A human proximal tubule-on-a-chip to study renal disease and toxicity

Abstract

Renal disease is a global problem with unsustainable health-care costs. There currently exists a lack of accurate human renal disease models that take into account the complex microenvironment of these tissues. Here, we present a reusable microfluidic model of the human proximal tubule and glomerulus, which allows for the growth of renal epithelial cells in a variety of conditions that are representative of renal disease states including altered glomerular filtration rate, hyperglycemia, nephrolithiasis, and drug-induced nephrotoxicity (cisplatin and cyclosporine). Cells were exposed to these conditions under fluid flow or in traditional static cultures to determine the effects of a dynamic microenvironment on the pathogenesis of these renal disease states. The results indicate varying stress-related responses (α-smooth muscle actin (α-SMA) expression, alkaline phosphatase activity, fibronectin, and neutrophil gelatinase-associated lipocalin secretion) to each of these conditions when comparing cells that had been grown in static and dynamic conditions, potentially indicating more realistic and sensitive predictions of human responses and a requirement for a more complex "fit for purpose" model.

FIG. 1.

(a) Device configuration highlighting the glomerular filter attachment. Device cross section is shown to highlight tubular and vascular compartments. (b) NGAL secretion (n = 3), and (c) fibronectin secretion (n = 3) normalized total protein. Unpaired Student’s t-tests were used to determine the significance between conditions (*p < 0.05).

FIG. 2.

Effects of shear stress on α-SMA expression. Cells were seeded into microfluidic devices and exposed to steady shear stress for 2 h. Cells were fixed and stained for α-SMA expression following (a) static, (b) physiological (0.8 dyn/cm²), and (c) high (5 dyn/cm²) steady shear stress. Scale bars = 100 μm. The results indicate a correlation between the amplitude of shear stress and the degree of α-SMA expression. (d) Quantitative results of α-SMA expression under increasing shear rates. The results are shown as % positive staining per field, mean ± SEM, n = 6. Unpaired Student’s t-tests were used to compare differences between means. **p < 0.01, ***p < 0.005, ****p < 0.001.

FIG. 3.

Effects of high glucose exposure on renal distress markers. HK-2 cells were adapted to low glucose (8mM) culture medium for 3 passages and then exposed to high glucose (30mM) medium for 12 h. (a) α-SMA protein (n = 9), (b) NGAL (n = 3), (c) Fibronectin (n = 3), and (d) alkaline phosphatase (AP) (n = 3) activity. Under fluidic conditions, cells were exposed to 0.2 dyn/cm² steady shear stress. The results are shown as mean ± SEM. An unpaired Student’s t-test was used to compare differences between means (*p < 0.05, **p < 0.01, ****p < 0.001).

FIG. 4.

Effects of calcium oxalate monohydrate (COM) exposure on renal distress markers. Static and fluidic (0.8 dyn/cm²) cultures were exposed to crystals for 2 h. (a) Scanning electron microscopy (SEM) image of calcium oxalate crystals demonstrating a diameter of approximately 3 μm. Scale bar = 1 μm. (b) Expression of α-SMA (results are shown in % positive staining for α-SMA per field, n = 4). (c) NGAL secretion (n = 3), (d) Fibronectin (n = 3), and (e) alkaline phosphatase (AP) (n = 4). Unpaired Student’s t-tests were used to determine the significance between treatments and controls (*p < 0.05, **p < 0.01).

FIG. 5.

Effects of pharmaceutical compounds on cell viability. (a) 24 h after seeding, cells in static cultures were exposed to cisplatin, cyclosporine, or vehicle control and then stained with calcein-AM live stain to determine the effects on cell viability. The results show mean ± SEM, n = 3 using a one-way ANOVA with Tukey’s post hoc testing. (b) Static and fluidic (0.2 dyn/cm²) cultures were exposed to (c) and (d) vehicle, (e) and (f) cyclosporine (0.33 mg/ml), or (g) and (h) cisplatin (0.414 mg/ml). NGAL and fibronectin concentrations were measured in the medium from plates and the effluent from devices and are shown as mean ± SEM, unpaired Student’s t-tests were used to compare controls and treatments (*p < 0.05, **p < 0.01, ***p < 0.005).