S100A8/A9 is abundantly expressed in neutrophils and monocytes and serves as a sensitive marker of innate immune activation. Its most extensively studied clinical application in nephrology is differentiating intrinsic (structural) from pre-renal (functional) AKI. This distinction is clinically useful, but urinary calprotectin should be interpreted as a marker of urinary tract or kidney inflammation rather than as a stand-alone diagnostic test. Its performance may be influenced by urinary tract infection, systemic inflammation, sampling time and the assay platform (34-36).

Multiple studies have evaluated urinary calprotectin for differentiating intrinsic from pre-renal AKI. An adult comparison study evaluated calprotectin together with neutrophil gelatinase-associated lipocalin (NGAL) (35). A cardiopulmonary-bypass proteomics study detected urinary S100A8 and provides perioperative biomarker data, but it did not evaluate calprotectin diagnostic accuracy (37). A meta-analysis and critical-care cohorts report favorable discrimination, although thresholds differ and external validation and standardization remain limited (38,39). In renal transplant recipients, urinary calprotectin can help distinguish pre-renal graft dysfunction from intrinsic acute allograft injury (40).

S100A8/A9 has also been studied in several clinical settings associated with AKI. In pediatric populations, urinary calprotectin has been investigated for differentiating AKI etiologies (41,42) and for predicting adverse outcomes. However, one pediatric AKI cohort reported poor mortality discrimination, with areas under the receiver operating characteristic curve (AUCs) of 0.55 for 30-day and 0.61 for 3-month mortality, whereas prediction of renal replacement therapy (RRT) yielded an AUC of 0.72 (43). After cardiac surgery, elevated plasma S100A8/A9 levels immediately following cardiopulmonary bypass are associated with subsequent AKI (44). Plasma S100A8/A9 has also been investigated for predicting postoperative AKI after high-risk procedures such as acute type A aortic dissection surgery (45). Mechanistic experiments in this setting implicate S100A8/A9-high macrophages in renal tubular epithelial cell damage (46). In sepsis-associated AKI, S100A8/A9 expression is time-dependently elevated in the kidney, and its inhibition ameliorates renal dysfunction in murine models, supporting a pathogenic role in that model (47). In drug-induced nephrotoxicity, urinary calprotectin has also been evaluated for detecting acute cisplatin-induced nephrotoxicity (48). Urinary calprotectin has additionally been studied in relation to kidney damage and mortality in COVID-19 (49). These plasma and urine studies use different sampling times and endpoints; thus a common diagnostic threshold cannot be assumed.

In CKD, S100A8/A9 has been studied primarily as a marker of systemic low-grade inflammation associated with disease progression and complications. Higher plasma calprotectin levels are prospectively associated with an increased risk of new-onset CKD in the general population, particularly when defined by urinary albumin excretion (50). CKD-related systemic inflammation has also been examined in relation to the gut-kidney axis: Elevated fecal calprotectin, a marker of intestinal inflammation, has been associated with higher mortality in patients with CKD, independent of traditional inflammatory markers (51). Related exploratory findings in children on RRT and elderly patients with moderate CKD support further investigation of this relationship (52,53). Intestinal barrier dysfunction and microbial products or uremic solutes may increase systemic inflammatory exposure. However, fecal calprotectin indicates intestinal inflammation; it does not establish transport of S100 proteins to the kidney or causation of CKD progression.

S100A8/A9 has also been studied in inflammatory glomerular diseases. In ANCA-associated vasculitis (AAV), serum and leukocyte surface S100A8/A9 reflect disease activity, decrease with treatment and may help predict relapse (54). A predictive model combining serum calprotectin, urinary soluble CD163 and hematuria has been proposed to improve detection of active renal vasculitis (55). In lupus nephritis, serum and urinary S100A8/A9 levels correlate with renal activity in the available cohorts (56,57). In IgA nephropathy, the accumulation of myeloid-related protein 8 (MRP8; S100A8)-expressing macrophages in glomeruli has been associated with sclerotic progression and unfavorable prognosis in children (58). These observations support S100A8/A9 as a marker of active myeloid inflammation, but larger prospective studies are needed to define its role in routine decision-making.

Evidence linking calprotectin to vascular calcification may be relevant to CKD-associated cardiovascular risk. A primary experimental study indicates that S100A8/A9 can promote osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) through RAGE and TLR4 signaling (16). Reviews and commentary provide broader mechanistic and clinical context (59,60). The extent to which circulating calprotectin is a causal mediator, a marker of vascular inflammation or both may vary among patient subgroups and requires prospective interventional study.

In DN, S100A8/A9 and S100B have been associated with disease status, progression and systemic complications. The evidence is most consistent for associations with albuminuria, inflammatory cell infiltration and diabetic complications, whereas their incremental value beyond established clinical markers remains insufficiently defined. DKD is used for the clinical disease category; DN is retained when describing studies that use that designation.

Serum S100A8/A9 levels increase across worsening stages of DN, from normo-albuminuria to macroalbuminuria, and correlate positively with urinary albumin-to-creatinine ratio and serum creatinine (4). The association with microalbuminuria has prompted evaluation of calprotectin as an early marker of microvascular damage and inflammation in type 2 diabetes (61). Single-cell sequencing studies have identified S100A9 as a candidate early molecular marker that is highly expressed in pro-inflammatory myeloid-derived suppressor cells that infiltrate the kidney in early DN (62). Machine-learning analyses have also nominated S100A9 as a candidate diagnostic biomarker for DKD (63). The gut-kidney axis is also relevant in DN, but a study of patients with type 1 diabetes found that fecal calprotectin did not differ significantly between progressive and stable DKD groups (64). These omics-derived signatures require independent protein-level validation and comparison with estimated glomerular filtration rate (eGFR) and albuminuria.

S100B, which is primarily expressed in glial cells, has been studied in relation to systemic complications of diabetes (65). Elevated serum S100B has been associated with diabetic peripheral neuropathy, a common comorbidity of DN (65). In patients with ESRD, which can result from DN, higher serum S100B levels are associated with neurologic and psychiatric complications, including cognitive impairment and depression (32,65-67). In high-glucose-stimulated mesangial cells, S100B contributes to pro-fibrotic responses (12). S100B is also under investigation as a potential marker for long-term complications in patients with DN who have a history of COVID-19 (68). These systemic associations do not establish S100B as a kidney-specific predictor of DKD progression. Studies of activated leukocyte cell adhesion molecule in DN and IgA nephropathy provide non-S100 biomarker comparators (69,70), not evidence of S100B signaling.

S100A4 and S100A12 are two S100 proteins with distinct candidate prognostic roles in kidney disease, related primarily to fibrosis and cardiovascular complications, respectively.

S100A4/FSP1 is a widely used marker of fibroblast activation in fibrotic kidneys, but its biological interpretation requires caution. In animal models of kidney fibrosis, including obstructive and remnant-kidney models, S100A4/FSP1 expression increases in interstitial fibroblast-like cells (71,72). Hypoxia and fibroblast-epithelial crosstalk may further promote FSP1-positive cell accumulation and fibrotic remodeling (71,73). In IgA nephropathy, interstitial FSP1-positive cell density has been associated with renal survival and corticosteroid responsiveness (21,74). However, extracellular FSP1 may also exert context-dependent protective effects through antioxidant pathways, indicating that FSP1 does not act solely as a fibrosis-promoting factor (13). Intracellular fibrogenic functions and extracellular protective effects should therefore be distinguished.

S100A12 (also known as EN-RAGE) is a pro-inflammatory RAGE ligand and a candidate marker of cardiovascular risk in advanced CKD. Plasma S100A12 levels are elevated in patients with ESRD undergoing hemodialysis or peritoneal dialysis (75,76), and several cohorts link higher levels to all-cause or cardiovascular mortality (77,78). These findings may have prognostic value, but they do not show that S100A12-directed therapy will improve outcomes. ABCC6 genetics provides non-S100 calcification context (79); the distinction between S100A12 as a risk marker and a causal factor remains important (80). Experimental studies support S100-associated vascular remodeling and calcification mechanisms (19,81).

The potential clinical value of S100 proteins should be assessed against established biomarkers, both individually and in combined panels. In AKI, urinary calprotectin provides information about inflammatory parenchymal injury that differs from tubular stress markers such as NGAL, kidney injury molecule-1 (KIM-1) and interleukin-18 (IL-18) (35,41,43). Its principal value may be etiological classification rather than universal AKI prediction. In CKD, S100 proteins are unlikely to replace albuminuria, estimated glomerular filtration rate or histology, but may add pathway-level information on inflammation, fibrosis or vascular risk.

In chronic and inflammatory kidney diseases, combining S100 proteins with other markers may improve diagnostic performance. In AAV, a model combining serum calprotectin with the macrophage marker urinary CD163 performed better for detecting active renal disease than either biomarker alone (55). In renal transplant recipients, urinary mRNA levels of NGAL, KIM-1 and IL-18 correlated with histological findings of rejection, whereas S100A8/A9 mRNA did not, indicating that biomarker choice may depend on the clinical question within the same patient population (82). In stable CKD, plasma calprotectin levels correlate with other inflammatory markers such as IL-6 and monocyte chemoattractant protein-1, as well as with levels of protein-bound uremic toxins, supporting its investigation as an indicator of the systemic inflammatory state associated with uremia (83). Integration of S100 proteins into panels for AKI differentiation, CKD assessment or prediction of inflammatory flares requires external validation and evidence of clinical benefit. Urinary transcript findings should not be extrapolated directly to calprotectin protein assays.

Across biomarker studies, the evidence base is heterogeneous. Urinary S100A8/A9 in AKI is supported by multiple clinical cohorts and meta-analytic evidence, whereas several other S100 members are supported mainly by small cohorts, tissue studies, animal models or bioinformatic analyses. Future studies should therefore report standardized assays, prespecified cutoffs, external validation cohorts and incremental predictive value beyond conventional markers.

Most urinary AKI studies used enzyme-linked immunosorbent assays with cohort- and assay-specific cut-offs (34,35,38). The 300 ng/ml value from the original adult study is a diagnostic threshold, not a normal range or treatment-selection threshold (34). Antibody specificity, calibrators, molecular forms, dilution, analytical range and urine-matrix effects limit comparability. A serum assay comparison required substantial calibration adjustment; this finding demonstrates the need for harmonization but does not quantify inter-assay bias in urine (84). Validation should address precision, recovery, linearity, interference and quantification limits, followed by independent validation of clinical thresholds and incremental value. Urinary concentrations and creatinine-normalized values should be reported separately.

Urinary tract infection, pyuria and contamination can confound interpretation. Bacteriuria can raise urinary calprotectin even without dipstick pyuria, thus a negative dipstick alone does not exclude infection-related elevation (36). Studies should document the collection method, catheter status, hematuria and suspected contamination, with urine culture or repeat sampling when indicated. Systemic infection, surgery and autoimmune activity also affect circulating markers independently of kidney injury (44,54,85). Studies should record eGFR, residual kidney function, dialysis status and sampling relative to dialysis; increased leukocyte S100A12 transcription does not exclude altered clearance (18).

Serum and plasma results should not be pooled: Specimen-dependent concentrations and delayed-processing effects have been demonstrated for circulating calprotectin (86). Collection-to-processing time, temperature, centrifugation, storage and freeze-thaw exposure should be controlled for each matrix and assay. Findings in blood cannot establish analyte stability in urine. Sampling should be defined relative to injury, surgery, treatment and recovery because these time points address different clinical questions (30,43,44).

A hallmark of kidney injury is inflammatory cell recruitment and activation. S100A8/A9 and S100A12 can act as chemo-attractants and immune activators, but their functions are not uniformly deleterious. In glomerulonephritis models, S100A8/A9 promotes inflammatory leukocyte-renal cell interactions, whereas in IRI it has been linked to macrophage-mediated repair responses (5,10). Distinguishing injury-amplifying from repair-associated S100 signaling is therefore essential for interpreting therapeutic studies.

These mechanisms occur across several nephropathies. In systemic lupus erythematosus, serum S100A8/A9 and S100A12 are elevated in patients with high disease activity and are associated with the presence of glomerulonephritis (85). In DKD, S100A8/A9-associated TLR4/NF-κB signaling contributes to inflammation and interstitial fibrosis in experimental models (11). In septic AKI, blockade of the S100A8/A9-TLR4 interaction with paquinimod ameliorates renal dysfunction and inflammatory injury (47). Human biomarker associations and experimental receptor effects provide different levels of evidence. S100A8- and S100A9-derived B-cell epitopes have also been evaluated as serological biomarkers in DN (87), but that diagnostic study does not establish a pathogenic signaling mechanism.

Recent single-cell transcriptomic studies have provided a more detailed cellular map of S100 expression. In IRI-induced AKI, inflammatory macrophage subsets with high S100A9 expression have been proposed as potential therapeutic targets (88). In DKD, S100A8/A9-high macrophages are also enriched in inflammatory niches (89). These datasets are valuable for hypothesis generation, but transcript abundance does not necessarily establish protein activity or causal function. Validation at the protein level and with spatial and functional studies remains important.

Together, these studies implicate myeloid recruitment and tubular stress as distinct contributors to inflammation. The relevant receptor, cell population and timing vary; an intervention should therefore be assessed for both early injury and later recovery (5,6,10,88).

TIF is a common feature of progressive kidney disease and contributes to irreversible loss of renal function. It is characterized by excessive accumulation of ECM, produced primarily by activated myofibroblasts. S100-associated mechanisms are implicated in this fibrotic process, with S100A4 and S100A8/A9 playing prominent roles.

S100A4/FSP1 is associated with fibroblast activation in fibrotic kidneys, although its expression is not fibroblast-specific. Niclosamide treatment, together with genetic S100A4 perturbation, attenuates fibroblast activation, collagen deposition and ECM accumulation in experimental renal fibrosis (90). In vitro, S100A4 overexpression promotes TGF-β1-induced fibroblast activation, whereas S100A4 knockdown is protective (90). S100A2 and S100A8/A9 are associated with additional profibrotic mechanisms: S100A2 promotes inhibitory FoxO1 phosphorylation and EMT-like changes in HK-2 cells, S100A8/A9 promotes fibrosis in obstructive nephropathy, and S100A8/A9 inhibition ameliorates diabetic renal interstitial fibrosis by suppressing TLR4/NF-κB signaling (11,15,91). S100A4 interacts with Smad3, stabilizes the Smad3/Smad4 complex and promotes its nuclear translocation, although niclosamide is not a selective mechanistic probe (90). S100A2-associated FoxO1 inhibition does not establish a S100A2-RAGE pathway.

The cellular origin of scar-producing myofibroblasts remains a matter of debate. S100A4/FSP1 expression in tubular epithelial cells or podocytes has often been interpreted as evidence for EMT in DN and human immunodeficiency virus-associated podocyte injury (24,92,93). However, lineage-tracing studies have shown that resident interstitial cells, particularly pericytes, rather than epithelial cells, are the dominant source of myofibroblasts in several models of kidney fibrosis (27). Thus, S100A4 expression should be interpreted as a marker of injury, dedifferentiation and profibrotic signaling rather than proof that epithelial cells become matrix-producing myofibroblasts in vivo.

S100A4-Smad3/Smad4, S100A2-FoxO1 and S100A16-associated Wnt or ERS mechanisms contribute to fibroblast activation, epithelial stress and tissue remodeling (14,15,90,94). This conclusion synthesizes findings across studies and does not demonstrate direct interactions among all S100 proteins. The kidney studies discussed in the present review do not establish direct S100-specific coupling to Hippo/YAP/TAZ; this relationship remains a testable hypothesis.

Podocytes are essential for maintaining the glomerular filtration barrier, and injury to these cells can result in foot-process effacement, detachment, proteinuria and progressive glomerulosclerosis. RAGE signaling is implicated in glomerular injury, although AGE-containing ligand preparations do not isolate S100-specific effects (1,8). In diabetic settings, RAGE signaling intersects with oxidative stress, inflammatory cytokines and ECM remodeling, contributing to podocyte vulnerability.

An EMT-like podocyte phenotype links S100A4 expression to proteinuria-associated cellular stress. TGF-β1 can reduce podocyte junctional proteins such as nephrin and ZO-1 while inducing mesenchymal markers including desmin and S100A4/FSP1; these changes are associated with increased albumin permeability (24). In DN, podocyte FSP1 expression correlates with podocyte depletion and glomerulosclerosis (92). These findings support a stress-associated phenotype but neither prove complete lineage conversion nor establish extracellular S100A4-RAGE causality. PLA2R binding to the annexin A2-S100A10 complex in human podocytes is a separate molecular observation whose contribution to barrier injury remains unresolved (95).

These observations link S100A4 expression to podocyte stress but do not establish a causal extracellular ligand-receptor mechanism. Cell-specific receptor perturbation and ligand rescue would be needed to establish whether a specific S100 pathway directly contributes to proteinuria.

Cardiovascular disease is the leading cause of mortality in patients with CKD, driven in part by accelerated atherosclerosis and vascular calcification (VC). The uremic environment, characterized by inflammatory mediators and mineral dysregulation, favors vascular remodeling. Within this setting, S100/RAGE-related signaling has been implicated in uremia-associated VC, an active, cell-mediated process that involves the osteogenic transition of VSMCs.

Mechanistic evidence supports a causal contribution of S100/RAGE signaling to vascular calcification. In CKD models, transgenic S100A12 expression directly mediates vascular remodeling and arterial calcification through RAGE-dependent pathways (19). Humanized mice expressing the S100A8/A9/A12 cluster develop left ventricular hypertrophy and aortic valve sclerosis with calcification during CKD, and this phenotype requires RAGE (81). Separate studies implicate RAGE in phosphate-associated calcification and mechanistic target of rapamycin complex 1 (mTORC1)/ERS in the uremic vascular environment; these findings do not establish one linear S100 pathway (96,97).

Calprotectin has emerged as a candidate mediator and therapeutic target in CKD-associated vascular calcification (16). Mechanistic experiments showed that calprotectin exacerbates calcification in human VSMCs and mouse aortic rings, whereas paquinimod reduced calcification by dampening inflammatory signaling (16). These findings link calprotectin measurements to an experimentally modifiable vascular mechanism, but clinical trials are needed before calprotectin can be regarded as a validated therapeutic target in CKD vascular disease. Pharmacological RAGE and TLR4 inhibition reduced pro-calcifying effects in that experimental setting, supporting receptor involvement without demonstrating treatment benefit in patients (16).

The vascular evidence links inflammatory ligand exposure to mineral stress in vascular cells. Human outcome associations remain observational, whereas intervention experiments provide support that is specific to each model; reduced inflammation or calcification in a model is not equivalent to improved cardiovascular outcomes.

Specific S100 proteins are potential therapeutic targets, but this strategy remains at an early stage. The S100A8/A9 heterodimer has been studied most extensively because of its biomarker performance and mechanistic links to vascular calcification and inflammation. Paquinimod reduced calprotectin-driven vascular calcification in experimental systems, providing preclinical proof of principle (16). However, the absence of clear efficacy in immune-mediated glomerulonephritis models indicates that S100A8/A9 inhibition may be disease- and compartment-specific rather than broadly renoprotective (100).

Preclinical studies have also examined pharmacological modulation of S100A4-associated renal fibrosis. In a mouse model of folic acid-induced nephropathy, niclosamide, an anthelmintic investigated as an S100A4 inhibitor, attenuated fibroblast activation, collagen deposition and ECM protein expression. S100A4 facilitated TGF-β1-induced fibroblast activation in this model, and niclosamide blocked this effect, supporting a link between S100A4 and a central fibrotic pathway (90). The pleiotropic effects of niclosamide limit attribution to selective S100A4 inhibition, and extracellular S100A4 can protect tubular cells in cisplatin injury models (13).

RAGE or its downstream pathways provide additional therapeutic targets. Preclinical studies have examined natural compounds that modulate RAGE-related signaling. For example, KIOM-79, a herbal extract, prevented S100B-induced expression of TGF-β1 and fibronectin in mouse mesangial cells by suppressing the activation of downstream effectors, including NF-κB and the MAP kinases p38 and ERK (31). An ethanolic extract from Scrophularia striata also produced protective effects in a rat model of DN by suppressing the renal expression of both RAGE and its ligand S100A8 (101). These studies show that RAGE-related expression or signaling can be reduced experimentally. These extracts are indirect modulators; reduced expression or downstream signaling does not establish direct receptor antagonism. Studies of sepsis-associated autophagy decline (102), β-chain-deficient complement component 4 binding protein [C4BP(β-)] and Benincasa hispida (103,104) provide broader injury and treatment context; none establishes selective S100 inhibition.

Despite the promise of targeting S100-associated mechanisms, several challenges remain. One concern is inhibitor specificity. Numerous small molecules, including paquinimod and niclosamide, may have off-target effects that contribute to their observed efficacy or toxicity. The discrepancy in paquinimod's effectiveness against vascular calcification vs. glomerulonephritis illustrates the challenge of translating findings from animal models to human disease (16,100). Animal models often represent a single, acute pathological process, which may not fully recapitulate the multifactorial nature and chronicity of human kidney disease. Furthermore, S100-associated signaling involves multiple ligands and receptors, and this redundancy may allow other components to compensate when one is inhibited.

Elevated S100 concentrations may reflect systemic inflammation rather than a treatment-responsive kidney pathway. Biomarker associations therefore require independent evidence of target dependence before they can justify treatment selection.

A staged research strategy should begin with assay qualification and validation of repeated measurements against relevant clinical variables. No kidney-specific S100 threshold for selecting treatment is established. A prespecified upper quantile defined for the assay and study population could be an exploratory enrichment criterion, but it would require validation and continuous biomarker analysis. Candidate populations include CKD with documented vascular calcification and DKD with persistent albuminuria and inflammatory evidence despite stable standard-of-care therapy. Infection, mineral metabolism, eGFR and dialysis-related confounding should be assessed before enrollment.

Early randomized, placebo-controlled add-on studies should prioritize safety, pharmacokinetics in kidney impairment and target engagement. Prespecified high- and low-biomarker strata would allow treatment-by-biomarker interaction analysis; a study restricted to the high-biomarker group cannot establish differential treatment benefit. Adaptive enrichment would require prespecified rules, adequate sample size and control of false-positive results. Ligand inhibition, RAGE blockade, expression modulation and intracellular complex disruption should be selected according to the demonstrated mechanism, rather than treated as interchangeable interventions (Table II).

For receptor-directed agents, pharmacodynamic assessment should measure ligand-receptor engagement or receptor activity using a functional assay. For intracellular targets, pharmacodynamic assessment should measure pathway responses in the relevant tissue. Total circulating S100 concentration may not fall after ligand blockade. eGFR trajectory, albuminuria and standardized calcification imaging can provide exploratory outcomes, with clinical benefit tested in later trials with adequate power. Infection, hematologic effects and impaired injury recovery should be monitored. This roadmap is a research proposal, not a validated clinical algorithm.