Mechanism Of CAMP/PKA Signaling Pathway In Lead Induced Kidney Injury

Objective The accumulation of lead in the human body can cause damage to multiple systems and organs.The kidney is an important target organ for lead toxicity injury,and the renal tubule is its important target site.Literature analysis shows that oxidative stress is the main manifestation of kidney damage caused by low-dose lead exposure.This study intends to explore the mechanism of oxidative stress injury in renal tissue caused by low-dose lead exposure,and provide the basis for risk assessment and lead nephrotoxicity mechanism of heavy metal lead.Methods(1)Construct an experimental mouse model close to the reference level of human blood lead.Thirty 4-week-old male ICR mice were random Ly divided into two groups according to their body weight.The normal control group drank sterile deionized water,and the lead-exposed group drank 200 mg/L sterile lead acetate water.After 90 days of exposure,the mice were anesthetized to collect blood from the heart,take the kidneys,and weigh the organ coefficients.The lead content in the blood and kidneys was measured by graphite furnace atomic absorption spectrometer,the morphological changes of kidney tissue were observed by HE staining,and the total kidney tissue was measured by the kit Oxide dismutase(T-SOD),copper zinc superoxide dismutase(Cu/Zn-SOD)activity,glutathione(GSH)and malondialdehyde(MDA)content,c AMP level,TUNEL method to detect kidney cells Apoptosis level,real-time fluorescence quantitative polymerase chain reaction method was used to measure the relative expression levels of PKA,NOX4 and apoptosis-related genes Bcl-2,Bax and Caspase-3 m RNA.(2)Rat renal tubular epithelial cells(NRK-52E)were exposed to different concentrations of lead acetate(Pb Ac)medium for 24 hours,the cell viability was detected by the CCK-8 method,and the Pb Ac concentration was selected for subsequent experiments.The kit was used to detect cell SOD viability,GSH,MDA content,flow cytometry to detect cellular reactive oxygen species(ROS)content,cell apoptosis rate,RT-q PCR method to detect apoptosis-related genes Bcl-2,Bax,Caspase-3 m RNA levels and the relative expression of PKA,Nox4 m RNA Level.(3)Choose appropriate concentrations of PKA inhibitors and activators,and measure the changes in the morphological structure of NRK-52 E cells,Nox4 m RNA levels,oxidation and antioxidant levels,apoptosis rate and apoptosis after lead exposure after PKA expression level changes.The relative expression levels of death-related factors Bcl-2,Bax and Caspase-3 m RNA.Results(1)Animal experiment: Compared with the control group,the blood lead and kidney lead content of mice in the lead exposure group increased significantly[(1.88±0.50)μg/L vs.(100.13±17.23)μg/L,(0.04± 0.01)μg/g vs.(2.58±0.21)μg/g](P<0.05),and the renal coefficient of mice in the lead exposure group increased(P<0.05);HE results showed that the structure of kidney tissue was significantly damaged,and renal tubular epithelial cells Shedding;GSH level,T-SOD and Cu/Zn-SOD activity in kidney decreased,MDA content increased,the difference was statistically significant(P<0.05);TUNEL results showed that the number of apoptotic cells doubled(P<0.05));The relative expression of Bcl-2 m RNA and the ratio of Bcl-2/Bax decreased,and the m RNA expression of Bax and Caspase-3 genes increased significantly(P<0.05);the content of c AMP in the renal tissue of the lead-exposed group,the levels of PKA and Nox4 genes The m RNA expression levels were all increased(P<0.05),and there was a positive correlation between the relative expression levels of Nox4 m RNA and c AMP content(r=0.468,P<0.05).(2)Cell experiment: As the exposure concentration of Pb Ac increases,the cell viability of NRK-52 E decreases(r=-0.988,P<0.01),and the IC50 is 93.248μM.The final concentration of Pb Ac in subsequent experiments is selected as 0,10,20,40,80μM.Compared with the control group,the content of ROS and MDA in the cells of the Pb Ac exposure group increased(P<0.05),and the SOD activity and GSH content of the 20,40,80μM Pb Ac exposure groups decreased(P<0.05);each Pb Ac exposure group The apoptosis rate was 1.4,4.2,6.6,and 8.7 times that of the control group,and they all increased significantly(P<0.05);in the 40,80μM Pb Ac-exposed groups,the relative expression of Bcl-2 m RNA decreased,and the levels of Bax and Caspase-3The relative expression of m RNA increased,and apoptosis was obvious(P<0.05);Pb Ac exposure up-regulated the relative expression of PKA and Nox4 m RNA(P<0.05);10μM PKA inhibitor H-89 significantly reduced the expression level of Nox4 m RNA in NRK-52 E cells.Compared with the Pb Ac-exposed group,the content of ROS and MDA in the Pb Ac+H-89 group decreased,SOD activity and GSH content increased(P<0.05),the relative expression of Bcl-2 m RNA increased,the relative expression of Caspase-3 m RNA decreased,and the apoptosis rate decreased(P<0.05);The 10μM PKA activator FSK significantly increased the relative expression of Nox4 m RNA.Compared with the Pb Ac-exposed group,the number of cells in the Pb Ac+FSK group decreased,the volume decreased,the number of intracellular lysosomes increased,cell apoptosis increased,and cell apoptosis rate Increased(P<0.05),the relative expression of Bcl-2 m RNA decreased,and the relative expression of Caspase-3 m RNA increased.Conclusion It shows that lead exposure can cause changes in Nox4 activity through the c AMP/PKA signaling pathway,leading to imbalance of oxidation and anti-oxidation,and oxidative stress damage to kidney tissue.