R adriamycin treatment (Fig. 5D to F), demonstrating that adriamycin-induced glomerular endothelial cell injury precedes that of podocytes in eNOS-deficient mice, suggesting that endothelial dysfunction may result in podocyte injury.Glomerular endothelial dysfunction precedes podocyte injury in ADR-induced kidney damage in Balb/c miceIt is believed that ADR-induced nephropathy is initiated by podocyte injury followed by overt proteinuria, glomerulosclerosis, Tazemetostat biological activity tubulointerstitial fibrosis and inflammation in ADR-susceptible mice [35,36]. In an attempt to address the role of endothelial dysfunction in the development and progression of ADR-induced podocyte injury, the expression of eNOS and NMS-E628 synaptopodin were examined by Western blotting in kidneys from Balb/c mice. Interestingly, the down-regulation of eNOS was significantlyGlomerular Endothelial Cell Injuryearlier than that of synaptopodin being prominent 24 hours and 7 days after ADR administration, respectively (Fig. 6A B). Confocal microscopy demonstrated that CD31 (Fig. 6C, D G) and synaptopodin (Fig. 6E, F H) were significantly decreased 7 days after ADR treatment. TUNEL demonstrated that glomerular endothelial cells (CD31+/TUNEL+) and podocytes (synaptopodin+/TUNEL+) undergoing apoptosis could be detected as early as 24 hours in glomerular endothelial cells (Fig. 7C E) but at 7 days in podocytes (Fig. 7D E) after ADR treatment compared with NS treatment. This suggests that glomerular endothelial dysfunction and damage precede podocyte injury in an ADR-susceptible mouse strain.eNOS overexpression 1527786 in endothelial cells protects podocytes from TNF-a-induced injuryTo further investigate the role of glomerular endothelial cells in the development and progression of podocyte injury, mouse microvascular endothelial cells (MMECs) over-expressing GFPtagged eNOS were generated. MMECs expressing GFP-tagged eNOS (GFP-eNOS+) were selected by FACS while GFPeNOS2MMECs were used as a negative control (Fig. 8A). Confocal microscopy demonstrated that the majority of the cultured GFP-eNOS+ MMECs expressed GFP-tagged eNOS (Fig. 8C) compared with GFP-eNOS2MMECs (Fig. 8B). Western blotting also confirmed the expression of GFP-eNOS and endogenous eNOS in MMECs (Fig. 8D). Conditioned medium from GFP-eNOS+ MMECs and GFP-eNOS2MMECs were added to podocytes in the presence or absence of TNF- a. Western blotting demonstrated that TNF-a significantly induced loss of synaptopodin in podocytes under conditioned medium from GFP-eNOS2MMECs while conditioned medium from GFPeNOS+ MMECs protected podocytes from TNF-a-induced loss of synaptopodin (Fig. 8E F), suggesting that eNOS over expression in MMECs may protect podocyte from inflammatory insult.DiscussionIn the present study using two mouse strains C57BL/6, an ADR resistant strain, and Balb/c, an ADR-susceptible strain, we have demonstrated that one of the important factors in driving ADRinduced nephropathy is the level of expression of eNOS. eNOS deficient C57BL/6 mice when treated with ADR developed overt proteinuria, persistent glomerular endothelial cell and podocyte injury, progressive glomerulosclerosis, tubulointerstitial fibrosis and inflammation. These results suggest that endothelial dysfunction may play a critical role in the development and progression of chronic kidney disease. We also demonstrated that glomerular endothelial cell injury precedes that of podocytes after ADR treatment in both ADR-resistant and ADR-susceptible strains. Using a reci.R adriamycin treatment (Fig. 5D to F), demonstrating that adriamycin-induced glomerular endothelial cell injury precedes that of podocytes in eNOS-deficient mice, suggesting that endothelial dysfunction may result in podocyte injury.Glomerular endothelial dysfunction precedes podocyte injury in ADR-induced kidney damage in Balb/c miceIt is believed that ADR-induced nephropathy is initiated by podocyte injury followed by overt proteinuria, glomerulosclerosis, tubulointerstitial fibrosis and inflammation in ADR-susceptible mice [35,36]. In an attempt to address the role of endothelial dysfunction in the development and progression of ADR-induced podocyte injury, the expression of eNOS and synaptopodin were examined by Western blotting in kidneys from Balb/c mice. Interestingly, the down-regulation of eNOS was significantlyGlomerular Endothelial Cell Injuryearlier than that of synaptopodin being prominent 24 hours and 7 days after ADR administration, respectively (Fig. 6A B). Confocal microscopy demonstrated that CD31 (Fig. 6C, D G) and synaptopodin (Fig. 6E, F H) were significantly decreased 7 days after ADR treatment. TUNEL demonstrated that glomerular endothelial cells (CD31+/TUNEL+) and podocytes (synaptopodin+/TUNEL+) undergoing apoptosis could be detected as early as 24 hours in glomerular endothelial cells (Fig. 7C E) but at 7 days in podocytes (Fig. 7D E) after ADR treatment compared with NS treatment. This suggests that glomerular endothelial dysfunction and damage precede podocyte injury in an ADR-susceptible mouse strain.eNOS overexpression 1527786 in endothelial cells protects podocytes from TNF-a-induced injuryTo further investigate the role of glomerular endothelial cells in the development and progression of podocyte injury, mouse microvascular endothelial cells (MMECs) over-expressing GFPtagged eNOS were generated. MMECs expressing GFP-tagged eNOS (GFP-eNOS+) were selected by FACS while GFPeNOS2MMECs were used as a negative control (Fig. 8A). Confocal microscopy demonstrated that the majority of the cultured GFP-eNOS+ MMECs expressed GFP-tagged eNOS (Fig. 8C) compared with GFP-eNOS2MMECs (Fig. 8B). Western blotting also confirmed the expression of GFP-eNOS and endogenous eNOS in MMECs (Fig. 8D). Conditioned medium from GFP-eNOS+ MMECs and GFP-eNOS2MMECs were added to podocytes in the presence or absence of TNF- a. Western blotting demonstrated that TNF-a significantly induced loss of synaptopodin in podocytes under conditioned medium from GFP-eNOS2MMECs while conditioned medium from GFPeNOS+ MMECs protected podocytes from TNF-a-induced loss of synaptopodin (Fig. 8E F), suggesting that eNOS over expression in MMECs may protect podocyte from inflammatory insult.DiscussionIn the present study using two mouse strains C57BL/6, an ADR resistant strain, and Balb/c, an ADR-susceptible strain, we have demonstrated that one of the important factors in driving ADRinduced nephropathy is the level of expression of eNOS. eNOS deficient C57BL/6 mice when treated with ADR developed overt proteinuria, persistent glomerular endothelial cell and podocyte injury, progressive glomerulosclerosis, tubulointerstitial fibrosis and inflammation. These results suggest that endothelial dysfunction may play a critical role in the development and progression of chronic kidney disease. We also demonstrated that glomerular endothelial cell injury precedes that of podocytes after ADR treatment in both ADR-resistant and ADR-susceptible strains. Using a reci.
