The Role Of MiR-155 In The Pathogenesis Of Diabetic Nephropathy

Background and objectiveIn recent years, diabetic nephropathy(DN) is the leading cause of end-stage renal disease(ESRD) and is the single strongest predictor of mortality in patients with diabetes in the western world, mainly due to the global increase in the morbidity diabetes mellitus(DM). In our country, the proportion is also increasing year by year. Glomerular basement membrane(GBM)thickening is the earliest histopathological lesion observed in patients with DN. Mesangial expansion and mesangial area broadening, abnormal accumulation of extracellular matrix(ECM)and the resulting glomerulosclerosis and tubulointerstitial fibrosis are the significant feature found in the progression of DN. However so far the pathogenesis involved in the process of DN is still not completely clear and the effective treatments to slow progression are still lacking, therefore it is of great significance for the prevention,diagnosis and treatment of DN to explore the pathogenesis of DN and search for new genes in relation to DN.MicroRNAs(mi RNAs) are a class of newly reconigized small, endogenous, non-coding single strand RNA with approximately 21-24 nucleotide sequence, which regulate gene expression through binding to the 3’untranslated region(UTR) of the target mRNA, thereby leading to mRNA degradation or repression of protein translation at the post-transeriptional level. Emerging research evidence suggests that mi RNAs are widely related to various physiological and pathological processes and play an important role in the pathogenesis of diverse diseases. Circulating mi RNAs which are found in a variety of body fluids such as serum, plasma, urine and other body fluids are a specific existence of extracellular mi RNAs. Many studies have shown that mi RNAs have the stability of fluid circulation, shuttle between cell-to-cell communication and derive from different tissues or organs. It is very suitable for circulating miRNAs as newly biological biomarkers which help the early diagnosis of many diseases and assessment of clinical curative effect and prognosis.At present, a large number of studies have conformed that mi RNA dysregulation is closely associated with the occurrence and development of DN. However, the specific mechanism by which mi RNAs contribute to the pathogenesis of DN is not very clear, and it’s still urgent to befurther studied. What’s more, our previous research through miRNA microarrays and RT-PCR showed that the expression of mi R-155 in the serum of patients with type 2 DN was significantly abnormal, indicating that miR-155 may be related to the pathogenesis of DN. And we further detected that the target gene of miR-155 was the transcriptional regulation factor-1(ETS1), the latter plays a critical role in matrix regulation by matrix metalloproteinases(MMPs). Therefore,the present study was to investigate mi R-155 and its target gene ETS1 expression levels in the kindey or serum of type 2 diabetic nephropahy db/db mice, and explore the potential relationship between miR-155 and ETS1, as well as renal histopathological features during the development of DN mice.MethodsType 2 diabetic db/db mice as experimental groups(4 weeks of age, n=20), and normal db/m mice as controls(4 weeks of age, n=20) were selected. Then the dynamic expression changes of serum mi R-155, renal tissue miR-155 and its target gene ETS1 were detected in each group. And the potential correlation between mi R-155 and renal histopathological features, as well as clinical biochemical indicators was explored during the development of DN mice.1. Detection of biochemical indicators Weekly monitor the random blood glucose(Glu). At the age of 4, 8, 12 and 16 weeks, record body weight(BW), take blood and urine samples to detect serum creatinine(SCR), blood urea nitrogen(BUN) and 24 h urine protein excretion(UAE)levels.2. Observation of renal tissue morphology Through HE, PASM and Masson staining, the kidney pathological changes of mice in each group were observed under optical microscope,which was to evaluate the pathological development of DN in db/db mice.3. Expression changes of mi R-155 and ETS1 mRNA Serum miR-155, renal tissue mi R-155 and its target gene ETS1 levels were detected by real-time PCR(RT-PCR).4. Expression of ETS1 protein The expression and distribution of ETS1 was evaluated by immunohistochemisty.Results1. Biochemical indicator changes of db/db mice Compared with the control db/m mice, BW in the db/db mice of 4 weeks was higher, while other biochemical indicators were no significant differences(P>0.05, respectively). The db/db mice of 8 weeks showed an obvious increase in BW,Glu,Scr,BUN(P<0.05,P<0.01), and proteinuria was detected, suggesting that DN had occurred at the 8 weeks of age in db/db mice. Thereafter with the increase of weeks of db/db mice,Glu,Scr,BUN, UAE levels had further increase.2. Kidney pathological changes of db/db mice Kidney histological observation had noobvious changes in the db/db mice of 4 weeks. After 8 weeks, glomerular hypertrophy, capillary basement membrane thickening and mesangial matrix expansion were gradually prominent.Furthermore, a segmental glomerular mesangial matrix hyperplasia and sclerosis, with renal tubular dilatation or atrophy, and inflammatory infiltration and fibrosis of renal interstitium were apparent in kidney obtained from db/db mice of 16 weeks.3. Expression of miR-155 in the kidney and serum of db/db mice Compared with the db/m mice, the expression levels of miR-155 in the kindey and serum of db/db mice at the age of 8, 12,16 weeks were up-regulated significantly with the development of DN(P<0.05).4. Expression of ETS1 mRNA in the kidney of db/db mice Compared with the db/m mice, the expression of ETS1 mRNA in the kidney was consistently decreased in db/db mice(P<0.05).5. Expression of ETS1 protein in the kidney of db/db mice Consistent with the expression of ETS1 mRNA in renal tissue, the staining of ETS1 protein was gradually weaked in db/db mice of 8,12 and 16 weeks. And we detected that ETS1 was mainly distributed in the glomerular cells,podocytes and renal epithelial cells.6. Correlation between serum miR-155 expression and biochemical indicators of db/db mice Analysis results showed that there was a positive correlation between the serum mi R-155 level of db/db mice and Scr, BUN, UAE, respectively.Conclusions1. The level of mi R-155 in the kidney and serum of db/db mice was gradually up-regulated significantly with the development of DN, indicating that mi R-155 may be related to the pathogenesis of DN, and serum mi R-155 may be used an newly biological biomarker for the early diagnosis of DN.2. The expression of ETS1 in the kidney of db/db mice at both mRNA and protein levels was gradually down-regulated, suggesting that miR-155 may be involved in the occurrence and development of DN by controlling the expression of ETS1.