The Regulatory Role Of Tristetraprolin In Diabetic Nephropathy And High Glucose-Induced Injury Of Podocytes

Background As the aging of population and the changes of people's diet structure,the incidence of diabetes increases annually around the world.Statistics data predict global diabetic patients will reach 366 million in 2030.Diabetic nephropathy is one of the most important microvascular complications of diabetes,and about 30-40% of the diabetic patients have diabetic nephropathy,which ranked the top cause of the end-stage renal disease(ESRD);it has also rose to the second place in China.Patients with diabetic nephropathy are often characterized by a large amount of proteinuria,severe edema,higher incidence of cardiovascular disease,and more complications related to vascular access that replacement treatment needs if developing ESRD.These all seriously affect the quality of diabetic patients' life,and bring larger burden to their family and the society.Clinicians and researchers are aiming to further explore the mechanism of the occurrence and development of diabetic nephropathy,as well as the effective measures to delay or reverse the progress of diabetic nephropathy.Abnormal circulation and abnormal expression of local renal inflammation factors are important factors in promoting the occurrence and development of diabetic nephropathy.Regulation on micro-inflammation of diabetic nephropathy may be an effective way of prevention and treatment of diabetic nephropathy.Tristetraprolin(TTP)and zinc finger protein 36(ZFP36)can adjust the expressions of multiple inflammatory factors via the post-transcription pathway,which is known as the “anti-inflammatory protein”.A literature has reported that lower expression of TTP is associated with obesity and some certain types of cancer.Our team has found in early research that patients with diabetic nephropathy had lower TTP expression in hemocytes and urine sediment,which was negatively correlated with the expression of inflammatory cytokines.Therefore,mouse podocytes and diabetic mice were adopted in this study to further explore the regulating mechanism of TTP in the inflammatory response of diabetic nephropathy.Part ? Changes of expression and function of Tristetraprolin in mouse podocytes cultured with high glucose medium and in the kidneys of db/db miceObjective To observe the expressions of Tristetraprolin(TTP)and inflammatory factors such as IL-6 and TNF-? in mouse podocytes cultured with high glucose medium and in renal cortex of db/db mice – the model of diabetic nephropathy,and to explore the influences of high glucose environment on the function of TTP.Methods 1.25mmol/L of glucose was used to stimulate mouse podocytes for different time periods,respectively.Real-time quantitative PCR and western blot were used to detect m RNA and protein expressions of TTP and ELISA was used to detect the secretion of IL-6,IL-18 and TNF-?.2.6-week-old male db/m mice and db/db mice were raised in SPF ventilated cage.Fluorescent quantitative PCR and western blot were used to detect m RNA and protein expressions of TTP,IL-6,IL-18 and TNF-? in mouse renal cortex at 10,14,18 and 22 weeks.3.RNA-binding protein immunoprecipitation was used to detect the influences of high glucose environment on the ability of TTP to combine with IL-6,IL-18 and TNF-? m RNA,namely the changes of TTP activity.Results 1.Compared with mouse podocytes cultured by 5.6mmol/L of glucose,TTP m RNA and protein expressions were both decreased in mouse podocytes stimulated by high glucose environment(P<0.05),and the secretions of IL-6,IL-18 and TNF-? were increased(P<0.05).2.Compared with the peer db/m mice,TTP m RNA and protein expressions were both decreased in renal cortex of db/db mice at 10,14,18,22weeks(P<0.05),and the expressions of IL-6,IL-18,TNF-? were increased(P<0.05).3.Compared with mouse podocytes cultured by 5.6mmol/L of glucose,the combination ability of TTP with IL-6 m RNA and TNF-? m RNA was weakened in mouse podocytes under high glucose environment(25mmol/L)(P<0.05).Conclusions Both TTP expression and activity are significantly decreased in mouse podocytes under high glucose environment.The expression of TTP are also decreased in renal cortex of db/db mice – the model of diabetic nephropathy.Part ? Influences of up-regulated or down-regulated tristetraprolin on phenotype and inflammation of mouse podocytesObjective To explore the role of down-regulated Tristetraprolin(TTP)in mouse podocyte damage via the in vitro experiments,so as to discuss whether over-expression of TTP has a relief on podocyte damage caused by high glucose environment.Methods 1.si RNA was used to down-regulate TTP expressions in mouse podocytes;ELISA was used to detect the secretions of IL-6,IL-18 and TNF-?,and western blot was used to detect the expressions of podocyte marker proteins nephrin and podocin,as well as dedifferentiation marker protein ?-SMA.2.Lentiviral vector with over-expression of TTP was used to up-regulate TTP expressions of mouse podocytes,ELISA was used to detect the secretions of IL-6 and TNF-?,IL-18 and western blot was used to detect the expressions of podocyte marker proteins nephrin and podocin,as well as dedifferentiation marker protein ?-SMA.Fluorescence microplate reader was used to detect the activities of ROS and NADPH.Results 1.Down-regulation of TTP resulted in the increase of IL-6,IL-18 and TNF-? in podocytes,reduction of nephrin and podocin and the increase of ?-SMA,and the differences were statistically significant(P<0.05).2.Over-expression of TTP can alleviate the increased expression of IL-6,IL-18 and TNF-? due to high glucose,reduce the generation of ROS;in addition,it can reduce the phenotypic changes,restore nephrin and podocin expression,and reduce the expression of ?-SMA(P<0.05).Conclusions TTP plays an important role in maintaining the normal physiological condition of podocytes,and overexpression of TTP can alleviate the oxidative stress reaction of podocytes and the phenotypic changes.Part ? Influences of over-expression of Tristetraprolin on proteinuria and kidney damage of diabetic nephropathyObjective To explore the role of over-expression of Tristetraprolin(TTP)in protecting db/db mouse kidneys via the in vivo experiments.Methods 1.6-week-old male db/m mice and db/db mice were raised in SPF ventilated cage.db/db mice were injected with TTP lentiviral vector and control vector at 10 weeks.And the injection was repeated at 14 weeks.2.The mice were detected for body weight,blood glucose,and urine albumin every month.Mice were sacrificed at 22 weeks,when creatinine and urea nitrogen were detected.PAS staining was used to observe renal structure,while immunohistochemistry and western blot were used to detect renal inflammatory factors and fibrosis index,as well as expression of podocyte marker proteins.The kits were used to detect SOD activity and MDA content.Results 1.The overexpression of TTP could reduce the excretion of urinary albumin in db/db mice in the early days but did not have significant effect on mouse body weight,blood glucose and renal functions.2.Overexpression of TTP could reduce the expressions of renal inflammatory factors and fibrosis index in db/db mice,increase the expressions of podocyte marker proteins and reduce the hyperplasia of extracellular matrix.3.Overexpression of TTP may reduce MDA content as well as increase SOD activity in db/db mice kidney.Conclusions Overexpression of TTP can delay the progress of diabetic nephropathy in the db/db mice and reduce the damage.Part ? Exploration on mechanism of abnormal Tristetraprolin expression and activity under high-glucose environmentObjective To study the influences of GSK3? on changes of TTP expressions and activity under high-glucose environment.Methods 1.GSK3? inhibitors were applied to pre-treat mouse podocytes respectively,or GSK3? si RNA was used to transfect mouse podocytes and then high glucose was used to treat each group of cells.Western blot was used to detect the expressions of TTP,GSK3?,and p-GSK3?.RIP detection was used to compare the pre-treatment group and high glucose group in the combination of TTP with TNF-? and IL-6 m RNA.2.GSK3? over-expressed plasmid was used to transfect podocytes cultured by normal glucose.Western blot was used to detect the expressions of TTP,GSK3?,and p-GSK3?.And RIP detection compared the combination of TTP with TNF-? and IL-6 m RNA.3.And co-immunoprecipitation was used to verify the interaction of TTP and GSK-3?.Results 1.GSK3? inhibitors,and GSK3? si RNA all could improve the decreased TTP and weakened combining ability to inflammatory factor m RNA due to high glucose.2.Over-expression of GSK3? can simulate the decreased content and activity of TTP in podocytes caused by high glucose.3.GSK3? and TTP can directly combine with each other.Conclusions GSK3? can both affect the expressions of TTP and affect its activity in podocytes under high glucose environment.It is one of the kinases that can regulate TTP activity.Conclusions for all High glucose caused decreased TTP expression and function both in vitro and in vivo.Restore TTP expression could alleviate high glucose induced-podocyte injury and also had a renoprotective role in diabetic mice.GSK-3? can both affect the expression of TTP and affect its activity in podocytes.