Predictive Value of 5-Methoxytryptophan on Clinical Outcome in Patients with Sepsis Associated Acute Kidney Injury

Introduction

Worldwide, the number of sepsis cases ranges from 1.9 million to 48.9 million each year.¹ Sepsis-associated acute kidney injury (SA-AKI) is a common and life-threatening complication in hospitalized and critically ill patients.² SA-AKI significantly increases in-hospital mortality³ and triples the risk of chronic kidney disease (CKD).⁴ Early detection of SA-AKI is challenging, and current diagnostic criteria rely on elevated serum creatinine and/or decreased urine output, which may lead to delayed dentification.^5,6 Several new biomarkers for SA-AKI, such as plasma and urinary neutrophil gelatinase-associated lipoproteins (NGAL) and urinary interleukin-18 (IL-18) have shown promise in predicting SA-AKI occurrence.^7–9 However, their clinical utility and prognostic value in SA-AKI remain uncertain. Further research is needed to identify reliable biomarkers for SA-AKI, aiding in early prevention strategies and long-term prognosis monitoring.

A recently discovered endogenous tryptophan metabolite, 5-Methoxytryptophan (5-MTP). Alterations in the serum levels of 5-MTP have been noted in different disease states. For instance, a significant decrease in serum 5-MTP concentration was observed in sepsis patients.¹⁰ In kidney diseases, the expression of serum 5-MTP in children with lupus nephritis (LN) was found to be positively correlated with disease activity and prognosis.¹¹ Moreover, investigations into patients with CKD revealed a decrease in the expression level of 5-MTP in these individuals.¹² According to our previously published data, 5-MTP serological expression levels in sepsis and SA-AKI patients have certain changes, which are correlated with renal function.¹³

However, the association between serum 5-MTP levels and renal function recovery and long-term clinical outcomes in SA-AKI patients remains unknown. Therefore, our study aimed to evaluate the predictive value of 5-MTP in long-term clinical prognostic characteristics after SA-AKI, and to help clinicians to assess the condition of patients early, improve the prognosis of SA-AKI patients.

Materials and Methods

Patients’ Cohort

A prospective study was conducted to investigate the association between serum 5-MTP levels, kidney function, and clinical outcomes in individuals suffering from sepsis and SA-AKI.The study included in patients aged 18 to 80 years admitted to the Affiliated Hospital of Qinghai University between March 2023 and February 2024.Participants included a healthy control group and a case group, with the latter diagnosed according to the Sepsis-3 criteria (International Consensus on the Definition of Sepsis and Septic Shock).¹⁴ Patients in the case group who met the diagnostic criteria for acute kidney injury as outlined by the Kidney Disease: Improving Global Outcomes (KDIGO) in 2012¹⁵ were included in the SA-AKI group.Healthy controls were individuals without cancer, acute or chronic kidney failure, liver disease, or neurological disease.Exclusion criteria utilized by the SA-AKI group consist of: 1) Patients with primary or secondary kidney diseases (eg, diabetic nephropathy, cardio-renal syndrome, hypertensive nephropathy); 2) Patients undergoing maintenance renal replacement therapy (peritoneal dialysis, hemodialysis); 3) Patients with autoimmune diseases and malignant tumors; 4) Cases of prerenal azotemia, such as severe blood loss or dehydration; 5) Patients who have undergone continuous hemofiltration therapy or received citric acid anticoagulation; 6) Pregnant women, children, and individuals over the age of 80.For Patient samples, our study complies with the Declaration of Helsinki and approved by the Ethics Committee of the Affiliated Hospital of Qinghai University (No:P-SL-2023--193).Informed consent was obtained from all the patients.

Sample Collection And Storage

Clinicians rigorously evaluate patients admitted to the ICU based on outpatient or emergency test results. Upon diagnosing sepsis, blood samples are promptly collected at the time of admission to prevent any potential errors in experimental results caused by medication or medical procedures during treatment. The medical team extracted 3mL of venous blood and placed it in a collection vessel containing EDTA. Following thorough mixing, the sample was centrifuged for 15 minutes. Post-centrifugation, the samples were visually inspected for signs of hemolysis. To prevent repeated freeze-thaw cycles, the plasma was divided into 100μL aliquots, transferred to labeled frozen tubes using a corresponding pipette, stored for subsequent analysis. Once all samples were collected, they were tested and analyzed together in the same batch to ensure consistency and avoid inter-batch variations.

Clinical Data Collection

General information(gender, age, underlying diseases, comorbidities), hemodynamics[MAP (mean arterial pressure), pressors], severity of the disease [Sequential Organ Failure Assessment(SOFA)score, mechanical ventilation, Acute Physiology and Chronic Health Evaluation(APACHE)II score], blood routine examination[white blood cell(WBC), Neutrophil(NEUT), lymphocyte(LY), Neutrophil-lymphocyte Ratio(NLR)], Inflammatory index [procalcitonin (PCT), IL-6 and C-reactive protein(CRP)], serum biochemical index(bilirubin, albumin), renal function[serum creatinine (Scr), blood urea nitrogen(BUN)].

Study Endpoint

Prospective follow-up was conducted at 1, 2, and 3 months from the date of selection through outpatient visits or telephone interviews. The study had two endpoints: primary endpoint was all-cause death, referring to the death of patients due to any cause, secondary endpoint was the recovery of renal function within 7 days in SA-AKI patients, the criteria of renal function recovery refer to previous literature.¹⁶

Determination of Plasma 5-MTP

The specific method of serum 5-MTP is described in our previously published protocol.¹³ LC-MS/MS analyze was commissioned Shanghai Bioprofile Technology Co., Ltd.

Statistical Analysis

All statistical analyses were conducted using SPSS 28.0 software. Continuous variables with a normal distribution were presented as mean ± standard () deviation.Two-independent sample t-tests were used for group comparisons, while one-way ANOVA was utilized for comparisons across multiple groups. Non-normally distributed data were presented as median (quartile)[M(P25, P75)], with non-parametric tests such as rank sum test used for group comparisons. A significance level of α=0.05 was employed for all tests. Categorical data were expressed as percentages and inter-group comparisons were performed using the chi-square test at α=0.05 level. Spearman correlation analysis was used to assess the relationship between serum 5-MTP levels and inflammatory, renal function, and other biomarkers. Logistic regression analysis was conducted to identify factors influencing renal function, and an ROC curve was generated to assess the predictive value of 5-MTP in SA-AKI. Cox regression analysis was employed to evaluate the impact of 5-MTP levels and related risk factors on renal function recovery and clinical outcomes, with Kaplan-Meier curves constructed for survival analysis. Statistical significance was set at P < 0.05.

Results

Comparison of General Information and Laboratory Indexes Among Subject

A total of 546 hospitalized patients admitted to the ICU were initially screened, with 136 patients meeting the diagnostic criteria for sepsis. Following the application of exclusion criteria, 78 sepsis patients were ultimately included, of which 39 met the diagnostic criteria for AKI. Additionally, 35 cases were selected as the healthy control group. After excluding 4 cases with incomplete basic data, a total of 31 cases were included for analysis. Statistical analysis revealed no significant differences in basic data such as age, sex, BMI, and comorbidities among the three groups (P>0.05), indicating comparable data across the groups. Refer to Supplementary Table 1 for more details.WBC, NEUT, LY, NLR, CRP, platelet, 5-MTP, Scr, BUN, eGFR, Cys-C, AST, total bilirubin, direct bilirubin, indirect total bilirubin, TP, albumin, APTT, PT, TT, FIB and D-dimer among subject were significant differences(P<0.05). Refer to Table 1 for detailed information.

Table 1 Comparison of Laboratory Indicators Among the Three Groups

Results of Serum 5-MTP Targeted Metabolomics

Figure 1A and B shows the overall distribution of samples. The consistency of the healthy control group was robust, displaying a clear clustering trend. In contrast, the data from the sepsis group and SA-AKI group exhibited slightly more scattered distribution compared to the control group, with some samples overlapping to a certain extent. This variation may be attributed to individual differences resulting from the primary infection focus in each group.

Figure 1 Metabolomic results of 5-MTP. (A) Principal component analysis diagram; (B) Principal component analysis 3D diagram;(C) Heat map;(D) metabolite importance analysis;(E) Comparison of 5-MTP expression levels among the three groups.*P < 0.05, **P < 0.01, ***P < 0.001 denote statistically meaningful differences among the groups.

Figure 1C illustrates the results of hierarchical clustering of metabolites across the three groups. Notably, the expression of 5-MTP in the SA-AKI group is markedly higher than in the sepsis and healthy control group.The metabolites of the key comparison are depicted in Figure 1D. The figure illustrates that 5-MTP is up-regulated in the SA-AKI group compared to the sepsis group (Fold Chang value = 1.440426, P<0.001). Among the detected tryptophan metabolites, 5-MTP emerges as the most crucial, with the highest variable importance in the Projection value. Figure 1E illustrates statistical differences in 5-MTP levels among the three groups (P<0.05). These findings, suggest that the change in 5-MTP levels is specific to the SA-AKI group.

Comparison of Primary Morbidity and Severity between Sepsis and SA-AKI Group

Statistical analysis revealed no significant differences in infection source, mechanical ventilation, and APACHEII scores between the sepsis group and SA-AKI group (P>0.05). However, there were significant disparities in SOFA score, IL-6 levels, and PCT levels between the two groups (P<0.05). The SA-AKI group exhibited higher SOFA score, IL-6 levels, and PCT levels compared to the sepsis alone group, indicating more severe inflammation, organ damage, complications, and overall disease severity in SA-AKI patients. Detailed data can be found in Table 2.

Table 2 Comparison of Primary Morbidity and Severity of Sepsis and SA-AKI Between the Two Groups

Correlation of 5-MTP with Renal Function and Inflammatory Markers

Figure 2A–C demonstrated a significant correlation between serum 5-MTP and Scr, BUN, and eGFR (P<0.05), with correlation coefficients of 0.402, 0.282, and −0.455, respectively. However, no significant differences were observed in cystatin C, PCT, CRP, IL-6, and other indicators (P>0.05), as depicted in Figure 2D–G.

Figure 2 Correlation of 5-MTP with renal function and inflammation and its predictive value for SA-AKI.Correlation between serum 5-MTP and Scr (A), eGFR (B), BUN (C), Cys-C (D), IL-6 (E), PCT (F) and CRP(G); (H) ROC curve of 5-MTP predicted AKI in sepsis patients(n=78). Abbreviations: r2, correlation coefficient; P, p value; AUC, Area Under the Curve.

Analysis of Influencing Factors of SA-AKI Occurrence

Patients with sepsis were categorized based on the development of AKI, with 0 representing sepsis without AKI and 1 representing SA-AKI patients. Various factors were considered as independent variables in logistic regression analysis. The study findings revealed that PCT, SOFA score, IL-6, PT, APTT, TT, MAP and 5-MTP were significant factors influencing the occurrence of SA-AKI (P<0.05), as presented in Table 3. Further multivariate analysis was conducted on clinically relevant indicators identified in the univariate analysis to elucidate the impact of 5-MTP on SA-AKI. The results demonstrated that serum PCT, 5-MTP, PT and APTT were independent influencing factors for the development of SA-AKI, as depicted in Table 4.In Figure 2H, the optimal cut-off value for serum 5-MTP was found to be 110.16 pg/mL. The area under the ROC curve was calculated as 0.736 (95% CI: 0.626–0.846, P<0.001), suggesting that 5-MTP could serve as a valuable biomarker for predicting the onset of SA-AKI.

Table 3 Univariate Logistic Regression Analysis

Table 4 Multivariate Logistic Regression Analysis

Comparison of Clinical Data between SA-AKI Patients with Renal Function Recovery and Those without Recovery within 7 Day

To further investigate the impact of 5-MTP on kidney injury, we assessed the recovery time of kidney injury in SA-AKI patients. Patients were categorized into a recovery group and a non-recovery group based on whether their kidney injury resolved within 7 days. A comparison was then made between the two groups in terms of basic data, laboratory indicators, and 5-MTP expression levels that could potentially influence the outcome. No statistically significant variances were observed in APACHEII score, SOFA score, and CRP (P>0.05). However, statistically significant differences were noted in mechanical ventilation, IL-6, PCT, and 5-MTP levels between the two groups (P<0.05). These results suggest that SA-AKI patients with mechanical ventilation are more likely to experience unrecovery in renal function, and a more severe inflammatory response is associated with a lower likelihood of renal function recovery. Conversely, SA-AKI patients exhibiting high levels of 5-MTP expression are more likely to recover from renal injury, as illustrated in Table 5.

Table 5 Comparison of Renal Function Recovery and Non-Recovery in SA-AKI Patients

Comparison of Clinically Relevant and Analysis of Influencing Factors indicators in Sepsis and SA-AKI patients with Different Prognoses

The findings revealed no significant variations in age, sex, CRP, WBC, NEUT, LY, and NLR between the survival and death groups in the overall sepsis cohort (P>0.05). However, significant differences were observed in 5-MTP, IL-6, PCT, SOFA, and APACHEII scores (P<0.05). In SA-AKI queue, significant differences were noted in 5-MTP, IL-6, PCT, SOFA score, and APACHEII score between the survival and death groups(P<0.05). These results suggest that patients in the death group exhibited more severe disease manifestations, characterized by higher IL-6, PCT levels, SOFA score, and APACHEII score. Notably, the serum levels of 5-MTP were significantly higher in the survival group compared to the death group, indicating that elevated 5-MTP levels were associated with a more favorable prognosis and reduced risk of mortality, as depicted in Table 6.

Table 6 Sepsis Cluster and SA-AKI Cohort Comparison Between Surviving and Dying Groups

In order to specifically assess the impact of 5-MTP on the all-cause death of patients with sepsis and SA-AKI, our study utilized univariate and multivariate COX models.The findings revealed that 5-MTP levels were linked to all-cause mortality in sepsis and SA-AKI patients, as depicted in Figure 3A–D. This suggests that 5-MTP plays a crucial role in determining the prognosis of SA-AKI. Subsequently, K-M survival curves were utilized to compare mortality rates in sepsis and SA-AKI based on 5-MTP expression levels. The optimal cutoff value for 5-MTP was determined to be 115.23 pg/mL using the Jorden index. Analysis of the K-M curve demonstrated that lower levels of 5-MTP expression were significantly associated with higher 90-day mortality rates in sepsis patients, as illustrated in Figure 3E and F). Similarly, in the SA-AKI subgroup, lower 5-MTP levels were strongly correlated with increased 90-day mortality, while higher 5-MTP levels were linked to better survival outcomes.

Figure 3 COX regression analysis and survival curves of sepsis group and SA-AKI group. (A)Sepsis group single-factor COX regression;(B) Sepsis group multi-factor COX regression; (C) SA-AKI subgroup single-factor COX regression;(D)SA-AKI subgroup multi-factor COX regression; (E)Sepsis group K-M survival curve;(F) SA-AKI subgroup K-M survival curve.

Discussion

In this study, we investigated the relationship between 5-MTP and SA-AKI by analyzing the serological expression level of 5-MTP. We also assessed the correlation between serological 5-MTP expression and the prognosis of patients with sepsis and SA-AKI. Our findings revealed a significant increase in serum 5-MTP expression in patients with SA-AKI, which showed correlations with Scr, BUN, and eGFR. Additionally, 5-MTP was identified as an independent influencing factor for all-cause mortality in patients with sepsis and SA-AKI. Higher levels of 5-MTP were associated with accelerated kidney function recovery, while lower levels were linked to higher 90-day all-cause mortality in SA-AKI patients. 5-MTP may have a protective role in the development of SA-AKI, with early increases in serological 5-MTP expression positively impacting the prognosis of sepsis and SA-AKI.

Up to 60% of individuals with sepsis develop AKI.¹⁷ Early detection of kidney injury is crucial for successful intervention and patient prognosis. However, the definition of AKI by KDIGO has limitations, including the need for baseline serum creatinine values for creatinine elevation determination and challenges in establishing pre-AKI baseline serum creatinine without direct data.^18,19 In summary, neither urine volume nor creatinine alone can reliably predict SA-AKI occurrence, nor can they serve as clinical indicators for patient prognosis assessment. In a previous study by our team, it was found that 5-MTP is a crucial metabolic product in sepsis and SA-AKI, showing a significant positive correlation with renal function indicators such as Scr and BUN. This means that as the levels of Scr and BUN increase, serum 5-MTP levels also increase. Previous studies had a limitation in lacking a healthy control group as a baseline. However, upon adding the healthy control group in this study, it was observed that the expression level of serum 5-MTP in the SA-AKI group was higher and related to renal function. These findings suggest that the early elevation of 5-MTP serves as an indicator of renal function impairment in sepsis patients. There are few studies investigating 5-MTP in SA-AKI. However, we have found that melatonin, along with 5-MTP, is an endogenous metabolite produced by the tryptophan metabolic pathway. Melatonin plays a therapeutic role in SA-AKI and is elevated in the disease state,²⁰ demonstrating a similar trend to that of 5-MTP as discussed in this paper.We propose that in the event of sepsis, an inflammatory outburst should trigger renal to release 5-MTP, which plays a crucial role in regulating the inflammatory response and mitigating kidney damage associated with sepsis. Utilizing the ROC curve, we assessed the impact of 5-MTP on SA-AKI, revealing that 5-MTP possesses a high predictive value for forecasting the occurrence of SA-AKI. Thus, it is suggested that 5-MTP may serve as a valuable tool for predicting AKI occurrence in patients with sepsis.

A retrospective cohort study revealed that a longer recovery time following AKI correlates with a heightened risk of CKD progression.The duration of recovery post-AKI emerges as a crucial predictor for CKD risk.²¹ Notably, individuals who experience complete reversal of AKI early in the disease exhibit more favorable clinical outcomes compared to those with persistent AKI.²² During our patient monitoring throughout hospitalization, we observed that serum 5-MTP expression levels were elevated in AKI patients with renal function recovery within 7 days, as opposed to those who did not recover within this timeframe. This was similar to previous findings in LN¹¹ and acute myocardial infarction,²³ where increased 5-MTP levels improved patient renal outcomes.This finding suggests a correlation between serological 5-MTP expression and renal function recovery, offering potential for predicting AKI reversal and introducing a novel target for SA-AKI treatment.

Clinical events of death or survival within 90 days were observed as an endpoint for all patients in our study. We discovered that patients with lower baseline concentrations of 5-MTP were more likely to experience adverse events such as death, both in the sepsis cohort and the SA-AKI subgroup cohort analysis. This is consistent with the results of regarding its effects on cardiovascular events.²³ These findings indicate that 5-MTP anti-inflammatory mediators play a significant role in the pathophysiological processes of sepsis patients. High serum levels of 5-MTP early on may inhibit the release of inflammatory factors and responses, leading to a positive impact on patient prognosis.

Our study aims to complement the array of biomarkers for SA-AKI, offering a fresh perspective to enhance outcomes in SA-AKI patients, and to advance the diagnosis and treatment of SA-AKI. However, our study does have limitations. Firstly, we only measured 5-MTP at one time point upon ICU admission, without continuous monitoring during treatment, which hindered a comprehensive evaluation of the impact of 5-MTP level fluctuations on SA-AKI prognosis.Secondly, in some cases, serological samples were not collected again at the conclusion of the 90-day follow-up, preventing a reevaluation of relevant kidney function indicators and the association between these indicators and 5-MTP at the end of the follow-up.

Conclusion

The results of this study suggest that tryptophan metabolic pathway changes significantly during SA-AKI disease, and endogenous 5-MTP is an important factor affecting the prognosis of critically ill patients with SA-AKI. 5-MTP is closely related to kidney function, and high concentrations of 5-MTP are associated with early recovery of kidney function. Low concentrations of 5-MTP increased the risk of death in SA-AKI patients.