The main function of the glomeruli is to filter fluids and electrolytes from the blood, while retaining plasma proteins. This activity happens at the level of the glomerular filtration barrier (GFB) and is coordinated by the interaction of two highly specialized glomerular cells (the fenestrated endothelium and the podocytes), which are separated by a thin layer of glomerular basement membrane (GBM4). Here, we describe a glomerulus-on-a-chip constituted by human podocytes and human glomerular endothelial cells (hGEC).
- Formation of 40 proximal glomerular filtration units in the OrganoPlate®
- Multi-layered structure without artificial membranes in between
- Glomerular cells generate layer of extracellular matrix composed by collagen IV trimer and laminin
- Recapitulates function of the GFB, including selective permeability and response to nephrotoxic compounds
- Responds to serum from individuals affected by different glomerular diseases, including membranous nephropathy
- Real-time readout of loss barrier 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® (figure 1a). An extracellular matrix (ECM) gel was injected to the middle lane of the 3-lane chip. Podocytes were seeded in channel C. After 24 h, human glomerular endothelial cells were added to the same channel C. After 24 h, the chip was placed under flow conditions on the OrganoFlow® platform. The two co- seeded cell types showed the ability to form clearly distinguishable layers, with selective expression of nephrin and CD31 (Figure 1b). Importantly,, the layers showed de novo deposition of glomerular matrix components collagen IV (COL4α3) and LAMA5 (Figure 1c-e), thus demonstrating that the model resembles the in vivo glomerular filtration barrier.
One of the most important characteristics of the glomerular filtration barrier is permselectivity, i.e. the capacity to filter molecules based on their size. Albumin is the most abundant protein in human plasma and under physiological conditions is retained within the bloodstream. Leakage of albumin in the urine is considered a sign of kidney dysfunction, and its levels (albuminuria) correlate with the severity of glomerular injury in mice and humans. Permselectivity was tested by adding a physiological concentration of FITC-conjugated albumin to the media in channel C. Podocytes (derived from 3 different sources hAKPC, hiPOD and hpPOD) in conjunction with the glomerular endothelium prevented albumin leakage at 5 and 60 min (Fig. 2a–c). For controls, the same experiment was repeated with (1) podocytes + human lung endothelial cells (HuLECs) (as negative control for glomerular endothelium (hGEC ,Fig. 3d), (2) human fibroblasts (as negative control for podocytes) + hGEC (Fig. 3e), or (3) devoid of cells (Fig. 3f). In all these conditions, FITC-conjugated albumin easily filled all three channels at both 5 and 60 min (Fig. 3d–g).
The glomerulus on a chip was exposed to sera from individuals affected by membranous nephropathy, a major cause of nephrotic syndrome in adults. After 24 h of exposure to serum from membranous nephropathy patients or healthy controls (Figure 3a), the chips with MN serum, but not control sera, showed an increase in albumin leakage, and a statistically significant loss of permselectivity (Figure 3b). The extent of albumin leakage (proteinuria) in our chips highly correlated with proteinuria measured in the same patients (R = 0.8901, p < 0.01, with a confidence of at least 95%, Figure 3c).
This project was fully performed at Children’s Hospital Los Angeles, by Laura Perin and Stefano Da Sacco and colleagues. Source: Nature communications, (2019) 10:3656