Effect Of Oxidative Stress Reaction On Podocin Expression In Glomerular Podocyte Slit Diaphragm

Objective The oxidative stress reaction in renal cortex and the podocin expression in glomerular podocyte slit diaphragm as well as the correlation of them were investigated to explore the role of oxidative stress in the injury of podocyte slit diaphragm proteinsMaterrial and Methods(1) Animals and Subgroups Thirty-two male Spraque- Dawley (SD) rats were randomly divided into control,low-dose ( 3.0mg/Kg ) ,medium-dose(7.5mg/Kg),large-dose(10.0mg/Kg)groups by the dosage of ADR injection respectively. Three weeks after the administration, 24 hours urine were collected and the rats were then killed to acquire the renal cortex.(2) Observation Index The level of 24h urinary protein and the contents of MDA,GSH-PX,.OH and SOD in renal cortex were measured; The expression of podocin was measured with immunohistochemistry and immuno-gold electron microscopy.Results(1) The 24h urinary protein level(mg/d):control group :7.98±3.01; low-dose group:14.35±9.72; medium-dose group:88.46±23.52; large-dose group:14.42±8.57. Compared with control group,the levels of 24-hour urinary protein in ADR groups increased,especially in medium-dose group ( P < 0.01).(2) The cortex ROS and anti-ROS content:①MDA(U/mg pr):control group: 1.45±0.12;low-dose group:1.87±0.16;medium-dose group:2.73±0.20; large-dose group:2.10±0.30.②SOD(U/mg pr):control group:504.45±43.84;low-dose group:425.37±35.93;medium-dose group:411.96±45.04; large-dose group:442.96±23.13.Compared with control group,the contents of MDA were increased,SOD were decresed in ADR groups,especially in medium-dose group( P <0.01);The contents of GSH-PX in ADR groups were higher than that in control group,The contents of .OH were no significant chang in four groups.(3)The expression of podocin:①IHC staining: in control group,podocin staining was a sable linear-like pattern along the capillary loops of glomerulus;In medium-dose group,podocin staining was a light tan discontinuous punctiform or short linear–like pattern along the capillary loops of glomerulus;Compared with control group,the score of podocin IHC staining was decreased in ADR groups,especially in medium-dose group(P<0.01).②In immuno-gold electron microscopy :podocin distributed at the base of the podocyte foot processes near the GBM.It primarily located at the cytoplasmic face of the plasmamembrane adjacent to the filtration slits.Small clusters of particles of podocin were occasionally found along the surface of the foot processes with a distance from the GBM ; In medium-dose group,No significant labelling was observed ,podocyte foot process confluenced;Compared with control group , the number of gold particle was obviously decreased,podocyte foot process were retraction.(4)There were some significant negative correlations between the score of podocin IHC staining and the levels of 24h urinary protein,the contents of MDA in renal cortex,There were some significant positive correlations between the score of podocin IHC staining and the contents of SOD in renal cortex.Conclusion(1)The expression of podocin in glomerular podocyte slit diaphragm was significant decreased,demonstrated that the development of proteinuria is closely corelated with podocyte SD protein podocin.(2)The renal cortex MDA contents were significant increased,SOD were significant decresed in ADR groups, podocin IHC staining score positively correlated with MDA and negatively with SOD that implied there were significant correlations between the reduction of podocin in glomerular podocyte slit diaphragm and oxidative stress reaction , especially lipid peroxidation.