Background: Type 2 diabetes, characterised by insulin resistance, is an everincreasing problem in the world. Diabetic nephropathy is a renal microvascular complication of diabetes that is a common cause of end-stage renal disease worldwide. Low levels of albumin can be detected in the urine at an early stage of diabetic nephropathy, and more severe albuminuria develops as the disease progresses. Insulin resistance is associated with albuminuria in patients with type 1 or type 2 diabetes. Also, the loss of podocytes plays a major role in the pathogenesis of diabetic nephropathy. The mechanisms underlying the development of insulin resistance and apoptosis in podocytes are still not fully understood. This study aimed to investigate the pathophysiological mechanisms leading to insulin resistance and podocyte apoptosis at the molecular level, and concentrated to the roles of CD2-associated protein (CD2AP) and SH2-domain containing 5’ inositol phosphatase 2 (SHIP2) in these processes. Results: We found that the lack of CD2AP in podocytes led to attenuated glucose uptake, without affecting PI3K-AKT-mediated insulin signalling. This led us to investigate the role of CD2AP in glucose transporter trafficking. Indeed, live-cell imaging revealed that internalized Glucose transporter 4 (GLUT4) was trapped in the perinuclear region of podocytes lacking CD2AP. CD2AP co-fractionated with known components of GLUT4 vesicles and interacted with clathrin and clathrin adaptor GGA2. These results suggest that CD2AP plays a role in GLUT4 vesicle trafficking. We further observed that the level and activity of SHIP2, an interaction partner of CD2AP, was increased in the absence of CD2AP in podocytes. Also, production of reactive oxygen species (ROS) and the rate of apoptosis were increased when CD2AP was lacking from podocytes. We hypothesized that inhibition of SHIP2 would decrease the production of ROS and apoptosis, but even though we detected reduced ROS production, inhibition of SHIP2 increased podocyte apoptosis in the absence of CD2AP. In the search for novel SHIP2 inhibitors we found an old anti-diabetic drug, metformin, to bind to and inhibit the activity of SHIP2 in silico, in vitro and in vivo. By inhibiting the activity of SHIP2, metformin increased glucose uptake and protected podocytes from apoptosis initiated by SHIP2 overexpression. SHIP2 exhibited higher activity in the kidney pieces received from nephrectomy patients with type 2 diabetes receiving non-metformin medication compared to nephrectomy patients without diabetes; in comparison, the activity of SHIP2 did not differ between metformin receiving patients with type 2 diabetes and people without diabetes. Patients with type 2 diabetes with metformin medication also showed reduced podocyte loss. Conclusions: In the light of our results we suggest a novel role for CD2AP in the regulation of sorting of internalized GLUT4 and regeneration of insulin responsive GLUT4 vesicle compartment. We also suggest that SHIP2 inhibitors, including metformin, can be used to reduce the oxidative stress of podocytes and to prevent podocyte loss, except in the case of patients suffering from reduced expression of CD2AP.
|Tilldelningsdatum||18 dec 2017|
|Status||Publicerad - 18 dec 2017|
|MoE-publikationstyp||G5 Doktorsavhandling (artikel)|
Bibliografisk informationM1 - 95 s. + liitteet
- 3111 Biomedicinska vetenskaper