stratified analyses revealed that the T allele led to a greater risk of developing AKI in cirrhosis with hepatic encephalopathy. Analyses of the liver and kidney function indicators of the AKI group with respect to genotype demonstrated that the SCr and BUN concentrations of patients with the TT genotype were significantly higher, and their eGFRs were significantly lower than those with the CC+CT genotype (p 0.05), indicating that among AKI patients with cirrhosis, those who had the TT genotype had significantly worse renal function than those who had the CC+CT genotype. Similarly, Tang et al. [36] found that compared with other genotypes, the SIRT1 rs4746720 TT genotype led to more severe diabetic nephropathy in Han Chinese patients with diabetes. Sarumaru et al. [40] showed that in Japanese patients with autoimmune thyroid disease, the thyroid autoantibody titer in patients with the SIRT1 rs4746720 T allele (i.e., the TT+TC genotype) was significantly higher than that in patients with the CC genotype, which may affect the expression of SIRT1 in inflammatory lesions of target tissues and increase susceptibility to disease. Zhang et al. [41] studied the relationship between SIRT1 functional loci and healthy aging in Han Chinese older adults and found that the participants exhibited a significantly higher frequency of the SIRT1 rs4746720 C allele than of the SIRT1 rs4746720 T allele, a higher frequency of the CC genotype than of the CT+TT genotypes and that those with the C allele may have a higher risk of unhealthy aging than those with the T allele. In addition, in the current study, SIRT1 rs4746720 was found to be most associated with AKI susceptibility in the hepatic encephalopathy subgroup, suggesting that gene-risk factors may jointly increase susceptibility to disease. MELD score, serum sodium concentrations, and hepatic encephalopathy are independent predictors of the risk of renal failure in cirrhosis cohorts, and hepatic encephalopathy is significantly associated with prognosis in patients with cirrhosis complicated by renal failure [42]. However, due to the small sample size of this study, further research is needed to confirm these findings.

Bioinformatics software was used to predict the miRNAs that may bind to rs4746720 in the 3${}^{\prime }$-UTR region of SIRT1, and this revealed that miR-599 should be capable of performing this binding. Given that the site bound by miR-599 is in the 3${}^{\prime }$-UTR, it is speculated that when the site has a T allele, it can be bound by miR-599, resulting in the inhibition of SIRT1 expression, whereas when the site has a C allele (due to a T-to-C mutation), it cannot be bound by miR-599, resulting in no effect on SIRT1 expression. The construction and use of a double luciferase reporter plasmid revealed that miR-599 could significantly inhibit the expression of luciferase by the T allele of rs4746720 but could not inhibit the expression of luciferase by the C allele of rs4746720, confirming that the polymorphic site of SIRT1 rs4746720 was likely to be the site where miR-599 acts. The recombinant overexpression plasmid of SIRT1 rs4746720 was constructed, and its use in experiments showed that the T allele of rs4746720 may mediate miR-599’s inhibition of the expression of SIRT1 mRNA and its downstream effectors PGC-1$\alpha$, NRF1, and TFAM.

The above-described results, combined with those from other studies, indicate that the T allele of SIRT1 rs4746720 may mediate the ability of miR-599 to target and inhibit the expression of SIRT1 and its downstream PGC-1$\alpha$/NRF1/TFAM signaling pathway, and may be involved in the pathogenesis of AKI in cirrhosis. The pathogenesis of AKI in cirrhosis involves the regulation of multiple genes, proteins, and signaling pathways. This is the first study to show the regulatory relationship between SIRT1 rs4746720 and miR-599. Current research on miR-599 has mainly focused on its involvement in the pathogenesis of lung cancer, liver cancer, kidney cancer, and other cancer-related pathogenesis, as miR-599 plays different roles in different tumor tissues. For example, in liver cancer, the expression of miR-599 is downregulated; this is manifested by changes in the abundance of tumor suppressor factors, which affect tumor production. By contrast, in lung cancer, the expression of miR-599 is upregulated, which promotes proliferation and invasion [43, 44]. However, the role of miR-599 in AKI has not been fully described, so further research is needed.