Figure 1.
Expression of NOS3, c-myc and p21 Cip1 following unilateral ureteral obstruction (UUO).
(A) Western blotting of NOS3 in sham operated and UUO kidneys; (B) Quantification of NOS3 expression relative to α-Tubulin; (C) Western blotting of C-myc and p21Cip1; and (D) Quantification of p21Cip1 expression relative to α-Tubulin. Data are mean ±SD from groups of 5 mice and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. ***p<0.001 versus sham operated group.
Figure 2.
Phosphorylation of JNK, the Smad3 linker region (T179 and S208) and the Smad3 C-terminal domain following unilateral ureteral obstruction (UUO) and in sham operated controls.
(A) Immunoprecipitation (IP) of Smad3 followed by Western Blotting (WB) identified phosphorylation of the Smad3 C-terminal domain (p-C-Smad3), phosphorylation of the Smad3 linker region (p-T179) in the UUO kidney. IP of Smad3 also pulled down phosphorylated JNK (p-JNK). Detection of total Smad3 confirms equal efficiency of Smad3 precipitation. The results are quantified in (B). (C) Direct WB of kidney lysates was used to analyse protein levels of TGF-β1, phosphorylation of Smad3 linker region at S208 (p-S208) and total p-JNK. The results are quantified in (D). Data are mean ±SD from groups of 5 mice and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001 versus sham operated group.
Figure 3.
Peritubular capillaries (PTC) and macrophage infiltration in unilateral ureteral obstruction (UUO).
Confocal microscopy identifies CD31+ (green) PTC endothelial cells in wild type (WT, A&C)) and NOS3 knockout (KO, B&D) mouse kidneys 7 days after sham (A&B) or UUO surgery (C&D). In UUO kidney, PTCs appear compressed and misshapen (C&D). Quantification of CD31+ PTC lumina/mm2 (E) and F4/80+ macrophages infiltration (F). Data are mean ±SD from groups of 5 mice and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. ***p<0.001 versus sham operated group. #p<0.05 versus WT operated group. Magnification, ×600 in A to D.
Figure 4.
NOS3 deficiency promotes renal interstitial fibrosis on day 7 following unilateral ureteral obstruction (UUO).
(A–D) Confocal microscopy identification of Ki67 (green), α-smooth muscle actin (α-SMA, red) and nuclear staining DAPI (blue) in wild type (WT) mouse kidney (A&C) or NOS3-/- (KO) kidney (B&D) following a sham operation (A&B) or UUO surgery (C&D). Quantification of: (E) Ki67+α-SMA+DAPI+ cells/mm2, and; (F) the area of α-SMA and collagen I staining in WT and KO mouse kidney in sham operated and UUO kidneys. (G–N) Confocal microscopy identifying α-SMA (G–J, red) and collagen I (K–N, green) staining in WT (G, H, K and L) or KO (I, J, M and N) mouse kidneys with sham (G, I, K and M) or UUO (H, J, L and N) surgery. Data are mean ±SD from groups of 5 mice and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *p<0.05, ***p<0.001 versus sham operated control.
Figure 5.
NOS3 deficiency enhanced phosphorylation of JNK and the Smad3 linker region on day 7 following unilateral ureteric obstruction (UUO), but not the Smad3 C-terminal domain.
(A) Immunoprecipitation (IP) of Smad3 followed by Western Blotting (WB) identified phosphorylation of the Smad3 C-terminal domain (p-C-Smad3) and phosphorylation of the Smad3 linker region (p-T179 and p-S208) in the UUO kidney. In addition, Smad3 IP pulled down phosphorylated JNK (p-JNK). Detection of total Smad3 confirms equal efficiency of Smad3 precipitation. (B) Quantification of blotting results. Data are mean ±SD from groups of 5 mice and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *p<0.05, ***p<0.001 versus sham operated control.
Figure 6.
RvD1 reduced renal interstitial fibrosis in the obstructed kidneys of wild type mice.
Mice underwent sham or unilateral ureteral obstruction (UUO) surgery. Two days after UUO surgery, mice received intraperitoneal injections of RvD1 for another two days and were killed on day 4. (A) Quantification of CD31+ peritubular capillary (PTC) lumina; (B) Western blotting analysis of kidney protein levels of NOS3, α-smooth muscle actin (α-SMA), fibronectin, collagen I and GAPDH. (C) Quantification of the relative abundance of NOS3, α-SMA, collagen I and GAPDH. Data are mean ±SD from groups of 6 mice and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001.
Figure 7.
RvD1 inhibited phosphorylation of JNK and the Smad3 linker region but not Smad3 c-terminal phosphorylation in the obstructed kidney.
Mice underwent sham or unilateral ureteral obstruction (UUO) surgery. Two days after UUO surgery, mice received intraperitoneal injections of RvD1 for another two days and were killed on day 4. (A) Immunoprecipitation (IP) of Smad3 followed by Western Blotting (WB) identified phosphorylation of the Smad3 C-terminal domain (p-C-Smad3) and linker region (p-T179 and p-S208) following unilateral ureteric obstruction (UUO) compared to the sham operated control. In addition, Smad3 IP pulled down phosphorylated JNK (p-JNK). (B) Western blot identifying phosphorylation of the Smad2 C-terminal domain (p-C-Smad2), total Smad2, total Smad4 and GAPDH. (C) Quantification of the relative levels of abundance of p-JNK, p-T179, p-S208, p-C-Smad3 compared to total Smad3, p-C-Smad2 compared to total Smad2 and total Smad4 compared to GAPDH. Data are mean ±SD from groups of 6 mice and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *** p<0.001; N.S., not significant.
Figure 8.
RvD1 prevents TNF-α induced down-regulation of NOS3 expression in mouse endothelial cells.
(A) Western blotting of NOS3 levels in mouse microvascular endothelial cells (MMEC) cultured under control conditions, with 10 ng/ml TNF-α or with TNF-α plus 4 ng/ml RvD1 for 12 hr. (B) Quantification of NOS3 expression relative to GAPDH. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *** p<0.001.
Figure 9.
L-NAME-treated media induces a pro-fibrotic response in renal fibroblasts (NRK49F cells).
(A) Western blot showing expression of fibronectin, α-smooth muscle actin (α-SMA), collagen I and GAPDH in NRK49F cells cultured with L-NAME-treated or control MMEC media in the presence or absence of TGF-β1 stimulation for 24 hrs. (B) Quantification of the relative expression of fibronectin, α-SMA and collagen I compared to GAPDH. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *p<0.05, **p<0.01. Experiments were repeated at least three times.
Figure 10.
L-NAME-treated media enhances PDGF-BB-induced proliferation of renal fibroblasts (NRK49F cells).
Sub-confluent NRK49F cells were incubated in L-NAME-treated or control MMEC media in the presence or absence of PDGF-BB stimulation for 24 hr. (A) Immunostaining of Ki67 (brown) with nuclear staining (blue) in NRK49F cells. (B) Quantification of the percentage of NRK49F cells positive for Ki67 immunostaining. (C) Quantification of cell proliferation using incorporation of bromodeoxyuridine (Brdu). Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. **p<0.01, ***p<0.001. Experiments were repeated at least three times.
Figure 11.
L-NAME-treated media induces phosphorylation of JNK and the Smad3 linker region in renal fibroblasts (NRK49F cells) which is inhibited by RvD1.
(A) Western blot (WB) showing phosphorylation of JNK and p-S208 in the Smad3 linker region in NRK49F cells incubated with L-NAME conditioned MMEC media which is inhibited by RvD1. (B) Immunoprecipitation (IP) of Smad3 followed by WB identified phosphorylation of the Smad3 C-terminal domain (p-C-Smad3) and linker region (p-T179) following culture of NRK49F cells in L-NAME conditioned MMEC media which is inhibited by RvD1. (C) Quantification of phosphorylation of JNK and the different sites on Smad3. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. *p<0.05, ***p<0.001, N.S., not significant. Experiments were repeated at least three times.
Figure 12.
RvD1 inhibits the pro-fibrotic response induced by L-NAME-treated media in renal fibroblasts (NRK49F cells).
(A) Western blot showing that incubation of NRK49F cells in L-NAME treated MMEC media for 24 hr up-regulates expression of fibronectin and collagen I which is prevented by RvD1. (B) Quantification of fibronecting and collagen I expression relative to GAPDH. ***p<0.001. (C) Quantification of bromodeoxyuridine (Brdu) uptake shows that L-NAME treated MMEC media can increase proliferation of NRK49F cells which is prevented by the addition of RvD1. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. ***p<0.001. Experiments were repeated at least three times.
Figure 13.
Regulation of collagen I promoter activity.
(A) NRK49F cells were transfected with a reporter plasmid in which luciferase production is driven by the collagen I promoter. Incubation of these cells with L-NAME treated MMEC media for 24 hr up-regulated collagen I promoter activity which was prevented by the addition of RvD1. (B) 293T cells were transfected with Smad3 plasmids containing point mutations of various phosphorylation sites plus a collagen I promoter plasmid. Twenty-four hours later, cells were cultured with or without TGF-β1 and luciferase activity measured 24 hr later. Data are mean ±SD and analysis by one-way ANOVA with post hoc analysis with Tukey's multiple comparison test. * p<0.05, ***p<0.001, N.S., not significant. Experiments were repeated at least three times.