Long non-coding RNA expression profile in the kidneys of male, low birth weight rats exposed to maternal protein restriction at postnatal day 1 and day 10

Abstract

Background: Long non-coding RNAs (lncRNAs), which are involved in a variety of biological functions and aberrantly expressed in many types of diseases, are required for postnatal development. In this study, we aimed to investigate the lncRNA profiles in low birth weight (LBW) rats with reduced nephron endowment induced by the restriction of maternal protein intake. LBW by reduced nephron endowment is a risk factor for hypertension and end-stage renal disease in adulthood.

Methods: Kidneys were obtained from LBW rats fed a low-protein diet throughout gestation and lactation as well as from normal control rats born from dams fed normal protein diets at postnatal day 1 (p1) and 10 (p10). The total number of glomeruli in the kidneys was counted at p10. LncRNA expression profiles were analyzed by sequencing and screening using the Agilent Rat lncRNA Array. Quantitative real-time PCR (qRT-PCR) analysis of these lncRNAs confirmed the identity of some genes.

Results: The total number of glomeruli per kidney at p10 was significantly lower in LBW rats than in controls. A total of 42 lncRNAs were identified to be significantly differentially expressed, with fold-changes ≥2.0, between the two groups. According to correlation analysis between the differentially expressed lncRNAs and mRNAs involved in kidney development, we randomly selected a number of lncRNAs for comparison analysis between LBW and control kidneys at the two time-points, p1 and p10, using qRT-PCR. Three lncRNAs (TCONS_00014139, TCONS_00014138, and TCONS_00017119), which were significantly correlated with the mRNA expression of mitogen-activated protein kinase 4, were aberrantly expressed in LBW rats, compared with controls, at both p1 and p10.

Conclusions: LncRNAs are aberrantly expressed in the kidneys of LBW rats, compared with controls, during nephron development, which indicates that lncRNAs might be involved in impaired nephron endowment.

Fig 1. Body weight and glomerular number in low birth weight rats compared with controls.

A: Body weight in low birth weight (LBW) rats compared with normal controls at postnatal day 1 (p1) and 10 (p10). Values are means with SD. B: Comparison of glomerular number between LBW and control rats at p10. Each circle represents an individual rat and the horizontal and vertical lines indicate the means and SD. p Value between LBW and control rats was calculated by the Mann-Whitney U-test, n = 7 for each group. C: Scatter plot showing the distribution of glomerular number based on birth weight. The number of glomeruli was positively correlated with body weight at birth. r = Spearman’s correlation coefficient. The open circles represent the three rats randomly selected for microarray analysis from each group.

Fig 2. Hierarchical clustering of lncRNAs differentially expressed in kidney between low birth weight and control rates.

A hierarchical clustered heat map showing the log₂ transformed expression values for differentially expressed lncRNAs (absolute fold-change ≥2; p ≤0.05) between low birth weight rats (L) and normal controls (C). Three rats were analyzed for each group. The intensity of the color scheme is calibrated to the log₂ expression values, where red refers to high relative expression and green refers to low relative expression. The bar code represents the color scale of the log₂ values.

Fig 3. Comparison of microarray and quantitative real-time PCR data for differentially expressed lncRNAs.

The qRT-PCR results (n = 7) in three lncRNAs are consistent with the microarray data in the kidney obtained at postnatal day 10. The qRT-PCR reactions were repeated three times for every lncRNA. The level of lncRNA was calculated relative to GAPDH. The data are expressed as the log-transformed mean fold changes (Low birth weight/Control).

Fig 4. LncRNAs expression in the kidneys of low birth weight rats compared with controls.

Comparison of the expression of lncRNAs in kidney between low birth weight (LBW) and control rats at postnatal day 1 (p1) and day 10 (p10) by quantitative real-time PCR. The level of lncRNA was calculated relative to GAPDH. The data are expressed as relative to the controls at p1. Values are means with SEM. p values for comparison of lncRNA level between LBW and control rats was calculated by the Student’s t-test (n = 7).

Fig 5. Correlation analysis of lncRNAs expression with glomerular number.

The number of glomeruli was positively correlated with the relative expression of lncRNAs TCONS_0014139 (A: r = 0.88; p <0.001; n = 14) and TCONS_00014138 (B: r = 0.77; p = 0.001; n = 14), but negatively with lncRNA TCONS_00017119 (C: r = -0.80; p = 0.001; n = 14) in the kidneys at postnatal day 10 (p10). Black circles, control rats (n = 7); open circles, low birth weight rats (n = 7). The level of lncRNA was calculated relative to GAPDH. The data are expressed as relative to the controls at p10. r = Spearman’s correlation coefficient.

Fig 6. Comparison of lncRNA expression in different tissues.

Comparison of the expression of lncRNA TCONS_00017119 in different tissues from low birth weight and control rats at postnatal day 10 by quantitative real-time PCR. The level of lncRNA was calculated relative to GAPDH. The data are expressed as relative to lncRNA level in the brain of normal control rats. Values are means with SEM. p Value was calculated by the Student’s t-test. Black bars, control rats (n = 5); open bars, low birth weight rats (n = 5). *p <0.05 vs. low birth weight rats, ^#p <0.05 vs. normal kidney.