Figure 1.
Pathological characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.
PAS staining of sections from NS (A&C) and ADR-injected (B&D) wild type (A&B) and eNOS-deficient (C&D) mice at day 28. Masson trichrome staining of sections from NS (E&G) and ADR-injected (F&H) wild type (E&F) and eNOS-deficient (G&H) mice at day 28. eNOS-deficient mice with ADR-induced nephropathy exhibited well developed exudative (fibrin-cap) lesions, glomerular sclerosis, interstitial fibrosis and inflammation at day 28. Original magnifications, 400 X.
Figure 2.
Functional characterization of ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.
A: Ratio of urinary 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. Two-way ANOVA; n = 5, data are means ± SD.
Figure 3.
Extracellular matrix products in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.
Collagen IV (A–D) and fibronectin (E–H) 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 ± SD. ***: vs WT NS, WT ADR and eNOS KO NS, P<0.001.
Figure 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–E) and podocyte synaptopodin (F–J) 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 ± SD. Vs NS day 28, * P<0.05; **P<0.01; ***P<0.001.
Figure 5.
Apoptosis in glomerular endothelial cells and podocytes in ADR-induced nephropathy in C57BL/6 mice with eNOS deficiency.
Apoptotic glomerular endothelial cells (A&B) and podocytes (D&E), triple labeled with terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labelling (TUNEL; A, B, D and E, green), anti-CD31 (A&B, red) and anti-synaptopodin (D&E, red), were detected at days 3 (B) and 7 (E) after ADR injection in eNOS-deficient mouse kidneys. Positive apoptotic cells (B&E) were counterstained with DAPI nuclear staining. Sections from NS-treated kidneys (A&D) were used as controls. Quantification of CD31+/TUNEL+ glomerular endothelial cells (C) and synaptopodin+/TUNEL+ podocytes in glomeruli (F). Original magnification, 600 X. Magnification in insets, 1200×. One-way ANOVA, n = 5, data are means ± SD. ***: vs NS day 28, P<0.001.
Figure 6.
Glomerular endothelial cell and podocyte injury in ADR-induced nephropathy in Balb/c mice.
(A) Western blotting detected expression of CD31 and synaptopodin in NS-treated and ADR-treated kidneys in Balb/c mice. (B) Quantification of CD31/α-Tubulin and synaptopodin/α-Tubulin in Western blotting. Immunostaining of CD31+ (glomerular endothelial cells) (D) and synaptopodin+ (podocytes) (F) in ADR-induced nephropathy. NS-treated kidneys were used as normal controls (C&E). Quantification of CD31 (G) and synaptopodin (H) staining in NS-treated and ADR-treated kidneys. One-way ANOVA, n = 6, data are means ± SD. *P<0.05.
Figure 7.
Apoptotic glomerular endothelial cells and podocytes in ADR-induced nephropathy in Balb/c mice.
Apoptotic glomerular endothelial cells (A&B) and podocytes (D&E), triple labeled with terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labeling (TUNEL; A, B, D and E, green), anti-CD31 (A&B, red) and anti-synaptopodin (D&E, red), were detected at days 1 (B) and 7 (D) after ADR injection in Balb/c mouse kidneys. Positive apoptotic cells (B&D) were counterstained with DAPI nuclear staining. Sections from NS-treated kidneys (A&C) were used as controls. Quantification of CD31+/TUNEL+ glomerular endothelial cells and synaptopodin+/TUNEL+ podocytes in glomeruli (E). Original magnification, 600 X. Magnification in insets, 1200 X. One-way ANOVA, n = 6, data are means ± SD. Vs NS day 7, *P<0.05; **P<0.01; ***P<0.001.
Figure 8.
eNOS overexpression protecting podocytes from TNF-α-induced loss of synaptopodin.
GFP eNOS – positive (GFP-eNOS+) and GFP-eNOS – negative (GFP-eNOS−) MMECs were obtained by FACS (A). Confocal microscopy of GFP in GFP-eNOS− (B) and GFP-eNOS+ (C) MMECs. (D) Western blotting using anti-eNOS and anti-GFP antibodies to detect endogenous eNOS and overexpression of GFP-eNOS in GFP-eNOS− and GFP-eNOS+ MMECs. (E) Conditioned media from GFP-eNOS− and GFP-eNOS+ MMECs was added to podocytes in the presence or absence of TNF-α, western blotting demonstrated expression levels of synaptopodin 36 hours after TNF-α stimulation. (F) Quantification of expression levels of synaptopodin by western blotting. One-way ANOVA, data are means ± SD.