A novel U-STAT3-dependent mechanism mediates the deleterious effects of chronic nicotine exposure on renal injury

Abstract

Previous data from our group have demonstrated (Arany I, Grifoni S, Clark JS, Csongradi, Maric C, Juncos LA. Am J Physiol Renal Physiol 301: F125-F133, 2011) that chronic nicotine (NIC) exposure exacerbates acute renal ischemic injury (AKI) in mice that could increase the risk for development and progression of chronic kidney disease (CKD). It has been shown that proximal tubules of the kidney can acquire characteristics that may compromise structural recovery and favor development of inflammation and fibrosis following injury. Chronic NIC exposure can amplify this epithelial process although the mechanism is not identified. Recently, the unphosphorylated form of signal transducer and activator of transcription-3 (U-STAT3) has emerged as a noncanonical mediator of inflammation and fibrosis that may be responsible for the effects of chronic NIC. We found that levels of transforming growth factor β-1 (TGF-β1), α-smooth muscle actin (α-SMA), fibronectin, monocyte chemotactic protein-1 (MCP-1), and expression of U-STAT3 were increased in the ischemic kidneys of NIC-exposed mice. Chronic NIC exposure also increased TGF-β1-dependent F-actin reorganization, vimentin, fibronectin, and α-SMA expression as well as promoter activity of α-SMA and MCP-1 without significant loss of epithelial characteristics (E-cadherin) in cultured renal proximal tubule cells. Importantly, transduction of cells with a U-STAT3 mimetic (Y705F-STAT3) augmented stress fiber formation and also amplified NIC+TGF-β1-induced expression of α-SMA, vimentin, fibronectin, as well as promoter activity of α-SMA and MCP-1. Our results reveal a novel, chronic NIC-exposure-related and U-STAT3-dependent mechanism as mediator of a sustained transcription of genes that are linked to remodeling and inflammation in the kidney during injury. This process may facilitate progression of AKI to CKD. The obtained data may lead to devising therapeutic methods to specifically enhance the protective and/or inhibit adverse effects of STAT3 in the kidney.

Fig. 1.

Chronic nicotine (NIC) exposure augments mediators and markers of inflammation and fibrosis in the postischemic kidney. A group of mice received nicotine in their drinking water for 4 wk while another group received the vehicle (2% saccharine) for the same time as described in materials and methods. Some mice underwent 18-min warm renal ischemia while others were sham operated. Kidneys were collected 24 h postischemia and lysed in RIPA buffer. Transforming growth factor (TGF)-β1 and monocyte-chemotactic protein (MCP)-1 content was determined by ELISA (A) and α-smooth muscle actin (SMA), fibronectin (fibro), and E-cadherin (Ecad) expression along with actin by Western blotting (B). C: densitometry results of Western blots in B. Protein expression was normalized to actin; n = 4 (sham) and n = 8 [ischemia-reperfusion (IR)]. *P < 0.05 compared with sham. #P < 0.05 compared with the corresponding value in the saccharine group.

Fig. 2.

Chronic NIC exposure augments signal transducer and activator of transcription-3 (U-STAT3) expression in the postischemic kidney. A: kidney lysates, as described in Fig. 1, were separated by SDS-PAGE, and levels of U-STAT3 along with actin were determined by Western blotting. B: densitometry of Western blots described in A. Levels of U-STAT3 were expressed as the U-STAT3/actin ratio. *P < 0.05 compared with sham or as indicated.

Fig. 3.

Chronic NIC exposure aggravates TGF-β1-induced stress fiber formation in a U-STAT3-dependent fashion in cultured renal proximal tubule cells. LLC-PK₁ cells were treated with 10 ng/ml TGF-β1 for 3 days (B–D). In a set of experiments, cells were pretreated with 200 μM NIC (C and D) for 24 h before treatment with TGF-β1. Cells in D were infected with a Y705F-STAT3 adenovirus for 24 h before treatment. Cells were fixed and stained with Alexa Fluor 488-labeled phalloidin plus 4′,6-diamidino-2-phenylindole (DAPI; magnification ×400). Results are representative of 3 separate experiments.

Fig. 4.

Chronic NIC exposure augments TGF-β1-mediated induction of profibrotic genes as well as activation of α-SMA promoter in cultured renal proximal tubule cells. A: LLC-PK₁ cells were treated with 10 ng/ml TGF-β1 for 3 days. In a set of experiments, cells were pretreated with 200 μM NIC for 24 h before treatment with TGF-β1. Expression of α-SMA, vimentin (vim), fibronectin, and E-cadherin was determined by Western blotting in cell lysates as described in materials and methods. B: densitometry results of blots shown in A; n = 3 independent experiments. Expression of those proteins were normalized to actin expression *P < 0.05 compared with none. #P < 0.05 compared with the TGF-β1 group. C: LLC-PK₁ cells were transfected with an α-SMA promoter-luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h. Some cells were treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1 for 24 h while other cells were treated with TGF-β1 only. Firefly (α-SMA promoter) and Renilla luciferase activities were determined as described in materials and methods. Values represent relative luciferase activities (firefly/Renilla) expressed as a percentage of controls. Values are means ± SD; n = 3 independent experiments. *P < 0.05 compared with none or as indicated.

Fig. 5.

Overexpression of the Y705F-STAT3 mutant augments TGF-β1- and NIC+TGF-β1-induced α-SMA expression as well as activation of α-SMA promoter in cultured renal proximal tubule cells. LLC-PK₁ cells were infected with either a negative control (AdNull) or with the Y705F-STAT3 adenovirus as described in materials and methods. Uninfected cells were also included. A: cells were treated with 10 ng/ml TGF-β1 for 3 days, and levels of α-SMA and STAT3 along with actin were determined by Western blotting. B: densitometry results of blots shown in A; n = 3 independent experiments. *P < 0.05 compared with none or as indicated. C: LLC-PK₁ cells were transfected with an α-SMA promoter luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h and then treated with 10 ng/ml TGF-β1 for 24 h. Firefly (α-SMA promoter) and Renilla luciferase activities were determined as described in materials and methods. Values are means ± SD and represent relative luciferase activities (firefly/Renilla) expressed as a percentage of controls; n = 3 independent experiments. *P < 0.05 compared with none or as indicated. D: cells were treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1 for 3 days, and levels of α-SMA and STAT3 along with actin were determined by Western blotting. E: densitometry results of blots shown in D; n = 3 independent experiments. *P < 0.05 compared with none or as indicated. N+T, NIC+TGF-β1. F: LLC-PK₁ cells were transfected with an α-SMA promoter luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h. Cells were treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1. Firefly (α-SMA promoter) and Renilla luciferase activities were determined as described in materials and methods. Values are means ± SD and represent relative luciferase activities (firefly/Renilla) expressed as a percentage of controls; n = 3 independent experiments. *P < 0.05 compared with none or as indicated.

Fig. 6.

Overexpression of the U-STAT3-mimetic Y705F-STAT3 mutant augments NIC+TGF-β1-induced activation of MCP-1 promoter as well as expression of profibrotic genes in cultured renal proximal tubule cells. A: LLC-PK₁ cells were transfected with an MCP-1 promoter luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h followed by infection with either an empty adenovirus (AdNull) or with the Y705F-STAT3 (ad705F) adenovirus as described in materials and methods. Cells were then treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1 for 24 h. Firefly (MCP-1) and Renilla luciferase activities were determined as described in materials and methods. Values are means ± SD and represent relative luciferase activities (firefly/Renilla) expressed as a percentage of controls; n = 3 independent experiments. *P < 0.05 compared with none or as indicated. B: LLC-PK₁ cells were transfected with adY705F and then treated with NIC and TGF-β1 as described in materials and methods. Expression of vimentin, fibronectin, and E-cadherin along with actin was determined by Western blotting. Results shown are representatives of 3 independent experiments. C: densitometry of results in B; n = 3. YF, adY705F-STAT3. *P < 0.05 compared with none. #P < 0.05 compared with the corresponding N+T values.