Exploring the significance of interleukin-33/ST2 axis in minimal change disease

Minimal change disease (MCD), a common cause of idiopathic nephrotic syndrome, has been postulated to exhibit an association with allergic conditions. Recent studies revealed the crucial role of interleukin (IL)-33 in type 2 innate immunity. We hypothesized that development of MCD involves an IL-33–related immune response. We examined 49 patients with biopsy-proven MCD, 6 healthy volunteers, and 29 patients in remission. In addition to clinical features, serum and urinary levels of IL-33 and soluble suppression of tumorigenicity 2 protein (sST2), a secreted form of the receptor of IL-33, were analyzed. Although IL-33 was barely detectable in either MCD or control samples, sST2 levels at diagnosis were elevated in MCD patients. Serum sST2 levels of MCD patients were correlated with serum total protein level (r =  − 0.36, p = 0.010) and serum creatinine level (r = 0.34, p = 0.016). Furthermore, the elevated sST2 levels were observed to decrease following remission. Immunofluorescence revealed IL-33 expression in the podocytes among MCD patients, with a significant increase compared with controls. In vitro, mouse podocyte cells incubated with serum from a MCD patient at disease onset showed increased IL-33 secretion. These results suggest an IL-33–related immune response plays a role in MCD.


Localization of IL-33 expression in glomeruli
We hypothesized that the IL-33/ST2 axis plays a role in kidney tissues in MCD, similar to that in the endobronchial tissue of asthma patients 20 .To determine the expression profile and potential cellular source of IL-33 in kidney tissue, we examined IL-33 protein expression in glomeruli using immunofluorescence analysis of sections of renal biopsy specimens.IL-33 was observed in the nucleus of endothelial cells among arteries or arterioles, including glomerular vascular poles (Fig. 3A).IL-33 expression was also detected in the cytoplasm of glomerular epithelial cells identified by positive staining for the podocyte nuclear marker 21 p57 (Fig. 3B).The number of cells double-positive for p57 and IL-33 per glomerular cross-section was significantly higher in MCD patients than controls (mean ± SEM = 0.345 ± 0.169 versus 0.065 ± 0.025; p = 0.027).

IL-33 expression in cultured glomerular epithelial cells stimulated with serum from an MCD patient
We speculated that some substance in the serum of MCD patients stimulates the secretion of IL-33 by glomerular epithelial cells.To consider the possible roles of IL-33 in glomerular epithelial cells, we investigated whether IL-33 expression was increased in cultured MPCs stimulated with serum obtained from a patient with MCD.Stimulation for 6 h with serum obtained at disease onset increased the secretion of IL-33 into the culture supernatant (p = 0.0100; Fig. 2C), whereas there was no significant increase in IL-33 secretion by cells stimulated with serum from a patient in remission.To comprehensive the regulatory mechanism underlying IL-33, we evaluated the gene expression of NLR family pyrin domain containing 3 (NLRP3) inflammasome, along with its subsequent signaling cascade.Treatment of podocytes with serum from a patient did not stimulate NLRP3 inflammasome signaling and resulted in the inverse effects on NLRP3, Casp1, or IL-1β gene expression levels (p = 0.022, p = 0.0034, and p = 0.015 respectively; Fig. 2D).We also examined expression of the genes encoding Angptl4 and CD80, which are considered mediators of proteinuria in MCD.Stimulation with serum obtained at disease onset increased Angptl4 gene expression (p = 0.0034; Fig. 2E).In contrast, there was no significant increase in CD80 gene expression (Fig. 2F).

Discussion
Although recent studies have revealed the mechanism of IL-33-related immune responses and type 2 immunity in allergic conditions, the role and function of IL-33 and ST2 in kidney diseases is poorly understood.We demonstrated that sST2 levels are elevated in patients with MCD and that serum sST2 levels are correlated with disease severity and activity of NS.Furthermore, IL-33 expression was detected on the surface of glomerular epithelial cells in kidney biopsy specimens, and upregulation of IL-33 was observed in cultured MPCs stimulated with serum obtained from an MCD patient.To the best of our knowledge, this is the first report to describe the potential role of the IL-33/ST2 axis in proteinuria of MCD.IL-33 is considered an important modulator of immune responses against invading pathogens or other environmental insults due to its predominant distribution in the epithelial barrier 22 .IL-33 released from damaged or necrotic cells acts as an alarmin that stimulates ST2-positive cells.Various studies have demonstrated the role of IL-33 in innate allergic airway responses using ST2-or IL-33-deficient mice and identified lung ILC2 as the primary target of IL-33 in the airway to induce a type 2 immune response.Clinical studies have reported elevated IL-33 protein levels in sputum 19 , bronchoalveolar lavage fluids, and serum 19 of bronchial asthma patients, with IL-33 levels generally correlating with disease severity 22 .We hypothesized that IL-33 plays a role in the proteinuria of MCD coexisting with an allergic condition.Counter to our expectation, IL-33 was rarely detected in MCD patients or healthy controls in this study.However, whether serum or urine IL-33 levels are elevated in patients with MCD should be carefully determined, as elevated levels of soluble ST2 in human serum have www.nature.com/scientificreports/been shown to interfere with IL-33 quantification by enzyme-linked immunosorbent assay (ELISA) 23 , and the sensitivity and specificity of available ELISA kits are not sufficient for evaluating serum IL-33 levels 24 .In addition, the potential effect of posttranslational modifications through cathepsins or elastase on IL-33 detection should also be considered 25 .Generally, discerning cytokines circulating at very low levels in blood is not consistently straightforward 26 .Regarding IL-13, a prototypical Type 2 cytokine, its circulating concentrations are not consistently high within the context of allergic conditions 26,27 .It is proposed that evaluating IL-13 concentrations locally at the diseased anatomical site holds greater relevance 26 .In this study, we measured urinary IL-13 levels in MCD patients, however, no significant differences were observed between HCs and MCD patients.Further study is needed to comprehensively assess their status.Some studies have reported that levels of serum sST2, the soluble form of the IL-33 decoy receptor, are elevated in patients with asthma and correlated with disease severity [17][18][19] .In this study, we demonstrated that sST2 levels were significantly elevated in serum and urine samples obtained from patients with MCD.Serum sST2 levels were positively correlated with serum total protein levels and negatively correlated with serum creatinine levels.Although we could not observe a correlation between sST2 levels and proteinuria directly, we speculated that serum sST2 levels are correlated with disease severity in NS.Furthermore, we found that serum and urine levels of sST2 were decreased after the onset of remission, corresponding with disease activity in patients with MCD.We speculated that sST2 expression is upregulated in IL-33-related immune responses as a form of www.nature.com/scientificreports/negative feedback, and these results might therefore reflect the severity of the type 2 immune response.Type 2 immunity induces a complex inflammatory response that could become an important disease driver.Préfontaine et al. reported that IL-33 expression by epithelial cells is increased in bronchial asthma 20 .In this study, we observed that IL-33 was expressed on glomerular epithelial cells confirmed as positive for expression of the podocyte nuclear marker p57 21 .IL-33 expression on glomerular epithelial cells was significantly increased among patients with MCD, suggesting that IL-33 plays a role in the blood-urine barrier in glomerular epithelial cells.IL-33 is a nuclear protein lacking a signal sequence, unlike conventional cytokines, and it functions as a rapid-acting alarmin that activates immune cells.Although excessive necrotic change is not thought to occur in glomerular epithelial cells in MCD, some studies have revealed that IL-33 can be released from the cytoplasm without cell death by increasing the intracellular calcium concentration through ATP administration 28 .Interestingly, we found that IL-33 expression was localized predominantly in the cytoplasm of glomerular epithelial cells rather than the nucleus.Gordon et al. demonstrated that IL-33 alternative splice variants localized to different cellular compartments in primary human airway epithelial cells 29 ; that is, full-length IL-33 localized to the cell nucleus, whereas variants resulting from deletion of exons 3 and 4 localized to the nucleus and cytoplasm.They showed that ∆exon 3, 4 variants are secreted by ionomycin without cell death to a greater extent than the fulllength protein and that this is strongly associated with airway expression of type 2 cytokine genes in asthma.Although the distribution of IL-33 was not identical to that in airway epithelial cells in terms of negativity in the nucleus, we speculate that IL-33 might be secreted from the cytoplasm of glomerular epithelial cells in the absence of cell death in MCD.
We also found that treatment of cultured mouse glomerular epithelial cells with serum obtained from a patient at the onset of MCD led to an increase in the IL-33 concentration in the supernatant.This result suggests that some substance in the serum stimulates the secretion of IL-33 by podocytes in MCD and confirms our hypothesis that IL-33 plays a role in the blood-urine barrier in glomerular epithelial cells.Multiple stressors can induce podocytes to acquire features of immune cells 30,31 .CD80 is a transmembrane protein normally expressed on antigen-presenting cells to regulate the immune response 30 .Expression of CD80 is reportedly also upregulated in podocytes of rodent NS models, thereby contributing to the pathogenesis of proteinuria 32 .Lai et al. reported that, in Wistar rats, overexpression of IL-13, a type 2 cytokine induced by IL-33, results in minimal change-like www.nature.com/scientificreports/nephropathy, which is characterized by severe proteinuria and fusion of podocyte foot processes with upregulation of CD80 expression in glomeruli 33 .Furthermore, Garin et al. showed that CD80 levels are elevated in urine from MCD patients 34 ; the source of this elevated CD80 could be podocytes 35 .Although the significance of CD80 in MCD remains controversial 30 , elucidating the function of this molecule could help explain the role of type 2 cytokines in MCD.In this study, we did not observe an upregulation of CD80 mRNA expression in cultured podocytes stimulated with serum from an MCD patient at disease onset, as opposed to Angptl4, another candidate mediator of MCD 2,36 .We speculated that IL-33 secreted by podocytes induces the activation of type 2 cytokines produced by ILC2 or Th2 cells to induce proteinuria (Fig. 4).
Recently, it has been sighted that the inflammasome component of NLRP3 regulate inflammation, pyroptosis, apoptosis, and fibrosis in kidney diseases 37 .R Fu et al. reported that NLRP3 inflammasome was activated in podocytes from lupus-prone mice and patients with LN 38 .Although there is not strong evidence for an interaction of the NLRP3 inflammasome and IL-33/ST2 axis 39 , some studies have proposed the NLRP3/IL-33/ST2 axis 40,41 .Ma et al. reported the positive correlation between the IL-33 and NLRP3 in patient with lupus nephritis 41 .In this study, we did not observe that treatment of podocytes with serum from a MCD patient enhanced NLRP3 inflammasome signaling.Further studies will be needed to fathom the association between NLRP3 and IL-33/ST2.
There are some limitations in this study.Firstly, because the allergic patients without MCD were not enrolled as controls, it is difficult to determine how much effect MCD without allergic conditions has on the levels of sST2.Secondly, due to the quantification of IL-33 levels utilizing a commercially procurable ELISA kit, the potential impact stemming from elevated sST2 concentrations within the serum or urine samples could not be mitigated.IL-33 biding with sST2 poses heightened complexity in its discernibility through the utilization of ELISA.Thirdly, our results of cell culture study were based on murine podocyte cell line.The more optimal approach for assessing the reaction of glomerular epithelial cells to human serum should be based on human podocyte.Finally, we could not definitively exclude the potential influence of complement factors on our in vitro findings, as we exposed human serum to experimentation without prior inactivation.
In conclusion, we demonstrated for the first time that levels of sST2, the soluble form of the IL-33 receptor, are elevated in the serum and urine of patients with MCD.IL-33 was detected in glomerular epithelial cells of MCD patients, and cell culture experiments revealed that serum obtained from an MCD patient could stimulate

Patients and samples
All patients and their legal guardians provided written informed consent regarding preservation of blood samples, urine samples, and kidney biopsy specimens.Opt-out methods were used to obtain informed consent regarding the measurement of IL-33/sST2 values at diagnosis and evaluation of IL-33 deposition.All enrolled patients agreed to participate in this study.The protocol of this study was established according to the Declaration of Helsinki and approved by the Showa University Research Ethics Review Board (No.3118).A total of 49 patients with MCD admitted to Showa University Hospital (Tokyo, Japan) between April 2009 and March 2022 were selected for the study (Table 1).The diagnosis of MCD was based on renal biopsy findings.All patients had proteinuria > 1.0 g/day.Blood samples were collected from all patients at the time of renal biopsy, and urine samples were obtained from 44 patients.Blood samples were collected from 29 patients in complete remission, and urine samples were obtained from 26 of these patients.

Enzyme-linked immunosorbent assay
The serum and urine concentrations of IL-13, IL-33 and sST2 were measured using commercially available ELISA kits (R&D Systems, San Diego, CA, USA), according to the manufacturer's instructions.All urine measurements were normalized to urinary creatinine (Cr) levels.

Immunofluorescence staining
We selected kidney biopsy specimens diagnosed as MCD (n = 7) with a sufficient number of glomeruli (> 5) and 0-h protocol biopsy specimens of kidney grafts (n = 7) without glomerular lesions, and the latter were used as normal controls.Formalin-fixed, paraffin-embedded kidney biopsy specimens were sectioned (3-µm thick), deparaffinized, and washed in phosphate buffered saline (PBS).Antigen-retrieval solution (0.01 mol/L citrate buffer; pH 6.0) was heated in a microwave oven at 500 W of power for 5 min, and then the tissue sections were placed in the solution and heated in the microwave oven at 500 W of power for 9 min.After cooling at room temperature for 30 min, the sections were washed three times with PBS for 2 min each.Background Buster ® (Innovex Biosciences, Richmond, CA, USA) was applied to the sections and incubated at room temperature for 15 min to block nonspecific background staining.The sections were then washed three times with PBS and incubated with goat polyclonal anti-human IL-33 antibody (working dilution 1:100; R&D Systems) at 4 °C overnight.After washing with PBS, the sections were incubated with donkey polyclonal anti-goat IgG H&L (Alexa Fluor ® 647) (working dilution 1:200; Abcam, Cambridge, UK) for 60 min at room temperature.The sections were then washed with PBS and incubated with mouse monoclonal anti-p57 Kip2 antibody (working dilution 1:600; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight.After washing, the sections were incubated with goat polyclonal anti-mouse IgG (H + L) (Alexa Fluor ® 488) (working dilution 1:200; Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 60 min.Finally, the sections were counterstained with 4′6-diamidino-2phenylindole (DAPI) (Vector Laboratories, Newark, CA, USA).The nuclei of arteriole or small artery endothelial cells, which typically stain positive, were used as internal controls for IL-33 staining 42 .

Quantification of immunostaining
Image acquisition was performed using a BZ-X800 all-in-one fluorescence microscope (Keyence, Osaka, Japan).All images were processed using ImageJ software, version 1.53a.Podocyte cells were identified based on p57 staining of nuclei 21,43 .The number of podocytes double-positive for IL-33 and p57 per glomerular cross-section was also determined.An average of 39.7 control glomeruli and 16.3 MCD glomeruli were assessed in this study.

Cell culture study
Conditionally immortalized mouse podocyte cells (MPCs) were kindly donated by Dr. Peter Mundel (Boston, MA, USA).MPCs were grown in RPMI-1640 (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% streptomycin-penicillin mixture (Thermo Fisher Scientific) in an atmosphere 5% CO 2 and 95% air.MPCs were cultured for propagation at 33 °C with 10 U/mL of recombinant mouse γ-interferon (Merck, Darmstadt, Germany).Upon reaching 80% confluence, the MPCs were shifted to 37 °C without γ-interferon for 10 days to induce differentiation.Differentiated MPCs were stimulated for 6 h with 15% serum from a patient with MCD (Supplemental Table 1).Serum was directly added to the MPCs without activation of complements 44 .Expression of the mouse angiopoietin-like protein 4 (Angptl4), cluster of differentiation (CD)80, NLR family pyrin domain containing 3 (NLRP3), Caspase 1 (Casp1), and GAPDH genes were analyzed using real-time reverse transcription-polymerase chain reaction as previously described 45 .Levels of mouse IL-33 in the supernatant were determined using an ELISA kit (R&D Systems), according to the manufacturer's instructions.

Statistical analysis
Quantitative data are expressed as mean and standard deviation (SD), median and interquartile range (IQR), or mean ± standard error of the mean (SEM), as appropriate.Qualitative data are presented as absolute numbers and percentages.Differences between the groups were evaluated using the Mann-Whitney U test or Kruskal-Wallis test.Correlations between various parameters were analyzed using the Spearman's rank correlation test.Differences in sST2 levels between the time of diagnosis and onset of remission were analyzed using the Wilcoxon

Figure 3 .
Figure 3. Localization of IL-33 and p57 in glomeruli.Representative images of kidney biopsy specimens of control kidney (CON) and MCD tissue stained for IL-33 (red) and p57 (green) and counterstained with DAPI (blue).(A) IL-33 was observed in the nucleus of endothelial cells of glomerular vascular poles, as well as arteries and arterioles (not shown).(B) In patients with MCD, IL-33 was detected in the cytoplasm of glomerular epithelial cells confirmed as positive for the podocyte nuclear marker p57.Original magnification × 400.

Table 1 .
Characteristics of patients with MCD at the time of renal biopsy.Data are expressed as mean (SD), median (IQR), or number (%). a Data were not available for 2 patients.b Data were not available for 4 patients.eGFR, estimated glomerular filtration rate.