Insights Into The Role Of The Transcription Factor GATA In The Signal Transduction Pathway Of Renal Fibrosis

Reserch background:At present,research on renal fibrosis is focused on its signal transduction pathway.Among these pathways,the Smad signal pathway is one of the most important and most understood.A study found that the bone morphogenetic protein-7(BMP-7) mediated the negative adjusting process of renal fibrosis in the Smad signal pathway.In contrast,in the common recognized factor-induced renal fibrosis,the transforming growth factor-β1(TGF-β1) is closely related with BMP-7 by means of positive and negative regulation mechanisms,in which one of the important negative feedbacks is the generation of the inhibitory Smads (Smad6/7).Samd-7 produced a marked effect by combining with protease to simultaneously facilitate the ubiquitination and degradation of the TGF-βreceptor and block the phosphorylation of Smad2 and Smad3. These block the signal transduction process of TGF-β,transforming it to become the negative feedback mechanism of TGF-β.By regulating the Smad protein binding site,TGF-βinduced the expression of Samd-7. Likewise,by means of multiple response original(BRE),BMP-7 induced the expression of Samd-7.In these responses,there were two low affinity binding sites:BRE-1/2 which was activated by a high-concentration BMP, and the high-affinity I-BRE which was activated in a low-concentration BMP.Another relevant observation was that BRE-1/2 combined with Smad1/4 complex,whereas I-BRE combined with Smad1/4/GATA.This suggests that the effect of blocking the TGF-βsignal transduction process as mediated by Samd-7 might be enhanced by the transcription factor GATA.Second,the production of Samd-7 could be reduced by a low-concentration BMP which started the negative feedback mechanism of renal fibrosis.In this study,we intend to use the GATA-3 antisense oligonucleotide to block the expression of GATA,observe the expression of GATA-3,observe BMP-7 and Samd-7 by means of animal experiment in the molecular and tissue levels,and explore the possible mechanism of action that can provide inspiration for research and therapy on fibrotic diseases.Objective:1.The present study was conducted to investigate the effect of targeted inhibition of the transcription factor GATA using the antisense oligonucleotide technology on the expression of Smad-7 and bone morphogenetic protein(BMP)-7 as well as on the disease development in a Unilateral Ureteral Obstruction(UUO) rat model of renal fibrosis, aiming at exploring the therapeutic potential of GATA antisense oligonucleotides. 2.through animal experiment to detect the clinical significance of transcription factor antisense oligonucleotides's intervention with fibrotic diseases.3.investigate the expression of GATA antisense oligonucleotides in the differents presulfided rats.Methods:1.Thirty six healthy,male,and certified pathogen-free Wistar rats, provided by the were randomly divided into two groups:the Unilateral Ureteral Obstruction group(UUO) and the normal control group,we made a one-sided ureteral obstruction model then divided this model into the GATA-3 antisense oligonucleotide group(AS group),the GATA-3 nonsense oligonucleotide group(NS group),and the normal saline group (Normal saline group).Each group had 6 rats.The 18 rats in the normal control group were divided into the AS group,NS group,and Normal saline group according to the same grouping method.The making of the animal model and the first-time administration were marked as time 0d. The duration of repeated dosing was 48 hours.At times 1d,7d,and 14d, respectively,2 rats were sacrificed for each treatment group in the UUO and normal control groups.2.After the observation and recording of the renal gross changes,the obstruction side renal in situ lavage for taking renal tissue specimens was put in place.One part of the tissue specimens was fixed in 10%neutral formalin and then embedded in paraffin for the HE and Masson dyeing observations. 3.The other tissue specimens were fixed in room temperature protection liquid preservation in -80℃for reverse transcription polymerase chain reaction(RT-PCR).4.The experimental data were represented by mean number±standard deviation(x±s).The results were analyzed by SPSS 13.0statistical software,and the completely random block and multifactor factorial design and variance analysis as well as the Spearman correlation coefficient method were used for the correlation analysis.P＜0.05 indicated a statistically significant difference.Results:1.GATA-3 Expression Results:The expressions of GATA-3 was observed in both UUO and normal control groups.The positive sites were the renal tubule and renal intersitium.The renal glomerus expressed it partly.The optic density of the renal glomerus was obviously higher than that of the renal tubule and interstitium.The result of immunohistochemistry showed that in the UUO group treat with antisense oligonucleotide's fibrosis obviously higher than the normal saline group,UUO group's expression is less than normal control groups.2.BMP-7 Expression Results:Expression of BMP-7 was observed in both UUO and normal control groups.Except in the renal glomerus, BMP-7 was expressed in the renal tubule,renal interstitium,proximal tubule,and collecting tubule.The result of immunohistochemistry showed that there was significant difference between the UUO and normal control groups,.UUO group's expression is less than normal control groups,and with the increase time of obstruction the expression correspondingly decrease.3.Smad-7 Expression Results:Significant differences between the UUO and normal control groups were observed.Antisense oligonucleotide GATA-3 was shown to inhibit the expressions of the transcription factor GATA-3 and Smad-7.The result of immunohistochemistry showed that there was significant difference between the UUO and normal control groups,UUO group's expression is less than normal control groups.Likewise,significant differences were observed among the different groups,treatments,and times.4.TGF-β1 Expression Results:Analysis on the fibrosis marked factor TGF-β1 showed that there was expression in the renal tubule,the differences of the positive areas of the UUO and normal control groups were significant,and the different times showed significant differences. These are consistent with the findings of similar research.The expression of TGF-β1 in the different treatments and times of UUO showed significant differences.And there was significant difference between the different treatment in the UUO groups.However,in the different treatments and times of the normal control group,no significant differences were observed.RT-PCR Result of GATA-3 and Samd-7In the rat renal tissues of both UUO and normal control groups,we observed the expressions of GATA-3 and Samd-7.In the expression of GATA-3 at Time 7d,the UUO group treated with antisense oligonucleotide showed significant differences compared with the normal control group.The expression of UUO was lower than that of the normal control group.The expression of Smad-7 analysis showed that there were significant differences between the UUO and normal control groups treated with antisense oligonucleotide at Times 7d and 14d.The expression of Samd-7 in the UUO group was significantly lower than that of the normal control group.Likewise,the differences among the different treatments were also significant.1.GATA-3 indeed played its role in the renal fibrosis signal pathway.2.The present research,which utilized the UUO animal model, established the pathological and physiological model of renal fibrosis,utilized antisense oligonucleotide to block the expression of GATA,and inhibited the combination of the high-affinity site I-BRE with BRE Smad1/4/GATA.As a result,the expression of Samd-7 decreased.3.The expression of Smad7 in the AS-treated rats was significantly lower than that of the two other treatment factors in both UUO and normal control groups.This is consistent with the results of immunohistochemical and RT-PCR.4.This study confirmed that it is feasible to affect renal fibrosis process through interference with the transcription factor of the renal fibrosis signal path,and may be that investigation way was bright.