Microphysiological Systems To Recapitulate The Gut–Kidney Axis
Mar 18, 2022
Laura Giordano,1,3 Silvia Maria Mihaila,1,3 Hossein Eslami Amirabadi,1,2 and Rosalinde Masereeuw
Chronic kidney disease (CKD) typically appears alongside other comorbidities, highlighting underlying complex pathophysiology that is thought to be vastly modulated by the bidirectional gut–kidney crosstalk. By combining advances in tissue engineering, fabrication, microfluidics, and biosensors, micro physiological systems (MPSs) have emerged as promising approaches for emulating the in vitro interconnection of multiple organs, while addressing the limitations of animal models. Mimicking the (patho)physiological states of the gut–kidney axis in vitro requires an MPS that can simulate not only this direct bidirectional crosstalk but also the contributions of other physiological participants such as the liver and the immune system. We discuss recent developments in the field that could potentially lead to in vitro modeling of the gut–kidney axis in CKD.
Contact: joanna.jia@wecistanche.com
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Chronic Kidney Disease: A Metabolic Disorder with Disrupted Inter-Organ and Inter-Organismal Signaling
Chronic kidney disease (CKD) is the most widespread kidney disease and is characterized by the gradual loss of organ function over time, which impairs the ability to filter metabolic waste products from the blood (Box 1). The kidneys have many highly specialized functions, such as blood filtration and active secretion for the removal of metabolic waste, reabsorption of essential nutrients, maintenance of blood volume and electrolyte homeostasis, and metabolic and endocrine activity [1].
The complex and enigmatic pathophysiology of CKD is thought to be modulated by kidney crosstalk with multiple organs and systems, particularly via bidirectional inter-organ communication with the gastrointestinal tract, referred to as the gut–kidney axis[2]. The human gut accommodates a complex community of microbes that live in a commensal relationship with their host [3] and provide significant and unique contributions to the human metabolome (see Glossary). In symbiosis, intestinal absorption ensures the uptake of beneficial microbial metabolites, whereas the kidneys maintain homeostasis by excreting potentially toxic metabolic end-products. Conversely, kidney failure results in the accumulation of gut microbiota-derived metabolites (i.e., uremic toxins) leading to the development of the uremic syndrome. This complication contributes to gut dysbiosis that adversely affects the inflammatory, endocrine, and neurological pathways involved in CKD onset and progression (Box 2 and Figure 1)[4]. Overall, CKD can be viewed as a metabolic disorder that reflects disrupted inter-organ and inter-organismal flow of metabolites and signaling molecules accompanied by overactivation of the immune system (Figure 2). Accordingly, the central role of gut–kidney remote signaling via uremic toxins [5] raises the need to further characterize the gut metabolome in CKD.
Traditionally, kidney disease research has largely relied on clinical [6] and animal studies [7] that offer limited control over experimental parameters and have high inter-species variability. Owing to the lack of suitable in vitro experimental models, there is currently an imperative need for cell culture systems that can capture the different aspects of in vivo organ function through the use of highly controlled and specialized culture microenvironments, including 3D scaffolds and microfluidics [8]. Given the advances in multicellular culturing and biomanufacturing, the integration of real-time monitoring features, and the independent control of experimental parameters, capturing the complexities of human physiology in vitro is certainly in sight. We provide a comprehensive overview of the most emblematic and recent advances in the field of 3D in vitro models and highlight their relevance for the development of a 3D bidirectional gut–kidney axis system. We also discuss the major hurdles that these entail, how to overcome them, and offer new insights into the current direction of this field in the context of CKD.
Microphysiological Models to Unravel Complex Organ Interconnections
The advent of micrography sociological systems (MPSs), also referred to as organ-on-chips (OOCs), has created novel possibilities to study the physiological processes involved in individual organs and inter-organ crosstalk. A range of different multi-MPSs is emerging that underpin a 'physiome-on- a-chip' approach for simulating the functional units of organs, as well as crosstalk between them, instead of aiming to reproduce the complete organ(s). From a technical standpoint, MPSs often consist of single or multiple microfluidic channels with cross-sectional dimensions of hundreds of micrometers in which small volumes (nanoliters to microliters) are (re)circulated. By ensuring close contact between the cells, these volumes allow dynamic cell-cell interplay to be captured while ensuring minimal reagent consumption and compound dilution [9,10]. Added laminar flow can continuously supply cells with fresh nutrients and oxygen while simultaneously removing waste products, and can generate accurate spatiotemporal chemical and mechanical gradients in their vicinity [11]. Physical isolation of different tissue analogs is achieved through compartmentalization into microchannels separated by thin porous membranes or layers of extracellular matrix (ECM) [12].
The creation of the lung-on-a-chip platform, in which mechanical strain and multiple cell types were combined to mimic the respiration of the lung, pioneered the development of biologically inspired MPSs [13]. Since then, progress in microfluidic handling has made it possible to connect different organ models and control their crosstalk within one or multiple devices [14,15]. The latest developments in the field prompt us to discuss these advances in relation to modeling the gut–kidney axis in CKD and explore the requirements of MPSs for supporting inter-organ communication, also taking into consideration the synergistic approach of combining them within silico models.
En Route to Replicating the Gut–Kidney Axis Using MPSs
Several gut-on-a-chip systems have been established by integrating microfluidics, tissue engineering, and microelectromechanical systems. Among the most representative configurations, the gut-on-a-chip from the Wyss Institute (USA) successfully emulated the dynamic human intestinal microenvironment through the application of physiologically relevant fluid fellow and peristalsis-like mechanical forces, and these supported cell differentiation into villus- and crypt-like structures, the formation of a thick epithelial monolayer, and enhanced cellular function (Figure 3A) [16–19]. Recently, topological features have emerged as being pivotal in directing cell function, but only a few studies have attempted to replicate the crypt-villus architecture in microfluidic systems that can now be easily obtained through 3D high-resolution stereolithography [20], photolithography [21], and micro-molding of crosslinked hydrogels [22]. At present, mimicking intestinal tubule-like structures has been addressed by culturing intestinal cells on the apical side of a per fusible hollow-fiber membrane system [23,24] or in the lumen of microchannels [25]. The addition of an ECM coating and unidirectional apical fellow resulted in a mature intestinal tubule phenotype with villus-like structures. Exposure to Clostridium difficile secreted toxin A, a natural gut inhabitant virulence factor and intestinal barrier disruptor in dysbiosis, or to the gut microbiota-derived metabolite, p-cresol, resulted in enhanced barrier permeability [23,24]. Concomitantly, p-cresol was converted to p-cresyl sulfate and p-cresyl glucuronide, the end-metabolites that accumulate in plasma during CKD progression, likely via cytochrome P450-mediated metabolism followed by conjugation, thus highlighting the contribution of the intestine to the biotransformation of gut microbiota-derived metabolites into uremic toxins [23].
The complexity and diversity of the intestinal epithelium can be reliably recapitulated by using 3D human tissue organoids [26,27]. However, their use has proved challenging because their closed, outside-in configuration hampers transport studies and exposure to commensal and pathogenic bacteria. Nevertheless, Thorne and coworkers showed that, through enzymatic dissociation of organoids, primary intestinal cells were able to self-organize and de novo segregate into undifferentiated or differentiated regions, forming niche-like compartments [28]. By integrating a separate microvascular endothelium cultured under independent fellow and cyclic deformation, the absorption properties of these cells were evaluated [29,30]. Recently, the inclusion of crypt-villus domains in a tube-shaped epithelium with per fusible lumen was demonstrated to sustain stereotypical cell patterning features with self-regenerative potential [31].
The in vitro study of host-microbiota interactions has been hampered by the inability of conventional models to sustain a viable complex microbiota for several days. Although the contribution of the mucus layer to host-microbiome interactions has often been overlooked, it was recently demonstrated that the integration of a thick mucus layer – acts as a physiological barrier between the bacteria and the intestinal epithelium – could delay barrier damage and paracellular permeability [32,33]. Accordingly, the sophisticated microfluidic model, HuMiX, enabled direct coculture of anaerobic bacteria and intestinal cells by incorporating a functional mucus layer as well as pulsatile fellow and mechanical stimulation (Figure 3B) [34].
The majority of the gut microbiota are obligate anaerobes that require <0.5% O2 growth conditions that are difficult to represent in vitro [19,35]. This limitation was overcome by engineering MPSs that incorporate physiologic oxygen gradients and support the dynamic interaction between intestinal and vascular endothelial layers. The chip consisted of an upper anaerobic epithelial chamber and a lower aerobic endothelial chamber, separated by a polydimethylsiloxane (PDMS) membrane. Through a radial oxygen gradient generated by the system, intestinal cells were oxygenated whereas anaerobic conditions allowed microbiota growth, as assessed by real-time monitoring via integrated noninvasive oxygen sensors [19,36]. Similar physiological hypoxia conditions were achieved by Zhang and coworkers who cocultured oxygen super-sensitive bacterial species using a differently designed MPS, the Gumi (Figure 3C) [37]. This platform induced a steep oxygen gradient through the addition of a long-term continuous fellow of anoxic apical medium and aerobic basal media. The use of polysulfone, which unlike PDMS is an oxygen-impermeable material, prevented any oxygen leakage.
The development of kidney-on-a-chip systems has also been challenging owing to the lack of functional cells to recapitulate in vitro the multicellular structure and functional complexity within the nephron. Accordingly, relative to the gut-on-a-chip device, the development of kidney-on-a-chip systems is to some extent lagging behind. To date, models of the glomerular, proximal tubule, and distal tubule physiology have been developed, but the integration of all components into a complete nephron-on-a-chip remains to be achieved [38]. To be physiologically relevant, in addition to cellular complexity, a biomimetic kidney-on-a-chip should integrate (i) cell-cell interactions such as those between podocytes or proximal tubule epithelial cells and the (micro)vascular endothelium, (ii) the transcellular electrochemical and osmotic pressure gradients that drive fluids and metabolites across the interstitial space, (iii) fluid fellow, and (iv) the structural arrangement of the kidney tubules, as well as (v) cellular metabolic and endocrine functions [38].
The proximal tubule plays a crucial role in metabolic waste excretion and biomolecule reabsorption and has therefore been a major focus of interest in the development of in vitro kidney-on-a-chip systems that recapitulate in vivo kidney tissue. The development of functional kidney tubules using proximal tubule cells with biofunctionalized hollow fibers enabled Jansen and coworkers to study the secretory clearance of gut microbiota-derived metabolites. This system enabled the researchers to demonstrate how, through remote sensing and signaling, proximal tubule cells sense elevated levels of indoxyl sulfate and accordingly adjust the expression of the transporters responsible for their excretion in an attempt to maintain stable metabolite levels and homeostasis [39].
Endothelium–interstitial space–epithelium interactions govern the continuous exchange of solutes between the circulatory and urine compartments. Lin and coworkers successfully developed a perfusable 3D vascularized proximal tubule that was able to simulate, via tubule–vasculature exchange of solutes, the active reabsorption function of the kidney [40]. This model allows quantity for certification of kidney albumin uptake and glucose reabsorption over time, offering a promising tool for investigating kidney (patho)physiological functions and pharmacology. Other than a solute exchange, the kidney interstitial space is also considered to be central to the development of kidney fifibrosis, a hallmark of CKD. This is thought to be caused by scarring of the tubule interstitial space as a result of interstitial myofibroblast activation and subsequent ECM deposition. Nevertheless, only a few studies have reported its integration into 3D in vitro systems. The validation of a simple and highly reproducible 3D tubule/interstitium microenvironment model for the study of kidney fifibrosis in a physiologically relevant in vitro system was reported by Moll and coworkers [41]. This study used cisplatin to successfully mimic acute tubular injury. The in vitro replication of the renal tubule/interstitium microenvironment was achieved by using human dermal fibroblasts instead of renal fibroblasts because the former express low levels of fibrotic markers under basal conditions. Nevertheless, despite this limitation, the system demonstrated that epithelial cells play a central role in triggering the activation and differentiation of myofifibroblasts. Moll and coworkers attempted to repeat this study using primary renal fibroblasts but encountered great variability in the results. Given the importance of the interstitial space in CKD, further 3D in vitro studies will be necessary to elucidate its role in disease onset and progression.
The kidneys also activate 25(OH)vitamin D by hydroxylation at the 1α position, resulting in 1,25 (OH)2vitamin D, an essential hormone that is often deficient among CKD patients and that can affect gut microbiota composition and barrier integrity. Recently, an on-chip representation of hepatic metabolism and kidney activation of vitamin D was developed by perfusing vitamin D-containing medium into a microfluidic chip, suggesting that complex inter-organ metabolic interactions are highly attainable using MPS technologies [42].
In CKD, the reduction in short-chain fatty acid (SCFA) production, complemented by the simultaneous increase in uremic toxin production and their systemic accumulation [4], is assumed to drive the chronic inflflammatory condition that is typical of CKD [4,43]. Indeed, SCFAs, particularly butyrate, have both nephroprotective and intestinal protective effects [4,44], and high levels of butyrate have been associated with gut barrier integrity and intestinal immunity improvements as a result of its anti-inflflammatory properties [45]. Nevertheless, this was recently contradicted by Trapecar and coworkers who, in a psychomimetic approach, demonstrated that SCFAs can exacerbate an inflflammatory response in a gut–liver model. By connecting two pneumatic plates separately representing the gut and the liver, CD4+ T cells and inflammatory type 17 T helper (Th17) cells could circulate within and between the two compartments. The opposing effects of SCFAs might correlate with the degree of inflflammation, with a heightened inflflammatory state producing a more deleterious effect [46].
To our knowledge, there is currently no MPS that addresses the effects of gut-derived metabolites on the gut, kidney, or other organs, with concomitant tracking of their biotransformation, in the context of CKD. Tuning the chip to faithfully recapitulate the bidirectionality of production and removal fluxes of metabolites will be a challenge. Integrating microbiota derived from stool samples of CKD patients into an intestinal microfluidic system would enable us to study alterations in microbial metabolism and analyze their (in)direct effects on remote organs, a feature that is not attainable through in vivo experiments.
Technological Advances Enhancing the In Vivo Translational Value of MPSs Designing an MPS is challenging and requires a multidisciplinary approach. It is important to note that no single MPS 'can do it all, and, depending on the application, different systems may be required. The advantages and limitations of available systems for tackling the gut–kidney axis are summarized in Table 1. One of the most common challenges in the field is to design a system that is biologically complex and sufficiently technically simple to be established in cell culture laboratories.
The Ingber group (Wyss Institute, USA) has established well-optimized protocols for cell culturing, connecting microfluidic components to the chip, and sampling [16–18,47]. Although technologically advanced, their microfluidic system requires significant training of non-technical operators, even for automatic microfluidic assays [47]. Pumpless multiorgan chips, pioneered by Shuler laboratory (Cornell University, USA), as well as by companies such as Hesperos Inc. (Figure 3E) and InSphero, increase the throughput at the expense of limited control over the fellow and device complexity [48] (https://hesperosinc.com/). Although limited in replicating biophysical cues, the MPS developed by the Griffith laboratory (Massachusetts Institute of Technology, USA) makes use of more conventional protocols, for example, by enabling direct access to the tissue analog and by using modified standard Transwell® inserts (Figure 3C, D) [37,49].
Innovative companies have similarly developed multiorgan platforms, such as TissUse®.Their on-chip pumps connect the organs and make the system less prone to bubble trapping and leakage. However, these devices offer limited microfluidic routing, for example, the apical fellow in the gut model is lacking, and customization of the tissue models is difficult.
Another major challenge is the chip material. PDMS is among the most frequently used materials because of its excellent oxygen permeability, optical clarity, and prototyping properties. However, oxygen permeability is a downside when coculturing the obligate anaerobe microbiome with intestinal cells [20,37]. In testing hydrophobic compounds, for example, in drug toxicity or efficacy studies, PDMS is not recommended because it absorbs small hydrophobic molecules. Hence, MPSs composed of more inert materials, that prevent nonspecific binding of compounds, are the most reliable. For instance, Edington and coworkers developed a polystyrene-based microfluidic platform of interconnected MPSs in an attempt to recreate a physiome-on-a-chip that can generate complex molecular distribution profiles for advanced drug discovery applications [50].
The development of platforms with integrated sensors (oxygen, urea, lactate, or glucose), and/or optical transparency, have facilitated real-time noninvasive cellular analytics (Box 3) [51,52]. A platform with fully integrated modular sensing has recently been developed. This operates MPS units in a continual, dynamic, and automated manner, and includes physical sensors to monitor the extracellular microenvironment, biochemical sensors to measure soluble biomarkers, miniature microscopes to capture morphological changes, and a microfluidic-routing breadboard to route fluids in a timely manner [53].
Computational analysis is also necessary to establish whether MPS-derived experimental data can be extrapolated to in vivo performance [54]. Thus, the integration of machine learning algorithms (in silico modeling) should become a strategic component of MPSs [55]. The computational model can be adjusted to help to resolve the limitations of experimentally linked MPSs and bring the data within the range of animal studies and extrapolation methods [54]. Predictions obtained from in silico studies can provide feedback to further improve MPS models [55]. For instance, in silico studies could be employed to model immune cell motility following intestinal barrier damage and predict cell behavior upon exposure to specific parameters or biomolecules [56–59].
Concluding Remarks and Future Perspectives
Over the next century, the prevalence of CKD is predicted to drastically increase worldwide, posing significant economic and societal challenges. Regardless of the country of origin, the annual healthcare and societal costs have been found to increase in parallel with the progression of CKD [60], highlighting the urgent need for a disease model platform in which to study CKD pathophysiology and identify potential therapeutic targets.
Nevertheless, many challenges remain to be addressed, and several issues must be resolved before MPSs can be developed that accurately model CKD (see Outstanding Questions). For example, the initial events that drive the onset of CKD remain unknown, making the modeling of CKD onset in MPS challenging. Kidney injuries that lead to the development of CKD are diverse in nature and often involve a cardiovascular component, making their representation even more difficult. In addition, the composition of the gut microbiome is complex and challenging to reproduce; nonetheless, it is an essential requirement for a CKD disease model. The latest successful developments of MPSs that integrate anaerobic bacteria have been made possible by the integration of biosensors for oxygen sensing, as well as by the inclusion of controlled flows and a mucus layer that reduce bacteria overgrowth and limit intestinal cell damage (Table 1). However, broad anaerobic bacterial consortiums within the systems remain to be achieved, although this will be necessary for a physiological representation of the gut microbiome. Issues with absorptive and air-permeable materials also represent a major hurdle in the field, challenging the suitability of the systems for the growth of anaerobic bacteria or for testing lipophilic compounds. The relevance of organ interconnections has been strongly highlighted in this review; it is therefore of pivotal importance to integrate circulatory and immune systems within MPSs, but these have been incorporated into only a few models.
By enhancing interdisciplinarity, the integration of bioprinting, biomaterials, and biosensors for real-time monitoring of the microenvironment could address the anatomical and biochemical features, as well as the complexity, of the systems that are necessary to increase their physiological relevance. As MPS technology progresses, alongside the current trend towards enhanced multi-disciplinary approaches, these unanswered questions will eventually be addressed.
Acknowledgments
This project received funding from the EU Horizon 2020 research and innovation program under the Marie SkłodowskaCurie grant agreement STRATEGY-CKD H2020-2019-ETN (860329), as well as the WIDESPREAD-05-2018-TWINNING call REMODEL (857491). This work was further supported by the Dutch Kidney Foundation (DKF, 17OI13). R.M. is a member of the ESAO/ERA-EDTA-endorsed Work Group EUTox.
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