Circulating and urinary microRNA profile in focal segmental glomerulosclerosis: a pilot study

Abstract

Background: MicroRNAs (miRNAs) are noncoding RNA molecules that play important roles in the pathogenesis of various kidney diseases. We investigated whether patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) have distinct circulating and urinary miRNA expression profiles that could lead to potential development of noninvasive biomarkers of the disease.

Materials and methods: Exosome miRNAs were extracted from plasma and urine samples of patients with primary FSGS (n = 16) or MCD (n = 5) and healthy controls (n = 5). Differences in miRNA abundance were examined using Affymetrix GeneChip miRNA 3.0 arrays. QRT-PCR was used to validate the findings from the array.

Results: Comparison analysis of FSGS versus MCD revealed 126 and 155 differentially expressed miRNAs in plasma and in urine, respectively. Only 38 of these miRNAs were previously cited, whereas the remaining miRNAs have not been described. Comparison analysis showed that a significant number of miRNAs were downregulated in both plasma and urine samples of patients with FSGS compared to those with MCD. Plasma levels of miR-30b, miR-30c, miR-34b, miR-34c and miR-342 and urine levels of mir-1225-5p were upregulated in patients with MCD compared to patients with FSGS and controls (P < 0.001). Urinary levels of mir-1915 and miR-663 were downregulated in patients with FSGS compared to MCD and controls (P < 0.001), whereas the urinary levels of miR-155 were upregulated in patients with FSGS when compared to patients with MCD and controls (P < 0.005).

Conclusions: Patients with FSGS and MCD have a unique circulating and urinary miRNA profile. The diagnostic and prognostic potential of miRNAs in FSGS and MCD warrants further studies.

Keywords: chronic kidney disease; circulatory miRNA; focal segmental glomerulosclerosis; minimal change disease; urinary miRNA.

Figure 1

Comparative analysis of miRNA expression levels in plasma and urine samples obtained from FSGS, MCD and control subjects. FSGS induces miRNA down-regulation in both plasma and urine. Significantly more miRNAs were down-regulated in both plasma and urine samples of FSGS patients compared to normal controls and MCD. On the other hand, miRNA expression was significantly up-regulated in both plasma and urine of MCD patients when compared to FSGS or controls. miRNAs with > 2-fold dysregulated expression are graphed for plasma and urine samples. The percentages of up- or down-regulated miRNAs are shown with corresponding up or down arrows on each graph.

Figure 2

Unsupervised hierarchical clustering over selected miRNAs (Pearson correlation, average linkage). Heatmap colors represent relative miRNA expression as indicated in the color key for each panel. Brackets on the top margins indicate samples from the same cohort. (A) Clustering of plasma samples classified as primary FSGS (n = 16, yellow), MCD (n = 5, magenta) and controls (n = 5, green) over 70 miRNAs. Samples are in columns, miRNAs in rows. Only miRNAs that survived multiple testing (FDR), and had a fold-change > 3 or < −3 and p < 0.05 are shown. (B) Clustering of urine samples classified as FSGS (n = 15, yellow), MCD (n = 5, magenta) and normal control (n = 5, green) over 42 miRNAs. Only miRNAs that survived multiple testing (FDR), and fold-change > 3 or < −3 and p < 0.05 are shown. (Not all of the differentially regulated miRNA are shown in the figure).

Figure 3

(A) Comparative analysis of miRNA expression levels in plasma and urine samples obtained from sex- and age-matched FSGS and control subjects. Significantly more miRNAs were down-regulated in urine samples of FSGS patients compared to normal controls. miRNAs with > 2-fold dysregulated expression are graphed for plasma and urine samples. The percentages of up- or down-regulated miRNAs are shown with corresponding up or down arrows on each graph. (B) Unsupervised hierarchical clustering over selected miRNAs (Pearson correlation, average linkage). Heatmap colors represent relative miRNA expression as indicated in the color key for each panel. Brackets on the top margins indicate samples from the same cohort. (Left) Clustering of plasma samples classified as primary FSGS (n = 16, yellow), MCD (n = 5, magenta) and controls (n = 5, green) over 33 miRNAs. Samples are in columns, miRNAs in rows. Only miRNAs that survived multiple testing (FDR), and had a fold-change > 3 or < −3 and p < 0.05 are shown. (Right) Clustering of urine samples classified as FSGS (n = 15, yellow), MCD (n = 5, magenta) and normal control (n = 5, green) over 65 miRNAs. Only miRNAs that survived multiple testing (FDR), and fold-change > 3 or < −3 and p < 0.05 are shown. (Not all of the differentially regulated miRNA are shown in the figure).

Figure 4

QRT-PCR verification of (A) plasma and (B) urine miRNAs expression results from microarray data. Blue bars represent the results from microarray, while red bars indicate the results from qRT-PCR. The error bars are the standard error of mean (SEM) for each analysis. Four representative miRNAs (miR-30b/c and miR-34b/c) were observed up-regulated in MCD plasma compared to FSGS and controls. miR-1225 found to be up-regulated specifically in MCD urine compared to FSGS and normal control. miR-1915 was significantly down-regulated in FSGS urine compared to MCD and controls, whereas miR-155 was up-regulated in FSGS urine compared to MCD and controls. *P<0.01.

Figure 5

Relation between levels of representative dysregulated plasma and urine miRNAs in samples from FSGS patients and: (A) glomerular filtration rate (GFR) and (B) proteinuria. Data are compared by Spearman’s rank correlation coefficient.