MicroRNA Expression and Target Prediction in Children with Nephrotic Syndrome

Introduction

Nephrotic syndrome is a common kidney syndrome in children.^1-4 Certain microRNAs have been experimentally validated in order to identify and select the specific function related to this condition.⁵ Almost 80% of children with idiopathic nephrotic syndrome show remission of proteinuria with corticosteroids (steroid sensitive), but nearly 40%–60% may tend to experience intermittent relapses.^6-8 Steroid resistance denotes the absence of remission following at least 4 weeks of prednisone therapy.⁹ While about 30% patients with steroid resistance have an underlying genetic etiology, the exact disease mechanisms for others remain elusive. Epigenetic modification of gene expression, including expression of noncoding RNA (microRNAs), might provide an etiological clue in patients without a definitive genetic etiology. Further research is needed to fully comprehend the implications of this discovery and explore potential interventions or treatments.¹⁰ Our study focuses on idiopathic nephrotic syndrome in children with steroid sensitive nephrotic syndrome (SSNS) and steroid resistant nephrotic syndrome (SRNS).

MicroRNAs (miRNAs), which regulate target genes in the post-transcriptional processes and play crucial roles in many disorders, are about 20 nucleotides in length. miRNAs have been demonstrated to function as noninvasive biomarkers in a variety of diseases, including renal disorders, in serum and urine.^11,12

Both microRNAs (miRNAs) and RNA binding proteins (RBPs) are crucial transcriptional regulators of gene expression. These two factors also act to reduce translation by translation repression or miRNA cleavage.¹³ They are found to be a biomarker not only in diseases such as viral infections, cardiovascular disorders, diabetes, and cancer, but also in kidney diseases.¹⁴

Kidney disease has been discovered to be significantly influenced by miR-155. It has been linked to a number of pathological processes, including fibrosis, immune response regulation, and inflammation, all of which are crucial for the onset and progression of kidney disorders.⁶ A study by Wang et al., has compared expression pattern for miR-424 between the nondiabetics along with the diabetic nephropathy patients and it was observed that the expression of miR-424 in the kidneys of diabetic nephropathy patients was considerably higher. Additionally, inhibiting miR-424 decreased kidney damage and fibrosis in vitro, indicating that miR-424 may be involved in the onset and progression of diabetic nephropathy in people.⁷ When compared to healthy controls, the researchers discovered miR-17 was noticeably elevated in the glomeruli of patients with membranous nephropathy. miR-17 overexpression increased albuminuria and podocyte death in cultured podocytes, pointing to a potential function for miR-17 in the onset and progression of membranous nephropathy.⁸

The present study investigated the differential expression of miR-17-5p, miR-155p, miR- 424 -5p, with miR-484-5p as endogenous control in urine samples between the study groups.

Materials and Methods

The study was conducted at Sri Ramachandra Institute of Higher Education and Research and the samples were collected from the Department of Nephrology, SRIHER, Chennai, India. The children who have met the criteria were enrolled for the study are within the age group 1–12 years. The sampling for the SRNS was taken before the starting of calcineurin inhibitors and then for the SSNS group, it was taken during the relapse. The study included 300 participants, 200 cases with SSNS and SRNS, and 100 age-matched controls. Informed consent was obtained from the parents or guardians of all participants. Ethics committee approval was obtained from the Institutional Ethics committee at Sri Ramachandra Institute of Higher Education and Research [IEC-NI/21/FEB/77/2].

Results

Target prediction analysis

The miRtarlink has revealed that miR-155p, miR-424-5p, and miR-17-5P are found to have stronger predication connected with the diseases pathogenesis and are represented in Figure 1a and b and miRWalk is represented in Table 1. The targeted microRNAs were also analyzed for the various pathways that are associated with the nephrotic syndrome. A detailed analysis involving various functions of candidate miRNAs has been broadly represented with their unique IDs in Table 1.

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Figure 1:

(a) Represents the gene ANLN and its targeting microRNAs. (b) Represents the microRNA(miR-424-5p) and its targeting genes. ANLN: Anillin

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Table 1: Target Prediction for the selected microRNAs and its biological function

microRNAs	Predicted Targets
has-miR-424-5p	VEGFA
has-miR-17-5p	IL8
has-miR-155-5p	IL8, AGTR1, TRPS1, NR3C1

Description	Term Id	Corrected P Value
Molecular Function
Cytokine receptor binding	GO: 0005126	3.952×10-4
Signaling receptor binding	GO: 0005102	1.025×10-3
Vascular endothelial growth factor receptor binding	GO: 0005172	1.902×10-3
Biological Process
Cell migration	GO: 0016477	1.620×10-2
Localization of cell	GO: 0051674	2.953×10-2
Cell motility	GO: 0048870	2.953×10-2
Cellular Components
Platelet alpha granule lumen	GO: 0031093	3.905×10-2
Reactome Pathway
Nephrin family interactions	REAC: R-HSA-37375	2.911×10-2
Wiki Pathway
Nephrotic syndrome	WP: WP4758	9.984×10-4
HP
Abnormal nephron morphology	HP: 001257	1.165×10-3
Focal segmental glomerulosclerosis	HP: 0000097	1.715×10-2
Abnormal renal glomerulus morphology	HP: 0000095	2.653×10-3
Abnormal renal glomerulus morphology	HP: 0000095	3.929×10-3
Proteinuria	HP: 0000093	5.157×10-3
Glomerular sclerosis	HP: 0000096	5.157×10-3
Abnormal urine protein level	HP: 0020129	5.649×10-3

RNA: Ribonucleic acid, VEGFA: Vascular endothelial growth factor A, AGTR: Angiotensin II Receptor Type 1, TRPS: Trichorhinophalangeal Syndrome, NR3C: Nuclear Receptor Subfamily 3 Group C Member (also known as Glucocorticoid Receptor), HP: Haptoglobin, REAC: Ras-Responsive Element-Binding Protein (also known as ATF2 - Activating Transcription Factor 2), R-HSA: Reactome Database (R) Stable identifier corresponding species (Homosapiens (HSA), WP: Wingless-related Pathway)

MicroRNA expression analysis

The basic demographic characteristics are shown in Table 1. In comparison to the control group, it was observed that miR-17, miR-424, and miR-155 were elevated in SSNS and SRNS as shown in Figure 2 a-f. Further, the microRNAs were compared within the case groups (SSNS, SRNS) to identify the fold change and found that miR-424 and miR-155 are upregualated (two-fold increase) in SRNS group while miR-17 is found to be downregulated in SRNS group as shown in Figure 3 and Table 2.

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Figure 2:

(a-f) Urinary microRNA expression of miR-155, miR-424 and miR-17 in children with SSNS and SRNS. SSNS: Steriod sensitive nephrotic syndrome, SRNS: Steriod resistant nephrotic syndrome.

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Figure 3:

(a-c) Bar diagrams showing fold change in the expression levels of miR-424.,miR-17 and miR-155 among the cases. SSNS: sensitive nephrotic syndrome, SRNS: steroid resistant nephrotic syndrome

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Table 2: Relative expression analysis of microRNAs between SSNS and SRNS

miRNA	Fold change	P value
miR-424	SSNS = 0.12 SRNS = 2.80	SSNS = <.0001 SRNS = 0.00056
miR-17	SSNS = 2.80 SRNS = 1.72	SSNS = 0.001779 SRNS = 0.329064
miR-155	SSNS = 1.33 SRNS = 2.95	SSNS = 0.179375 SRNS = 0.0009

RNA: ribonucleic acid, SSNS: steroid sensitive nephrotic syndrome, SRNS: steroid resistant nephrotic syndrome

Discussion

Many studies have reported the importance of microRNAs and its potential roles in apoptosis, cell differentiation and cell proliferation with the inclusion of the kidney disease. Differential expression miRNAs studies has been helpful in genome-wide profiling studies.^10-14 In kidney injury, miR-21 and miR-155 were found to show decreased level of expression in blood and urine samples.¹⁵ By using a micro-RNA array, Luo et al. showed that miRNAs mir30a05p, mir-151-3p, and miR-191 showed high level of expression in urine and serum samples in NS children when compared to the healthy individuals.¹⁶

In this study, we investigated the expression of miR-17 on the pathogenesis of nephrotic syndrome. The expression pattern observed for miR-17 was downregulated in our study, while a study by Zang et al. (2020), showed that mir-17-5p was found to be significantly increased in childhood nephrotic syndrome when compared with the healthy subjects.¹⁷ The miR-17 deregulation has been seen in the kidneys of nephrotic syndrome patients in numerous investigations. The authors hypothesized that elevated miR-17 expression may aid in FSGS development by encouraging podocyte damage and dysfunction.¹⁷

Our findings revealed that miR-155 expression was significantly upregulated in the glomeruli of patients with active minimal change disease (MCD), a common cause of nephrotic syndrome in children. These results are consistent with previous studies by Ramezani, et al.,¹⁸ and Zhang et al. (2020).¹⁹

Our findings are in contrast to the study by Shaffi SK, et al. that has investigated the expression levels of miR-424 in urine and plasma samples renal parenchymal diseases which has shown down regulation.²⁰

The limitation of the study is that the biopsy correlation with miRNA expression was not performed because our study was not focused on the kidney biopsy results; no correlation analysis was performed for the data on the kidney biopsy.

Conclusion

This was the first study to report the expression pattern of the microRNAs which specifically plays an important role in the podocyte junction of actin cytoskeleton. miR-424-5p and miR-155p is upregulated in SRNS group while miR-17-5p is downregulated. Combined analysis of gene expression along with the studied candidate microRNAs can give a better understanding in bringing out the knowledge in the childhood nephrotic syndrome in future studies.