Fluorescein-Labeled Dexamethasone (F-DEX)

Human Glucocorticoid Receptor (huGCR)

Glucocorticoid (GC)

Glucocorticoid Sensitivity (GCS)

Glucocorticoid Sensitivity Index (GCSI)

Glucocorticoid Resistance (GC resistance)

Idiopathic Nephrotic Syndrome (INS)

Peripheral Blood Mononuclear Cells (PBMNC)

Nephrotic syndrome is a heterogeneous group of disorders characterized by proteinuria with hypoalbuminemia, edema and hyperlipidemia [1]. Idiopathic Nephrotic Syndrome (INS) is the most common of the primary glomerular diseases in children and represents about 90% of the cases of nephrotic syndrome presenting between 1 to 10 years of age [2]. The prevalence is about 16 cases per 100,000 children, with an incidence rate of about 2-7 in 100,000 children. Even though INS affects children in all age groups, it is most commonly diagnosed between 3 to 9 years of age.  There is a slight predominance in males versus females with a ratio of 2:1 [3,4]. INS includes minimal change nephrotic syndrome (MCNS), focal segmental glomerulosclerosis (FSGS) and diffuse mesangial hypercellularity (DMH) [4, 5].  Secondary causes of nephrotic syndrome can be due to underlying disease entities such as lupus and HIV [1].

Response to steroid therapy is used to characterize childhood INS as either steroid sensitive (SSNS), steroid dependent (SDNS), Frequent relapse (FR) , Infrequent relapse (IFR) or steroid resistant (SRNS), a classification system that has been shown to have better prognostic value than renal histology [4]. The majority of patients (over 90%) with MCNS respond to glucocorticoids (prednisone). However, only 20- 25% of patients with FSGS respond to glucocorticoid therapy [6]. The children are identified as steroid resistant, if they do not respond to 4-6 weeks of daily steroid therapy. These children may suffer from significant side effects related to the steroids including hypertension, hyperglycemia, as well as salt and water retention [2, 5].

Currently, there is no diagnostic test to determine resistance to steroid therapy in patients with INS.  Various in-vitro tests have been used in trying to anticipate the patient’s response to steroids, but none were found to be predictive enough to be used when deciding on a patient’s treatment with steroids.  Therefore, there is a serious need to find a method that can identify those patients who may benefit from steroid immunosuppressive treatment versus patients who will not benefit from such treatment to minimize its major side effects.

We hypothesize that an in-vitro Glucocorticoid Sensitivity (GCS) assay can be used to predict the response to glucocorticoid treatment in cases of INS. To test this hypothesis, we evaluated GCS in 10 children with INS and compared the results with 31 normal controls using a novel in-vitro fluorescein labeled-dexamethasone (F-DEX)-mononuclear cell (-MNC)-binding assay and correlated this with their clinical response to steroid treatment.

Patients: The present study included 10 children (12 ± 4.5 yrs), 4 males and 6 females, with a clinical diagnosis of INS, who were being followed at the Children’s Kidney Center at SUNY Downstate Health Science University, Brooklyn, NY. The study was a Case Control study approved by the Institutional Review Board at SUNY Downstate Health Science University (Protocol Number 10-183).  Informed assent and consent were obtained from each subject and her/his parents or guardian.

Patients were eligible for inclusion in this study, if they met the following criteria: (1) diagnosis of INS, (2) completion of daily 4-6 weeks of prednisone therapy at 2 mg/kg per day followed by alternate day therapy for another 4-6 weeks. Exclusion criteria consisted of: (1) use of any other immunosuppressive treatments prior to starting steroids, (2) secondary causes of nephrotic syndrome.

We collected data, which included age of presentation, race, sex, blood pressure, edema, serum creatinine, serum albumin, serum hemoglobin, cholesterol level, urinary protein/creatinine ratio and eGFR (modified Schwartz formula) at baseline and during follow-up [7].

Controls: A total 31 of controls were recruited, 11 children (8.3 ± 3.0 yrs.), 9 adolescents (16.1 ± 2.5 yrs.) and 11 young adults (28.8 ± 2.1 yrs.). The controls had no history of kidney disease or any disorders involving the glucocorticoid pathway.

• Nephrotic-range proteinuria: first morning or 24-hour urine protein to urine creatinine (uPCR) ≥2 mg/mg (or 200 mg/mmol or 3+ dipstick).
• Nephrotic Syndrome (NS): nephrotic-range proteinuria and either hypoalbuminemia (serum albumin <3 g/dL) or edema when albumin level is not available.
• Secondary SRNS; SSNS patient at disease onset who at subsequent relapse fails to achieve remission after 4 weeks of prednisone or prednisolone at standard dose.

Initial treatment: Patients were treated with oral prednisone starting at 60 mg/m²/day or 2 mg/kg/day to a maximum 60 mg/day for 4-6 weeks followed by alternate-day medication as a single daily dose at 40 mg/m² or 1.5 mg/kg (maximum 40 mg) and continued for another 4-6 weeks [6,8,9].

Indications for biopsy [6,8,9]:

• Subsequent failure to respond to corticosteroids in steroid-sensitive nephrotic syndrome (secondary steroid-resistant nephrotic syndrome).
• SRNS
• Subsequently, decreasing kidney function in children receiving calcineurin inhibitors (CNI) or prolonged exposure to CNI in patients with frequent relapses or steroid dependence.

In vitro GCS was assessed in all patients and controls using an F-DEX-MNC binding assay. The lab personnel performing the in vitro test were blinded with respect to the patient’s clinical characteristics and response to steroids.

GCS Binding Protocol: F-DEX-MNC binding assay using Peripheral Blood Mononuclear Cells (PBMC)

The complete methodology is described in reference [11].  In short, blood was drawn into heparinized tubes, diluted with normal saline and layered over a Ficoll 400 -Hypaque-solution (density 1.078g/ml) (Pharmacia -GE HealthCare). The interphase enriched for PBMCs was isolated and the cells were washed with normal saline. The final cell pellet was resuspended in 5mL of complete Culture Medium (cCM) and incubated at 37^oC in a shaking water bath for 30 minutes. The cells were then resuspended in 5mL of fresh cCM and counted using Trypan blue solution to establish the cells’ viability and number.  From the PBMCs of each subject, the monocytes were then purified by negative sorting to allow the direct observation of F-DEX binding to the mononuclear cells [11].

The monocytes obtained were diluted to 2x10⁵/mL of which 0.2 ml were distributed into each well of a 96-well high-affinity tissue culture plate (CTCp) and allowed to adhere for 1h at 37^oC followed by incubation in cCM (0.2ml/well) at 37^O in an incubator providing a 5% CO2-95% air mixture. The next day the cells were washed twice with PBS and once with non-fluorescent Binding buffer (Bb). The buffer was then replaced in triplicate wells with 0.1 mL PBS/well containing F-DEX (Invitrogen Life sciences) at concentrations from 400nM to 6400nM.  After 1h at 37^oC, fresh PBS (0.2 mL) was added to each well and the unbound F-DEX was washed off to assure that only the cell-bound F-DEX was detected by reading the fluorescein-intensity at OD488nm bound to the monocytes in each well in a Microplate Fluorimeter Series 7600 reader (Cambridge Technology, Bedford, MA). Testing was performed in triplicates to control for intra assay variation.

Statistical analysis

The GCSI was calculated as AUC of F-DEX-MNC binding for control and subjects. AUC was determined by using the linear Trapezoidal Rule [10]. Student t-tests were used to compare the GCSI between subjects and controls (Excel v 14.3.8). A result was considered statistically significant if p < 0.05.

Clinical characteristics

A normative GCSI was calculated as 325 ± 30.6 (mean ± SDS) from 31 controls. GCSI was obtained by the F-DEX-MNC binding assay and was reproducible when measured on different days with PBMCs from the same controls [11]. There were no differences in the GCSI between children and young adults nor between males and females in the control group.  A normal GCSI was between 265-385. A lower GCSI represents GC resistance. Subjects were classified as GC resistant, if the GCSI ≤ 264. The subjects with a GCSI ≥ 386 were considered to have increased GC sensitivity (GC Sensitive) [11].

Patients were divided into 3 subgroups according to the GCSI obtained from the in vitro study. Out of 10 patients with INS: Three patients (30 %) were found to be GC resistant with a GCSI of 223.6 ± 27, Three patients (30 %) had normal GC sensitivity with a GCSI 343.5 ± 36.8 and 4 patients (40 %) had increased GC sensitivity(GC Sensitive) with a GCSI of 484.5 ±52.8 (Figure 1, 2, 3). There was no difference in mean age, serum albumin, hemoglobin, serum creatinine, cholesterol and eGFR at baseline amongst these three groups (Tables 1, 2,3).

All patients underwent treatment with prednisone as per standard guidelines [6]. Analysis of the data before and 6 weeks after prednisone treatment revealed significant improvement in serum albumin, total cholesterol and urinary protein /creatinine ratio in the increased GC sensitivity (GC Sensitive) and normal GC sensitive groups. There was no improvement in serum albumin, total cholesterol and urinary protein /creatinine ratio in the GC resistant group after prednisone treatment (Table 3). Among the different groups, the mean urinary protein /creatinine excretion was higher in the GC resistant group compared to the normal GC sensitive and increased GC sensitivity (GC Sensitive) groups after treatment. The serum albumin and the difference in serum albumin after treatment were lower in the GC resistant group compared to the other two groups (Table 3).

Histopathology examination of kidney biopsies after the steroid treatment course was concluded, showed that 3 patients of 3 in the GC resistant group had FSGS, 2 patients of 4 in the increased GC sensitivity (GC Sensitive) group had FSGS and 1 patient of 3 in the normal GC sensitive group had FSGS. Overall, 50 % of patients with FSGS were found to have glucocorticoid resistance (Tables 1,2).

GCSI positively correlated with the difference in albumin (R² =0.45, p=0.03) and negatively correlated with the difference in urinary protein/creatinine ratio (R² =0.56, p=0.01) (Figure 4, 5).

Figure 1: Distribution of GCSI in the INS subgroups: 40% GC Sensitive, 30 % GC Resistant and 30 % Normal GC sensitivity.

Figure 2: Comparison of GCSI in INS patients Subgroups (GC sensitive, GC resistant, normal GC sensitive) with controls.

^p < 0.05 between Normal GCS and GC Sensitive subgroup.

#p < 0.05 between Normal GCS and GC Resistant subgroup.

$p < 0.05 between GC Sensitive and Control subgroup.

&p < 0.05 between GC Sensitive and GC Resistant subgroup.

*p < 0.05 between GC Resistant and Control subgroup.

Figure 3:  The F-DEX-Monocyte Binding Assay (F-DEX-MBA) results of the INS group (10 patients) represented by the results for each individual patient and ‘control of the day’. The patients are grouped according to their GC sensitivity as resistant, normal and sensitive as defined by our measurements. The red line shows the result for the individual patient; the black line shows the result obtained on the ‘control of the day’. X-axis:  concentrations of F-DEX (400 to 6400 nm) added to triplicate wells. Y- axis: bound F-DEX as per fluorimeter read out at OD488nm.

Figure 4:  In the INS group the GCSI correlated with the difference in albumin (R²=0.45, P=0.03). X-axis: GCSI, Y-axis: difference in albumin. GC sensitive group is shown with red dots, GC Resistant group with yellow dots and Normal GCS group with blue dots.

Figure 5: In the INS group the GCSI negatively correlated with the difference in protein/creatinine ratio (R²=0.56, P=0.01). X-axis: GCSI, Y-axis: difference in urinary protein/creatinine ratio. GC sensitive group is shown with red dots, GC Resistant group with yellow dots and Normal GCS group with blue dots.

Table 1:  Individual patient demographics and biochemical characteristics. Patients are grouped according GCSI.  Values are prior to treatment.

AA -African American, H -Hispanic

ESRD- End-stage renal disease, FSGS- Focal segmental glomerulosclerosis, MCNS- minimal change nephrotic syndrome, DMH - Diffuse mesangial hypercellularity.

Table 2:  Individual patient demographics and biochemical characteristics. Patients are grouped according GCSI.  Values are after treatment.

AA – African American, H – Hispanic

ESRD – End-stage renal disease, FSGS - Focal segmental glomerulosclerosis, MCNS - minimal change nephrotic syndrome, DMH - Diffuse mesangial hypercellularity.

Table 3: Differences in biochemical characteristics between groups, Mean ± SD and p value.

#p<0.05 normal GCS vs GC Resistant

*p<0.05 GC Sensitive vs GC Resistant

&p<0.05 normal GCS vs GC Sensitive

¹p<0.05 normal GCS before and after treatment

²p<0.05 GC Sensitive before and after treatment

³p<0.05 GC Resistant before and after treatment

INS is the most common cause of nephrotic syndrome in children. It is defined by the combination of nephrotic syndrome (proteinuria, hypoalbuminemia, hyperlipidemia, and edema) and non-specific histological abnormalities of the kidney including MCD, FSGS and DMP. Glucocorticoids, predominantly prednisone, are the first line therapy for nephrotic syndrome in children.

Clinical response to steroids has been a predictor of the overall prognosis in children with INS. Patients who are resistant to the initial course of steroids are considered to have a worse overall prognosis, with 36%–50% progressing to end-stage renal disease within 10 years [4, 12]. There are currently no reliable laboratory tests to predict steroid resistance. In this study, we evaluated whether the in-vitro F-Dex-MNC binding assay can be used to predict a clinical response to steroids in the INS patient population. This assay has already been validated in our previous publication regarding PCOS, adrenarche and controls[11].

As per the results of in vitro testing, we divided the patients into three groups based on the GCSI: GC resistant, increased GC sensitivity (GC Sensitive) and normal GC sensitive.  Patients in the GC resistant group did not respond to steroid therapy clinically, while the patients in the increased GC sensitivity (GC Sensitive) and normal GC sensitive groups responded well to steroids (Tables 1, 2, 3; Figure 4,5). This strongly indicated that the clinical steroid resistance response correlated with the GCSI obtained in vitro. In our study of 10 children with INS, 7 patients had normal or increased sensitivity to steroids.  In all these patients, there was a significant improvement in proteinuria after 4-6 weeks of treatment. There was a statistically significant correlation of the GCSI score and serum albumin with a R² of 0.45. The GCSI correlated better with the urinary proteinuria. Three out of the seven patients (42%) in the increased GC sensitivity (GC Sensitive)and normal GC sensitive groups did eventually progress to ESRD.  We can hypothesize several reasons for this; 1) these patients are of African American background, which puts them at high risk of ESRD [12]; 2) the number of patients progressing to ESRD may have been confounded by our small pilot population. We also believe that there is a group of patients, who are initially steroid responsive that eventually become secondarily steroid resistant. In one study looking at recurrence of nephrotic syndrome in patients who received renal transplant for ESRD, 28 of 150 (19%) of these patients were initially steroid responsive and ultimately became steroid resistant and developed ESRD [14].  Given that this is a pilot study, this unexpected higher rate of ESRD seen in this population may not be seen in a larger population.  To that effect, we plan to do a larger study to examine this patient population further.  Three patients with in vitro steroid resistance showed no improvement in proteinuria with worsening of their renal function at follow up.

The ages of patients in this group were heterogeneous, ranging from 3-18 years of age. Similar to what is described in the literature, for our patients between 3-5 years, three had MCNS and one had DMH; whereas the six patients who were 9.5 years and older had FSGS. Our data in this pilot study indicate that overall, 30% of patients with INS did not respond to steroid therapy.  Several previous publications have revealed that resistance to steroid therapy is present in 20 - 30 % of INS patients, which were found to have mostly FSGS [12, 15-17]. The results of the present study, in which 30% of children with GC resistance also had FSGS, substantiates the previous findings. However, not all of the patients with FSGS were steroid resistant.  All of the GC resistant patients in our study showed no improvement in proteinuria with worsening of renal function at follow-up.

At present, non-response to steroid therapy in patients with INS can only be assessed empirically by observing the clinical response after 4-6 weeks of therapy [15, 19]. This may lead to unnecessary use of steroids and drug related complications and even may delay other forms of essential treatment. Currently, many groups are looking at ways to predict the steroid response in this patient population. These investigations involve identifying various biomarkers, metabolomics and proteomic profiles [15, 19]. However, these studies are still in the investigative stages. 

Some studies have looked at in-vitro steroid sensitivity patterns in patients with renal and extra renal disease [20, 21]. In a study by Hearing et al [18] examining the steroid response in patients with ulcerative colitis (UC), it was found that in vitro steroid resistance is an important marker in determining response to steroid treatment in patients with severe UC and may in fact be more predictive of a patient’s outcome than disease severity [20]. However, the methodology used here required the use of the radioactive nucleoside [methyl-H³] thymidine in order to measure the sensitivity of proliferation to suppression of dexamethasone. Radioactive nucleosides are more difficult to use, given the overall safety measures that must be taken and are costlier to purchase and to dispose of Carlotti et al. [21, 22] have described that altered number and affinity of GC receptors may be involved in tissue sensitivity to GC in INS patients using in vitro studies. Bagdasarova et al. [23] also demonstrated that the number of GC receptors was increased in clinically steroid sensitive patients and decreased in steroid resistant patients. In contrast, Haack et al. demonstrated that there were no changes in the density and binding affinity of GC receptors in SR INS patients, using a [H³] dexamethasone binding assay [22].

There are multiple advantages of the F-DEX-MNC in vitro binding assay used in this study. In comparison to the biomarker studies mentioned, the F-DEX-MNC binding assay results correlated with steroid response in a patient with nephrotic syndrome independent of disease entity (FSGS, MCD or DMP) and it can be done anytime within the patient’s treatment course, even for patients that are on steroid therapy. The methodology has a washout procedure that clears the GC receptor, so that the F-DEX can occur without interference from any endogenous or exogenous steroids.  Previously described binding studies [22] using [H³] Dexamethasone, a radioisotope, are more difficult to work with and are significantly more expensive. Also, the F-DEX-MNC binding assay has been validated in other patient populations including predicting steroid sensitivity in patients with PCOS and premature adrenarche [11].

In spite of the fact that the study is limited by the overall small sample size and our group was not able to analyze in vitro sensitivity of the patients prior to starting steroid therapy, the results obtained with the F-DEX-MNC binding assay were strongly predictive of the response to therapy. We plan to do further studies to study this population further.

In conclusion, the present study indicates that the clinical response to corticosteroid therapy in patients with INS can be predicted accurately using an F-DEX-MNC binding assay as an in vitro test. Furthermore, the F-DEX-MNC binding assay has clear advantages over other methodologies used to predict steroid response in this patient population. This can help clinicians choose the appropriate treatment for these patients, while avoiding side effects of steroids.  

There was no funding disclosures for this study.

All authors contributed to the study concept and design. Steven Ghanny, Svetlana Ten, Anil Mongia, Josef Michl, and Amrit Bhangoo wrote the initial draft of the manuscript. Anil Mongia was involved in clinical care of the participants and their follow-up. All authors were involved in editing and revision of the manuscript.  All authors read and approved the final version of the manuscript.

The study was a Case Control study approved by the Institutional Review Board at SUNY Downstate Health Science University (Protocol Number 10-183).

Informed assent and consent were obtained from each subject and her/his parents or guardian.

It was not needed as per Institutional Review Board at SUNY Downstate Health Sciences University.

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