Effect Of PRL-3 On Podocyte Epithelial To Mesenchymal Transition In Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN),one of the major complications caused by diabetes involving capillaries,is clinically characterized by progressively increasing proteinuria and continuous renal function damage.Now,it has become the main cause of end-stage renal disease(ESRD).Podocytes are the key structure of glomerular filtration barrier.Kidney disease,which is mainly caused by podocyte abnormalities,is usually characterized by severe proteinuria.At present,research suggests that DN is a kind of "Podocyte disease",the podocyte damage is mainly referring to the podocyte hypertrophy,foot process fusion,epithelial-to-mesenchymal transition(EMT)in situ as well as reduction in the number of podocytes.When EMT occurs in podocytes,the original molecular markers of mature podocytes will be lost and mesenchymal cytoplasm-like phenotypic markers will be expressed.Studies have shown that both in vivo and in vitro hyperglycemia environment can induce the development of EMT in podocytes through Snail,which can affect the expression of podocyte-specific protein,disrupt the cytoskeleton structure and the morphology of podocyte,and further cause the destruction of the filtration barrier and even cause apoptosis of podocyte,thus accelerating the development of DN.Phosphatase of regenerating liver-3(PRL-3)belongs to the family of non-transmembrane protein tyrosine phosphatase,which was first discovered to be overexpressed in rectal cancer cells and can promote the invasion and metastasis of rectal cancer tumor cells.The morphological changes of tumor cells play an important role in the process of invasion and metastasis,one of the important mechanisms involved in the morphological changes of tumor cells is EMT.EMT can reduce the ability of intercellular adhesion,gradually loses polarity and turn into interstitial state,which makes the cells more mobile.In recent years,PRL-3 has been confirmed to be highly expressed in a variety of tumor cells and can induce the occurrence of tumor cell EMT by Snail.However,whether the expression of prl-3 in renal tissue is affected by high glucose environment and whether it is related to the occurrence of podocyte EMT has not been studied.PurposeTo investigate the expression of PRL-3 in renal tissues,the effect of high glucose environment on prl-3 expression and the role of PRL-3 in podocyte EMT.Methods(1)Breeding db/db and db/m mice for 1-2 weeks under the condition of the SPF standard,monitoring blood weight,glucose,the ratio of urinary total proterin to creatinine(g/g)and other related indicators to determine whether the model of diabetic nephropathy are successfully established.(2)At 0,4,8 and 12 weeks after the successful modeling,3 mice were randomly selected to be sacrificed and the kidney tissue was taken.The normal kidney tissue was used as the control group.The expression position and level of PRL-3 in the kidney tissue of the diabetic nephropathy mouse were detected by immunohistochemical staining.The expression level of PRL-3 in renal tissue of diabetic mice was detected by Western blot again.(3)Cultured of immortalized human glomerular podocytes in vitro.Cells were divided into four groups and stimulated for 24 h with normal glucose concentration(5.6mmol/L,Control grouph);low glucose concentration(20mmol/L,Hg20 grouph);high glucose concentration(40mmol/L,Hg40 grouph);high osmolar pressure(5.6 mmol/L glucose + 34.4 mmol/L mannitol,Mannitol grouph).Proteins were extracted from the cells,the expression levels of PRL-3 and EMT-related proteins(Nephrin,Podocin,?-SMA)in each group were detected by Western blot.(4)The immortalized human glomerular podocytes were transfected with the pre-mixed Lipofectamine2000+ specific shRNA(3:1)transfection solution,Using the normal glucose concentration group as control group,detecting the expression level of PRL-3 in the scramble RNA group(scrRNA)and shRNA interference group by Western blot.(5)After specific shRNA silencing PRL-3 expression,the podocytes were divided into four groups: Control grouph,Hg40 grouph,shRNA + Hg40 grouph,scrRNA + Hg40 grouph.After co-culture for 24 hours,the expression of Nephrin,Podocin,?-SMA in podocytes was detected by Western blot.Results(1)PRL-3 was mainly expressed in the glomeruli of renal tissues,and the expression level of PRL-3 in diabetic nephropathy mice was significantly increased compared with the normal group(P<0.05),which was time-dependent.(2)Compared with the Control grouph,the expression level of PRL-3 was significantly increased in the Hg20 group and the Hg40 group(P<0.05),the effect was concentration-dependent.PRL-3 expression in Mannitol grouph was not significantly changed compared with that in Control grouph.(3)Compared with the Control grouph,the expression levels of Nephrin and Podocin in the Hg20 group and the Hg40 group were significantly decreased(P<0.05),and the expression of ?-SMA was significantly increased(P<0.05).The effect was concentration-dependent.The expression of Nephrin,Podocin,?-SMA in the Mannitol grouph was not significantly different from that in the Control grouph.(4)After the specific shRNA was transfected into podocytes,the molecular level showed that the expression level of PRL-3 was significantly decreased.PRL-3 expression was not significantly changed in the scrRNA group compared with the Control grouph.(5)When using shRNA to interfer the expression of PRL-3,the expression of Nephrin and Podocin in Hg40+shRNA group was restored(P<0.05)and the expression level of ?-SMA was significantly decreased(P<0.05)compared with the Hg40 group.Besides,the expression levels of PRL-3,Nephrin,Podocin and ?-SMA in the scrRNA+ Hg40 group were not significantly changed compared with Hg40 group,so the influence of scramble sequence was excluded.ConclusionOur data demonstrate that PRL-3 is mainly expressed in the glomeruli,the expression of PRL-3 is increased under high glucose conditions and can promote the occurrence of EMT in podocytes.Inhibition of PRL-3 expression can alleviate podocyte injury.