The role of inflammation and ROS in CaOx kidney stones

Abstract

Kidney stones are a common urological disorder, and their formation involves a complex interplay of multiple factors. Recently, numerous studies have concentrated on the role of reactive oxygen species (ROS) and inflammatory responses in stone formation, offering new insights into their mechanisms. ROS are chemically reactive oxygen-derived molecules generated during normal aerobic metabolism through redox reactions. These molecules are crucial in regulating cellular signaling, cell cycle progression, and cell death. However, excessive production of ROS leads to oxidative stress (OS), which subsequently triggers cellular damage and inflammatory responses. Inflammation, as a protective reaction of the body to injury or infection, may paradoxically promote the formation and progression of kidney stones. This review summarizes the roles of ROS and inflammation in the formation of calcium oxalate (CaOx) kidney stones, focusing on the sources of ROS, their role in stone formation, and the activation of inflammation in relation to kidney stones. Furthermore, it discusses the interplay between OS and inflammation in facilitating stone formation. A deeper understanding of these mechanisms could provide a theoretical reference and scientific basis for the development of novel strategies to prevent and treat CaOx kidney stones.

Introduction

Kidney stones are a prevalent condition that poses a substantial challenge to human health. Epidemiological studies on kidney stones worldwide have revealed a marked increase in their incidence over the past few decades, particularly in Western countries, with projections indicating a continued rise in the future [1–3]. This trend is primarily attributed to increased salt and protein consumption, a higher prevalence of metabolic syndrome, malnutrition, and dehydration [4]. Current research suggests that kidney stone formation is influenced by genetic, dietary, environmental, and lifestyle factors. Dietary habits, such as high salt, high oxalate, and low fluid intake, as well as metabolic disorders like hypercalciuria, hyperoxaluria, and metabolic syndrome, significantly increase the risk of calcium oxalate (CaOx) stone formation [5, 6].

Although surgical interventions, such as extracorporeal shock wave lithotripsy (ESWL), ureteroscopy (URS), and percutaneous nephrolithotomy (PCNL), effectively alleviate clinical symptoms and facilitate stone expulsion, the high recurrence rate of kidney stones remains an unresolved clinical challenge [7]. Recurrent kidney stones not only increase the physical and emotional burden on patients, but also impose a significant strain on healthcare systems due to repeated surgeries. Comparatively, dietary and pharmacological interventions aimed at preventing and managing stone formation represent the most promising therapeutic strategies. Consequently, elucidating the mechanisms underlying renal stone formation is of great significance for optimizing clinical treatment strategies in patients with nephrolithiasis.

In recent years, numerous studies have demonstrated that reactive oxygen species (ROS) and inflammatory responses may play pivotal roles in the formation of CaOx kidney stones. ROS are highly reactive molecules that participate in various physiological and pathological processes within living organisms. Under normal conditions, ROS regulate cellular signal transduction and immune responses. However, their excessive production induces oxidative stress (OS), leading to exaggerated inflammatory responses and cellular damage [8]. Both in vivo and in vitro experiments have shown that CaOx crystals induce renal epithelial cell damage through excessive ROS production, triggering OS responses [9]. Kidney biopsy specimens from patients with kidney stones reveal widespread cellular damage and interstitial fibrosis around crystal obstructions, with the stone matrix containing inflammation-related proteins [10]. Basic studies using high oxalate concentrations or CaOx crystals have further emphasized the significance of OS and inflammation in kidney stone formation by demonstrating renal tubular epithelial cell damage and CaOx crystal deposition in animal models [9, 11–13].

In conclusion, existing studies suggest a potential role of inflammation and OS in kidney stone formation. However, the precise mechanisms by which they contribute to this process remain unclear, necessitating further investigation. Additionally, the generalizability of findings from animal models to humans requires validation through future research. This review aims to synthesize recent studies on the etiology of ROS and inflammatory overproduction in individuals with renal calculi. It seeks to elucidate the mechanisms underlying renal stone formation, as well as the interplay between ROS and inflammation in this context. By clarifying the pivotal roles of ROS and inflammation in renal calculi pathogenesis, this review aims to enhance our comprehension of their involvement and offer insights for the development of novel therapeutic approaches and identification of potential clinical drug targets for renal calculi.

Formation of CaOx stones

Crystallization of CaOx in the kidney

Kidney stones are classified by their primary components: CaOx, calcium phosphate (CaP), cystine, uric acid, ammonium magnesium phosphate, and mixed types [14]. Calcium oxalate (CaOx) stones represent about 80% of cases and consist mainly of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) [10, 14–16]. These two forms exhibit similarities in their initial crystal formation stages, with Randall’s plaque being a key factor in their genesis. The development of COD and COM is significantly influenced by various factors, including the solution environment, temperature, ion concentration, and hydration levels. Given that both crystal forms involve Randall’s plaque in their early formation stages, this text does not clearly differentiate between them. The formation of CaOx stones is a complex, multi-step process that includes urine supersaturation, crystal nucleation, growth, and aggregation [17]. Within the renal tubules, CaOx crystal formation begins with the supersaturation of calcium and oxalate ions in the urine, which combine to form a crystal core once a critical concentration is reached. This process is modulated by renal tubular epithelial cells, particularly through their interaction with CaOx crystals [18].

Contribution of Randall’s plaques to the nucleation and growth of CaOx crystals

The Randall's plaques (RPs), first proposed by Alexander Randall in 1937, are defined as calcified deposits located at the tip of the human renal papilla. Its primary constituents are CaP crystals intermixed with an organic matrix rich in proteins and lipids, including membrane-bound vesicles (or exosomes), collagen fibers, and other extracellular matrix components [19]. This plaque serves as a crucial pathological foundation for the formation of CaOx kidney stones.

Microscopic examination of renal papillae in mice, rats, and pigs, using various modeling approaches, has consistently demonstrated the presence of RPs, papillary tip endothelial damage, and tissue injury. Although Drosophila melanogaster lacks renal papillae and does not form RPs, this model can still be used to investigate the fundamental mechanisms of crystal formation in a simplified tubular environment [20–23]. A recent study, employing endoscopy and biopsy, confirmed that CaOx kidney stone patients exhibit two distinct phenotypes: one characterized by mineral deposits in the papillary interstitium, and the other by tubular obstruction [24]. These findings suggest that, despite substantial discrepancies in renal physiological characteristics between mammalian model animals (mice, rats, pigs) and humans, RPs are consistently detectable across species—further indicating that these plaques constitute a key pathological basis for CaOx stone formation.

Existing research has established that factors such as low urine volume, low urine pH, and high urinary calcium excretion can lead to cell hypoxia, ROS production, activation of NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasomes, and the release of inflammatory mediators like interleukin-1β (IL-1β). This creates an inflammatory microenvironment that promotes the formation of kidney stones by driving an “osteogenesis-like” phenotypic transformation of renal tubular epithelial cells. This transformation activates signaling pathways typically associated with bone formation, altering cell behavior and leading to the deposition of calcium salts, which can promote the development of RPs [25–27].

The transformation of RPs into CaOx stone foci is believed to occur in at least four distinct stages. First, there are initial crystal deposits in the papillary interstitium, around the renal tubules and blood vessels. This is followed by a second stage of growth and expansion of these deposits. In the third stage, the epithelium covering the plaque is destroyed, exposing it to the urine [19]. The fourth stage involves the deposition of CaOx on the plaque, where the outer layer of RPs is replaced by CaOx or CaOx nucleated on a crystal-related organic matrix [28, 29].

In our discussion, we focus primarily on CaOx stone formation on Randall’s plaque (RPs), which acts as a nidus for crystal deposition in the kidney. RPs not only serve as a nucleation site for CaOx crystals, but also play a role in inducing OS and fibrosis by interacting with the surrounding renal tissues [19, 30, 31]. Recent research indicates that high intake of calcium and vitamin D could enhance RPs' formation, thereby elevating kidney stone risk. Understanding the role of ROS and inflammation in RPs' formation is crucial for comprehending the early stages of stone development.

The role of ROS in CaOx nephrolithiasis

Generation and sources of ROS

ROS, including superoxide anions (O₂•−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH), are byproducts of cellular metabolism. Under normal physiological conditions, the generation of ROS and the counteracting antioxidant defense systems exist in a dynamic balance, which is critical for maintaining homeostasis in the body [32].

During the formation of CaOx stones, ROS are primarily released from the cytoplasm and mitochondria. Under physiological conditions, mitochondria are the predominant source of ROS, particularly during electron leakage in oxidative phosphorylation. In stress conditions, such as hyperoxaluria, ROS production disrupts mitochondrial integrity, leading to blurred mitochondrial membranes, disappearance of internal cristae, chromatin condensation, and marginalization, ultimately resulting in mitochondrial dysfunction. This mitochondrial dysfunction further promotes excessive ROS production, causing oxidative damage to renal cells and facilitating crystal retention and growth [33] (Fig. 1).

Fig. 1.

This schematic diagram outlines the origins of reactive oxygen species and the possible pathways that result in renal tubular injury. Subsequent to tubular injury, renal tubular epithelial cells encounter urine containing elevated levels of calcium and oxalate, facilitating the creation of Randall's plaque. The formation of kidney stones on this plaque could potentially serve as a mechanism contributing to nephrolithiasis

Further studies have demonstrated that nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase enzymes (NOX), especially NOX4, are major contributors to ROS production. NOX4 is widely expressed in renal tubular cells and generates superoxide anions upon activation. Under conditions of hyperoxaluria, increased NOX activity leads to elevated ROS levels, accompanied by cellular injury, reduced antioxidant defenses, and pro-inflammatory responses, collectively promoting the formation of CaOx stones [34] (Fig. 1).

CaOx crystals directly damage renal tubular epithelial cells. When these crystals adhere to and interact with renal epithelial cells, they cause both physical and biochemical injury, leading to further ROS production. This crystal–cell interaction induces mitochondrial stress, compromises cellular integrity, and activates inflammatory pathways, exacerbating OS, aggravating cellular damage, and facilitating stone development [35].

Additionally, multiple studies have found the involvement of immune cells, particularly macrophages and neutrophils, in ROS production. In response to CaOx crystal-induced damage, these immune cells are recruited to renal tissues and produce ROS as part of their innate immune function to clear CaOx crystals. However, this inflammatory response amplifies OS, causing further cellular and tissue damage [36] (Fig. 1).

Xanthine oxidase has also been implicated in ROS generation. This enzyme produces superoxide anions as byproducts during purine metabolism and is activated in response to oxidative and metabolic stress. Elevated xanthine oxidase activity in the kidneys results in ROS accumulation, tissue damage, and stone formation [37] (Fig. 1).

A recent study demonstrated that CaOx crystals activate the angiotensin II type 1 receptor (AT1R), promoting calcium ion release. The subsequent elevation of intracellular calcium levels further increases ROS production, inducing OS and promoting CaOx crystal deposition [38] (Fig. 1).

Collectively, studies indicate that CaOx crystals engage in the high ROS environment within kidney tissue via multiple pathways, initiating cell damage and creating a feedback loop that amplifies ROS production. This process further damages cells and promotes stone formation and development [35, 36].

Mechanisms of ROS-induced damage and related molecular pathways

ROS-induced renal cell membrane damage and mitochondrial dysfunction

ROS play a critical role in apoptosis, lipid peroxidation, mitochondrial dysfunction, and inflammation signaling within renal tissues. Studies using animal models of kidney stones have demonstrated that ROS can increase the hydrophilicity of cell surfaces through oxidative reactions, making CaOx crystals more likely to adhere to the surface of renal tubular epithelial cells. This process damages membrane lipids, producing reactive aldehydes such as malondialdehyde (MDA), which further harm proteins and DNA. These effects are closely linked to the oxidation of phospholipids in the cell membrane and changes in extracellular matrix components. Lipid peroxidation compromises membrane integrity, leading to cellular dysfunction, inflammation, and ultimately promoting stone formation [39, 40] (Fig. 1).

Previous research has shown that mitochondria are highly susceptible to ROS-induced damage. Deficiency or dysfunction in mitochondrial energy production exacerbates metabolic processes in the tricarboxylic acid cycle and the electron transport chain, resulting in increased ROS generation and reduced antioxidant enzyme activity. Excessive mitochondrial ROS damage both the inner and outer mitochondrial membranes [41, 42]. This, in turn, triggers the release of pro-inflammatory factors such as ROS, cytochrome c, and calcium ions into the cytoplasm. Elevated cytoplasmic ROS levels further damage cell membranes, enhancing the deposition of crystals on the surface of renal tubular cells [42].

Impairment of antioxidant signaling pathways may also play a significant role in the formation of kidney stones. In vivo, the nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway serves as a major defense mechanism against oxidative and inflammatory diseases [43]. Under physiological conditions, Nrf2 is tightly bound to its repressor Keap1 in the cytoplasm and is degraded via the ubiquitin–proteasome pathway [44]. In the OS state, ROS alter Keap1, freeing Nrf2 to translocate to the nucleus and initiate transcription of antioxidant genes like heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1) [40].

In one study, Zhu Jianning and Wang Qing utilized dimethyl fumarate (DMF) to induce Nrf2 expression and activate this signaling pathway, which reduced oxidative damage in oxalate-exposed rat renal epithelial-like cell lines, increased cell viability, and decreased the expression of inflammation, apoptosis, and osteogenic-related proteins. In a rat kidney stone model, DMF enhanced Nrf2 expression, inhibited the expression of NADPH oxidase subunits Nox4 and p22 induced by hyperoxaluria, alleviated OS in renal cells, corrected mitochondrial metabolic abnormalities, suppressed inflammatory responses, differentiation, and apoptosis, and significantly reduced stone deposition in renal tissues [45].

Additionally, Zhou Donghui et al. found that gallic acid could reduce ROS levels and oxidative damage by inducing the antioxidant Nrf2/HO-1 pathway [46]. These studies indicate that dysfunction in the Nrf2-Keap1 antioxidant signaling pathway, leading to elevated ROS levels in vivo, may significantly contribute to kidney stone formation (Fig. 1).

Moreover, the apoptosis signaling cascade activated by cytochrome c exacerbates renal tubular cell damage, facilitating crystal adhesion and retention within the kidneys. Retained crystals further grow and aggregate, promoting the formation and progression of stones [47].

Studies have also shown that ROS can trigger apoptosis in renal cells by activating caspases and disrupting mitochondrial function. ROS initially cause mitochondrial membrane depolarization, leading to the release of cytochrome c and the activation of caspase-3, ultimately resulting in apoptosis of renal tubular epithelial cells. This cell death causes tubular damage and fibrosis, creating favorable conditions for the aggregation and growth of CaOx crystals [45, 48] (Fig. 1).

This complex interplay of mitochondrial dysfunction, impaired antioxidant defenses, and ROS-mediated apoptosis underscores the central role of ROS in cellular damage, inflammation, and the pathogenesis of CaOx kidney stones.

Autophagy-mediated kidney stone formation by ROS

Studies have demonstrated that oxalate can activate autophagy, leading to ferroptosis in renal tubular epithelial cells [49]. Key signaling pathways, such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)/mTOR, have been identified as regulators of autophagic responses in renal tubular cells. Oxalate activates these pathways, promoting autophagy and contributing to the formation of CaOx stones [50]. CaOx crystals induce apoptosis in renal tubular cells by increasing OS levels and intracellular calcium concentrations [51].

Activation of inflammatory signaling pathways

ROS are potent activators of inflammatory signaling pathways, particularly nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK). NF-κB is a major pro-inflammatory transcription factor family that regulates inflammation, immune responses, and tissue homeostasis. ROS activate the NF-κB pathway, leading to the upregulation of pro-inflammatory cytokines such as interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) [49, 52] (Fig. 1). These cytokines not only promote local inflammatory responses, but also enhance the recruitment of immune cells, exacerbating kidney damage and fibrosis. Particularly in cases of renal tubular epithelial cell injury, the increase in pro-inflammatory cytokines intensifies renal inflammation, thereby promoting stone formation [51].

ROS activate MAPK signaling cascades, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38-MAPK. These MAPK pathways mediate critical cellular processes such as survival, apoptosis, and inflammation, in addition to their regulation by the transcription factor NF-κB [50] (Fig. 1). Excessive ROS production and the physical stimulation of CaOx crystals induce the activation of the NLRP3 inflammasome, further exacerbating renal tubular cell damage. This accelerates the deposition of CaOx crystals and facilitates stone formation [53, 54] (Fig. 1).

Fibrosis and tissue remodeling

Chronic fibrosis has been identified as a critical factor contributing to the long-term increase in OS. Additionally, it plays a pivotal role in the pathogenesis of kidney stone formation. Fibrosis can enhance the microenvironment conducive to stone nucleation, promote stone deposition, and increase the risk of stone recurrence [45]. Fibrosis is characterized by excessive deposition of extracellular matrix (ECM) during tissue repair. Studies have shown that ROS promote kidney fibrosis by increasing OS, especially in the context of chronic inflammation. This process significantly affects the structure and function of the kidney [49]. Functional loss of renal tubular epithelial cells, excessive ECM accumulation, and altered vascular permeability during this process make CaOx crystals more likely to be retained in the kidneys, further promoting stone formation [51].

Inflammatory responses in CaOx kidney stone formation

Role of immune cell in kidney stone formation

The available evidence suggests that immune cells, particularly macrophages, may play a crucial role in the formation of kidney stones. This is primarily reflected in the recognition and phagocytosis of crystals by macrophages. When crystals are deposited in the kidney, macrophages recognize them through surface receptors, such as Toll-like receptors (TLRs), and become activated [55]. Activated TLR4 not only enhances the phagocytic function of macrophages, but also induces the release of cytokines, such as IL-1β and TNF-α, which further promote the local inflammatory response [56]. Furthermore, CaOx stimulation of renal tubular epithelial cells promotes the expression of S100A9 in M1 macrophages by secreting IL-6 [57]. S100A9 then promotes nephritis injury by activating the TLR4–p38/MAPK–Lipocalin 2 (LCN2) pathway, which in turn facilitates CaOx stone formation [58]. These findings collectively suggest that TLRs play a crucial role in recognizing and mediating the inflammatory response during the early stages of kidney stone formation.

In addition to their immune functions, macrophages contribute to the remodeling of the renal microenvironment during stone formation. During the early stages of stone formation, macrophages secrete various cytokines that activate adjacent renal tubular epithelial cells and stromal cells, thereby promoting stone formation [56]. Clinical studies reveal elevated serum levels of pro-inflammatory cytokines TNF, IL-1β, and IL-6 in patients with renal calculi compared to those without [59]. Quantitative PCR analyses indicate significant increases in inflammatory molecules such as C–C Chemokine Ligand 2 (CCL2), CCL5, CCL7, C–C Chemokine Receptor 2 (CCR2), Cluster of Differentiation 40 (CD40), Colony-Stimulating Factor 1 (CSF1), C–X–C Chemokine Ligand 9 (CXCL9), CXCL10, and Fas ligand in the papilla tip tissues of these patients, alongside upregulation of TLR3 expression [60]. During inflammation, immune cells like macrophages intensify local responses by secreting pro-inflammatory cytokines and chemokines, including IL-1β, TNF-α, CCL2, and CCL5. These factors not only facilitate crystal aggregation but also promote the release of matrix vesicles containing calcium and phosphorus, which serve as nucleation sites for crystal formation [61, 62]. This process accelerates stone formation by enhancing crystal adhesion to the tubular epithelium [19, 63]. Cucurbitacin A was identified as a promising lead compound for stone treatment, with studies further revealing interactions between the IL-6/JAK/STAT3 inflammatory pathway and endogenous oxalate metabolism in stone pathogenesis [64]. In vitro studies using animal models demonstrate that dark plum extract can protect renal function and inhibit kidney stone formation by suppressing monocyte chemoattractant protein-1 in renal tissue [65].

As stone formation progresses, macrophage functions shift. M1 macrophages, which are pro-inflammatory, exacerbate renal tissue damage, while M2 macrophages, which are anti-inflammatory, contribute to immune tolerance and crystal clearance. Stimulated by factors such as CSF-1, interleukin-4 (IL-4), and interleukin-13 (IL-13), M2 macrophages can phagocytose CaOx crystals, thereby inhibiting stone formation. The signaling mechanisms underlying M2 macrophage-mediated crystal clearance include the PPAR-mir-23-IRF1/Pknox1, miR-93-TLR4/IRF1, and androgen receptor (AR)-miR-185-5p/CSF1 pathways [56].

Activation of the aryl hydrocarbon receptor (AhR) has been shown to reduce M1 macrophage polarization and promote M2 macrophage polarization via the AhR-miR-142a-IRF1/HIF-1α pathway, thereby inhibiting CaOx deposition in the kidney [66]. Similarly, Sirtuin 1 (Sirt1) induces M2 polarization by deacetylating Forkhead Box Protein O1. M2 macrophages, in turn, alleviate OS-induced damage to renal tubular epithelial cells by downregulating NADPH oxidase activation [67] (Fig. 2). A previous study of patients with kidney stones [61]. corroborated these findings. Microarray analysis of gene expression in renal papillary tissue revealed that genes associated with the pro-inflammatory M1 macrophage phenotype were upregulated, while genes linked to the anti-inflammatory M2 phenotype were downregulated in kidney stone patients compared to non-stone controls. These results suggest that modulating M2 polarization could inhibit CaOx crystal deposition, potentially mitigating excessive inflammation and promoting tissue repair. However, the involvement and specific roles of other immune cell types in kidney stone formation require further investigation.

Fig. 2.

Inflammatory responses in CaOx kidney stone formation

Role of renal parenchymal cells in kidney stone formation

Exposure of renal tubular epithelial cells to CaOx crystals can upregulate the expression of pro-inflammatory factors such as CCL2 and prostaglandin E2 (PGE2). This upregulation not only intensifies the local inflammatory response but also serves as crystal promoters, thereby exacerbating the deposition of CaOx crystals in the kidney [68, 69]. Inflammation can also impact the expression of mineralization modulators. In conditions like hyperoxaluria and in animal models of CaOx-induced kidney stones, there may be alterations in the expression of mineralization inhibitors such as matrix Gla protein (MGP), osteopontin (OPN), and fetuin A. Reduced expression of these modulators may heighten susceptibility to stone formation [70–72]. Furthermore, research indicates that inflammation may promote the formation of CaOx crystals in urine and their accumulation in the kidneys [63].

The activation of NLRP3 inflammasomes likely plays a significant role in kidney stone formation. Expression levels of NLRP3 and its downstream molecules in peripheral blood mononuclear cells of patients with stone disease were notably higher compared to those of healthy individuals, implying the involvement of NLRP3 inflammasomes in stone formation [73]. Studies on NLRP3 knockout mice have shown a significant reduction in stone burden during kidney stone formation, providing further evidence of the crucial role of NLRP3 activation in stone formation [53].

The activation of the NLRP3 inflammasome typically occurs in two stages: the priming signal and the secondary signal. During kidney stone formation, urinary crystals such as CaOx and uric acid act as stimuli to trigger the activation of the priming signal. These crystals elicit stress responses in renal tubular epithelial cells, activating the sensing domain of the NLRP3 protein and promoting its interaction with apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase-1 [74]. Upon NLRP3 inflammasome activation, Caspase-1 is activated, leading to the maturation and release of pro-inflammatory cytokines such as IL-1β and interleukin-18 (IL-18). These cytokines amplify inflammatory responses, exacerbating renal tissue damage [75] (Fig. 2).

NLRP3 knockout mice on a high oxalic acid diet showed reduced expression of hyaluronidase, a cell membrane crystal adhesion factor, in renal tubular epithelial cells, resulting in significantly decreased CaOx stone formation compared to normal mice. This indicates that NLRP3 inflammasomes facilitate stone formation by modifying cellular activation of CaOx crystal adhesion [76].

The existing evidence suggests that NLRP3 activation may contribute to the development of CaOx nephrolithiasis through two potential mechanisms: direct cytotoxicity toward renal tubular epithelial cells and modulation of the crystal adhesion properties of these cells. However, the precise underlying mechanisms warrant further investigation.

Inflammation and OS mutually promote kidney stone formation

ROS promote inflammatory responses

Under the stimulation of high oxalate, high calcium, or CaOx crystals, renal tubular epithelial cells generate excessive ROS through various pathways. If the antioxidant defense mechanisms fail to clear the excess ROS, their accumulation in renal tissues activates signaling pathways such as NF-κB and MAPK. This activation stimulates the production of inflammatory cytokines and pro-inflammatory immune cells, including M1 macrophages and neutrophils, triggering an inflammatory cascade.

M1 macrophage polarization is regulated by the transcription factor NF-κB. Activated NF-κB induces the differentiation of circulating monocytes into M1 macrophages, further amplifying the inflammatory response and promoting stone formation. In addition, activated NF-κB upregulates the expression of hypoxia-inducible factor-1α (HIF-1α), which exacerbates inflammation [77, 78].

Recent proteomic and metabolomic studies have demonstrated that CaOx crystals induce OS through the Akt, ERK1/2, and P38/MAPK pathways, promoting inflammation and causing renal injury by altering amino acid metabolism and fatty acid β-oxidation [79]. The MAPK pathway, in particular, is a key regulator of macrophage activation and polarization. Activation of this pathway drives macrophages to polarize into the M1 phenotype, which contributes to stone formation [80]. Genome-wide expression profiling of renal papillary tissue demonstrated significant upregulation of genes related to the mitogen-activated protein kinase and Akt–phosphatidylinositol 3-kinase pathways in plaque-associated tissue compared to normal tissue [81].

Furthermore, elevated ROS prompts renal tubular cells to secrete interleukin-8 (IL-8), a cytokine that attracts neutrophils to the kidney’s damaged region, intensifying the local inflammatory response [82, 83]. ROS promote the release of endogenous damage-associated molecular patterns (DAMPs), which interact with TLRs to further amplify inflammatory responses. In this process, ROS act as both an initial trigger and an amplifier, causing the inflammatory response to expand and worsen in local tissues, thereby accelerating stone formation [84].

A recent study by Parveen Kumar et al. revealed that oxalate impairs metabolic and immune responses by modulating IL-10 signaling and mitochondrial ROS production in monocytes and macrophages. Macrophages exposed to oxalate exhibit a significant decrease in IL-10 protein expression and secretion, inhibiting the transition of macrophages from the M1 to the M2 phenotype and thus promoting stone formation [85].

Research indicates that urinary oxalate exacerbates renal inflammation by inducing OS, activating the NLRP3 inflammasome, and promoting IL-1β and IL-18 secretion [75]. These findings imply that CaOx crystal deposition harms renal tubular epithelial cells, generating excessive ROS. This process exacerbates renal tissue damage and promotes stone formation by activating numerous inflammatory cells and factors (Fig. 3).

Fig. 3.

Inflammation and OS mutually promote kidney stone formation

Inflammatory responses enhance ROS production

The deposition of CaOx crystals triggers a local inflammatory response, where the recruitment and activation of inflammatory cells serve as key sources of ROS production. Macrophages and neutrophils, in particular, continuously release inflammatory cytokines that upregulate the expression of NADPH oxidase, leading to sustained ROS generation [86]. These ROS not only directly damage renal tubular epithelial cells but also contribute to extracellular matrix remodeling and alterations in cell–cell adhesion, further exacerbating crystal adherence [36]. The persistent crystal attachment and growth can further intensify the inflammatory response, creating a vicious cycle of “inflammation–ROS–crystal deposition”.

M1 macrophages play a pivotal role in this process, as they promote the release of ROS and inflammatory cytokines and induce local cell death, thereby aggravating renal damage [87]. Moreover, studies have shown that M1 macrophages enhance the activation of the NLRP3 inflammasome, a process closely associated with kidney stone formation [56].

In contrast, M2 macrophages exhibit a protective role by directly suppressing NADPH oxidase activation, reducing ROS production, and decreasing p38-MAPK expression. Through the increased phosphorylation of Akt, M2 macrophages enhance cell proliferation and repair, mitigating tissue damage [88]. These findings highlight the potential of M2 macrophages in inhibiting stone formation (Fig. 3).

The feedback loop between ROS and inflammation forms a self-amplifying cycle. This cycle not only accelerates the formation of CaOx stones, but also poses the risk of irreversible structural and functional damage to the kidneys [89]. Breaking this feedback mechanism could serve as a promising strategy for the prevention and treatment of kidney stones.

Summary and outlook

Recent studies have preliminarily elucidated the role of ROS and the inflammatory response in CaOx stone formation. CaOx crystals can induce increased ROS levels in cells through various pathways. ROS subsequently activate signaling cascades including NF-κB and MAPK, triggering an OS response that may lead to cell damage or death, thereby creating conditions conducive to crystal deposition and stone formation. Concurrently, the regulatory role of macrophage polarization in the inflammatory response has gained attention, as the dynamic changes in macrophage phenotypes can directly influence the intensity and duration of inflammation. Furthermore, the NLRP3 inflammasome may also participate in the regulation of CaOx stone formation, although its precise mechanisms require further elucidation.

Existing in vitro and animal model studies have provided evidence supporting the involvement of immune cells and inflammatory responses in the pathogenesis of CaOx stones. However, clinical data remain limited. The specific regulatory mechanisms underlying macrophage recruitment and polarization, as well as the roles of ROS and inflammasomes in the immune response within the urinary lithogenic microenvironment, are not yet fully elucidated. Future studies should further explore the potential of immunomodulatory strategies to promote macrophage polarization and enhance stone clearance, clarify the role of NLRP3 inflammasomes in renal stone formation, and investigate the feasibility of blocking stone plaque growth and promoting its regression.

The molecular mechanisms underlying human CaOx stone pathogenesis remain incompletely understood. Existing studies have primarily focused on in vitro cell experiments and animal stone models, while systematic research data from clinical patients are relatively limited. Although these models provide important insights into the underlying mechanisms, they may not fully recapitulate the complex physiological microenvironment of the human body, and the molecular pathways and regulatory mechanisms revealed may differ from the actual clinical scenario. From an evidence-based medicine perspective, most long-term clinical trials of antioxidants have failed to demonstrate clear clinical benefits [90], further highlighting the research translation gap between existing animal models and clinical patients. Therefore, the development of animal models that more closely reflect the pathological characteristics of human kidney stone formation is of great significance to advance research translation. Current preliminary findings from cell and animal models require further experimental validation and clinical data to confirm their relevance to patient care. Large-scale, multicenter clinical studies are essential for accurately evaluating the roles of ROS and inflammatory responses in renal calculi formation, thus providing robust evidence for optimizing clinical treatments.

This article specifically addresses the involvement of ROS and inflammation in CaOx stone formation. Future research should also focus on urine abnormalities, genetic factors, anatomical variations, metabolic diseases, sex hormones, and intestinal flora in kidney stone development. A comprehensive understanding of renal calculi pathogenesis could lead to targeted interventions, offering safer and more effective clinical treatments for patients.

Author contributions

J.H.B and T.L. wrote the manuscript. Z.T.Z prepared Figs. 1, 2 and 3. All authors reviewed the manuscript.

Funding

This study was supported by The First Affiliated Hospital of Air Force Military Medical University Aviation Clinical Medicine Special Research Project (HKLCZX03).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.