Higher Urine Exosomal miR-193a Is Associated With a Higher Probability of Primary Focal Segmental Glomerulosclerosis and an Increased Risk of Poor Prognosis Among Children With Nephrotic Syndrome

Abstract

Background: In children, focal segmental glomerulosclerosis (FSGS) is one of the most common primary glomerular diseases leading to end-stage renal disease. Exosomes facilitate communication between cells by transporting proteins and microRNAs. We aimed to investigate the utility of urine exosomal miR-193a for diagnosis and prognosis estimation among patients with primary FSGS, and preliminarily explore the regulation mechanism of exosome secretion from podocytes. Methods: Specimens of urine were obtained from patients with primary FSGS, minimal change nephropathy (MCN) and IgA nephropathy (IgAN), followed by exosome isolation. We quantified urine exosomal miR-193a based on quantitative reverse transcription-polymerase chain reaction, and evaluated its applicability using area-under-receiver-operating-characteristics curves (AUROCs). The semiquantitative glomerulosclerosis index (GSI) was used to evaluate the degree of glomerulosclerosis according to the method of Raij et al. We further used FAM-labeled miR-193a-5p to examine exosome shuttling using confocal microscopy for visualization, and explored the regulation mechanism of exosomes release from podocytes using Fluo-3AM dye. Results: Urine exosomal miR-193a levels were significantly higher in patients with primary FSGS than those with MCN and IgAN. The AUROCs for discriminating between primary FSGS and MCN or IgAN were 0.85 and 0.821, respectively. Urine exosomal miR-193a levels positively correlated with GSI in patients with primary FSGS. We further found that kidney tissues from these patients had increased CD63 expression involving podocytes in non-sclerotic tufts. Exosomes from cultured podocytes could transport miR-193a-5p to recipient cells, potentially through a calcium-dependent release mechanism. Conclusion: Urine exosomal miR-193a might be harnessed as a non-invasive marker for diagnosis and outcome assessment among patients with primary FSGS. Exosomes were potential vehicles for miRNAs shuttling between podocytes, and released from podocytes in a calcium-dependent manner.

Keywords: exosomes; focal segmental glomerulosclerosis; microRNA-193a; nephrotic syndrome; podocyte.

FIGURE 1

Identification and characterization of exosomes. (A) Urine exosomes were isolated from urine by the ExoQuick exosome precipitation solution. Urine vesicles, showing the characteristic exosomal cup-shape and size, are shown in the representative electron micrographs; scale bar = 200 nm. (B) Expression of exosomal markers (HSP70, CD9) was assessed by immunoblotting in total protein extracts from isolated urinary exosomes.

FIGURE 2

Relative expression levels of urine exosomal miR-193a between patients with FSGS patients and controls. Relative expressions were shown which was calculated with the equation 2^{–delta Ct}, normalized for RNU6 and relative to the control average. Scatter plot (middle line: median). *P < 0.05.

FIGURE 3

Diagnostic values of urine exosomal miR-193a levels in patients with primary FSGS. ROC curve analysis of miR-193a expression to discriminate between primary FSGS patients and MCN or IgAN patients. The areas under the ROC curve were 0.85 and 0.821, respectively.

FIGURE 4

Prognostic values of urine exosomal miR-193a levels in patients with primary FSGS. (A) Representative photomicrographs of the renal biopsy with Masson’s trichrome staining (200×). (B) The correlation between the expression level of urine exosomal miR-193a with GSI in patients with primary FSGS.

FIGURE 5

Representative micrographs show renal CD63 staining (IHC, 400×). The expression level of CD63 was significantly up-regulated in patients with FSGS compared with TBMD, which was mainly located in the cytoplasm of podocytes within non-sclerotic tuft segments (indicated by black arrows).

FIGURE 6

Visualization of exosomes shuttling FAM-labeled miR-193a-5p to recipient podocytes. Recipient podocytes were incubated with donor podocytes-derived exosomes (10 μg/ml). Yellow fluorescent “specs” (indicated by white arrow) represent N-Rh-PE labeled exosomes containing FAM-miR-193a-5p taken up by the recipient podocytes.

FIGURE 7

Monensin stimulates exosome release by a calcium-dependent mechanism. (A) Podocytes were loaded with Fluo-3AM, and intracellular Ca²⁺ concentration was measured. Shown are changes in Ca²⁺ concentration elicited by 7 μM MON. Data are representative of at least three independent experiments. (B) Podocytes were incubated for 7 h in the presence of 1.5 mM EGTA, 30 μM BAPTA-AM, 7 μM MON, or the combination of 1.5 mM EGTA/7 μM MON (MON + EGTA) or 30 μM BAPTA-AM/7 μM MON (MON + BAPTA-AM). The secreted exosomes were collected and quantitated by measuring the AChE activity. ^∗ significantly different from the control, P < 0.05. # significantly different from the MON-treated cells, P < 0.05.