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BackgroundCigarette 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.MethodsTo 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.ResultsHuman 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.ConclusionsNicotine 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.

Morphine has been reported to accelerate the progression of chronic kidney disease. However, whether morphine affects slit diaphragm (SD), the major constituent of glomerular filtration barrier, is still unclear. In the present study, we examined the effect of morphine on glomerular filtration barrier in general and podocyte integrity in particular. Mice were administered either normal saline or morphine for 72 h, then urine samples were collected and kidneys were subsequently isolated for immunohistochemical studies and Western blot. For in vitro studies, human podocytes were treated with morphine and then probed for the molecular markers of slit diaphragm. Morphine-receiving mice displayed a significant increase in albuminuria and showed effacement of podocyte foot processes. In both in vivo and in vitro studies, the expression of synaptopodin, a molecular marker for podocyte integrity, and the slit diaphragm constituting molecules (SDCM), such as nephrin, podocin, and CD2-associated protein (CD2AP), were decreased in morphine-treated podocytes. In vitro studies indicated that morphine modulated podocyte expression of SDCM through opiate mu (MOR) and kappa (KOR) receptors. Since morphine also enhanced podocyte oxidative stress, the latter seems to contribute to decreased SDCM expression. In addition, AKT, p38, and JNK pathways were involved in morphine-induced down regulation of SDCM in human podocytes. These findings demonstrate that morphine has the potential to alter the glomerular filtration barrier by compromising the integrity of podocytes.

Tubular cell HIV-infection has been reported to manifest in the form of cellular hypertrophy and apoptosis. In the present study, we evaluated the role of mammalian target of rapamycin (mTOR) pathway in the HIV induction of tubular cell protein synthesis. Mouse proximal tubular epithelial cells (MPTECs) were transduced with either gag/pol-deleted NL4-3 (HIV/MPTEC) or empty vector (Vector/MPTEC). HIV/MPTEC showed enhanced DNA synthesis when compared with Vector/MPTECs by BRDU labeling studies. HIV/MPTECs also showed enhanced production of β-laminin and fibronection in addition to increased protein content per cell. In in vivo studies, renal cortical sections from HIV transgenic mice and HIVAN patients showed enhanced tubular cell phosphorylation of mTOR. Analysis of mTOR revealed increased expression of phospho (p)-mTOR in HIV/MPTECs when compared to vector/MPTECs. Further downstream analysis of mTOR pathway revealed enhanced phosphorylation of p70S6 kinase and associated diminished phosphorylation of eEF2 (eukaryotic translation elongation factor 2) in HIV/MPTECs; moreover, HIV/MPTECs displayed enhanced phosphorylation of eIF4B (eukaryotic translation initiation factor 4B) and 4EBP-1 (eukaryotic 4E binding protein). To confirm our hypothesis, we evaluated the effect of rapamycin on HIV-induced tubular cell downstream signaling. Rapamycin not only attenuated phosphorylation of p70S6 kinase and associated down stream signaling in HIV/MPTECs but also inhibited HIV-1 induced tubular cell protein synthesis. These findings suggest that mTOR pathway is activated in HIV-induced enhanced tubular cell protein synthesis and contributes to tubular cell hypertrophy.