CYP enzymes are not only crucial in the elimination of foreign compounds but also play a pivotal role in the generation of ROS that are associated with cell damage 22
. We have identified and localized CYP2B1 to rat GEC. CYP2B1 is expressed at a very low basal level in primary hepatocytes 23
and in GEC, as shown by real time RT-PCR and western blot analysis. In cultured rat hepatocytes, phenobarbital induction of CYP2B1 is most pronounced 24,25
. However, we have not been able to demonstrate increased induction of CYP2B1 in phenobarbital-treated kidney glomeruli and GEC (data not shown), consistent with the observations of Guengerich et al 13,26
. Hence our experiments in the current study were performed in GEC infected with an adenoviral vector that encodes the rat CYP2B1 cDNA.
ROS play an important role in the alteration of the GEC morphology leading to proteinuria 27,28
. Utilizing CYP inhibitors we have demonstrated marked reduction in ROS generation with attenuation of proteinuria in a PAN model of nephrotic syndrome and PAN-induced GEC cytotoxicity 14,15
. The current study extends those findings by gene-specific targeting of CYP2B1. Both in vivo
and in vitro
studies have shown that CYP2B1 induction by Phenobarbital (PB) treatment was selectively associated with oxidative stress 29,30
. Recent studies indicated that the ability of phenobarbital to selectively induce in vivo
oxidative stress was related to decreases in glutathione peroxidase and pyridine nucleotides, which normally protect cells from ROS 13,26
. In the current study, ROS generation in GEC overexpressing CYP2B1 was significantly higher than in control cells even in the basal state (.). Moreover, exposure of these CYP2B1 overexpressing cells to PAN markedly increased ROS generation and significantly increased cytotoxicity. These results suggest that the changes resulting from CYP2B1 overexpression are gene specific ( and ). Silencing of CYP2B1 attenuated PAN-induced ROS generation and cytotoxicity. Therefore, CYP2B1 is indeed the source of generation of intracellular H2
leading to oxidant injury.
The heme moiety of the CYP may serve as an important source of catalytic iron capable of catalyzing free radical reactions 9,31–34
. CYP inactivation in the PAN-treated cells may involve the formation of active oxygen species accompanied by bleaching of the heme protein as a result of heme loss or degradation 35
. The CYP would then be inactivated and catabolized with the release of heme and catalytic iron, which promotes the generation of OH· and induces lipid peroxidation. In the present study the marked increase in OH· generation in PAN-treated GEC was significantly decreased upon silencing of CYP2B1 (), confirming the role of CYP2B1 in the generation of the OH· and subsequent injury.
The induction of CYP2B1 mRNA by phenobarbital can be modulated in a redox sensitive manner 22
. Redox regulation of CYP2B1 mRNA in GEC is also suggested in the present study, where CYP2B1 mRNA levels were increased 4-fold within 1 h of PAN treatment, at which time CYP2B1 protein was significantly decreased (). This increase in CYP2B1 mRNA may be a compensatory effect to replace the CYP2B1 protein. Furthermore, these results indicate a post-translational rather than a post-transcriptional effect of PAN on the loss of CYP2B1 protein.
Agents that promote the induction of the heme-degrading enzyme HO-1 cause a release of the heme from CYP, which in turn leads to activation of HO-1 36
. HO-1 was induced in PAN-treated glomeruli and GEC following marked generation of H2
and degradation of CYP2B1, which was attenuated by CYP inhibitors 14,15
. In our current study HO-1 induction was markedly increased in PAN-treated GECs with a significant decrease in the CYP2B1-silenced cells (). Thus, breakdown of the CYP2B1 heme protein leads to the release of heme, which in turn induces HO-1. A similar protective effect was observed following stabilization of CYP by carbon monoxide, thus preventing its degradation, induction of HO-1 and oxidative stress 10
Caspases are cysteine proteases that play an important role in programmed cell death 37,38
. Fogo et al have shown a marked increase in the active form of caspase 3 in PAN-induced apotosis 39
. Caspase 3 activity was markedly increased following PAN treatment but was significantly decreased in the CYP2B1-silenced cells (). Caspase 12 is an ER-specific caspase that participates in apoptosis under ER stress 20,40,41
. It is an initiator caspase that undergoes activation in response to apoptotic stimuli and in turn activates downstream effector caspases that are responsible for the cleavage of a wide variety of physiologic substrates 42
. The PAN-induced increase in the active form of caspase 12 was significantly ameliorated in the GEC transfected with CYP2B1 siRNA (). Caspase 12 and caspase 3 could be functioning in conjunction with each other or independently.
Actin cytoskeleton is a major constituent of the glomerular foot processes and reorganization of the actin filaments leads to effacement of the foot processes 17
. There is increasing evidence to indicate that PAN induces actin cytoskeletal depolarization that can result in structural changes of the podocyte and leakage of the protein through the slit diaphragm 43–46
. CYP inhibitors enhance the preservation of individual foot processes of the podocytes and protect from PAN-induced proteinuria 15
. In the current study CYP2B1 gene silencing prevented the disruption of the actin cytoskeleton induced by PAN (), confirming that the disorganization of the actin cytoskeleton is mediated by CYP2B1 induced oxidative stress in an in vitro
model of PAN-induced cytotoxicity.
Experimental animal models are commonly utilized to predict the mechanism of podocyte injury in the humans despite species variability 47
. Majority of the rat strains are prone to PAN induced injury with effacement of foot processes and development of proteinuria while most of the mice strains have been traditionally resistant 48,49
. Differences in the genetic traits including prior presence of hypercholesterolemia have been associated to the development of PAN induced proteinuria 50–52
. Harris et al. have shown that PAN induces reversible proteinuric injury in transgenic mice expressing COX-2 in the podocytes 53
. Expression of COX-2 in the vascular endothelial cells has been linked to the CYP 54
. Prostanoids have also been shown to down regulate PB induced CYP 2B1 gene expression 55
. There are marked specific inter and intra tissue differences in the expression and inducibility of the CYP in the different strains and species 56
. This may determine the generation of H2
, breakdown of the heme protein, release of catalytic iron and the formation of the hydroxyl radical leading to disruption of the cytoskeleton and proteinuria based on the interaction between PAN and CYP2B1. We have not been able to demonstrate the expression of the CYP2B1 in the 129/SV strain of mice glomeruli both by immuno histochemistry and western blot analysis ( unpublished data ). Transgenic mice with expression of CYP2B1 in the podocyte may provide better understanding of the pathogenesis of PAN induced nephrotic syndrome. Autophagy represents an important protective mechanism that attempts to rescue cells from apoptosis 57,58
. In a recent communication Huber et al observed significant level of autophagy in the podocytes in mice under pathophysiological conditions and this loss of autophagy resulted in increased susceptibility to injury in models of glomerular disease (Renal Week 2009, San Diego, CA, F-FC 275).
In conclusion, utilizing CYP2B1 gene specific silencing we have confirmed that CYP2B1 plays a crucial role as the primary site for the generation of ROS. Furthermore, we have shown that PAN acts at the level of CYP2B1 protein to increase production of ROS and induce degradation of CYP2B1 protein with the release of heme and iron. The free iron participates in the OH· generation while the heme causes induction of HO-1. The resultant cytotoxicity and cell death is mediated through an ER stress-associated apoptotic pathway. These findings may facilitate the identification of specific CYP forms in human kidney that exert functions similar to CYP2B1 and thus offer a valuable therapeutic target in the treatment of minimal change disease in humans.