P0975EFFECT OF REGULATED INTRAMEMBRANE PROTEOLYSIS ON MEGALIN EXPRESSION DURING OXIDATIVE STRESS EXPOSURE
Authors: Kurosaki Yoshifumi , Ikarashi Futoshi , Imoto Akemi , Kawakami Fumitaka , Yokoba Masanori , Takenaka Tsuneo , Katagiri Masato , Ichikawa Takafumi , Nielsen Rikke , Ishii Naohito 
Published: June 2020

Abstract

Background and Aims
Megalin, an endocytic receptor in proximal tubular cells, plays a critical role in renal tubular protein reabsorption. We previously reported that oxidative stress induced the temporally increase in renal megalin expression through the PI3K/AKT signaling pathway, but that megalin elevation is normalized or decreased during long term exposure to oxidative stress (hydrogen peroxide). However, the underlying mechanisms are unclear. Studies have addressed that megalin is subjected to regulated intramembrane proteolysis (RIP). Intracellular megalin COOH-terminal fragment (MCTF) is produced by protein kinase C-regulated, metalloprotease-mediated ectodomain shedding and further cleavage by gamma-secretase to produce the soluble megalin intracellular domain (MICD). The MICD in turn translocates to the nucleus where it decreases expression of the Lrp2 gene encoding megalin. In the present study, we evaluated the effect of megalin RIP on the oxidative stress-regulated megalin expression.

Method
HK-2 cells were cultured with hydrogen peroxide (0.4 mmol/l) for 4.5 or 24 h, followed by treatment with gamma-secretase inhibitor, Compound E (5 mmol/L) or PKC activator, Phorbol 12-myristate 13-acetate (PMA, 0.5 mmol/L). Megalin expression was determined by performing western blotting or real-time PCR. The MCTF in medium was detected by western blotting. In animal experiments, Sprague-Dawley rats were randomly divided into two groups (n = 5): (i) STZ group (diabetic phenotype induced by streptozotocin administration) and (ii) sham group (vehicle). Urine was collected at two weeks after STZ administration, and the excretion of MCTF in urine was analyzed.

Results
Treatment of HK-2 cells with hydrogen peroxide (0.4 mmol/L) significantly increased megalin protein and mRNA levels at 4.5 h. Pretreatment of Compound E showed further increase in megalin expression in hydrogen peroxide-exposed cells. It was also found that presenilin-1 and -2, which are components of gamma-secretase, double knockdown with siRNA increased megalin expression in hydrogen peroxide treated-cells. On the other hand, PMA treatment inhibited the increase in both megalin protein and mRNA levels. In the cells treated with hydrogen peroxide for 24 h, megalin mRNA levels were normalized, but pretreatment of Compound E kept the elevation in megalin mRNA levels at 24 h after the treatment with hydrogen peroxide. Interestingly, megalin MCTF in the medium was increased by hydrogen peroxide treatment in a dose-dependent manner. Furthermore, megalin MCTF excretion in the urine of STZ-induced diabetes was significantly increased compared to sham rats.

Conclusion
These results suggested that oxidative stress-induced megalin upregulation was inhibited by RIP activation of megalin, suggesting that megalin RIP plays a role as a negative feedback system to oxidative stress-induced megalin upregulation. Furthermore, our data indicate that oxidative stress induces urinary excretions of MCTF in diabetic rats during the normoalbuminuric stage and potentially act as a marker of diabetic kidney disease.