Geliştirilen Üç Boyutlu Böbrek Proksimal Tübül Modelinde Nefrotoksisite Değerlendirmesi

Abstract

Acute kidney injury (AKI) is an important health problem causes the death of 2 million people worldwide every year with high morbidity and mortality. One of the important causes of AKI is drug-induced nephrotoxicity. After organ transplantation, nephrotoxic effect of tacrolimus, which is used as an immunosuppressant treatment, limits its clinical use. In modeling nephrotoxicity that primarily targets the proximal tubule (PT) epithelium, two-dimensional in vitro models can’t provide microenvironment, polarity, cell-cell interactions and tubular flow, therefore three-dimensional (3D) ex vivo models are needed. It was assumed that the real-time antiproliferative dose of tacrolimus on human proximal tubule epithelial cells (PTEC) can determined and the effects of the drug could be evaluated structurally and functionally on the 3D-PT model developed with bioengineering approaches in ex vivo conditions. To determine the antiproliferative effective dose (ED50), 1x10-1, 1, 1x101, 1x102, 1x103, 1x104 µg/ml concentrations of tacrolimus were applied to human PTEC’s (HK-2) and evaluated by real-time impedance-based proliferation analysis. In order to obtain the 3D-PT model, a platform was produced with 3D printing techniques while a perfusable channel was made and HK-2 cells were cultivated into the channel. Additionally, a 3D-PT model was created on a commercial microfluidic culture platform, and perfusion provided with an intermittent release device after HK-2 cells were cultivated. In the control and tacrolimus-treated experimental groups, ZO-1 and acetylated-α-tubulin immunolabeling, cell viability, albumin reuptake function and barrier integrity were evaluated comparatively in 3D-PT model which created on the microfluidic culture platform. As a result of real-time proliferation analysis, the ED50 of tacrolimus in HK-2 cells was 44.69 µg/ml at 48th hour. The canal designed, created and perfusion was achieved on the platform produced by 3D printing, but HK-2 cells didn’t show any attachment tendency in the canal. In the microfluidic culture platform, a 3D-PT model with perfusion was successfully created. Tacrolimus significantly decreased cell viability in static conditions, 3D-PT model. When the control and tacrolimus-treated experimental groups were compared in the 3D-PT model on the microfluidic culture platform, it was shown that tacrolimus disrupted the 3D-PT structure by reducing ZO-1 and acetylated-α-tubulin immunolabeling. Compared with the control group in the 3D-PT model, tacrolimus impaired the albumin reuptake function. Tacrolimus increased the permeability by disrupted the barrier integrity of PT epithelium, to 155 kDa dextran molecule 2.3-fold and to 20 kDa dextran molecule 1.4-fold compared to the control group. As a result, the real-time ED50 of tacrolimus in HK-2 cells was calculated and a 3D-PT model was created in the perfusable microfluidic plate, and the effect of tacrolimus on 3D-PT was evaluated structurally and functionally under ex vivo conditions.