Kidney Proximal Tubule
Kidney: Perfused proximal tubule
The proximal tubule is a component of the kidney nephron that plays a crucial role in reabsorbing salt, water, and organic solutes such as glucose from the glomerular filtrate as well as eliminating endogenous and exogenous waste products from the body. The active transport across the epithelial barrier as well as increased concentration of xenobiotics, make the proximal tubule sensitive to drug-induced toxicity. The proximal tubule is furthermore a segment of the kidney that gets damaged in a range of diseases including renal ischemia and poly cystic kidney disease. Here we show how the proximal tubule gets modulated in the OrganoPlate®.
Formation of 40 proximal tubules in the OrganoPlate®
Real-time readout of loss barrier function
No artificial membranes; cells are directly against the extracellular matrix
Transport activity assays show transporter function
For all experiments an OrganoPlate® 3-lane was used. The top part of this plate is a standard 384 well microtiter plate with a modified glass bottom with 40 microfluidic chips embedded. Each chip consists of three channels separated by ridges, the phaseguides. An extracellular matrix (ECM) gel was injected to the middle lane of the 3-lane chip. Human proximal tubule cells were seeded to against the extracellular matrix gel. The plate was placed on the OrganoFlow® platform enducing flow though the channels by passive leveling. Tubes were formed within six days (Figure 1b), having a perfusable lumen that show primary cilia (Figure 1c) and brush borders (Figure 1d).
Cells were exposed to concentration ranges of the nephrotoxicant cisplatin. Leakage of a FITC labeled dye allows to probe the integrity of the barrier function (Figure 2a-b.) Barrier integrity corresponds to cell viability and Lactate Dehydrogenase (LDH) release.
Transporter activity can be demonstrated as shown in Figure 3 for the P-glycoprotein (P-gp) transporter. Calcein-AM was perfused through the lumen of the proximal tubule and taken up by the cells, making the cells green-fluorescent. In presence of a P-gp inhibitor, cyclosporin A, P-gp efflux of calcein is reduced, resulting in accumulation of green-fluorescent signal inside the cells (figure 3c).