This case control cross-sectional study was conducted at the Department of Biochemistry and Department of Paediatric, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India, over the period of two years from July 2018 to June 2020 after obtaining approval from the Institutional Ethics Committee vide no 850/IEC/IGIMS. The sample size was calculated to a minimum of 104 (52 cases and 52 control) assuming the proportion of cases with hypothyroidism to be about 30%; the proportion of control with hypothyroidism to be about 2% found in a previous study [7]. Written informed consent was obtained from parents of children prior to enrollment. Consecutive children of age between 1 to 18 years coming to the OPD of the Paediatric Department who fulfilled the definition of SRNS [9] were enrolled in the study. Children with Non-nephrotic proteinuria, Congenital NS, NS secondary due to lupus nephritis, Henoch-Schönlein purpura nephritis, metabolic, infectious, vascular, malignant and cardiac disease, and autoimmune thyroiditis were excluded. Other groups of children were enrolled as control, from the outpatient Department of Paediatrics with undetectable thyroid swelling, no evidence of hypothyroidism or hyperthyroidism, an autoimmune disorder, not on any thyroid hormone or drug like Carbimazole with absence of proteinuria. Age and sex were matched and informed consent from parents was obtained. Characteristic finding in NS are heavy proteinuria, hypoalbuminemia (serum albumin <2.5 g/dL), hyperlipaemia (serum cholesterol >200 mg/dL), and oedema [10]. When the urine protein is 3+/4+ on a dipstick test, spot protein/creatinine ratio >2 mg/mg, or urine albumin >40 mg/m² per hour (on a timed urine sample), proteinuria is considered to be in the nephrotic range. Most of the cases, the finding of 3+/4+ proteinuria (on dipstick or boiling test) is sufficient for defining nephrotic range proteinuria. Precise quantitative assessment of proteinuria was not essential and a 24 hour urine protein measurement was not required for the diagnosis of NS [9].

SRNS is defined as the absence of remission despite therapy with four weeks of daily prednisolone with a dose of 2 mg/kg per day. Initial resistance is defined as a lack of remission within the first episode of NS. Late resistance is defined as the patient remains steroid sensitive initially, but demonstrating steroid resistance during further relapse. Complete remission in SRNS is defined as serum albumin >2.5 gm/dL, urine protein:urine creatinine <0.2 g/g and without oedema. Partial remission in SRNS is defined as serum albumin >2.5 gm/dL, urine protein:urine creatinine between 0.2 to 2.0 g/g or oedema. No remission in SRNS is defined as serum albumin <2.5 gm/dL, urine protein:urine creatinine >2 g/g and or oedema [7,11].

In the condition when low Free T4 (normal: 0.7-2 ng/mL) and elevated serum TSH above the upper limit of the reference range (>4.5 mIU/L) is defined as overt hypothyroidism [12]. When there is an elevation in serum TSH above the upper limit of the reference range with a normal serum FT4 concentration defined as subclinical hypothyroidism. Subclinical hypothyroidism is sub classified as follows-Grade 1: subclinical hypothyroidism is defined as TSH >4.5 mIU/L but <6 mIU/L, Grade 2: TSH between 6-12 mIU/L, Grade 3: TSH >12 mIU/L; with normal FT4 concentration [13]. Serum albumin level and proteinuria in NS patients correlated with subclinical hypothyroidism. The low serum albumin level has a high likelihood of subclinical hypothyroidism [14]. As per Indian Paediatric Nephrology Group guidelines children with SRNS were investigated and treated [9]. The following demographic and clinical parameters were recorded: age of onset of NS, age at the time of presentation, sex, duration of disease, oedema, height, weight, and body mass index, type of immunosuppressants advised, type of steroid resistance, remission state. Fasting intravenous blood sample and first morning urine sample collected and following laboratory parameters were analysed in both groups, T3, T4, Free T3, Free T4, Serum albumin, Serum cholesterol, blood urea, Serum creatinine, urine protein: urine creatinine ratio, anti-thyroglobulin, anti-TPO antibodies and estimated Glomerular Filtration Rate (eGFR).

Tightly stopper was applied at the tube immediately after collecting sample. Samples were stored tightly stoppered at room temperature (15 to 30°C) for up to 18 hours. If the assay was not be completed within 18 hours, the samples were refrigerated at 2 to 8°C. If the assay was not be completed within seven days, freeze at -20°C or colder. Antibodies against TPO and thyroglobulin were measured in those patients having abnormal thyroid profile reported. FT3 and FT4 measurements were performed by competitive immunoassay using direct chemiluminescent technology (Beckman coulter). Intra-assay coefficient of variation was <3.5% for TSH, 2.1% for FT4 and 6.8% for FT3. The inter-assay coefficient of variation was <2.1% for TSH, 2.4% for FT4, and 11.9% for FT3.

All measurements and calculations are mentioned as mean±Standard Deviation (SD). MedCalc statistical software Version 19.2.6 was used to do statistical analysis. Between group differences were assessed by the student’s t-test and Wilcoxon test for non-parametric distribution. Pearson correlation coefficient was used for correlation analysis between thyroid hormone profiles and serum albumin or urinary protein/creatinine ratio. The p-value of <0.05 was considered to be statistically significant.

Sixty five paediatric patients were evaluated with SRNS for eligibility, 10 were excluded from the study as per exclusion criteria, out of which 9 were having secondary SRNS, 1 was having hypothyroidism of autoimmune origin as anti-TPO antibody titre was high and three were excluded because consent was not given by parents, 52 children’s with SRNS were found eligible, which include 26 (50%) male and 26 (50%), female child. Demography and clinical profile are shown in [Table/Fig-1]. The mean age at the onset of NS was 4.9±3.2 years and the mean age at the time of thyroid hormone profile analysis was 8.1±3.7 years. Comparison of clinical, laboratory, and thyroid hormone profile between cases and controls shown in [Table/Fig-2]. Serum albumin level 2.5±1.0 g/dL and 3.7±0.3 g/dL found respectively in case and control group and was significantly lower in case group (p<0.01). BMI, Serum Creatinine, Serum Cholesterol were significantly different between case and control. The prevalence of hypothyroidism (overt and subclinical) among the case and control group found 38.46% (n=20) and 1.92% (n=1) respectively. Overt hypothyroidism found in 4 (7.6%) and subclinical hypothyroidism found in 16 (30.76%) in SRNS patients. Out of 16 subclinical hypothyroid patients, two cases with grade 1, 12 cases with grade 2, and two cases with grade 3 found. Cases with overt hypothyroidism (four cases) and grade 3 subclinical hypothyroidism (two cases) were treated with levothyroxine therapy. Serum levels of FT4 were within normal range and TSH value in case and control group was 4.5±4.7 mIU/L and 1.8±1.1 mIU/L, respectively which was significantly different (p< 0.01). Anti-TPO and anti-thyroglobulin titre were in normal range in children with hypothyroidism. Oedema were present in 41 (78.84%) and 2 (3.84%) in cases and controls respectively at the time of presentation. Types of chemotherapy and corticosteroids administered are shown in [Table/Fig-3]. No significant difference was found between the occurrence of hypothyroidism and the type of chemotherapy, corticosteroids, or the number of chemotherapy cycles. When correlating urinary protein/Cr ratio with thyroid hormone profiles, urinary protein/Cr ratio negatively correlated with serum T3, T4, and free T4 levels (r=-0.5874, p=0.002; r=-0.5695, p=0.004; r=-0.5425, p=0.006), respectively. The urinary protein/Cr ratio positively correlated with serum TSH levels (r=0.5102, p=0.013) [Table/Fig-4].

Interpretation of the interactions between thyroid and renal function may be a challenge for paediatricians doing the treatment of patients with thyroid and renal disease. In cases of NS, thyroid function gets affected through low circulating thyroid hormone concentration, insufficient binding to carrier proteins or altered iodine storage in the thyroid gland [15]. Among different subtypes of NS, SRNS has a chronic and progressive course. In present study 38.46% of all SRNS patients found to have hypothyroidism. A 30.76% had subclinical hypothyroidism and 7.6% had overt hypothyroidism. The prevalence of hypothyroidism in SRNS in this study was more than reported by a single centre study from north India done by Kapoor K et al., and Marimuthu V et al., from south India were 30% and 33.3% respectively [7,16]. Few other studies have evaluated the prevalence of hypothyroidism in SRNS. Dagan A et al., found in case series of five children with SRNS, who on follow-up for the period of (5-42 months) developed non-autoimmune hypothyroidism. All the five children subsequently developed End Stage Renal Disease (ESRD) and required dialysis and/or transplantation [8]. Guo QY et al., studied on 164 patients and found that the incidence of abnormal thyroid function was 44.51% of paediatric NS patients [13]. Sharma S et al., studied on 50 children with SRNS prevalence of subclinical hypothyroidism was 20% with a positive correlation between TSH level and proteinuria [4]. Studies have also shown that serum albumin and serum TSH to be negatively correlated. The incidence of hypothyroidism increases as the GFR decreases [6,17].

Children having exclusively idiopathic SRNS were enrolled in the present study in order to ensure homogeneity and therefore excluded secondary SRNS. In case of IgA nephropathy, membranous nephropathy and membranoproliferative glomerulonephritis thyroid dysfunction has been earlier reported with and is mainly due to autoimmune mechanisms in these disorders [18]. In present study the prevalence of non-autoimmune acquired hypothyroidism only were evaluated; and ruled out autoimmune causes.

The decision to administer thyroxine to overt and grade 3 subclinical hypothyroidism was based on the important effect of thyroid hormone on growth and metabolism, the cardiovascular and central nervous system and the skeleton in health and disease. Accordingly, other authors reported the administration of thyroxine in infants who have hypothyroidism [16].

The study design was cross sectional and so follow-up levels of serum T3, T4 and TSH levels were not measured. It was also not observed that whether there was any development of overt or subclinical hypothyroidism. Molecular and biochemical mechanisms for subclinical hypothyroidism e.g., estimation of urinary loss of T3, T4 and TSH levels were also not assessed. The present study did not examined the relationship between histopathological profile, duration of the disease and thyroid status. Once the mechanism of hypothyroidism in NS is established through large-scale prospective studies, it will help to establish a reasonable basis to develop a treatment protocol to start levothyroxine administration.

On the basis of the findings of present study, subclinical non-autoimmune hypothyroidism was detected in a significant number of children with SRNS. Evaluation of thyroid profile should be done routinely in case of SRNS seems to be rational approach. This can help in planning preventive and therapeutic approach for early recognition of hypothyroidism in SRNS.