Kidney Res Clin Pract.  2018 Sep;37(3):210-221. 10.23876/j.krcp.2018.37.3.210.

Puromycin aminonucleoside triggers apoptosis in podocytes by inducing endoplasmic reticulum stress

• Min SY
• Ha DS
• Ha TS

• KMID: 2447160
• DOI: http://doi.org/10.23876/j.krcp.2018.37.3.210

Abstract

BACKGROUND
Puromycin aminonucleoside (PAN) is a known podocytotoxin. PAN-induced nephrosis is a widely used animal model for studying human idiopathic nephrotic syndrome. Abnormal protein accumulation associated with podocyte-specific endoplasmic reticulum (ER) stress damages cells structurally and functionally, which in turn induces apoptosis and severe proteinuria. In the present study, we investigated the effect of PAN on ER stress and apoptosis in podocytes in vitro.
METHODS
Mouse podocytes were cultured and treated with various concentrations of PAN. ER stress markers were then evaluated by western blotting, and apoptosis was evaluated by fluorescence-activated cell sorting (FACS) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays.
RESULTS
PAN treatment increased ER stress markers such as activating transcription factor (ATF) 6α and caspase-12 in a dose-dependent manner at 12 and 24 hours, respectively. These markers were reduced by chemical chaperones, such as sodium 4-phenylbutyric acid and tauroursodeoxycholic acid. PAN treatment also increased 78 kD glucose-regulated protein (GRP78)/binding immunoglobulin protein (BiP) at the earlier stage of 12 hours. PAN significantly induced podocyte apoptosis in concentration- and time-dependent manners, as seen using FACS and TUNEL assays. This result was improved by Nox4 siRNA, ATF6 siRNA, and chemical chaperones. LY294002, a PI3-kinase inhibitor, significantly boosted ER stress and apoptosis. PAN-induced ER stress increased oxidative stress and subsequently induced apoptosis, and could be mitigated by inhibition of PI3-kinase signaling.
CONCLUSION
Our findings suggest that PAN induces ER stress in podocytes mainly through the GRP78/BiP, ATF6α, and caspase-12 pathways, which trigger apoptosis via induction of oxidative stress. This stress is mitigated by inhibiting PI3-kinase signaling.

Keyword

Apoptosis; Endoplasmic reticulum stress; Podocyte; Proteinuria; Puromycin aminonucleoside

MeSH Terms

Animals
Apoptosis*
Blotting, Western
Caspase 12
DNA Nucleotidylexotransferase
Endoplasmic Reticulum Stress*
Endoplasmic Reticulum*
Flow Cytometry
Humans
Immunoglobulins
In Situ Nick-End Labeling
In Vitro Techniques
Mice
Models, Animal
Nephrosis
Nephrotic Syndrome
Oxidative Stress
Phosphatidylinositol 3-Kinases
Podocytes*
Proteinuria
Puromycin Aminonucleoside*
Puromycin*
RNA, Small Interfering
Sodium
Transcription Factors
Caspase 12
DNA Nucleotidylexotransferase
Immunoglobulins
Phosphatidylinositol 3-Kinases
Puromycin
Puromycin Aminonucleoside
RNA, Small Interfering
Sodium
Transcription Factors