Expression Changes Of RNA-binding Proteins Tristetraprolin And Human Antigen R In Diabetic Kidney Disease And Their Mechanisms Involved In Podocyte Injury

BackgroundDiabetic kidney disease(DKD)is a common cause of chronic kidney disease(CKD)and end stage renal disease(ESRD).Its occurrence and development is a special process involving autoimmune response and multiple signaling pathways,which is regulated by multiple levels of gene expression.The expression of mammalian RNA is regulated at several post-transcriptional levels,including splicing,transport,localization,degradation and translation.The RNA molecule is transferred from the nucleus to the cytoplasm of the messenger ribonucleoprotein complex,and dynamically binds to RNA binding proteins(RBPs).These RNA-binding proteins,which are frequently altered in tissue-specific diseases,are a powerful and widely-used regulator.By recognizing the interaction between specific RNA domains,they mediate the key steps of post-transcriptional regulation of gene expression from RNA processing to final attenuation in the cytoplasm.Human antigen R(HuR)and zinc finger protein 36(TTP)are ubiquitous R BPs in vertebrates.HuR can enhance the stability of RNA and inhibit its degradation,while TTP is the opposite.TTP and HuR are known as anti-inflammatory proteins and pro-inflammatory proteins,respectively.Does the two play a corresponding role in diabetic nephropathy?Changes in glomerular filtration rate and proteinuria are the main clinical manifestations of diabetic kidney disease.As an important barrier of glomerular filtration membrane,podocyte damage is a key factor of glomerular filtration barrier damage,and has been considered as an important cause of proteinuria in diabetic kidney disease.The change of podocyte junction is also one of the main reasons for the change of permeability of filtration barrier.Tight junction,as an important intercellular adhesion mode between podocytes,plays an indispensable role in building a paracellular barrier and preventing water and solute from freely passing through the podocyte gap.Claudins are a class of transmembrane proteins that exist in many organisms,such as humans,and are key participants in the formation of tight connections.The expression of tight junction protein claudin-1 is one of the determinants of podocyte-to-podocyte junction.Previous studies have shown that high expression of claudin-1 gene in mice can lead to the injury of podocyte cleft membrane protein complex,which can lead to podocyte injury and proteinuria.Inflammation plays an important role in the process of podocyte injury.Observing the expression of inflammatory mediators can help us understand the process.As a multipotent cytokine,IL-17 participates in tissue inflammation by inducing the expression of chemokines,proinflammatory cytokines and matrix metalloproteinases.Previous studies have confirmed that IL-17 is also a factor causing damage to podocyte-damaged renal filtration system.Claudin-1 and IL-17 are co-regulated by HuR and TTP in the study of meningitis and other diseases,but does this phenomenon also exist in diabetic kidney disease? In addition,the affinity of HuR and TTP to RNA was affected by their phosphorylation level.Glycogen synthase kinase 3 beta(GSK-3 beta)is a serine and threonine kinase involved in the phosphorylation of various proteins.Both HuR and TTP may have phosphorylation sites of GSK-3 beta.Does it also participate in HuR and TTP phosphorylation in diabetic kidney disease?ObjectiveTo observe the changes of HuR/TTP expression in diabetic kidney disease,analyze the correlation between them and podocyte injury in diabetic kidney disease,and explore the possible relationship between GSK-3?and HuR/TTP.Method1.To observe the morphological changes and the expression of HuR/TTP and related indicators during the development of diabetic kidney disease in human renal tissues.Kidney tissue samples were split into normal control group,diabetic non-nephropathy group and diabetic kidney disease group.The normal control group and the diabetic non-nephrotic group were consulted on the non-cancerous tissues surrounding the cancer tissue of the urology department of the First Affiliated Hospital of Zhengzhou University.The normal control group was no diabetes,proteinuria(n = 10),diabetes.The non-nephrotic group was diabetic(n = 6)with diabetes mellitus and negative urine protein.The specimens of the diabetic kidney disease group were obtained from the renal biopsy of the Department of Nephrology,the First Affiliated Hospital of Zhengzhou University,and the remaining renal puncture tissue of the diabetic kidney disease was confirmed by the pathologist(n = 15).The paraffin sections of the kidney tissue were stained with PAS,and the thickness of the basement membrane and the damage of podocytes were observed by transmission electron microscopy.Immunofluorescence/immunohistochemistry was utilized to observe the localization and expression of HuR,TTP,IL-17 and claudin-1 in renal tissues.2.Results of verification of human kidney tissue in kidney tissue of type 2 diabetes model miceThe db/m mice were used to simulate the normal control group,the 14-week-old db/db mice were used to simulate the diabetic group,the 22-week-old db/db mice were used to simulate the diabetic kidney disease group,and the above experiments performed on human kidney tissues were repeated to verify Whether a pathological change similar to that of a diabetic patient's kidney tissue occurs in the kidney tissue of a type 2 diabetes model mouse.3.Further detect the expression changes of HuR/TTP and related indicators in renal tissues of diabetic kidney disease,and explore possible mechanisms.Quantitative analysis of HuR/TTP and related indicators by immunoblot analysis in kidney tissue of type 2 diabetic mice that have been shown to have similar pathological changes to diabetic kidney disease,and detection of GSK-3? and HuR by immunoprecipitation /TTP combination.Results1.Pathological changes such as glomerular hypertrophy,foot process fusion,basement membrane thickening,and mesangial matrix increase in diabetic kidney disease such as PAS staining and transmission electron microscopy.2.Compared with the normal control group and the simple diabetes group,the location of HuR,IL-17 and claudin-1 in the kidney cells of diabetic kidney disease patients increased,and the localization of TTP in podocytes increased.3.Similar results to the above 1,2 were obtained in the kidney tissue of type 2 diabetes model mice.4.Western blot showed that compared with the normal control group,HuR,IL-17,claudin-1 and GSK-3? were significantly increased in the kidney tissue of 22-week-old db/db mice,and the expression of TTP,nephrin and podocin was decreased.5.Co-immunoprecipitation showed that GSK-3? could bind to HuR and TTP,respectively.Conclusion1.HuR/TTP may participate in podocyte injury and proteinuria by affecting the expression levels of claudin-1 and IL-17.2.HuR/TTP expression activity may be affected by the phosphorylation level of GSK-3?.