The role of locally activated renin-angiotensin system in albumin permeability in glomerular endothelial cells under high glucose condition

Abstract

Background: Previous studies have demonstrated that local renin-angiotensin system (RAS) is activated in proximal tubular cells, mesangial cells, and podocytes under diabetic conditions. However, the role of RAS within glomerular endothelial cells (GECs) in the pathogenesis of diabetic nephropathy has not been fully explored. In this study, I investigated the existence and changes of RAS components in high glucose (HG)-stimulated GECs and the role of local RAS in morphological and functional changes of GECs under diabetic conditions. Methods: In vitro, GECs were exposed to 5.6 mM glucose (NG), NG+24.4 mM mannitol, or 30 mM glucose (HG) with or without 10-7 M L-158,809, an angiotensin II type I receptor (AT1R) blocker, for 24 hours. In vivo, 32 2 Sprague-Dawley rats were injected with diluents (n=16, C) or streptozotocin intraperitoneally (n=16, DM), and 8 from each group were treated with 1 mg/kg/day of L-158,809 by oral gavage for 3 months. Real-time PCR, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA) using cultured GECs were carried out to examine the activation of local RAS. Immunofluorescent (IF) staining for VE-cadherin and heparin sulfate glycosaminoglycans (HS-GAG) was also performed. In addition, the permeability of GECs to macromolecules was assessed by measuring the passage of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) across the monolayer of GECs. Moreover, the morphological changes were evaluated by scanning electron microscopy (SEM). Results: There were 4.9- and 3.4-folds increases in angiotensinogen mRNA and protein expression in HG-stimulated GECs compared to NG cells, respectively (P<0.01). The concentrations of angiotensin I (AI) and AII in HG-conditioned cultured media were also significantly higher than those in NG media (P < 0.05). In contrast, there were no differences in

the mRNA and protein expression of angiotensin-converting enzyme, renin, AT1R, and AT2R among the groups. IF staining showed that HS-GAG protein expression was significantly decreased (P < 0.01), while there was no change in VE-cadherin protein expression in GECs exposed to HG medium. The permeability to albumin assessed by FITC-BSA permeability assay was significantly higher in GECs cultured under HG conditions (P < 0.001), and L-158,809 treatment significantly abrogated the 3 increase in albumin permeability in HG cells (P < 0.01). On SEM examination, the mean size of fenestrae was significantly greater in HG-stimulated GECs (P < 0.01), and the enlarged fenestrae in HGcells were significantly ameliorated by AT1R blocker (P <0.05). In vivo, urinary albumin excretion and the size of GECs fenestrae were also significantly greater in DM rats compared to C rats (P <0.01), and these increases were significantly attenuated by L-158,809 (P <0.05). Conclusion: Local RAS within GECs was activated under HG conditions, and this locally activated RAS seemed to be associated with the alteration in GEC fenestration along with a decrease in HS-GAG, resulting in the development of albuminuria in diabetic nephropathy.