The Effect Of Long Non-coding RNA Sonabnormal Nephron Development In Low Birth Weight Rats

Background and Objective: In recent years, long non-coding RNAs（lnc RNAs）, which are involved in the occurrence and development of a variety of major diseases, have gradually become the focus in the various fields, such as malignant tumors, metabolic diseases, and chronic diseases. Studies have shown that lnc RNAs play an important role of gene regulation in the process of organ development and diseases. However, research on lnc RNAs involved in kidney development was rarely reported. In this study, we aimed to investigate the lnc RNA profiles in low birth weight（LBW） rats with reduced nephron endowment induced by the restriction of maternal protein intake, to explore the expression pattern of lnc RNAs in LBW rats during nephron development, and to analyze the role of lnc RNAs in LBW rats associated with nephron endowment.Mthods: Pregnant rats were randomly fed either a normal protein diet（22% protein） or an isocaloric low-protein diet（6% protein） to induce LBW during the pregnancy. LBW was defined as a birth weight <2 standard deviation below the mean of the control rats. Control rats born from dams fed normal protein diets had normal birth weight. Body and kidney weights were measured on postnatal day 1（p1） and day 10（p10） of life. Lnc RNA expression profiles were analyzed by sequencing and screening using the Agilent Rat lnc RNA Array. Quantitative real-time PCR（q RT-PCR） analysis of these lnc RNAs confirmed the identity of some genes on the tissues of rat kidney, liver, lung, and brain. The protein abundance of MAPK10, ERK1/2, P38 MAPK and P-p38MAPK（mitogen activated protein kinase） was examined using semiquantitativeimmunoblotting at p1 and p10. Apoptosis in the kidneys was detected using the TUNEL assay. The total number of glomeruli per kidney was determined using the modified maceration method at p10. Result:（1） Both birth weight and kidney weight in LBW rats were significantly lower than in controls at p1 and p10（P<0.05）.（2） The total number of glomeruli per kidney at p10 was significantly lower in LBW rats than in control rats（P=0.001）, and glomerular number was closely related to birth weight（r=0.718, P=0.009）.（3） The apoptosis cell number in glomeruli of LBW rats was higher than in controls at two time points.（4） A total of 42 lnc RNAs and 129 m RNAs were identified to be differentially expressed with more than 2-fold change in the Array. Among them, 17 and 25 lnc RNAs were up and down expressed, 41 and 88 m RNAs were up and down expressed in LBW rats compared with normal controls, respectively.（5） Differentially expressed lnc RNAs and m RNAs correlation analysis showed that six differentially expressed lnc RNAs were closely associated with MAPK10 m RNA; and lnc RNA TCONS₀0031111 and MAPK10 m RNA were positively related（r=0.951, p=0.004）.（6） Verifying 16 differentially expressed lnc RNAs by q RT-PCR showed that the q RT-PCR results in the expression of the majority of lnc RNAswere consistent with the microarray data; and the expression of most proven lnc RNAs increased gradually with the increase of the age in rats during nephron development.（7） The expression of 16 lnc RNAs at p1 and p10 was compared among rat kidney, liver, lung, and brain tissues. Lnc RNA TCONS₀0031111 had the highest expression in the kidney; and the comparison of TCONS₀0031111 expression between LBW and control groups was statistically significant.（8） Nephron number was closely related to TCONS₀0031111 expression in kidney（r=0.888, P<0.001）.（9）The protein levels of both ERK1/2 and MAPK10 were not different in the kidneys of LBW rats when compared with controlsat two time points. The protein level of P-p38 MAPK, but not p38 MAPK, was significantly higher in the kidneys of LBW rats when compared with controlsat both p1 and p10.（10） In the rat kidney, phosphorylated p38 MAPK protein expression was closely related to TCONS₀0031111 expression at p10（r=-0.851, P<0.001）. Conclusion:（1） A number of lnc RNAs were aberrantly expressed during nephron development in the kidneys of LBW rats with reduced nephron endowment compared with normal controls, indicating that these lnc RNAs might play a potential role in impaired nephron endowment.（2） Lnc RNA TCONS₀0031111, which had a high expression in normal kidneys and was significantly lower in LBW rats, was significantly positively related to the nephron number in kidneys, which suggest that lnc RNA TCONS₀0031111 might be involved in abnormal nephron development.（3） Lnc RNA TCONS₀0031111 was closely related to P-p38 MAPK protein expression in rat kidneys; suggesting that lnc RNA TCONS₀0031111 may be involved in nephron endowment through p38 MAPK signaling pathway.（4） This study provides a basis for future investigations of lnc RNAs on nephron development and a potential therapeutic target for reduced nephron number associated with LBW.