Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction

Abstract

Tubulointerstitial fibrosis is the final common result of a variety of progressive injuries leading to chronic renal failure. Transforming growth factor-beta (TGF-beta) is reportedly upregulated in response to injurious stimuli such as unilateral ureteral obstruction (UUO), causing renal fibrosis associated with epithelial-mesenchymal transition (EMT) of the renal tubules and synthesis of extracellular matrix. We now show that mice lacking Smad3 (Smad3ex8/ex8), a key signaling intermediate downstream of the TGF-beta receptors, are protected against tubulointerstitial fibrosis following UUO as evidenced by blocking of EMT and abrogation of monocyte influx and collagen accumulation. Culture of primary renal tubular epithelial cells from wild-type or Smad3-null mice confirms that the Smad3 pathway is essential for TGF-beta1-induced EMT and autoinduction of TGF-beta1. Moreover, mechanical stretch of the cultured epithelial cells, mimicking renal tubular distention due to accumulation of urine after UUO, induces EMT following Smad3-mediated upregulation of TGF-beta1. Exogenous bone marrow monocytes accelerate EMT of the cultured epithelial cells and renal tubules in the obstructed kidney after UUO dependent on Smad3 signaling. Together the data demonstrate that the Smad3 pathway is central to the pathogenesis of interstitial fibrosis and suggest that inhibitors of this pathway may have clinical application in the treatment of obstructive nephropathy.

Figures

Figure 1

Smad3-null mice maintain the renal architecture after UUO and have reversed EMT. (a) Obstructed kidneys from WT and Smad3-null (KO) mice at day 14 after UUO. (b and c) Hematoxylin and eosin staining of the obstructed kidneys at day 14 after UUO in WT (b) and KO (c) mice. Scale bars: 20 μm. (d–g) Dual immunofluorescence of E-cadherin (green) and α-SMA (red) in obstructed kidneys of WT (d and e) and KO (f and g) mice at day 7 (d and f) and day 14 (e and g) after UUO. DAPI (blue) was used for nuclear staining. Scale bars: 20 μm. (h) Immunoblot of E-cadherin (E-cad) and α-SMA with extracted proteins from kidneys of WT and KO mice with UUO and sham-operated WT mice (Sham). (i) Northern blot of Snail mRNA in kidneys of WT and KO mice with UUO and sham-operated counterparts (Sham).

Figure 2

In situ hybridization of α-SMA, Snail, and TGF-β1. (a–d) De novo expression of Snail (a) and α-SMA (c) mRNA in the renal tubular epithelial cells of WT mice at day 7 after UUO. There are no positive signals for Snail (b) or α-SMA (d) mRNA in Smad3-null (KO) counterparts. (e and f) Signals for TGF-β1 mRNA in WT (e) and KO (f) mice at day 14 after UUO. Insets, negative controls reacted with sense probe. Counterstained in nuclear fast red solution. Scale bars: 20 μm. Similar results were obtained from three additional experiments.

Figure 3

Lack of Smad3 prevents renal fibrosis, monocyte influx, and TGF-β1 upregulation. (a and b) Immunofluorescence of type I collagen in obstructed kidneys of WT (a) and Smad3-null (KO) (b) mice at day 14 after UUO. (c) Hydroxyproline content in obstructed kidneys from WT and KO mice and sham-operated WT mice (Sham). (d and e) Immunofluorescence of F4/80 antigen, a mouse monocyte marker, in obstructed kidneys from WT (d) and KO (e) mice at day 14 after UUO. DAPI (blue) was used for nuclear staining. Scale bars: 20 μm. (f) Number of monocytes per unit area in obstructed kidneys from WT and KO mice with UUO and sham-operated WT mice (Sham). (g) Northern blot of TGF-β1 mRNA in kidneys from WT and KO mice with UUO and sham-operated counterparts (Sham). (h) Active and total TGF-β1 concentrations as determined by immunoassay in kidneys of WT and KO mice and sham-operated mice (Sham). Results are means ± standard deviation of four to five samples. *P < 0.01 compared with Sham or KO.

Figure 4

Smad3-mediated EMT in cultured renal tubular epithelial cells. (a–d) Phase-contrast microscopy of epithelial cells from WT (a and b) and Smad3-null (KO) (c and d) mice in the absence (a and c) or presence (b and d) of TGF-β1 (10 ng/ml) for 24 hours. Scale bars: 100 μm. (e–h) Dual immunofluorescence of E-cadherin (green) and α-SMA (red) in epithelial cells from WT (e and f) and KO (g and h) mice in the absence (e and g) or presence (f and h) of TGF-β1 (10 ng/ml) for 24 hours. Scale bars: 20 μm. (i) Immunoblot of E-cadherin (E-cad) and α-SMA with extracted protein from epithelial cells of WT and KO mice in the absence (–) or presence (+) of TGF-β1 (10 ng/ml) for 24 hours. (j) Northern blot of Snail mRNA in the epithelial cells from WT and KO mice in the absence (–) or presence (+) of TGF-β1 (10 ng/ml) for 8 hours. Similar results were obtained from three additional experiments.

Figure 5

Smad3-mediated autoinduction of TGF-β1 in cultured renal tubular epithelial cells. (a) Concentration of total TGF-β1 in culture medium of renal tubular epithelial cells from WT and Smad3-null (KO) mice. Results are means ± standard deviation of four to five samples. *P < 0.05 compared with KO. (b) Northern blot of TGF-β1 mRNA in epithelial cells from WT and KO mice in the absence (–) or presence (+) of TGF-β1 (10 ng/ml) for 24 hours. Cells without TGF-β1 were further treated with a neutralizing antibody against TGF-β (20 μg/ml) to exclude any effects of endogenous TGF-β1. The same amount of normal IgG was added to the medium of TGF-β1-treated cells. Results are means ± standard deviation of four samples. *P < 0.01 compared with WT (–), KO (–) or KO (+).

Figure 6

Epithelial-mesenchymal transition and TGF-β1 upregulation in an environment of mechanical stretch. (a–d) Dual immunofluorescence of E-cadherin (green) and α-SMA (red) in renal tubular epithelial cells derived from WT (a and b) and Smad3-null (KO) mice (c and d) stretched for 24 hours in the absence (a and c) or presence (b and d) of neutralizing anti–TGF-β1 (20 μg/ml). Scale bars: 20 μm. (e) Northern blot of Snail mRNA in the epithelial cells either stretched for 24 hours or nonstretched, in the absence or presence of neutralizing anti–TGF-β1. Similar results were obtained from additional two experiments. (f) Northern blot of TGF-β1 mRNA in primary culture of the epithelial cells either stretched for 24 hours or nonstretched, in the absence or presence of a neutralizing anti–TGF-β1 antibody. Results are means ± standard deviation of five samples. *P < 0.01 compared with other experimental groups. (g) Total TGF-β1 concentration in culture medium of epithelial cells either stretched or nonstretched. Results are means ± standard deviation of five samples. *P < 0.05 compared with nonstretched counterparts.

Figure 7

Role of exogenous monocytes in EMT of renal tubular epithelial cells. (a–d) Dual immunofluorescence of E-cadherin (green) and α-SMA (red) in renal tubular epithelial cells and bone marrow monocytes cocultured for 48 hours. (a) WT epithelial cells and WT monocytes. (b) WT epithelial cells and Smad3-null (KO) monocytes. (c) KO epithelial cells and WT monocytes. (d) KO epithelial cells and KO monocytes. (e–l) Three days after transplantation of monocytes into the subcapsular space of the kidney with UUO. Dotted lines indicate the border between the subcapsular space (left) and the renal cortex (right). (e–h) Immunofluorescence of F4/80 antigen (green). (i–l) Dual immunofluorescence of E-cadherin (green) and α-SMA (red). (e and i) Transplantation of WT monocytes into WT kidneys. (f and j) Transplantation of KO monocytes into WT kidneys. (g and k) Transplantation of WT monocytes into KO kidneys. (h and l) Transplantation of KO monocytes into KO kidneys. DAPI (blue) was used for nuclear staining. Scale bars: 20 μm. Similar results were obtained from four additional experiments.