Chronic nicotine exposure exacerbates acute renal ischemic injury

Abstract

Recent epidemiological reports showed that smoking has a negative impact on renal function and elevates the renal risk not only in the renal patient but perhaps also in the healthy population. Studies suggested that nicotine, a major tobacco alkaloid, links smoking to renal dysfunction. While several studies showed that smoking/chronic nicotine exposure exacerbates the progression of chronic renal diseases, its impact on acute kidney injury is virtually unknown. Here, we studied the effects of chronic nicotine exposure on acute renal ischemic injury. We found that chronic nicotine exposure increased the extent of renal injury induced by warm ischemia-reperfusion as evidenced by morphological changes, increase in plasma creatinine level, and kidney injury molecule-1 expression. We also found that chronic nicotine exposure elevated markers of oxidative stress such as nitrotyrosine as well as malondialdehyde. Interestingly, chronic nicotine exposure alone increased oxidative stress and injury in the kidney without morphological alterations. Chronic nicotine treatment not only increased reactive oxygen species (ROS) production and injury but also exacerbated oxidative stress-induced ROS generation through NADPH oxidase and mitochondria in cultured renal proximal tubule cells. The resultant oxidative stress provoked injury through JNK-mediated activation of the activator protein (AP)-1 transcription factor in vitro. This mechanism might exist in vivo as phosphorylation of JNK and its downstream target c-jun, a component of the AP-1 transcription factor, is elevated in the ischemic kidneys exposed to chronic nicotine. Our results imply that smoking may sensitize the kidney to ischemic insults and perhaps facilitates progression of acute kidney injury to chronic kidney injury.

Fig. 1.

Chronic nicotine (NIC) exposure exacerbates injury of the kidney after acute ischemia-reperfusion (IR) and cultured proximal tubule cells after treatment with H₂O₂. A: expression of kidney injury molecule (KIM)-1 in kidney lysates from C57BL/6J mice that underwent renal IR injury was determined by Western blotting. One group received saccharine, while the other NIC as described in materials and methods. B: densitometry of results from A. Values are means ± SD of ratios of KIM-1/actin; n = 4 (sham) and n = 8 (IR). *P < 0.05 compared with sham. #P < 0.05 compared with saccharine group. @P < 0.05 compared with the saccharine group. C: TKPTS cells were pretreated or not with 200 μM NIC for 24 h before treatment with 200 μM H₂O₂. Lactate dehydrogenase (LDH) release was determined 24 h after treatment with H₂O₂. Values represent percentage of released LDH and expressed as means ± SD; n = 3. *P < 0.05 compared with untreated (none).

Fig. 2.

Effects of chronic NIC exposure on kidney morphology and integrity of cultured proximal tubule. Sections of kidneys form saccharine+sham (A), saccharine+IR (B), NIC+sham (C), and NIC+IR (D) were stained with PAS and evaluated for injury as described in materials and methods. TKPTS cells were treated or not with 200 μM NIC for 24 h followed by 200 μM H₂O₂ for 3 h and stained with calcein/ethidium as described in materials and methods. Pictures are representatives of 3 independent experiments. E: control, untreated. F: H₂O₂-treated; G: NIC alone; H: NIC+H₂O₂.

Fig. 3.

Chronic NIC exposure increases oxidative stress in the kidney. A: expression of nitrotyrosine (NTyr) in kidney lysates from C57BL/6J mice that underwent renal IR injury was determined by Western blotting. One group received saccharine while the other NIC as described in materials and methods. B: densitometry of results from A. Values are means ± SD of NTyr/actin ratios; n = 4 (sham) and n = 8 (IR). *P < 0.05 compared with sham. #P < 0.05 compared with saccharine group. @P < 0.05 compared with the saccharine group. C: renal malondialdehyde (MDA) content of kidney lysates from C57BL/6J mice that underwent renal IR injury was determined by spectrophotometry as described in materials and methods. One group received saccharine while the other NIC as described in materials and methods. Values represent MDA content in nmol/mg protein and expressed as means ± SD; n = 4 (sham) and n = 8 (IR). *P < 0.05 compared with sham. #P < 0.05 compared with saccharine group. @P < 0.05 compared with saccharine group.

Fig. 4.

Chronic NIC exposure exacerbates H₂O₂-induced reactive oxygen species (ROS) production and resultant injury of cultured proximal tubule cells. A: TKPTS cells were pretreated or not with 200 μM NIC for 24 h, and then 200 μM H₂O₂-induced ROS production was determined. Values represent ROS production as percentage of the corresponding untreated values and are expressed as means ± SD; n = 3. *P < 0.05 compared with untreated. B: NIC-exposed TKPTS cells were pretreated with the xanthine oxidase inhibitor allopurinol (Allo; 100 μM), the NADPH inhibitor diphenilenediodium (DPI; 5 μM), or the mitochondrial inhibitor antimycin A (AntA; 10 μM) for 30 min before treatment with 200 μM H₂O₂. ROS production was expressed as percentage of NIC+H₂O₂-treated values; n = 3; means ± SD. *P < 0.05 compared with NIC+H₂O₂-treated value. C: TKPTS cells were pretreated with 100 μM N-acetylcysteine (NAC) 1 h before treatment with 200 μM NIC for 24 h followed by 200 μM H₂O₂ for 24 h; then, LDH release was determined. Similarly, some cells were infected with a dominant-negative JNK (dnJNK) adenovirus for 24 h before treatment with 200 μM NIC for 24 h followed by 200 μM H₂O₂. In these cells, LDH release was also determined. Values represent the pcrcentage of the control values and are expressed as means ± SD; n = 3. *P < 0.05 compared with untreated.

Fig. 5.

Chronic NIC exposure exacerbates acute renal ischemia-induced phosphorylation of JNK and c-jun in the mouse kidney. A: levels of phospho-JNK and JNK in kidney lysates from C57BL/6J mice that underwent renal IR injury were determined by Western blotting. One group received saccharine while the other NIC as described in materials and methods. B: densitometry of results from A. Values are expressed as means ± SD of ratios of pJNK/JNK; n = 4 (sham) and n = 8 (IR). *P < 0.05 compared with sham. #P < 0.05 compared with saccharine group. @P < 0.05 compared with the saccharine group. C: levels of phospho-c-jun and actin were also determined as described in A. D: densitometry of results from C. Values are expressed as means ± SD of p-c-jun/actin ratios; n = 4 (sham) and n = 8 (IR). *P < 0.05 compared with sham. #P < 0.05 compared with saccharine group. @P < 0.05 compared with the saccharine group.

Fig. 6.

Oxidative stress mediates phosphorylation of JNK leading to activation of activator protein-1 (AP-1) in cultured proximal tubule cells. A: TKPTS cells were pretreated with 100 μM NAC before treatment with 200 μM NIC for 24 h followed by treatment with 200 μM H₂O₂ for 30 min. Phosphorylation of JNK was determined by Western blotting. The blot shown is representative of 3 independent experiments. B: TKPTS cells were infected with a dnJNK adenovirus for 24 h before exposure to 200 μM NIC for 24 h. In these cells, 200 μM H₂O₂-induced ROS production was determined. Values represent changes in DCFDA fluorescence/30 min/0.5 × 10⁶ cells as described in materials and methods and expressed as means ± SD (n = 3). *P < 0.05 compared with untreated. C: TKPTS cells were transiently transfected with an AP-1 luciferase plasmid together with a Renilla luciferase. After 24 h, some cells were infected with the dnJNK adenovirus and some with a negative (empty) adenovirus. These cells were treated with 200 mM H₂O₂ or 200 μM NIC for 24 h. In another set of experiments, cells were pretreated with 200 μM NIC for 24 h followed by treatment with 200 μM H₂O₂ for 24 h, and firefly (AP-1) as well as Renilla luciferase activities were determined. Values represent relative luciferase activities (firefly/Renilla) expressed as percentage of their own controls in means ± SD; n = 3. *P < 0.05 compared with the negative virus-infected counterparts.

Fig. 7.

Summary of results. Chronic NIC exposure exacerbates acute renal IR-induced production of ROS, which in turn aggravates phosphorylation of JNK. The resultant increase in JNK activation leads to increased AP-1 activity and consequent increase in AKI. Ultimately, this increase in AKI could lead to increased progression to chronic kidney disease (CKD) in smokers.