Morphine has been reported to cause kidney cell injury in both in vitro
and in vivo
. In the current study, we observed that administration of morphine contributed to albuminuria which appeared to be glomerular in origin and a consequence of the loss of slit diaphragm integrity 
. Both in vivo
and in vitro
data confirmed that morphine inflicted podocyte injury in the form of attenuated expression of SDCMs ( and ); these findings further confirmed that loss of integrity of SD contributed to albuminuria in morphine-receiving mice. Since these SDCMs are actin-associated molecules, the reduced level of expression of these molecules by morphine may change the actin cytoskeleton organization. Transmission electron microscopy of the mouse kidney sections showed effacement of podocyte foot process in morphine-receiving mice (), which might be due to the change of the actin cytoskeleton organization. However, further studies are needed to explore this aspect.
In previously reported studies, morphine has been demonstrated to exert bimodal effects (both apoptosis and proliferation) on glomerular epithelial cells 
; however the role of opiate receptors was not evaluated in these studies. In the current study, we found that KOR and MOR, but not DOR, were expressed in both human and mouse podocytes ( and ). We also found that stimulating either KOR or MOR in podocytes could activate related kinases and down-regulate SDCMs. To our knowledge, this is the first report on the opiate receptors in podocytes. Weber et al
reported that although both KOR and MOR were expressed in glomerular mesangial cells, only KOR played the function to activate STAT3 and led to the proliferation and glomerulopathy 
. These findings indicate that podocytes behave differently from mesangial cells in morphine milieu.
Oxidative stress is a common cause of cellular injury. Several investigators have demonstrated that enhanced ROS generation leads to albuminuria by damaging SD components 
. Morphine has been demonstrated to stimulate the production of superoxide by macrophage and mesangial cells 
. As we previously reported, generation of ROS induces apoptosis of rat glomerular epithelial cells 
. Here we also found that morphine increased ROS generation in human podocytes in a dose-dependent manner (). Addition of H2
to the media decreased the expression of SDCMs (), while free radical scavengers prevented this damaging effect of morphine (). All these findings strongly suggest the role of ROS in morphine-induced down-regulation of SDCMs. One potential possibility is that ROS induced injury hampers the expression of SDCMs. Further investigation into the detailed underlying mechanisms need to be carried out in future studies.
Investigating the kinase or transcription factor pathways involved in morphine-induced kidney injury may provide insight into new potential targets for therapy. STAT3, AKT and mitogen-activated protein (MAP) kinases, including ERK1/2, JNK, and p38, have been implicated in podocyte injury and the progression of chronic kidney diseases (CKD) 
. All these kinases or transcription factors may also be activated by morphine in various cells 
. We examined the effect of morphine on the activation of these kinases and factors in podocytes. Our results revealed that morphine stimulated the phosphorylation of AKT, JNK and p38 (), but could not activate Erk1/2 or STAT3 (data not shown). These observations were further confirmed by the use of KOR and MOR specific agonists on human podocytes in morphine milieu (data not shown).
Takano et al
reported that activation of AKT suppressed the expression of nephrin 
; Ikezumi et al
claimed that activation of JNK or p38 decreased the expression of nephrin and podocin, while inhibiting these kinases restored their expressions 
. Consistent with these reports, we also found that inhibiting AKT, JNK, and p38 could partially prevent the morphine-induced decrease of SDCMs (), indicating the regulation of these three kinases in morphine-induced SD damage.
In conclusion, we have demonstrated that morphine has the potential to directly impair the SDCMs in podocytes, which will contribute to acute kidney injury. The effects of morphine on podocytes are mediated through both MOR and KOR. These impairments are through generation of ROS, and are regulated by AKT, JNK, and p38 pathways. Our study provides insight into new mechanisms involved in morphine-induced podocyte damage, and highlights some new therapeutic targets for morphine induced kidney injury.