Protective Mechanism Of Dexamethasone On Podocyte Cytoskeleton Injury

The nephrotic syndrome (NS) is the most common glomerular disease of childhood,75%～90%of which is minimal change nephrotic syndrome. In spite of massive studies in human disease and animal model being in progress, the pathogenesis of NS and mechanisms of proteinuria are still unclear.Corticosteroid therapy has been used in childhood nephrotic syndrome since the1950s. Of children who present with their first episode, the majority of children have minimal change disease and90～95%will respond to steroid therapy. With steroid therapy, mortality has fallen from35%to3%because of a reduction in serious infections. Because of this dramatic before-after evidence that corticosteroids improve the outcome of nephrotic syndrome, oral corticosteroids are the first line treatment of a child presenting with idiopathic nephrotic syndrome. Nephrotic syndrome is a potentially chronic disease with about60%of patients suffering a relapsing course and being at risk of the adverse effects of steroid treatment. Therefore to unravel the etiology and pathogenesis of NS and the cause of proteinuria is a big challenge to pediatric nephrologists all the times.1974Shalhoub first advanced the "soluble mediator hypothesis" that minimal-change nephrotic syndrome (MCNS) is a disorder of the immune system in which release of T-lymphocyte-derived circulating factor(s) results in the pathophysiologic changes associated with nephrotic syndrome. This hypothesis has remained the dominant paradigm for subsequent investigations into the pathogenesis of nephrotic syndrome, and as a result, glucocorticoid therapy has been presumed to act in nephrotic syndrome by repression of a T-lymphocyte-mediated response. However, identification of a circulating factor responsible for proteinuria in nephrotic syndrome has remained elusive. Several circulating factors have been shown to induce proteinuria in experimental animals, including several serum factors and a variety of cytokines. However, none of the identified factors have fully satisfied the criteria of being (1) universally present in elevated levels in the serum or urine of patients with MCNS in relapse, but not of patients in remission, and (2) able to induce proteinuria in experimental model systems.The characteristic ultrastructural changes observed during the development of nephrotic syndrome occur in the kidney glomerulus, where the distal (foot) processes of podocytes retract and efface, resulting indisplacement and disruption of the specialized slit diaphragms that span the filtration slits between foot processes. Foot process effacement, often with detachment from the underlying glomerular basement membrane (GBM), correlates closely with the development of proteinuria. These alterations in the highly differentiated morphology of podocytes have also been closely associated with dramatic rearrangements of the actin filaments in foot processes. Recent studies have identified mutations in the genes encoding podocyte proteins as responsible for some cases of inherited forms of nephrotic syndrome, and gene knockout experiments in mice have identified additional podocyte molecules that are required for normal glomerular function, together providing strong evidence suggesting that nephrotic syndrome is fundamentally a disease of the podocyte. Slit diaphragms play two additional critical roles:as a signaling platform, and as an interface for the actin cytoskeleton. Recent studies have shown that glucocorticoid receptors are present in glomerular cells, including podocytes, and translocate to the nucleus upon dexamethasone treatment. Glucocorticoids have also been shown to increase the stability of actin filaments, increase actin polymerization, and activate cytoskeleton-associated kinases in glomerular mesangial cells. As showed above glucocorticoids can ameliorate nephrotic proteinuria.In our previous studies, by using Affymetrix GeneChip technology we found the mRNA level of ANGPTL3was significantly increased in kidneys of children with minimal change nephrotic syndrome compared to that of the normal control. And the mRNA and protein level of ANGPTL3was increased in the glomerulus of adriamycin rats along with the development of proteinuria by laser microdissection. The level of ANGPTL3in glomeruli of MCD and MN were significantly higher than those of normal control, TBMN or FSGS respectively. The expression of ANGPTL3in cytoplasm of cultured podocyte was examined by reverse transcription and western blotting analysis. These preliminary data suggested that ANGPTL3secreted by podocyte in glomeruli may regulate biological function of glomerular filtration barrier and may take part in development of proteinuria.So we hypothesized that dexamethasone can reorganize the actin cytoskeleton and change podocyte associated molecules in vitro. ANGPTL3may take part in the process of podocyte injury and/or dexamethasone protective effects.Cultured murine podocytes in vitro were treated with puromycin aminonucleoside (PAN)-induced podocyte injury, pretreatment with or without dexamethasone. The distribution levels of F-actin were revealed by immunofluorescence staining under laser scanning confocal microscope. Paracelluar permeability of podocytes on Millicell-PCF Inserts was evaluated by measuring FITC-BSA passage through podocytes monolayer. Compared with control group, PAN-induced podocyte injury showed evidently increased paracelluar permeability to FITC-BSA and the fluorescenc intensity of F-actin was significantly lower, while pretreatment with dexamethasone, paracelluar permeability decreased and the fluorescenc intensity of F-actin returned to nearly normal intensity.To study mechanism of dexamethason protection on PAN-induced podocyte injury, we detected podocyte-associated molecules by real-time PCR and Western blotting. We found that PAN-induced podocyte injury could change the mRNA and protein expression of podocyte associated molecules compared to control group while dexamethasone could invert the above changes. This indicated that dexamethason protective effects on podocyte maybe associated with podocyte associated molecules.Our data also showed the mRNA and protein expression of ANGPTL3evidently increased in PAN group while pretreatment with dexamethasone, the mRNA and protein expression of ANGPTL3decreased. Using transient tansfection by ANGPTL3-overexpression plasmid and ANGPTL3-miRNA to treat podocytes, we found the similar changes in F-actin and paracelluar permeability as PAN-induced podocyte injury and dexamethasone still could invert changes.To explore further mechanism of dexamethasone asssociated with ANGPTL3and podocyte molecules, we found overexpression of ANGPTL3induced changes of podocyte molecules were different from those of PAN induced and dexamethasone could not completely invert the changes.Accordingly, in this study we investigated the direct effect of dexamethasone on podocyte injury induced by PAN and ANGPTL3, our results showed that dexamethasone could protect podocytes from jinjury which may at some extent be associated with podocyte associated molecules.