Nicotine Induces Podocyte Apoptosis through Increasing Oxidative Stress

Cigarette smoking plays an important role in the progression of chronic kidney disease (CKD). Nicotine, one of the major components of cigarette smoking, has been demonstrated to increase proliferation of renal mesangial cells. In this study, we examined the effect of nicotine on podocyte injury.To determine the expression of nicotinic acetylcholine receptors (nAChR subunits) in podocytes, cDNAs and cell lysate of cultured human podocytes were used for the expression of nAChR mRNAs and proteins, respectively; and mouse renal cortical sections were subjected to immunofluorescant staining. We also studied the effect of nicotine on podocyte nephrin expression, reactive oxygen species (ROS) generation (via DCFDA loading followed by fluorometric analysis), proliferation, and apoptosis (morphologic assays). We evaluated the effect of nicotine on podocyte downstream signaling including phosphorylation of ERK1/2, JNK, and p38 and established causal relationships by using respective inhibitors. We used nAChR antagonists to confirm the role of nicotine on podocyte injury.Human podocytes displayed robust mRNA and protein expression of nAChR in vitro studies. In vivo studies, mice renal cortical sections revealed co-localization of nAChRs along with synaptopodin. In vitro studies, nephrin expression in podocyte was decreased by nicotine. Nicotine stimulated podocyte ROS generation; nonetheless, antioxidants such as N-acetyl cysteine (NAC) and TEMPOL (superoxide dismutase mimetic agent) inhibited this effect of nicotine. Nicotine did not modulate proliferation but promoted apoptosis in podocytes. Nicotine enhanced podocyte phosphorylation of ERK1/2, JNK, and p38, and their specific inhibitors attenuated nicotine-induced apoptosis. nAChR antagonists significantly suppressed the effects of nicotine on podocyte.Nicotine induces podocyte apoptosis through ROS generation and associated downstream MAPKs signaling. The present study provides insight into molecular mechanisms involved in smoking associated progression of chronic kidney disease

Nicotine decrease nephrin expression in podocyte.

<p>Differentiated human podocytes were treated with nicotine (1 and10 μM) for 48 h. Cell lysates were then collected and subjected for Western blotting to detect nephrin expression. A. Representative gels are displayed. B. Quantification of the expression of nephrin in <b>A,</b> and the results (mean ± SD) represent three independent samples. * p < 0.05 compared with control (0 μM).</p

Nicotine treatment affects apoptosis related proteins in podocyte.

<p>Differentiated human podocytes were treated with nicotine (0.1–10 μM) for 24 h. Cell lysates were then collect and were subjected for Western blotting. A. Representative results were selected to show the Western blottings. B-D. Quantification of the expression of Bcl-2 (<b>B</b>), Bax (<b>C</b>), and Cleaved caspase-3 (<b>D</b>) in <b>A,</b> and the results (mean ± SD) represent three independent samples. * p < 0.05 compared with control (0 μM).</p

Primer sequences for Nicotine receptor subunits (nAChRs).

<p>Primer sequences for Nicotine receptor subunits (nAChRs).</p

MAPKs regulated nicotine-induced podocyte apoptosis.

<p>A. Differentiated Human podocytes were starved in serum free medium for 12 h, and then 0.1 μM nicotine was added. Cell lysates were collected at different time points, and Western blotting was performed to detect the phosphor-ERK1/2, JNK, and p38. Total proteins and actin were used as loading control. <b>B</b>. Human podocytes were pretreated with of PD98059 (PD, 5 μM), SB203580 (SB, 3 μM), or SP600125 (SP, 5 μM) for 1 h before 10 μM nicotine was added. After incubation at 37°C for another 48 h, apoptotic cells were then determined and counted by using Hoechst staining. Results (mean ± SD) were calculated from 20 pictures of each treatment. * p < 0.05 compared with blank control, while # p < 0.05 compared with morphine treatment alone.</p

Nicotine increases ROS generation in podocyte.

<p>A. Differentiated human podocytes were treated with 0.01 to 10 μM nicotine for 12 h, and were labeled with DCFH for 30 min. After washing with PBS, the cells were incubated at room temperature, and the ROS generation was determined after different periods of time. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control (0 μM). B. Differentiated human podocytes were pre-treated with VAS2870 (10 μM) for 1 h, followed by treatment with 10 μM nicotine for another 12 h. Subsequently, the cells were labeled with DCFH and the ROS generation was determined at 1 hour as described above. *p< 0.05 compared with control (0 μM) while #p<0.05 compared with nicotine treatment alone.</p

Nicotine receptor subunits (nAChRs) are expressed in podocytes.

<p>A. Total RNAs were prepared from differentiated human podocytes, and were used for RT-PCR to detect the expression of nAChR subunits. GAPDH was used as internal control. B. Cell lysate was collected from differentiated human podocytes, and was subjected to Western blotting to detect the expression of nAChRs. C. Paraffin sections were prepared from 2-month-old mice kidneys, and immunofluorescence staining was performed to detect the expression of nAChR subunits. Synaptopodin was used as a marker of podocytes.</p

Nicotine induces podocyte apoptosis.

<p>Differentiated human podocytes were treated with nicotine (0.1–10 μM) for 48 h. Apoptotic cells were then determined and counted by using Hoechst staining, as describe in Materials and Methods. A. Representative pictures were selected to show the apoptotic cells (red arrow). Scar bar was for 50 μm. B. Results (mean ± SD) were calculated from 20 pictures of each treatment. *, p < 0.05 compared with control (0 μM).</p

ROS scavengers attenuate nicotine-induced podocyte apoptosis.

<p><b>A</b>. Differentiated human podocytes were treated with nicotine (10 μM) for 48 h in the presence or absence of NAC (100 μM) or TEMPO (10 μM). Apoptotic cells were then determined and counted by using Hoechst staining, as described in Materials and Methods. Representative results (mean ± SD) were calculated from 20 pictures of each treatment. <b>B</b>. Differentiated human podocytes were pretreated with NAC (100 μM) or TEMPO (10 μM) for 1 h before 10 μM nicotine was added. After another 24 h incubation at 37°C, the cell lysates were collected for Western blotting. <b>C</b>. Quantification of the expression of Cleaved caspase-3 in <b>A,</b> and the results (mean ± SD) represent three independent samples. * p < 0.05 compared with blank control, while # p < 0.05 compared with nicotine treatment alone. C, control; N, NAC; T, TEMPO.</p