| PART ONE:PURPOSE: Renal fibrosis,which is pathologically characterized by excessive accumulation of extracellular matrix,is the key factor that mediates almost all progressive renal diseases.Although the function of GAS5,a long non-coding RNA,in organism development and tumorigenesis has received intense scrutiny,its role in renal fibrosis remains unclear.Therefore,this part will focus on the expression as well as function of GAS5 in renal fibrosis.METHODS: Unilateral ureteral obstruction(UUO)surgery mice served as a model of renal fibrosis and sham surgery mice served as control for 7 days before kidney tissues were collected.GAS5 expression and localization were detected by in-situ immunohybridization.Collagen 3(Col3)and fibronectin 1(FN1)deposition were measured by immunohistochemistry,and expression of GAS5 and Col3/FN1 were studied by real-time PCR.Protein expression of Col3 and FN1 in TGF-β-stimulated tubular epithelial cells(NRK-52E)were detected by Western Blot.Also,real-time PCR and fluorescence in situ hybridization were separately performed to measure m RNA expression of Col3 or FN1 and GAS5 expression.The fibrosis scores of renal biopsy tissues from patients were determined according to Masson staining,and GAS5 expression was detected by in situ immunohybridization.In ex vivo experiment,after up-or down-regulation of GAS5 expression with overexpression plasmids or si RNA,protein expression of Col3 and FN1 were detected by Western Blot.RESULTS: 1)Compared with control group,GAS5 expression significantly decreased in UUO group,mainly deposited on renal tubules;2)GAS5 expression decreased in TGF-β-stimulated NRK-52 E cells;3)The level of GAS5 reduced in the renal biopsy of patients with renal fibrosis compared with the control individuals;4)After transfection with GAS5 plasmid,the experimental group showed increased GS5expression accompanied by decreased Col3 and FN1 expression in the extracellular matrix than control group;5)After downregulated GAS5 expression with GAS5-si RNA,the expression level of Col3 and FN1 were markedly higher than that in the control group.CONCLUSIONS: This part reveals that decreased GAS5 expression in cellular and animal models of renal fibrosis,as well as clinical specimens,may explain the increased expression of extracellular matrix proteins and following progression to renal fibrosis.PART TWO:PURPOSE: With the results in part one suggesting that GAS5 inhibited renal fibrosis,the underlying mechanism would be further explored.It was proved that GAS5 regulated target gene expression by modulating the expression of micro RNAs.There are several mi RNA binding to GAS5 sequence,among which mi R-21 positively regulates renal fibrosis.Therefore,this part will focus on whether GAS5 inhibits renal fibrosis progression by regulating mi R-21 and its downstream target genes.METHODS: The well-established model of renal fibrosis was achieved by performing unilateral ureteral obstruction surgery for 7 days,and sham surgery mice were served as control group.mi R-21 expression was detected by real-time PCR in kidney tissues and TGF-β-stimulated NRK-52 E cells.Expression of mi R-21 and GAS5 was detected in NRK-52 E cells transfected with mimic mi R-21 or mi R-21-inhibitor.Also,mi R-21 expression in NRK-52 E cells was detected after transfection with GAS5 plasmid or si RNA.Potential binding sites of GAS5 and mi R-21 were mutated,and wild-type mi R-21 or mutated mi R-21 and luciferas reporter plasmid containing GAS5 sequence were transfected in cells.With overexpression of mi R-21 or GAS5,the transcription and protein expression of MMP2/9 in NRK-52 E cells were detected by luciferase assay and Western blot,respectively.RESULTS: 1)mi R-21 expression increased in UUO kidney and TGF-β-stimulated cells;2)Overexpression of GAS5 inhibited mi R-21 expression;3)Mutant mi R-21 was unable to inhibit GAS5 expression after mutating the potential binding site to GAS5 in the mi R-21 sequence;4)mi R-21 inhibited the expression and transcription of MMP2 and MMP9 protein;5)GAS5 upregulated the expression of MMP2 and MMP9.CONCLUSION: The mechanism by which GAS5 negatively regulates renal fibrosis may be that GAS5 negatively modulates the activity of mi R-21,and then alleviates the inhibition of mi R-21 on its downstream MMP2 and MMP9,increases the expression of MMP2 and MMP9,and augments the degradation of extracellular matrix,ultimately alleviates the progression of renal fibrosis.PART THREE:PURPOSE: Renal fibrosis is a common pathological process in the progression of various chronic kidney diseases to end-stage renal disease.Early diagnosis and intervention of renal fibrosis are of great clinical importance.Currently,the gold standard for the diagnosis of renal fibrosis is histological testing by renal biopsy.However,renal biopsy is invasive and has a certain possibility of complications.Therefore,it is extremely important to find non-invasive biomarkers of renal fibrosis.It has been found that long non-coding RNA-GAS5 plays an important role in the formation of renal fibrosis.In this part,we will investigate the relationship between plasma and urine GAS5 and renal fibrosis to explore whether they could predict renal fibrosis.METHODS: General and clinical data as well as blood and urine samples were collected from patients who underwent renal biopsy at the Department of Nephrology,Tongji Hospital,Shanghai,China from October 2017 to December 2020.Blood and urine samples were collected from sex-and age-matched healthy controls.Sections of kidney biopsy specimens were tested by Masson staining and then scored for fibrosis by two specialized nephropathologists using Image J software independently.The expression level of GAS5 in plasma and urine were detected with real-time PCR method.Urine creatinine level was measured to standardize urine GAS5 level.The correlation between plasma and urine GAS5 levels and renal fibrosis scores was statistically analyzed.RESULTS: 1)There were 20 healthy controls.After excluding incomplete basic data,missing blood and urine specimens,or acute lesions,a total of 198 patients with renal biopsy were included.There were 61 cases in non renal fibrosis group and 137 cases in renal fibrosis group,including 55 cases with mild fibrosis(fibrosis score <25%),66 cases with moderate fibrosis(25% ≤ fibrosis score <50%),and 16 cases with severe fibrosis(fibrosis score ≥ 50%);2)The main pathological diagnosis in non renal fibrosis group was membranous nephropathy,followed by minimal change disease(including minor renal lesions)and Ig A nephropathy,while the pathological diagnosis of renal fibrosis was most common in Ig A nephropathy,diabetic nephropathy,and membranous nephropathy;3)The expression of plasma GAS5 was significantly higher in renal fibrosis group than that in healthy control group as well as that in non-renal fibrosis group.Also,in the subgroup with higher renal fibrosis scores,the plasma GAS5 expression levels was higher;4)Urinary GAS5 expression was significantly lower in renal fibrosis group than that in healthy controls and non-renal fibrosis group,while GAS5 expression levels were lower in the moderate and severe renal fibrosis groups;5)Correlation analysis showed that plasma GAS5 expression was positively correlated with the occurrence of renal fibrosis,and urinary GAS5 expression was negatively correlated with the incidence of renal fibrosis.After adjusting possible confounding factors(hypertension history,systolic blood pressure,diastolic blood pressure,urea nitrogen,creatinine,e GFR,uric acid and other factors),diabetic history,plasma GAS5 and urine GAS5 could affect the score of renal fibrosis,among which urine GAS5 had the highest correlation coefficient.Compared with e GFR and plasma GAS5,urine GAS5 had stronger predictive significance for renal fibrosis.CONCLUSION: Plasma and urine GAS5 expression are correlated with the presence and extent of renal fibrosis,with urine GAS5 having a stronger correlation.Urine GAS5 may be a non-invasive biomarker of renal fibrosis and could assist in the diagnosis of renal fibrosis in its early stages. |
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