Mice overexpressing latent TGF-beta1 are protected against renal fibrosis in obstructive kidney disease

Abstract

Transforming growth factor (TGF)-beta1, once activated, binds to its receptors and mediates renal fibrosis via the downstream Smad signaling pathway. We reported here that mice overexpressing latent TGF-beta1 in keratinocytes were protected against renal fibrosis in a model of obstructive kidney disease. In normal mice, both transgenic (Tg) and wild-type (WT) mice had normal renal histology and function, despite a 10-fold increase in plasma latent TGF-beta1 in Tg mice. A severe renal fibrosis was developed in WT mice at 7 days after urinary obstruction. Unexpectedly, renal fibrosis was prevented in Tg mice, although levels of latent TGF-beta1 in both circulation and renal tissues remained high. Compared with the WT mice, quantitative real-time PCR showed that upregulation of renal alpha-smooth muscle actin (SMA), collagen I, and collagen III mRNA was inhibited in Tg mice (60-70% reduced, all P < 0.01). These were further confirmed by immunohistochemistry with a marked inhibition of tubulointerstitial accumulation of alpha-SMA+ fibroblasts, collagen I, and collagen III matrix in Tg mice (all P < 0.001). Further studies showed that inhibition of renal fibrosis in Tg mice was associated with a significant reduction in renal TGF-beta1 and CTGF (60% reduced, P < 0.05), an increase in renal Smad7, a suppression of TSP-1 (a critical factor for TGF-beta1 activation), and an inhibition of Smad2/3 activation (all P < 0.001). In conclusion, latent TGF-beta may play a protective role in renal fibrosis. Inhibition of renal TGF-beta1 expression and activation, thereby blocking the downstream TGF-beta signaling pathway, may be a critical mechanism by which latent TGF-beta1 protects against renal fibrosis.

Fig. 1.

Histological features of kidneys of latent transforming growth factor (TGF)-β1 transgenic (Tg) mice and wild-type (WT) mice in normal and obstructive kidney. A and B: kidneys from both K5.TGFβ1^wt Tg and WT mice show normal renal histology. C: representative obstructive kidney from a WT mouse with severe tubulointerstitial fibrosis as evident by abundant elongated fibroblasts and extracellular matrix accumulation at day 7 after ureteral ligation. D: representative obstructive kidney from a Tg mouse shows relatively normal histology at day 7 after the ureteral ligation. Tissue sections are stained with PAS. Magnification ×200. UUO, unilateral ureteral obstruction.

Fig. 2.

Immunohistochemistry shows that mice overexpressing latent TGF-β1 are protected against a marked accumulation of α-smooth muscle actin (SMA)+ myofibroblasts within the tubulointerstitium at day 7 after UUO. Normal kidney from a WT mouse (A), normal kidney from a Tg mouse (B), UUO kidney from a WT mouse (C), UUO kidney from a Tg mouse (D), and quantitative analysis (E). Note that both normal WT and Tg mice show a few α-SMA+ myofibroblasts (dark black) within the interstitium (A, B), but severe renal fibrosis with a prominent α-SMA+ myofibroblasts (dark black) within the tubulointerstitium is developed in WT mice with UUO (C), which is significantly inhibited in Tg mice (D). Sections are stained with the anti-α-SMA antibody as described in materials and methods. Each bar represents means ± SE for a group of 6 (normal) or 8 (UUO) mice. ***P < 0.001 compared with the normal control. ###P < 0.001 when compared with the WT UUO. Magnification ×200.

Fig. 3.

Immunohistochemistry shows that mice overexpressing latent TGF-β1 are protected against renal fibrosis as evident by a marked inhibition of collagen I accumulation within the tubulointerstitium at day 7 after UUO. Normal kidney from a WT mouse (A), normal kidney from a Tg mouse (B), UUO kidney from a WT mouse (C), UUO kidney from a Tg mouse (D), and quantitative analysis (E). Note that both normal WT and Tg mice show few collagen I (dark black) accumulation within the interstitium (A, B), but severe renal fibrosis with an abundant collagen I accumulation within the tubulointerstitium is developed in WT mice with UUO (C), which is significantly inhibited in Tg mice (D). Sections are stained with the anti-collagen I antibody as described in materials and methods. Each bar represents means ± SE for a group of 6 (normal) or 8 (UUO) mice. ***P < 0.001 compared with the normal control. ###P < 0.001 when compared with the WT UUO. Magnification ×200.

Fig. 4.

Immunohistochemistry shows that mice overexpressing latent TGF-β1 are protected against renal fibrosis as evident by a marked inhibition of collagen III accumulation within the tubulointerstitium at day 7 after UUO. Normal kidney from a WT mouse (A), normal kidney from a Tg mouse (B), UUO kidney from a WT mouse (C), UUO kidney from a Tg mouse (D), and quantitative analysis (E). Note that both normal WT and Tg mice show few collagen III (dark black) accumulation within the interstitium (A, B), but severe renal fibrosis with an abundant collagen I accumulation within the tubulointerstitium is developed in WT mice with UUO (C), which is significantly inhibited in Tg mice (D). Sections are stained with the anti-collagen III antibody as described in materials and methods. Each bar represents means ± SE for a group of 6 (normal) or 8 (UUO) mice. ***P < 0.001 compared with the normal control. ###P < 0.001 when compared with the WT UUO. Magnification ×200.

Fig. 5.

Real-time PCR shows that a marked upregulation of α-SMA, collagen I, and collagen III mRNA expression in WT mice with UUO at day 7 is prevented in latent TGF-β1 Tg mice. α-SMA mRNA expression (A), collagen I mRNA expression (B), collagen III mRNA expression (C). Each bar represents means ± SE for a group of 6 mice. **P < 0.01, ***P < 0.001 compared with the normal control. ##P < 0.01, ###P < 0.001 when compared with the WT UUO.

Fig. 6.

Real-time PCR shows that a marked upregulation of TGF-β1 and CTGF mRNA expression in WT mice with UUO at day 7 is prevented in latent TGF-β1 Tg mice. TGF-β1 mRNA expression (A), CTGF mRNA expression (B). Each bar represents means ± SE for a group of 6 mice. **P < 0.01, ***P < 0.001 compared with the normal control. #P < 0.05, ##P < 0.01, when compared with the WT UUO.

Fig. 7.

Immunohistochemistry shows that upregulation of renal TGF-β1 protein is prevented in mice overexpressing latent TGF-β1 at day 7 after UUO. Normal kidney from a WT mouse (A), normal kidney from a Tg mouse (B), UUO kidney from a WT mouse (C), UUO kidney from a Tg mouse (D), and quantitative analysis (E). Note that renal TGF-β1 (dark black) is markedly upregulated with severe tubulointerstitial fibrosis in WT mice with UUO (C), which is significantly inhibited in Tg mice (D). Each bar represents means ± SE for a group of 6 (normal) or 8 (UUO) mice. *P < 0.05 compared with the normal control. #P < 0.05; ###P < 0.001 when compared with the WT UUO. Magnification ×200.

Fig. 8.

Immunohistochemistry shows that mice overexpressing latent TGF-β1 are protected against a marked activation of Smad2/3 within the fibrotic kidney at day 7 after UUO. Normal kidney from a WT mouse (A), normal kidney from a Tg mouse (B), UUO kidney from a WT mouse (C), UUO kidney from a Tg mouse (D), and quantitative analysis (E, F) in the glomerulus and tubulointerstitium. Note that both normal WT and Tg mice show a few phosphorylated Smad2/3 (p-Smad2/3) within the glomerulus and tubulointerstitium as evident by nuclear location (dark black; A and B), but a strong activation of p-Smad2/3 within areas of severe tubulointerstitial fibrosis is found in WT mice with UUO (C), which is significantly inhibited in Tg mice (D). Sections are stained with the anti-p-Smad2/3 Ab as described in materials and methods. Each bar represents means ± SE for a group of 6 (normal) or 8 (UUO) mice. *P < 0.05; ***P < 0.001 compared with the normal control. ###P < 0.001 when compared with the WT UUO. Magnification ×200.

Fig. 9.

Two-color immunohistochemistry shows that upregulation of renal Smad7 in Tg mice prevents activation of Smad2/3 at day 7 after UUO. Smad7 is labeled as brown, whereas phosphorylated Smad2/3 (p-Smad2/3) is stained as blue within the nucleus. Normal kidney from a WT mouse (A), normal kidney from a Tg mouse (B), UUO kidney from a WT mouse (C), and UUO kidney from a Tg mouse (D). Note that there is a strong activation of p-Smad2/3 (blue nuclei) with low expression level of Smad7 in the diseased kidney of WT mice (C). In contrast, renal Smad7 (brown) is markedly upregulated in Tg mice in both normal and UUO kidneys (B, D), which is associated with a significant inhibition of p-Smad2/3 nuclear translocation (blue nuclei). Magnification ×400.

Fig. 10.

TSP-1 is upregulated locally with the diseased kidney and increased systemically in plasma in WT mice, which is inhibited in Tg mice at day 7 after UUO. Normal kidney from a WT mouse (A), normal kidney from a Tg mouse (B), UUO kidney from a WT mouse (C), UUO kidney from a Tg mouse (D), quantitative analysis of TSP-1 immunostaining (E), and quantitative analysis (F) of plasma TSP-1 by ELISA. Note that renal TSP-1 (dark black) is markedly upregulated with severe tubulointerstitial fibrosis in WT mice with UUO, which is significantly inhibited in Tg mice. Each bar represents means ± SE for a group of 6 (normal) or 8 (UUO) mice. Arrows, TSP-1-positive cells. ***P < 0.05 compared with the normal control. ###P < 0.001 when compared with the WT UUO. Magnification ×200.