New Insights into a Novel Metabolic Biomarker and Therapeutic Target for Chronic Kidney Disease

Chronic kidney disease (CKD) affects approximately 13% of the global population.1 CKD contributes to kidney dysfunction and eventually progresses to end-stage kidney disease. In its early stages, CKD is often asymptomatic, with kidney function decline and proteinuria occurring in later stages. However, once CKD advances, kidney function is significantly reduced, necessitating dialysis or a transplant.2 This underscores the importance of an early-stage diagnosis of CKD. In clinical settings, the estimated glomerular filtration rate (eGFR) is used to distinguish the stages of CKD, which is calculated using serum creatinine. However, traditional biomarkers, such as serum creatinine and proteinuria, are susceptible to interference from non-renal processes, highlighting the urgent need for the identification of novel CKD biomarkers. The development of reliable biomarkers is crucial not only for early diagnosis but also for improving the precision of treatment strategies, potentially enhancing patient outcomes significantly. Through untargeted metabolomic approaches, primary disturbances in serum galactose metabolism and glycerolipid metabolism have been identified in patients with diabetic kidney disease (DKD).3 Mechanistic research employing spatial metabolomics has revealed the facilitative role of endogenous adenine in DKD progression, with adenine inhibition proving effective in mitigating kidney hypertrophy and injury in DKD mice.4 Targeted metabolomics has identified increased lactate production as a consequence of kidney injury, with urinary lactate emerging as a potential biomarker for kidney disease progression.5 Moreover, in a hyperuricemia mouse model, serum betaine and biotin have been identified as potential biomarkers.6 Previous studies have underscored the indispensable role of metabolomics in biomarker identification for CKD. Using untargeted metabolomics, our research elucidated the underlying mechanisms of advanced CKD in rats, revealing disturbances in the serum metabolome that promote inflammation, oxidative stress, and fibrosis in the kidney.7 Additionally, through a combined untargeted and targeted metabolomics approach, serum 5-methoxytryptophan (5-MTP), homocysteine, and citrulline were identified as biomarkers in patients with advanced DKD, demonstrating satisfactory accuracy, sensitivity, and specificity.8 These findings underscore the critical importance of metabolomics in biomarker identification and mechanism investigation in the context of kidney disease. Our recent article published in Nature Communications9 represents a significant advancement in the identification of serum biomarkers for early CKD and the exploration of potential therapeutic targets through metabolomic analysis. This study utilized both untargeted and targeted metabolomics to analyze 703 serum samples from patients across CKD stages 1–5, resulting in the identification of five serum biomarkers: 5-MTP, canavaninosuccinate, acetylcarnitine, tiglylcarnitine, and taurine. These biomarkers exhibited strong correlations with the clinical indices of kidney disease. Notably, four biomarkers (5-MTP, canavaninosuccinate, tiglylcarnitine, and taurine) displayed negative correlations with CKD progression, while acetylcarnitine showed a positive correlation. External validation of these five metabolites was conducted using serum samples from both healthy individuals and patients in CKD stages 1–2. Furthermore, a longitudinal cohort analysis was performed to validate the efficacy of these biomarkers over time, while a treatment cohort evaluation assessed their performance following drug treatment. The results demonstrated that the five biomarkers could effectively distinguish between healthy donors and patients across CKD stages 1–5, achieving classification accuracies of 95.5% and 92.0% using supervised random forest and support vector machine classification, respectively. Validation in an independent cohort comprising 30 healthy donors, 30 patients with CKD stage 1, and 30 patients with CKD stage 2 yielded a classification accuracy of 100% for early-stage DKD diagnosis using both supervised random forest and support vector machine classification. Additionally, longitudinal cohort validation of these biomarkers in 31 subjects before and after CKD development demonstrated high sensitivity, specificity, and area under the curve (AUC) values. Further validation in a cohort of 114 patients with CKD stage 2 who were treated with enalapril, prednisone, and Wuling San confirmed the diagnostic utility of these five biomarkers for early CKD detection. This comprehensive validation underscores the reliability and potential clinical significance of the identified biomarkers for the early diagnosis and management of CKD. The gradual reduction of 5-MTP with CKD progression prompted further investigation into its potential protective effect in both animal and cell models of CKD. We found that in a unilateral ureteral obstruction (UUO) mouse model, a significant reduction in kidney 5-MTP levels was observed, and an intraperitoneal injection of 5-MTP led to the inhibition of renal inflammation and fibrosis. Similarly, in renal tubular epithelial cells stimulated with transforming growth factor beta 1 (TGF-β1) and mesangial cells stimulated with lipopolysaccharide (LPS), 5-MTP demonstrated a substantial suppression of inflammatory and fibrotic injuries. These findings represent the first evidence of the protective role of 5-MTP in CKD. Given that 5-MTP is an endogenous metabolite10 whose production is not easily influenced directly, our investigation turned toward tryptophan hydroxylase-1 (TPH-1), the rate-limiting enzyme in endogenous 5-MTP synthesis. TPH-1 expression was found to be decreased in both CKD animal and cell models. Furthermore, overexpression of TPH-1 attenuated inflammatory and fibrotic injuries, while knockdown of TPH-1 exacerbated renal injury. These results suggest a protective role for TPH-1 in kidney function by promoting 5-MTP production through the inhibition of nuclear factor kappa B (NF-κB) and the activation of the nuclear factor-erythroid-2-related factor 2 (Nrf2) pathway. These findings provide valuable insights into the potential therapeutic targets for CKD treatment, highlighting the importance of the TPH-1/5-MTP pathway in mitigating renal inflammation and fibrosis associated with CKD progression. There are some limitations to the present study. We used eGFR to distinguish kidney function rather than measuring GFR using the inulin clearance method, which is impractical in a larger population-based study. Additionally, the five biomarkers were validated only in the Chinese Han population. However, the positive results of 5-MTP in cell and animal models suggest the possible generalization of 5-MTP in other populations. In conclusion, our recent study9 successfully identified five metabolites as biomarkers for early-stage CKD, with subsequent investigations revealing that one of these metabolites, 5-MTP, exerts inhibitory effects on renal inflammation and fibrosis. Importantly, our findings shed light on TPH-1, the rate-limiting enzyme responsible for 5-MTP synthesis, as a promising therapeutic target for CKD. This study serves as a compelling example of utilizing metabolomic approaches for biomarker identification and mechanistic investigations in the context of CKD.