This framework is consistent with hallmarks of renal scarring that characterize advanced nephropathy in MWF rats when vasculature rarefaction was strongly evident

This framework is consistent with hallmarks of renal scarring that characterize advanced nephropathy in MWF rats when vasculature rarefaction was strongly evident. increased and endothelin-1 gene expression with age. Notably, 10-week inhibition of the renin-angiotensin system regenerated kidney vasculature and normalized and endothelin-1 gene expression in aged MWF rats. These changes were associated with reduced apoptosis, increased Flavoxate endothelial cell proliferation, and restoration of Nrf2 expression, suggesting mechanisms by which angiotensin II antagonism mediates regeneration of capillary segments. These results have important implications in the clinical setting of chronic renal insufficiency. microCT in 50- and 60-week-old MWF rats compared with 50-week-old Wistar rats and representation of intermediateC and smallCsized capillary beds. Flavoxate (C) Quantification of vascular and kidney volume (top panel), vessels with diameter ranging from 80 to 180 and related receptors or angiopoietin-1 and -2, did not change between MWF60 and Wistar rats, whereas genes related to fibrosis, inflammation, and extracellular matrix remodeling were differentially expressed between the two strains. This framework is consistent with hallmarks of renal scarring that characterize advanced nephropathy in MWF rats when vasculature rarefaction was strongly evident. Upregulated profibrotic genes included the three isoforms, with protein in glomeruli and the cortical interstitium and paralleled the reduction in urinary excretion of ET-1, which likely reflects the renal synthesis of the SCA14 peptide.4,6,7 ET-1, synthesized predominantly (although not exclusively) in endothelial cells (ECs), exerts proinflammatory, mitogenic, and profibrotic effects through the ETA receptor (ETAR).8,9 At the level of glomerular microcirculation, it has been reported that podocyte-specific activation of TGF-signaling results in the release of ET-1 by visceral epithelial cells that act as paracrine stimulus for EC dysfunction through ETAR activation, setting in motion a vicious cycle that leads to podocyte depletion that eventuates in segmental glomerular damage.10 This evidence prompted us to investigate the role of endothelial deregulation of ET-1/ETAR signaling in MWF rats, a well known model of progressive endothelial and podocyte loss.5,11 To identify the cellular sources of ET-1, we performed multiple immunostaining and found that ET-1 protein was highly expressed by both ECs and podocytes, which were documented by costaining of Reca1 and and ET-1/ETAR signaling likely explains the beneficial effect of RAS blockade on the regeneration of kidney vasculature in advanced nephropathy. Because on RAS blockade, new vessel segment and capillary formation occurs, we further explored how the balance between apoptosis and proliferation at the EC level could account for vessel regeneration. Activated caspase3 was strongly upregulated in Reca1-positive ECs in MWF rats at 50 weeks of age and even more at 60 weeks of age (Figure 4A). However, ECs were induced to proliferate, which was confirmed by the increased number of Ki67-positive cells in the renal vascular compartment, possibly to counterbalance the onset of the apoptotic events (Figure 4B). Angiotensin II inhibition significantly reduced the number of apoptotic cells and sustained the compensating protective mechanism fostering EC proliferation, which could account for the important increase in the density and length of the microvessels observed by microCT (Figure 1, C and D) in treated MWF rats. NF-E2Crelated factor2 (Nrf2) has been recently recognized as a critical intracellular regulator of EC dynamics, governing angiogenic sprouting and vascular branching.16 Such pathways can be theoretically targeted therapeutically to counteract the effects of oxidative stress and TGF-analysis was adopted to estimate statistical significance of between groups comparisons. Statistical significance was defined as em P /em 0.05. Study Approval Animal care and treatment were conducted in accordance with the institutional guidelines and compliance with national (DL 116, GU 18/2/1992, Circ. 8, and G.U 14/7/1994) and international laws and policies (Dir. 2010/63/EU and 9/22/2010). All animal studies were approved by the Institutional Animal Care and Use Committee of Istituto di Ricerche Farmacologiche Mario Negri. Disclosures None. Supplementary Material Supplemental Data: Click here to view. Acknowledgments Flavoxate We thank Dr. Lucia Condorelli, Dr. Katia Passera, and Daniela Cavallotti for excellent assistance during scanning electron microscopy and image processing. We also thank the Associazione Ricerca sulle Malattie Rare Research Association for supporting P.R. with.