The Effect And Mechanism Of HDAC5 On Renal Tubular Epithelial-mesenchymal Transition In Diabetic Nephropathy

Objective: Histone deacetylase 5(HDAC5)is a class IIa histone deacetylase which involved in the onset and progression of tumors,metabolic diseases and heart diseases etc.Related reports indicate that the expression of HDAC5 is elevated in the kidneys of animals and patients with diabetic nephropathy.However,whether HDAC5 is involved in the processes of renal tubular epithelial-mesenchymal transition and related mechanisms have not known yet.Therefore,we aim to explore the effect of HDAC5 on renal tubular epithelial-mesenchymal transition of diabetic nephropathy.Methods:1.HDAC5 expression was assessed by the methods of Western blot and immunohistochemistry in diabetic mice which induced by STZC57BL/6J mice were randomly divided into two groups,diabetic mice group and normal mice group,the first group injected streptozotocin by intraperitoneal in order to induce diabetes,Three days later,mice with random blood glucose greater than 16.7 mmol/L were regarded as diabetic models.Mice were sacrificed for the related detections after two months,four months and six months respectively.The expression of HDAC5 was detected by Western blot and immunohistochemistry,immunohistochemistry was used to detect the expression of TGF-?1.2.The expression of HDAC5 was detected by the methods of Western blot,quantitative PCR and immunofluorescence in high glucose medium-cultured HK-2 cellsHK-2 cells were randomly divided into two groups,one was cultured in normal glucose medium which contained 12 mmol/L glucose and one was cultured in high glucose medium which contained 40 mmol/L glucose,After 48 hours,proteins and m RNA were extracted from those cells for related detections,HDAC5 expression was detected by the methods of Western blot,quantitative PCR and immunofluorescence.3.Western blot,immunofluorescence and q PCR were used to detect the expression of TGF-?1 and extracellular matrix after knocking down HDAC5 in HK-2 cellsUnder normal glucose and high glucose environments,The expression of HDAC5 in HK-2 cells were knocked down,and the expression of TGF-?1,?-SMA were detected by the methods of Western blot and immunofluorescence.In addition,the expression of Col 1,Col 3 and FN m RNA were detected by quantitative PCR.In order to understand the role of TGF-?1 in HDAC5-induced renal tubular epithelial-mesenchymal transition,we did this: under the high glucose environment,HK-2 cells were treated by p Genesil-1,p Genesil-1-HDAC5,p Genesil-1-HDAC5 + TGF-?1(for 24 hours and 48 hours)respectively,After that,The expression of ?-SMA and HDAC5 was assessed by Western blot.Results:1.HDAC5 expression was upregulated in the kidneys of diabetic mice which induced by STZWestern blot results showed that the expression of HDAC5 in kidneys of STZ-induced diabetic mice was 2.58 times of that in normal mice(P < 0.05).At the same time,immunohistochemical staining results showed that HDAC5 was expressed in the cytoplasm of glomerulus,renal tubules,and renal interstitial.Compared with normal mice,the expression of HDAC5 in the renal tubules of diabetic mice was significantly increased(P < 0.05).2.HDAC5 expression was upregulated in high glucose medium-cultured HK-2 cellsWhat we can learn from Western blot results is that the expression of HDAC5 in the high glucose medium-cultured HK-2 cells was increased 44.80% compared with that in normal glucose(P < 0.05),moreover,the level of HDAC5 m RNA in HK-2 cells treated with high glucose was 31.17 times of that in normal glucose(P < 0.05).The result of immunofluorescence was consistent with the results of Western blot and q PCR.3.HDAC5 regulates the expression of ?-SMA and extracellular matrix in HK-2 cells through TGF-?1 signalning1)Under normal glucose conditions,The expression of HDAC5,TGF-?1 and ?-SMA after knocking down HDAC5 in HK-2 cells were significantly decresed by 44.10%,58.57% and 47.77% compared with control group(P < 0.05).And then,we detected these factors under high glucose conditions and find that the expression of HDAC5,TGF-?1 and ?-SMA after knocking down HDAC5 in HK-2 cells were significantly decresed by 41.02%,42.37% and 59.95% compared with control group(P < 0.05).We can see the same decreasing trend in immunofluorescence.The expression of Col 1,Col 3 and FN m RNA were downregulated in high glucose medium-cultured HK-2 cells which knocked down HDAC5.2)Under high glucose environment,the expression of ?-SMA in HK-2 cells after knocking down HDAC5 and with TGF-?1 treatment for 48 h was increased,and it's expression was 2.08 times of that in which only knocking down HDAC5 without the treatment of TGF-?1(P < 0.05).Interestingly,TGF-?1 treatment also induced the expression of HDAC5 upregulated of which knocking down HDAC5.Conclusions:1.The expression of HDAC5 was upreglulated in the kidneys of STZ-induced diabetic mice and in HK-2 cells treated with high glucose,indicating that HDAC5 may involved in the progress of diabetic nephropathy.2.High glucose induced upregulation of the expression of HDAC5,which may lead to renal tubular epithelial-mesenchymal transition through TGF-?1 signaling,thus extracellular matrix accumulation occurs.