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Learn more: PMC Disclaimer | PMC Copyright Notice Ren Fail . 2026 Apr 9;48(1):2653964. doi: 10.1080/0886022X.2026.2653964 Search in PMC Search in PubMed View in NLM Catalog Add to search Predictive value of CD44 in membranous nephropathy with focal segmental glomerular sclerosis lesions treated with calcineurin inhibitors Hong-Guang He Hong-Guang He a Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Writing – original draft, Writing – review & editing Find articles by Hong-Guang He a , Yi-Yun Huang Yi-Yun Huang a Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China Data curation, Resources Find articles by Yi-Yun Huang a , Kun Ye Kun Ye a Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China Conceptualization, Supervision, Validation Find articles by Kun Ye a , Qing-Mei Lu Qing-Mei Lu a Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China Data curation, Resources, Software Find articles by Qing-Mei Lu a , Qin-Qing Liang Qin-Qing Liang b Department of Pathology, The People’s Hospital of Guangxi Zhuang Autonomous Region Resources, Software, Visualization Find articles by Qin-Qing Liang b , Qiu-Rong Ye Qiu-Rong Ye b Department of Pathology, The People’s Hospital of Guangxi Zhuang Autonomous Region Resources, Visualization Find articles by Qiu-Rong Ye b , Qiu-Xia Wu Qiu-Xia Wu a Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China Conceptualization, Investigation, Methodology Find articles by Qiu-Xia Wu a , Yan-Wu You Yan-Wu You a Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Writing – review & editing Find articles by Yan-Wu You a, ✉ Author information Article notes Copyright and License information a Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China b Department of Pathology, The People’s Hospital of Guangxi Zhuang Autonomous Region ✉ CONTACT Yan-Wu You [email protected] Department of Nephrology, The People’s Hospital of Guangxi Zhuang Autonomous Region, 6 Taoyuan Road, Qingxiu District, Nanning 530000, China Roles Hong-Guang He : Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Writing – original draft, Writing – review & editing Yi-Yun Huang : Data curation, Resources Kun Ye : Conceptualization, Supervision, Validation Qing-Mei Lu : Data curation, Resources, Software Qin-Qing Liang : Resources, Software, Visualization Qiu-Rong Ye : Resources, Visualization Qiu-Xia Wu : Conceptualization, Investigation, Methodology Yan-Wu You : Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Validation, Writing – review & editing Received 2025 Jul 23; Accepted 2026 Feb 6; Collection date 2026. © 2026 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/ ), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent. PMC Copyright notice PMCID: PMC13072700 PMID: 41958046 Abstract Calcineurin inhibitors (CNIs) are effective immunosuppressive agents for idiopathic membranous nephropathy (iMN). Focal segmental glomerular sclerosis (FSGS) lesions, frequently observed in MN, have been identified as indicators of poor prognosis. This retrospective study enrolled 71 patients with biopsy-proven iMN accompanied by FSGS lesions, all of whom were initially treated with CNIs. This study identified independent risk factors for renal function progression and assessed the predictive significance of CD44. The primary outcome was defined as a 50% decline in the eGFR. Fifty-eight patients (81.7%) had CD44+ visceral epithelial cells (VECs), with 36 (36/71, 50.7%) demonstrating a linear positive pattern, of whom 19 patients showed a strong positive (3+) linear distribution in VECs. The patients with a strong linear distribution of CD44+ VECs had a higher rate of progression to the primary outcome (13/19 vs. 6/52, p < 0.001). The model based on age (OR, 1.134; p = 0.004), failure to achieve CR with CNIs treatment (non-CR) (OR, 6.924; p = 0.042), and strong linear CD44+ VECs (OR, 37.139; p < 0.001) showed good discrimination in predicting renal function decline, with a receiver operating characteristic AUC of 0.929 (0.871–0.986) ( p < 0.001). Therefore, patients with strong linear CD44+ VECs showed a lower remission rate and a higher rate of progression to the primary outcome. The model, which incorporated age, non-CR status, and strongly linear CD44+ VECs, demonstrated effective discrimination in predicting renal function deterioration. Keywords: Idiopathic membranous nephropathy, focal segmental glomerular sclerosis, calcineurin inhibitors, CD44, prognosis Introduction The prevalence of idiopathic membranous nephropathy (iMN) among glomerular diseases has significantly increased [ 1 , 2 ], often manifesting as nephrotic syndrome [ 3 ]. Calcineurin inhibitors (CNIs) have advantages in clinical applications because they help patients achieve remission faster and avoid the side effects of high-dose glucocorticoids and cyclophosphamide (CTX) toxicity [ 4 , 5 ]. Studies have indicated that patients with FSGS+ iMN may have an increased risk of renal function deterioration [ 6 , 7 ], especially those with non-tip FSGS [ 6 ] and those treated with CNIs [ 7 ]. Therefore, identifying effective predictive factors is essential to improve the prognosis of iMN patients with FSGS lesions treated with CNIs. Clinical remission is a crucial predictor of renal outcome [ 7 , 8 ], but its predictive value lacks timeliness for initial treatment. Therefore, identifying optimal baseline predictive indicators [ 9 , 10 ], or molecular markers of renal biopsies, may offer earlier and more precise insights into long-term outcomes. In MN, increased intraglomerular pressure [ 11 ], and complement activation triggered by epithelial immune complexes [ 12 ], contributes to podocyte injury. In response, podocyte precursor parietal epithelial cells (PECs) proliferate and are activated during repair. However, excessive activation of PECs may result in the development and progression of FSGS lesions [ 13 ]. CD44, a glycoprotein expressed by activated PECs and involved in extracellular matrix interactions, cell migration and adhesion [ 14 , 15 ], may facilitate the migration of PECs into the glomerular tuft to compensate for the detached podocytes [ 14 ]. A previous study demonstrated that CD44 may serve as a pathological marker of CNIs-induced nephrotoxicity [ 16 ]. This study focused on evaluating the outcomes of patients with FSGS+ iMN initially managed with CNIs. We identified the risk factors contributing to renal function decline, and the prognostic significance of CD44. Materials and methods Research objects Adult patients diagnosed with MN using renal biopsy between 2011 and 2022 at the Department of Nephrology, People’s Hospital of Guangxi Zhuang Autonomous Region, were enrolled in this study. The patients did not undergo immunosuppressive therapy before the renal biopsy and presented with nephrotic syndrome. The initial immunosuppressive treatment administered was CNIs-, and all enrolled patients had FSGS lesions. The exclusion criteria included drugs associated with MN, malignant tumors, autoimmune diseases, hepatitis virus infections, diabetes, follow-up durations < 6 months, baseline estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73 m 2 , coexisting with other glomerular diseases, or insufficient tissue for CD44 staining. All participants or their legal representatives provided written informed consent. This study was approved by the Ethics Committee of the People’s Hospital of Guangxi Zhuang Autonomous Region (approval numbers: KY-KJT-2023-213). Research methods All enrolled patients initially received treatment with CNIs, unless adverse events prevented treatment continuation. Baseline laboratory and clinical data were collected from all participants, including disease duration before renal biopsy, age, sex, hypertension, serum albumin, blood urea nitrogen (BUN), serum creatinine (Scr), triglyceride, cholesterol, and 24-h urinary protein levels. The eGFR was determined using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [ 17 ]. Pathological data included pathological stage, FSGS classification based on the Columbia FSGS classification system [ 18 ], percentage of global sclerosis and renal tubulointerstitial fibrosis areas, and arterial and arteriolar lesions. Tubulointerstitial fibrosis refers to the accumulation of extracellular matrix in the cortical region. Grades of arterial intimal thickening and arteriolar hyaline were evaluated using a scale ranging from 0 to 3 [ 19 ]. CD44 immunohistochemical staining Biopsies were stained for CD44 using 3 μm-thick sections of formalin-fixed paraffin-embedded tissues. The tissue sections were deparaffinized in xylene and dehydrated using an ethanol gradient. Antigen retrieval was performed using a microwave. Endogenous peroxidase activity was blocked using hydrogen peroxide (10 min). Anti-CD44 antibody(abcam156-3C11, 1:1000) was incubated overnight at 4 °C, followed by reaction enhancer treatment (37 °C, 20 min). Sections were then incubated with secondary antibody (Zhongshan Jinqiao universal detection system) (37 °C, 20 min). Freshly prepared diaminobenzidine (DAB) was used as a chromogenic substrate, followed by hematoxylin counterstaining (1 min). The subsequent steps included differentiation (2 s in acidic alcohol), bluing (5 min in alkaline solution), ethanol dehydration, air -drying, and coverslip mounting for microscopic analysis. All glomeruli were assessed for epithelial cell staining for CD44 over the glomerular tuft at the anatomical location of visceral epithelial cells (VECs), and parietal epithelial cells (PECs) along the inner surface of the Bowman’s capsule. A positive distribution can also occur simultaneously between PECs ( Figures 1a , and b) and VECs [ 16 ] ( Figures 1 b- f). Staining in endothelial cell locations, interstitium, or capillary lumen was excluded from the evaluation. Tonsil samples stained with CD44 were used as positive controls. Renal tissues from 21 patients with MCD (excluding those with FSGS and early FSGS) were used as negative controls. Figure 1. Open in a new tab Distribution characteristics of CD44 in iMN with focal segmental glomerulosclerosis (FSGS) lesions. a. CD44 positivity was evident in both parietal epithelial cells (solid arrow) and visceral epithelial cells (hollow arrow), without a CD44+ linear pattern in VECs distribution (original magnification× 400). b. Activated parietal epithelial cells were observed migrating toward the capillary loops (solid arrow); a multinucleated cell was visible at the 3 o’clock position, accompanied by a weak CD44+ linear pattern distributed along the capillary loops (hollow arrow) (original magnification× 400). c. Moderate CD44+ linear pattern distributed along the capillary loops (hollow arrow) (original magnification× 400). d. Strong CD44+ linear distribution was noted in visceral epithelial cells around the region of sclerosis; speckled positivity was also observed in segmental sclerosis areas (original magnification× 400). e. In non-sclerotic glomeruli, a strong linear positivity of visceral epithelial cells was observed (original magnification× 400). f. Activated parietal epithelial cells were noted to migrate toward the near capillary tuft, exhibiting a strong linear positivity along the loops (indicated by the solid arrow), while a weak speckled positivity was observed in the areas of segmental sclerosis (indicated by the hollow arrow) (original magnification× 400). Among the 21 negative controls (MCD), one patient showed CD44+ PECs and one patient showed CD44+ VECs. Of the 71 enrolled patients, PECs exhibited CD44 positivity in 27 patients, whereas 58 patients showed CD44+ expression in VECs. A linear CD44+ VECs pattern was observed in 36 cases, and the linear expression of CD44 was semi-quantitatively scored on a scale of 0 to 3+ based on the immunohistochemical staining intensity: 0 (negative; Figure 1a ), 1+ (weakly positive; Figure 1b ), 2+ (moderately positive; Figure 1c ), and 3+ (strongly positive; Figures 1d-f ), as previously reported[ 16 ]. It was observed that the strong positivity of linear CD44 showed greater continuity than weak and moderate positivity. Renal tissue specimens were examined by two pathologists blinded to the patients’ clinical conditions. Treatment plan A total of fifty-eight patients were administered cyclosporine A (CSA) at an initial dosage of (2.8 ± 0.6) mg/(kg·d). Additionally, thirteen patients received tacrolimus, with an initial dose of (0.055 ± 0.021) mg/(kg·d). Both calcineurin inhibitors (CNIs) were administered orally, twice daily at 12-h intervals. It was necessary to reduce the dosage of CNIs if the eGFR declined by more than 30% compared with baseline. The administration of CNIs was stopped if renal function showed no signs of improvement or further deterioration. After 6–12 months, a gradual reduction was considered. Sixty-six patients received glucocorticoids at an initial dosage of 0.34 (0.23, 0.45) mg/(kg·d) (calculated as prednisone) for one month, which was gradually tapered to 5–10 mg per day after six months. Definition and outcomes CR is characterized by a reduction in 24-h urine protein < 0.3 g, accompanied by stable renal function during the initial phase of immunosuppressive therapy. Partial remission (PR) in iMN is defined as a reduction of over 50% in 24-h urine protein levels to < 3.5 g, with stable renal function. The primary outcome was defined as either a ≥ 50% decline in eGFR from baseline or progression to end-stage renal disease (ESRD). ESRD is defined as an eGFR below 15 mL/(min·1.73 m 2 ) or a requirement for renal replacement therapy. Complete relapse is described as the recurrence of 24-h urine protein ≥ 3.5 g, along with serum albumin levels dropping below 30 g/L, following a period of remission. The follow-up endpoint date was December 31, 2024. Statistical analysis Continuous data were presented as mean ± standard deviation or medians with interquartile ranges, depending on whether the data were parametric or nonparametric. Differences were assessed using either the t-test or the Mann-Whitney U test. Categorical variables were analyzed using the χ 2 test or Fisher’s exact test. The reproducibility of CD44 staining features was assessed using the intraclass correlation coefficient (ICC). ICC values were interpreted as follows: < 0.40 indicated poor intraclass reliability; 0.40–0.59, moderate reliability; 0.60–0.79, substantial agreement; and ≥ 0.80, outstanding. Multivariate logistic regression analysis was performed to identify independent predictors of the primary outcome. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the area under the curve (AUC). Statistical significance was determined using a two-sided P -value of less than 0.05. Statistical analyses were conducted using SPSS software (version 26.0, SPSS Inc., Chicago, IL). Results Baseline characteristics A total of 71 patients were enrolled in the study. The median follow-up duration was 38 months (the first and third interquartile range: 18–58 months), ranging from 6 to 126 months, and the age at diagnosis was 52 ± 13 years. At the baseline, patients had an average serum albumin level of 20.7 ± 4 g/L, and the 24-h urinary protein excretion was 8.5 ± 4.4 g. There were 22 patients with tip lesions and 36 with NOS lesions. ( Table 1 ). Table 1. Comparison of clinicopathological features between patients with and without progression to the primary outcome. Total ( n = 71) Non-Primary outcome ( n = 52) Primary outcome ( n = 19) p value Male, n (%) 45 (63.4%) 32 (61.5%) 13 (68.4%) 0.782 HT n (%) 47 (66.2%) 32 (61.5%) 15 (78.9%) 0.258 Age(years) 52 ± 13 50 ± 12 59 ± 13 0.009 Duration before biopsy (months) 4 (1, 7) 4 (1, 8) 4 (1, 6) 0.984 S-ALB (g/L) 20.7 ± 4 21 ± 3.6 19.9 ± 4.9 0.313 U-pro (g/24h) 8.5 ± 4.4 8.8 ± 4.6 7.8 ± 3.7 0.423 CHOL (mmol/L) 9.7 ± 2.7 9.9 ± 2.8 8.9 ± 2.2 0.164 TG (mmol/L) 2.8 ± 2.1 2.8 ± 1.9 2.9 ± 2.5 0.948 BUN (mmol/L) 4.98 (3.83, 6.61) 4.52 (3.7, 5.60) 6.61 (4.55, 7.95) 0.004 Scr (μmol/L) 87 ± 25 83 ± 24 97 ± 25 0.036 eGFR (mL/min per 1.73 m 2 ) 83 ± 24 87 ± 24 72 ± 22 0.015 Initial treatment (CsA / TAC) 58/13 43/9 15/4 0.736 Initial dosage of GCs [mg/(kg·d)] 0.34(0.23, 0.45) 0.35(0.25, 0.46) 0.32(0.17, 0.43) 0.128 Stage (I/II/III/IV) 15/44/12/0 10/35/7/0 5/9/5/0 0.275 Positive PlA2R in Renal tissue (%) 57/71 (80.3%) 42/52 (80.8%) 15/19 (78.9%) 1 Glomerular sclerosis (%) 3.4 (0, 8.8) 3.2(0, 7.3) 4.5(0, 16.7) 0.534 FSGS (Tip/NOS/Peri/CELL/COL) 22/36/11/1/1 17/25/8/1/1 5/11/3/0/0 0.881 Mes (0/1/2/3) 23/38/10/0 18/29/5/0 5/9/5/0 0.189 TF area (%) 9.5 ± 11.8 8.1 ± 10.9 13.3 ± 13.4 0.101 Thickening A(0/1/2/3) 14/38/14/5 13/28/9/2 1/10/5/3 0.020 Hyalinosis A(0/1/2/3) 36/28/6/1 27/20/4/1 9/8/2/0 0.756 Complete relapse (%) 19 (26.8%) 11 (21.1%) 8 (42.1%) 0.239 Remission (CR/PR/NR) 27/25/19 25/18/9 2/7/10 0.003 Infections (%) 6 (8.5%) 3 (5.8%) 3 (15.8%) 0.332 SDM (%) 4 (5.6%) 3 (5.8%) 1 (5.3%) 1 Open in a new tab Abbreviations: Hypertension, HT; Serum albumin, S-ALB; Urine protein content, U-pro; Cholesterol, CHOL; Triacylglycerol, TG; Blood urea nitrogen, BUN; Serum creatinine, Scr; Glucocorticoids, GCs; Pathologic Stage, Stage; Tip variant FSGS, Tip; Not otherwise specified FSGS, NOS; Perihilar variant FSGS, Peri; Cellular variant FSGS, CELL; Collapsing variant FSGS, COL; Mesangial area expansion, Mes; Tubulointerstitial fibrosis area, TF area; Arteries intimal thickening, Thickening A; Hyalinosis of arterioles, Hyalinosis A; Complete remission, CR; Partial remission, PR; Non-remission, NR; Sencondary diabetes mellitus, SDM. Treatment outcomes Treatment progression of the 71 patients is shown in Figure 2 . Fifty-two patients achieved remission following the initial treatment, including 27 with CR and 25 with PR. Among the 25 patients who achieved PR, seven progressed to the primary outcome, and four developed ESRD (including three patients who discontinued follow-up for more than two years). Among the 19 patients who did not achieve remission, 10 progressed to the primary outcome, and subsequently three progressed to ESRD. Two patients who switched to CTX combined with glucocorticoid therapy did not achieve remission. Figure 2. Open in a new tab Prognostic outcomes of the enrolled patients with iMN. Among the 19 patients with recurrence, eight were retreated with CNIs. Of these, one patient achieved CR, whereas the remaining seven achieved PR. Among the eight patients who received rituximab (RTX) treatment during follow-up, one was lost to follow-up, two achieved CR, three achieved PR, and two exhibited improved and stable renal function without proteinuria remission. CD44 staining and renal outcome A linear CD44+ VECs pattern was observed in 36 cases, comprising 19 with strong positivity ( Figures 1d–f ), eight with moderate positivity ( Figure 1c ), and nine with weak positivity ( Figure 1b ). Among the 27 patients exhibiting a linear moderate to strong positive distribution of VECs, 14 (CR 4, PR 10) achieved remission, whereas 38 (CR 23, PR 15) out of 44 patients with weak or non-linear CD44 distributions achieved remission ( p = 0.002). Furthermore, 13 of 19 patients with a strong CD44+ linear VECs progressed to the primary outcome. In contrast, only six patients (6/52) of all other patients without strong positivity progressed to the primary outcome ( p < 0.001) ( Figure 3 ). We assessed the inter-rate correlation coefficient according to the linear positive intensities of CD44+ VECs and found that strong positivity had substantial agreement reproducibility (0.751, p < 0.001), whereas the intra-rate correlation coefficient was 0.804. Figure 3. Open in a new tab Analysis of initial treatment response and primary outcome based on CD44 staining. a. The remission cases according the intensity of linear CD44+ VECs. b. Patients with a strong CD44+ linear VECs had a higher rate of progression to the primary outcome. * p < 0.05; *** p < 0.001. Among patients with CD44+ PECs, 20 of 27 patients achieved remission after the initial treatment. In comparison, 32 out of 44 patients without CD44+ PECs achieved remission ( p = 1.0). Nine of 27 cases with CD44+ PECs progressed to the primary outcome, compared to 10 of 44 cases without CD44+ PECs ( p = 0.410). Comparison between the stable and progressive renal function groups Nineteen patients had progressed to the primary outcome. Compared with patients with stable renal function, those progressing to the primary outcome had a higher baseline age ( p = 0.009), BUN levels ( p = 0.004), Scr levels ( p = 0.036), and lower baseline eGFR ( p = 0.015). The CR rate during the initial treatment in patients progressing to the primary outcome (2/19) was lower compared to those without the primary outcome (25/52) ( p = 0.005) ( Table 1 ). Predictors of the renal outcomes The baseline predictors of the primary outcome identified through univariate analysis included: age (OR, 1.07; 95% CI, 1.013–1.129; p = 0.015), blood urea nitrogen (BUN) levels (OR, 1.618; 95% CI, 1.179–2.221; p = 0.003), Scr levels (OR, 1.023; 95% CI, 1.001–1.046; p = 0.043), eGFR (OR, 0.971; 95% CI, 0.947–0.995; p = 0.019), arterial intimal thickening (OR, 2.25; 95% CI, 1.132–4.472; p = 0.021), and the presence of a strong linear CD44+ VECs ( vs without CD44 linear; OR, 16.792; 95% CI, 4.054–69.558; p < 0.001). Failure to achieve CR during the initial treatment (non-CR) was also a significant predictor of progression to the primary outcome (OR, 7.87; 95% CI, 1.649–37.562; p = 0.01) ( Table 2 ). Table 2. Univariate logistic regression analysis of renal function progression to the primary outcome. p value OR (95% CI) Male, n (%) 0.595 1.354 (0.443–4.138) HT, n (%) 0.177 2.344 (0.681–8.07) Age (years) 0.015 1.07 (1.013–1.129) MN Duration(months) 0.237 1.03 (0.981–1.083) S-ALB (g/L) 0.310 0.933 (0.816–1.067) U-pro (g/24h) 0.419 0.947 (0.829–1.081) CHOL (mmol/L) 0.165 0.859 (0.694–1.065) TG (mmol/L) 0.947 1.009 (0.783–1.298) BUN (mmol/L) 0.003 1.618 (1.179–2.221) Scr (μmol/L) 0.043 1.023 (1.001–1.046) eGFR (mL/min per 1.73 m 2 ) 0.019 0.971 (0.947–0.995) Initial treatment (CsA vs TAC) 0.718 0.785 (0.210–2.927) Initial dosage of GCs [mg/(kg·d)] 0.073 0.037 (0.001–1.358) Stage I 1 (referent) II 0.316 0.514 (0.14–1.886) III 0.656 1.429 (0.297–6.877) Glomerular sclerosis (%) 0.165 1.051 (0.98–1.127) FSGS (NOS) 0.465 1.485 (0.514–4.29) Mes (0/1/2/3) 0.161 1.794 (0.792–4.062) TF area (%) 0.119 1.034 (0.991–1.079) Thickening A (0/1/2/3) 0.021 2.25 (1.132–4.472) Hyalinosis A (0/1/2/3) 0.851 1.074 (0.512–2.253) Non-CR 0.01 7.87 (1.649–37.562) PEC CD44+ 0.329 1.7 (0.585–4.939) VEC CD44+ 0.718 0.785 (0.21–2.927) Linear CD44 intensity 0 1 (referent) 1+ 0.979 0.969 (0.095–9.908) 2+ 0.932 1.107 (0.107–11.491) 3+ <0.001 16.792 (4.054–69.558) Open in a new tab Abbreviations: Hypertension, HT; Serum albumin, S-ALB; Urine protein content, U-pro; Cholesterol, CHOL; Triacylglycerol, TG; Blood urea nitrogen, BUN; Serum creatinine, Scr; Glucocorticoids, GCs; Pathologic Stage, Stage; Not otherwise specified FSGS, NOS; Mesangial area expansion, Mes; Tubulointerstitial fibrosis area, TF area; Arteries intimal thickening, Thickening A; Hyalinosis of arterioles, Hyalinosis A; Failure to achieve complete remission during the initial treatment, Non-CR; Parietal epithelial cells, PECs; Visceral epithelial cells, VECs; Odds ratio, OR; Confidence interval, CI. Multivariate analysis showed that age, non-CR and strong linear CD44+ VECs were independent predictors of the primary outcome ( Table 3 ). The model based these three variables achieved an AUC of 0.929 (0.871–0.986) ( p < 0.001). The AUC of the model was better than each of the three risk factors alone, including age ( p = 0.001, AUC difference 0.221 [0.089–0.352]), non-CR ( p < 0.001, AUC difference 0.241 [0.141–0.34]), and strong linear CD44+ VECs ( p = 0.004, AUC difference 0.144 [0.047–242]) ( Figure 4 ). Table 3. Multivariate logistic regression analysis of renal function progression to the primary outcome. Wald p value OR (95% CI) Age (years) 8.337 0.004 1.134 (1.041–1.235) Non-CR 4.133 0.042 6.924 (1.072–44.731) Strong linear CD44+ VECs 14.565 <0.001 37.139 (5.803–237.703) BUN (mmol/L) 0.074 Scr (μmol/L) 0.482 eGFR (mL/min per 1.73 m 2 ) 0.559 Thickening A 0.373 Open in a new tab Age, BUN, Scr, eGFR, strong linear CD44+ VECs, arteries intimal thickening, and non-CR were analyzed using stepwise method in Multivariate Logistic regression. Abbreviations: Failure to achieve complete remission during the initial treatment, Non-CR; Visceral epithelial cells, VECs; Blood urea nitrogen, BUN; Arterial intimal thickening, Thickening A; Odds ratio, OR; Confidence interval, CI. Figure 4. Open in a new tab The ROC curve for predicting the primary outcome. Discussion The natural prognosis of MN varies significantly [ 20 ], and patients with FSGS lesions are often associated with poor outcomes [ 21 ], particularly in patients undergoing treatment with CNIs [ 7 ]. However, for patients with FSGS+ iMN treated with CNIs, the protocol may help avoid the adverse effects associated with glucocorticoids and CTX while achieving remission earlier [ 5 ]. In the present study, patients in the worsening renal function group were older, had higher baseline BUN and Scr levels, and exhibited more significant arterial intimal thickening. Univariate analysis identified baseline age, BUN levels, serum creatinine levels, eGFR, and arterial intimal thickening as predictors of renal function decline. Additionally, CD44 linear strong positivity in glomerular VECs and failure to achieve CR during initial treatment were also predictors of renal function decline. Blood urea nitrogen levels can be influenced by various factors, including glomerular filtration rate, tubular reabsorption of urea, dietary protein intake, metabolic catabolism, and fluid volume status [ 22 ]. In the present study, elevated BUN levels, along with Scr and eGFR, could be used as predictors of renal function progression [ 23 ]; however, when combined with other factors, their predictive value weakened. Previous studies have identified age as a significant predictor of renal function progression in patients with iMN [ 24 , 25 ]. Older age is often accompanied by pronounced chronic pathological damage [ 19 , 25 ], a higher ratio of hypertension, and lower eGFR [ 25 ]. The limited predictive value of tubulointerstitial fibrosis in this study may be attributed to the relatively small sample size and mild severity of chronic tubulointerstitial lesions in the enrolled patients. Moreover, the risk of renal function deterioration attributable to CNI nephrotoxicity may attenuate the predictive value of tubulointerstitial fibrosis. In glomerular diseases, PECs play roles in podocyte regeneration and pathogenic activation. Activated PECs (aPECs), for which CD44 can serve as a marker, possess pathogenic characteristics such as excessive proliferation and migration, potentially leading to glomerulosclerosis and fibrosis [ 26 , 27 ]. CD44, which is involved in cell-cell and cell-matrix interactions [ 28 ], may facilitate the invasion of aPECs into the glomerular tuft, contributing to scarring in glomerular diseases [ 29 , 30 ]. CD44 can be used as a marker of PEC activation, and its expression in PECs and VECs can be used to distinguish early FSGS lesions [ 15 , 30 , 31 ]. The serum level of CD44 can be significantly elevated in animals treated with CSA [ 32 ]. CD44 may serve as a biomarker for the early identification of CNI-induced nephrotoxicity in kidney transplant recipients [ 33 ]. The present study revealed that in cases of FSGS+ iMN, CD44 expression was absent in VECs in 10 cases (19%), and no expression of PECs or VECs markers was observed in 8 cases (15%), consistent with previous research findings [ 15 ]. These findings may be attributed to the limitations inherent in the observational scope of different tissue sections, as well as the underlying mechanisms and stages of FSGS lesion development. CD44 has also been observed to exhibit a linear positive expression in VECs [ 30 , 34 ], positively distributed in PECs [ 30 ], and a speckled distribution in the segmental sclerosis areas. This suggests that CD44 positivity may occur at different stages of disease progression. The presence of strong (3+) linear CD44 immunostaining with marked continuity in the VECs suggests that these PEC-derived VECs lack an intricately organized filtration barrier structure composed of podocyte foot processes and slit diaphragm. This phenotypic alteration may indicate severe podocyte injury in the capillary loops concomitant with aPEC hyperactivation, invasion, and excessive extracellular matrix (ECM) deposition. Furthermore, calcineurin inhibitor (CNI) administration potentially enhances ECM proliferation [ 35 ] and upregulates CD44 expression [ 32 ], which forms a positive feedback loop to promote glomerulosclerotic progression [ 36 ]. Moreover, CNIs may exacerbate preexisting extracellular matrix proliferation, contributing to subsequent glomerulosclerosis. The expression of CD44 can be blocked by CD9 depletion, which may help improve renal function and proteinuria [ 37 ], and the inhibition of the MIF-CD74/CD44 signaling pathway by the ribosomal protein S19 can suppress PEC-mediated glomerular crescent formation [ 38 ]. Based on these studies, PEC-targeted therapy may emerge as a novel strategy for the treatment of FSGS or other glomerular diseases with FSGS lesions [ 39 ]. Notably, of the patients who achieved partial remission (PR) under initial CNI treatment, three were lost to follow-up and subsequently developed ESRD, highlighting the critical importance of monitoring treatment adherence in these patients. This may be related to chronic damage to the renal interstitium and blood vessels that occurred after the initial treatment. Persistent proteinuria and uncontrolled blood pressure continued to aggravate the preexisting lesions, leading to progressive deterioration of renal function [ 40 , 41 ]. In this study, we observed that patients unresponsive to CNIs or who experienced relapse achieved a favorable remission rate with stable renal function after switching to RTX. These results were consistent with previous studies [ 42 , 43 ]. Conversely, patients resistant to CNIs did not attain remission after switching to the CTX-combined glucocorticoid regimen, resulting in further deterioration of renal function. However, this finding differs from the conclusions of previous studies [ 44 ]. The findings of the present study suggest that RTX may serve as an alternative or first-line treatment for patients with FSGS+ iMN [ 3 ]. A predictive model incorporating age, non-CR, and strong linear CD44+ VECs demonstrated a better predictive value for renal function decline than individual variables alone. This study has several limitations. First, this was a retrospective analysis conducted at a single center, and the pathological definitions were determined solely by two researchers from a single center, which may have restricted the generalizability of the findings. Second, the study spanned a decade, leading to variability in drug-dosing regimens and subsequent immunosuppressive treatment strategies within the same cohort. Additionally, baseline serum levels of PLA2R antibodies were not available in the early cases of the study, limiting comprehensive analysis. Finally, the potential impact of patients lost to follow-up may have influenced the overall results, thus adding another layer of uncertainty. In summary, the presence of strong linear CD44+ in glomerular VECs is a significant predictor of renal function progression in patients with FSGS+ iMN undergoing CNIs treatment. When combined with age and non-CR, the model showed better discrimination for predicting renal function deterioration. Acknowledgements The authors express gratitude toward all patients involved in this study. Funding Statement This work was supported by the Natural science Foundation Program of Guangxi [2023GXNSFAA026112, 2023 GXNSFAA026005, 2023 GXNSFAA026059], and the Scientific Research Project of Guangxi health commission [S2020086, Z-A20230046]. Ethical approval This study was approved by the ethics committee of the People’s hospital of Guangxi Zhuang autonomous Region (No.KY-KJT-2023-213). 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