The concept of RP was derived from the systematic examination of 429 paired kidneys at autopsy by Alexander Randall, published in 1937 (5). Randall documented subepithelial calcified lesions on the renal papillae in ~17% of specimens, of which 28 specimens harbored CaOx stones directly adherent to the plaque surface. A key mineralogical observation was that the plaque core consisted predominantly of HAP, largely carbonate-substituted apatite (carbapatite), whereas the overlying stones were CaOx (5). Modern mineralogical and imaging studies have since confirmed this two-phase mineral pattern, with carbapatite accounting for ~97.6% of the plaque mineral (15,16,25,26). Randall's original hypothesis posited a sequential process in which progressive HAP accumulation within the papillary interstitium breaches the overlying renal papillary epithelium, exposing a mineralized HAP surface to urinary constituents and thereby providing a heterogeneous nucleation site for CaOx crystal attachment and growth (5).

The advent of digital ureteroscopy and intraoperative papillary mapping transformed RP from a postmortem observation into a clinically assessable lesion. In an early endoscopic mapping study, Low and Stoller (6) identified papillary RP in 42 of 57 patients (74%) undergoing stone removal and in 3 of 7 patients (43%) undergoing endoscopic procedures unrelated to stone disease. Due to the small non-stone comparison group, these statistics should be interpreted as descriptive endoscopic observations rather than a definitive population-prevalence estimate. Direct endoscopic evidence of calculus attachment to RP was subsequently provided in a study by Matlaga et al (7), which examined 24 kidneys and 172 renal papillae in 23 idiopathic CaOx stone formers; all examined kidneys contained papillary plaque and 11 patients (48%) had attached calculi. Quantitative mapping provided more informative evidence of plaque burden. Kuo et al (17) mapped the papillae of 14 idiopathic CaOx stone formers and four non-stone controls and identified significantly higher mean polar fractional plaque coverage in the stone formers (7.4 vs. 0.5%; P=0.012). However, within idiopathic CaOx stone formers, plaque burden was heterogeneous. Wang et al (18) divided 42 patients into a low-plaque group with <5% plaque coverage per papilla (mean, 1.5%; n=32; 76.2%) and a high-plaque group with ≥5% coverage (mean, 10.5%; n=10; 23.8%). Compared with high-plaque stone formers, low-plaque stone formers were more frequently obese (50 vs. 10%; P=0.03) and had a more frequent history of urinary tract infection (34 vs. 0%; P=0.04), but were less likely to have experienced at least four previous stone events (22 vs. 80%; P=0.002) and had lower mean 24-h urinary calcium excretion (187±86 vs. 291±99 mg/day; P<0.01). The stones in the low-plaque stone formers also lacked a calcium phosphate (CaP) core on micro-CT, and their papillary biopsies demonstrated less interstitial and basement-membrane punctate crystallization. Therefore, these observations supported a major role for plaque-associated stone attachment in a subset of idiopathic CaOx stone formers, while also demonstrating clinical and morphological heterogeneity consistent with alternative or plaque-limited pathways.

Among idiopathic CaOx stone formers with measurable plaque, plaque surface coverage associates positively with stone burden: The RP-to-papilla area ratio scales with stone number after adjusting for disease duration (27) and a 'papillary calcification index' demonstrates a linear relationship with cumulative stone mass (28). This association is stone-type specific; struvite stone formers exhibit RP prevalences statistically indistinguishable from those of non-stone controls (29), supporting a closer plaque-stone relationship in idiopathic CaOx disease.

Micro-CT and multimodal imaging studies have extended these observations to the microstructural level by enabling non-destructive three-dimensional visualization of mineral continuity within intact papillae and stones (11-16). Williams et al (12) identified annular HAP-rich deposits at the apex of small CaOx calculi; Miller et al (11) demonstrated that free-floating, unattached CaOx stones also harbor internal HAP-rich regions whose mineral signature and spatial orientation are consistent with remnant RP. Multimodal imaging combining micro-CT, confocal autofluorescence microscopy and electron energy-loss spectroscopy has delineated a multi-stage progression model: Nascent HAP crystallites arise within the interstitial matrix, coalesce into plaque, extend through the renal papillary epithelium and are then colonized by CaOx from pelvic urine (13,14,16,30). This continuum of mineralization from the inner medullary interstitium to the stone surface provides structural evidence (level A) that a subset of idiopathic CaOx stones forms on exposed RP.

A notable finding by Evan et al (19) localized the earliest RP mineral deposits to the basement membranes of the thin limbs of the loop of Henle in the inner medulla. This anatomical specificity carries physicochemical significance: The inner medullary interstitium is a uniquely pro-calcific compartment. The countercurrent multiplication system generates interstitial calcium concentrations several-fold higher compared with that in plasma, while acid loading, through bicarbonate reabsorption and proton secretion, paradoxically alkalinizes the interstitial fluid compared with that in the tubular lumen (19,31). Thus, the combination of elevated calcium activity and high local pH drives CaP supersaturation to levels that favor spontaneous HAP nucleation (32,33). Low medullary blood flow and high tissue osmolality further concentrate solutes, compounding supersaturation. Therefore, these conditions make the inner medullary interstitium the sole site in the kidney where spontaneous apatite nucleation is thermodynamically favorable. The inner medullary interstitium is also where RP consistently originates.

A key observation that any microenvironment-centric model of RP should address is the apparent histological normalcy of plaque-bearing papillary tissue. In a 2003 biopsy study, Evan et al (19) noted no overt evidence of cell injury, inflammation or fibrosis in the papillary interstitium of idiopathic CaOx stone formers at the light-microscopic level. This observation may contrast the molecular injury signatures (for example, oxidative stress, immune activation and ECM remodeling) described below.

Canela et al (22) provide a notable molecular atlas of the stone-forming papilla. However, these data should not be interpreted as a plaque-exclusive transcriptome. As noted in the original study, an intratubular plug present in one analyzed specimen was not separately segmented from the plaque-focused region (22). The single-nucleus and spatial signatures may therefore include cells adjacent to ductal plugging as well as cells associated with interstitial plaque. In the present review, these findings are classified as level B evidence for molecular remodeling in the stone-forming papilla, rather than as direct proof that each identified pathway is intrinsic to interstitial plaque. This limitation reinforces the need to distinguish interstitial from intratubular pathology in future spatial studies, as discussed later in section 7.

This apparent discordance may reflect molecular remodeling that precedes changes detectable by routine light microscopy; however, the cross-sectional biopsy data cannot distinguish an early transient response from a stable subclinical state. Longitudinal papillary biopsies with matched multi-omics profiling (technically challenging; however, increasingly feasible) will be warranted to distinguish these possibilities in the future.

Prior to discussing specific molecular events, the present review briefly revisits the physicochemical conditions of the inner medullary interstitium, which provide the thermodynamic foundation for RP formation. As described in section 2, the countercurrent multiplication system elevates interstitial calcium concentrations to levels several-fold higher compared with that in plasma, while bicarbonate reabsorption and proton secretion paradoxically alkalinize the interstitial fluid compared with that in the tubular lumen (19,31). The combination of high calcium activity and elevated local pH induces CaP supersaturation beyond the threshold for spontaneous HAP nucleation (32,33). Low medullary blood flow and high tissue osmolality compound the problem by further concentrating solutes. These conditions are sufficient to promote spontaneous HAP nucleation in the absence of any biological catalyst. Idiopathic hypercalciuria, which increases calcium delivery to the medulla, amplifies this baseline supersaturation and is the metabolic phenotype most closely associated with interstitial plaque formation (19,26). These are the baseline conditions within which the molecular events described below unfold.

Evidence directly obtained from RP tissue for oxidative stress remains limited. In human RP tissue, dihydroethidium staining combined with E-cadherin and CD34 immunofluorescence demonstrated increased oxidative stress signal in renal tubules adjacent to calcium deposits (level A) (38). In parallel, microdissected RP transcriptomic data and subsequent analyses of RP datasets have identified oxidative stress-associated expression signatures (level B) (24,39). These observations support an association between RP-related tissue and oxidative stress responses; however, they do not identify the responsible nephron segment, establish temporal order or demonstrate that oxidative stress initiates interstitial HAP deposition.

By contrast, CaOx- or oxalate-exposed proximal tubular human kidney-2 cells, porcine proximal tubular Lilly Laboratories culture-porcine kidney 1 cells and Madin-Darby canine kidney cells, as well as high-calcium normal rat kidney-52E cells and vitamin D-associated rodent models, have reported ROS-associated epithelial injury responses (level C) (40-42). These systems do not reproduce the thin loop basement membrane and papillary interstitial environment in which human RP initially develops (19). Although CaOx crystals may contact tubular luminal surfaces in certain stone formers, these exposure paradigms do not model the initial interstitial HAP mineralization of RP. Thus, these studies have been retained only to generate the verifiable hypothesis that epithelial cells anatomically adjacent to plaque may exhibit associated injury responses; these studies are not interpreted as direct evidence of ROS-mediated plaque initiation.

Whether epithelial or interstitial cells adjacent to early HAP deposits in the human papilla display oxidative stress responses in vivo remains to be elucidated. Addressing this question warrants spatially resolved analyses of human papillary tissue and experimental systems that reproduce the local interstitial mineral environment in the future.

Cell death is a key process; however, surviving cells undergo changes as well, and what these cells develop into may markedly impact long-term plaque growth. Injury-associated inflammatory signals may favor osteoblast-like reprogramming of resident fibroblasts and tubular cells in experimental systems; whether this transition initiates or drives human RP remains to be investigated in the future.

The renal papillary interstitium harbors tissue-resident macrophages that actively patrol the medullary space (81,82). Macrophages are present in the papillary interstitium and may participate in local inflammatory responses. Proposed interactions between macrophages, oxidative-stress signals and fibroblast remodeling are derived mainly from crystal injury models and warrant plaque-specific validation in future research. Whether these interactions favor mineralization or resolution may depend partly on the balance between classically activated, pro-inflammatory M1 macrophages and alternatively activated, repair-associated M2 macrophages.

Interstitial calcification does not solely produce a kidney stone. For stones forming via the interstitial-plaque route, focal disruption of the overlying renal papillary epithelium exposes the interstitial HAP deposit to pelvic urine and permits heterogeneous CaOx crystal nucleation and overgrowth (5,19,20). The interstitium-to-lumen transition phase in the RP-to-stone sequence remains to be elucidated. The following section examines the available evidence.

The systemic and microbiome factors discussed in sections 4 and 5 are hypothesized modifiers of the papillary microenvironment rather than established determinants of RP presence or burden. No systemic or microbiome factor has been reported to alter endoscopic or histological RP burden in humans to date. Current tissue-level evidence directly derived from RP-related human papillary specimens is limited to level B molecular observations, including altered fatty acid-binding protein 4 (FABP4) and α-Klotho expression (35,111); other proposed associations are derived from level C cell or animal studies and level D stone epidemiology or clinical associations. Fig. 2 distinguishes these evidence sources and presents only proposed routes of influence.

Beyond the epidemiological association between dyslipidemia and nephrolithiasis, an important question is how abnormal lipid metabolism may modify the papillary interstitial milieu. Epidemiological data consistently associate dyslipidemia with nephrolithiasis risk. Cross-sectional analyses identify abdominal adiposity (112), elevated RC (113) and reduced high-density lipoprotein (114) as independent risk factors, with RC exhibiting a dose-response relationship when low-density lipoprotein cholesterol is controlled (113). These associations hold across Japanese (115) and American populations (112,114), identifying dyslipidemia as a robust systemic risk factor for nephrolithiasis.

In vitro co-culture studies have begun to explore how a metabolic-syndrome-like environment may enhance tubular inflammatory responses and CaOx crystal adhesion. Ichikawa et al (116) demonstrated that adipocyte-tubular cell co-culture enhances CaOx crystal adhesion and upregulates IL-6 expression in tubular cells. Zuo et al (117) extended this system to include macrophages, demonstrating that the tri-cellular metabolic milieu markedly upregulates monocyte chemoattractant protein-1, OPN and TNF-α, consistent with an M1-promoting inflammatory milieu associated with crystal retention. These findings position adipose tissue-derived paracrine signals as potential contributors to the interstitial inflammatory cascade, although the specific lipid mediators responsible have not been fully identified.

Notably, Taguchi et al (111) performed transcriptomic profiling of microdissected human RP tissue and identified marked enrichment of differentially expressed genes in lipid metabolism pathways. Among these, FABP4 was markedly downregulated in plaque tissue. Functional validation in Fabp4^−/− mice subjected to glyoxylate-induced hyperoxaluria demonstrated that FABP4 deficiency markedly exacerbated both interstitial and intratubular CaOx crystal deposition compared with wild-type controls (111). This level C glyoxylate-model phenotype suggests a candidate protective role for FABP4 in experimental renal calcification; its contribution to human RP burden remains to be elucidated.

FABP4 exhibits a notable tissue-specific paradox: Although decreased renal expression promotes local calcification, elevated serum FABP4 levels are associated with the severity of vascular calcification (118), and FABP4 inhibition has been explored as a therapeutic strategy for cardiovascular disease (119). The inverse relationship suggests that circulating FABP4 reflects systemic metabolic disturbance (acting as a biomarker), while interstitial FABP4 functions as a local calcification suppressor (acting as a protective effector). The mechanistic basis of this dichotomy (intracellular lipid-chaperoning vs. extracellular signaling) has not been investigated, to the best of our knowledge.

A meta-analysis of cohort studies reported a 16% higher kidney-stone risk among participants with diabetes mellitus than among those without diabetes (RR=1.16); the pooled analysis did not consistently stratify diabetes by type (120). Gestational diabetes mellitus (GDM), a distinct form of hyperglycemia first recognized during pregnancy, has also been associated with future kidney-stone risk (121), while Mendelian randomization supports a causal association between type 2 diabetes mellitus and kidney stones (122). The risk of kidney-stone formation is not confined to overt diabetes mellitus: Multiple insulin resistance indices, including the metabolic score for insulin resistance (METs-IR) (123), homeostatic model assessment of insulin resistance (HOMA-IR) (123) and triglyceride-glucose-body mass index (TyG-BMI) (123), were positively associated with both kidney-stone occurrence and recurrence in individuals without diagnosed diabetes, with each 1-unit increase in METs-IR corresponding to odds ratios of 1.020 for kidney-stone occurrence and 1.033 for recurrence, relative to a METs-IR value one unit lower (123). These data support insulin resistance as a metabolic correlate that may precede overt diabetes

A key route associating dysglycemia with renal crystal injury involves tubular epithelial stress in experimental systems (level C) (48,50,124). High-glucose exposure has been associated with increased apoptosis and reduced autophagic activity in HK-2 cells (level C) (48). In parallel, optineurin-enhanced mitophagy reduced mtROS accumulation and NLRP3 inflammasome signaling in high-glucose-treated murine renal tubular epithelial cells (level C) (50). These findings support a candidate tubular stress pathway; however, these findings do not establish high-glucose-induced pyroptosis or ferroptosis in the models cited in the present review, nor do these findings demonstrate relevance to human RP. Anan et al (124) reported that SGLT2 silencing in HK-2 cells attenuated high-glucose-induced OPN and CD44 upregulation and reduced CaOx crystal adhesion. In vivo, phlorizin attenuated ethylene glycol-induced renal stone formation in rats and downregulated kidney injury molecule-1 and OPN without altering water intake or urine volume, whereas Sglt2^−/− mice were resistant to glyoxylic acid-induced CaOx deposition (124). Therefore, level D stone associations and level C SGLT2-related experimental findings identify a candidate tubular injury pathway; however, whether SGLT2 inhibition alters human RP burden remains to be elucidated. In a distinct vascular model, high-glucose exposure promoted vascular smooth muscle cell calcification and senescence through BMF-AS1/BMF-associated signaling, providing cross-disease evidence that hyperglycemic stress can promote ectopic mineralization outside the kidney (125). However, these vascular findings do not establish a corresponding mechanism in human RP.

Kidney stone formers frequently exhibit lower bone mineral density (BMD) compared with non-stone controls, and clinical studies and reviews support an association between calcium stone disease and bone demineralization (126-128). Stone formers may also exhibit increased urinary calcium and altered bone-remodeling markers (129-132). In a cross-sectional study, Zhu et al (133) reported an inverse association between BMD and kidney stones and identified that elevated ALP was independently associated with the coexistence of osteoporosis/osteopenia and kidney stones.

ALP hydrolyzes PPi in mineralizing systems, thereby reducing an endogenous inhibitor of HAP nucleation. ALP expression is also upregulated in osteogenically induced RIF cultures (66). Aged bone-matrix-derived extracellular vesicles have been reported to promote osteogenic phenotypic switching and vascular calcification in experimental systems (134). Whether bone-derived extracellular vesicles reach or influence the renal papillary interstitium remains to be elucidated.

α-Klotho serves a key role in the bone-kidney axis. Reduced circulating soluble α-Klotho has been associated with CKD-related mineral and vascular abnormalities; however, methodological and clinical interpretation limitations remain (135). In a cross-sectional National Health and Nutrition Examination Survey (NHANES) analysis of middle-aged and older adults with diabetes mellitus (diabetes type was not stratified in the cited analysis), serum α-Klotho levels were inversely associated with prevalent kidney stones (136). KLOTHO G395A polymorphism has been associated with kidney-stone susceptibility in a previous case-control study (137). In one study of RP-related human papillary tissue, α-Klotho expression was reduced, and complementary cell experiments showed that tubular epithelial cell-derived α-Klotho inhibited Wnt/β-catenin signaling and osteogenic differentiation in neighboring RIFs (35). Reduced α-Klotho expression in RP-related tissue (level B) and α-Klotho-RIF interactions in experimental systems (level C) support a potential association between anti-calcific signaling and the papillary interstitium. Whether systemic mineral homeostasis directly alters human RP burden remains to be investigated in future research.

The fibroblast growth factor 23 (FGF23)-parathyroid hormone (PTH)-vitamin D axis regulates CaP homeostasis through coordinated renal and intestinal effects. FGF23 and PTH decrease proximal tubular phosphate reabsorption and thereby promote phosphaturia; FGF23 suppresses renal 1α-hydroxylase and 1,25-dihydroxyvitamin D production, whereas PTH stimulates vitamin D activation. Active 1,25-dihydroxyvitamin D, in turn, increases intestinal calcium and phosphate absorption (138). Dysregulation of the FGF23-Klotho-mineral axis is also implicated in CKD-associated mineral abnormalities and ectopic vascular calcification (139); however, it remains to be elucidated whether variation within this axis modifies kidney-stone risk or human RP burden independently of established metabolic abnormalities.

Metabolic syndrome and hypertension are associated with kidney-stone risk (140,141). Vascular calcification is considered primarily as a cross-disease mechanistic comparison rather than an established epidemiological predictor of stone disease in the present review. The molecular parallels between vascular calcification and RP are extensive enough to suggest overlapping disease mechanisms beyond what epidemiological co-occurrence alone would imply (detailed in section 3).

Vascular calcification and RP-related experimental systems share several candidate features, including osteogenic signaling, inflammatory pathways and altered calcification inhibitors (35,43,44,56,62,72-74,84,142,143); however, the evidence is heterogeneous and does not establish a shared causal mechanism in human RP. These cross-disease parallels nominate, rather than validate, potential therapeutic targets for future RP research. Furthermore, regarding causal directionality, it remains to be elucidated whether systemic vascular calcification drives renal interstitial calcification through circulating mediators or if both processes independently result from a shared metabolic insult.

A recent global meta-analysis estimated urolithiasis prevalence at 12.93% in men and 8.91% in women; however, the magnitude of the sex difference varied substantially according to population, diagnostic method and geographical region (144). In a population-based study from Rochester, the men-to-women incidence ratio for symptomatic nephrolithiasis decreased from 3.1 in 1970 to 1.3 in 2000 (level D) (145). In three large prospective cohorts, stone incidence remained higher in men than in women, with rates of 271 and 159 per 100,000 person-years, respectively; differences in lifestyle factors and urine chemistry provided partial explanations of the excess male risk (level D) (146). These epidemiological patterns alone do not establish a direct causal role for sex steroids. Clinical findings regarding estrogen status have also been reported to be inconsistent. Natural menopause was not independently associated with incident kidney stones in the Nurses' Health Study (147), whereas randomized Women's Health Initiative trials reported that estrogen therapy increased nephrolithiasis risk (148). Therefore, estrogen should not be described as uniformly protective in clinical stone disease. Furthermore, the mechanistic studies discussed below primarily focus on urinary solute metabolism, tubular CaOx crystal adhesion or injury and experimental crystal deposition; the findings do not establish that androgen or estrogen signaling alters the burden, mineral composition or molecular phenotype of morphologically verified human interstitial RP.

Circulating testosterone levels should not be treated as a direct surrogate for intrarenal androgen receptor (AR) activity. In a cross-sectional NHANES analysis, using the highest serum testosterone quartile as the reference, men in the lower three quartiles had higher adjusted odds of kidney stones (ORs, 1.375, 1.348 and 1.472 for the first, second and third quartiles, respectively), with the inverse association being most evident in men aged >40 years (level D) (149). A prospective controlled study of male patients with CaOx stones identified higher blood AR mRNA and plasma miR-185-5p levels, along with lower blood CSF1 mRNA expression, compared with that in healthy controls; circulating androgen levels did not differ markedly between the groups (150). These non-RP human observations are associative and do not localize the pathway to renal papillary tissue.

Mechanistic evidence has been derived from COM-exposed renal tubular epithelial cells and experimental CaOx deposition models. In HK-2 and HKC-8 cells, AR bound an androgen-response element within the miR-185-5p promoter and increased miR-185-5p transcription; miR-185-5p in turn directly targeted the 3'-untranslated region of CSF1 mRNA, reducing CSF1 expression and secretion. AR depletion increased CSF1-dependent macrophage recruitment, CD163/CD206-associated M2-like polarization and COM-crystal phagocytosis, whereas CSF1 suppression or miR-185-5p restoration partially reversed these effects (level C) (96). Consistently, renal tubule-specific loss or pharmacological degradation of AR increased renal CSF1 and M2-like macrophage accumulation and reduced CaOx deposition in glyoxylate-treated mice and hydroxy-L-proline-treated rats (level C) (96). This pathway aligns with independent evidence that CSF1 deficiency reduces renal M2-like macrophages and increases experimental CaOx deposition, whereas recombinant CSF1 or M2-macrophage transfer promotes crystal clearance (level C) (93). However, these systems model hyperoxaluria-associated intrarenal CaOx deposition rather than the initiation of chronic interstitial HAP plaque, and the AR-miR-185-5p-CSF1 axis has not been demonstrated in morphologically verified human RP.

A mechanistic study reported two anatomically distinct ERβ-dependent effects in experimental CaOx deposition. In HepG2 hepatocytes, ERβ bound estrogen-response elements within the promoter of alanine-glyoxylate aminotransferase 1 (AGT1), increased AGT1 expression and reduced oxalate production. In oxalate-exposed HK-2 and HKC-8 renal tubular epithelial cells, ERβ suppressed NADPH oxidase 2 (NOX2) transcription, ROS and H₂O₂ production and cell injury; NOX2 knockdown partially reversed the oxidative stress and injury responses caused by ERβ depletion. In vivo, genetic loss or pharmacological inhibition of ERβ increased urinary oxalate excretion, renal oxidative stress and CaOx deposition in glyoxylate-treated mice and hydroxy-L-proline-treated rats, whereas pharmacological NOX inhibition attenuated experimental crystal deposition (level C) (151). These findings support a dual hepatic AGT1 and renal tubular NOX2 mechanism in experimental CaOx disease. However, the renal systems model oxalate-exposed tubular injury and intrarenal CaOx deposition rather than early thin loop basement membrane HAP nucleation, and neither ERβ activity nor AGT1/NOX2 signaling has been demonstrated in morphologically verified human interstitial RP. Independently, 17β-estradiol exposure in MDCK renal tubular cells reduced CaOx crystal-binding capacity and promoted cell migration and tissue-healing phenotypes, providing additional experimental evidence that estrogen signaling can modify tubular crystal interactions (152).

Another candidate mechanism involves cross-talk between ER signaling, Wnt/β-catenin and fibroblast plasticity, although the available evidence is indirect and context-dependent. In established osteoblast-precursor cell lines and primary osteoblast progenitors, selective activation of kinase-mediated ER signaling stimulated Wnt and BMP2 pathways and promoted osteogenic differentiation, whereas conventional 17β-estradiol suppressed BMP2-induced osteoblast commitment (level C) (153). These opposing effects indicated that ER-Wnt interactions depend on the ligand, signaling mode and cellular context rather than exhibiting a uniformly pro- or anti-osteogenic effect of estrogen. In renal injury models, tubule-specific blockade of Wnt secretion reduced β-catenin activation, renal fibroblast proliferation and myofibroblast expansion, while tubule-derived Wnts promoted renal fibroblast activation in vitro (level C) (154). Furthermore, in the context of RP, HK-2-derived α-Klotho inhibited osteogenic differentiation of hRIFs by suppressing Wnt/β-catenin signaling (level C) (35), while activation of this pathway and reduced expression of its antagonists were observed in RP-related tissue (level B) (35). Therefore, these findings suggested that estrogen or ER signaling could modify epithelial-fibroblast Wnt communication or the osteogenic plasticity of RIFs. However, no study has directly investigated estrogen or ER pathway modulation in RP-derived RIFs, and ER-Wnt/β-catenin cross-talk has not been spatially demonstrated in morphologically verified human RP. This proposed association therefore remains a cross-model, hypothesis-generating inference.

The traditional assumption that urine from the healthy urinary tract is sterile has been challenged by expanded quantitative culture and sequencing studies demonstrating resident urinary microbial communities in individuals without clinical urinary tract infection (156-164). The concept of a urinary microbiome has recently been extended to the kidney parenchyma itself. Agudelo et al (155) reported that murine kidneys harbor a low-biomass, metabolically active microbiota in microniches between glomeruli and tubules. Cefazolin, a perioperative antibiotic extensively used in stone surgery, shifted the population away from uroprotective Lactobacillus species and toward pro-lithogenic Enterobacteriaceae; the effect was duration-dependent and reversible after cessation. In vitro, Lactobacillus crispatus inhibited CaOx crystallization, whereas stone-associated Escherichia coli promoted it. RNA sequencing and RNA fluorescence in situ hybridization imaging of human kidney tissue confirmed microbial signatures in both glomerular and tubular compartments (155). These findings raise the possibility that perioperative antibiotics may alter the renal microbiome in ways relevant to stone biology. Expanded quantitative urine culture and 16S ribosomal RNA sequencing studies have countered the dogma of sterile urine and identified resident urinary microbial communities in adults (156-164). In urinary stone disease, Zampini et al (165) reported urinary and gut dysbiosis in stone formers compared with controls, including altered Lactobacillus- and Enterobacteriaceae-associated microbial community patterns; however, a direct effect on papillary interstitial RP remains to be investigated in the future.

Urease-producing bacteria can alkalinize urine and favor phosphate precipitation (173). In vitro, selected Enterobacteriaceae can modify CaOx crystal interactions. E. coli-associated citrate depletion has also been proposed as a lithogenic mechanism (174). However, the direct influence of these microbial effects on papillary interstitial RP remains to be elucidated.

If the urinary microbiome acts locally, the gut microbiome influences stone risk distally, primarily through its effects on oxalate handling and systemic inflammation. Oxalate homeostasis is regulated by intestinal oxalotrophic bacteria, particularly Oxalobacter formigenes, which degrades dietary oxalate via formyl-CoA transferase and oxalyl-CoA decarboxylase (167-170). Reduced O. formigenes colonization has been associated with altered intestinal oxalate regulation and higher urinary oxalate in certain studies (167-170). However, these observations are associative and do not establish that antibiotic exposure or dietary change uniformly increases urinary oxalate through loss of O. formigenes.

Comparative gut microbiome profiling revealed extensive dysbiosis in stone formers. Bacteroides is ~3.4-fold more abundant in patients with kidney stones, while Prevotella is 2.8-fold more prevalent in controls (171). In a small pilot cohort study, Eubacterium abundance demonstrated an inverse trend with urinary oxalate, whereas Escherichia abundance exhibited an inverse trend with urinary citrate (171). These exploratory associations warrant validation in larger cohorts in the future.

Although much research has focused on O. formigenes, the broader gut microbial community may also contribute to intestinal oxalate metabolism and kidney-stone susceptibility (166,172). Miller et al (166) used metagenomic analysis to describe a network of bacterial taxa that co-occur with O. formigenes in non-stone-forming controls and are underrepresented in stone formers, suggesting that oxalate homeostasis depends on a consortium rather than a single species. A two-sample Mendelian randomization study reported no causal association between genus Oxalobacter and kidney stones, but identified protective effects for Actinomycetales, Clostridiaceae and Hungatella, and risk associations with Haemophilus and Subdoligranulum (172). Miller et al (166) suggested that community-level oxalate-degrading capacity, including genes such as Oxc and Frc, may be more informative than the presence of any single taxon.

Beyond oxalate degradation, the gut microbiome may influence the renal interstitium through systemic inflammatory routes, although this hypothesis has not been validated in an RP model. In a cross-disease context, CKD-mineral and bone disorder includes vascular calcification as a recognized complication (175), whereas circulating endotoxemia in CKD is associated with systemic inflammation and cardiovascular disease (176). The contribution of gut-derived inflammatory signals to papillary interstitial mineralization in stone formers remains inferential. Short-chain fatty acids (SCFAs), whose production depends on gut microbial composition, regulate macrophage polarization in multiple organs, and fecal SCFA levels differ between stone formers and controls (177). Whether any of these gut-derived signals reach the renal papillary interstitium and modulate NLRP3 activation or M1/M2 macrophage balance remains to be elucidated. Future research is warranted to compare macrophage polarization and osteogenic markers in renal papillae of germ-free vs. conventionally housed mice under hyperoxaluric challenge.

Chemically induced hyperoxaluria remains one of the most widely used strategies for experimentally inducing renal CaOx crystal deposition. Inducing agents, such as ethylene glycol and glyoxylic acid, are administered to experimental animals via dietary supplementation or injection (178-181). These protocols produce predictable hyperoxaluria and renal crystal deposition; however, the mineral phase and anatomical distribution of deposits depend on the inducing regimen (179-181). Single-agent hyperoxaluria models are limited by crystal transience: Deposits resolve upon cessation of the inducing stimulus, contrasting with the more persistent and progressive nature of human RP (178-181). This divergence restricts the utility of these models in studying chronic plaque maturation and limits their predictive validity for therapeutic durability.

Combined metabolic disruptions can increase renal crystal deposition in experimental models; however, the mineral phase, compartment and chronicity vary across protocols. Such models may approximate selected metabolic combinations observed in kidney stone disease; however, these models do not reproduce the full progressive interstitial plaque sequence of human RP (182,183). Therapeutically, induced models have identified druggable targets. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone reduces glyoxylic acid-induced CaOx deposition via suppression of M1 macrophage polarization (179), while phlorizin attenuates ethylene glycol-induced renal stone formation in rats and Sglt2 deficiency reduces glyoxylic acid-induced CaOx deposition in mice (124). These findings provide experimental proof-of-concept in targeting tubular injury and inflammatory responses; however, neither model reproduces human interstitial RP.

The present review classifies the genetic and hereditary models according to the location and inducibility of their mineral phenotype (Fig. 3); Table SII summarizes the corresponding mechanisms and limitations. This grouping is intended to facilitate comparison of model phenotypes and should not be interpreted as an absolute biological classification. Certain models, particularly Thp^−/−, are present near the boundaries between groups; therefore, the categories should not be viewed as absolute.

The limitations of rodent models have promoted research using human-derived and biomimetic in vitro platforms. Patient-derived primary cultures of RIFs and tubular epithelial cells have contributed to the aforementioned osteogenic-differentiation studies (35,58,78), while biomimetic RP models using acidic biopolymer matrices have been developed to examine the effects of organic matrix components on crystal nucleation (199). Renal organoids and kidney-on-chip systems may provide additional human-background platforms; however, no available system recapitulates the complete adult papillary interstitial niche, including sustained medullary gradients and coordinated epithelial, interstitial, immune, vascular and ECM components, to date. These platforms should therefore be viewed as complementary tools rather than replacements for animal models in the future.

Available translational opportunities, stratified by biomarker readiness and therapeutic evidence level, are summarized in Fig. 4. Evan et al (19) localized the earliest RP mineral to the basement membranes of the thin loops of Henle; however, the temporal coordination of immune cell recruitment, fibroblast activation and matrix remodeling at this site remains to be elucidated. In a previous study by Canela et al (22), the integration of single-nucleus RNA sequencing with spatial transcriptomics revealed that the injury signature in stone-bearing papillae, including immune activation, oxidative stress and ECM remodeling, extends beyond focal mineral deposits. This pattern is compatible with molecular remodeling beyond visible plaque but does not establish that such remodeling temporally precedes plaque formation. Furthermore, as noted in section 2, the dataset did not separately resolve interstitial from intratubular mineralization. Therefore, future studies should adopt a morphology-first, lesion-staged design rather than including further candidate pathways to an undifferentiated list. Based on established RP morphology and mineral-mapping studies (19,20,36,53,54), it may be proposed to classify serial papillary sections as non-mineralized tissue, early thin-loop basement-membrane HAP, expanding interstitial plaque, exposed plaque or intratubular plug before spatial profiling. Spatial transcriptomics combined with multiplexed protein imaging, such as multiplexed ion beam imaging by time of flight or co-detection by indexing, may then define cell identities, subcellular protein localization, cellular neighborhoods and distance-dependent molecular gradients around each lesion class (200,201). Comparisons may be proposed among plaque-adjacent cells, distance-matched cells from the same papilla, non-stone controls and plug-dominant papillae, informed by prior papillary spatial profiling and emerging spatial multi-omics approaches (22,202,203), so that plaque-associated remodeling can be distinguished from generalized stone-related or intratubular injury (Fig. 4).

Candidate mechanisms should be evaluated as prespecified biological modules associated with the aforementioned framework described in section 3. A mechanotransduction-osteogenic module should assess PIEZO1 abundance, YAP/TAZ subcellular localization, local matrix properties and RUNX2-related output. A stress-resolution module should examine oxidative-stress signals along with autophagy- and mitophagy-associated proteins. However, fixed-tissue abundance of LC3-β, sequestosome 1/p62, PINK1 or Parkin can identify spatially associated machinery but these proteins are unable to establish altered autophagic or mitophagic flux, which requires dynamic assays in viable ex vivo or experimental systems (204). A senescence-immune module should combine cell-cycle-arrest markers, loss of nuclear-envelope integrity, SASP mediators and macrophage phenotypes rather than defining senescence from a single p16 or p21 signal (100). Lastly, morphology-guided laser-capture microdissection could isolate plaque-adjacent RIFs and epithelial cells for targeted analysis of KL promoter methylation, DNMT activity, m⁶A abundance and transcript-specific m⁶A regulation. Emerging spatial methylome-transcriptome and spatial m⁶A technologies provide a potential route to retain the tissue microenvironment; however, neither platform has yet been validated in human papillary RP (202,203).

It may be proposed that a candidate mechanism should be considered plaque-associated when it is reproducibly enriched in defined cells adjacent to early interstitial HAP, varies with lesion stage or distance from the mineral and is unevenly present in non-mineralized or plug-dominant tissue. Such spatial associations could then be tested functionally in papillary cell co-cultures, organotypic tissue systems or plaque-relevant mineral-exposure models using pathway-specific disruption. Temporal inference could combine standardized endoscopic plaque mapping, clinically obtained papillary specimens, longitudinal biospecimens and subsequent plaque-growth or recurrence data, rather than depending on routine repeated papillary biopsies (19,22,202,203). This staged framework could distinguish a fixed sequence of events from a coupled network present at plaque initiation and from patient-specific entry into different nodes of the network.

Oxidative stress, immune remodeling and osteogenic changes have been observed in RP-related tissue or experimental models (24,35,38,39,56,62), whereas dyslipidemia, dysglycemia and microbiome alterations are supported mainly by stone-related experimental or epidemiological evidence (111-124,155-174); however, the hierarchical relationship between these processes remains to be elucidated. To address whether lipid-induced inflammation and hyperglycemia-driven oxidative stress contribute independently or converge on pathways such as NLRP3, future studies could combine multicellular co-culture systems previously used to model metabolic inflammatory interactions (116,117) with controlled metabolic perturbation. Human cohorts could additionally be stratified by metabolic phenotype and studied using matched endoscopic plaque quantification together with papillary multi-omics or spatial profiling (22,202,203) to identify candidate subgroups for targeted investigation. Another key question is whether these drivers can be ranked by importance, and if the associations established to date reflect causal relationships. Both of these issues warrant similar methodological advances in future studies.

The metabolic syndrome-stone association rests predominantly on cross-sectional and retrospective data. Mendelian randomization has strengthened the causal association for type 2 diabetes mellitus (122) but has not been systematically applied to individual metabolic-syndrome components (dyslipidemia and insulin resistance) or specific microbial taxa, to the best of our knowledge. Prospective cohort studies using serial endoscopic papillary assessment combined with longitudinal multi-omics profiling of urine, blood and stool are warranted to establish temporal precedence. Interventional studies (randomized trials of SGLT2 inhibitors, M2-promoting agents or microbiome-targeted therapies) with plaque burden as the primary endpoint would provide notable causal evidence.

The transient crystal deposition of chemically induced models and the single-gene focus of KO models capture only a fragment of multifactorial human RP. Next-generation models should incorporate multiple simultaneous targets. For example, crossing Cldn2^−/− mice onto a high-fat diet background would combine hypercalciuria with metabolic syndrome and allow analysis of pathway interactions in vivo. Longitudinal intravital two-photon imaging of the rodent papilla could resolve the temporal sequence of cell death, immune infiltration and mineral nucleation that may not be captured by endpoint histology. For human-relevant platforms, papilla-on-chip devices incorporating primary patient-derived cells under medullary concentration gradients, combined with immune cell co-culture, represent the most promising path toward in vitro models that recapitulate the complexity of the human mineralization-permissive milieu. As a proposed next-generation human-relevant platform, a multicellular perfused 3D papillary co-culture incorporating papillary epithelial/tubular cells, RIFs and immune cells under medullary-like chemical gradients could enable controlled investigation of mineralization-associated cell-cell interactions.