| Renal fibrosis is the common pathological pathway for the progression of chronic kidney disease to end stage of renal disease.Renal interstitial fibrosis is an particular part of renal fibrosis.Its pathophysiological mechanism is complex,included multiple signaling pathways and cytokines.It also involves various responses such as inflammatory response,oxidative stress,and immune damage.These factors interact with each other,jointly promote the development of renal fibrosis.A number of studies have shown that Epithelial-mesenchymal transition of renal tubular epithelial cells is one of the key pathogenesis of tubulointerstitial fibrosis .Meanwhile,some studies have shown that transforming growth factor-β(TGF-β)is the main cytokine that triggers epithelial-mesenchymal transition of renal tubular epithelial cells by activating multiple signaling pathways,which is related to renal fibrosis,the classical one to renal fibrosis is the Smads pathway.In recent years,the pathological role of high-concentration urea in the body has been gradually recognized,and its induction of ROS production isclosely related to the occurrence of atherosclerosis and insulin resistance associated with chronic kidney disease.High concentration of urea can induce a number of production of reactive oxygen species(ROS)in vitro and in vivo,causing damage to proteins,lipids and DNA,and promoting the progressive deterioration of renal function in patients with chronic kidney disease.Cyanate is a hydrolysis product of non-enzymatic reaction of urea,which is higher in patients with chronic kidney disease.Cyanate alters the conformation of proteins by carbamylation to form carbamylated hemoglobin,albumin,lipoproteins,etc.These carbamylated proteins are important pathological mechanisms that trigger CKD with atherosclerosis.At the same time,it has been pointed out that cyanate itself can directly lead to vascular endothelial dysfunction through oxidative stress,thereby initiating the pathological process of atherosclerosis.Cyanate dissolved in the blood is circulating in the body.It acts on these main organs and various systems.The kidney is a organ which included with a large number of small blood vessels.It is worth to discuss whether the high-concentration of cyanate level in plasma causes damage to the kidney,further exacerbating kidney function and promoting the progress of renal fibrosis.Combined with the mechanism of action of cyanate in other systems,we hypothesized that increased cyanate concentration in the blood ofpatients with CKD may directly stimulate renal tubular epithelial cells and trigger epithelial-mesenchymal transition,thereby accelerating renal fibrosis.The process may be accompanied by the occurrence of oxidative stress and changes in TGF-β.ObjectiveIn this paper,human renal epithelial cells(HK-2)were used as research objects,and loaded with different concentrations of cyanate.The main aim of the study is to explore the effects of different concentrations of cyanate on the activity of renal tubular epithelial cells,the effects of reactive oxygen species and the expression of TGF-β,and to explore the changes of the expression level of epithelial-mesenchymal transition markers,in order to find the key target for renal fibrosis.MethodsCyanate was loaded to renal tubular epithelial cells(HK-2)with different concentrations,and its effect on cell viability was detected by CCK8 method;cell morphology was observed by inverted microscope;ROS level was detected by DCFH-DA method;cell immunofluorescence and Western blot were used to detect E-cadherin,Fibronectin,α-SMA expression.Western blot was used to detect the expression level of TGF-β.Results1.The results of inverted microscope showed that 1 mmol/L of cyanate resulted in a triangular shape on the surface of individual cells.When the concentration of cyanate reached 2 mmol/L,the number of cells decreased significantly while the number of shrinkage cells increased.The cells were shown as long and shuttle shape.2.The results of CCK-8 showed that the concentration of cyanate(0.25,0.5,1 mmol/L)caused a slight decrease in cell viability,but there was no significantly difference.When the concentration of cyanate reached2 mmol/L,the cell viability level dropped to about 56% with a significantly difference(P<0.05).3.DCFH-DA method was used to detect the ROS level in cells: with the increase of cyanate concentration,the ROS level in HK-2 cells increased gradually,1 mmol/L cyanate(P<0.05)and 2 mmol/L cyanate group were a significantly difference in ROS level(P <0.01).4.Western blot was used to detect the EMT markers: 0.5 mmol/L cyanate can cause the expression level of epithelial cell marker E-cadherin in HK-2 cells decreased(P<0.05),and the expression level was not influenced by the increasing cyanate concentration.At the same time,the expression levels of Fibronectin and α-SMA gradually increased from 0.5mmol/L cyanate,and the expression level of Fibronectin increased significantly at 1 mmol/L cyanate concentration(P<0.05),there was asignificantly difference in the concentration of 2 mmol/L cyanate(P<0.01);the increase in α-SMA expression was statistically significant at 2 mmol/L cyanate concentration(P<0.05).5.Cellular immunofluorescence was used to detect the EMT markers:Fibronectin,α-SMA showed no fluorescence signal in normal group.E-cadherin red fluorescence signal was strong and mainly distributed in cytoplasm.E-cadherin red fluorescence signal in cyanate group cells significantly reduced,while the fluorescence signal of Fibronectin andα-SMA in the cytosol was significantly enhanced.6.The level of TGF-β was detected by Western blot: there was a significantly difference at 1 mmol/L cyanate concentration(P<0.05),and there was a significantly difference at 2 mmol/L cyanate concentration(P<0.01).ConclusionThe effect of cyanate on human renal tubular epithelial cells caused changes in cell morphology and viability.With the increase of ROS expression,the expression of various EMT markers and TGF-β was up-regulated in a concentration-dependent manner.The above results suggest that cyanate triggers EMT of renal tubular epithelial cells and accelerates renal fibrosis by TGF-β overexpression.Mental illness is common in patients with chronic kidney disease(CKD),including depression,dementia,snoring,anxiety,personality disorder,and drug abuse.Depression is the most common mental problem in patients with end-stage renal disease(ESRD).In different studies,different populations use different assessment tools,and the prevalence of depression in CKD patients varies widely.Some studies have reported a prevalence of up to 30%.The symptoms of depression are divided into cognitive and physical symptoms.The physical symptoms of depression are very similar to those of uremia,such as anorexia,sleep disorders,fatigue,and gastrointestinal diseases.These similarities make it difficult to understand hemodialysis combined with depression.The etiology of depression is complex,and there is no clear pathophysiological mechanism.A large number of studies have shown that the mechanism of depression is closely related to immune disorders.A recent study showed a correlation between LL-37 levels and depressive symptoms in older women.Although depression is common in HD patients,its association with LL-37 has not been clearly elucidated.LL-37 is the only Cathelicidin antimicrobial peptide in the human body that is released into the blood by immune cells.Recent findings underscore the role of LL-37 in regulating immune system function inaddition to bactericidal action and as a bridge between innate and adaptive immune responses,thus participating in a variety of physiological and pathological processes.Studies have shown that it is involved in the pathophysiology of autoimmune diseases.ObjectiveThis research was recruited 32 dialysis patients with depression,33 dialysis without depression,32 healthy without depression.Medical history,serum,relevant clinical data were collected,serum was subjected to ELISA test,and statistical analysis was performed to find out the correlation between depression status and plasma LL-37 level.MethodsIn this study,from June 2013 to December 2015,32 patients with depressive symptoms and 33 patients with non-depressive HD(HDND)were recruited from the Second Affiliated Hospital of Chongqing Medical University.At the same time,32 healthy volunteers(HV)were randomly selected as a control group.In this study,the Hamilton Depression Rating Scale(HAMD)was used to assess depression status.To ensure the accuracy of depressive symptoms,HD patients were divided into two groups;patients with a score of 0-17 were included in the non-depressed group,while patients with a score greater than 17 were included.Inclusion in the depression group.After blood samples were collected,serum was obtained by centrifugation,then aliquoted and stored at-80 ℃ prior to analysis.The concentration of LL-37 in the serum was evaluated using an ELISA kit.All detected values were repeated three times.Statistical analysis results were then performed and P < 0.05 was considered statistically significant.ConclusionSerum LL-37 was significantly elevated in hemodialysis patients withdepressive symptoms compared to those with nondepressive symptoms.Adjusted multivariable regression analysis revealed that age,education level,urea levels and creatinine levels were significant predictors of depressive symptoms.A significantly positive correlation was shown between serum LL-37 and HAMD score. |
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