Differential microRNA expression in the serum of patients with nephrotic syndrome and clinical correlation analysis

Abstract

Many different microRNAs existed in nephrotic syndrome patients, and they may be involved in nephrotic syndrome occurrence. In order to further clarify miRNAs expression changes in nephrotic syndrome patients and their correlation with clinical features, this study investigated differential microRNA expression in the peripheral serum of patients with nephrotic syndrome and analyzed the correlation between miRNA with largest overexpression level and clinical features. miRNAs microarray was applied to screen different expressed miRNAs in nephrotic syndrome patients. Real-time PCR was performed to verify miRNA expression level. SPSS software was used to analyze correlation between miRNA expression and clinical features. Compared with healthy subjects, 35 miRNAs overexpressed and 24 miRNAs down-regulated in patients. After real-time PCR verification, 6 miRNAs up-regulated in nephrotic syndrome patients, including hsa-miR-181a, hsa-miR-210, hsa-miR-30a, hsa-miR-942, hsa-miR-192 and hsa-miR-586. miRNA-30a significantly overexpressed in nephrotic syndrome patients and with no difference between genders. miRNA-30a expression level in drug resistant nephrotic syndrome patients was obviously higher than the drug sensitive patients. miRNA-30a up-regulated most significantly in mesangial proliferative glomerulonephritis among different pathology types, while it decreased most obviously in glomerular lesions. miRNA differently expressed in the serum of nephrotic syndrome patients. miRNA-30a could be treated as the molecular marker in predict drug resistance and pathological type of nephrotic syndrome.

Introduction

Nephrotic Syndrome (NS) is a comprehensive disease characterized as glomerulus basement membrane permeability increase by multiple factors (such as oxidative stress, immune, etc.). Clinical characteristics mainly include proteinuria, hypoalbuminemia, edema, and hyperlipidemia. At present, the diagnosis of nephrotic syndrome pathology mainly relies on the renal biopsy. However, renal biopsy is an invasive examination but lack of sensitivity and specificity. Therefore, searching for specific and sensitive method to identify the pathological types of nephrotic syndrome is of great significance.

MicroRNAs is a kind of non-coding RNA about 19-24 nucleotides that exist in eukaryotes. It could degrade mRNA or inhibit mRNA transcription through the 3’-UTR region of the target mRNAs. Because of its conservative feature in evolution and important role in the physiological function, it could be treated as predictors for disease classification and clinical process based on specific expression. Many studies have shown that microRNAs could regulate kidney cells in a variety of different pathological types of nephrotic syndrome. For example, miRNA-192 is the only miRNA that has been found expressed in glomerular mesangial cells, and it may affect pathological occurrence by participating in extracellular matrix proteins accumulation. In addition, the miRNA-29c expression changes causes transforming growth factor β1 (TGF-β1) abnormal expression, leading to renal interstitial fibrosis occurrence [1-3]. A variety of miRNAs play an important role in the initiation and progression of kidney disease [4]. Meanwhile, miRNAs in peripheral blood might be treated as the molecular biomarkers of human disease [5]. Zhang C investigated serum miRNA expression in focal segmental glomerulosclerosis (FSGS) patients, and found that miR-186 expression was correlated with proteinuria level and might be served as biomarkers for FSGS [6]. MiRNAs changes in the urine also can be used as biomarkers for nephrotic syndrome [7]. MiR-29a, miR-192 and miR-200c showed characteristic expression changes in the urine of nephrotic syndrome adults, and can be used as markers for disease diagnosis and severity evaluation [8]. At present, serum is the easiest accessed biological sample with individual physiology information [9,10]. Compared with healthy control, overexpressed miRNAs in the serum could be more easily detected than down-regulated miRNAs. This study investigated miRNAs expression in the serum of nephrotic syndrome patients and discussed the correlation between overexpressed miRNA and clinical features, providing auxiliary examination indexes for nephrotic syndrome.

Materials and methods

Taqman low density miRNA microarray

800 μl blood was extracted from 25 cases of nephrotic syndrome patients and 20 cases of health controls. RNA was extracted and stored at -80°C. MiRNA expression level was detected by ABI Company. U6 was selected as control. The study protocol was approved by the Research Ethics Committee of our hospital, and all patients gave their informed consent before study commencement.

Serum RNA extraction

RNA was extracted and stored at -80°C for RT-PCR.

Real-time PCR

MiRNA was reverse transcripted to DNA using PCR kit (Takara, Japan). Each real-time RT-PCR reaction (in 20 µL) contained 2.5×SYBR Green Mixture, 5 µM primers and 2 µL template cDNA. The cycling conditions consisted of an initial, single cycle of 30 s at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 55°C, and 60 s at 72°C. PCR amplifications (ABI 7500 PCR amplifier, Applied Biosystem) were performed in three duplicates for each sample.

Statistical analysis

Numerical data were presented as means and standard deviation (± SD). All statistical analyses were performed using SPSS13.0 software (SPSS Inc., USA). Differences between multiple groups were analyzed by t-test or one-way ANOVA. P < 0.05 was considered as significant difference.

Results

Microarray result analysis

Taqman low density microarray was applied to detect serum miRNA expression changes in nephrotic syndrome patients and healthy controls. Screening standard of differentially expressed miRNAs was as follows: hybridization signal strength ratio between nephrotic syndrome patients and control > 1 and P < 0.05 was defined as up-regulation; while the ratio < 1 and P < 0.05 was defined as down-regulation. Compared with healthy subjects, 35 miRNAs overexpressed and 24 miRNAs down-regulated in patients. Has-miR-181a expression level changes have been reported in the literature. We listed the miRNAs with most obviously up-regulation (Table 1) and down-regulation (Table 2).

Table 1.

Up-regulated miRNAs in the serum of nephrotic syndrome patients

miRNA ID	NS patient signal strength	Healthy subjects signal strength	Fold change
hsa-miR-923	17130	10324	1.66
hsa-miR-942	10286	6584	1.56
hsa-miR-181a	14987	10643	1.41
hsa-miR-1308	1467	364	4.03
hsa-miR-151	1044	548	1.90
hsa-miR-222	887	432	2.05
hsa-miR-191	765	345	2.22
hsa-miR-210	11243	10189	1.10
hsa-miR-30a	2340	870	2.69
hsa-miR-231	1743	786	2.22

Table 2.

Down-regulated miRNAs in the serum of nephrotic syndrome patients

miRNA ID	NS patient signal strength	Healthy subjects signal strength	Fold change
hsa-miR-16	2443	2614	0.93
hsa-miR-510	843	2170	0.39
hsa-miR-172	2659	3430	0.78
hsa-miR-1245	2848	2934	0.97
hsa-miR-633	3445	5676	0.61
hsa-miR-320	545	789	0.69
hsa-miR-1324	2911	3455	0.84
hsa-miR-626	3004	3435	0.87
hsa-miR-383	31426	53435	0.87
hsa-miR-422a	2736	3812	0.72

Real-time PCR verification

Real-time PCR was used to detect serum miRNA that overexpressed in Taqman low density microarray. U6 was selected as control and the screening standard was fold change ≥ 1.5 and P < 0.05. 6 miRNAs up-regulated in nephrotic syndrome patients, including hsa-miR-181a, hsa-miR-210, hsa-miR-30a, hsa-miR-942, hsa-miR-192 and hsa-miR-586 (Figure 1). miR-30a exhibited the largest overexpression level.

Figure 1.

qRT-PCR verification. **P < 0.05 compared with healthy subjects.

Correlation analysis between miR-30a expression and NS patients’ clinical features

A total of four kinds of different pathological types including mesangial proliferative glomerulonephritis (MPGN), podocyte lesion (PCL), and glomerular interstitial nephritis (IGN), and glomerular lesions (GMC) according to the kidney biopsy. MiR-30a expression level in different pathological types was listed in Table 3. MiRNA-30a significantly overexpressed in nephrotic syndrome patients and with no difference between genders. MiRNA-30a expression level in drug resistant nephrotic syndrome patients was obviously higher than the drug sensitive patients. miRNA-30a up-regulated most significantly in mesangial proliferative glomerulonephritis among different pathology types, while it decreased most obviously in glomerular lesions.

Table 3.

Correlation analysis between miR-30a expression and NS patients’ clinical features

Index	NS	Healthy subjects	P value
Gender
Male	7.8 ± 4.5	2.3 ± 1.3	0.023
Female	7.2 ± 3.6	1.5 ± 0.9	0.001
Drug resistance
Hormone sensitive	6.3 ± 3.6	2.0 ± 1.3	0.003
Hormone resistance	8.6 ± 4.5**	2.0 ± 1.3	P < 0.001
Pathological type
MPGN	9.8 ± 4.5	2.0 ± 1.3	0.023
PCL	7.2 ± 3.6&&	2.0 ± 1.3	P < 0.001
IGN	2.4 ± 4.5&&,##	2.0 ± 1.3	0.431
GMC	2.2 ± 3.6&&,##	2.0 ± 1.3	0.329

^** P < 0.05, compared with hormone sensitive;

^&& P < 0.05, compared with MPGN;

^## P < 0.05, compared with PCL.

Discussion

Several researches suggested that miRNAs participate in a variety of disease by inhibiting mRNA expression and can be used as molecular diagnostic markers [11-13]. MiRNA-192 expression level in kidney was significantly higher than that of the bone marrow, while it plays an important role in the renal epithelial sodium ion transport. It has been found that kidney disease can lead to specific circulating miRNA expression change. Studies have suggested that miR-16 and miR-320 expression level significantly elevated in patients with acute kidney disease [14]. At the same time, serum miRNA showed stronger stability than the cells. Gui J et al. found that serum miR885-5p might be a potential biomarker for liver pathology [15]. MiR-126 differentially expressed in multiple tumors including renal cell carcinoma, and can be used as the marker to differentiate transparent cell carcinoma and papillary carcinoma [16]. Thus, quantitative detection of miRNAs in the blood can be treated as a new method to detect and monitor kidney disease.

In this study, TaqMan low density microarray was applied to detect serum miRNAs expression in patients with nephrotic syndrome. Since the microarray may have false positive result, real-time PCR was used for validation. 35 miRNAs up-regulated in nephrotic syndrome patients such as hsa-miR-30a, hsa-miR-221, and hsa-miR-181a, of them miRNA-181a has been reported. Sui W et al. found that miR-181a, miR-483-5p, and miR-557 differentially expressed in nephrotic syndrome, and might be used as peripheral blood biomarkers for diagnosis [17]. Zhu et al. showed that after transfection with Anti-miRNA-181a, tubular epithelial cells apoptosis degree reduced treated by DDP, indicating miR-181a may down-regulate BAX expression and impact kidney disease [18]. Our study also detected 24 down-regulated miRNAs, such as hsa-miR-320 and hsa-miR-510. Johan M Lorenzen suggested that plasma miR-320 and miR-16 expression level decreased, while miR-210 increased (P < 0.0001) in patients with acute renal failure [14]. They could prompt patient’s survival rate and act as new biomarkers. In the study about kidney transparent cell carcinoma, miR-210 overexpressed significantly in the cancer tissue and adjacent normal tissue, and obviously correlated with shorter overall survival and disease recurrence [19].

Real-time PCR was used for miRNA verification. The results showed that serum hsa-miR-181a, hsa-miR-210 and hsa-miR-30 still up-regulated in patients compared with the healthy control. The pathogenesis of primary nephrotic syndrome is still unclear. Mathieson PW study revealed that nephrotic syndrome occurrence may be associated with glomerular layer sertoli cell defects [20]. When the Dicer enzyme was knockout in sertoli cells, adult mice appeared significant proteinuria in four or five weeks after birth, following with kidney failure. Some pathological changes such as sertoli cell damage, capillary expansion were due to the lack of miRNAs [21,22], suggesting miRNAs play an important role in maintaining normal renal function. At the same time, it was found that miR-30a plays an important effect on sertoli cell structure and function. Thus, it is reasonable to speculate that miR-30a plays an important role in nephrotic syndrome occurrence. Current studies also found that serum miR-30a-5p, miR-151-3p, miR-150, miR-191, and miR-19b increased in children with nephrotic syndrome, while urine miR-30a-5p expression also up-regulated [23]. Also, some researchers found that [24], miR-30a may affect kidney development and regulate kidney maturity by targeting transcription factor Xlim1/Lhx1.

Correlation analysis between miRNA-30a and kidney disease clinicopathological characteristics showed that miR-30a failed to present difference between different genders, while it was different between drug resistant or sensitive cases. MiRNA-30a overexpressed more significantly in drug resistant cases. In patients with different pathological types, miR-30a showed obvious statistical difference in mesangial proliferative glomerulonephritis and sertoli cell lesions with glomerular interstitial nephritis and glomerular lesions. It may be because of the miR-30a influence on sertoli cell apoptosis and proliferation.

Based on previous studies results, miRNA-30a overexpressed in kidney development [25]. Together with our results, it suggested that miR-30a may help diagnose drug resistance and pathological type. It still needs further investigation about the mechanism of miR-30a influence on nephrotic syndrome occurrence and different pathological types. At the same time, since the population study might be affected by many factors, further in vitro study is needed.

Multiple miRNAs abnormal expressed in the serum of nephrotic syndrome patients, suggesting that miRNAs could be treated as the serum biomarkers to differentiate pathological classification and drug resistance. This study only studied miR-30a, whether other miRNAs changes exist in the serum of patients with nephrotic syndrome and related in different pathological types still need further research.

Disclosure of conflict of interest

None.