Application of miR-135a in screening / preparing drugs to improve fetal glomerulosclerosis

Abstract

This invention discloses the application of miR-135a in screening / preparing drugs to improve fetal glomerulosclerosis. Based on the fact that exposure to exogenous substances (dexamethasone) during pregnancy can lead to podocyte phenotype inhibition in offspring and the development of glomerulosclerosis in adulthood, a fetal glomerulosclerosis model was constructed. In this model, miR-135a expression levels were significantly increased compared to normal controls. Based on mice exposed to dexamethasone during pregnancy, offspring were treated with 20 mg / kg antisense miR-135a every 3 days from week 1 to week 4 after birth, effectively improving podocyte phenotype inhibition and the occurrence of glomerulosclerosis. miR-135a can serve as an early prevention and treatment target for fetal glomerulosclerosis, and can be used to prepare drugs to improve fetal glomerulosclerosis, thus providing a new approach to the treatment of fetal glomerulosclerosis.

Description

Technical Field

[0001] This invention relates to the function and application of non-coding RNA, and particularly to the application of miR-135a in screening / preparing drugs to improve fetal glomerulosclerosis. Background Technology

[0002] Glomerulosclerosis is a clinicopathological syndrome characterized by glomerular hyaline degeneration, with massive proteinuria and progressive deterioration of renal function as the main clinical manifestations. Glomerulosclerosis is one of the most common causes of chronic kidney disease among glomerular diseases. For example, glomerulosclerosis accounts for 5% of cases of adult end-stage renal disease (ESRD), and even 12% of cases of pediatric ESRD [1]. In recent years, epidemiological surveys have shown that the incidence of glomerulosclerosis in children with low birth weight is significantly higher than that in children with normal birth weight [2]. In addition, animal experiments have also confirmed that intrauterine growth retardation (IUGR) may lead to glomerulosclerosis [3]. Previous animal experiments in our laboratory have also found that exposure to various exogenous substances (caffeine, dexamethasone, and ethanol, etc.) during pregnancy can induce fetal renal dysplasia and glomerulosclerosis in adult offspring, suggesting that glomerulosclerosis has an intrauterine origin. However, the pathogenesis and early prevention and treatment targets of fetal glomerulosclerosis are still unclear.

[0003] Podocytes are visceral epithelial cells of the glomerulus, which are differentiated from metanephric mesenchymestem cells (MMSCs) and are an important component of the glomerular filtration barrier structure. It has been confirmed that glomerulosclerosis is one of the podocyte diseases, and podocyte damage is the initiating factor of glomerulosclerosis [4]. Its occurrence is related to the inhibition of the podocyte phenotype. Podocytes are terminally differentiated cells and do not regenerate after differentiation and maturation. Their number depends to a large extent on their development during the embryonic period. Nephrin, WT1, and Podocin are important phenotypic proteins of podocyte structure and function. Their normal expression is of great significance for maintaining the function and number of podocytes [5]. Epidemiological data show that the number of podocytes in the kidneys of people with low birth weight is significantly reduced, and the risk of developing glomerular diseases in adulthood is increased [6]. The above suggests that the inhibition of the podocyte phenotype is closely related to glomerulosclerosis.

[0004] Kruppel-like factor 4 (KLF4) is mainly expressed in tissues and organs containing epithelial cells. It is abundant in both fetal and adult kidneys and participates in promoting podocyte development and maintaining its phenotype [7]. It has been confirmed that Nephrin, WT1, and Podocin are downstream target genes of KLF4, and KLF4 can maintain the podocyte phenotype by upregulating the expression of Nephrin, WT1, and Podocin [8]. miRNA is a class of non-coding small RNAs that are widely present in organisms. It is abundant in kidney tissue and can participate in regulating fetal kidney development and kidney physiological function [9]. It has been reported that abnormal expression of miRNAs participates in mediating various kidney diseases, especially playing an important pathological role in podocyte diseases

[10] . Literature shows that miR-135a is a known upstream regulator of KLF4 and can lead to its degradation by binding to the 3'-UTR end of KLF4

[11] . Previous animal experiments in our laboratory have confirmed that treatment of rats with dexamethasone (0.2 mg / kg / day) from gestational age 9-20 can cause fetal renal dysplasia and glomerulosclerosis in adult rat pups. However, whether prenatal dexamethasone exposure (PDE) can induce podocyte developmental toxicity in offspring, whether miR-135a and KLF4 are involved in mediating PDE-induced podocyte developmental toxicity and the occurrence of fetal-derived adult glomerulosclerosis, and whether miR-135a can serve as an early prevention and treatment target for fetal-derived glomerulosclerosis, still need further investigation.

[0005] References:

[0006] 1. Spino, C., et al., Changing the Paradigm for the Treatment and Development of New Therapies for FSGS. Front Pediatr, 2016.4: p.25.

[0007] 2.Tanaka,M.,et al.,Pregnancy Is a Risk Factor for Secondary FocalSegmental Glomerulosclerosis in Women with a History of Very Low BirthWeight.Intern Med,2017.56(12):p.1537-1541.

[0008] 3.Baum,M.,Role of the kidney in the prenatal and early postnatalprogramming of hypertension.Am J Physiol Renal Physiol,2010.298(2):p.F235-47.

[0009] 4.Nagata,M.,Podocyte injury and its consequences.Kidney Int,2016.89(6):p.1221-30.

[0010] 5.May,C.J.,M.Saleem,and G.I.Welsh,Podocyte dedifferentiation:aspecialized process for a specialized cell.Front Endocrinol(Lausanne),2014.5:p.148.

[0011] 6.Conti,G.,et al.,Low birth weight is a conditioning factor forpodocyte alteration and steroid dependance in children with nephroticsyndrome.J Nephrol,2018.31(3):p.411-415.

[0012] 7.Sidaway,P.,Glomerular disease:KLF4 promotes podocytedifferentiation.Nat Rev Nephrol,2014.10(7):p.362.

[0013] 8.Hayashi,K.,et al.,KLF4-dependent epigenetic remodeling modulatespodocyte phenotypes and attenuates proteinuria.J Clin Invest,2014.124(6):p.2523-37.

[0014] 9. Cerqueira, DM, M. Tayeb, and J. Ho, MicroRNAs in kidney development and disease. JCI Insight, 2022.7(9).

[0015] 10. Trionfini, P. and A. Benigni, MicroRNAs as Master Regulators ofGlomerular Function in Health and Disease. J Am Soc Nephrol, 2017.28(6):p.1686-1696.

[0016] 11.van Battum,EY,et al.,An Image-Based miRNA Screen IdentifiesmiRNA-135s As Regulators of CNS Axon Growth and Regeneration by Targeting Krüppel-like Factor 4.J Neurosci, 2018.38(3):p.613-630. Summary of the Invention

[0017] To address the deficiencies and shortcomings of the prior art, the present invention aims to determine the relationship between changes in renal miR-135a expression and fetal glomerulosclerosis, and to provide an application of miRNA-miR-135a as a drug target in screening drugs to improve fetal glomerulosclerosis, as well as the application of miR-135a antisense nucleotides in the preparation of drugs to improve glomerulosclerosis.

[0018] The above objective is achieved through the following technical solution:

[0019] In a first aspect, the present invention provides the application of miR-135a as a drug intervention target in screening / preparing drugs to improve fetal glomerulosclerosis.

[0020] Secondly, the present invention provides the application of miR-135a inhibitors in screening / preparing drugs for early intervention in fetal glomerulosclerosis.

[0021] Preferably, the miR-135a inhibitor is the antisense miR-135a (miR-135a antagomir).

[0022] The technical principles and research process of this invention are as follows:

[0023] This invention establishes a fetal glomerulosclerosis model through exposure to exogenous substances (such as dexamethasone) during pregnancy. In this fetal glomerulosclerosis model, the expression level of miR-135a was significantly increased compared to the normal control group.

[0024] Meanwhile, this invention clarified the effect of dexamethasone on the directed differentiation of MMSCs into podocytes at the cellular level. By detecting the expression of podocyte phenotypic genes (nephrin and podocin), it was found that dexamethasone in the range of 20 nM to 500 nM could significantly inhibit the expression of these genes. This invention further examined miR-135a / KLF4, and the results showed that high concentrations of dexamethasone could significantly promote miR-135a expression and reduce the mRNA and protein expression levels of KLF4, suggesting that dexamethasone inhibits KLF4 expression by upregulating miR-135a, thereby inhibiting the expression of podocyte phenotypic genes.

[0025] Furthermore, this invention involved transfecting MMSCs with either a KLF4 overexpression plasmid or a miR-135a inhibitor plasmid for 24 hours, and observing the effect of dexamethasone on the podocyte-directed differentiation of MMSCs. By detecting the expression of podocyte phenotype genes, it was found that transfection with the KLF4 overexpression plasmid significantly increased the mRNA and protein expression levels of the podocyte phenotype gene nephrin. Conversely, transfection with the miR-135a inhibitor reversed the protein expression of both KLF4 and the podocyte phenotype gene nephrin. Therefore, this further confirms, from a reverse perspective, that miR-135a upregulation mediates dexamethasone-induced podocyte phenotype inhibition by suppressing KLF4 expression.

[0026] Finally, at the overall level, based on mice exposed to dexamethasone during pregnancy, the present invention administered 20 mg / kg miR-135a antagomir to offspring every 3 days from week 1 to week 4 after birth. The expression of miR-135a, KLF4, and podocyte phenotype genes (nephrin and podocin) in the offspring was detected at puberty (week 7 after birth) and adulthood (week 24 after birth). The results showed that inhibiting miR-135a expression could effectively alleviate the inhibition of KLF4 expression and podocyte phenotype in the offspring kidneys caused by dexamethasone exposure during pregnancy, thereby reducing the occurrence of glomerulosclerosis.

[0027] Therefore, miR-135a can serve as an early prevention and treatment target for fetal glomerulosclerosis, providing a new approach for its treatment.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] 1. This invention discovers a novel function of miR-135a in the kidney, namely, that high expression of miR-135a can cause downstream KLF4 degradation, inhibit podocyte phenotype, and thereby induce fetal glomerulosclerosis.

[0030] 2. Based on the adverse effects of high expression of miR-135a in fetal glomerulosclerosis, it provides a target for the development of drugs to improve fetal glomerulosclerosis.

[0031] 3. miR-135a inhibitors can be used to prepare drugs for early improvement of fetal glomerulosclerosis.

[0032] 4. This invention discovers a novel function of miR-135a antisense nucleotide, which can be used to prepare drugs that improve fetal glomerulosclerosis. Attached Figure Description

[0033] Figure 1 Dexamethasone exposure during pregnancy can cause glomerular sclerosis in adult offspring rats.

[0034] Figure 1 In the middle: AB: HE staining of offspring kidneys at 28 weeks of age (magnification ×400); CD: PAS staining of offspring kidneys at 28 weeks of age (magnification ×400); EF: Morphological observation of podocytes in offspring kidneys at 28 weeks of age by transmission electron microscopy (magnification ×10000); G: Semi-quantitative analysis to assess the glomerular sclerosis index; HI: Serum creatinine (SCr) and blood urea nitrogen (BUN) levels; J: mRNA levels of kidney podocyte phenotypic genes (nephrin, podocin, and desmin).

[0035] Figure 2 Dexamethasone exposure during pregnancy can cause poor development of forefoot cells in offspring rats at birth.

[0036] Figure 2 In the middle: AD: HE staining of intrauterine kidneys (magnification ×100, ×400); EF: Morphological observation of intrauterine kidney podocytes by transmission electron microscopy (magnification ×10000); GH: Western blot detection of intrauterine podocyte phenotypic genes (WT1 and nephrin).

[0037] Figure 3 Pregnancy-induced dexamethasone exposure can lead to phenotypic suppression of pedicle cells in offspring rats after birth.

[0038] Figure 3 In the middle: A: Detection of phenotype genes (nephrin and podocin) in offspring kidney podocytes at 6 weeks after birth; BC: Western blot detection of phenotype genes (WT1 and nephrin) in offspring podocytes at 6 weeks after birth.

[0039] Figure 4 Pregnancy-induced dexamethasone exposure can lead to high expression of miR-135a and low expression of KLF4 in offspring rats before and after birth.

[0040] Figure 4 In the study: AI: RT-qPCR and Western blotting were used to determine the expression of KLF4 in the kidneys of offspring in utero, at 6 weeks and 28 weeks after birth; JL: miR-135a expression in the kidneys of offspring in utero, at 6 weeks and 28 weeks after birth was detected.

[0041] Figure 5 Dexamethasone can suppress the expression of podocyte phenotypic genes in MMSCs.

[0042] Figure 5 In the study: AC: Detection of podocyte phenotypic genes (nephrin and podocin) in MMSCs after podocyte differentiation following dexamethasone treatment.

[0043] Dex: Dexamethasone; Wnt4: podocyte-directed differentiation inducing factor.

[0044] Figure 6 Dexamethasone inhibits KLF4 expression by upregulating miR-135a, thereby suppressing the podocyte phenotype of MMSCs.

[0045] Figure 6 In the middle: AB: Detection of miR-135a and KLF4 after podocyte differentiation of MMSCs treated with dexamethasone; CF: Detection of KLF4 and nephrin expression by RT-qPCR and Western blot after KLF4 overexpression; GH: Detection of KLF4 and nephrin expression by RT-qPCR and Western blot after miR-135a inhibition.

[0046] Figure 7 Early postnatal miR-135a antagomir intervention in progeny mice exposed to dexamethasone during pregnancy improved podocyte phenotype suppression in 7-week-old progeny.

[0047] Figure 7 In the study: A: HE staining of offspring kidneys after intervention at 7 weeks of age (magnification ×400); BE: Detection of miR-135a, KLF4 and podocyte phenotypic genes in offspring kidneys after intervention at 7 weeks of age; FH: Western blot detection of KLF4 and nephrin in offspring after intervention at 7 weeks of age.

[0048] Figure 8 Early postnatal miR-135a antagomir intervention in progeny mice exposed to dexamethasone during pregnancy improved podocyte phenotype suppression at 24 weeks of age and reduced the incidence of glomerulosclerosis.

[0049] Figure 8 In the middle: A: HE staining of offspring kidneys after intervention at 24 weeks of age (magnification ×400); BC: PAS staining of offspring kidneys after intervention at 24 weeks of age (magnification ×400); DG: Detection of miR-135a, KLF4 and podocyte phenotypic genes in offspring kidneys after intervention at 24 weeks of age; HJ: Western blot detection of KLF4 and nephrin in offspring after intervention at 24 weeks of age. Detailed Implementation

[0050] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, so as to better understand the features and advantages of the present invention.

[0051] The reagents used in the experiments of this invention were purchased from domestic or international markets, or prepared by the user according to the formula in the instruction manual; the experimental methods not specifically described are all conventional methods known in the art.

[0052] Example 1: miR-135a can serve as an early intervention target for fetal-derived glomerulosclerosis

[0053] 1. Laboratory animals

[0054] SPF-grade Wistar rats were purchased from the Hubei Provincial Center for Disease Control and Prevention Animal Experimentation. Females weighed 200±20g, and males weighed 280±20g. The animals were housed and used for subsequent experiments at the Animal Experimentation Center of Wuhan University (room temperature: 18-22℃; humidity: 40%-60%; light and dark alternation for 12 hours). This experimental center has passed the international accreditation for laboratory animal care (AAALAC International). This experiment was conducted strictly in accordance with the relevant principles and guidelines of the China Animal Welfare Committee regarding the use and care of animals in experiments, and the operating procedures have been approved by the Animal Experimentation Ethics Committee (License No.: 201719).

[0055] 2. Animal handling

[0056] After one week of acclimatization, experimental animals were mated daily at 18:00 at a female:male ratio of 2:1. Vaginal smears were examined under a microscope at 6:00 the following morning. Sperm detection was recorded as gestational day 0 (GD0). Pregnant rats were then randomly divided into a PDE group and a control group. Starting on GD9, the PDE group received a subcutaneous injection of 0.2 mg / kg / day, while the control group received an equal volume of physiological saline. This invention only used pregnant rats with litters of 10–14 pups as the experimental group.

[0057] Fetal rat experiment: Pregnant GD20 mice in the PDE group (n=10) and the control group (n=10) were randomly anesthetized and euthanized. After euthanizing the male fetal rats, both fetal kidneys were removed on ice. The entire right kidney (4 kidneys randomly selected from each group) was fixed with 4% formaldehyde fixative for HE and PAS staining. Three additional fetal kidneys (3 randomly selected from each group) were fixed in electron microscopy fixative for ultrastructural examination. The remaining kidneys were stored at -80°C for further analysis.

[0058] Offspring rat experiments after birth: The remaining pregnant rats delivered naturally. Four weeks after birth (postnatal week 4, PW4), after weaning, two male rats were randomly selected from each litter of offspring rats in the PDE group (n=10) and the control group (n=10), and fed to PW6 and PW28. The rats were sacrificed after isoflurane anesthesia, and both kidneys were harvested and weighed. Half of the right kidney (four kidneys were randomly selected from each group) was fixed with 4% formaldehyde fixative for morphological changes; 1 mm of the kidney was fixed with electron microscopy fixative. 3 Several pieces of kidney tissue of varying sizes were collected from the cortical region; the remaining kidney tissue was stored at -80°C for later analysis.

[0059] 3. Detection indicators and methods

[0060] 3.1 Observation of morphological changes in offspring kidneys using hematoxylin and eosin (HE) staining

[0061] After embedding and sectioning kidney tissue, the sections were dewaxed to water, stained with hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, and then blued with 0.6% ammonia solution, rinsed with running water. Stained with eosin for 1-3 minutes, dehydrated, and mounted. Microscopic examination was performed, and images were acquired and analyzed.

[0062] 3.2 Periodic acid Schiff (PAS) staining to observe morphological changes in offspring glomeruli

[0063] After embedding and sectioning the kidney tissue, the sections were dewaxed to water, rinsed with periodic acid for 10 minutes, washed with tap water, then immersed in Schiff's solution for 10 minutes, rinsed with running water, stained with hematoxylin for 3-8 minutes, rinsed with running water, dehydrated, and mounted. The sections were then examined under a microscope, and images were acquired and analyzed.

[0064] 3.3 Transmission electron microscopy observation of ultrastructural changes in offspring kidneys

[0065] Freshly extracted fetal kidney and adult offspring kidney tissue (operated on ice) were cut into pieces approximately 1 mm in size using a scalpel. 3Small pieces were pre-fixed in 2.5% glutaraldehyde solution (0.2M dimethylarsine buffer, pH 7.4) for several hours, then fixed with osmium tetroxide for about 1.5 hours. After rinsing with distilled water, ethanol (50%, 70%, 90%), ethanol-acetone (1:1), and acetone (90%, 100%) were added sequentially for dehydration, each stage requiring 15-20 minutes. The pieces were embedded in epoxy resin, sectioned into 50 nm pieces using an ultramicrotome, and double-stained with 1.25% uranium acetate and 0.4% lead citrate. Transmission electron microscopy was used to observe the number and structure of foot processes, the thickness of the glomerular basement membrane, and the morphology of mitochondria and autophagosomes.

[0066] 3.4 Cell Culture

[0067] MMSCs were extracted from Wistar rats on day 13 of gestation, at a ratio of 2 × 10⁶ cells / well. 5 MMSCs were seeded at a density of 100 g / mL in 6-well plates. After adherence, the medium was replaced with fresh medium when the concentration was 30%-40%. Wnt4 100 ng / mL was added to induce differentiation into podocytes. After successful differentiation, the cells were treated with different concentrations of dexamethasone (0, 20, 100, 500, 2500 nM) or in combination with KLF4 overexpression plasmid or miR-135a inhibitor plasmid for 72 h to detect the expression of miR-135a, KLF4 and podocyte phenotypic genes.

[0068] 3.5 Western blotting technique for detecting KLF4 and podocyte phenotypic gene protein expression

[0069] A suitable amount of kidney tissue or treated cells was harvested, washed with PBS, and then lysed with 200 μl of RIPA lysis buffer and 1 mM PMSF. After homogenization, the mixture was placed on ice and incubated for 30 min. Centrifugation was then performed to extract total protein from the tissue or cells. The labeled proteins were subjected to gel electrophoresis, transferred to a membrane, blocked, and incubated with a specific primary antibody. The membrane was then developed using a spectrophotometer for relative quantitative analysis of protein levels.

[0070] 3.6 RT-qPCR technology was used to detect the expression levels of related genes and miR-135a mRNA.

[0071] Total RNA was extracted from intrauterine cells, kidneys at 6 and 28 weeks postnatal time, and from processed cells. cDNA was then reverse transcribed and quantified by RT-PCR to analyze the expression of related genes. The miR-135a sequence was provided by Guangzhou Ruibo Biotechnology Co., Ltd., and primer sequences for other genes are shown in Table 1.

[0072] Table 1: RT-PCR primer sequences

[0073]

[0074] 4. Data processing and statistical methods

[0075] The collected data were analyzed using Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Quantitative data are expressed as mean ± standard error (Mean ± SEM). Independent samples unpaired t-tests were used for comparisons between two groups; one-way ANOVA was used for comparisons among multiple groups, followed by post-hoc Dunnett's t-test or Tukey's test for multiple comparisons. P < 0.05 was used as the criterion for statistical significance.

[0076] 5. Experimental Results

[0077] 5.1 Pregnancy-induced dexamethasone exposure can cause glomerular sclerosis in adult offspring rats.

[0078] like Figure 1 As shown, compared with the control, offspring exposed to dexamethasone during pregnancy showed glomerular hyperplasia and Bowman's capsule stenosis in adulthood (PW28). Figure 1 (AB); Focal glomerulosclerosis of the kidneys accompanied by thickening of the capillary basement membrane and capillary ring occlusion, among other pathological changes. Figure 1 (Medium CD). Semi-quantitative analysis also showed a significant increase in the glomerular sclerosis index in the PDE group ( Figure 1 (G). Under electron microscopy, the glomerular basement membrane in the PDE group showed abnormal thickening, and large-area fusion or disappearance of foot processes. Figure 1 In addition, the levels of SCr and BUN in the PDE group were significantly higher than those in the control group (EF). Figure 1 The presence of dexamethasone during pregnancy (HI) indicates impaired renal function. Further analysis of the mRNA expression levels of podocyte phenotype genes (nephrin, podocin, and desmin) revealed that prenatal dexamethasone exposure significantly increased the expression of desmin (a sensitive early marker of podocyte injury) in offspring, while significantly decreasing the expression of the other phenotype genes, nephrin and podocin. Figure 1 (J). In summary, PDE can induce glomerular sclerosis in adult offspring rats.

[0079] 5.2 Pregnancy-induced dexamethasone exposure can cause dysplasia of forepocket cells in offspring rats at birth.

[0080] Secondly, this invention observed and detected the kidney structure and podocyte phenotype genes (WT1 and nephrin) expression in prenatal (GD20) fetal rats. Figure 2 As shown, compared with the control group, the PDE group showed significantly thinner fetal renal cortex and nephrotic area, with a significantly reduced number of mature glomeruli. Figure 2 (AB); glomerular Bowman's capsule is empty, capillary network is underdeveloped ( Figure 2(CD); thickening of the glomerular basement membrane, extensive fusion or disappearance of foot processes, significant reduction in the number of foot processes, and the appearance of autophagosomes in podocytes (CD); Figure 2 (EF). Western blotting results showed that the expression of phenotypic genes (WT1 and nephrin) in fetal rat podocytes of the PDE group was significantly reduced. Figure 2 (Glucose in the middle). The above results suggest that PDE can cause dysplasia of fetal rat foot cells.

[0081] 5.3 Pregnancy dexamethasone exposure can cause inhibition of podocyte phenotype in offspring rats after birth.

[0082] Subsequently, this invention observed the effect of PDE on the phenotype of podocytes in PW6 (adolescent) progeny. For example... Figure 3 As shown, compared with the control, the expression of phenotype genes (nephrin, podocin, and WT1) in the kidney podocytes of PW6 progeny was significantly reduced after dexamethasone exposure during pregnancy. Figure 3 (AC). The above results suggest that PDE can lead to phenotype suppression of PW6 progeny podocytes.

[0083] 5.4 Pregnancy dexamethasone exposure can lead to high expression of miR-135a and low expression of KLF4 in offspring rats before and after birth.

[0084] In investigating the mechanism by which PDE suppresses the expression of phenotypic genes in offspring kidney podocytes, this invention examined the expression of KLF4 in offspring kidneys from intrauterine to postnatal periods. For example... Figure 4 As shown, in GD20, PW6, and PW28, the expression of KLF4 in the progeny kidneys of the PDE group was significantly lower than that in the control group. Figure 4 (Chinese AI). This suggests that PDE may suppress the podocyte phenotype by inhibiting KLF4 expression in offspring kidneys. To further investigate the mechanism by which PDE inhibits KLF4, this invention examined the changes in miR-135a expression in offspring kidneys before and after birth. Compared with the control group, the expression of miR-135a in the PDE group was significantly higher at GD20, PW6, and PW28 than that in the control group (…). Figure 4 (JL). This suggests that PDE can cause programming alterations that result in persistently high expression of miR-135a in offspring kidneys.

[0085] 5.5 Dexamethasone can inhibit the expression of podocyte phenotypic genes.

[0086] To clarify the effect of dexamethasone on the podocyte-directed differentiation of MMSCs at the cellular level, this invention, in addition to Wnt4-induced differentiation, simultaneously treated cells with different concentrations of dexamethasone. By detecting the expression of podocyte phenotypic genes (nephrin and podocin), it was found that dexamethasone in the range of 20 nM to 500 nM significantly inhibited the expression of these genes. Figure 5In B-C). The above results suggest that dexamethasone can inhibit the expression of podocyte phenotypic genes.

[0087] 5.6 Dexamethasone inhibits the expression of KLF4 by upregulating miR-135a, thereby inhibiting the expression of podocyte phenotypic genes

[0088] To explore the mechanism by which dexamethasone inhibits podocyte phenotype, this invention observed the effects of dexamethasone on the expression of miR-135a and KLF4 in a cell differentiation model. After treating cells with different concentrations of dexamethasone for 72 hours, the expression of miR-135a was significantly upregulated ( Figure 6 in A), and the expression of KLF4 was significantly decreased ( Figure 6 in B). Inhibition of miR-135a or overexpression of KLF4 could reverse the inhibition of the expression of podocyte phenotypic gene (nephrin) caused by dexamethasone ( Figure 6 in C-H). It is suggested that dexamethasone inhibits the expression of KLF4 by upregulating miR-135a, thereby inhibiting podocyte phenotype.

[0089] Example 2: Application of miR-135a intervention target in the preparation of drugs for fetal glomerulosclerosis

[0090] 1. Experimental animals

[0091] SPF-grade healthy male and female C57BL / 6 mice were provided by the Hubei Provincial Center for Disease Control and Prevention, license number: SCXK(E)2009-2011. The experimental animals were raised and subsequent experiments were carried out in the Animal Experiment Center of Wuhan University (room temperature: 18-22 °C; humidity: 40%-60%; light time: 12-hour dark and light alternation). This experimental center has passed the international accreditation for laboratory animal assessment and care.

[0092] 2. Animal treatment

[0093] After one week of acclimatization, male and female mice were grouped in a 2:1 ratio at 6 PM daily. Vaginal plugs were checked the following morning; pregnancy was classified as GD0. The birth status of pregnant mice was checked daily at 9 AM; if pups were born, it was recorded as postnatal day 0 (PD0). On day 9 of gestation, the PDE group began subcutaneous injections of 0.25 mg / kg / day of dexamethasone, while the control group received the same volume of saline. Pregnant mice were managed until natural delivery. Mice with litters of 6-10 pups were included in the experiment. To ensure nutritional balance, each litter was adjusted to 6 pups (3 males and 3 females). Pups were weighed one day after birth and once a week. They were breastfed until weaned at 4 weeks of age, and after separation of males and females, they continued to be fed a normal diet. Male offspring were divided into three groups at 4 weeks of age: a control group, a PDE group, and a PDE+miR-135aantagomir group, with n=8 in each group. The PDE+miR-135aantagomir group received miR-135aantagomir every 3 days from week 1 to week 4 at a dose of 20 mg / kg. Male offspring were sacrificed at week 7 and week 24, and kidneys were harvested. Half of the right kidney (4 kidneys were randomly selected from each group) was fixed in 4% formaldehyde fixative for morphological changes. The remaining kidneys were stored at -80°C for analysis.

[0094] 3. Detection indicators and methods

[0095] The expression of miR-135a, KLF4 and podocyte phenotype genes (nephrin and podocin) in the progeny kidneys was detected according to the method in Example 1, and morphological changes in the kidneys were observed.

[0096] 4. Experimental Results

[0097] 4.1 Pregnancy dexamethasone exposure in offspring mice followed by early postnatal miR-135a antagomir intervention improved podocyte phenotype suppression and reduced the incidence of glomerulosclerosis in adult offspring.

[0098] like Figure 7 As shown, compared with the control, glomerular stenosis of offspring exposed to dexamethasone during pregnancy was observed at 7 weeks after birth, but this pathological phenomenon was improved after intervention with miR-135a antagomir. Figure 7 (A). Further examination of the expression of miR-135a, KLF4, and podocyte phenotype genes (nephrin and podcoin) revealed that early administration of miR-135a antagomir to offspring during pregnancy significantly downregulated miR-135a expression in offspring exposed to dexamethasone during pregnancy, while significantly increasing the expression of podocyte phenotype genes (nephrin and podcoin) and KLF4 at 7 weeks postnatally. Figure 7(BH). This suggests that miR-135a antagomir intervention improves the phenotypic suppression of PDE progeny podocytes by inhibiting miR-135a expression and reducing downstream KLF4 expression. Furthermore, such as... Figure 8 As shown, similar phenomena were also observed in adult offspring mice exposed to dexamethasone during pregnancy at 24 weeks of age. Treatment with miR-135a antagomir improved glomerular Bowman's capsule emptiness in PDE offspring and reduced the incidence of glomerulosclerosis. Figure 8 (AC). Compared with the PDE group, the expression of nephrin and KLF4 was significantly increased in the PDE+miR-135aantagomir group (AC). Figure 8 (E,F,H,I,J).

[0099] In summary, early miR-135a antagomir intervention for dexamethasone exposure during pregnancy can improve podocyte phenotype suppression in offspring after birth and reduce glomerular sclerosis in adult offspring.

Claims

1. The application of miR-135a in screening / preparing drugs to improve fetal glomerulosclerosis, characterized in that: miR-135a is used as a drug intervention target to screen for drugs that improve fetal glomerulosclerosis; miR-135a refers to microRNA-135a-5p.

2. The application of a miR-135a inhibitor in screening / preparing drugs to improve fetal glomerulosclerosis, characterized in that: The miR-135a refers to microRNA-135a-5p; the miR-135a inhibitor refers to antisense miR-135a.

Images