Mmittee,Cell CultureMouse podocyte cell culture. Podocytes between passage 10 and 15 were maintained in RPMI 1640 medium supplementGlomerular Endothelial Cell InjuryFigure 2. Functional characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. A: Ratio of urinary 1081537 protein/ creatinine; B: Body weight; C: Ratio of kidney /body weight; D: Serum creatinine and E: Systolic blood pressure in NS- and ADR-injected mice. Twoway ANOVA; n = 5, data are means 6 SD. doi:10.1371/journal.pone.0055027.gwith 10 fetal bovine serum (FBS) and 1 streptomycin/ penicillin solution [33]. Cells were propagated in 10 U/ml murine IFNc at 33uC and then differentiated by culture for 7 days at 37uCin the absence of IFNc [34]. Differentiated podocytes showed prominent cytoplasmic processes and expressed synaptopodin.Glomerular Endothelial Cell InjuryFigure 3. Extracellular 4-IBP custom synthesis matrix products in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. Collagen IV (A ) and fibronectin (E ) staining sections from NS- (A, C, E G) and ADR-injected (B, D, F H) wild type (A, B, E F) and eNOS-deficient (C, D, G H) kidneys at day 28. Graph showing quantification of the area of staining for collagen IV and fibronectin. One-way ANOVA, n = 5, data are means 6 SD. ***: vs WT NS, WT ADR and eNOS KO NS, P,0.001. doi:10.1371/journal.pone.0055027.gMouse microvascular endothelial cell (MMEC) culture and generation of eNOS over-expression MMECs. MMECswere purchased from ATCC (Manassas, VA ) and cultured in 5 CO2 atmosphere at 37uC in Dulbecco’s modified MedChemExpress MK 8931 Eagle’s medium (Life Technologies BRL, Gaithersburg, MD) containing 10 FBS. To generate eNOS over-expression in MMECs, MMECs were transfected with pcDNA3-eNOS-GFP plasmid (Addgene Plasmid 22444) using FuGENE HD (Roche, Hawthorn, Austrialia). Seven days after transfection, two rounds of fluorescence activated cell sorting (FACS) (FACsDiva, Flowcore, Clayton, Australia) were employed to obtain eNOS-GFP-positive and eNOS-GFP-negative MMECs. MMEC conditioned mediae. NOS-GFP-positive and eNOS-GFP-negative MMECs were separately seeded intowell-tissue culture plates at a density of 36106 cells/well. The cells were incubated for 12 hours then washed three times with PBS prior to fresh media being added to the cells. The supernatant was collected 24 hours later and is referred to as eNOS-GFPpositive and eNOS-GFP-negative media, respectively. TNF-a treated podocyte cell culture. Podocytes were seeded in 6 well-plates at a density of 16106 cells per well and cultured initially at 33uC (propagating condition) prior being cultured at 37uC (differentiating condition). Five days after differentiation had commenced, conditioned media was added to the cells. The medium was changed to 0.1 FBS on day 7. Podocytes were stimulated with 10 ng/ml TNF- a for 36 hours before harvesting.Glomerular Endothelial Cell InjuryFigure 4. Glomerular endothelial cell and podocyte damage in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. Time course of glomerular endothelial cell CD31 (A ) and podocyte synaptopodin (F ) staining sections from NS-treated kidneys at day 28 (A F), ADR-treated kidneys at days 3 (B G), 7 (C H), 14 (D I) and 28 (E J). Graph showing quantification of the area of CD31(K) and synaptopodin (L) staining. One-way ANOVA, n = 5, data are means 6 SD. Vs NS day 28, * P,0.05; **P,0.01; ***P,0.001. doi:10.1371/journal.pone.0055027.gHistological assessmentA coronal slice of kidney tissue was fixed in 4 par.Mmittee,Cell CultureMouse podocyte cell culture. Podocytes between passage 10 and 15 were maintained in RPMI 1640 medium supplementGlomerular Endothelial Cell InjuryFigure 2. Functional characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. A: Ratio of urinary 1081537 protein/ creatinine; B: Body weight; C: Ratio of kidney /body weight; D: Serum creatinine and E: Systolic blood pressure in NS- and ADR-injected mice. Twoway ANOVA; n = 5, data are means 6 SD. doi:10.1371/journal.pone.0055027.gwith 10 fetal bovine serum (FBS) and 1 streptomycin/ penicillin solution [33]. Cells were propagated in 10 U/ml murine IFNc at 33uC and then differentiated by culture for 7 days at 37uCin the absence of IFNc [34]. Differentiated podocytes showed prominent cytoplasmic processes and expressed synaptopodin.Glomerular Endothelial Cell InjuryFigure 3. Extracellular matrix products in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. Collagen IV (A ) and fibronectin (E ) staining sections from NS- (A, C, E G) and ADR-injected (B, D, F H) wild type (A, B, E F) and eNOS-deficient (C, D, G H) kidneys at day 28. Graph showing quantification of the area of staining for collagen IV and fibronectin. One-way ANOVA, n = 5, data are means 6 SD. ***: vs WT NS, WT ADR and eNOS KO NS, P,0.001. doi:10.1371/journal.pone.0055027.gMouse microvascular endothelial cell (MMEC) culture and generation of eNOS over-expression MMECs. MMECswere purchased from ATCC (Manassas, VA ) and cultured in 5 CO2 atmosphere at 37uC in Dulbecco’s modified Eagle’s medium (Life Technologies BRL, Gaithersburg, MD) containing 10 FBS. To generate eNOS over-expression in MMECs, MMECs were transfected with pcDNA3-eNOS-GFP plasmid (Addgene Plasmid 22444) using FuGENE HD (Roche, Hawthorn, Austrialia). Seven days after transfection, two rounds of fluorescence activated cell sorting (FACS) (FACsDiva, Flowcore, Clayton, Australia) were employed to obtain eNOS-GFP-positive and eNOS-GFP-negative MMECs. MMEC conditioned mediae. NOS-GFP-positive and eNOS-GFP-negative MMECs were separately seeded intowell-tissue culture plates at a density of 36106 cells/well. The cells were incubated for 12 hours then washed three times with PBS prior to fresh media being added to the cells. The supernatant was collected 24 hours later and is referred to as eNOS-GFPpositive and eNOS-GFP-negative media, respectively. TNF-a treated podocyte cell culture. Podocytes were seeded in 6 well-plates at a density of 16106 cells per well and cultured initially at 33uC (propagating condition) prior being cultured at 37uC (differentiating condition). Five days after differentiation had commenced, conditioned media was added to the cells. The medium was changed to 0.1 FBS on day 7. Podocytes were stimulated with 10 ng/ml TNF- a for 36 hours before harvesting.Glomerular Endothelial Cell InjuryFigure 4. Glomerular endothelial cell and podocyte damage in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency. Time course of glomerular endothelial cell CD31 (A ) and podocyte synaptopodin (F ) staining sections from NS-treated kidneys at day 28 (A F), ADR-treated kidneys at days 3 (B G), 7 (C H), 14 (D I) and 28 (E J). Graph showing quantification of the area of CD31(K) and synaptopodin (L) staining. One-way ANOVA, n = 5, data are means 6 SD. Vs NS day 28, * P,0.05; **P,0.01; ***P,0.001. doi:10.1371/journal.pone.0055027.gHistological assessmentA coronal slice of kidney tissue was fixed in 4 par.