Urinary acetylated protein as a biomarker of lupus nephritis: a prospective cohort study

Abstract

Objective

Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE). Kidney biopsy is the gold standard for diagnosing LN. Post-translational lysine acetylation modifications have emerged as potential biomarkers. This study explored using urinary lysine-acetylated peptides as novel biomarkers for diagnosing LN and assessing activity.

Methods

Urine samples were collected from patients with LN (n = 30), SLE without nephritis (n = 30), antineutrophil cytoplasmic antibody-associated glomerulonephritis (AAV-GN; n = 11), and healthy controls (n = 25). The experiment involved two steps: screening through liquid chromatography–mass spectrometry and quantifying five lysine-acetylated peptides through multiple reaction monitoring. Receiver operating characteristic analysis was used to assess the ability of these peptides to differentiate LN from other conditions. Additionally, their diagnostic value for histopathological activity was evaluated.

Results

Among the five acetylated peptides analyzed, three (ALB-K36, ALB-K161, ALB-K402) were significantly elevated in LN compared to SLE, AAV-GN, and healthy controls. Urinary ALB-K36 exhibited the highest diagnostic accuracy for LN versus AAV-GN (AUC = 0.947). These peptides remained elevated in LN patients with low proteinuria (UPCR < 500 mg/g), suggesting a potential role in early disease detection. Combining ALB-K36 with UPCR improved the diagnostic performance of histological activity index > 2 (AUC = 0.706).

Conclusion

Urinary lysine-acetylated peptides are promising novel biomarkers for LN diagnosis and histologic assessment, complementing traditional markers, such as UPCR, and highlighting their clinical potential; however, further validation in larger cohorts is required.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13075-025-03684-8.

Introduction

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by recurrent flares, leading to progressive deterioration of multiple organ systems, including the kidneys, skin, joints, lungs, bowel, and central nervous system [1]. Among these manifestations, lupus nephritis (LN) develops in approximately 40% of all SLE patients, with 10% progressing to end-stage renal disease (ESRD), which is associated with a poor prognosis [2]. Currently, the definitive diagnosis of lupus nephritis relies on a kidney biopsy [3]. This procedure allows for assessing glomerular damage, the degree of interstitial inflammation, and the extent of fibrosis and atrophy [4]. However, a kidney biopsy is contraindicated in patients with a solitary kidney, those with coagulopathy, or those who cannot discontinue anticoagulation therapy [5]. Meanwhile, a kidney biopsy poses bleeding risks and other complications even in eligible patients [6]. Consequently, there has been growing interest in identifying alternative diagnostic methods, particularly by studying biomarkers [7].

Urinary biomarkers, defined as specific molecular or protein levels detected in urine, have been investigated in inflammatory diseases and various conditions such as cancer and diabetes [8, 9]. Urine serves as an ideal diagnostic source due to its non-invasive collection process. Additionally, urinary proteins reflect substances filtered from the bloodstream or produced by kidney cells, making them applicable for diagnosing kidney-specific and systemic diseases [10]. Therefore, extensive research has focused on chemokines, cytokines, adhesion molecules, and other analytes that can be measurable in urine [7, 11, 12]. Although these analytes are potential biomarkers in lupus nephritis, their diagnostic accuracy remains insufficient for widespread clinical application.

Post-translational modifications (PTMs) of proteins, such as acetylation, methylation, and phosphorylation, alter the structural stability, enzymatic activity, and subcellular localization of proteins [13]. These modifications consequently influence critical biological processes, including gene transcription, signal transduction, and DNA damage repair. In a proteome-wide analysis of lysine acetylation sites in normal human urine, a study identified 629 lysine acetylation sites across 315 proteins [14]. These proteins were most commonly associated with organismal injury and abnormalities, endocrine system disorders, and cell-to-cell signaling and interaction disruptions. Despite these findings, studies on urinary lysine acetylation in kidney disease, including lupus nephritis, remain notably limited. Therefore, this study aimed to identify novel urinary biomarkers for diagnosing lupus nephritis by investigating changes in protein lysine acetylation.

Materials and methods

Experimental design and population

Urine samples were obtained from the registry cohort of a single tertiary center in Seoul, Republic of Korea. We prospectively collected urine samples from patients with newly diagnosed LN in this center. The patients included in this study underwent sampling between January 2019 and April 2023. SLE was diagnosed according to the 2012 Systemic Lupus International Collaborating Clinics Criteria [15] and 2019 European League Against Rheumatism/American College of Rheumatology (EULAR/ACR) Classification Criteria [16]. LN was diagnosed and classified according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification system [17]. A diagnosis of antineutrophil cytoplasmic antibody-associated vasculitis (AAV) was determined based on the criteria of the International Chapel Hill Consensus Conference on the Nomenclature of Systemic Vasculitides [18]. Glomerulonephritis in AAV (AAV-GN) was confirmed via kidney biopsy. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The Institutional Review Board of Asan Medical Center approved the study (IRB No. 2014 − 0568). Informed written consent was obtained from all participants.

Data collection

The following information was collected from electronic medical records: age, sex, laboratory data including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), creatinine, urine protein/creatinine ratio (UPCR), complement levels, anti-dsDNA levels, and histologic findings from kidney biopsy specimens.

Outcomes

The primary outcome included assessing the ability of each peptide to distinguish LN from SLE and AAV-GN. Additionally, as a secondary outcome, we evaluated the predictive performance of these peptides for histological activity indices in LN. A previous study has reported that a renal activity index exceeding 2 is associated with a significantly lower 10-year renal survival rate [ 19]. Based on this evidence, we analyzed the activity index using a cutoff of ≤ 2 versus >2.

Preparation of urine for multiple reaction monitoring

A total of 100 µL of urine was dried to concentrate the sample and subsequently lysed using a lysis buffer containing 10% sodium dodecyl sulfate (SDS) and 50 mM triethylammonium bicarbonate (pH 8.5). The lysate was then boiled at 80 °C for 10 min, followed by probe sonication at 20% amplitude for 5 min to facilitate protein extraction. The sample was centrifuged at 13,000 rpm for 30 min, and the resulting supernatant was collected. Protein concentration was determined using a bicinchoninic acid (BCA) assay. As previously described, 50 µg of protein was subjected to enzymatic digestion using the S-trap-based protocol with trypsin/LysC [20]. The resulting peptides were lyophilized using a SpeedVac and stored at −80 °C until liquid chromatography–mass spectrometry (LC–MS) analysis.

Multiple reaction monitoring for the acetylated urine peptides

Stable isotope-labeled internal standard (SIS) peptides for 5 acetylated peptides (albumin at K36, K161, and K402; serotransferrin at K588; and C11orf40 at K45) were synthesized using C13 and N15 heavy isotope-labeled lysine or arginine residue at C-terminal of each tryptic peptide with 98% purity (Synpeptide, China). A list of possible b- and y-series product ions was theoretically generated for those peptides using Skyline software (64 bits, version 19.1.0.193, University of Washington, MacCoss Lab) with the m/z range from 300 to 1400. Those theoretical transitions were optimized using default instrument parameters of the HPLC system (1290 Infinity, Agilent Technologies) and triple quadrupole mass spectrometry (MS) with a jet stream ESI source (6495 Agilent, Agilent Technologies). The MS source settings were set as follows: the gas temperature was 250 °C, the gas flow was 15 L/min, the nebulizer was 30 psi, the sheath gas temperature was 350 °C, the sheath gas flow was 10 L/min, the capillary was 3500 V for positive ion funneling, nozzle voltage was 300 V, iFunnel parameters for high-pressure RF was 90 V and low-pressure RF was 60 V. Mixture of light peptide and SIS peptide was injected directly into a reversed-phase analytical column (Zorbax Eclipse Plus C18 rapid resolution HD, 2.1 × 100 mm, 1.8-micron column, Agilent Technologies) with 40 °C oven temperature. The peptide sample was separated and eluted at 0.3 mL/min on a linear gradient of mobile phase B from 10% to 80% B in 15 min (mobile phase A: water/0.1% FA; mobile phase B: acetonitrile/0.1% FA); the gradient was ramped to 100% B for 2 min; 10% B for 5 min to equilibrate the column for the next run. This resulted in the top four or five most intensive transitions for each peptide selected for collision energy optimization and retention time determination, respectively. Targeted MS acquisitions were performed using detection windows of 3 min and a 1000 msec cycle time. Dynamic multiple reaction monitoring (MRM) was performed using 9 points of concentration samples ranging from 0 to 500 fmol with a blank matrix to determine the response curve. The limit of detection (LoD), which used 3 times the standard deviation plus the average peak area of three blank runs, and the limit of quantitation (LoQ), which used 10 times the standard deviation plus the average peak area of three blank runs were determined using the reverse response curve. In total, 5 µg of urine peptide spiked with 25 fmol of 7 SIS peptides were analyzed using the established MRM method. The peak area of each transition for the representative peptide was extracted using Skyline and normalized with the peak area of the spiked heavy peptide. Urinary lysine-acetylated peptide levels were measured in duplicate for each patient sample to ensure reproducibility.

Statistical rationale

Categorical variables are presented as frequencies and percentages (n, %), and continuous variables are expressed as the median and interquartile ranges (IQRs). Outliers exceeding six standard deviations were excluded from the analysis. One-way analysis of variance (ANOVA) was used to compare lysine-acetylated peptide levels between groups, followed by Tukey’s multiple comparisons test for post-hoc analysis. An independent t-test was used to compare proteinuria levels between the AAV-GN and LN groups. To evaluate the diagnostic performance of each lysine-acetylated peptide, receiver operating characteristic (ROC) analysis was performed. All statistical analyses and graph generation were performed using RStudio (version 4.3.3; Posit, PBC, Boston, MA, USA), SPSS (version 21.0; IBM, Armonk, NY, USA), or Prism (version 8.4.0; GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant.

Results

Selection of urinary PTM site

We analyzed samples from 11 healthy controls, 12 patients with SLE without nephritis, and 7 newly diagnosed LN patients for the initial screening (Supplementary Table 1). A total of 30 samples were subjected to LC–MS/MS analysis with two technical replicates per sample, resulting in the identification of 1685 proteins. Among these, 62 lysine-acetylated peptides were identified in 22 proteins. Of these peptides, 18 exhibited significant group differences as determined by one-way ANOVA (Table 1, Supplementary Fig. 1). The five most significantly upregulated lysine-acetylated peptides in LN patients, which were reliably measurable, were selected for further analysis.

Table 1.

The number of lysine-acetylated peptides and types of proteins identified in urine

Uniprot Accession ID	Protein names	Gene names	# of acetyl_K_peptides (significant peptides)
P02768	Albumin	ALB	35 (12)
P04264	Keratin, type II cytoskeletal 1	KRT1	4
P02787	Serotransferrin, Transferrin	TF	4 (1)
P02760	Protein AMBP	AMBP	1
Q8WZ69	Putative uncharacterized protein C11orf40, Ro/SSA1-related protein	C11orf40	1 (1)
Q5BKX8	Muscle-related coiled-coil protein	CAVIN4	1
P42357	Histidine ammonia-lyase	HAL	1 (1)
P01857	Immunoglobulin heavy constant gamma 1	IGHG1	1
P01834	Immunoglobulin kappa constant	IGKC	1
P01705	Immunoglobulin lambda variable 2–23	IGLV2-23	1
Q14626	Interleukin-11 receptor subunit alpha	IL11RA	1 (1)
P04259	Keratin, type II cytoskeletal 6B	KRT6B	1
Q08380	Galectin-3-binding protein	LGALS3BP	1
Q96L73	Histone-lysine N-methyltransferase	NSD1	1
P61970	Nuclear transport factor 2	NUTF2	1 (1)
P02763	Alpha-1-acid glycoprotein 1	ORM1	1
P35030	Trypsin-3, 3.4.21.4	PRSS3	1
P07998	Ribonuclease pancreatic	RNASE1	1
P06702	Protein S100-A9	S100A9	1
P01009	Alpha-1-antitrypsin	SERPINA1	1
P07911	Uromodulin	UMOD	1
Q9HAH1	Zinc finger protein 556	ZNF556	1 (1)

Baseline characteristics of patients

After identifying five acetylated peptides, these findings were validated in an expanded cohort. Urinary samples were collected from 25 healthy controls, 30 patients with LN, 30 with SLE, and 11 with AAV-GN. The baseline characteristics of the patients are summarized in Table 2. The median age of lupus nephritis patients was 41.0 years (IQR, 27.5–48.0), and 76.7% were female. The median UPCR at the time of kidney biopsy was 1315.5 mg/g in patients with LN, 127.2 mg/g in patients with SLE, and 913.4 mg/g in patients with AAV-GN, who served as disease controls for kidney involvement in inflammatory conditions. Proteinuria levels did not differ significantly between the AAV-GN and lupus nephritis groups (p = 0.425).

Table 2.

Baseline characteristics of healthy controls and patients with LN, SLE, and AAV-GN

	HC (n = 25)	LN (n = 30)	SLE (n = 30)	AAV-GN (n = 11)
Age	33.0 (25.5–39.0)	41.0 (27.5–48.0)	42.0 (34.8–57.3)	66.0 (62.0–77.0)
Sex, Female	22 (88.0)	23 (76.7)	24 (80.0)	4 (36.4)
ESR		29.5 (13.8–50.3)	17.5 (12.0–27.5)	81.0 (53.0–120.0)
CRP		0.25 (0.10–0.76)	0.17 (0.10–0.68)	7.06 (0.39–10.71)
Creatinine		0.73 (0.64–0.99)	0.68 (0.55–0.82)	1.46 (0.91–4.94)
UPCR		1315.5 (575.6–2585.6)	127.2 (70.4–216.2)	913.4 (573.9–2448.8)
C3		68.8 (45.8–89.4)	97.2 (68.2–119.4)
C4		11.9 (4.9–22.1)	21.0 (13.1–27.4)
Anti-ds DNA		69.0 (45.8–386.5)	6.8 (5.1–13.1)
LN class
Proliferative		18 (60.0)
Non-proliferative		12 (40.0)
Activity index		3.0 (1.0–6.0)
Chronicity index		3.0 (1.0–3.0)

Proliferative LN includes class III and IV disease according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification system; non-proliferative LN includes other classes

HC Healthy control, LN Lupus nephritis, SLE Systemic lupus erythematosus, AAV-GN Anti-neutrophil cytoplasmic antibody-associated vasculitis with glomerulonephritis, ESR Erythrocyte sedimentation rate, CRP C-reactive protein, UPCR Urine protein/creatinine ratio 

Elevated acetylation of urinary protein level in lupus nephritis

Among the five acetylated peptides, C11orf40-K45 did not show significant differences across any of the groups (Fig. 1). In healthy controls, TF1-K588 exhibited significantly lower acetylation levels than SLE and AAV patients. Three acetylated peptides were specifically elevated in patients with LN: ALB-K36, ALB-K161, and ALB-K402. The levels of these acetylated peptides were significantly higher in patients with LN compared to healthy controls, patients with SLE, and AAV-GN. Notably, ALB-K161 and ALB-K402 showed higher levels in patients with AAV-GN compared to healthy controls, though the levels were markedly higher in LN patients. Furthermore, no significant differences were observed when we compared the acetylated peptide levels between LN samples with UPCR values below 500 mg/g and those above 500 mg/g (Supplementary Fig. 2).

Fig. 1.

Abundance of lysine-acetylated peptides in urine samples from healthy controls and patients with LN, SLE, and AAV-GN. Urinary lysine-acetylated peptide levels were measured in duplicate for each patient sample. One-way analysis of variance (ANOVA) was used to compare peptide levels among groups, followed by Tukey’s multiple comparisons test for post-hoc analysis. LN: lupus nephritis; SLE: systemic lupus erythematosus; AAV-GN: anti-neutrophil cytoplasmic antibody-associated vasculitis with glomerulonephritis

ROC analysis of urinary acetylated peptide for distinguishing lupus nephritis from systemic lupus erythematosus

Subsequently, we evaluated these biomarkers for their potential to distinguish LN from SLE (Fig. 2). Four of the five acetylated peptides—excluding C11orf40-K45—demonstrated statistically significant differentiation between LN and SLE patients. Notably, ALB-K36, ALB-K161, and ALB-K402 exhibited AUC values exceeding 0.94, indicating strong discriminatory power. However, the AUC of the UPCR for distinguishing LN from SLE was also high at 0.916.

Fig. 2.

Receiver operating characteristic (ROC) curve of urinary acetylated peptides for distinguishing lupus nephritis from systemic lupus erythematosus

ROC analysis of urinary acetylated peptides for distinguishing lupus nephritis from AAV-GN

Therefore, to assess whether lysine-acetylated peptides could function as biomarkers beyond UPCR, we evaluated their diagnostic performance in distinguishing LN from AAV-GN, which served as a disease control characterized by inflammatory renal involvement and comparable UPCR levels (Fig. 3). Similar to previous findings, four of the five acetylated peptides—excluding C11orf40-K45—were significantly elevated in patients with LN compared to those with AAV-GN. Among these, ALB-K36 demonstrated the highest discriminatory power with an AUC of 0.946, comparable to its performance in differentiating LN from SLE. In contrast, the UPCR showed no significant diagnostic value for distinguishing LN from AAV-GN, with an AUC of 0.555.

Fig. 3.

Receiver operating characteristic (ROC) curve of urinary acetylated peptides for distinguishing lupus nephritis from AAV-GN. AAV-GN: anti-neutrophil cytoplasmic antibody-associated vasculitis with glomerulonephritis

ROC analysis of urinary acetylated peptide for distinguishing high activity and chronicity index from kidney biopsy in patients with lupus nephritis

After confirming the specific elevation of urinary lysine-acetylated peptides in LN, we further investigated whether these peptides could enhance the diagnostic performance for distinguishing between levels of histopathological activity within patients with LN. Previous studies have reported that an activity index (AI) > 2 is associated with worse renal survival and increased LN flares. Based on these findings, the UPCR alone demonstrated an AUC of 0.662 for discriminating AI > 2 (Fig. 4). However, the diagnostic performance improved when combined with a single acetylated peptide. Specifically, combining UPCR with ALB-K36 increased the AUC to 0.706 for discriminating AI > 2.

Fig. 4.

Receiver operating characteristic (ROC) curve of urinary acetylated peptide for distinguishing high activity (AI > 2) (A: UPCR, B: UPCR + ALB-K36) based on kidney biopsy findings in patients with lupus nephritis. AI: activity index, UPCR: urine protein/creatinine ratio

ROC analysis of urinary acetylated peptides to distinguish between proliferative and non-proliferative lupus nephritis based on kidney biopsy classification

Moreover, we subdivided LN patients into proliferative (n = 18) and non-proliferative (n = 12) forms and conducted an analysis. Expectedly, most of the non-proliferative LN cases (10 of 12) exhibited a low activity index (AI ≤ 2). The discriminative ability of the UPCR to differentiate between proliferative and non-proliferative LN was modest (AUC = 0.583); however, adding acetylated peptides slightly improved performance. The UPCR and ALB-K36 combination, as well as the UPCR and ALB-K402 combination, achieved AUCs of 0.625 and 0.627, respectively (Fig. 5), indicating that urinary acetylated peptides may enhance the diagnostic utility of the UPCR in distinguishing between proliferative and non-proliferative LN patients.

Fig. 5.

Receiver operating characteristic (ROC) curve of urinary acetylated peptide to distinguish between proliferative lupus nephritis and non-proliferative forms based on kidney biopsy classification (A, UPCR; B, UPCR + ALB-K36; C, UPCR + ALB-K402). UPCR, urine protein/creatinine ratio

Discussion

LN is a severe SLE manifestation involving immune-mediated inflammation of the kidneys. Thus, to improve the prognosis of LN, extensive research has been conducted on early detection strategies and identifying novel biomarkers that could complement or potentially replace conventional markers such as UPCR and kidney biopsy In this context, the present study provides novel evidence that acetylated peptides are specifically elevated in patients with LN. These peptides were uniquely increased in LN patients compared to SLE patients without nephritis and those with other inflammatory kidney diseases, such as AAV-GN. Furthermore, while these peptides alone have limited predictive power for assessing the activity index of LN, we observed that their combination with UPCR significantly enhances predictive accuracy.

Previous studies have examined acetylation in kidney diseases, with most focusing on histone modifications and intracellular signaling. Histone deacetylases have been implicated as either pathogenic or protective in acute kidney injury [21], and increased acetylation of p65 and STAT3 has been reported in diabetic kidney tissue [22]. In chronic kidney disease, elevated circulating acetylated amino acids were linked to disease, although urinary levels showed no association with kidney failure [23]. In SLE, prior work has largely centered on histone acetylation, showing increased acetylation in apoptotic microparticles during active LN [24], enhanced autoantibody reactivity to acetylated histone peptides in plasma [25], and transcriptional regulation by acetylation/deacetylation [26]. In contrast, our study highlights lysine acetylation of urinary proteins, particularly albumin, as a potential non-invasive biomarker for LN, with the additional ability to distinguish it from other inflammatory renal conditions. Although determining whether the same acetylated peptides are detectable in serum and how these peptides relate to systemic disease activity is of interest, this study was specifically designed to characterize urinary acetylation profiles associated with renal involvement. Thus, future comparative analyses between serum and urine proteomes should further clarify the systemic versus renal origins of these acetylated peptides.

This study used MRM methods to measure urinary peptide acetylation. This targeted mass spectrometry approach enables precise and reproducible quantification of post-translationally modified peptides across biological samples. Although the MRM assay may appear technically demanding, this assay is a well-established and standardized analytical platform that can be employed in large-scale studies. Meanwhile, internal reference standards and quality-control samples were included for every analytical batch in our workflow to ensure measurement consistency, with inter-run variability maintained within the accepted limits for quantitative proteomic assays. Furthermore, the overall process—from sample digestion to peptide quantification—can be adapted to multi-well plate formats compatible with automated liquid-handling systems, allowing high-throughput analysis of up to approximately 144 urine samples per instrument per day. Notably, similar MRM-based quantification methods are currently employed in Clinical Laboratory Improvement Amendments (CLIA)-certified clinical laboratories for peptide biomarkers such as thyroglobulin and amyloid-β [27, 28], thereby supporting the reproducibility, scalability, and potential clinical applicability of this technique.

A key question that arises from these findings is why lysine acetylation of albumin is increased in LN. Acetylation can occur in histone and non-histone proteins, with histone acetylation playing a role in gene transcription, whereas acetylation of pre-existing proteins is considered a PTM. Although research on PTMs, particularly lysine acetylation, in LN remains limited, lysine acetylation is reported to be directly associated with acetyl-CoA levels [13, 29]. Notably, acetyl-CoA synthetase 2 was upregulated in an acute kidney injury mouse model, where it regulates NLRP3 inflammasome activation, thereby contributing to pyroptosis and inflammation in renal tubular epithelial cells [30]. Furthermore, studies using lupus mouse models have reported that pyroptosis of kidney tubular epithelial cells plays a significant role in disease progression, and inhibition of this process leads to alleviation in disease activity [31]. In contrast, while pyroptosis has been extensively studied in various kidney diseases, including LN, its role in AAV-GN remains largely unreported [32]. Based on these findings, it can be hypothesized that acetyl-CoA synthetase contributes to kidney inflammation in LN, increasing acetyl-CoA levels and subsequently enhancing lysine acetylation. However, the increased albumin lysine acetylation in LN may not be solely a result of elevated acetyl-CoA levels but could also involve more direct involvement in disease pathogenesis. Hence, further studies are required to validate whether this acetylation is a mere consequence of increased acetyl-CoA or plays a more integral role in the underlying mechanisms of LN.

An interesting finding in this study is that LN patients with a UPCR below 500 mg/g exhibited higher acetylated peptide levels, similar to those observed in LN patients with a UPCR above 500 mg/g, than AAV-GN, SLE without nephritis, or healthy controls. This suggests that lysine acetylation of albumin may precede the rise in UPCR, highlighting its potential as an early marker for detecting LN. Previous studies have reported that when the 24-hour urine protein level is below 500 mg, its correlation with UPCR is inconsistent [33]. Additionally, LN was confirmed in 76% of patients with UPCR or 24-hour urine protein levels below 1000 mg [34]. Another study demonstrated that treating low-proteinuria LN patients significantly reduced the long-term progression to ESRD, suggesting that early detection and intervention are beneficial [35]. In this context, it can be anticipated that albumin lysine acetylation, independently or in conjunction with proteinuria, may contribute to the early diagnosis of LN, particularly in patients with low proteinuria below 500 mg daily.

Our study has several limitations. First, it was conducted with a relatively small sample size and limited to a single-center cohort of a single ethnic group. In particular, although it was an interesting observation that LN patients with low UPCR levels also exhibited elevated lysine-acetylated albumin peptide levels comparable to those with higher UPCR, only four LN patients in our cohort had a UPCR below 500 mg daily. This corresponded to a total of eight samples when measured in duplicate, underscoring the need for larger-scale validation in external cohorts. Second, serial urine samples from the same patients were unavailable, thereby preventing assessment of intra-individual variation or temporal changes in the acetylated peptide levels. Thus, longitudinal monitoring could be performed to clarify whether these peptides fluctuate with disease activity or treatment response. Finally, while we identified lysine acetylation at specific sites of certain proteins as a potential LN-specific marker, we could not elucidate its precise role in the pathogenesis of LN. Nevertheless, this study provides novel insights by investigating the clinical relevance of urinary PTMs in LN.

Our findings show that urinary lysine-acetylated peptides are significantly elevated in LN patients, with strong discriminatory power compared to healthy controls, SLE patients, and AAV-GN patients. These peptides may serve as disease-specific biomarkers, particularly when traditional markers, such as UPCR, lack specificity. Additionally, the ability of these peptides to enhance the predictive performance of UPCR for histopathological indices highlights their potential in assessing LN disease activity. These results underscore the value of urinary lysine-acetylated peptides as non-invasive biomarkers for diagnosing and monitoring LN.

Authors’ contributions

All authors contributed to the design and implementation of the research, to the analysis of the results, and to the writing of the manuscript, reviewed the results, and approved the final version.

Funding

This work was supported by the National Research Foundation of Korea [RS-2024-00348380, RS-2022-NR069963], and Asan Institute for Life Sciences, Asan Medical Center [2024IF0022]. This research was also supported by a grant from the MD–PhD/Medical Scientist Training Program through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea.

Data availability

The data that support the findings of this study are available on request from the corresponding author, YG Kim. However, due to ethical review board restrictions, these data are not publicly available, as information is contained that could compromise the privacy of the research participants.

Declarations

Ethics approval and consent to participate

This study was performed in accordance with the Declaration of Helsinki and its later amendments. The Institutional Review Board of Asan Medical Center approved this study (IRB No. 2014 − 0568).

Consent for publication

Informed written consent was obtained from all participants.

Competing interests

The authors declare no competing interests.