Heart failure (HF) is a complex clinical syndrome caused by structural and/or functional abnormalities within the heart, characterized by the heart’s inability to pump sufficient blood and oxygen to meet the metabolic demands of other organs. The World Health Organization estimates that approximately 64.3 million individuals, constituting 1% to 2% of the global population, are afflicted by HF. Overactivation of the sympathetic and renin-angiotensin-aldosterone systems (RAAS) leads to water and sodium retention, resulting in extracellular volume expansion and a significant deterioration in a patient’s condition [1, 2]. Key symptoms such as lung congestion, peripheral oedema, and elevated jugular venous pressure are commonly seen in patients with HF [3]. Consequently, diuretics serve as fundamental therapeutic agents aimed at alleviating symptoms and signs attributable to water and sodium retention [4, 5, 6].

As the frequency and dosage of diuretic administration increase, the effectiveness of diuresis diminishes, leading to the development of diuretic resistance (DR). DR can be succinctly defined as either a diminished or complete absence of response to loop diuretics [7]. Specifically, a commonly used measure is the fractional excretion of sodium. The fractional excretion of sodium refers to the proportion of the filtered sodium load that is excreted from the body in the form of sodium (mmol/time) [8]. DR is present when this fraction is less than 0.2. Under normal physiological conditions, the kidneys filter the sodium in the blood, reabsorb part of the filtered sodium as needed, and excrete the rest in the urine. When loop diuretics are used, they normally promote sodium excretion and increase the fractional excretion of sodium. However, in the case of DR, even if loop diuretics are administered, the fractional excretion of sodium remains below 0.2%, indicating that the diuretic’s effect of promoting sodium excretion has not been effective. Approximately one-third of patients experience DR [9], characterised by an inability to enhance renal sodium and water excretion through diuretic therapy, resulting in persistent symptoms of volume overload and edema [5, 10, 11]. The DR is a refractory phenotype in the progression of HF, often necessitating frequent hospital admissions and stays in the intensive care unit. It is independently associated with worsening renal function and death [12, 13, 14]. Diagnosis of DR typically occurs after a patient’s non-responsiveness to escalated diuretic doses, often with a considerable time lag. Therefore, identifying reliable biomarkers for the timely detection of DR is crucial. This could facilitate prompt intervention and potentially improve the prognosis.

Introduced by Professor Nicolson in 1999, metabolomics has emerged as a crucial element within the realm of systems biology. The primary objective of this analysis is to discern the relative associations between metabolites and pathological alterations [15]. With advancements in technology and metabolic databases, metabolomics can uncover insights into cardiovascular disease and identify potential new biomarkers [16, 17, 18]. In patients with HF, changes in circulating metabolites reflect metabolic alterations in both the heart and peripheral tissues, with these peripheral metabolic changes being an integral part of the pathogenesis and disease progression of HF [19, 20]. Therefore, this prospective study used metabolomics techniques to identify differential metabolites as biomarkers for diagnosing DR in HF.

This study employed untargeted metabolomics analysis via LC-MS/MS to identify 192 differential metabolites distinguishing between diuretic-resistant and non-diuretic-resistant groups. Among them, 164 metabolites were up-regulated and 28 were down-regulated. Notably, the top five significant differential endogenous metabolites were Hydroxykynurenine, Perillic acid, Adrenic acid, 5-Acetamidovalerate, and Adipic acid. The diagnostic potential of these five key metabolites was evaluated through ROC curve analysis, suggesting their promising utility as early markers for diagnosing the DR phynotype of HF. For example, in routine blood tests of patients, if the levels of these biomarkers can be monitored, when abnormal changes are detected, even if the patient has not yet exhibited traditional symptoms of DR, doctors can preliminarily determine that the patient is at a high risk of developing DR based on the changes in these biomarkers. During the treatment process, continuous monitoring of the levels of these biomarkers can reflect the effectiveness of the treatment plan in real time, enabling timely adjustment of the treatment plan.

In the KEGG analysis, the pathway demonstrating the most pronounced disparities was the neuroactive ligand-receptor interaction. This pathway exhibited a notable concentration of ligands and receptors on the plasma membrane, indicating a potential close association between the pathogenesis of DR and intracellular as well as extracellular ionic pathways and signal transduction.

Imbalances in various amino acids have been observed in diuretic-resistant and non-diuretic-resistant groups. Amino acids play vital roles in numerous cellular biosynthetic and metabolic processes, some of which have been linked to HF [38]. Additionally, amino acid metabolism is closely associated with the progression of DR. Among the dysregulated metabolites identified in metabolomics analyses, certain compounds exhibit neurotransmitter properties or neuroactive functions. For instance, GABA, a prominent inhibitory neurotransmitter in the nervous system, acts by binding to specific transmembrane receptors on the plasma membrane of both pre- and postsynaptic neurons. Alterations in GABAergic input to the paraventricular nucleus in patients with chronic HF maintain a sympathetic vasodilatory tone. Elevated GABA levels might offer novel insights into the neurological influences on disease progression in HF [39]. However, further biological experiments are warranted to elucidate the detailed mechanisms involved. Furthermore, several metabolites categorised as dipeptides have been identified as dysregulated from the stage of HF to DR. Examples include prolylhydroxyproline, glutamylphenylalanine, and threonylleucine. These compounds are typically regarded as breakdown products of protein digestion or proteolytic metabolism, with some dipeptides serving physiological or cellular signalling functions [40]. Given the presence of cardiac structural alterations spanning from HF to DR, these dipeptides might stem from protein digestion within abnormal cardiac cells, holding promise as potential diagnostic biomarkers.

Differences were also observed in central carbon metabolism in cancer between the two groups, suggesting a potential link between severe HF and cancer. First, factors such as heightened oxidative stress, low-level inflammatory response, activation of the neurohormonal system, and immune system dysregulation might collectively contribute to the development of HF and cancer. Second, alterations in the cardiac extracellular matrix influence tumour stroma. As HF progresses, the stroma undergoes significant changes, becoming more fibrotic [41, 42]. This shift in the microenvironment not only precipitates pathological changes such as cardiomyocyte hypertrophy and abnormal energy metabolism but also indirectly stimulates other organs, including tumor tissues, via the bloodstream. This stimulation occurs through the release of paracrine or endocrine growth factors, cytokines, and chemokines. Therefore, future studies can explore HF biomarkers based on those associated with cancer.

There are differences in the pathways associated with protein digestion and absorption between the two groups. Patients with HF often experience concomitant digestive and absorption dysfunction, resulting in inadequate protein intake and various adverse effects such as hypoproteinaemia [43, 44]. Consequently, hypoproteinaemia limits the ability of diuretics by impeding their ability to reach appropriate concentrations at their target sites, including distal tubular adaptation, among others. Aminoacyl-tRNA plays a pivotal role in shuttling amino acids to ribosomes for protein synthesis [45, 46]. Aminoacyl-tRNA synthetases (ARSs) are widely distributed in organisms. Disturbances in aminoacyl-tRNA biosynthesis observed in patients with DR might be associated with disturbances in the metabolism of numerous amino acids. Several mutations might result in compromised aminoacylation or editing activity or altered gene expression levels of ARSs [47]. The adenosine triphosphate-binding cassette (ABC) transporter protein family comprises crucial efflux-type transporter proteins in the human body, with P-glycoprotein, breast cancer resistance protein, and multidrug resistance protein representing prominent members [48]. The administration of diuretics in various clinical diseases can impact the function or expression of ABC transporter proteins, subsequently affecting the in vivo dynamic processes and efficacy of co-administered chemotherapeutic drugs.

Apart from these identified differential metabolites and metabolic pathways, traditional markers are of great significance in DR. The pathophysiological mechanism underlying DR involves complex interactions at multiple levels. From the perspective of the cardiorenal axis, a reduction in cardiac output leads to insufficient renal perfusion and a gradual impairment of renal function. Some clinical guidelines recommend continuous measurement of natriuresis in patients with acute heart failure (AHF) to monitor DR [49]. A previous study has shown that long-term use of loop diuretics leads to a weakened natriuretic response [50]. The reasons are as follows: first, the relative or absolute reduction in extracellular fluid volume reduces the transport of solutes to the proximal renal tubules through the mechanisms mediated by the RAAS and the sympathetic nervous system; second, long-term exposure to loop diuretics induces adaptive epithelial hypertrophy and hyperfunction in the distal renal units, manifested as hypertrophy of distal tubular cells [51]. This structural change in cells leads to a compensatory increase in sodium reabsorption, alters the renal response to diuretics, and ultimately results in a decrease in blood sodium levels during chronic loop diuretic treatment [52]. Clinicians should closely monitor the renal function of long-term users and adjust the use of diuretics accordingly.

In the in-depth study of metabolic changes in HF patients, the liver dysfunction in AHF and its metabolic impact are significant. Research [53] indicates that AHF patients’ hepatorenal dysfunction is complex. At admission, 82% had an elevated Model of End-Stage Liver Dysfunction (MELD)-XI score, with the prevalence of different levels of dysfunction changing over time. Hepatorenal-dysfunction patients have unique clinical features, and the MELD-XI score-prognosis correlation shows the importance of liver function in disease progression. Pathophysiologically, liver congestion causes liver dysfunction, affecting drug metabolism, disrupting metabolic balance via abnoral kidney interactions, and potentially impacting amino acid, nucleotide, and energy metabolism. MELD-XI in AHF is crucial for risk stratification and treatment decisions related to liver and kidney function, and is predictive of treatment response. In view of this, the differential metabolites in our study may be affected by liver dysfunction, so their specificity and accuracy as DR biomarkers need further verification. Future research should more comprehensively assess liver function.

In patients with HF, accurately assessing mortality risk and assessing disease severity are crucial for effective clinical management and targeted interventions. DR frequently precipitates hospitalisation due to congestion and exacerbation of symptoms, although it is sometimes preventable or reversible. A recent study suggests that urine sodium can be used to identify patients with DR, and it may play an important role in guiding individual treatment [54, 55]. However, the real clinical value for patients with DR remains to be investigated. Hence, the development of novel diuretics, strategies, or combinations is imperative to overcome DR.

We used an untargeted LC-MS/MS metabolomics approach to analyze blood metabolites from HF patients and identify metabolic differences between diuretic-resistant and non-diuretic-resistant cases. Key differential metabolites included Hydroxykynurenine, Perillic acid, Adrenic acid, 5-Acetamidovalerate, and Adipic acid. Significant metabolic pathways that were affected included amino acid, energy, and nucleotide metabolism. Further evidence-based diagnostic testing is needed to fully understand the roles of these pathways in DR. This study highlights the potential of non-targeted metabolomics in improving the identification and management of DR in patients with HF.

All data involved in the article have been submitted as part of the results section. All data reported in this paper will also be shared by the lead contact upon request.

In the design and conceptualization phase of this study, YPY, QY and HL took the lead. CLY, XDS and DTT were responsible for the extraction of serum samples and data analysis. QHP and XS provided assistance in image processing. YPY, QY and CLY collaboratively wrote the first draft. XDS, DTT and XS modified the manuscript. QHP and HL critically reviewed the manuscript for key knowledge and verified the accuracy of the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was carried out in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of The First Affiliated Hospital of Hunan University of Chinese Medicine (No. HN-LL-SZR-2021-10). It was registered with the China Clinical Trials Registry (ChiCTR2100053587), and written informed consent was obtained from all participants prior to their involvement in the study. No sex-based or race/ethnicity-based differences were present.

This work was supported by the grants of the Natural Science Foundation of Hunan Province (No. 2022JJ40300); the National Natural Science Foundation of China (No. 82274411, No. 82204986); the Changsha Natural Science Foundation (No. kq2403157); the Leading Research Program of Hunan University of Chinese Medicine (No. 2022XJJB002) and the Science & Technology Innovation Program of Hunan Province (No. 2022RC1021).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/RCM27001.