Renal miR-148b is associated with megalin down-regulation in IgA nephropathy

Abstract

Megalin is essential for proximal tubule reabsorption of filtered proteins, hormones, and vitamins, and its dysfunction has been reported in IgA nephropathy (IgAN). miR-148b has been shown to regulate renal megalin expression in vitro and in animal models of kidney disease. We examined a potential role of miR-148b and other miRNAs in regulating megalin expression in IgAN by analyzing the association between megalin and miR-148b, miR-21, miR-146a, and miR-192 expression. Quantitative PCR (qPCR) analysis identified a marked increase in renal levels of several miRNAs, including miR-148b, miR-21, miR-146a, and a significant decrease in megalin mRNA levels in IgAN patients when compared with normal controls. By multiple linear regression analysis, however, only renal miR-148b was independently associated with megalin mRNA levels in IgAN. Proximal tubule megalin expression was further evaluated by immunofluorescence labeling of biopsies from the patients. The megalin expression was significantly lower in patients with highest levels of renal miR-148b compared with patients with lowest levels. To examine the direct effects of the miRNAs on megalin and other membrane proteins expression, proximal tubule LLC-PK1 cells were transfected with miR-148b, miR-21, miR-146a, or miR-192 mimics. Transfection with miR-148b mimic, but not the other three miRNA mimics inhibited endogenous megalin mRNA expression. No significant effect of any of the four miRNA mimics was observed on cubilin or aquaporin 1 (AQP1) mRNA expression. The findings suggest that miR-148b negatively regulates megalin expression in IgAN, which may affect renal uptake and metabolism of essential substances.

Keywords: IgA nephropathy; cell transfection; megalin; microRNA; renal proximal tubule.

Figure 1. Megalin mRNA as well as miR-148b, miR-21, miR-146a, and miR-192 expression in kidney tissue from patients with IgAN

(A) Megalin mRNA levels were decreased by 94% when compared with biopsy controls. (B–E) In contrast, levels of miR-148b, miR-21, and miR-146a were increased by 85%, 32%, and 80%, respectively, while a non-significant trend of increased miR-192 was observed in IgAN patients when compared with biopsy controls kidney tissue. (F–I) Megalin mRNA expression correlated inversely with levels of miR-148b, miR-21, miR-146a, and miR-192 in patients with IgAN. Renal megalin mRNA, miR-148b, miR-21, miR-146a, and miR-192 levels were detected by qPCR and normalized to 18S and U6, respectively. IgAN, n=70; biopsy controls, n=20. The horizontal lines (A–E) from top down represent 75th percentile, median, and 25th percentile; the vertical lines represent interquartile range. Megalin mRNA levels were logarithmically transformed to fit a normal distribution.

Figure 2. Correlations between renal miR-148b and eGFR or proteinuria in patients with IgAN.

(A,B) Renal miR-148b levels correlated significantly with eGFR, but not with 24-h urinary protein excretion in patients with IgAN. Renal miR-148b levels were measured by qPCR and normalized to U6.

Figure 3. Renal megalin protein expression by immunofluorescence in biopsy controls and in IgAN patients with the highest or the lowest expression of renal miR-148b

The images show double staining for megalin (red) and LTL (green) in kidney tissue from biopsy controls (A), IgAN patients with the lowest levels of renal miR-148b (B) or patients with the highest levels (C). Scale bars = 40 μm. (D) Higher magnification images from a patient belonging to the group with the highest levels of renal miR-148b. Some tubular profiles show intense labeling for both LTL and megalin (arrows), while others, that also show significant labeling for LTL, reveal much weaker labeling for megalin (arrowheads). Scale bars = 40 μm. (E) Quantitative analysis of megalin protein expression showing the ratios of megalin staining intensity to that of LTL in each section (n=3 for biopsy controls and n=10 for each of the two IgAN groups). Each box plot represents median, 25th and 75th percentiles. Whiskers represent 1.5-times interquartile range.

Figure 4. The effects of miR-148b, miR-21, miR-146a, and miR-192 mimics on the expression of megalin, cubilin, and AQP1 in LLC-PK1 cells

(A) The expression of miR-148b, miR-21, miR-146a, and miR-192 was increased similarly in LLC-PK1 cells transfected with the corresponding miRNA mimics when compared with respective negative control (miRNA mimic NC). (B–D) Transfection with miR-148b mimic resulted in a significant decrease in endogenous megalin mRNA levels, but had no effect on cubilin or AQP1 mRNA levels when compared with its negative control. In contrast, no significant effects on the levels of endogenous megalin, cubilin, or AQP1 mRNA were observed following transfection with miR-21, miR-146a, or miR-192 mimic. The membrane proteins (megalin, cubilin, and AQP1) and miRNAs (miR-148b, miR-21, miR-146a, and miR-192) levels were measured by qPCR and normalized to GAPDH or U6. All values were presented as means ± S.D.; n=6 for each group. *P<0.001 when comparing corresponding miRNA mimic to the miRNA mimic NC.