Disruption of Smad7 promotes ANG II-mediated renal inflammation and fibrosis via Sp1-TGF-β/Smad3-NF.κB-dependent mechanisms in mice

Abstract

Smad7 is an inhibitory Smad and plays a protective role in obstructive and diabetic kidney disease. However, the role and mechanisms of Smad7 in hypertensive nephropathy remains unexplored. Thus, the aim of this study was to investigate the role and regulatory mechanisms of Smad7 in ANG II-induced hypertensive nephropathy. Smad7 gene knockout (KO) and wild-type (WT) mice received a subcutaneous infusion of ANG II or control saline for 4 weeks via osmotic mini-pumps. ANG II infusion produced equivalent hypertension in Smad7 KO and WT mice; however, Smad7 KO mice exhibited more severe renal functional injury as shown by increased proteinuria and reduced renal function (both p<0.05) when compared with Smad7 WT mice. Enhanced renal injury in Smad7 KO mice was associated with more progressive renal fibrosis with elevated TGF-β/Smad3 signalling. Smad7 KO mice also showed more profound renal inflammation including increased macrophage infiltration, enhanced IL-1β and TNF-α expression, and a marked activation of NF-κB signaling (all p<0.01). Further studies revealed that enhanced ANG II-mediated renal inflammation and fibrosis in Smad7 KO mice were also associated with up-regulation of Sp1 but downregulation of miR-29b expression. Taken together, the present study revealed that enhanced Sp1-TGF-β1/Smad3-NF-κB signaling and loss of miR-29 may be mechanisms by which deletion of Smad7 promotes ANG II-mediated renal fibrosis and inflammation. Thus, Smad7 may play a protective role in ANG II-induced hypertensive kidney disease.

Figure 1. Deletion of Smad7 enhances ANG II-induced renal injury.

A: Systolic blood pressure. B: Proteinuria. C: Serum creatinine. D: Creatinine clearance (CCR). E: Histological damage [periodic acid-Schiff (PAS)-stained sections]. Note that disruption of Smad7 enhances ANG II-mediated renal injury, including higher levels of proteinuria and serum creatinine, a greater fall in CCR, and histological damage such as glomerular hypercellularity, vascular sclerosis (arrows), and ECM deposition when compared with Smad7 WT mice, despite equal levels of high blood pressure. Values are means ± SE for groups of 6 mice. Scale bar, 50 µM. *P<0.05, **P<0.01, ***P<0.001 compared with saline (SL) control mice. ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with ANG II-infused Smad7 WT mice.

Figure 2. Deletion of Smad7 enhances ANG II-induced renal fibrosis.

A: Collagen I. B: α-SMA. Immunohistochemistry (IHC, i, ii), real-time PCR (iii), and Western blot (WB, iv, vi) analyses show that deletion of Smad7 enhances ANG II-induced renal fibrosis when compared with Smad7 WT mice. Each bar represents means ± SE for groups of 6 mice. Scale bar, 50 µM. *P<0.05, **P<0.01, ***P<0.001 compared with saline (SL) control mice. ^# P<0.05, ^## P<0.01, ^### P<0.001 compared with ANG II-infused Smad7 WT mice.

Figure 3. Deletion of Smad7 enhances ANG II-induced renal inflammation.

A: TNFα. B: IL-1β. C: F4/80⁺ macrophages. Immunohistochemistry (IHC, i, ii) and real-time PCR (iii) analyses show that deletion of Smad7 enhances ANG II-induced renal inflammation with up-regulation of TNFα and IL-1β, and an increased macrophage infiltrate when compared with Smad7 WT mice. Each bar represents means ± SE for groups of 6 mice. Scale bar, 50 µM. *P<0.05, **P<0.01, ***P<0.001 compared with saline (SL) control mice. ^# P<0.05, ^## P<0.01 when compared with ANG II-infused Smad7 WT mice.

Figure 4. Deletion of Smad7 enhances ANG II-induced activation of TGF-β1/Smad3 signaling in the kidney.

A: TGF-β1 expression detected by immunohistochemistry (i ii) and real-time PCR (iii). B: Activation of Smad3 determined by immunohistochemistry for phospho-Smad2/3 nuclear translocation (i, ii) and Western blots for phosphorylation levels of Smad3 in the kidney (iii, iv). C: Smad7 protein levels. Note that ANG II induces degradation of renal Smad7 protein in the Smad7 WT. Each bar represents means ± SE for groups of 6 mice. Scale bar, 50 µM. *P<0.05, **P<0.01, ***P<0.001 compared with saline (SL) control mice. ^# P<0.05, ^## P<0.01, ^### P<0.001 when compared with ANG II-infused Smad7 WT mice.

Figure 5. Deletion of Smad7 enhances ANG II-induced activation of NF-κB/p65 in the kidney.

A: Immunohistochemistry detects that deletion of Smad7 enhances phospho-NF-κB/p65 nuclear translocation in the hypertensive kidney. B: Western blot analysis shows that disruption of Smad7 promotes IκBα degradation through phosphorylation, thereby enhancing NF-κB activation as determined by significantly increasing phosphorylation of p65 in the hypertensive kidney. C. Real-time PCR shows that deletion of Smad7 significantly inhibits IκBα mRNA expression in mice after ANG II infusion. Each bar represents means ± SE for groups of 6 mice. Scale bar, 50 µM. *P<0.05, ***P<0.001 compared with saline (SL) control mice. ^# P<0.05, ^## P<0.01 when compared with ANG II-infused Smad7 WT mice.

Figure 6. Deletion of Smad7 enhances upregulation of Sp1 but dowregulates miR-29b expression in ANG II-induced hypertensive nephropathy.

A. Western blot and real-time PCR analysis show that disruption of Smad7 enhances ANG II-induced upregulation of Sp1 at both protein and mRNA levels. B. Real-time PCR detects that disruption of Smad7 results in a further inhibition of miR-29b expression in the hypertensive kidney. Each bar represents means ± SE for groups of 6 mice. *P<0.05, **P<0.01, ***P<0.001 compared with saline (SL) control mice. ^# P<0.05, ^## P<0.01 when compared with ANG II-infused Smad7 WT mice.