Corticosteroids augment cyclosporine nephrotoxicity in pediatric nephrotic syndrome: potential role of alternatively activated macrophages.

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Tomomi Kondoh, Yuji Matsumoto, Takuma Andoh, Masahiro Kaneko, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7737561/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jan, 2026 Read the published version in Pediatric Nephrology → Version 1 posted 5 You are reading this latest preprint version Abstract Background: Cyclosporine A (CsA) is an effective steroid-sparing agent for steroid-dependent nephrotic syndrome (SDNS); however, long-term administration of CsA can induce chronic kidney injury (CsA nephropathy). We previously reported that alternatively activated macrophages (M2-type macrophages) are closely linked to the pathogenesis of interstitial fibrosis in progressive kidney disease. In this study, we investigated the possible involvement of M2-type macrophages in CsA nephropathy in SDNS. Materials and Methods: A total of 33 children diagnosed with SDNS and who were treated with CsA for more than 2 years were investigated. Fourteen biopsy specimens from age-matched SDNS children who had not received CsA treatment were used as the control. Renal fibrosis was assessed by Masson chrome staining of biopsy sections. Sections were also stained for α-smooth muscle actin (α-SMA), type I collagen, CD68 (total macrophages), CD163 (M2 marker) and CCL2. Urine levels of CCL2 were measured by cytometric bead array kit. Results: The CsA-treated group showed significant interstitial fibrosis (12.2±7.3 vs 7.6±2.1%, p<0.001) with accumulation of interstitial CD163 + CD68 + macrophages (10.8 vs 7.9/HPF; p<0.001) compared with SDNS control patients. There was a significant correlation between the degree of interstitial fibrosis and the number of interstitial CD163 + cells (p<0.001), and between interstitial fibrosis and the cumulative steroid dose used during CsA treatment (p<0.001), while no correlation was found between the cumulative steroid dose used before CsA treatment and histological changes. Immunostaining revealed significant expression of CCN2 and CCL2 in biopsies from the CsA group which co-localized with CD163 + macrophages. In addition, the urinary CCL2/creatinine ratio was significantly elevated in the CsA group compared to controls, both at relapse (1012±641.2 vs. 239.9±226.9 pg/mg; p = 0.02) and at remission (202.0±178.4 vs. 77.7±127.3 pg/mg; p = 0.04). Conclusion: Our findings suggest that CD163 + M2-type macrophages may participate in the development of interstitial fibrosis induced by CsA. Steroid treatment during CsA treatment appears to augment CsA nephrotoxicity through the production of pro-fibrotic factors. Cyclosporine Fibrosis Nephrotic syndrome M2-type macrophage CCL2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Nephrotic syndrome (NS) is one of the most common kidney diseases in children, characterized by heavy proteinuria, hypoalbuminemia, and edema. Glucocorticoid steroids, particularly prednisolone (PSL), are the first-line treatment for pediatric idiopathic NS, inducing remission in approximately 90% of cases. However, 80% of these patients experience relapse, with 30–40% developing frequently relapsing NS (FRNS) or steroid-dependent NS (SDNS) [ 1 , 2 ]. In patients with FRNS, increased PSL use leads to significant steroid-related adverse effects. To reduce or discontinue PSL, immunosuppressive agents are recommended for relapse prevention. Cyclosporine A (CsA) is one of the most commonly used and effective immunosuppressive agents for steroid-sparing treatment in children with FRNS or SDNS. However, many patients relapse after CsA discontinuation, reverting to FRNS or SDNS [ 3 ]. Consequently, long-term CsA administration often becomes necessary, increasing the risk of tubulointerstitial damage, including microvascular injury and fibrosis, collectively referred to as CsA nephropathy. Nevertheless, the factors contributing to the development of tubulointerstitial lesions remain unclear [ 4 ]. Macrophage accumulation in the kidneys is a universal phenomenon observed in all progressive renal diseases, regardless of whether they are inflammatory or non-inflammatory [ 5 ]. We previously reported the involvement of alternatively activated (M2 phenotype) macrophages in the pathogenesis of chronic allograft kidney injury, which develops under long-term concomitant use of steroids and calcineurin inhibitors such as CsA and tacrolimus [ 6 ]. This suggests a crucial role for M2-type macrophages in the progression of graft fibrosis. It is further hypothesized that a similar pathogenic mechanism may be involved in CsA nephropathy, which arises under analogous conditions of steroid and calcineurin inhibitor administration. This study aimed to investigate the role of M2-type macrophages in CsA nephropathy, with a particular focus on tubulointerstitial fibrosis in patients with FRNS or SDNS, in order to better understand the mechanisms underlying the associated histological damage. Materials and Methods Patients Among 131 children diagnosed with idiopathic NS between 1998 and 2020 at the Department of Pediatrics, Niigata University Medical and Dental Hospital, or the Department of Pediatrics, Fujita Health University Hospital, who underwent renal biopsy, 33 children with FRNS or SDNS and biopsy-confirmed minimal change disease (MCD) and who received CsA for at least 2 years were retrospectively analyzed. Additionally, 14 age-matched children with FRNS/SDNS who underwent renal biopsy prior to CsA initiation served as controls. Children with pathological findings other than MCD, including focal segmental glomerulosclerosis (FSGS), membranous nephropathy, or proliferative glomerulonephritis, as well as those who developed steroid-resistant NS (SRNS) during the course of treatment, were excluded. All patients included in this study were classified as steroid-sensitive NS (SSNS), defined as achieving remission with standard steroid therapy within 4 weeks [ 2 ]. FRNS was defined as ≥ 2 relapses within 6 months or ≥ 4 relapses within 12 months among SSNS patients. SDNS was defined as two consecutive relapses during steroid tapering or within 2 weeks after cessation of therapy. SRNS was defined as failure to achieve remission within 4 weeks of standard treatment. Treatment protocol Treatment protocol Initial treatment for idiopathic NS consisted of daily PSL at 60 mg/m²/day or 2 mg/kg/day (maximum 60 mg/day) for 4 weeks, followed by alternate-day PSL at 40 mg/m² or 1.5 mg/kg/day for another 4 weeks. Upon relapse, patients were treated with a single daily dose of PSL (2 mg/kg/day or 60 mg/m²/day, maximum 60 mg) until complete remission, defined as three consecutive days of negative dipstick results. This was followed by alternate-day PSL (1.5 mg/kg or 40 mg/m² per dose, maximum 40 mg), with gradual tapering over the next 2–3 months. For relapses in FRNS/SDNS patients, PSL was tapered daily after achieving complete remission. CsA was administered at an initial dose of 2.5–5 mg/kg/day in two divided doses, with subsequent dose adjustments based on blood drug concentration monitoring: trough levels of 60–100 ng/mL or C2 levels of 500–700 ng/mL. Renal biopsy indications in children with NS included: (1) age < 1 year, (2) persistent or gross hematuria, (3) hypertension or renal dysfunction, (4) hypocomplementemia, and (5) extrarenal symptoms (e.g., purpura), as these patients are more likely to have histological types other than MCD. Additional indications included: (6) steroid resistance or FRNS/SDNS prior to immunosuppressant initiation, and (7) long-term (> 2 years) CsA therapy, even in the absence of renal dysfunction, to assess nephrotoxicity. Informed consent was obtained from all patients for the use of residual biopsy tissue for research purposes following completion of diagnostic evaluation. Clinical and pathologic data Urinary protein concentration (Up), serum creatinine (sCr), and urine creatinine (Ucr) levels were measured for each patient at the time of renal biopsy. The estimated glomerular filtration rate (eGFR) was calculated using an equation specific to Japanese children [ 7 ]. Medical records were retrospectively reviewed to determine the cumulative steroid dose from the onset of NS to the time of biopsy. In the CsA group, the steroid dose was assessed both before and after CsA initiation, expressed as PSL dose per body weight. Additionally, the number of relapses occurring before and after CsA initiation was extracted from the medical records. Kidney biopsy specimens were fixed in Carnoy’s solution, embedded in paraffin, and sectioned for staining with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), periodic acid-methenamine silver (PAM), or Masson’s trichrome (MT). An independent pathologist, blinded to clinical data and macrophage staining results, reviewed the stained sections. The degree of interstitial fibrosis was quantified by image analysis of MT-stained sections, as previously described [ 8 , 9 ]. The number of interstitial macrophages was assessed using immunohistochemical staining, as detailed below. The extent of fibrosis and macrophage infiltration in the tubulointerstitial region was evaluated in relation to the clinical parameters described above. Antibodies Primary antibodies used in this study included, 10D6: anti-human CD163 (mouse IgG1; Visionbiosystems, Benton Lane, UK), PG-M1: anti-CD68, labeling human monocytes and macrophages (mouse IgG3; DAKO, Santa Clara, CA, USA), 1A4: anti-human smooth muscle actin (α-SMA/Acta2; mouse IgG2a; DAKO), Anti-collagen type I (rabbit polyclonal; Novus Biologicals, Littleton, CO, USA), 2154-60: anti-human cellular communication network factor 2 (CCN2, formerly known as connective tissue growth factor or CTGF; mouse IgM; Acris Antibodies, Hiddenhausen, Germany), Anti-C–C motif chemokine ligand 2 (CCL2 also known as monocyte chemoattractant protein-1 or MCP-1; rabbit polyclonal; Bioss Antibodies, Woburn, MA, USA). Secondary antibodies included, FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL, USA), TRITC-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates), FITC-conjugated goat anti-rabbit IgG (MP Biomedical, Santa Ana, CA, USA). Immunohistochemistry Macrophages, α-SMA, and type I collagen were detected in 2 µm tissue sections fixed with Carnoy’s solution, using the VECTASTAIN Elite ABC HRP Kit (Vector Laboratories, Burlingame, CA, USA). After dewaxing, antigen retrieval was performed by microwave heating for 10 minutes in 0.1 M sodium citrate buffer—pH 6.0 for general targets and pH 9.0 specifically for α-SMA—using Target Retrieval Solution (DAKO). Following PBS washes, sections were blocked for 30 minutes at room temperature with a solution containing 10% normal goat serum and 1% bovine serum albumin (BSA) in PBS. Primary antibodies were diluted in PBS supplemented with 10% normal human serum and 1% BSA, and incubated overnight at 4°C. After rinsing with PBS, endogenous peroxidase activity was suppressed using 0.3% hydrogen peroxide in methanol for 20 minutes. Sections were then incubated sequentially with biotinylated secondary antibodies and the VECTASTAIN Elite ABC reagent, each for 30 minutes at room temperature. Chromogenic development was carried out using diaminobenzidine (DAKO) for brown staining of macrophages, TrueBlue Substrate (KPL, Gaithersburg, MD, USA) for blue staining of α-SMA, and Vina Green Chromogen Kit (Biocare Medical, Pacheco, CA, USA) for green staining of type I collagen. Quantification of CD68- or CD163-positive cells was performed in at least 12 glomeruli per patient. For interstitial regions, positive cells were counted in a minimum of five consecutive high-power fields (×400 magnification). For dual-color immunostaining, CD163 was first visualized using microwave-based antigen retrieval to minimize cross-reactivity, followed by staining with either α-SMA or type I collagen antibodies, each developed with distinct chromogenic substrates. Urinary CCL2 measurement To investigate the mechanism of macrophage infiltration in the kidney, urinary levels of CCL2/MCP-1, a key macrophage chemoattractant, were measured using a cytometric bead array kit (BD Biosciences, San Jose, CA, USA). Assays were performed according to the manufacturer’s instructions. Statistical analyses Comparisons were made between two groups by Mann–Whitney test (GraphPad 8.0, San Diego, CA) or Fisher’s exact test. Data are shown as the mean ± 1 SD. Correlation analysis used the Spearman correlation coefficient. We declared a finding to be statistically significant if the p-value was less than 0.05. Results Comparison of patient profiles between the CsA-treated and control groups Patient profiles at the time of biopsy are summarized in Table 1 . There were no significant differences between the two groups in clinical parameters, including gender, age at initial onset, age at biopsy, duration from NS onset to biopsy, degree of proteinuria, kidney function (eGFR), cumulative prednisolone (PSL) dose from onset to biopsy, or the number of relapses prior to biopsy. In the CsA-treated group, 8 out of 33 patients (24.2%) were pathologically diagnosed with CsA nephropathy, whereas no patients in the control group had this diagnosis. However, the difference between the groups was not statistically significant (p = 0.084). Table 1 Comparison of clinical parameters at biopsy. CsA-treated group CsA non-treated group p-value Number of patients 33 14 ― Gender (M:F) 25 : 8 11 : 3 > 0.99 Age at onset (years) 5.9 ± 3.6 7.1 ± 4.6 0.62 Age at biopsy (years) 11.3 ± 3.9 11.2 ± 4.4 0.94 Time from onset to biopsy (years) 5.4 ± 3.3 4.2 ± 2.6 0.29 Urinary protein/Cr ratio (g/gCr) 2.2 ± 8.9 0.7 ± 1.8 0.45 eGFR (ml/min/1.73m 2 ) 118.8 ± 23.4 125.1 ± 20.7 0.47 Cumulative PSL dose up to biopsy (mg/kg) 743.7 ± 423.0 659.1 ± 299.2 0.94 Number of relapses from onset to biopsy 10.7 ± 6.9 9.5 ± 6.1 0.22 Number of children with CsA nephropathy 8 (24.2%) 0 0.084 Degree of interstitial fibrosis (Masson positive area %) 12.2 ± 7.3 7.6 ± 2.1 0.0012 Number interstitial CD68 + cells (/HPF) 13.2 ± 6.2 9.3 ± 3.2 0.0048 Number interstitial CD163 + cells (/HPF) 13.4 ± 6.9 8.5 ± 2.9 < 0.0001 CsA: cyclosporine, eGFR: estimated glomerular filtration rate, PSL: prednisolone, HPF: high power field Data is shown as mean ± 1 SD. Renal Histology No changes were observed in glomerular morphology in either group, with all cases classified as minimal change disease (MCD) ( Fig. 1 a-b ) . However, interstitial fibrosis, quantified by the area of Masson’s trichrome staining, was significantly greater in the CsA group compared to the control group (Fig. 1 c-d, Table 1 ). In addition, the accumulation of CD163 + cells in areas of interstitial fibrosis shown by the presence of interstitial a-SMA + cells and increased interstitial collagen I deposition, was evident in the CsA-treated group ( Fig. 1 e-h ) . Indeed, pathological findings revealed significantly greater interstitial fibrosis, along with higher numbers of interstitial CD68⁺ and CD163⁺ cells, in the CsA-treated group ( Table 1 Fig. 1 i-k ) . Table 2 summarizes the correlations between interstitial fibrosis based on Masson trichrome staining and clinical parameters in the CsA-treated group. Significant correlations were found between the extent of interstitial fibrosis and the cumulative PSL dose during CsA treatment (r = 0.63, p < 0.0001), duration of CsA administration (r = 0.48, p < 0.005), cumulative PSL dose from disease onset (r = 0.40, p < 0.05), and number of relapses during CsA treatment (r = 0.41, p < 0.05). Interstitial fibrosis also significantly correlated with the degree of CD163⁺ cell infiltration (r = 0.56, p < 0.001) and CD68⁺ cell infiltration (r = 0.50, p < 0.005). In contrast, cumulative PSL dose (r = − 0.11, p = ns) and number of relapses (r = − 0.10, p = ns) prior to CsA initiation showed no significant correlation with interstitial fibrosis. Notably, the degree of interstitial accumulation of CD163⁺ cells significantly correlated with both the extent of interstitial fibrosis and the cumulative PSL dose during CsA administration ( Fig. 2 a, d ) . However, no significant correlations were found between interstitial CD163 + cell accumulation and the cumulative PSL dose from disease onset to biopsy or with the PSL dose prior to CsA initiation ( Fig. 2 b, c ) . Table 2 Relationship between interstitial fibrosis measured by measured by Masson's trichrome staining and clinicopathological parameters in the CsA group Parameters Mean ± SD Spearman r (p-value) Age at onset (years) 5.9 ± 3.6 -0.13 (0.48) Age at biopsy (years) 11.3 ± 3.9 0.16 (0.38) Time from onset to biopsy (years) 5.4 ± 3.3 0.11 (0.56) eGFR (ml/min/1.73m 2 ) at biopsy 118.8 ± 23.4 -0.08 (0.67) Cumulative PSL dose from onset to biopsy (mg/kg) 743.7 ± 423.0 0.40 (0.021) Cumulative PSL dose prior to CsA initiation (mg/kg) 424.0 ± 290.0 -0.11 (0.53) Cumulative PSL dose during CsA administration (mg/kg) 319.7 ± 328.8 0.63 (< 0.0001) Duration of CsA treatment (years) 3.2 ± 2.0 0.33 (0.06) Number of relapses from onset to biopsy 13.2 ± 6.2 0.21 (0.24) Number of relapses before CsA administration 6.7 ± 4.8 0.12 (0.51) Number of relapses during CsA administration 4.0 ± 2.5 0.41 (0.017) Number interstitial CD68 + cells (/HPF) 13.2 ± 6.2 0.50 (0.003) Number interstitial CD163 + cells (/HPF) 13.4 ± 6.9 0.56 (0.001) CsA: cyclosporine, eGFR: estimated glomerular filtration rate, PSL: prednisolone, HPF: high power field. Data is shown as mean ± 1 SD. Immunofluorescence study Immunofluorescence analysis of biopsies from the CsA-treated group revealed that CD163⁺ cells also expressed the pan-macrophage marker CD68, indicating that these cells were M2-type macrophages ( Fig. 3 a–c ) . CD163⁺ cells were localized to interstitial regions expressing type I collagen ( Fig. 3 d–f ) , suggesting their involvement in tubulointerstitial fibrosis. Dual immunostaining demonstrated that focal infiltrates of CD163⁺ cells co-localize with expression of the pro-fibrotic factor, CCN2/CTGF, and overlap of stains indicate that some CD163 + cells express CCN2/CTGF ( Fig. 3 g–i ) . Renal Expression of CCL2/MCP-1 CCL2/MCP-1 is a well-characterized monocyte chemokine whose expression in the kidney is associated with macrophage accumulation [ 5 ]. Urinary CCL2/MCP-1 levels were measured in 11 patients from the CsA-treated group and 7 patients from the control group during the initial onset, relapse, and remission phases. In the CsA group, urinary MCP-1 levels were significantly higher than those in the control group during both active disease and remission phases. Notably, MCP-1 levels during the active disease phase were approximately five-fold higher than those during the remission phase in both groups ( Fig. 4 a,b ) . In addition, strong CCL2/MCP-1 expression was observed in the tubular epithelial cells of the CsA-treated group. CD163⁺ cells were seen around the tubules expressing CCL2/MCP-1, and the overlap of stains identified that some CD163 + cells also express CCL2/MCP-1 ( Fig. 4 c ) . Discussion One of the key findings of this study is that, although idiopathic NS has traditionally been considered a non-inflammatory disease, significant macrophage infiltration was observed in the renal tissue of pediatric patients. In cases requiring CsA treatment for FRNS or SDNS, macrophage infiltration in the tubulointerstitial area was particularly prominent. Notably, many of these infiltrating macrophages expressed CD163, a marker of M2-type macrophages, and were localized in regions of interstitial fibrosis. These findings suggest that the pathophysiological mechanism of CsA nephropathy may resemble that of interstitial fibrosis mediated by M2-type macrophages, as previously reported by our group in chronic allograft injury [ 6 ]. It could be argued that the increased fibrosis and macrophage infiltration observed in the CsA group were due to the histologically refractory nature of the cases requiring CsA treatment. However, as shown in Table 1 , there were no significant differences between the two groups in the number of relapses or cumulative steroid dose prior to CsA initiation. Therefore, CsA use itself is strongly implicated as the cause of the observed histological differences. Furthermore, the significant correlation between cumulative steroid dose after CsA initiation and both interstitial fibrosis and macrophage count suggests that CsA and steroid administration may have jointly contributed to the histological changes, rather than steroid therapy alone. Another interesting finding was that only eight cases in the CsA-treated group were pathologically diagnosed with CsA nephropathy, showing no statistically significant difference compared to the control group. However, quantitative studies revealed a significant increase in fibrotic lesions and interstitial accumulation of CD163-positive M2 macrophages in the CsA group. These findings suggest that tubulointerstitial lesions are already progressing before the typical pathological lesions of cyclosporine nephropathy become apparent [ 10 ]. Nevertheless, such changes—considered early indicators of CsA nephrotoxicity—may be overlooked in routine pathological assessments. From this perspective, the present study’s finding that the extent of M2 macrophage infiltration correlates with the degree of tubulointerstitial fibrosis indicates that M2 macrophage infiltration may serve as a useful marker for evaluating CsA-induced nephrotoxicity. In our previous study, we also demonstrated M2-type macrophage infiltration in IgA nephropathy, where these cells may contribute to the development of chronic lesions such as glomerular matrix expansion, sclerosis, and tubulointerstitial fibrosis [ 8 , 9 ]. Importantly, M2-type macrophages are known to be activated by corticosteroids. In vitro experiments showed that dexamethasone stimulation of human blood monocytes induced CD163 expression, along with upregulation of CCL2/MCP-1 and profibrotic factors such as FGF8, FGF21 and CCN2/CTGF (Table S1 ) . CCN2/CTGF expression was also observed in renal biopsy specimens from patients with IgA nephropathy, and some CD163-positive M2 macrophages co-expressed CCN2/CTGF. These findings support the hypothesis that M2 macrophages may play a role in interstitial fibrosis in IgA nephropathy [ 8 , 9 ]. In the present study, we further demonstrated CCN2/CTGF expression in CsA nephropathy. Moreover, a significant correlation was found between cumulative PSL dose during CsA treatment and the number of interstitial M2-type macrophages. This strongly suggests that steroid-activated M2 macrophages may be involved in the development of interstitial fibrosis in CsA nephropathy. To investigate the mechanism of macrophage accumulation in the interstitial area, we also examined CCL2/MCP-1 as a potential mediator of macrophage recruitment. CCL2/MCP-1 is a key regulator of innate immunity and tissue inflammation [ 11 ]. Monocyte-derived cultured macrophages express CCL2/MCP-1 upon stimulation with dexamethasone (Table S1 ) . Upon binding to its receptor, CCL2/MCP-1 promotes the recruitment, migration, activation, and differentiation of lymphocytes and natural killer cells, as well as the infiltration of monocytes and macrophages [ 12 ]. In this study, CsA treatment was associated with elevated urinary CCL2/MCP-1 levels and strong CCL2/MCP-1 expression in renal tubular epithelial cells. These findings suggest that tubular epithelial cells, damaged by CsA-induced vascular endothelial injury and subsequent ischemia, release CCL2/MCP-1, which in turn promotes macrophage recruitment and infiltration. Additionally, some interstitial M2-type macrophages appeared to express CCL2/MCP-1, consistent with our previous in vitro findings (Table S1 ) , indicating a possible autocrine or paracrine amplification loop. Taken together, these results suggest that CCL2/MCP-1 plays a central role in the recruitment of macrophages to CsA-injured regions, contributing to fibrotic progression. This study has several limitations. First, its retrospective design inherently limits the ability to establish causal relationships. Second, the small sample size reduces statistical power and may not fully capture the heterogeneity of clinical presentations or histopathological findings in pediatric NS. Third, the lack of longitudinal follow-up data restricts our understanding of the long-term impact of CsA-induced macrophage infiltration and fibrosis. Additionally, while our previous in vitro findings support the proposed mechanisms, they may not fully reflect the complexity of in vivo immune responses. Further validation using animal models and well-designed prospective clinical studies is essential to confirm these hypotheses and to explore the temporal dynamics of macrophage polarization and chemokine expression. In recent years, the use of CsA has declined due to the availability of alternative treatments such as mycophenolate mofetil and rituximab [ 13 ]. Nevertheless, in the absence of nephrotoxicity concerns, CsA remains a valuable option for short-term prevention of NS recurrence. From this perspective, M2 macrophages represent a promising therapeutic target. Future research should explore preventive strategies targeting M2 macrophages using existing immunosuppressants or novel inhibitors [ 8 , 14 – 16 ]. In conclusion, this study highlights the potential role of M2-type macrophages in the development of interstitial fibrosis in pediatric patients with idiopathic NS undergoing CsA therapy. The findings suggest that steroid-induced polarization of macrophages, coupled with MCP-1-mediated recruitment, may contribute to fibrotic changes in the kidney. Targeting M2 macrophages may offer a novel therapeutic approach to mitigate CsA nephropathy and improve long-term renal outcomes in affected children. Declarations Acknowledgments. This work was partially supported by a research grant from the Aichi Kidney Foundation to T.K, and by JSPS KAKENHI Grant Number JP24K10988 to Y.I. Authors’ contributions. Y.I. and D.J.N-P. conceived and designed the study. T.K., T.A., Y.M., N.K., M.K., H.H., and T.Y. contributed to patient management and data collection. T.K. and Y.I. performed data analysis and interpretation. T.K., D.J.N-P., and Y.I. drafted and revised the manuscript. All authors reviewed and approved the final version of the manuscript. Compliance with ethical standards Conflict of interest. The authors declare that they have no conflicts of interest. Ethical approval. All procedures involving human participants were performed in accordance with the requirements of the institutional research committee (IRB approval HM23-471) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 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Biomed Pharmacother 177:117079. https://doi:10.1016/j.biopha.2024.117079 Supplementary Files GraphicalAbstractfinal.pptx SupplementalFigurefinal.pptx Cite Share Download PDF Status: Published Journal Publication published 21 Jan, 2026 Read the published version in Pediatric Nephrology → Version 1 posted Editorial decision: Major Revisions Needed 30 Oct, 2025 Reviewers agreed at journal 01 Oct, 2025 Reviewers invited by journal 01 Oct, 2025 Editor assigned by journal 01 Oct, 2025 First submitted to journal 28 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7737561","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":523330749,"identity":"42817b77-20f2-486d-b811-a9a79b24f83d","order_by":0,"name":"Tomomi Kondoh","email":"","orcid":"","institution":"Fujita Health University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tomomi","middleName":"","lastName":"Kondoh","suffix":""},{"id":523330750,"identity":"e8719281-132d-410c-8295-2a361c309d07","order_by":1,"name":"Yuji Matsumoto","email":"","orcid":"","institution":"Fujita Health University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuji","middleName":"","lastName":"Matsumoto","suffix":""},{"id":523330751,"identity":"8a705e52-34d9-4f99-8cb8-b2a338910206","order_by":2,"name":"Takuma Andoh","email":"","orcid":"","institution":"Fujita Health University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takuma","middleName":"","lastName":"Andoh","suffix":""},{"id":523330752,"identity":"181388bf-6aa3-44f8-90eb-de7069f29790","order_by":3,"name":"Masahiro Kaneko","email":"","orcid":"","institution":"Niigata University Medical and Dental Hospital","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Kaneko","suffix":""},{"id":523330753,"identity":"7faa646e-8de5-4e29-ab0b-003b21199d99","order_by":4,"name":"Hiroya Hasegawa","email":"","orcid":"","institution":"Niigata University Medical and Dental Hopital","correspondingAuthor":false,"prefix":"","firstName":"Hiroya","middleName":"","lastName":"Hasegawa","suffix":""},{"id":523330754,"identity":"284a06a6-3fc2-4ec4-87bd-df3b2f716555","order_by":5,"name":"Takeshi Yamada","email":"","orcid":"","institution":"Niigata University Medical and Dental Hopital","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Yamada","suffix":""},{"id":523330755,"identity":"500fa48c-c8a7-4e17-8bce-fdf0de1f1f16","order_by":6,"name":"Naonori Kumagai","email":"","orcid":"","institution":"Fujita Health University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Naonori","middleName":"","lastName":"Kumagai","suffix":""},{"id":523330756,"identity":"e6abb296-867f-4bce-85e8-7e87e309798f","order_by":7,"name":"David J Nikolic-Paterson","email":"","orcid":"","institution":"Monash University Department of Medicine, Monash Medical Centre","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"J","lastName":"Nikolic-Paterson","suffix":""},{"id":523330757,"identity":"2c55cbe1-2046-4186-950c-fb3590130c5a","order_by":8,"name":"Yohei Ikezumi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACNh4wJSHH3sCQcADISoCI8xDWYsxzAKjlADFaYHKJPSArEFrwAD6ew4c/8+ZYpPdIJDw8/KGGIc/gAPPDDwwyd3A7jLctwZh3m0QuUAvQYccYig0OsBlLMPA8w62Fn8cgGaRlP1gL2//EDQcYzIDuPYxHC/+Hw0At6TxgLf8YgFrYv+HXwtvD2AzUkgDWcrANpIWHgC08x4wZ526TMOzheZBw4GwfQ+LMwzzFQBNw+0W+J/nxh7fb6uR52HOSP1R8Y0jsO96+8cPHHtwhhgR4EiA0MwMsmggCdmRVP4jSMgpGwSgYBSMDAAA/VlX8qKm8LQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6460-1188","institution":"Fujita Health University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yohei","middleName":"","lastName":"Ikezumi","suffix":""}],"badges":[],"createdAt":"2025-09-29 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1","display":"","copyAsset":false,"role":"figure","size":562379,"visible":true,"origin":"","legend":"\u003cp\u003eHistological findings in children with nephrotic syndrome (NS) from the CsA-treated group (a, c, e, g) in comparison to children with NS controls (b, d, f, h). (a and b) Periodic acid-Schiff staining. (c and d) Masson-trichrome staining (e and f). Two-color immunostaining showing CD163\u003csup\u003e+\u003c/sup\u003e cells (brown) in areas with α-smooth muscle actin+ myofibroblasts (α-SMA, blue). \u0026nbsp;(g and h) Two-color immunohistochemistry staining for CD163\u003csup\u003e+\u003c/sup\u003e cells (brown) and collagen type I (green). Original magnification: ×100. Quantitative comparison of interstitial changes between the control and CsA-treated groups: (i) degree of interstitial fibrosis, (j) number of interstitial CD68⁺ cells, (k) number of interstitial CD163⁺ cells.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-7737561/v1/ad44667db4e4a98d649a328c.png"},{"id":93556322,"identity":"bb7e25fa-eec3-4dc1-ac8d-11137d18e3a9","added_by":"auto","created_at":"2025-10-15 06:42:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49028,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between the number of interstitial CD163⁺ cells and (a) the degree of interstitial fibrosis, (b) cumulative prednisolone (PSL) dose from disease onset to biopsy, (c) cumulative PSL dose prior to cyclosporine A (CsA) initiation, and (d) cumulative PSL dose during CsA administration.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-7737561/v1/a25411c1b4ab9df11e1826af.png"},{"id":93558307,"identity":"873d19a8-6b38-4fdd-9530-4a1a8868f362","added_by":"auto","created_at":"2025-10-15 06:58:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":472567,"visible":true,"origin":"","legend":"\u003cp\u003eDual-color immunofluorescence (IF) staining in children with nephrotic syndrome from the CsA-treated group. (a–c) Dual IF staining shows that most interstitial CD163⁺ cells (green) co-express CD68 (red). (d–f) Dual IF staining reveals type I collagen expression (green), co-localized with CD163⁺ macrophages (red). (g–i) Dual IF staining demonstrates the presence of Cellular communication network factor 2 (CCN2; green) in areas rich in CD163⁺ macrophages (red), with many CD163⁺ cells showing co-expression of CCN2 in the merged image. Original magnification: ×100.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-7737561/v1/864cd44bf7a809946c6aa695.png"},{"id":93556329,"identity":"d036b322-e5fe-474a-ba56-c83e5563ae1c","added_by":"auto","created_at":"2025-10-15 06:42:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":263347,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of urinary MCP-1 expression between CsA-treated and non-treated children with nephrotic syndrome during the (a) active disease phase, and (b) remission phase. (c) Dual IF staining shows C–C motif chemokine ligand 2 (CCL2; green) expression by some tubular epithelial cells in areas rich in CD163⁺ macrophages (red), with some CD163⁺ cells also showing co-expression of CCL2 in the merged image (arrowheads).\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-7737561/v1/b5cfb71c37675d78dc2e8fac.png"},{"id":101152032,"identity":"94d365f3-5f29-4346-b399-f251d492ff9d","added_by":"auto","created_at":"2026-01-26 16:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2050226,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7737561/v1/d74b01ac-ba46-41d7-8c2b-2c47edecf991.pdf"},{"id":93556324,"identity":"0fa14068-48c6-486c-97ef-06502daffcb8","added_by":"auto","created_at":"2025-10-15 06:42:44","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":89859,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractfinal.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7737561/v1/e65cc9bb060e7e8105386c61.pptx"},{"id":93557435,"identity":"f365e333-4c2e-4275-9407-12ef098cf7b3","added_by":"auto","created_at":"2025-10-15 06:50:44","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":55434,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigurefinal.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7737561/v1/a8fd549656977762f4916db2.pptx"}],"financialInterests":"","formattedTitle":"Corticosteroids augment cyclosporine nephrotoxicity in pediatric nephrotic syndrome: potential role of alternatively activated macrophages.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNephrotic syndrome (NS) is one of the most common kidney diseases in children, characterized by heavy proteinuria, hypoalbuminemia, and edema. Glucocorticoid steroids, particularly prednisolone (PSL), are the first-line treatment for pediatric idiopathic NS, inducing remission in approximately 90% of cases. However, 80% of these patients experience relapse, with 30\u0026ndash;40% developing frequently relapsing NS (FRNS) or steroid-dependent NS (SDNS) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In patients with FRNS, increased PSL use leads to significant steroid-related adverse effects. To reduce or discontinue PSL, immunosuppressive agents are recommended for relapse prevention. Cyclosporine A (CsA) is one of the most commonly used and effective immunosuppressive agents for steroid-sparing treatment in children with FRNS or SDNS. However, many patients relapse after CsA discontinuation, reverting to FRNS or SDNS [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, long-term CsA administration often becomes necessary, increasing the risk of tubulointerstitial damage, including microvascular injury and fibrosis, collectively referred to as CsA nephropathy. Nevertheless, the factors contributing to the development of tubulointerstitial lesions remain unclear [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMacrophage accumulation in the kidneys is a universal phenomenon observed in all progressive renal diseases, regardless of whether they are inflammatory or non-inflammatory [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. We previously reported the involvement of alternatively activated (M2 phenotype) macrophages in the pathogenesis of chronic allograft kidney injury, which develops under long-term concomitant use of steroids and calcineurin inhibitors such as CsA and tacrolimus [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This suggests a crucial role for M2-type macrophages in the progression of graft fibrosis. It is further hypothesized that a similar pathogenic mechanism may be involved in CsA nephropathy, which arises under analogous conditions of steroid and calcineurin inhibitor administration.\u003c/p\u003e\u003cp\u003eThis study aimed to investigate the role of M2-type macrophages in CsA nephropathy, with a particular focus on tubulointerstitial fibrosis in patients with FRNS or SDNS, in order to better understand the mechanisms underlying the associated histological damage.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatients\u003c/h2\u003e\u003cp\u003eAmong 131 children diagnosed with idiopathic NS between 1998 and 2020 at the Department of Pediatrics, Niigata University Medical and Dental Hospital, or the Department of Pediatrics, Fujita Health University Hospital, who underwent renal biopsy, 33 children with FRNS or SDNS and biopsy-confirmed minimal change disease (MCD) and who received CsA for at least 2 years were retrospectively analyzed. Additionally, 14 age-matched children with FRNS/SDNS who underwent renal biopsy prior to CsA initiation served as controls. Children with pathological findings other than MCD, including focal segmental glomerulosclerosis (FSGS), membranous nephropathy, or proliferative glomerulonephritis, as well as those who developed steroid-resistant NS (SRNS) during the course of treatment, were excluded.\u003c/p\u003e\u003cp\u003eAll patients included in this study were classified as steroid-sensitive NS (SSNS), defined as achieving remission with standard steroid therapy within 4 weeks [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. FRNS was defined as \u0026ge;\u0026thinsp;2 relapses within 6 months or \u0026ge;\u0026thinsp;4 relapses within 12 months among SSNS patients. SDNS was defined as two consecutive relapses during steroid tapering or within 2 weeks after cessation of therapy. SRNS was defined as failure to achieve remission within 4 weeks of standard treatment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTreatment protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eTreatment protocol\u003c/div\u003e\u003cp\u003eInitial treatment for idiopathic NS consisted of daily PSL at 60 mg/m\u0026sup2;/day or 2 mg/kg/day (maximum 60 mg/day) for 4 weeks, followed by alternate-day PSL at 40 mg/m\u0026sup2; or 1.5 mg/kg/day for another 4 weeks. Upon relapse, patients were treated with a single daily dose of PSL (2 mg/kg/day or 60 mg/m\u0026sup2;/day, maximum 60 mg) until complete remission, defined as three consecutive days of negative dipstick results. This was followed by alternate-day PSL (1.5 mg/kg or 40 mg/m\u0026sup2; per dose, maximum 40 mg), with gradual tapering over the next 2\u0026ndash;3 months. For relapses in FRNS/SDNS patients, PSL was tapered daily after achieving complete remission.\u003c/p\u003e\u003cp\u003eCsA was administered at an initial dose of 2.5\u0026ndash;5 mg/kg/day in two divided doses, with subsequent dose adjustments based on blood drug concentration monitoring: trough levels of 60\u0026ndash;100 ng/mL or C2 levels of 500\u0026ndash;700 ng/mL.\u003c/p\u003e\u003cp\u003eRenal biopsy indications in children with NS included: (1) age\u0026thinsp;\u0026lt;\u0026thinsp;1 year, (2) persistent or gross hematuria, (3) hypertension or renal dysfunction, (4) hypocomplementemia, and (5) extrarenal symptoms (e.g., purpura), as these patients are more likely to have histological types other than MCD. Additional indications included: (6) steroid resistance or FRNS/SDNS prior to immunosuppressant initiation, and (7) long-term (\u0026gt;\u0026thinsp;2 years) CsA therapy, even in the absence of renal dysfunction, to assess nephrotoxicity. Informed consent was obtained from all patients for the use of residual biopsy tissue for research purposes following completion of diagnostic evaluation.\u003c/p\u003e\n\u003ch3\u003eClinical and pathologic data\u003c/h3\u003e\n\u003cp\u003eUrinary protein concentration (Up), serum creatinine (sCr), and urine creatinine (Ucr) levels were measured for each patient at the time of renal biopsy. The estimated glomerular filtration rate (eGFR) was calculated using an equation specific to Japanese children [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Medical records were retrospectively reviewed to determine the cumulative steroid dose from the onset of NS to the time of biopsy. In the CsA group, the steroid dose was assessed both before and after CsA initiation, expressed as PSL dose per body weight. Additionally, the number of relapses occurring before and after CsA initiation was extracted from the medical records.\u003c/p\u003e\u003cp\u003eKidney biopsy specimens were fixed in Carnoy\u0026rsquo;s solution, embedded in paraffin, and sectioned for staining with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), periodic acid-methenamine silver (PAM), or Masson\u0026rsquo;s trichrome (MT). An independent pathologist, blinded to clinical data and macrophage staining results, reviewed the stained sections. The degree of interstitial fibrosis was quantified by image analysis of MT-stained sections, as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The number of interstitial macrophages was assessed using immunohistochemical staining, as detailed below. The extent of fibrosis and macrophage infiltration in the tubulointerstitial region was evaluated in relation to the clinical parameters described above.\u003c/p\u003e\n\u003ch3\u003eAntibodies\u003c/h3\u003e\n\u003cp\u003ePrimary antibodies used in this study included, 10D6: anti-human CD163 (mouse IgG1; Visionbiosystems, Benton Lane, UK), PG-M1: anti-CD68, labeling human monocytes and macrophages (mouse IgG3; DAKO, Santa Clara, CA, USA), 1A4: anti-human smooth muscle actin (α-SMA/Acta2; mouse IgG2a; DAKO), Anti-collagen type I (rabbit polyclonal; Novus Biologicals, Littleton, CO, USA), 2154-60: anti-human cellular communication network factor 2 (CCN2, formerly known as connective tissue growth factor or CTGF; mouse IgM; Acris Antibodies, Hiddenhausen, Germany), Anti-C\u0026ndash;C motif chemokine ligand 2 (CCL2 also known as monocyte chemoattractant protein-1 or MCP-1; rabbit polyclonal; Bioss Antibodies, Woburn, MA, USA).\u003c/p\u003e\u003cp\u003eSecondary antibodies included, FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL, USA), TRITC-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates), FITC-conjugated goat anti-rabbit IgG (MP Biomedical, Santa Ana, CA, USA).\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eMacrophages, α-SMA, and type I collagen were detected in 2 \u0026micro;m tissue sections fixed with Carnoy\u0026rsquo;s solution, using the VECTASTAIN Elite ABC HRP Kit (Vector Laboratories, Burlingame, CA, USA). After dewaxing, antigen retrieval was performed by microwave heating for 10 minutes in 0.1 M sodium citrate buffer\u0026mdash;pH 6.0 for general targets and pH 9.0 specifically for α-SMA\u0026mdash;using Target Retrieval Solution (DAKO).\u003c/p\u003e\u003cp\u003eFollowing PBS washes, sections were blocked for 30 minutes at room temperature with a solution containing 10% normal goat serum and 1% bovine serum albumin (BSA) in PBS. Primary antibodies were diluted in PBS supplemented with 10% normal human serum and 1% BSA, and incubated overnight at 4\u0026deg;C. After rinsing with PBS, endogenous peroxidase activity was suppressed using 0.3% hydrogen peroxide in methanol for 20 minutes. Sections were then incubated sequentially with biotinylated secondary antibodies and the VECTASTAIN Elite ABC reagent, each for 30 minutes at room temperature. Chromogenic development was carried out using diaminobenzidine (DAKO) for brown staining of macrophages, TrueBlue Substrate (KPL, Gaithersburg, MD, USA) for blue staining of α-SMA, and Vina Green Chromogen Kit (Biocare Medical, Pacheco, CA, USA) for green staining of type I collagen.\u003c/p\u003e\u003cp\u003eQuantification of CD68- or CD163-positive cells was performed in at least 12 glomeruli per patient. For interstitial regions, positive cells were counted in a minimum of five consecutive high-power fields (\u0026times;400 magnification). For dual-color immunostaining, CD163 was first visualized using microwave-based antigen retrieval to minimize cross-reactivity, followed by staining with either α-SMA or type I collagen antibodies, each developed with distinct chromogenic substrates.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eUrinary CCL2 measurement\u003c/h2\u003e\u003cp\u003eTo investigate the mechanism of macrophage infiltration in the kidney, urinary levels of CCL2/MCP-1, a key macrophage chemoattractant, were measured using a cytometric bead array kit (BD Biosciences, San Jose, CA, USA). Assays were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eComparisons were made between two groups by Mann\u0026ndash;Whitney test (GraphPad 8.0, San Diego, CA) or Fisher\u0026rsquo;s exact test. Data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;1 SD. Correlation analysis used the Spearman correlation coefficient. We declared a finding to be statistically significant if the p-value was less than 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eComparison of patient profiles between the CsA-treated and control groups\u003c/h2\u003e\u003cp\u003ePatient profiles at the time of biopsy are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. There were no significant differences between the two groups in clinical parameters, including gender, age at initial onset, age at biopsy, duration from NS onset to biopsy, degree of proteinuria, kidney function (eGFR), cumulative prednisolone (PSL) dose from onset to biopsy, or the number of relapses prior to biopsy. In the CsA-treated group, 8 out of 33 patients (24.2%) were pathologically diagnosed with CsA nephropathy, whereas no patients in the control group had this diagnosis. However, the difference between the groups was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.084).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of clinical parameters at biopsy.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCsA-treated group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCsA non-treated group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ep-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of patients\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e―\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender (M:F)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25 : 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 : 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;0.99\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge at onset (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge at biopsy (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime from onset to biopsy (years) \u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUrinary protein/Cr ratio (g/gCr)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeGFR (ml/min/1.73m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e118.8\u0026thinsp;\u0026plusmn;\u0026thinsp;23.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e125.1\u0026thinsp;\u0026plusmn;\u0026thinsp;20.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCumulative PSL dose up to biopsy (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e743.7\u0026thinsp;\u0026plusmn;\u0026thinsp;423.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e659.1\u0026thinsp;\u0026plusmn;\u0026thinsp;299.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of relapses from onset to biopsy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of children with CsA nephropathy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8 (24.2%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.084\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDegree of interstitial fibrosis (Masson positive area %)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0012\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber interstitial CD68\u003csup\u003e+\u003c/sup\u003e cells\u003c/p\u003e\u003cp\u003e(/HPF)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.0048\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber interstitial CD163\u003csup\u003e+\u003c/sup\u003e cells (/HPF)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eCsA: cyclosporine, eGFR: estimated glomerular filtration rate, PSL: prednisolone, HPF: high power field\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eData is shown as mean \u0026plusmn; 1 SD.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRenal Histology\u003c/h2\u003e\u003cp\u003eNo changes were observed in glomerular morphology in either group, with all cases classified as minimal change disease (MCD) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b\u003cb\u003e)\u003c/b\u003e. However, interstitial fibrosis, quantified by the area of Masson\u0026rsquo;s trichrome staining, was significantly greater in the CsA group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, the accumulation of CD163\u0026thinsp;+\u0026thinsp;cells in areas of interstitial fibrosis shown by the presence of interstitial a-SMA\u0026thinsp;+\u0026thinsp;cells and increased interstitial collagen I deposition, was evident in the CsA-treated group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-h\u003cb\u003e)\u003c/b\u003e. Indeed, pathological findings revealed significantly greater interstitial fibrosis, along with higher numbers of interstitial CD68⁺ and CD163⁺ cells, in the CsA-treated group \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-k\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the correlations between interstitial fibrosis based on Masson trichrome staining and clinical parameters in the CsA-treated group. Significant correlations were found between the extent of interstitial fibrosis and the cumulative PSL dose during CsA treatment (r\u0026thinsp;=\u0026thinsp;0.63, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), duration of CsA administration (r\u0026thinsp;=\u0026thinsp;0.48, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), cumulative PSL dose from disease onset (r\u0026thinsp;=\u0026thinsp;0.40, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and number of relapses during CsA treatment (r\u0026thinsp;=\u0026thinsp;0.41, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Interstitial fibrosis also significantly correlated with the degree of CD163⁺ cell infiltration (r\u0026thinsp;=\u0026thinsp;0.56, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and CD68⁺ cell infiltration (r\u0026thinsp;=\u0026thinsp;0.50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). In contrast, cumulative PSL dose (r = \u0026minus;\u0026thinsp;0.11, p\u0026thinsp;=\u0026thinsp;ns) and number of relapses (r = \u0026minus;\u0026thinsp;0.10, p\u0026thinsp;=\u0026thinsp;ns) prior to CsA initiation showed no significant correlation with interstitial fibrosis. Notably, the degree of interstitial accumulation of CD163⁺ cells significantly correlated with both the extent of interstitial fibrosis and the cumulative PSL dose during CsA administration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, d\u003cb\u003e)\u003c/b\u003e. However, no significant correlations were found between interstitial CD163\u0026thinsp;+\u0026thinsp;cell accumulation and the cumulative PSL dose from disease onset to biopsy or with the PSL dose prior to CsA initiation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRelationship between interstitial fibrosis measured by measured by Masson's trichrome staining and clinicopathological parameters in the CsA group\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSpearman r (p-value)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge at onset (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.13 (0.48)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge at biopsy (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.16 (0.38)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTime from onset to biopsy (years) \u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.11 (0.56)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeGFR (ml/min/1.73m\u003csup\u003e2\u003c/sup\u003e) at biopsy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e118.8\u0026thinsp;\u0026plusmn;\u0026thinsp;23.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.08 (0.67)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCumulative PSL dose from onset to biopsy (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e743.7\u0026thinsp;\u0026plusmn;\u0026thinsp;423.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.40 (0.021)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCumulative PSL dose prior to CsA initiation (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e424.0\u0026thinsp;\u0026plusmn;\u0026thinsp;290.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e-0.11 (0.53)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCumulative PSL dose during CsA administration (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e319.7\u0026thinsp;\u0026plusmn;\u0026thinsp;328.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.63 (\u0026lt;\u0026thinsp;0.0001)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDuration of CsA treatment (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.33 (0.06)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of relapses from onset to biopsy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.21 (0.24)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of relapses before CsA administration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.12 (0.51)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of relapses during CsA administration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.41 (0.017)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber interstitial CD68\u003csup\u003e+\u003c/sup\u003e cells (/HPF)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.50 (0.003)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber interstitial CD163\u003csup\u003e+\u003c/sup\u003e cells (/HPF)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.56 (0.001)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eCsA: cyclosporine, eGFR: estimated glomerular filtration rate, PSL: prednisolone, HPF: high power field. Data is shown as mean \u0026plusmn; 1 SD.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence study\u003c/h2\u003e\u003cp\u003eImmunofluorescence analysis of biopsies from the CsA-treated group revealed that CD163⁺ cells also expressed the pan-macrophage marker CD68, indicating that these cells were M2-type macrophages \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u0026ndash;c\u003cb\u003e)\u003c/b\u003e. CD163⁺ cells were localized to interstitial regions expressing type I collagen \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u0026ndash;f\u003cb\u003e)\u003c/b\u003e, suggesting their involvement in tubulointerstitial fibrosis. Dual immunostaining demonstrated that focal infiltrates of CD163⁺ cells co-localize with expression of the pro-fibrotic factor, CCN2/CTGF, and overlap of stains indicate that some CD163\u0026thinsp;+\u0026thinsp;cells express CCN2/CTGF \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u0026ndash;i\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRenal Expression of CCL2/MCP-1\u003c/h2\u003e\u003cp\u003eCCL2/MCP-1 is a well-characterized monocyte chemokine whose expression in the kidney is associated with macrophage accumulation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Urinary CCL2/MCP-1 levels were measured in 11 patients from the CsA-treated group and 7 patients from the control group during the initial onset, relapse, and remission phases. In the CsA group, urinary MCP-1 levels were significantly higher than those in the control group during both active disease and remission phases. Notably, MCP-1 levels during the active disease phase were approximately five-fold higher than those during the remission phase in both groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b\u003cb\u003e)\u003c/b\u003e. In addition, strong CCL2/MCP-1 expression was observed in the tubular epithelial cells of the CsA-treated group. CD163⁺ cells were seen around the tubules expressing CCL2/MCP-1, and the overlap of stains identified that some CD163\u003csup\u003e+\u003c/sup\u003e cells also express CCL2/MCP-1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOne of the key findings of this study is that, although idiopathic NS has traditionally been considered a non-inflammatory disease, significant macrophage infiltration was observed in the renal tissue of pediatric patients. In cases requiring CsA treatment for FRNS or SDNS, macrophage infiltration in the tubulointerstitial area was particularly prominent. Notably, many of these infiltrating macrophages expressed CD163, a marker of M2-type macrophages, and were localized in regions of interstitial fibrosis. These findings suggest that the pathophysiological mechanism of CsA nephropathy may resemble that of interstitial fibrosis mediated by M2-type macrophages, as previously reported by our group in chronic allograft injury [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt could be argued that the increased fibrosis and macrophage infiltration observed in the CsA group were due to the histologically refractory nature of the cases requiring CsA treatment. However, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, there were no significant differences between the two groups in the number of relapses or cumulative steroid dose prior to CsA initiation. Therefore, CsA use itself is strongly implicated as the cause of the observed histological differences. Furthermore, the significant correlation between cumulative steroid dose after CsA initiation and both interstitial fibrosis and macrophage count suggests that CsA and steroid administration may have jointly contributed to the histological changes, rather than steroid therapy alone.\u003c/p\u003e\u003cp\u003eAnother interesting finding was that only eight cases in the CsA-treated group were pathologically diagnosed with CsA nephropathy, showing no statistically significant difference compared to the control group. However, quantitative studies revealed a significant increase in fibrotic lesions and interstitial accumulation of CD163-positive M2 macrophages in the CsA group. These findings suggest that tubulointerstitial lesions are already progressing before the typical pathological lesions of cyclosporine nephropathy become apparent [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nevertheless, such changes\u0026mdash;considered early indicators of CsA nephrotoxicity\u0026mdash;may be overlooked in routine pathological assessments. From this perspective, the present study\u0026rsquo;s finding that the extent of M2 macrophage infiltration correlates with the degree of tubulointerstitial fibrosis indicates that M2 macrophage infiltration may serve as a useful marker for evaluating CsA-induced nephrotoxicity.\u003c/p\u003e\u003cp\u003eIn our previous study, we also demonstrated M2-type macrophage infiltration in IgA nephropathy, where these cells may contribute to the development of chronic lesions such as glomerular matrix expansion, sclerosis, and tubulointerstitial fibrosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Importantly, M2-type macrophages are known to be activated by corticosteroids. In vitro experiments showed that dexamethasone stimulation of human blood monocytes induced CD163 expression, along with upregulation of CCL2/MCP-1 and profibrotic factors such as FGF8, FGF21 and CCN2/CTGF \u003cb\u003e(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. CCN2/CTGF expression was also observed in renal biopsy specimens from patients with IgA nephropathy, and some CD163-positive M2 macrophages co-expressed CCN2/CTGF. These findings support the hypothesis that M2 macrophages may play a role in interstitial fibrosis in IgA nephropathy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the present study, we further demonstrated CCN2/CTGF expression in CsA nephropathy. Moreover, a significant correlation was found between cumulative PSL dose during CsA treatment and the number of interstitial M2-type macrophages. This strongly suggests that steroid-activated M2 macrophages may be involved in the development of interstitial fibrosis in CsA nephropathy.\u003c/p\u003e\u003cp\u003eTo investigate the mechanism of macrophage accumulation in the interstitial area, we also examined CCL2/MCP-1 as a potential mediator of macrophage recruitment. CCL2/MCP-1 is a key regulator of innate immunity and tissue inflammation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Monocyte-derived cultured macrophages express CCL2/MCP-1 upon stimulation with dexamethasone \u003cb\u003e(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. Upon binding to its receptor, CCL2/MCP-1 promotes the recruitment, migration, activation, and differentiation of lymphocytes and natural killer cells, as well as the infiltration of monocytes and macrophages [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In this study, CsA treatment was associated with elevated urinary CCL2/MCP-1 levels and strong CCL2/MCP-1 expression in renal tubular epithelial cells. These findings suggest that tubular epithelial cells, damaged by CsA-induced vascular endothelial injury and subsequent ischemia, release CCL2/MCP-1, which in turn promotes macrophage recruitment and infiltration. Additionally, some interstitial M2-type macrophages appeared to express CCL2/MCP-1, consistent with our previous in vitro findings \u003cb\u003e(Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e, indicating a possible autocrine or paracrine amplification loop. Taken together, these results suggest that CCL2/MCP-1 plays a central role in the recruitment of macrophages to CsA-injured regions, contributing to fibrotic progression.\u003c/p\u003e\u003cp\u003eThis study has several limitations. First, its retrospective design inherently limits the ability to establish causal relationships. Second, the small sample size reduces statistical power and may not fully capture the heterogeneity of clinical presentations or histopathological findings in pediatric NS. Third, the lack of longitudinal follow-up data restricts our understanding of the long-term impact of CsA-induced macrophage infiltration and fibrosis. Additionally, while our previous in vitro findings support the proposed mechanisms, they may not fully reflect the complexity of in vivo immune responses. Further validation using animal models and well-designed prospective clinical studies is essential to confirm these hypotheses and to explore the temporal dynamics of macrophage polarization and chemokine expression.\u003c/p\u003e\u003cp\u003eIn recent years, the use of CsA has declined due to the availability of alternative treatments such as mycophenolate mofetil and rituximab [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Nevertheless, in the absence of nephrotoxicity concerns, CsA remains a valuable option for short-term prevention of NS recurrence. From this perspective, M2 macrophages represent a promising therapeutic target. Future research should explore preventive strategies targeting M2 macrophages using existing immunosuppressants or novel inhibitors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn conclusion, this study highlights the potential role of M2-type macrophages in the development of interstitial fibrosis in pediatric patients with idiopathic NS undergoing CsA therapy. The findings suggest that steroid-induced polarization of macrophages, coupled with MCP-1-mediated recruitment, may contribute to fibrotic changes in the kidney. Targeting M2 macrophages may offer a novel therapeutic approach to mitigate CsA nephropathy and improve long-term renal outcomes in affected children.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u0026nbsp;\u003c/strong\u003eThis work was partially supported by a research grant from the Aichi Kidney Foundation to T.K, and by JSPS KAKENHI Grant Number JP24K10988 to Y.I.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions.\u0026nbsp;\u003c/strong\u003eY.I. and D.J.N-P. conceived and designed the study. T.K., T.A., Y.M., N.K., M.K., H.H., and T.Y. contributed to patient management and data collection. T.K. and Y.I. performed data analysis and interpretation. T.K., D.J.N-P., and Y.I. drafted and revised the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest.\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval.\u0026nbsp;\u003c/strong\u003eAll procedures involving human participants were performed in accordance with the requirements of the institutional research committee (IRB approval HM23-471) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTarshish P, Tobin JN, Bernstein J, Edelmann CM Jr. (1997) Prognostic significance of the early course of minimal change nephrotic syndrome: report of the International Study of Kidney Disease in Children. 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Biomed Pharmacother 177:117079. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.biopha.2024.117079\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.biopha.2024.117079\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pediatric-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pnep","sideBox":"Learn more about [Pediatric Nephrology](http://link.springer.com/journal/467)","snPcode":"467","submissionUrl":"https://www.editorialmanager.com/pnep/default2.aspx","title":"Pediatric Nephrology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cyclosporine, Fibrosis, Nephrotic syndrome, M2-type macrophage, CCL2","lastPublishedDoi":"10.21203/rs.3.rs-7737561/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7737561/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Cyclosporine A (CsA) is an effective steroid-sparing agent for steroid-dependent nephrotic syndrome (SDNS); however, long-term administration of CsA can induce chronic kidney injury (CsA nephropathy). We previously reported that alternatively activated macrophages (M2-type macrophages) are closely linked to the pathogenesis of interstitial fibrosis in progressive kidney disease. In this study, we investigated the possible involvement of M2-type macrophages in CsA nephropathy in SDNS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods:\u003c/strong\u003e A total of 33 children diagnosed with SDNS and who were treated with CsA for more than 2 years were investigated. Fourteen biopsy specimens from age-matched SDNS children who had not received CsA treatment were used as the control. Renal fibrosis was assessed by Masson chrome staining of biopsy sections. Sections were also stained for α-smooth muscle actin (α-SMA), type I collagen, CD68 (total macrophages), CD163 (M2 marker) and CCL2. Urine levels of CCL2 were measured by cytometric bead array kit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The CsA-treated group showed significant interstitial fibrosis (12.2±7.3 vs 7.6±2.1%, p\u0026lt;0.001) with accumulation of interstitial CD163\u003csup\u003e+\u003c/sup\u003eCD68\u003csup\u003e+\u003c/sup\u003e macrophages (10.8 vs 7.9/HPF; p\u0026lt;0.001) compared with SDNS control patients. There was a significant correlation between the degree of interstitial fibrosis and the number of interstitial CD163\u003csup\u003e+\u003c/sup\u003e cells (p\u0026lt;0.001), and between interstitial fibrosis and the cumulative steroid dose used during CsA treatment (p\u0026lt;0.001), while no correlation was found between the cumulative steroid dose used before CsA treatment and histological changes. Immunostaining revealed significant expression of CCN2 and CCL2 in biopsies from the CsA group which co-localized with CD163\u003csup\u003e+ \u003c/sup\u003emacrophages. In addition, the urinary CCL2/creatinine ratio was significantly elevated in the CsA group compared to controls, both at relapse (1012±641.2 vs. 239.9±226.9 pg/mg;\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026nbsp;= 0.02) and at remission (202.0±178.4 vs. 77.7±127.3 pg/mg;\u0026nbsp;\u003cem\u003ep\u003c/em\u003e\u0026nbsp;= 0.04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our findings suggest that CD163\u003csup\u003e+ \u003c/sup\u003eM2-type macrophages may participate in the development of interstitial fibrosis induced by CsA. Steroid treatment during CsA treatment appears to augment CsA nephrotoxicity through the production of pro-fibrotic factors.\u003c/p\u003e","manuscriptTitle":"Corticosteroids augment cyclosporine nephrotoxicity in pediatric nephrotic syndrome: potential role of alternatively activated macrophages.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 06:42:39","doi":"10.21203/rs.3.rs-7737561/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-10-30T14:50:58+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-10-01T11:04:44+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-01T11:01:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-01T07:50:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pediatric Nephrology","date":"2025-09-28T23:39:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pediatric-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pnep","sideBox":"Learn more about [Pediatric Nephrology](http://link.springer.com/journal/467)","snPcode":"467","submissionUrl":"https://www.editorialmanager.com/pnep/default2.aspx","title":"Pediatric Nephrology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cb631248-f860-43f5-95b7-fdacbc93a832","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:05:33+00:00","versionOfRecord":{"articleIdentity":"rs-7737561","link":"https://doi.org/10.1007/s00467-026-07160-6","journal":{"identity":"pediatric-nephrology","isVorOnly":false,"title":"Pediatric Nephrology"},"publishedOn":"2026-01-21 15:57:50","publishedOnDateReadable":"January 21st, 2026"},"versionCreatedAt":"2025-10-15 06:42:39","video":"","vorDoi":"10.1007/s00467-026-07160-6","vorDoiUrl":"https://doi.org/10.1007/s00467-026-07160-6","workflowStages":[]},"version":"v1","identity":"rs-7737561","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7737561","identity":"rs-7737561","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}