A noninvasive method of diagnosing membranous nephropathy using exosomes derived from urine
Article information
, Taewoon Kim2,*, Kwang Sik Kim2,*, Kyusoon Shin2, Jin-Ho Paik3,4, Jee Yoon Park5, Luke P. Lee,6,7,8,9,10,11,†, Jong Wook Hong,2,12,13,†, Sejoong Kim,1,14,15,†
Abstract
Background
Membranous nephropathy (MN) is a specific autoimmune disease affecting kidneys. It is characterized by the accumulation of immune complexes in the glomerular basement membrane. Renal biopsy is currently the standard procedure to confirm the diagnosis, although the presence of autoantibodies against the phospholipase A2 receptor (PLA2R) can also help diagnose. In this study, we aimed to investigate the potential of urinary exosomes as noninvasive markers for diagnosing MN.
Methods
Exosomes were extracted from urine samples of five patients with MN and four healthy controls. The concentration of PLA2R was measured in both urine and isolated exosomes using enzyme-linked immunosorbent assay techniques. The measurements were adjusted based on the urine creatinine (UCr) level of each participant.
Results
The levels of PLA2R/UCr were investigated in urine and urine-derived exosomes from patients and controls. Results of the analysis revealed significantly higher expression of PLA2R/UCr in patients compared to the control group (p < 0.05). Furthermore, the expression level of PLA2R/UCr was higher in urine-derived exosomes than in urine samples. Additionally, a positive correlation was observed between the expression levels of PLA2R/UCr and the urine protein-to-creatinine ratio, with urine-derived exosomes exhibiting a stronger correlation than urine samples.
Conclusion
Studies have indicated that measuring exosomal PLA2R/UCr levels in urine could be a noninvasive method for diagnosing MN. Using urine-derived exosomes could also reduce the burden of performing a biopsy on patients and facilitate follow-up treatment, such as monitoring for future recurrence.
Introduction
Membranous nephropathy (MN) is a pathological disorder characterized by specific features observed using light microscopy and immunofluorescence techniques, such as the presence of subepithelial immune complexes, diffuse thickening of the glomerular basement membrane, and deposits of immunoglobulin (Ig) G and complement [1–3]. Approximately 80% of cases are classified as primary MN (pMN), predominantly affecting the kidneys, while the remaining 20% are secondary MN associated with other systemic diseases or exposures [4].
pMN is a renal-specific autoimmune disease caused by circulating autoantibodies that specifically target antigens on glomerular podocytes, resulting in the deposition of immune complexes in the glomerular basement membrane. This process damages the glomerular filtration barrier, increasing urine protein levels [5,6]. pMN is the leading cause of nephrotic syndrome in adults, and approximately one-third of patients with pMN progress to end-stage renal disease [3,7]. Managing patients with MN is challenging due to the heterogeneous nature of the disease, with one-third of patients experiencing spontaneous remission, while a significant proportion (20%–30%) progress to chronic kidney disease, thus necessitating renal replacement therapy [8].
The best-known autoantigen associated with MN is phospholipase A2 receptor (PLA2R). Approximately 70% of patients with MN exhibit circulating anti-PLA2R antibodies, confirming its role as the primary autoantigen in the MN [9]. Although the presence of autoantibodies confirms the diagnosis of MN, testing serum PLA2R antibodies (PLA2R Ab) is known to be specific but not sensitive [10]. As is well known, PLA2R Ab levels are correlated with the timing of antibody measurements throughout the disease [11]. A newly identified scenario states that early in the course of the disease, PLA2R Ab is generated, attaches to the PLA2R antigen on podocytes, and is removed from the bloodstream more quickly than generated [11]. The kidneys function as an “immunological sink” in this hypothesis [12,13]. Serum levels eventually grow to detectable levels when the antigen is saturated. The “reservoir effect,” which states that the kidney must be saturated for the antibody to be detected in the blood, explains this [12]. Nevertheless, renal biopsy remains the standard procedure for most patients because it provides valuable information regarding prognosis and the identification of concurrent diseases [2,8]. Although renal biopsy is the gold standard for diagnosing and evaluating patient response to treatment, it is invasive, costly, and associated with several potentially serious complications [2,14].
Urine samples offer a more specific reflection of kidney injury than blood samples and directly indicate kidney alterations and damage [15]. The urine anti-PLA2R antibody titer strongly correlates with serum anti-PLA2R Ab level in patients with pMN [15]. Although the autoantibody PLA2R Ab plays an essential role in developing pMN [16], some studies have revealed a positive correlation between the strength of glomerular PLA2R and the urinary PLA2R Ab titer [15,17]. Moreover, several studies have demonstrated the presence of immune-intact soluble PLA2R in healthy plasma [18,19] and urine exosomes [20]. Recently, a study showed that urinary exosomal PLA2R antigen showed good consistency with the PLA2R antigen level in renal specimens [10].
Exosomes have emerged as promising candidates for biomarker discovery, offering diagnostic and pathophysiological insights without requiring invasive tissue biopsies [21]. Urinary extracellular vesicles (uEVs) are a promising material for liquid biopsies to assess histological injury patterns and severity in the kidney. The unique signature of uEVs presents an opportunity to complement existing diagnostic methods for kidney diseases using noninvasive approaches [22]. The correlation between protein levels in uEVs and kidney function makes them an attractive source of noninvasive biomarkers for studying kidney physiology and disease(s) [22]. The mechanism by which PLA2R is secreted is unknown, but one study has shown that the PLA2R/annexin2/S100A10 complex was discovered on the plasma membrane of podocytes and in secreted extracellular vesicles [23]. This complex is related to the actin skeleton’s organization, suggesting that PLA2R may play a crucial role in tight junction assembly and actin cytoskeleton reorganization processes known to be affected early in the proteinuric MN [24]. Furthermore, the ability of podocytes to release PLA2R-containing vesicles provides a pathway for the interaction of PLA2R with the immune system [23].
Our study presents a noninvasive approach for diagnosing MN by utilizing exosomes isolated from the urine samples obtained from both patients with MN and healthy controls (HCs). The characteristics of these exosomes were analyzed. The correlation between PLA2R levels and clinical parameters related to renal function, such as urine protein-to-creatinine ratio (UPCR), was used to explore the relationship between the clinical results. Furthermore, we investigated the possibility of diagnosing MN using urine exosomes as a source of biomarkers.
Methods
Patients and data collection
Our study utilized urine samples from five patients diagnosed with MN obtained from a glomerulonephritis cohort study at the Seoul National University Bundang Hospital (Institutional Review Board [IRB] No. B-1408-264-003). Urine samples (first-morning urine) from HCs were collected. The MN diagnosis was based on light microscopy, immunofluorescence staining, and electron microscopy. Additionally, exosomes were isolated and analyzed from four urine samples collected from HCs. Patients’ medical records were reviewed for demographic and clinical data, including laboratory findings and pathological reports. Blood and urine tests were performed immediately before the biopsy was collected. Blood chemistry data included serum creatinine (SCr), estimated glomerular filtration rate (eGFR), and albumin levels. The urinalysis data had urine creatinine (UCr) levels and UPCR. The eGFR was calculated using the creatinine equation developed by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) [25]. Renal pathology data included light microscopy findings, including the percentage of global and segmental sclerosis, severity of tubular atrophy, and interstitial fibrosis. For immunofluorescence, the extent of IgG and C3 staining was calculated as the intensity of the staining. Semiquantitative reports of the immunofluorescence staining results included negative, trace, and 1 to 4 positive results. Electron microscopy revealed electron-dense deposits in the subepithelial space. In patients with MN, the relationship between PLA2R and anti-PLA2R Abs was separated through the clinical results at the time of diagnosis, and exosomes were compared and analyzed.
This study was approved by the IRB of Seoul National University Bundang Hospital, Republic of Korea (IRB No. B-2301-807-301). Written informed consent was obtained from participants. The study complied with principles of the Declaration of Helsinki.
Glomerular staining of phospholipase A2 receptor
PLA2R staining was performed for all MN patients using the renal tissue samples stored in Seoul National University Bundang Hospital. The biopsies were stained for subsequent immunohistochemistry analysis of the paraffin sections using rabbit anti-PLA2R as the primary antibody (Sigma-Aldrich) and ultraView Universal DAB Detection kit (ROCHE/VENTANA) as the secondary antibody. The formalin-fixed paraffin-embedded blocks were immunostained on the Benchmark ULTRA (Ventana Medical Systems) using the ultraView Universal DAB Detection Kit (material No, 5269806001) under the condition of CC1 (antigen retrieval solution) for 64 minutes at 100 ℃, and antibody incubation for 40 minutes. Positive deposition of glomerular PLA2R was defined as a strong pattern of staining distributed along the glomerular capillary wall in a granular pattern [26]. We defined no reactivity as a score of 0, faint reactivity as indicated by a score of 1+, moderate reactivity as a score of 2+, and strong reactivity as denoted by a score of 3+ [27]. For PLA2R, scores of 0 or 1+ were regarded as negative due to the faint PLA2R immunoexpression.
Urinary exosome isolation
Urinary PLA2R, urinary exosomal PLA2R (urinary exosome [uEx] PLA2R), urine anti-PLA2R Ab, and urinary exosomal anti-PLA2R Ab levels were evaluated. For downstream analysis, exosomes were isolated from approximately 500-μL urine samples using a biologically intact exosome separation technology (BEST) system, as described previously [28]. First, the urine samples were thawed in a water bath at 37 °C for 3 minutes. After thawing, the sample and buffer were injected into the sample and buffer channels. The sample, buffer, and suction flow rates were set to 5:95:75. Exosome-sized particles were separated from the other particles and maintained at 4 °C during separation.
Nanoparticle tracking analysis
Nanoparticle tracking analysis (NTA) was utilized to characterize the size distribution and particle concentration of the isolated exosomes. Initially, exosomes were diluted in Dulbecco’s phosphate-buffered saline to a concentration that allowed the examination of 20 particles per frame. Subsequently, 500 μL of the diluted sample was injected into the sample chamber of a NanoSight LM10 instrument (Malvern Panalytical Ltd.) equipped with a 642-nm laser. The temperature of the sample chamber was maintained at 22 °C during the analysis. The Brownian motion of the particles was recorded using a camera, and NTA 3.1 software was used to analyze the particle movement and determine the size distribution and particle concentration. Each sample was analyzed three times for 30 seconds per analysis.
Western blot
Western blot (WB) analysis was performed to detect the PLA2R (MA5-24608; Invitrogen) and specific exosome markers, including TSG101 (NB200-112; Novus Biologicals) and CD63 (NBP2-32830; Novus Biologicals). First, the exosomes were lysed using radio-immunoprecipitation assay (RIPA) lysis buffer, and the protein concentration was measured using the bicinchoninic acid method. After normalization and electrophoretic separation, the proteins were transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature and incubated overnight at 4 °C with primary antibodies against PLA2R, TSG101, and CD63. The membrane was then washed thrice with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with the corresponding horseradish peroxidase-labeled secondary antibodies at room temperature for 2 hours. After washing thrice with TBST, an enhanced chemiluminescence solution and a ChemiDoc XRS (Bio-Rad Laboratories, Inc.) were used to detect and visualize the signals. Images were analyzed using ImageJ software (National Institutes of Health).
Enzyme-linked immunosorbent assay
The anti-PLA2R IgG and PLA2R enzyme-linked immunosorbent assay (ELISA) assays were conducted using the anti-PLA2R ELISA (IgG) (EA 1254-9601G; EUROIMMUN AG) and PLA2R ELISA kit (MBS3801010; MyBioSource, Inc.), respectively. Exosome samples were diluted 1:1 in RIPA lysis buffer for 20 minutes at 4 °C. Following the manufacturer’s instructions, microplates were incubated with urine and exosome samples. After each incubation and washing step, the optical density of the wells was measured at 450 nm using a Synergy HTX multimode reader (BioTek Instruments, Inc.).
Statistical analysis
Statistical analysis was performed using Microsoft Excel, including descriptive statistics and an independent two-tailed t test (α = 0.05). Data are presented as means ± standard deviation (SD) or median (interquartile range [IQR]), with four HCs and five patients with MN. Correlations between PLA2R/UCr and other parameters were analyzed using Pearson correlation coefficient and multivariate logistic regression analysis. Sensitivity, specificity, positive predictive value, negative predictive value, and the Youden index for urinary exosomal PLA2R/UCr were computed across various cutoff values using OriginPro (OriginLab Corporation). The optimal value was established using a receiver operating characteristic (ROC) curve. Significance was defined as p-value of <0.05.
Results
Characterization of urine-derived exosomes
As shown in Fig. 1A and Supplementary Fig. 1 (available online), exosomes were isolated from the urine samples of patients with MN and HCs using an advanced microfluidic platform called BEST [28], and a PLA2R ELISA test was performed to diagnose MN. First, the isolated uExs were characterized using NTA and WB techniques.
Characterization of urine-derived exosomes from patients with MN and HCs.
(A) Exosomes were isolated and collected from the urine of patients and controls using biologically intact exosome separation technology. (B) Mechanism of exosome formation with PLA2R membrane protein in urine. (C) The size distribution and particle concentration of exosomes were measured via nanoparticle tracking analysis. (D) Comparison of particle concentrations in urine and urinary exosomes (uEXs) from MN and HC groups. There was no significant difference in exosome particle concentration between MN and HC groups. (E) Western blotting of exosomal protein markers TSG101 and CD63. (F) Quantitative graph of TSG101 protein expression level. (G) Quantitative graph of CD63 protein expression level. All data are presented as means ± standard error and analyzed by paired t test and independent two-tailed t test.
GBM, glomerular basement membrane; HC, healthy controls; MN, patients with membranous nephropathy; NS, not significant; PLA2R, phospholipase A2 receptor.
*p < 0.05, **p < 0.01, ***p < 0.001.
The uEx size peaks from the HC and MN groups were 175 and 125 nm, respectively, which met the size definition of exosomes (approximately 30–200 nm), as shown in Fig. 1C. The particle concentrations of urine and uEx from HCs and patients with MN are demonstrated in Fig. 1D. This indicates that the exosomes were isolated by minimizing particle loss using this method. Next, WB analysis revealed that uExs from both HCs and patients with MN were positive for TSG101 and CD63 markers (Fig. 1E), confirming that the isolated particles were exosomes. The band intensities of these markers are shown in Fig. 1F and G and appeared darker in patients with MN than in HCs.
Correlation analysis of phospholipase A2 receptor/urine creatinine and clinical parameters
As shown in Fig. 2A, PLA2R was detected in urine samples and uExs from patients with MN and HCs using WB. All samples reacted with PLA2R; however, the expression level of PLA2R was higher in MN patients than in HCs (Fig. 2B). PLA2R was also detected using commercially available ELISA kits, and its expression level was calibrated using UCr, as shown in Fig. 2C. Significant differences were observed between the HC and MN groups.
Immunoblot analysis comparing the levels of PLA2R between MN and HC groups.
(A) Immunoblot analysis was conducted to compare the levels of PLA2R between MN and HC groups. (B) Quantitative graph for the amount of PLA2R expression, demonstrating higher expression in patients with MN than HCs. (C) Dot plot representing the expression level of PLA2R/UCr using enzyme-linked immunosorbent assay.
HC, healthy controls; MN, patients with membranous nephropathy; PLA2R, phospholipase A2 receptor; UCr, urine creatinine; uEx, urinary exosome.
As shown in Fig. 3, Correlations between PLA2R/UCr levels and various clinical parameters, including eGFR, SCr, UPCR, and glomerular PLA2R intensity were further investigated. The PLA2R/UCr ratio demonstrated a negative correlation with eGFR (urine: r = –0.235, p = 0.54; uEx: r = –0.307, p = 0.42), weak correlations were observed between PLA2R/UCr and SCr (urine: r = –0.102, p = 0.79; uEx: r = 0.003, p = 0.99). However, the obtained p-values, which were >0.05, suggested no statistically significant association between PLA2R/UCr levels and the eGFR and SCr variables. Subgroup analysis of the urine samples revealed a positive correlation between PLA2R/UCr and UPCR (r = 0.803, p = 0.009). Similarly, a statistically significant difference between PLA2R/UCr in uEx and UPCR (r = 0.908, p < 0.001) was observed. Overall, uEx demonstrated stronger correlations with clinical parameters than urine. While our analysis included a limited sample size of five patients, notable trends were observed. Except for one patient (MN 1), who showed no reactivity in glomerular PLA2R staining, the remaining patients (MN 2–MN 5) demonstrated positive glomerular PLA2R staining, ranging from +1 to +3 (Table 1).
Correlation analysis of PLA2R/UCr and clinical parameters.
Correlation analysis was performed to assess the relationship between PLA2R/UCr and (A) eGFR, (B) SCr, (C) UPCR, and (D) glomerular PLA2R intensity in urine and urine-derived exosomes. A stronger correlation was observed in urine-derived exosomes compared to urine. All data are presented as means± standard error and analyzed by paired t test and independent two-tailed t test.
eGFR, estimated glomerular filtration rate by Chronic Kidney Disease Epidemiology Collaboration equation; HC, healthy controls; MN, patients with membranous nephropathy; PLA2R, phospholipase A2 receptor; RU, relative units; SCr, serum creatinine; UCr, urine creatinine; UPCR, urine protein-to-creatinine ratio; uEx, urinary exosome.
Renal biopsy results and clinical characteristics of patients with MN
Association between phospholipase A2 receptor/urine creatinine level and clinical outcomes in patients with membranous nephropathy
Baseline characteristics of patients with MN and HCs are summarized in Table 2. The mean (± SD) age of patients with MN and HCs was 50 ± 20 and 31 ± 12 years, respectively. The median SCr levels were 0.8 mg/dL (IQR, 0.8–1.3 mg/dL) in patients with MN and 1.0 mg/dL (IQR, 0.9–1.0 mg/dL) in HCs, and eGFR values 80.7 mL/min/1.73 m2 (IQR, 54.0–103.5 mL/min/1.73 m2) in patients with MN and 110.6 mL/min/1.73 m2 (IQR, 105.6–113.4 mL/min/1.73 m2) in HCs. Patients with MN exhibited more severe proteinuria, with UPCR levels (4.39 g/g Cr [IQR, 1.30–5.63 g/g Cr] vs. 0.04 g/g Cr [IQR, 0.03–0.04 g/g Cr] in HCs, p = 0.05). Additionally, those with MN exhibited significantly lower serum albumin levels (2.8 g/dL [IQR, 1.8–2.9 g/dL]) vs. 4.7 g/dL [IQR, 4.5–4.9 g/dL] in HCs, p = 0.02). The MN group presented slightly higher urine and uEx anti-PLA2R IgG concentrations (11.6 relative units [RU]/mL [IQR, 11.6–11.7 RU/mL] vs. 11.5 RU/mL [IQR, 11.5–11.5 RU/mL] in HCs urine, p = 0.004; and 11.8 RU/mL [IQR, 11.7–12.1 RU/mL] vs. 11.6 RU/mL [IQR, 11.6–11.7 RU/mL] in HCs uEx, p = 0.15). PLA2R concentrations of MN presented as 12.1 RU/mL (IQR, 11.6–12.4 RU/mL) in urine and 28.8 RU/mL (IQR, 28.5–29.8 RU/mL) in uEx, which were similar to those of HCs, 12.1 RU/mL (IQR, 11.4–12.5 RU/mL) in urine and 30.3 RU/mL (IQR, 29.5–31.0 RU/mL) (p = 0.88 in urine and p = 0.11 in uEx). Calibrated anti-PLA2R IgG and PLA2R levels using UCr have changed their relationship between MN and HCs. Anti-PLA2R IgG/UCr level of MN was 2.7 folds higher than that of HCs, both in urine (15.7 RU/mg [IQR, 14.9–16.7 RU/mg] vs. 5.8 RU/mg [IQR, 4.6–6.9 RU/mg], p = 0.045) and in uEx (15.9 RU/mg [IQR, 15.5–17.4 RU/mg] vs. 5.9 RU/mg [IQR, 4.7–7.1 RU/mg], p = 0.04). PLA2R/UCr level with calibration was also higher in MN (15.6 RU/mg [IQR, 15.5–20.5 RU/mg] vs. 5.8 RU/mg [IQR, 4.3–7.5 RU/mg] in urine and 40.1 RU/mg [IQR, 36.3–43.1 RU/mg] vs. 15.1 RU/mg [IQR, 12.3–18.1 RU/mg] in uEx).
Baseline characteristics of patients with MN and HC
To investigate the association between PLA2R/UCr and clinical results, blood and urine test results, such as those for SCr, eGFR, albumin, and UPCR at the time of diagnosis, were reviewed. Table 1 summarizes patient data according to uEx levels. There was no significant difference in the concentration of anti-PLA2R IgG levels even after uEx separation. Notably, uEx PLA2R/UCr demonstrated an approximately 2.6-fold increase in levels compare with urinary PLA2R/UCr, while PLA2R Ab levels were similar in both urine and uEx. The increase in urine PLA2R observed when separated into uEx was similar in both the pMN and secondary MN groups. At the time of diagnosis, uEx PLA2R/UCr ratios were not associated with SCr or eGFR; however, a correlation was observed between UPCR and hypoalbuminemia.
Histopathological examination indicated the absence of a significant correlation between the percentage of global or segmental sclerosis and uEx PLA2R levels. All patients with MN consistently exhibited focal tubular atrophy and interstitial fibrosis, and virtually all showed positive IgG and C3 staining in immunofluorescence analysis. Furthermore, a tendency of higher intensity in IgG and C3 is observed as the uEx PLA2R/UCR ratio increases. Despite inter-individual variations in histological findings, abnormal glomerular and subepithelial electron-dense deposits were consistently observed in all patients.
The optimal cutoff value of urinary exosomal phospholipase A2 receptor/urine creatinine for diagnosing membranous nephropathy
Current clinical practice sets a typical positive threshold for anti-PLA2R in serum at 20 RU/mL [29]. We have introduced a novel diagnostic tool, uEx PLA2R/UCr, for diagnosing MN. We used the ROC curve analysis to validate the optimal cutoff value for uEx PLA2R/UCr [30]. As shown in Table 3, when the cutoff value was set at 28.4 RU/mg, we achieved a sensitivity of 80.0% and a specificity of 100.0%. The Youden index, which indicates the best overall performance, was 0.80. Furthermore, as depicted in Fig. 4, the area under the ROC curve was 0.95 (95% confidence interval, 0.805–1.095; p < 0.05).
The efficiency of urinary exosomal PLA2R/UCr for diagnosing MN at different cutoff values
Receiver operating characteristic curve of urinary exosomal PLA2R/UCr in diagnosing membranous nephropathy.
AUC, area under receiver operating characteristic curve; CI, confidence interval; PLA2R, phospholipase A2 receptor; UCr, urine creatinine.
Discussion
The conventional clinical diagnosis of MN involves invasive procedures, such as renal biopsy and serum anti-PLA2R IgG ELISA. To alleviate patient burden, our study aimed to establish a noninvasive approach for diagnosing MN using urine samples. Urine, being easily accessible, was explored for increased diagnostic sensitivity by isolating exosomes from urine. We proposed a novel MN diagnostic marker using urinary PLA2R ELISA instead of the conventional serum anti-PLA2R IgG ELISA. Recent research suggests that urinary exosomal PLA2R detection could be a sensitive method for diagnosing PLA2R-MN [10]. This approach offers potential advantages by avoiding invasive tissue biopsy and improving diagnosis accuracy through isolated exosomes.
In this study, we explored a noninvasive method for diagnosing MN using exosomes isolated from the urine samples of patients with MN and HCs. Characterization of urine-derived exosomes using NTA and WB confirmed their size distribution, particle concentration, and expression of specific exosomal markers (TSG101 and CD63). The isolated exosomes exhibited significant differences in PLA2R/UCr levels between MN patients and HCs, as demonstrated by ELISA. Particularly, the uEx PLA2R/UCr values, regardless of pMN or secondary MN, were consistently approximately 2.6 times higher than urine PLA2R/UCr, suggesting the potential use of uEx as a more sensitive diagnostic biomarker. This contradicts the current use of anti-PLA2R Ab for pMN diagnosis. Podocytes are known to express PLA2R, and a recent study generally hypothesized that podocyte injury would lead to an increase in the production of urinary exosomal PLA2R, as shown in Fig. 1B [10,31]. Therefore, urinary exosomal PLA2R can be considered a promising biomarker for assessing podocyte damage in primary and secondary MN.
Furthermore, we investigated the relationship between uEx PLA2R/UCr levels at the diagnosis and clinical parameters related to renal function, particularly UPCR and hypoalbuminemia. Results revealed varying levels of uEx PLA2R/UCr among patients with MN, which was correlated with the severity of proteinuria and hypoalbuminemia at the time of diagnosis. Interestingly, calibration of anti-PLA2R IgG and PLA2R levels using UCr significantly altered the relationship between patients with MN and HCs. After calibration, PLA2R/UCr level showed a significant increase in MN patients compared to HCs in both urine and uEx. Reflecting a more pronounced distinction between MN patients and healthy individuals, these calibrated levels underscore the enhanced diagnostic potential of anti-PLA2R IgG and PLA2R when considering urinary creatinine. These results demonstrate the potential of uEx-derived biomolecules, specifically exosome PLA2R/UCr levels, as noninvasive diagnostic markers for MN.
Histopathological examination revealed no significant correlation between global or segmental sclerosis percentage and uEx PLA2R levels. This suggests that uEx PLA2R levels may not directly reflect kidney damage or disease chronicity. Our data showed patients with MN exhibited positive IgG and C3 staining in immunofluorescence analysis. Furthermore, a tendency of higher intensity in IgG and C3 is observed as the uEx PLA2R/UCR ratio increases. This is consistent with previous studies that state that C3 deposits in pMNs are often present with pathogenic IgG deposits and are associated with increased proteinuria [32–34].
Our study aimed to assess the correlation between glomerular PLA2R staining intensity and PLA2R/UCr levels in uEx among patients with MN. We observed a notable increase in uEx PLA2R/UCr levels in all patients with MN, and MN 2 to MN 5 exhibited positive glomerular PLA2R staining, exceeding our suggested urinary exosomal PLA2R/UCr cutoff value of 28.4. This indicates their potential as diagnostic indicators for MN. However, our study did not find a significant correlation between uEx PLA2R/UCr levels and subtle gradations of glomerular PLA2R staining intensity (ranging from 1+ to 3+). This limitation may arise from the intricate influence of various biological and technical factors on urinary exosomal PLA2R expression, which might not directly reflect the nuances in staining intensity. Additionally, variability in staining outcomes, possibly due to antigenicity reduction in older biopsy specimens over time [35,36], could affect the correlation between glomerular PLA2R staining intensity and uEx PLA2R/UCr levels. This variability poses a critical limitation in interpreting our study’s findings. Nevertheless, the exceedance of PLA2R/UCr levels beyond the established cutoff value could be a significant indicator for MN diagnosis.
Despite numerous studies investigating anti-PLA2R antibodies, our research primarily focused on PLA2R antigens [11,37]. Analysis of exosome PLA2R/UCr levels benefits patient comfort and safety compared to invasive renal biopsies through noninvasive urine sample collection [38]. Additionally, it presents the potential for clinicians to identify patients at higher risk for disease progression and to potentially benefit from early intervention and/or more aggressive treatment strategies. A recently published study centrifuged 25 mL of urine for uEx isolation [10], whereas our study used approximately 500 μL of urine sample. Our study differs from previous studies by using much smaller amounts of urine samples than previous studies to isolate uEx, demonstrating its potential for diagnostic use. Determining an optimal cutoff value for uEx PLA2R/UCr is crucial for the diagnostic utility of our proposed noninvasive tool for MN. Conducting ROC curve analysis, we found that setting the cutoff value at 28.4 RU/mg achieved a notable sensitivity of 80.0% and an impressive specificity of 100.0%, with a Youden index of 0.80. Integration of this cutoff value into clinical practice has the potential to enhance the reliability of MN diagnosis, offering a more patient-friendly alternative to current invasive procedures. However, further validation studies must confirm its applicability across diverse patient populations.
It is important to acknowledge the limitations of this study. First, the sample size of patients with MN was relatively small, which may have limited the statistical power to detect significant differences in specific outcomes. Nevertheless, more investigation and confirmation are needed on this topic because HC and MN showed a noticeable difference in uEx levels. Second, the lack of follow-up uEx data has led to difficulties in evaluating clinical courses and responses to treatment in MN patients. Third, due to the unavailability of testing at the time of biopsy, the lack of serum anti-PLA2R Ab measurements in our MN patient cohort represents a gap that future studies should aim to fill. Lastly, we focused on PLA2R antigen, but MN is associated with various antigens beyond proven pathogenic endogenous antigens, such as PLA2R [39]. Multiple domains within PLA2R can serve as epitopes [39,40], necessitating further studies to explore their potential role in disease activity and pathogenicity, verifying the usefulness of uEx. Larger cohorts and longer-term follow-up studies are necessary to validate the diagnostic utility of uEx-derived biomarkers and evaluate their predictive value for disease progression and treatment response. Future studies should explore the potential of other biomolecules within uEx to provide additional insights into the pathophysiology and prognosis of MN.
In conclusion, our study demonstrates the potential of uEx-derived biomolecules, specifically exosome PLA2R/UCr levels, as promising markers for the noninvasive diagnostic of MN. The correlation between exosome PLA2R/UCr levels and clinical parameters related to renal function suggests their potential utility in assessing disease severity and prognosis. Further research and validation studies are required to establish these findings’ clinical significance comprehensively and advance the field of noninvasive diagnostics for MN.
Supplementary Materials
Supplementary data are available at Kidney Research and Clinical Practice online (https://doi.org/10.23876/j.krcp.23.208).
Notes
Conflicts of interest
All authors have no conflicts of interest to declare.
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (2022R1A2C1093134), and a grant (RS-2024-00331844) from Ministry of Food and Drug Safety in 2024.
Data sharing statement
The data presented in this study are available from the corresponding author upon reasonable request.
Authors’ contributions
Conceptualization: KSK, JWH, SK
Data curation, Software: GY, TK, KSK
Formal analysis, Validation: GY, TK, KSK, SK
Funding acquisition: JWH
Investigation: GY, TK, KSK, JHP
Methodology: GY, TK, KSK, JHP, JWH, SK
Project administration: JYP, JWH, SK
Resources: JHP, JWH, SK
Supervision: LPL, JWH, SK
Visualization: GY, TK
Writing–Original Draft: GY, TK
Writing–Review & Editing: All authors
All authors read and approved the final manuscript.
