Roles of Serum NLRP3 Inflammasome and Associated Cytokines in Gout-Induced Kidney Injury Open Access

Gout is a form of inflammatory arthritis caused by the deposition of monosodium urate crystals in joints and tissues. It is characterized by recurrent episodes of intense joint pain, swelling, and redness, often accompanied by systemic symptoms such as fever and malaise [1, 2]. While traditionally regarded as a disease of affluent societies due to its association with dietary factors and sedentary lifestyles, gout has emerged as a global health concern with rising prevalence across diverse populations [3]. Gout is often accompanied by dyslipidemia, metabolic disorders, cardiovascular disease, and kidney disease [4]. Despite advances in understanding the pathophysiology and management of gout, there remains a significant clinical gap in the early detection and management of renal complications, particularly in patients with goat-related renal injury.

Gout-related kidney injury (GRI) is caused by a disorder in purine metabolism, leading to supersaturation of urate salts in the blood. Moreover, repeated gout attacks can damage kidney function, further exacerbating hyperuricemia and triggering additional gout attacks. These conditions are mutually causal and closely related [5]. GRI further complicates the management of gout by limiting treatment options and increasing the risk of drug-related adverse events. Long-term urate crystal deposition in the kidneys can cause the formation of uric acid (UA) stones, interstitial nephritis, tubular damage, glomerular sclerosis, and ultimately renal failure if left untreated [6]. Therefore, early clinical diagnosis and intervention in GRI are of great significance.

Currently, glomerular filtration rate (GFR) and urine microalbumin analysis are the primary diagnostic tools for kidney disease, but their accuracy and clinical utility are limited [7, 8]. GFR estimation can be influenced by factors such as muscle mass, age, and ethnicity, compromising accuracy and sensitivity in detecting early or mild kidney damage [9]. Urine microalbumin analysis, while useful for identifying glomerular damage, lacks specificity and can be affected by systemic factors unrelated to kidney disease, leading to false positives. Moreover, urinary albumin excretion if often detectable only after significant kidney damage has occurred, limiting its utility as an early biomarker [10]. These limitations are particularly pronounced in GRI, where inflammatory and structural changes develop before detectable declines in kidney function. Traditional diagnostic tools fail to capture the molecular and inflammatory processes underlying GRI, underscoring the need for novel biomarkers such as nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasomes, which provide insights into early inflammatory changes and enable earlier and more precise diagnosis.

In addition to the NLRP3 inflammasome, several other molecular pathways have been investigated in the context of GRI, including the renin-angiotensin-aldosterone system (RAAS), oxidative stress mechanisms, and endothelial dysfunction. The activation of RAAS is a well-recognized contributor to renal injury, promoting vasoconstriction, inflammation, and fibrosis within the renal microenvironment. Oxidative stress, characterized by an imbalance between reactive oxygen species and antioxidant defenses, has also been implicated in kidney injury, exacerbating cellular damage and inflammation. While these pathways play important roles in the progression of renal damage, their involvement is often indirect or nonspecific, which complicates their use as early diagnostic or therapeutic targets. Although RAAS activation, endothelial dysfunction, and inflammation are key contributors to GRI, inflammation plays a major role in renal damage [11]. Specifically, the NLRP3 inflammasome represents a more direct link to the inflammatory processes driving GRI. Its activation triggers the release of cytokines such as interleukin-1 beta (IL-1β) and interleukin-18 (IL-18), which drive the inflammatory pathology of both gout and renal impairment [12‒14]. This makes NLRP3 not only a valuable biomarker for early detection but also a promising therapeutic target for mitigating kidney damage in gout patients. In the pathology of renal damage, extensive fusion, apoptosis, and shedding of podocyte processes can be observed, and multiple cytokines are involved in this progression.

Research has shown that serum levels of NLRP3 inflammasomes, an intracellular complex along with ASC and caspase-1, can be activated in various aseptic inflammatory conditions, triggering a series of inflammatory reactions that result in apoptosis of podocytes [15]. The induced secretion of molecules such as caspase-1, IL-18, and IL-1β participated in the injury of the glomerulus together [12]. Our study aimed to explore the relationship between serum NLRP3, IL-1β, IL-18 levels, and GRI to elucidate the molecular mechanisms underlying renal impairment in gout.

The diagnosis of GRI was determined based on the guidelines outlined in Kidney Disease: Improving Global Outcomes (KDIGO) (2012). The diagnostic criteria included albuminuria (albumin excretion rate AER ≥30 mg/24 h; albumin-to-creatinine ratio ACR ≥30 mg/g [≥3 mg/mmol]); urine sediment abnormalities; electrolyte and other abnormalities due to tubular disorders; histological abnormalities; structural abnormalities detected by imaging; history of kidney transplantation; decreased GFR, defined as GFR <60 mL/min/1.73 m². The severity of kidney injury was evaluated based on the classification from KDIGO guideline. GFR <30 mL/min/1.73 m² indicated severely decreased and GFR <15 mL/min/1.73 m² was defined as kidney failure.

Inclusion criteria were clinical diagnosis of gout, age ≥18 years, patient’s written informed consent.

Exclusion criteria were (1) acute gout attacks; (2) patients with known primary kidney disease (for example, those with minimal change disease, membranous nephropathy, membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, and focal segmental glomerulosclerosis were also excluded) or secondary hyperuricemia, severe renal, hepatic, pulmonary, cardiovascular, cerebrovascular diseases, tumors, infections, immune, or psychiatric disorders; (3) patients who have used urate-lowering, anti-inflammatory, or antioxidant drugs outside the hospital.

This study was a retrospective study that included a total of 279 eligible gout patients, among whom 96 had renal injury and 183 did not. In addition, 100 age-matched healthy physical examination participants were included in the study. The healthy controls had normal laboratory parameters and no history of gout, kidney, liver, lung, heart, cerebrovascular diseases, infections, tumors, or immune disorders. These parameters were tested for significant differences as they are all related to gout and renal impairment, allowing us to rule out confounding factors such as age and comorbidities. Qingdao Municipal Hospital’ Ethics Committee approved the study protocol and the study was conducted according to the principles outlined in the Helsinki Declaration. Participants were presented with a comprehensive overview of the study objectives, and their informed consent was obtained in written form.

All patients provided 3 mL of fasting venous blood in the morning, while healthy individuals undergoing routine physical examination provided fasting 3 mL of venous blood on the day of the examination. The samples were centrifuged at 3,500 rpm for 10 min to separate serum, and the serum levels of NLRP3, IL-1β, and IL-18 were determined using corresponding enzyme-linked immunosorbent assays (ELISA; purchased from Wuhan Fine Biotech). NLRP3, IL-1β, and IL-18 were chosen for their direct involvement in inflammasome activation, which is central to the pathogenesis of GRI.

The statistical analysis was performed by IBM SPSS Statistics R26. Continuous variables were presented as mean ± standard deviation (SD) or median, and compared using Student’s t test, Mann-Whitney U test, analysis of variance (ANOVA), or Kruskal-Wallis test as appropriate. Correlations between variables were assessed using Pearson’s correlation coefficient or Spearman’s rank correlation coefficient. The significance level was set at p < 0.05.

This retrospective study included a total of 279 eligible gout patients, 96 of whom had renal injury, while 183 did not. To compare clinical characteristics between the two groups, Table 1 presents comparisons of parameters such as age, gender, BMI, gout duration, and clinical medication use. The results revealed significant differences in gout duration (p < 0.001), eGFR (p < 0.001), and serum UA levels (p < 0.001) between the two groups.

We first examined the correlation between serum concentrations of NLRP3, IL-1β, and IL-18 in gout patients. Gout patients were classified into two subgroups: those with kidney injury (GKI, n = 96) and those without (GNKI, n = 183). Additionally, 100 age-matched healthy individuals served as controls. Our results showed consistently and significantly elevated serum concentrations of NLRP3, IL-1β, and IL-18 in both GKI and GNKI patients compared to the control group (p < 0.001), with GKI patients exhibiting significantly higher concentrations than GNKI (p < 0.001) (Fig. 1a–c). Additionally, our analysis revealed significant positive correlations among NLRP3, IL-1β, and IL-18 in gout patients (n = 279). Spearman's correlation analysis revealed correlation coefficients for serum NLRP3 and IL-1β (Fig. 1d, r = 0.34, p < 0.001), NLRP3 and IL-18 (Fig. 1e, r = 0.47, p < 0.001), and IL-1β and IL-18 (Fig. 1f, r = 0.37, p < 0.001). Our results confirm a positive association between gout and inflammatory markers, particularly NLRP3, IL-1β, and IL-18.

As serum NLRP3, IL-1β, and IL-18 showed positive correlations with gout, particularly in patients with GKI, we conducted a receiver operating characteristic (ROC) analysis to assess their diagnostic value. The individual ROC curves for serum NLRP3, IL-1β, and IL-18 revealed AUC values of 0.72 (95% CI: 0.66–0.79), 0.76 (95% CI: 0.69–0.82), and 0.69 (95% CI: 0.62–0.75) (Fig. 2), respectively. These values highlight the moderate diagnostic performance of each biomarker independently, with IL-1β showing the highest individual predictive value.

To enhance diagnostic accuracy, a combined prediction model was constructed using the formula: Compute (Combine) = 0.562 * Serum NLRP3 + 0.065 * Serum IL-1β + 0.023 * Serum IL-18 – 6.103. This model was designed to integrate the predictive contributions of all three biomarkers. Based on the maximum Youden index, the optimal cutoff values were determined as 2.87 ng/mL for NLRP3, 45.82 pg/mL for IL-1β, and 47.77 pg/mL for IL-18. The corresponding sensitivity and specificity values for each biomarker are summarized in Table 2, with serum NLRP3 exhibiting sensitivity of 61.46% and specificity of 77.05%, serum IL-1β showing sensitivity of 68.75% and specificity of 76.50%, and serum IL-18 achieving sensitivity of 68.75% and specificity of 62.30%.

The combined prediction model demonstrated a marked improvement in diagnostic performance, achieving an AUC of 0.85 (95% CI: 0.81–0.89), significantly surpassing the AUC values of the individual biomarkers (p < 0.001). The model also showed superior sensitivity and specificity, reaching 75% and 82.51%, respectively. This represents a substantial enhancement compared to the individual markers, which had lower sensitivity and specificity values. These results underscore the potential utility of combining NLRP3, IL-1β, and IL-18 as a robust predictive tool for diagnosing GKI, offering a more accurate and comprehensive diagnostic approach than using any single biomarker.

Since eGFR is widely considered to be a key indicator to evaluate kidney function, correlations between eGFR and serum concentrations of NLRP3, IL-1β, and IL-18 were explored in GKI patients (n = 96). Our data showed statistically significant negative correlations between NLRP3 (p < 0.01, r = −0.43), IL-1β (p < 0.01, r = −0.39), and IL-18 (p < 0.01, r = −0.35) and eGFR (Fig. 3a–c). As decreased eGFR indicates renal dysfunction, our data highlight the correlations of NLRP3, IL-1β, and IL-18 with gout-induced kidney damage in patients.

Due to the high level of serum UA being the main mechanism leading to gout, and serum NLRP3, IL-1β, and IL-18 have been demonstrated to be positively related to GRI, we further evaluated whether the serum NLRP3, IL-1β, and IL-18 levels relate to serum UA level (Fig. 4a–c). The results of Spearman’s correlation analysis indicated a positive correlation between serum IL-18 concentration and serum UA level (p = 0.009, r = 0.27) (Fig. 4c). However, no significant statistical level correlation between serum UA level and either serum NLRP3 (p = 0.053, r = 0.20) (Fig. 4a) or IL-1β (p = 0.157, r = 0.15) (Fig. 4b).

Gout, which is characterized by hyperuricemia and the subsequent deposition of urate crystals in joints and tissues, has garnered increasing recognition as a systemic inflammatory disorder with implications for various organs, particularly the kidneys. Early stages of kidney injury from urate crystal deposition in renal structures like the medulla, papilla, and cortex result in a cascade of functional kidney declines, notably tubulointerstitial inflammation [17, 18]. Unfortunately, the onset of kidney injury is often hidden, lacking clear symptoms that would prompt early clinical intervention [19]. Consequently, many patients, despite undergoing routine tests for gout, fail to detect early kidney complications, resulting in diagnoses only at the stage of renal failure. Serum biomarker detection methods present notable advantages over traditional diagnostic methods due to their speed, non-invasive nature, and repeatability. Earlier investigations have identified serum biomarkers associated with kidney injury; however, most of these markers are linked to already impaired kidney function, such as our previous study, where Cys-C reflected GFR and beta-2 microglobulin was identified [20]. Recognizing the limitations of existing clinical indicators in accurately assessing the risk of early GRI, there is still an urgent need for early detection and interventions in GRI patients. Thus, we specially divided the gout patients into GNKI and GKI subgroups in this study, and healthy individuals for comparison at the same time.

The inflammasome, a complex multiprotein signaling platform, is triggered by environmental stress or pathogenic infections. NLRP3 stands out as the most extensively studied inflammasome in inflammatory conditions. Upon activation, NLRP3 can cause cellular damage through processes like endoplasmic reticulum stress, lysosomal disruption, and mitochondrial dysfunction, making it a key mechanism of kidney disease progression [21]. NLRP3 processes the caspase-1 precursor into its mature form, leading to the maturation and heightened expression of pro-inflammatory cytokines like IL-1β and IL-18, which play pivotal roles in GRI. Our study compared serum NLRP3 concentrations between GNKI and GKI patients, with those from earlier studies corroborating our findings. We demonstrated a significant elevation in NLRP3 inflammasome concentrations in gout patients within an inflammatory microenvironment. Even higher concentrations were observed in GKI patients. Analysis of downstream IL-1β and IL-18 concentrations with NLRP3 revealed similar patterns, confirming a positive correlation between NLRP3 concentrations and IL-1β/IL-18 levels in all gout patients, underscoring the downstream activation role of NLRP3 in gout. Excessive activation of the NLRP3 inflammasome in kidney tissues not only amplifies inflammatory signaling through IL-1β and IL-18 but also disrupts the integrity of the renal epithelial barrier, leading to proteinuria and accelerating tubulointerstitial damage. Additionally, sustained inflammasome activation leads to heightened oxidative stress [22] and mitochondrial dysfunction, further exacerbating renal cell apoptosis and fibrosis, and contributing to the progressive decline in kidney function observed in GRI.

Under normal circumstances, NLRP3 inflammasome activation aids in maintaining tissue homeostasis by eliminating stressed cells and initiating robust inflammatory responses. However, excessive NLRP3 activation contributes to the onset and progression of various inflammatory diseases, such as acute gouty arthritis, particularly under conditions of high UA levels. Moreover, NLRP3-activated inflammatory pathways implicated in GRI are known to initiate in the early stages. Additionally, we showed the negative correlation of eGFR with NLRP3, IL-1β or IL-18, emphasizing the potential pathogenic mechanisms in GKI patients.

Notably, further validation through ROC curves in this study confirmed the diagnostic utility of NLRP3, IL-1β, and IL-18 in gout patients, with enhanced diagnostic efficacy when combining these indicators. Consequently, the relationship between these markers and GRI warrants further exploration. Despite the promising diagnostic value of NLRP3, IL-1β, and IL-18 demonstrated in this study, it is essential to consider their role in the broader context of existing biomarkers for kidney injury [13, 14].

For instance, biomarkers such as serum cystatin C, NGAL, and KIM-1 have been widely studied for their sensitivity in detecting early kidney damage, particularly in acute kidney injury and chronic conditions [23]. However, these markers primarily reflect general renal injury and may lack specificity for GRI. By contrast, the NLRP3 inflammasome and its downstream cytokines not only serve as biomarkers but also provide mechanistic insights into the inflammatory pathways driving GRI. This dual functionality strengthens their clinical relevance as both diagnostic tools and potential therapeutic targets.

It is important to note that high UA levels are not the sole determinant of gout, with only a minority of individuals with hyperuricemia developing the condition [24]. Nonetheless, evidence indicates a positive correlation between UA levels and kidney inflammation, impacting both glomerular and tubular levels, as well as vascular changes in renal arteries, leading to functional impairments [25, 26]. In our study of GKI patients, we further explored the correlation between UA and NLRP3, IL-1β, and IL-18, revealing a positive correlation only with IL-18. This suggests a distinct mechanism may exist for IL-18-induced kidney damage compared to other inflammatory pathways activated by NLRP3. Particularly in acute kidney injury cases, IL-18 alone has also emerged as a rapid-response biomarker for diagnosis [13, 14]. Considering that UA levels accelerate gout attacks in GRI patients, IL-18 may be associated with the severity of kidney damage as a potential therapeutic target. Still, many studies have suggested that targeting NLRP3 activation and modulating the NLRP3/IL-1 axis are promising avenues for treating inflammatory-related diseases [27], with NLRP3 inhibitors emerging as potential candidates for kidney disease treatment [28, 29], highlighting NLRP3 as a prospective therapeutic target for gouty nephropathy patients.

A key limitation of this study is its retrospective design, which may introduce selection and information bias due to reliance on pre-existing and potentially incomplete data. While this limits the ability to establish causal relationships, rigorous inclusion criteria and robust statistical methods were employed to mitigate these issues. A further limitation of this study lies in its reliance on serum biomarkers without histological confirmation of kidney injury. While serum markers such as NLRP3, IL-1β, and IL-18 provide valuable insights, they may not fully capture the structural and cellular changes occurring within the kidneys, which can only be confirmed through renal biopsy or advanced imaging techniques [13, 14]. To address these limitations, further prospective studies are required to verify the findings of our study and facilitate clinical translation of this new diagnostic method.

Serum NLRP3 inflammasome and associated cytokines (IL-1β and IL-18) are markedly elevated in gout patients, particularly those with GRI. These inflammatory markers show strong potential as diagnostic indicators and potential therapeutic targets for managing renal complications in gout. The study findings suggested that monitoring serum NLRP3, IL-1β, and IL-18 levels could enhance early detection of kidney involvement in gout patients, facilitating timely interventions to mitigate renal damage. Targeting the NLRP3 inflammasome pathway represents a promising strategy for treating gout-related nephropathy and improving patient outcomes. Future research should focus on prospective, multi-center clinical trials to validate the diagnostic performance of these biomarkers across diverse patient populations. Additionally, investigating their utility in predicting treatment response and disease progression could further establish their clinical significance. Incorporating these biomarkers into clinical practice could aid in personalized treatment decisions, such as tailoring anti-inflammatory therapies to specifically target NLRP3 activation and its downstream cytokines. Furthermore, routine monitoring of these markers may help clinicians assess treatment efficacy and adjust therapeutic strategies proactively to prevent the progression of kidney damage.

The study was approved by the Ethics Committee of Qingdao Municipal Hospital (#WYQ-2010). The study was performed in strict accordance with the Declaration of Helsinki, Ethical Principles for Medical Research Involving Human Subjects. All patients have given the written informed consent.

The authors declare that there is no conflict of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

The authors declare that they have no funding for this work.

All authors contributed to the study conception and design, commented on previous versions of the manuscript, and read and approved the final manuscript. Material preparation, data collection, and analysis were performed by Xiaoqing Xu, Juanjuan Zhang, and Yanqun Wu. The first draft of the manuscript was written by Yanqun Wu.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from Y.Q.W. upon reasonable request.