Our clinical and laboratory data suggest that levamisole should be added to the list of immunotherapeutic agents that have direct actions on podocytes and point to the usefulness of levamisole in the treatment of adult as well as paediatric patients.

CLINICAL PERSPECTIVES

  • We aimed to add to the evidence base for the use of levamisole as a second-line drug in steroid-dependent nephrotic syndrome in adults and to understand its mode of action on cultured human podocytes, the cell type which is the main target of injury in nephrotic syndrome.

  • Our clinical results show that levamisole was helpful in reducing or stopping steroids or calcineurin antagonists in the majority of cases and was generally well-tolerated, although significant adverse effects occurred in a minority of patients. Our laboratory work suggested that levamisole's effects on podocytes are related to, but different from, those of steroids.

  • RCTs are needed to establish the role of levamisole in nephrotic syndrome in adults: our results provide prima facie evidence that this drug can be useful and highlight pathways in podocytes that should be the target for new drug design.

INTRODUCTION

Minimal change disease is the third most common cause of primary nephrotic syndrome in adults (10–15% of the total) [1]. The treatment of minimal change nephropathy (MCN) in adults remains problematic; although the majority of patients respond to an initial course of corticosteroids, relapse is common when the dosage of steroids is reduced and steroid-dependence and/or frequently relapsing nephrotic syndrome are common, so that cumulative steroid toxicity is a major clinical problem. Second-line agents are of variable effectiveness, have major potential toxicities and are not supported by robust clinical trial evidence [2]. The literature on children with steroid-sensitive nephrotic syndrome (SSNS) is somewhat stronger so that the treatment policy in adults is often developed by extrapolation from the paediatric experience [2]. One agent that has been shown to be effective in randomized controlled clinical trials (RCTs) in children with SSNS is levamisole [37] but there are no reports on the use of this agent in adults with nephrotic syndrome [1]. Levamisole is an imidazothiazole derivative, originally used as an anti-helminthic treatment, but later shown to have an effective role as an adjuvant to chemotherapy with 5-fluorouracil in the treatment of colonic carcinoma [8] as well as its role in children with nephrotic syndrome where it is used as a steroid-sparing agent. In addition to reducing the dose of steroids required to maintain remission, it also reduces the frequency and severity of relapses [911]. This steroid-sparing capacity of levamisole is highly desirable due to the cumulative adverse consequences of long-term steroid use.

Levamisole's modes of action are poorly understood; in the treatment of certain cancers it is considered to be an immune-stimulant, but as an explanation for its effects in nephrotic syndrome this is counter-intuitive since most other therapies that are effective in nephrotic syndrome are immune-suppressive. An alternative hypothesis is that levamisole's usefulness is because of direct actions on the kidney podocyte, as has been suggested for other agents whose use was initially motivated by their effects on the immune system [1215]. In the healthy kidney, proteinuria is prevented by the glomerular capillary wall, a complex structure comprising podocytes (glomerular epithelial cells) on the urinary aspect, glomerular endothelial cells (GnECs) on the luminal aspect and the glomerular basement membrane in between. Podocytes are widely accepted to be the major target of injury in nephrotic syndrome [16]. These cells have a very limited capacity for repair or regeneration, so that therapeutic strategies aimed at preventing or limiting podocyte injury and/or at promoting podocyte repair or regeneration therefore have major potential clinical and economic benefits [12].

We report a study in two related parts, made possible by the academic possibilities provided by the multidisciplinary team in Bristol consisting of both clinicians and basic scientists. First, we report our clinical experience in the use of levamisole in adults with MCN/SSNS, and secondly we report our laboratory studies using human podocytes which allowed us to explore mechanisms of the effects observed in our clinical studies. We have found that patients greatly welcome being told that their clinical treatment is being linked to active research on the subject. Together, our clinical and laboratory data support the usefulness of levamisole in adults with MCN and indicate that its effects include direct actions on intracellular signalling in podocytes. Further work is needed, including RCTs in adults with nephrotic syndrome and further detailed laboratory investigation, to establish whether levamisole's mode of action in SSNS is attributable to its direct effects on podocytes.

MATERIALS AND METHODS

Clinical

Standard management of renal biopsy-confirmed MCN in adults [2] is that initial treatment is with oral prednisolone at a starting dose of 1 mg/kg of body weight up to a maximum of 80 mg/day. Once remission is achieved, the prednisolone dosage is progressively reduced, aiming to withdraw it altogether. If relapse occurs, second-line agents are considered after a discussion with the patient about the risks and benefits of the various agents available. The specialist nephrotic syndrome clinic in Bristol often receives referrals of patients from other clinics in the city or further afield. All patients described in the present report were under follow-up in the Bristol clinic, but most received their initial treatments elsewhere. Renal biopsies had been performed some years earlier and were not repeated. Based on the paediatric literature, levamisole is one of the second-line options considered in this clinic if an adult with MCN has frequently relapsing and/or steroid-dependent nephrotic syndrome. The dosage used is 1.25 mg/kg/day (based on the standard paediatric dosage of 2.5 mg/kg on alternate days; http://www.bnf.org/bnf/index.htm). Drugs used in the treatment of nephrotic syndrome are typically used ‘off-licence’ on the basis of informed consent from individual patients; this was the case with levamisole use in this clinic, with no formal ethical approval being sought because this was not a clinical trial.

Laboratory

Reagents

Unless otherwise stated, all materials were purchased from Sigma–Aldrich. Primary antibodies used for Western blotting were obtained from Cell Signaling Technology (all anti-rabbit). Mineralocorticoid receptor (MR) antibody was a kind gift from Dr B. Conway-Campbell and Professor S. Lightman (University of Bristol). Mouse anti-β-actin was from Sigma. Species-specific secondary horseradish peroxidase (HRP)-linked antibodies were from GE Healthcare.

Levamisole was purchased from Sigma–Aldrich. Concentrations used for cell experiments were based on calculations of ‘therapeutic’ concentrations in vivo. The mean peak concentration following a single 2.5 mg/kg dose has been reported to be 0.48±0.22 μg/ml and therefore we used 1 and 10 μM which fall in this range [17].

Cell lines

Wild-type conditionally immortalized human podocytes, developed at the Academic Renal Unit, were cultured as previously described [18,19].

Protein extraction, SDS/PAGE and Western blotting

Protein extraction, SDS/PAGE and Western blotting were carried out as described in [20].

Nuclear and cytoplasmic extraction

Nuclear and cytoplasmic extraction was carried out using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) as per the manufacturer's instructions.

RNA extraction, cDNA synthesis and RT-PCR

These analyses were carried out as described previously [20]. Primer sequences used are listed below. Primers for real-time reverse transcription (RT)-PCR: cluster of differentiation (CD) 80 forward primer 5′-TGCCTGACCTACTGCTTTGC-3′, reverse primer 5′-TGCTTCTGCGGACACTGTTAT-3′; NADPH oxidase 4 (Nox4), forward primer 5′-GTTAAACACCT-CTGCCTGTTCA-3′, reverse primer 5′-GTGATACTCTGGCC-CTTGGT-3′; p22phox, forward primer 5′-TACTATGTT-CGGGCCGTCCT-3′, reverse primer 5′-CAGCCGCCAGT-AGGTAGATG-3′; p47phox, forward primer 5′-GTCGCCATCAAGGCCTACAC-3′, reverse primer 5′-ACGTCGTCTTTCCTGATGACC-3′; apoptosis-inducing factor (AIF), forward primer 5′-GACAAATGGCTAGCTCTGGTG-3′, reverse primer 5′-TAGGCACCAGCTCCTACTGT-3′; β-actin, forward primer 5′-ATGTGGCCGAGGACTTTGATT-3′, reverse primer 5′-AGTGGGGTGGCTTTTAGGATG-3′; dual-specificity protein phosphatase 1 (DUSP1), forward primer 5′-AGGACATTTGGGCTGTGTGT-3′, reverse primer 5′-TTGGACTGAGAGAGGAGCGT-3′; FK506-binding protein 5 (FKBP5), forward primer 5′-AGGGGGAACTGGCAAAAGTG-3′, reverse primer 5′-TCTTGAGTGGTAATGGGCACC-3′.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde (Sigma) for 15 min and then permeabilized with 0.2% Triton X-100 (BDH Laboratory Supplies) for 5 min. Non-specific binding was blocked with 3% BSA. Primary antibodies were applied at 1:400 dilutions in blocking buffer for 1 h at room temperature. After washing cells were incubated with appropriate Alexa Fluor-labelled secondary antibodies diluted 1:500 in blocking buffer for 1 h. Three further PBS washes were performed before mounting on to microscope slides (Fisherbrand) with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Images were obtained using a Leica MM AF microscope, Leica DFC350FX camera, with Leica Application Suite Advanced Fluorescence (LAS AF) software platform.

Statistics and computing

Experiments were carried out a minimum of n=3 times on separate passages of cells. Densitometry was carried out on separate blots from each experiment using QuantityOne software. Excel software (Microsoft Corporation) was used for normalization of raw densitometry data obtained from QuantityOne. Data were normalized to β-actin. Prism 5.01 (GraphPad Software) was used for generation of descriptive statistics S.D. for error bars on graphs and ANOVAs. Two-tailed Student's unpaired t test was used in the comparison between two groups. One way ANOVAs were used where there were more than two groups to compare. Bonferroni post-hoc tests for selected columns, following ANOVA to correct for small sample size, were used to identify statistical significance. P<0.05 was taken to indicate statistical significance.

RESULTS

Clinical series

Fifteen adult patients (nine male, six female; age range at referral 17–62 years, mean 38.7 years) with SSNS were treated with levamisole as a second-line agent because of frequent relapses/steroid-dependence and/or toxicity of alternative therapies. The presenting features, previous treatments and clinical outcomes are summarized in Table 1. In 14 of the 15 patients (93.3%), levamisole led to significant benefit in terms of reduction or stopping of other more toxic medications and/or reduced relapse frequency and/or better quality of life (Figure 1). Ten patients, in whom steroid withdrawal had previously led to relapse, were able to stop steroids altogether, including one patient in whom cyclosporine was also withdrawn; furthermore, three patients that had been treated with tacrolimus alone were able to reduce and stop it without relapse. One patient was unable to stop steroids altogether but remission was maintained on a lower dose than previously. Serious adverse effects were seen in three out of 15 patients (20%, cases 5, 6 and 11). In two patients (cases 6 and 11) neutropenia led to the cessation of levamisole; one patient (case 6) was rechallenged and the neutropenia recurred. The other (case 11) was not rechallenged because it was felt that the levamisole had already served its purpose in allowing tacrolimus withdrawal. The single patient that did not benefit (case 5) had probable levamisole allergy with a febrile reaction that recurred on rechallenging. Furthermore, one patient (case 14) developed a rash that might have been attributable to levamisole allergy although he was on multiple other drugs and a skin biopsy and dermatological opinion were inconclusive about causation. Two patients (cases 3 and 4) attributed gastrointestinal symptoms to levamisole, both improved when the dosage was reduced, allowing continuation of the drug.

Table 1
Patient data

Abbreviations: alt, alternate; CyA, cyclosporin A; CyAD, cyclosporin A-dependent; CyP, cyclophosphamide; dose-dep, dose-dependent; F, female; F/U, follow-up; FR, frequently relapsing; FRNS, frequently relapsing nephrotic syndrome; FSGS, focal segmental glomerulosclerosis; GI, gastrointestinal; L, levamisole; M, male; mes hyper, mesangial hypercellularity; mes scl, mesangial sclerosis; MCN, minimal change nephropathy; MMF, mycophenolate mofetil; ND, not done; NS, nephrotic syndrome; PR, partial response; Pred, prednisolone; rechall, rechallenge; rel, relapse; rem, remission; Rx, initial treatment; S, sex; SD, steroid-dependent; SDNS, steroid dependent nephrotic syndrome; SS, steroid-sensitive; Tac, tacrolimus; TacD, tacrolimus-dependent; y, years; x2, two times.

CaseAge at referral (y)SClinical featuresRenal biopsyRxEffectLevamisole benefitAdverse effectsLatest F/U
61 NS bladder cancer MCN Pred Rem, rel Steroid withdrawal None 14 y rem 
17 NS MCN (mes hyper) Pred Rem, FR Steroid withdrawal SS rel x2 None 10 y rem 
40 NS MCN Pred Rem, FR, SD SS, rem on low dose Pred GI 5 y rem 
37 NS MCN (mes scl) Pred then CyA, CyP FR, SD Rem off Pred GI (dose-dep) 14 y rem 
36 NS MCN Pred, CyA, CyP FR, CyAD Fever, nausea, recurrent on rechall Probable allergy CyAD MMF, FR 
18 SDNS since 7 y ND Pred 25 alt days SD Steroid withdrawal Neutropenia (recurrent on rechall) SD, FR 
17 SSNS ND Pred FR Steroid withdrawal None 6 y rem 
46 FRNS since 7 y MCN (mes hyp) Pred, CyA, CyP x4 FR, SD Steroid withdrawal None 4 y rem, SS 2 y rem 
19 FRNS MCN Pred, CyA, MMF FR Steroid withdrawal None 3 y rem 
10 45 SDNS since 2 y FSGS Pred CyA CyP SD, FR Steroid and CyA withdrawal None 2 y rem 
11 48 SSNS FSGS Pred, Tac Mania, TacD Tac withdrawal Neutropenia, L stopped 1 y rem 
12 54 NS FSGS Pred, Tac PR, Tac intol Steroid and Tac withdrawal None 2 y rem 
13 25 NS, single kidney MCN (IgM, C1q) Pred PR Steroid withdrawal None 2 y rem 
14 62 NS FSGS Pred CyA Tac TacD Tac withdrawal Rash 2 y rem 
15 55 SDNS since 7 y ND Pred CyA Tac TacD Tac withdrawal None 1 y rem 
CaseAge at referral (y)SClinical featuresRenal biopsyRxEffectLevamisole benefitAdverse effectsLatest F/U
61 NS bladder cancer MCN Pred Rem, rel Steroid withdrawal None 14 y rem 
17 NS MCN (mes hyper) Pred Rem, FR Steroid withdrawal SS rel x2 None 10 y rem 
40 NS MCN Pred Rem, FR, SD SS, rem on low dose Pred GI 5 y rem 
37 NS MCN (mes scl) Pred then CyA, CyP FR, SD Rem off Pred GI (dose-dep) 14 y rem 
36 NS MCN Pred, CyA, CyP FR, CyAD Fever, nausea, recurrent on rechall Probable allergy CyAD MMF, FR 
18 SDNS since 7 y ND Pred 25 alt days SD Steroid withdrawal Neutropenia (recurrent on rechall) SD, FR 
17 SSNS ND Pred FR Steroid withdrawal None 6 y rem 
46 FRNS since 7 y MCN (mes hyp) Pred, CyA, CyP x4 FR, SD Steroid withdrawal None 4 y rem, SS 2 y rem 
19 FRNS MCN Pred, CyA, MMF FR Steroid withdrawal None 3 y rem 
10 45 SDNS since 2 y FSGS Pred CyA CyP SD, FR Steroid and CyA withdrawal None 2 y rem 
11 48 SSNS FSGS Pred, Tac Mania, TacD Tac withdrawal Neutropenia, L stopped 1 y rem 
12 54 NS FSGS Pred, Tac PR, Tac intol Steroid and Tac withdrawal None 2 y rem 
13 25 NS, single kidney MCN (IgM, C1q) Pred PR Steroid withdrawal None 2 y rem 
14 62 NS FSGS Pred CyA Tac TacD Tac withdrawal Rash 2 y rem 
15 55 SDNS since 7 y ND Pred CyA Tac TacD Tac withdrawal None 1 y rem 

Benefits and adverse effects of levamisole in frequent relapses/steroid-dependent patients

Figure 1
Benefits and adverse effects of levamisole in frequent relapses/steroid-dependent patients

In 14 of the 15 patients, levamisole led to significant benefit in terms of reduction or stopping of other more toxic medications and/or reduced relapse frequency and/or better quality of life (see details in Table 1). Notation: −/+, negative/positive adverse effects or levamisole benefit; /, female/male; the number within the sex symbol indicates the case number.

Figure 1
Benefits and adverse effects of levamisole in frequent relapses/steroid-dependent patients

In 14 of the 15 patients, levamisole led to significant benefit in terms of reduction or stopping of other more toxic medications and/or reduced relapse frequency and/or better quality of life (see details in Table 1). Notation: −/+, negative/positive adverse effects or levamisole benefit; /, female/male; the number within the sex symbol indicates the case number.

In six patients, the impact of levamisole was particularly dramatic (cases 1, 2, 3, 4, 7 and 9; see the statement below written by patient 4).

In general, levamisole was well-tolerated and straight forward to monitor. It proved less toxic, as well as considerably cheaper, than alternative second-line agents and was clinically useful in the vast majority of cases. On the basis of our experience we continue to recommend its use in adult steroid-sensitive or steroid-dependent nephrotic syndrome but clearly there is a need for suitably powered RCTs of this and other agents in this condition.

Patient 4 (female, aged 37 years at referral), on learning that we were planning to publish our experience with levamisole, wanted to write a statement on her own experience. The full text is available on request but the statement concludes: “…Professor Mathieson introduced me to levamisole. I must have been one of the most pessimistic patients he had ever encountered. I explained trials with cyclosporine and cyclophosphamide had failed. After a dubious start including diarrhoea and stomach cramps my medication is at such a low level that to me, there are no obvious side effects. I have not relapsed in years and am now leading a normal life. Pleasures such as evening meals and travelling abroad are a possibility without worry. My new quality of life has benefited myself, immediate family and friends.”

Laboratory

Levamisole increases expression of glucocorticoid receptor and activates glucocorticoid signalling in human podocytes

As detailed above, levamisole is a steroid-sparing agent since it is able to reduce the dosage of steroids used to treat patients and it can prolong the duration of remission in patients. Due to its steroid-sparing effect, we hypothesized that levamisole may exert its function by mimicking the actions of glucocorticoids. In human podocytes treated with levamisole for 24 h, we observed that levamisole induced a 1.5-fold increase in GR (glucocorticoid receptor) expression (Figure 2A). As a comparison, at the same time we also measured the expression level of the MR. Unlike GR, MR was not induced by levamisole (Figure 2B).

GR expression and activation in podocytes in response to levamisole
Figure 2
GR expression and activation in podocytes in response to levamisole

(A) Levamisole increases podocyte GR protein expression as shown by Western blotting. Cells were treated with Lev (levamisole) or Dex (dexamethasone) for 24 h at the indicated concentrations (n=4). (B) Levamisole does not affect MR expression in podocytes. (C) Levamisole partially reverses Dex-induced down-regulation of GR. Podocytes were treated with 1 μM Lev, 0.1 μM Dex alone or in combination for 6 h (n=3). (D) Levamisole rapidly increases GR phosphorylation. Podocytes were treated with Lev (1 μM) or Dex (0.1 μM) for the indicated times (n=5). (E) Nuclear fractionation analysis shows an increase in nuclear GR in response to levamisole (10 μM). The Western blot is representative of at least three independent experiments. Densitometric analysis of the blots was performed and the ratios of GR and β-actin or phosphorylated and total GR are plotted as fold changes relative to the controls. Values are the means ± S.E.M. for at least three independent experiments. (F) Levamisole treatment (10 μM) in 30 min leading to GR phosphorylation and accumulation in the nucleus was confirmed by immunofluorescence staining, compared with Dex (0.1 μM). GR/phospho-GR localization was monitored using anti-GR/pGR antibody (green); nuclei (blue) were visualized with DAPI. (G) Levamisole up-regulates FKBP5 and DUSP1 mRNA expression. Cells were treated with Lev or Dex for 24 h then RNA was extracted and analysed by real-time RT-PCR. Values were normalized to β-actin and fold changes compared with controls are plotted as means ± S.E.M. from triplicates in three independent experiments. *Lev or Dex treatment compared with control, #Dex compared with combined treatment of Lev and Dex. Significance: */#P<0.05, **/##P<0.01, ***/###P<0.001.

Figure 2
GR expression and activation in podocytes in response to levamisole

(A) Levamisole increases podocyte GR protein expression as shown by Western blotting. Cells were treated with Lev (levamisole) or Dex (dexamethasone) for 24 h at the indicated concentrations (n=4). (B) Levamisole does not affect MR expression in podocytes. (C) Levamisole partially reverses Dex-induced down-regulation of GR. Podocytes were treated with 1 μM Lev, 0.1 μM Dex alone or in combination for 6 h (n=3). (D) Levamisole rapidly increases GR phosphorylation. Podocytes were treated with Lev (1 μM) or Dex (0.1 μM) for the indicated times (n=5). (E) Nuclear fractionation analysis shows an increase in nuclear GR in response to levamisole (10 μM). The Western blot is representative of at least three independent experiments. Densitometric analysis of the blots was performed and the ratios of GR and β-actin or phosphorylated and total GR are plotted as fold changes relative to the controls. Values are the means ± S.E.M. for at least three independent experiments. (F) Levamisole treatment (10 μM) in 30 min leading to GR phosphorylation and accumulation in the nucleus was confirmed by immunofluorescence staining, compared with Dex (0.1 μM). GR/phospho-GR localization was monitored using anti-GR/pGR antibody (green); nuclei (blue) were visualized with DAPI. (G) Levamisole up-regulates FKBP5 and DUSP1 mRNA expression. Cells were treated with Lev or Dex for 24 h then RNA was extracted and analysed by real-time RT-PCR. Values were normalized to β-actin and fold changes compared with controls are plotted as means ± S.E.M. from triplicates in three independent experiments. *Lev or Dex treatment compared with control, #Dex compared with combined treatment of Lev and Dex. Significance: */#P<0.05, **/##P<0.01, ***/###P<0.001.

As previously reported, prolonged treatment with dexamethasone causes a down-regulation of GR (Figure 2A) [21,22]. This down-regulation is partially but significantly blocked by co-treatment of podocytes with both dexamethasone and levamisole (Figure 2C).

Phosphorylation of GR is required for its translocation and activation. It is clear that there is an increase in GR phosphorylation and translocation into the nucleus in levamisole-treated podocytes as compared with the untreated control (Figures 2D–2F). Moreover, the increase occurred within 30 min of levamisole treatment. An accumulation of GR in nucleus was also confirmed by immunofluorescence analysis (Figure 2F). Furthermore we demonstrate that, like dexamethasone, levamisole induces the expression of DUSP1 and FKBP5, two genes which are known to be induced by GR activation [23,24] (Figure 2G).

Levamisole prevents podocyte damage induced by puromycin aminonucleoside

PAN (puromycin aminonucleoside) induces podocyte injury in vitro and in vivo. Low-dose PAN in vivo induces nephrotic range proteinuria and morphological features analogous to MCN [2530]. We used PAN-treated human podocytes as an in vitro model of MCN to investigate the effects of levamisole on podocyte injury, in terms of four pathways known to be altered by PAN treatment, induction of CD80 (also termed B7-1), overproduction of reactive oxygen species (ROS), translocation of AIF from the mitochondria to the nucleus and decreased phosphorylation of protein kinase B (AKT) [3145].

In our cells, as expected, PAN was able to induce dramatic increase in CD80 mRNA expression. This increase in CD80 expression was significantly blocked by both levamisole and dexamethasone (Figure 3A).

Levamisole inhibits expression of PAN-induced genes, which is GR dependent
Figure 3
Levamisole inhibits expression of PAN-induced genes, which is GR dependent

Cells were treated with Lev (levamisole) (1 μM) or Dex (dexamethasone) (1 μM) for 1 h following the 1-h pre-treatment with GR antagonist RU486 (1 μM). Cells were then exposed to PAN (30 μg/ml) treatment for 48 h. Total RNA was extracted and analysed by real-time RT-PCR for mRNA expression analysis of CD80 (A), Nox4 (B), p22phox (C), p47phox (D) and AIF (E) (n=4). (F) Analysis of AIF protein expression was performed by Western blotting. The Western blot is representative of three independent experiments. Values were normalized to β-actin and fold changes compared with controls are plotted as means ± S.E.M. *PAN treatment compared with control, #PAN compared with PAN plus Lev or Dex treatment, PAN plus Lev (or Dex) compared with PAN plus Lev (or Dex) in the presence of GR antagonist RU486. Significance: */#/†P<0.05, **/##/††P<0.01, ***/###/†††P<0.001. (G) Localization of AIF in podocytes was detected by immunofluorescence staining with anti-AIF antibody (green) after 3-day exposure to 30 μg/ml PAN. Nuclei are shown in blue.

Figure 3
Levamisole inhibits expression of PAN-induced genes, which is GR dependent

Cells were treated with Lev (levamisole) (1 μM) or Dex (dexamethasone) (1 μM) for 1 h following the 1-h pre-treatment with GR antagonist RU486 (1 μM). Cells were then exposed to PAN (30 μg/ml) treatment for 48 h. Total RNA was extracted and analysed by real-time RT-PCR for mRNA expression analysis of CD80 (A), Nox4 (B), p22phox (C), p47phox (D) and AIF (E) (n=4). (F) Analysis of AIF protein expression was performed by Western blotting. The Western blot is representative of three independent experiments. Values were normalized to β-actin and fold changes compared with controls are plotted as means ± S.E.M. *PAN treatment compared with control, #PAN compared with PAN plus Lev or Dex treatment, PAN plus Lev (or Dex) compared with PAN plus Lev (or Dex) in the presence of GR antagonist RU486. Significance: */#/†P<0.05, **/##/††P<0.01, ***/###/†††P<0.001. (G) Localization of AIF in podocytes was detected by immunofluorescence staining with anti-AIF antibody (green) after 3-day exposure to 30 μg/ml PAN. Nuclei are shown in blue.

Oxidative stress is another mechanism that causes podocyte injury. Therefore we analysed the expression of subunits of Nox, an important ROS producer [41]. Nox4, p22phox and p47phox are all induced by PAN treatment and, like CD80, their inductions are blocked by both levamisole and dexamethasone (Figures 3B–3D).

PAN has been shown to result in the translocation of AIF from the mitochondria to the nucleus where it binds to DNA to cause DNA fragmentation [42,43]. In our cells PAN increased the expression of AIF at both the mRNA and protein levels (Figures 3E and 3F) and caused the translocation of this protein to the nucleus (Figure 3G). These effects were blocked by levamisole (Figures 3E–3G).

Finally PAN treatment of podocytes resulted in a decrease in the phosphorylation of AKT at both of its key regulatory sites, Thr308 and Ser473, at both 1 h and 24 h, which was restored by levamisole treatment (Figures 4A and 4B).

Levamisole restores PAN-induced reduction in phosphorylation of AKT, which is blocked by GR antagonist RU486
Figure 4
Levamisole restores PAN-induced reduction in phosphorylation of AKT, which is blocked by GR antagonist RU486

Podocytes were first pretreated with 1 μM of RU486 for 1 h and then treated with Lev (levamisole) (1 μM) for another 1 h. Cells were injured with 30 μg/ml PAN for 1 h (A) or 24 h (B). Western blot analysis determined protein expression of phosphorylated and total AKT. The Western blot is representative of three independent experiments. Densitometric analysis of the blots was performed and the ratios of phosphorylated and total AKT are plotted as fold changes relative to the controls. Values are the means ± S.E.M. for three (n=3) independent experiments. *PAN treatment compared with control, #PAN compared with PAN plus Lev treatment, PAN plus Lev compared with PAN plus Lev in the presence of RU486. Significance: */#/†P<0.05, **/##/††P<0.01, ***/###/†††P<0.001.

Figure 4
Levamisole restores PAN-induced reduction in phosphorylation of AKT, which is blocked by GR antagonist RU486

Podocytes were first pretreated with 1 μM of RU486 for 1 h and then treated with Lev (levamisole) (1 μM) for another 1 h. Cells were injured with 30 μg/ml PAN for 1 h (A) or 24 h (B). Western blot analysis determined protein expression of phosphorylated and total AKT. The Western blot is representative of three independent experiments. Densitometric analysis of the blots was performed and the ratios of phosphorylated and total AKT are plotted as fold changes relative to the controls. Values are the means ± S.E.M. for three (n=3) independent experiments. *PAN treatment compared with control, #PAN compared with PAN plus Lev treatment, PAN plus Lev compared with PAN plus Lev in the presence of RU486. Significance: */#/†P<0.05, **/##/††P<0.01, ***/###/†††P<0.001.

Our collective results suggest that levamisole may activate the GR signalling pathway. To further clarify the role played by GR in the beneficial effects of levamisole we pre-treated cells with a GR antagonist (RU486) [46]. Interestingly, we found that RU486 blocked the effects of both levamisole and dexamethasone (Figures 3 and 4), suggesting that the effects of levamisole are mediated by the GR signalling pathway.

DISCUSSION AND CONCLUSIONS

The clinical usefulness of levamisole in paediatric nephrotic syndrome is widely accepted and it seemed to us that there was no reason that the same would not be true in adults, leading to its use in the adult patients described in the present report. However, clearly these are heterogeneous diseases and RCTs are needed before claims of efficacy can be accepted. Our results are certainly encouraging and suggest that there is an a priori case for more systematic study of levamisole in adults with nephrotic syndrome. Such studies are also required to assess risk/benefit relationships; in our experience, monitoring of levamisole therapy is straightforward and it is usually well tolerated. Of course, neutropenia, if neglected, could be a serious adverse effect and, as with all drugs, allergic reactions can occur. Levamisole is generally less toxic than other second-line agents which are used when steroids alone are inadequate in achieving control of nephrotic syndrome.

There is scant knowledge about the mechanism of action of levamisole. It is thought to have an immunostimulatory activity, which is presumed to underlie its beneficial effects as an anti-helminth and as an adjuvant in colonic cancer. Whether these actions are responsible for its effectiveness in SSNS remains speculative. A range of immunological abnormalities have been described in MCN [19], but it is difficult to discern whether these abnormalities are causal or merely a secondary consequence of the nephrotic state. These abnormalities include: hypogammaglobulinaemia, with both impaired IgG production and increased IgG catabolism; a type 2 biased cytokine response, dominated by interleukin 4 in particular; elevated serum IgE; and an association with atopy [47].

Levamisole is thought to augment a type 1 cytokine response and down-regulate a type 2 response [7]. Type 2 cytokines are involved in antibody-mediated immunity; in MCN this is manifest by class switching of B-cells to the production of IgG4 and IgE. Type 1 responses predominate in cell-mediated immunity, involving interferon γ in particular, although levamisole has been shown to selectively induce gene transcription of the type 1 cytokine interleukin 18 [48].

An alternative hypothesis is that levamisole's usefulness in nephrotic syndrome is because of direct actions on the podocyte, as has been suggested for other agents whose use was initially motivated by their effects on the immune system [1215]. The renal glomerulus is the region of the kidney that forms the biological sieve. This allows water and small solutes to freely pass from the circulation into the urine, but is relatively impermeable to macromolecules, such as albumin. Albumin loss into the urine (albuminuria) is prevented by the glomerular filtration barrier (GFB). The GFB consists of three layers: GnECs, the glomerular basement membrane and podocytes which lie adjacent to the urinary space. Podocytes are widely accepted to be the major target of injury in nephrotic syndrome [16]. These cells have a very limited capacity for repair or regeneration, so that therapeutic strategies to protect podocytes and/or promote their repair/replacement are greatly sought after [16]. There has been huge progress in understanding of modes of action of drugs that are effective in proteinuric kidney diseases [12]. By studying our unique human podocyte cell lines we reported [13] direct effects of glucocorticosteroids (GCs, another form of drug that is very effective in patients with nephrotic syndrome, but whose use is empirical because little is known of its mode of action) on human podocytes. We demonstrated that dexamethasone at therapeutic levels altered GR expression, podocyte maturation and the expression of a number of critical podocyte proteins [13]. More recent studies have reported that GCs protect cultured podocytes against injury via actin filament stabilization [49] and prevent apoptosis induced by PAN [42,43], confirming direct effects on podocytes. Functional responses to GCs in cultured murine and human podocytes have also been suggested by expression analyses reporting several GC-regulated genes [5052]. This is not an isolated example of drugs directly influencing the podocyte [12]. For example, cyclosporine, a calcineurin inhibitor used as an additional immunosuppressive agent in nephrotic syndrome, has been shown to stabilize the podocyte actin cytoskeleton [14] and even rituximab, considered a highly specific anti-CD20 agent, has ‘off-target’ effects on podocytes [15].

Our data suggest that levamisole's mode of action in SSNS/MCN is attributable to its direct effects on podocytes via activation of GR. Due to its steroid-sparing effect, we hypothesized that levamisole may exert its function by mimicking the actions of GCs. GCs, as the ligands, rely on binding to GR to activate GR signalling. The ligand-bound GR then translocates to the nucleus where GR-responsive genes are positively or negatively modulated through diverse mechanisms. In the present study, we demonstrated that in our podocytes, levamisole increases GR expression, blocks its down-regulation and activates GR signalling.

Low-dose PAN in vivo induces nephrotic range of proteinuria and morphological features analogous to MCN [2530]. CD80 (also termed B7-1) is induced in podocytes during injury and is required for inducing proteinuria [31]. MCN is reported to be associated with expression of CD80 in podocytes and increased excretion of CD80 in the urine [32,33]. In in vitro models of podocyte injury CD80 expression is increased and this is blocked by treatment of the cells with dexamethasone [34]. In our cells, as expected, PAN was able to induce dramatic increase in CD80 mRNA expression. This increase in CD80 expression was significantly blocked by both levamisole and dexamethasone.

Oxidative stress is another mechanism that causes podocyte injury. Overproduction of ROS has been reported in several types of experimental models of nephrotic syndrome including PAN [3539] and GCs have been reported to activate glomerular antioxidant enzymes thus protecting glomeruli from injury [40]. Therefore we analysed the expression of subunits of Nox, an important ROS producer [41]. We showed that Nox4, p22phox and p47phox are all induced by PAN treatment and, like the expression of CD80, this is blocked by both levamisole and dexamethasone.

Dexamethasone has also been shown to prevent PAN-induced podocyte apoptosis by inhibition of AIF translocation from the mitochondria to the nucleus where it binds to DNA to cause DNA fragmentation [42,43]. To examine levamisole's effects on this protein, we looked at both mRNA and protein expressions of AIF upon PAN treatment. In our cells, PAN increased AIF expression at both mRNA and protein levels and caused the translocation of this protein to the nucleus. These effects were blocked by levamisole.

Finally PAN treatment has been reported to result in a decrease in the phosphorylation of AKT, a key regulator of podocyte survival [44,45]. Our data shows that PAN decreases AKT phosphorylation at both of its key regulatory sites, Thr308 and Ser473, at both 1 h and 24 h, and this is restored by levamisole treatment.

Most importantly, our current results are of particular interest because in our cells, the protective effects that levamisole has shown above were eliminated by a GR antagonist, indicating that GR signalling may contribute to the beneficial effects of levamisole in podocyte injury and this may explain the steroid-sparing effects of this compound.

In summary, our clinical and laboratory data for the first time suggest that levamisole should be added to the list of immunotherapeutic agents that have direct actions on podocytes and point to the usefulness of levamisole in the treatment of adult as well as paediatric patients.

Abbreviations

     
  • AIF

    apoptosis-inducing factor

  •  
  • CD

    cluster of differentiation

  •  
  • DUSP1

    dual-specificity protein phosphatase 1

  •  
  • FKBP5

    FK506-binding protein 5

  •  
  • GC

    glucocorticosteroid

  •  
  • GFB

    glomerular filtration barrier

  •  
  • GnEC

    glomerular endothelial cell

  •  
  • GR

    glucocorticoid receptor

  •  
  • MCN

    minimal change nephropathy

  •  
  • MR

    mineralocorticoid receptor

  •  
  • Nox

    NADPH oxidase

  •  
  • PAN

    puromycin aminonucleoside

  •  
  • RCT

    randomized controlled trial

  •  
  • ROS

    reactive oxygen species

  •  
  • SSNS

    steroid-sensitive nephrotic syndrome

AUTHOR CONTRIBUTION

The original idea of the study was by Peter Mathieson. Peter Mathieson and Gavin Welsh participated in research design. Peter Mathieson took clinical care of all the patients. Lulu Jiang, Jenny Hurcombe, Heather Colyer and Gavin Welsh conducted the experiments. Lulu Jiang, Jenny Hurcombe, Ishita Dasgupta and Gavin Welsh performed data analysis. Lulu Jiang, Peter Mathieson and Gavin Welsh wrote the manuscript.

For much discussion and useful advice we thank Dr B. Conway-Campbell and Professor S. Lightman (University of Bristol).

FUNDING

This work was funded by the Kids Kidney Research (to G.W. and P.W.M.); and the University of Bristol Ph.D. Scholarship (to L.J.).

References

References
1
Palmer
 
S.C.
Nand
 
K.
Strippoli
 
G.F.
 
Interventions for minimal change disease in adults with nephrotic syndrome
Cochrane Database Syst. Rev.
2008
pg. 
CD001537
 
[PubMed]
2
Kidney Disease: Improving Global Outcomes
KDIGO Clinical Practice Guideline for Glomerulonephritis
Kidney Int. Suppl.
2012
, vol. 
2
 
Suppl. 2012
(pg. 
139
-
274
)
3
Elmas
 
A.T.
Tabel
 
Y.
Elmas
 
O.N.
 
Short- and long-term efficacy of levamisole in children with steroid-sensitive nephrotic syndrome
Int. Urol. Nephrol.
2012
, vol. 
45
 (pg. 
1047
-
1055
)
[PubMed]
4
Hodson
 
E.M.
Alexander
 
S.I.
 
Evaluation and management of steroid-sensitive nephrotic syndrome
Curr. Opin. Pediatr.
2008
, vol. 
20
 (pg. 
145
-
150
)
[PubMed]
5
Hodson
 
E.M.
Craig
 
J.C.
Willis
 
N.S.
 
Evidence-based management of steroid-sensitive nephrotic syndrome
Pediatr. Nephrol.
2005
, vol. 
20
 (pg. 
1523
-
1530
)
[PubMed]
6
Hodson
 
E.M.
Willis
 
N.S.
Craig
 
J.C.
 
Non-corticosteroid treatment for nephrotic syndrome in children
Cochrane Database Syst. Rev.
2008
, vol. 
10
 pg. 
CD002290
 
[PubMed]
7
Pravitsitthikul
 
N.
Willis
 
N.S.
Hodson
 
E.M.
Craig
 
J.C.
 
Non-corticosteroid immunosuppressive medications for steroid-sensitive nephrotic syndrome in children
Cochrane Database Syst. Rev.
2013
, vol. 
10
 pg. 
CD002290
 
[PubMed]
8
Moertel
 
C.G.
Fleming
 
T.R.
Macdonald
 
J.S.
Haller
 
D.G.
Laurie
 
J.A.
Goodman
 
P.J.
Ungerleider
 
J.S.
Emerson
 
W.A.
Tormey
 
D.C.
Glick
 
J.H.
, et al 
Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma
N. Engl. J. Med.
1990
, vol. 
322
 (pg. 
352
-
358
)
[PubMed]
9
Anon
 
 
Levamisole for childhood nephrotic syndrome
Lancet
1991
, vol. 
337
 pg. 
1574
 
[PubMed]
10
Davin
 
J.C.
Merkus
 
M.P.
 
Levamisole in steroid-sensitive nephrotic syndrome of childhood: the lost paradise?
Pediatr. Nephrol.
2005
, vol. 
20
 (pg. 
10
-
14
)
[PubMed]
11
Dayal
 
U.
Dayal
 
A.K.
Shastry
 
J.C.
Raghupathy
 
P.
 
Use of levamisole in maintaining remission in steroid-sensitive nephrotic syndrome in children
Nephron
1994
, vol. 
66
 (pg. 
408
-
412
)
[PubMed]
12
Mathieson
 
P.W.
 
The podocyte as a target for therapies–new and old
Nat. Rev. Nephrol.
2011
, vol. 
8
 (pg. 
52
-
56
)
[PubMed]
13
Xing
 
C.Y.
Saleem
 
M.A.
Coward
 
R.J.
Ni
 
L.
Witherden
 
I.R.
Mathieson
 
P.W.
 
Direct effects of dexamethasone on human podocytes
Kidney Int.
2006
, vol. 
70
 (pg. 
1038
-
1045
)
[PubMed]
14
Faul
 
C.
Donnelly
 
M.
Merscher-Gomez
 
S.
Chang
 
Y.H.
Franz
 
S.
Delfgaauw
 
J.
Chang
 
J.M.
Choi
 
H.Y.
Campbell
 
K.N.
Kim
 
K.
, et al 
The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A
Nat. Med.
2008
, vol. 
14
 (pg. 
931
-
938
)
[PubMed]
15
Fornoni
 
A.
Sageshima
 
J.
Wei
 
C.
Merscher-Gomez
 
S.
Aguillon-Prada
 
R.
Jauregui
 
A.N.
Li
 
J.
Mattiazzi
 
A.
Ciancio
 
G.
Chen
 
L.
, et al 
Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis
Sci. Transl. Med.
2011
, vol. 
3
 pg. 
85ra46
 
[PubMed]
16
Patrakka
 
J.
Tryggvason
 
K.
 
New insights into the role of podocytes in proteinuria
Nat. Rev. Nephrol.
2009
, vol. 
5
 (pg. 
463
-
468
)
[PubMed]
17
Awadzi
 
K.
Edwards
 
G.
Opoku
 
N.O.
Ardrey
 
A.E.
Favager
 
S.
Addy
 
E.T.
Attah
 
S.K.
Yamuah
 
L.K.
Quartey
 
B.T.
 
The safety, tolerability and pharmacokinetics of levamisole alone, levamisole plus ivermectin, and levamisole plus albendazole, and their efficacy against Onchocerca volvulus
Ann. Trop. Med. Parasitol.
2004
, vol. 
98
 (pg. 
595
-
614
)
[PubMed]
18
Saleem
 
M.A.
O'Hare
 
M.J.
Reiser
 
J.
Coward
 
R.J.
Inward
 
C.D.
Farren
 
T.
Xing
 
C.Y.
Ni
 
L.
Mathieson
 
P.W.
Mundel
 
P.
 
A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression
J. Am. Soc. Nephrol.
2002
, vol. 
13
 (pg. 
630
-
638
)
[PubMed]
19
Hale
 
L.J.
Hurcombe
 
J.
Lay
 
A.
Santamaria
 
B.
Valverde
 
A.M.
Saleem
 
M.A.
Mathieson
 
P.W.
Welsh
 
G.I.
Coward
 
R.J.
 
Insulin directly stimulates VEGF-A production in the glomerular podocyte
Am. J. Physiol. Renal Physiol.
2013
, vol. 
305
 (pg. 
F182
-
F188
)
[PubMed]
20
Harris
 
J.J.
McCarthy
 
H.J.
Ni
 
L.
Wherlock
 
M.
Kang
 
H.
Wetzels
 
J.F.
Welsh
 
G.I.
Saleem
 
M.A.
 
Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1
J. Pathol.
2013
, vol. 
229
 (pg. 
660
-
671
)
[PubMed]
21
Wallace
 
A.D.
Cidlowski
 
J.A.
 
Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
42714
-
42721
)
[PubMed]
22
Dong
 
Y.
Poellinger
 
L.
Gustafsson
 
J.A.
Okret
 
S.
 
Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms
Mol. Endocrinol.
1988
, vol. 
2
 (pg. 
1256
-
1264
)
[PubMed]
23
Clark
 
A.R.
Lasa
 
M.
 
Crosstalk between glucocorticoids and mitogen-activated protein kinase signalling pathways
Curr. Opin. Pharmacol.
2003
, vol. 
3
 (pg. 
404
-
411
)
[PubMed]
24
Paakinaho
 
V.
Makkonen
 
H.
Jaaskelainen
 
T.
Palvimo
 
J.J.
 
Glucocorticoid receptor activates poised FKBP51 locus through long-distance interactions
Mol. Endocrinol.
2010
, vol. 
24
 (pg. 
511
-
525
)
[PubMed]
25
Ryan
 
G.B.
Karnovsky
 
M.J.
 
An ultrastructural study of the mechanisms of proteinuria in aminonucleoside nephrosis
Kidney Int.
1975
, vol. 
8
 (pg. 
219
-
232
)
[PubMed]
26
Lowenborg
 
E.K.
Jaremko
 
G.
Berg
 
U.B.
 
Glomerular function and morphology in puromycin aminonucleoside nephropathy in rats
Nephrol. Dial. Transplant.
2000
, vol. 
15
 (pg. 
1547
-
1555
)
[PubMed]
27
Whiteside
 
C.I.
Cameron
 
R.
Munk
 
S.
Levy
 
J.
 
Podocytic cytoskeletal disaggregation and basement-membrane detachment in puromycin aminonucleoside nephrosis
Am. J. Pathol.
1993
, vol. 
142
 (pg. 
1641
-
1653
)
[PubMed]
28
Messina
 
A.
Davies
 
D.J.
Dillane
 
P.C.
Ryan
 
G.B.
 
Glomerular epithelial abnormalities associated with the onset of proteinuria in aminonucleoside nephrosis
Am. J. Pathol.
1987
, vol. 
126
 (pg. 
220
-
229
)
[PubMed]
29
Inokuchi
 
S.
Shirato
 
I.
Kobayashi
 
N.
Koide
 
H.
Tomino
 
Y.
Sakai
 
T.
 
Re-evaluation of foot process effacement in acute puromycin aminonucleoside nephrosis
Kidney Int.
1996
, vol. 
50
 (pg. 
1278
-
1287
)
[PubMed]
30
Bertram
 
J.F.
Messina
 
A.
Ryan
 
G.B.
 
In vitro effects of puromycin aminonucleoside on the ultrastructure of rat glomerular podocytes
Cell Tissue Res.
1990
, vol. 
260
 (pg. 
555
-
563
)
[PubMed]
31
Reiser
 
J.
von Gersdorff
 
G.
Loos
 
M.
Oh
 
J.
Asanuma
 
K.
Giardino
 
L.
Rastaldi
 
M.P.
Calvaresi
 
N.
Watanabe
 
H.
Schwarz
 
K.
, et al 
Induction of B7-1 in podocytes is associated with nephrotic syndrome
J. Clin. Invest.
2004
, vol. 
113
 (pg. 
1390
-
1397
)
[PubMed]
32
Garin
 
E.H.
Diaz
 
L.N.
Mu
 
W.
Wasserfall
 
C.
Araya
 
C.
Segal
 
M.
Johnson
 
R.J.
 
Urinary CD80 excretion increases in idiopathic minimal-change disease
J. Am. Soc. Nephrol.
2009
, vol. 
20
 (pg. 
260
-
266
)
[PubMed]
33
Garin
 
E.H.
Mu
 
W.
Arthur
 
J.M.
Rivard
 
C.J.
Araya
 
C.E.
Shimada
 
M.
Johnson
 
R.J.
 
Urinary CD80 is elevated in minimal change disease but not in focal segmental glomerulosclerosis
Kidney Int.
2010
, vol. 
78
 (pg. 
296
-
302
)
[PubMed]
34
Shimada
 
M.
Ishimoto
 
T.
Lee
 
P.Y.
Lanaspa
 
M.A.
Rivard
 
C.J.
Roncal-Jimenez
 
C.A.
Wymer
 
D.T.
Yamabe
 
H.
Mathieson
 
P.W.
Saleem
 
M.A.
, et al 
Toll-like receptor 3 ligands induce CD80 expression in human podocytes via an NF-kappaB-dependent pathway
Nephrol. Dial. Transplant.
2012
, vol. 
27
 (pg. 
81
-
89
)
[PubMed]
35
Thakur
 
V.
Walker
 
P.D.
Shah
 
S.V.
 
Evidence suggesting a role for hydroxyl radical in puromycin aminonucleoside-induced proteinuria
Kidney Int.
1988
, vol. 
34
 (pg. 
494
-
499
)
[PubMed]
36
Kinra
 
S.
Rath
 
B.
Kabi
 
B.C.
 
Indirect quantification of lipid peroxidation in steroid responsive nephrotic syndrome
Arch. Dis. Child.
2000
, vol. 
82
 (pg. 
76
-
78
)
[PubMed]
37
Diamond
 
J.R.
Bonventre
 
J.V.
Karnovsky
 
M.J.
 
A role for oxygen free radicals in aminonucleoside nephrosis
Kidney Int.
1986
, vol. 
29
 (pg. 
478
-
483
)
[PubMed]
38
Kojima
 
K.
Matsui
 
K.
Nagase
 
M.
 
Protection of alpha(3) integrin-mediated podocyte shape by superoxide dismutase in the puromycin aminonucleoside nephrosis rat
Am. J. Kidney Dis.
2000
, vol. 
35
 (pg. 
1175
-
1185
)
[PubMed]
39
Wang
 
J.S.
Yang
 
A.H.
Chen
 
S.M.
Young
 
T.K.
Chiang
 
H.
Liu
 
H.C.
 
Amelioration of antioxidant enzyme suppression and proteinuria in cyclosporin-treated puromycin nephrosis
Nephron
1993
, vol. 
65
 (pg. 
418
-
425
)
[PubMed]
40
Kawamura
 
T.
Yoshioka
 
T.
Bills
 
T.
Fogo
 
A.
Ichikawa
 
I.
 
Glucocorticoid activates glomerular antioxidant enzymes and protects glomeruli from oxidant injuries
Kidney Int.
1991
, vol. 
40
 (pg. 
291
-
301
)
[PubMed]
41
Gill
 
P.S.
Wilcox
 
C.S.
 
NADPH oxidases in the kidney
Antioxid. Redox Signal.
2006
, vol. 
8
 (pg. 
1597
-
1607
)
[PubMed]
42
Wada
 
T.
Pippin
 
J.W.
Marshall
 
C.B.
Griffin
 
S.V.
Shankland
 
S.J.
 
Dexamethasone prevents podocyte apoptosis induced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins
J. Am. Soc. Nephrol.
2005
, vol. 
16
 (pg. 
2615
-
2625
)
[PubMed]
43
Wada
 
T.
Pippin
 
J.W.
Nangaku
 
M.
Shankland
 
S.J.
 
Dexamethasone's prosurvival benefits in podocytes require extracellular signal-regulated kinase phosphorylation
Nephron Exp. Nephrol.
2008
, vol. 
109
 (pg. 
e8
-
e19
)
[PubMed]
44
Zhu
 
J.
Sun
 
N.
Aoudjit
 
L.
Li
 
H.
Kawachi
 
H.
Lemay
 
S.
Takano
 
T.
 
Nephrin mediates actin reorganization via phosphoinositide 3-kinase in podocytes
Kidney Int.
2008
, vol. 
73
 (pg. 
556
-
566
)
[PubMed]
45
Zuo
 
Y.
Yang
 
H.C.
Potthoff
 
S.A.
Najafian
 
B.
Kon
 
V.
Ma
 
L.J.
Fogo
 
A.B.
 
Protective effects of PPARγ agonist in acute nephrotic syndrome
Nephrol. Dial. Transplant.
2012
, vol. 
27
 (pg. 
174
-
181
)
[PubMed]
46
Bertagna
 
X.
Bertagna
 
C.
Luton
 
J.P.
Husson
 
J.M.
Girard
 
F.
 
The new steroid analog RU 486 inhibits glucocorticoid action in man
J. Clin. Endocrinol. Metab.
1984
, vol. 
59
 (pg. 
25
-
28
)
[PubMed]
47
Mathieson
 
P.W.
 
Immune dysregulation in minimal change nephropathy
Nephrol. Dial. Transplant.
2003
, vol. 
18
 
Suppl. 6
(pg. 
vi26
-
vi29
)
[PubMed]
48
Szeto
 
C.
Gillespie
 
K.M.
Mathieson
 
P.W.
 
Levamisole induces interleukin-18 and shifts type 1/type 2 cytokine balance
Immunology
2000
, vol. 
100
 (pg. 
217
-
224
)
[PubMed]
49
Ransom
 
R.F.
Lam
 
N.G.
Hallett
 
M.A.
Atkinson
 
S.J.
Smoyer
 
W.E.
 
Glucocorticoids protect and enhance recovery of cultured murine podocytes via actin filament stabilization
Kidney Int.
2005
, vol. 
68
 (pg. 
2473
-
2483
)
[PubMed]
50
Ransom
 
R.F.
Vega-Warner
 
V.
Smoyer
 
W.E.
Klein
 
J.
 
Differential proteomic analysis of proteins induced by glucocorticoids in cultured murine podocytes
Kidney Int.
2005
, vol. 
67
 (pg. 
1275
-
1285
)
[PubMed]
51
Liu
 
H.
Gao
 
X.
Xu
 
H.
Feng
 
C.
Kuang
 
X.
Li
 
Z.
Zha
 
X.
 
alpha-Actinin-4 is involved in the process by which dexamethasone protects actin cytoskeleton stabilization from adriamycin-induced podocyte injury
Nephrology
2012
, vol. 
17
 (pg. 
669
-
675
)
[PubMed]
52
Guess
 
A.
Agrawal
 
S.
Wei
 
C.C.
Ransom
 
R.F.
Benndorf
 
R.
Smoyer
 
W.E.
 
Dose- and time-dependent glucocorticoid receptor signaling in podocytes
Am. J. Physiol. Renal Physiol.
2010
, vol. 
299
 (pg. 
F845
-
F853
)
[PubMed]

Author notes

1

These authors contributed equally to this work and are co-senior authors.