Induction of epithelial-to-mesenchymal transition in proximal tubular epithelial cells on microfluidic devices

doi:10.1016/j.biomaterials.2013.10.070

Biomaterials

Volume 35, Issue 5, February 2014, Pages 1390-1401

https://doi.org/10.1016/j.biomaterials.2013.10.070 Get rights and content

Abstract

In proteinuric nephropathy, epithelial-to-mesenchymal transition (EMT) is an important mechanism that causes renal interstitial fibrosis. The precise role of EMT in the pathogenesis of fibrosis remains controversial, partly due to the absence of suitable in vitro or in vivo models. We developed two microfluidic and compartmental chips that reproduced the fluidic and three-dimensional microenvironment of proximal tubular epithelial cells in vivo. Using one microfluidic device, we stimulated epithelial cells with a flow of healthy human serum, heat-inactivated serum and complement C3a, which mimicked the flow of urine within the proximal tubule. We observed that epithelial cells exposed to serum proteins became apoptotic or developed a mesenchymal phenotype. Incubating cells with C3a induced similar features. However, cells exposed to heat-inactivated serum did not adopt the mesenchymal phenotype. Furthermore, we successfully recorded the cellular morphological changes and the process of transmigration into basement membrane extract during EMT in real-time using another three-dimensional microdevice. In conclusion, we have established a cell-culture system that mimics the native microenvironment of the proximal tubule to a certain extent. Our data indicates that EMT did occur in epithelial cells that were exposed to serum proteins, and C3a plays an essential role in this pathological process.

Introduction

Persistent proteinuria is an independent risk factor for the progression to end-stage renal disease in a variety of primary glomerular diseases, partly due to causing renal interstitial fibrosis [1], [2]. Among the mechanisms responsible for this process, epithelial-to-mesenchymal transition (EMT) has become a popular hypothesis [3]. The primary events of EMT include the adoption of a mesenchymal-like cellular phenotype and migration through the tubular basement membrane toward the tubular interstitium [4]. Recently, however, the EMT hypothesis has been questioned as the results of genetic fate mapping studies have been published [5], [6]. Taking advantage of this technology, genetically labeled epithelial cells could not be detected in the interstitium of mouse models of fibrosis. In contrast, primary renal epithelial cells incubated with TGF-β in vitro from these transgenic mice still demonstrated characteristics of EMT [7]. Importantly, no suitable experimental model is yet available that would enable the process of EMT to be monitored both temporally and spatially. The existing in vivo models cannot provide direct and real-time observations of EMT, and routinely used in vitro models lack the microenvironment that exists around proximal tubular epithelial cells (PTECs), and therefore cannot accurately reflect the pathological progress in vivo.

Recent technological advances have lead to great progress in the field of biomimetics [8], [9]. For example, microfluidics bring new opportunities for spatial and temporal control of cell growth and stimuli, and microfabricated devices have been used to facilitate basic research concerning the biology of cells [10], [11], [12], [13], [14]. The successful reconstitution of organ level lung tissue on a microfluidic device indicates that biomimetic microsystems might potentially serve as a replacement for animal testing [13].

Using microfluidic technology, we successfully established two devices that imitate the microenvironment of renal proximal tubular epithelia to investigate the process of EMT in detail. One unique environment of the renal tubular epithelia in vivo is that their brush borders are washed with a stimulus of flowing urine. Hence, this study established a two-dimensional (2D) integrated cell culture device to stimulate PTECs using the flow of liquid.

It has been established that transforming growth factor β1 (TGF-β1) is a strong fibrogenic cytokine [15]. Our previous work has shown that TGF-β1 alone can induce EMT of human PTECs (HK-2) in vitro [16]. Thus we chose TGF-β1 as a stimulator and successfully induced EMT of HK-2 cells, indicating the feasibility of our approach to study EMT of PTECs.

Previous studies of renal interstitial fibrosis usually focused on single biological factors, such as albumin or complement [17], [18]. However, it should be noted that in many human proteinuria renal diseases, various factors that include albumin, globulin, complement, TGF-β1 and other cytokines, leak through injured glomeruli into the tubules. Therefore, in this present study, low concentrations of healthy human serum (HHS), rather than albumin or complement alone, were used to approximate the initial proteinuria. Although some studies have shown that complement C3a is an important factor that promotes the progression of renal interstitial fibrosis [18], it remains unclear whether C3a plays a key role as one of the components of proteinuria. We therefore used HHS, heat-inactivated HHS and C3a as different inducers of EMT to investigate their effect on phenotypic changes and apoptosis of HK-2 cells.

Renal tubular epithelia consist of a single layer of cells that are connected to each other by a girdle of intercellular junctions on the apical side and are attached to a tubular basement membrane on the basal side. As such, we next developed a three-dimensional (3D), compartmental microdevice to simulate the microstructure of PTECs and interstitial environment in vivo. The EMT hypothesis supposes that PTECs damage the integrity of the tubule basement membrane and migrate toward the interstitium [4]. However, no model exists that can visualize the process. We treated HK-2 cells with TGF-β1, C3a and heat-inactivated HHS to verify the process of PTECs horizontally migrating across the basement membrane extract (BME) without the interference of gravitational force.

In this study, we established two biomimetic microdevices to investigate dynamic changes of cellular phenotype and migratory behavior during EMT of HK-2 cells. This culture system provided significance information regarding the role of complement C3a to mediate the progression of EMT.

Section snippets

Microfluidic cell culture system design and fabrication

The schematic of the proximal tubular fluid microenvironment device is shown in Fig. 1A. This device is composed of two layers, in which the top polydimethylsiloxane (PDMS) layer contained twelve microchannels. Each channel includes three cell culture chambers and a fluid buffer area, fabricated in PDMS using a rapid prototyping technique [19]. Each unit includes one inducer input, and a winding channel followed by three cell culture chambers. The winding channel is sufficient to avoid cells

Construction of a two-dimensional microfluidic device

Microfluidic devices with twelve cell culture units were prepared and loaded with HK-2 cells (Fig. 1). Each unit was designed to connect with a common output that was linked to the syringe pump to apply a medium flow rate. Our data showed that they did not interfere with each other (Fig. 1A and B). Not only HK-2 cells could grow in the flowing culture media, but the system also facilitated the treatment of cells with multiple stimuli simultaneously. The fluid in channels could reproduce the

Discussion

EMT is an orchestrated, highly regulated process that consists of four key steps: loss of epithelial cell adhesion, de novo α-SMA and FSP-1expression, disruption of the tubular basement membrane, and enhanced cell migration and invasion [27]. Recently, the occurrence of EMT during chronic renal tubulointerstitial fibrosis was called into question, partly because conventional in vivo models and technology limit the observation of the whole process to be visualized and observed in real-time [28].

Conclusions

In this study, we used two microdevice cell culture systems to monitor the process of EMT. We observed HK-2 cells transmigrating across basement membrane extracts accompanied with phenotypic changes from an epithelial to myofibroblastic phenotype. Our devices provide new insights into the complex and dynamic interactions among PTECs, proteinuria and tubular basement membranes in vitro. We interpret our data to directly confirm that PTEC transition is an important source of myofibroblasts

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (NSFC) (No. 81070564 to H. Lin), the National Basic Research Program of China (No. 2011CB944000 to H. Lin), the Knowledge Innovation Program of the Chinese Academy of Sciences (KJCX2-YW-H18 to J. Qin), and the National Natural Science Foundation of China (No. 81201689 to J. Qin).

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Induction of epithelial-to-mesenchymal transition in proximal tubular epithelial cells on microfluidic devices

Abstract

Introduction

Section snippets

Microfluidic cell culture system design and fabrication

Construction of a two-dimensional microfluidic device

Discussion

Conclusions

Acknowledgments

Am J Pathol

Am J Pathol

J Biol Chem

Exp Toxicol Pathol

Am J Kidney Dis

Epithelial-mesenchymal transdifferentiation of renal tubular epithelial cells induced by urinary proteins requires the activation of PKC-alpha and betaI isozymes

Cell Biol Int

Slowing nephropathy progression: focus on proteinuria reduction

Clin J Am Soc Nephrol

Mechanisms of disease: fibroblasts–a new look at an old problem

Nat Clin Pract Nephrol

The basics of epithelial-mesenchymal transition

J Clin Invest

The origin of interstitial myofibroblasts in chronic kidney disease

Pediatr Nephrol

The origins and the future of microfluidics

Nature

Cells on chips

Nature

Microscale culture of human liver cells for drug development

Nat Biotechnol

A microfluidic device for characterizing the invasion of cancer cells in 3-D matrix

Electrophoresis

Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device

Lab Chip

Reconstituting organ-level lung functions on a chip

Science

Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers

Lab Chip

Blocking core fucosylation of TGF-beta1 receptors downregulates their functions and attenuates the epithelial-mesenchymal transition of renal tubular cells

Am J Physiol Renal Physiol