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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2018  |  Volume : 29  |  Issue : 3  |  Page : 591-597
Evaluation of interleukin-18 in children with steroid-sensitive nephrotic syndrome before and after using levamisole


1 Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Biochemistry, Faculty of Medicine, Zagazig University, Zagazig, Egypt

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Date of Submission09-May-2017
Date of Decision18-Jul-2017
Date of Acceptance19-Jul-2017
Date of Web Publication28-Jun-2018
 

   Abstract 

Levamisole is often discussed as the first alternative to steroids. It is an antihelminthic drug that has been used for steroid-sensitive nephrotic syndrome (SSNS) for more than 20 years. Interleukin (IL)-18, a member of the IL-1 cytokine superfamily, is recognized as an important regulator of immune responses. The aim of the study was to investigate the IL-18 levels in serum from children with SSNS during relapse and remission after using levamisole or three months in a trial to test the efficacy of its action in reducing frequency of relapses in SSNS. This study was done on 23 children with frequently relapsing SSNS treated with levamisole besides steroids, then followed up three months; 16 males and seven females (mean age: 7.96 years and median 8 years). Clinical and laboratory assessments were done before starting therapy and after three months including cumulative dose of steroids and serum IL-18. We found that IL-18 level showed a significant elevation after three months of levamisole therapy compared to its level before initiation of levamisole therapy, with no relapses in these three months, no reported side effect, and significant reduction of cumulative dose of steroids. Levamisole effectiveness in reduction of relapses of SSNS may be due to resetting of the type 1/type 2 imbalance, proved by induction of IL 18 may be useful in the therapy.

How to cite this article:
Youssef DM, Abd Al-atif AM, El-Khateeb SS, Elshal AS. Evaluation of interleukin-18 in children with steroid-sensitive nephrotic syndrome before and after using levamisole. Saudi J Kidney Dis Transpl 2018;29:591-7

How to cite this URL:
Youssef DM, Abd Al-atif AM, El-Khateeb SS, Elshal AS. Evaluation of interleukin-18 in children with steroid-sensitive nephrotic syndrome before and after using levamisole. Saudi J Kidney Dis Transpl [serial online] 2018 [cited 2022 May 17];29:591-7. Available from: https://www.sjkdt.org/text.asp?2018/29/3/591/235173

   Introduction Top


Interleukin-18 (IL-18) is a unique cytokine that plays a critical role in various immuno-logic responses; it is one of the most important promising urinary biomarkers under investigation being a newly discovered pro-inflammatory cytokine that is structurally and functionally related to the IL-1 family and has recently been demonstrated to have an active role in a variety of renal diseases' processes including ischemia-reperfusion (I/R), allograft rejection, infection, autoimmune conditions, and malignancy.[1] Levamisole is a medication used to treat parasitic worm infection; about 3% of an oral dose is eliminated unchanged in the 24 h urine of humans.[2] Some studies reported that children with active steroid-sensitive nephrotic syndrome (SSNS) seem to have a shift to type-2 cytokine production, and IL-18 expression is significantly correlated with this type-2 immune response.[3] Levamisole is effective in reducing the number of relapses in children with frequently relapsing and steroid-dependent nephrotic syndrome (SDNS).[4] Levamisole shifts the immune response in BN rats away from the type-2 end of the spectrum and is capable of blunting the type-2 biased immune response.[5] As there are only a very few researches studying the effect of using of levamisole in the treatment of SSNS and on the cytokine levels in these patients, we aimed this study to evaluate IL-18 in children with SSNS during active disease and then in remission, before and after using levamisole.


   Subjects and Methods Top


The study was conducted at Pediatric Nephrology Unit, Zagazig University Hospitals in the period of January 2016 to March 2017. This cohort study was carried out on 23 children of SSNS aged from three to 12 years examined twice and treated with levamisole introduced at a dose of 2.5 mg/kg every other day beside steroids treated with the standard initial steroid therapy, consisting of daily dosages of prednisone, 60 mg/m2 body surface area, until remission was achieved, followed by 40 mg/m2 given on alternate days, and finally various steps tapering-off over the next four to eight weeks, then followed up three months after using levamisole.

The study was approved by the local Ethical Committee, and a written informed consent was obtained from all patients' parents before starting the clinical study.

The study included 23 cases of frequent relapsing SSNS admitted to Pediatric Nephrology Unit, Zagazig University Hospitals.

Frequent relapsing SSNS is defined in KDIGO guidelines as patients with two or more relapses within six months of the initial response or with four or more relapses in any 12-month period.[6]

We included children with frequent relapsing SSNS aged from three to 12 years in activity new starting using levamisole and three months after using levamisole. We excluded children with steroid-resistant NS and children with active infection.

All particiants enrolled in the study were subjected to the following; clinical assessment (complete history taking: age and sex, duration of illness, and consanguinity or similar conditions in the family), physical examination (abdominal examination included liver, spleen, and kidney and thorough clinical examination included edema in the whole body, especially lower limb and eye lid puffiness), and laboratory investigations (complete blood count, serum urea and creatinine, serum total proteins and albumin, serum cholesterol, and urine sample for estimating proteins in urine).

Steroid cumulative load three months before using levamisole and three months after using it were measured. Serum IL-18 was measured as follows.

The Sunred human IL-18 kit is an in vitro a solid phase sandwich enzyme-linked immu-nosorbent assay for the quantitative measurement of human IL-18. A monoclonal antibody specific for human IL-18 has been coated into the wells of the microtiter strips provided. Samples including standards of known human IL-18 content, control specimens, and unknowns are pipetted into these wells and IL-18 present in a sample is bound to the immobilized antibody-coated wells.

The wells are washed and biotinylated antihuman IL-18 antibody is added. During the first incubation, the human IL-18 antigen binds to the immobilized (captured) antibody on one site and to the solution phase biotinylated antibody on a second site.

After removal of excess second antibody, streptavidin-peroxidase (enzyme) is pipetted to the wells. This binds to the biotinylated antibody to complete the four-member sandwich. After a second incubation and washing to remove the entire unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color which is measured at 450 nm. The intensity of this colored product is directly proportional to the concentration of human IL-18 present in the original specimen.


   Statistical Analysis Top


Data were analyzed by Statistical Package for the Social Sciences (SPSS) version 22.0 for Windows (SPSS Inc., Chicago, IL, USA). Continuous data are presented as the mean (standard deviation) if normally distributed and the median (range) if not normally distributed; nominal data were presented by the count and percentage. Normality is checked by Shapiro-Wilk test. The paired samples t-test (dependent t-test) is used to compare the means between two related groups on the same continuous, dependent variable. Wilcoxon Signed-Rank test (nonparametric alternative to paired t-test) is used to compare two sets of data that come from the same participants. McNemar exact test: is used to determine if there are differences on a dichotomous dependent variable between two related groups when there are sparse data (some cells <5). Pearson's correlation test is used to estimate the strength of linear relationship between two continuous variables; at least one of the two variables must follow a normal distribution. Spearman's correlation is a nonparametric measure of the strength and direction of the association between two continuous variables; at least one of the two variables violated from a normal distribution; in all tests, P <0.05 was considered to indicate significance.


   Results Top


The demographic and characteristics of the studied participants are shown in [Table 1]. We noticed no relapses in our 23 cases after using levamisole for three months and no reported side effects as well.
Table 1: Baseline characteristics of the studied children.

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Moreover, we found a highly significant decreases (P <0.05) in weight and blood pressure measurements after three months of levamisole therapy compared to their measurements before initiation of levamisole therapy, a highly significant difference (P = 0.001) in the proportion of protein in urine before initiation of levamisole therapy compared to after three months of levamisole therapy [Table 2]. A negative correlation but not statistically significant correlations (P >0.05) were found between IL-18 and steroid load either before or after levamisole therapy [Table 3] and [Figure 1].
Figure 1: Line graphs show correlations between interleukin-18 level and steroid load before initiation and after 3 months of levamisole therapy.

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Table 2: Comparison between clinical findings and laboratory data in the studied children before initiation of levamisole therapy and after 3 months of levamisole therapy.

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Table 3: Correlations between interleukin-18 level and steroid load before and after 3 months of levamisole therapy.

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   Discussion Top


To reduce steroid toxicity, other immuno-suppressive agents have been proposed for frequently relapsing NS patients. Since the early 1980s, levamisole, an antihelminthic agent that has immune-modulator properties, has also been used. Experiments conducted in Brown Norway rats suggest that levamisole may act by augmenting the type 1 response and reciprocally by downregulating the type 2 response by selective induction of gene transcription of the key cytokines IL-18.[7] It was suggested also that levamisole's mode of action is attributable to its direct effects on podocytes; as it induced expression of glucoc-orticoid receptor (GR) and activated GR signaling in podocytes. Furthermore, leva-misole was able to protect against podocyte injury in a puromycin aminonucleoside-treated cell model.[8]

We studied 23 children treated with leva-misole as an add-on therapy for steroid-sparing for three months in cases with SSNS with frequent relapses; we tested the patients' clinical characteristics (including their cumulative steroid dose) and laboratory results including serum IL-18 before and after its usage. As there is a paucity of literature regarding the role of IL-18 in disease process and on reviewing the literature, we found no study about effect of using levamisole on IL-18 in SSNS human children. Therefore, to the best of our knowledge, this will be one of these early studies.

In our study, none of our patients experienced relapse in the three-month duration of the study, and we did not notice any of the levamisole side effects. This matches with Donia et al[9] who suggested that levamisole reduces the frequency of relapses and cortico-steroid doses in children with frequently relapsing nephrotic syndrome (FRNS) and SDNS. In most cases, levamisole therapy did not cause severe side effects such as reversible leukopenia, sporadically liver toxicity, seizures, gastrointestinal symptoms, psoriasis-like cutaneous reaction, and vasculitis.

In our study, IL-18 level showed a highly significant elevation after three months of levamisole therapy compared to its level before initiation of levamisole therapy. This elevation should be greatly emphasized considering that IL-18 is a unique cytokine that can trigger both type 1 and type 2 immune responses depending on the predominant cytokines. Printza et al[10] who found that IL-18 levels were increased in all stages of SSNS especially. In contrary to Shalaby et al,[3] who evaluated type 1/type 2 cytokine profile in children during different stages of primary SSNS and investigated the involvement of IL-18; they reported that children with active SSNS seem to have a shift to type 2 cytokine production, and IL-18 expression is significantly correlated with this type-2 immune response. Our finding can be explained by levamisole effect by resetting the immune balance toward a type 1 response through induction of IL-18 as it has been known as an immune-stimulatory agent rather than immu-nosuppressive and it has been hypothesized to normalize deficient cell-mediated immunity.[11] It enhances T-cell responses by stimulating T-cell activation and proliferation, Furthermore, it potentiates monocyte and macrophage functions, including phagocytosis and chemotaxis, and increases neutrophil mobility, adherence, and chemotaxis.[12] Szeto et al[13] showed that the upregulation of interferon-c at day 3 after levamisole was accompanied by marked upregulation of IL-18 expression; so we hypothesized that the drug acts by boosting cell-mediated immunity through induction of type-1 cytokines and elevating IL-18 is a mark of this action.

In our study, steroid cumulative load three months after initiation of levamisole therapy shows a highly significant decrease when compared to its level before initiation of levamisole therapy in our patients, our finding is in agreement with Madani et al.[14] Both studies found that the cumulative steroid dose given before levamisole therapy could be reduced following the introduction of levamisole. Furthermore, Madani et al study[14] demonstrated that levamisole therapy significantly reduced relapse rate and steroid dose in SDNS and FRNS.

Our study showed a negative correlation but not statistically significant correlations between IL-18 and steroid load either before or after levamisole therapy.


   Conclusion Top


We conclude that levamisole is effective in reducing the number of relapses in children with frequently relapsing and SDNS. We propose that the effect of levamisole is through shifting of type 2 to type 1 immune response proved by rise of serum IL-18 after 3 months of therapy.


   Limitation of the study Top


A number of patients are limited, and the duration of the study is short, so we recommend a study with longer duration on larger number of patients.

Conflict of interest: None declared.

 
   References Top

1.
Bonomini F, Foglio E, Rodella LF, Rezzani R. Clinical biomarkers in kidney diseases. Front Biosci (Schol Ed) 2010;2:591-615.  Back to cited text no. 1
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2.
Baselt R. Disposition of Toxic Drugs and Chemicals in Man. 9th ed. Seal Beach, CA: Biomedical Publications; 2011. p. 901-2.  Back to cited text no. 2
    
3.
Shalaby SA, Al-Edressi HM, El-Tarhouny SA, Fath El-Bab M, Zolaly MA. Type 1/type 2 cytokine serum levels and role of interleukin-18 in children with steroid-sensitive nephrotic syndrome. Arab J Nephrol Transplant 2013;6: 83-8.  Back to cited text no. 3
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Kuzma-Mroczkowska E, Skrzypczyk P, Paήczyk-Tomaszewska M. Levamisole therapy in children with frequently relapsing and steroid-dependent nephrotic syndrome: A single-center experience. Cent Eur J Immunol 2016;41:243-7.  Back to cited text no. 4
    
5.
Conti B, Jahng JW, Tinti C, Son JH, Joh TH. Induction of interferon-gamma inducing factor in the adrenal cortex. J Biol Chem 1997;272: 2035-7.  Back to cited text no. 5
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Biographic and disclosure information. Kidney Int Suppl (2011) 2012;2:252-7.  Back to cited text no. 6
    
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Striz I, Krasna E, Honsova E, et al. Interleukin 18 (IL-18) upregulation in acute rejection of kidney allograft. Immunol Lett 2005;99:30-5.  Back to cited text no. 7
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8.
Jiang L, Dasgupta I, Hurcombe JA, et al. Levamisole in steroid-sensitive nephrotic syndrome: Usefulness in adult patients and laboratory insights into mechanisms of action via direct action on the kidney podocyte. Clin Sci (Lond) 2015;128:883-93.  Back to cited text no. 8
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9.
Donia AF, Amer GM, Ahmed HA, et al. Levamisole: Adjunctive therapy in steroid dependent minimal change nephrotic children. Pediatr Nephrol 2002;17:355-8.  Back to cited text no. 9
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Printza N, Papachristou F, Tzimouli V, Taparkou A, Kanakoudi-Tsakalidou F. IL-18 is correlated with type-2 immune response in children with steroid sensitive nephrotic syndrome. Cytokine 2008;44:262-8.  Back to cited text no. 10
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Robinson RF, Nahata MC, Mahan JD, Batisky DL. Management of nephrotic syndrome in children. Pharmacotherapy 2003;23:1021-36.  Back to cited text no. 11
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Renoux G. The general immunopharmacology of levamisole. Drugs 1980;20:89-99.  Back to cited text no. 12
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13.
Szeto C, Gillespie KM, Mathieson PW. Levamisole induces interleukin-18 and shifts type 1/type 2 cytokine balance. Immunology 2000;100:217-24.  Back to cited text no. 13
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Madani A, Fahimi D, Taghaodi R, et al. An estimation of steroid responsiveness of idio-pathic nephrotic syndrome in Iranian children. Iran J Pediatr 2010;20:199-205.  Back to cited text no. 14
[PUBMED]    

Top
Correspondence Address:
Dr. Doaa Mohammed Youssef
Department of Pediatrics, Faculty of Medicine, Zagazig University, Zagazig
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-2442.235173

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