Kidney-on-a-Chip

C H A P T E R

82 Kidney-on-a-Chip: Technologies for Studying Pharmacological and Therapeutic Approaches to Kidney Repair Rosalinde Masereeuw1, Jelle Vriend2 and Martijn J. Wilmer2 1

Utrecht University, Utrecht, The Netherlands 2Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands

82.1 INTRODUCTION Chronic kidney disease (CKD) is a global health problem with significant morbidity and mortality, and affecting 5% 7% of the world population. Furthermore, patients are faced with major socio-economic and public health implications.1 3 Despite increased knowledge on factors involved in disease progression and advances in treatments of patients with CKD, the problem endures as the number of patients with renal disease still rises. This emphasizes the need to explore new avenues for slowing down or reversing renal disease progression. It should be noted that, especially in acute injury, the kidney has a high regenerative capacity and can overcome substantial damage by mechanisms currently not well understood. The complexity of endogenously triggered kidney regeneration, the shortage of donor organs and the increasing life span of humans have created a demand for regenerative nephrology, a field rapidly evolving. For functional restoration of the kidney, it is essential to understand how injured segments can be repaired and/or replaced.4,5 This requires uniting multidisciplinary and translational research strengths in the development of therapies for CKD patients. The combined efforts within tissue engineering and regenerative medicine have recently resulted in innovative technologies to study functional kidney repair. These include bioartificial kidney development as renal replacement therapy, and the directed differentiation of embryonic stem cells to kidney cells, (mesenchymal) stem cell therapy, kidney bioengineering with decellularized scaffolds, 3D printing, and cells cultured in microfluidic chips for studying regeneration.6 8 The latter approach allows cell culturing in a dynamic, 3D environment, enhancing maintenance of the tissues’ microenvironment and optimal communication between cells. This is essential for proper tissue functioning and mediated through paracrine and autocrine factors, as well as biomechanic and chemotactic processes, all influencing cell proliferation, migration, and differentiation. The “kidney-on-a-chip” incorporates living cultured kidney-derived cells in miniature systems to study kidney development and functioning in an organ-specific context, but allows developing in vitro disease models as well.9 These models will be applicable for pharmacological studies, with the ultimate goal to discover and develop new treatments for renal disorders. Where “classical” pharmacological approaches concentrate on the potential of small molecules to support kidney diseases and diminish progression, regenerative (nephro-) pharmacology focuses on accelerating, optimizing, and characterizing the development, maturation, and function of bioengineered and restoring kidneys.10 The current chapter gives an overview of recent developments in these fields appropriate for studying such pharmacological and therapeutic approaches in regenerative nephrology.

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82.2 IN SEARCH FOR TRANSLATIONAL CELL MODELS For more than a century, investigators have cultured human cells to model the human body in their laboratories. With increasing knowledge of culture systems and cell biology, the idea of regenerating a human organ is becoming a realistic goal. Despite sophisticated technological culture systems that copy the cellular environment including flow, mechanical stress, and 3D extracellular matrices (ECM), the basis of all models is to create a living cell that relates to the in vivo phenotype as closely as possible. By building a kidney-on-chip, researchers are faced with an extra challenge: The human kidney contains many different cell types that form one functional unit, the nephron. The exact physiological architecture of a complete functional nephron, including a complex vasculature system, is still subject of ongoing research. Consequently, the design of a kidney-on-a-chip is usually a simplified model developed for a specific goal, hence, is often mimicking only a part of the nephron. As in architecture, form follows function. Many kidney-on-a-chip designs cover only one, and occasionally two, cell types. The proximal tubule epithelium (PTE; Fig. 82.1) is an important player in the field of kidney repair and Urine

Blood

Nephrotoxic biomarkers (KIM1, NGAL)

OCTN1/2

OAT1

OAT4

OAT3

URAT1

OCT2

MATE 1/2-k ATP

P-gp

ADP + Pi

SLCO4C1

ATP MRP2/4 Na,KATPase

ADP + Pi ATP

ATP

ADP + Pi

ADP + Pi

BCRP

Receptor mediated endocytosis

SLC transporter

ABC transporter

FIGURE 82.1

The proximal tubule epithelial cells (PTEC) as prototypical cell model often applied in kidney-on-a-chip devices. Transport of xenobiotics is mediated via membrane transporters, including SLC and ABC transporters, located at the apical and basolateral membrane. Receptor mediated endocytosis is the major pathway for reabsorption of vital compounds from the ultrafiltrate. A proton gradient is the driving force for many transporters in PTEC, established by Na, K-ATPase. Intracellular signaling upon a toxic event can result in excretion of nephrotoxic biomarkers such as KIM-1 or NGAL. II. KIDNEY BIOENGINEERING AND REGENERATION

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1121

regenerative medicine, and cell isolates from this nephron segment are most widely applied in kidney-on-achips. The function of PTE cells (PTEC) is affected in CKD and cannot be replaced by current dialysis therapy, in contrast to the filtration function of the kidneys by the glomerulus. Hence, building a kidney-on-a-chip to study kidney repair requires a relevant PTEC model.

82.2.1 Relevant PTEC Characteristics For decades, homogeneous monocultures of PTEC have provided detailed information on cell functionality or intracellular regulation. However, no cell model has, as of yet, mimicked the complete in vivo functionality of PTEC, most likely because PTEC is a highly active cell population with diverse functions. PTEC are involved in the secretion of waste products and in the reabsorption of vital compounds from the glomerular filtrate. Active elimination of waste products from blood by the renal PTE is facilitated by influx transporters at the basolateral membrane, which are members of the solute carrier family (SLC).11 13 According to their substrate specificity, influx transporters can be divided in two groups: Organic cation transporters (predominantly organic cation transporter 2 (OCT2; SLC22A2) and anion transporters (organic anion transporter 1 (OAT1; SLC22A6, OAT3; SLC22A8 and organic anion transporting peptide 4C1 (OATP4C1; SLCO4C1); Fig. 82.1). Knowledge on the involvement of the OATP4C1 transporter in xenobiotic disposition is still emerging and may be of significant importance in the renal clearance of uremic toxins and the elimination of drugs such as digoxin, sitagliptin, and metotrexate.14 16 The final step in the elimination of xenobiotics from blood via the PTE is efflux into the tubular lumen via the apical membrane. So far, apical membrane transporters belonging to the SLC family (organic cation/carnitine transporter 1 (OCTN1; SLC22A4), OCTN2 (SLC22A5), OAT4 (SLC22A9), urate transporter (URAT1; SLC22A12), multidrug and toxin extrusion 1 (MATE1; SLC47A1), MATE2-K (SLC47A2)), and ABC transporters (P-glycoprotein (Pgp; ABCB1), multidrug resistance-associated proteins 2 (MRP2; ABCC2), MRP4 (ABCC4), and breast cancer resistance protein (BCRP; ABCG2)) have been described.12,17 19 The ABC transporters derive their energy from adenosine triphosphate (ATP) to mediate transport of their substrates across the membrane into the proximal tubular lumen, while the SLC transporters are gradient driven, mostly enabled via Na/K-ATPase abundantly expressed at the basolateral membrane. The active or facilitated elimination processes taking place at the renal PTE is influenced further by intracellular metabolism. Historically, the enzymes involved in drug metabolism are divided in two classes: Phase I metabolizing enzymes, predominantly comprising isoenzymes of the cytochrome P450 system,20 are responsible for oxidation, reduction, and hydrolyzation of xenobiotics, whereas the Phase II enzymes catalyze conjugation reactions, including glucuronidation, sulfation, methylation, acetylation, and glutathione or amino acid conjugation. The conjugated products are generally more hydrophilic, which facilitates their excretion and reduce potential toxicity. In human kidney, only few CYP enzymes have been described, including CYP1B1, CYP2D6, CYP3A4, CYP4A11, and CYP4F2,21 that is in contrast to a broad CYP expression in rat kidney.22,23 Especially CYP3A4 and CYP4A11 enzymes are expressed at relatively high levels in the proximal tubules. In addition to the CYP enzymes, other phase I enzymes expressed by the human kidney are flavin-containing monooxygenases (FMO), such as FMO1 and FMO5.24 The phase II metabolic enzymes are a very large family of enzymes, of which many are expressed in the human kidney.25 High RNA expression levels were found for glutathione S-transferases (GSTA1/2/4/5, GSTM1/2/3/4/5, GSTP1, GSTT1/2 and GSTZ), UDP-glucuronosyltransferases (UGT1A1/6/9, UGT2B7/17 and UGT8), and sulfotransferases (SULT1A1/3, SULT1B1/2).25,26 Usually, the conjugation reaction results in detoxification of the substrates by the formation of an inert product and/or enhanced excretion via drug transporters. When studying therapeutic interventions for kidney repair, it is evident that xenobiotic metabolism and transporter functionalities should be intact and considered during evaluation of cellular models that are used in a kidney-on-a-chip. In addition, PTEC have a range of specific epithelial markers that are frequently used to characterize their origin (when isolated from tissue or urine) or to investigate their differentiation stage, when derived from embryonic tissue, stem cells, or during culturing (Fig. 82.1). Widely used characteristics include brush border enzymes aminopeptidase N (CD13) and gamma-glutamyltransferase (GGT). Often the cobblestone morphology and tight junction formation (including expression of zona occludens-1; ZO-1) provide a quick scan for the epithelial character. Upon exposure to toxicants, the proximal tubular epithelium can express several biomarkers for renal toxicity, such as kidney injury molecule 1 (KIM1) or neutrophil gelatinase-associated lipocalin (NGAL). These biomarkers can be detected in urine of patients, but are also shedded in supernatant of cultured PTEC and relevant for evaluating cells models appropriate for a kidney-on-a-chip design.27 II. 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82.2.2 Animal Derived Models Widely used animal models with a renal origin are the commercially available dog (Madine Darby Canine Kidney, MDCK) or pig (Lilly Laboratories Cell Porcine Kidney, LLC-PK1) cells that demonstrated their value in renal physiology, pharmacology, and toxicology. MDCK cells form polarized tight monolayers with expression of ZO-1 and the epithelial marker E-cadherin, allowing demonstrating differential regulation during epithelial tubulogenesis without loss of cell-cell contact.28 In renal pharmacology, MDCK overexpressing specific drug transporters were pivotal in the elucidation of their role in drug disposition. For example, MDCK cells expressing MRP2 were used to demonstrate ATP-dependent glutathione conjugates export activity at the apical membrane in polarized monolayers.29 In a double-transfected MDCK cell line expressing both OCT2 and MATE1, transepithelial basolateral-to-apical transport could be demonstrated for metformin, together with a clinically relevant drug interaction with cimetidine.30 Furthermore, triple- and quadruple-transfected MDCK cells demonstrated coordinated activity of uptake transporters, drug-metabolizing enzymes and efflux pumps, thereby delivering a model predictive for drug disposition.31,32 Despite that the LLC-PK1 cell line demonstrated poor applicability in transporter transfections due to misrouting, the renal epithelial cell is positively applied to evaluate drug-induced toxicity.33 Furthermore, the long-term use of LLC-PK1 in a bioartificial renal tubule device demonstrated continued proliferation and glucose transporter expression for more than 3 weeks, when polysulfone membranes were used as scaffold.34 Regardless of these significant roles for LLC-PK1 and MDCK in renal physiology, pharmacology, toxicology, and biotechnology, their major drawback in a kidney-on-a-chip for studying regeneration relates to their nonhuman origin. Prediction of pharmacological and therapeutic responses or even preclinical applications will be limited because of species differences in drug response and drug handling, which stimulated researchers to develop robust and functional human derived cell lines.

82.2.3 Human Derived Models Two cell lines, human embryonic kidney 293 (HEK293) and human kidney 2 (HK2) have frequently been used for many years in kidney related research. The disputable origin of HEK293, with very limited renal characteristics, makes this cell line unsuitable to build a kidney-on-a-chip. Nevertheless, this cell line can easily be transfected and is therefore an important biotechnological tool. The limitations of HEK293 together with LLC-PK1 were demonstrated in comparison to 3D models of mouse proximal tubule, in terms of nephrotoxic biomarker expression.35,36 The origin of HK-2 cells is indeed proximal tubular and proliferation was maintained by immortalization with the human papilloma virus-16 E6/E7 (HPV16 E6/E7).37 Although this cell line has some renal characteristics, such as brush border and epithelial markers (aminopeptidase N), the functionality of relevant drug transporters is incomplete.38 Especially, neither uptake transporters OAT1/3 and OCT2 nor efflux transporter BCRP could be detected. Only the efflux mediated by MRPs and P-glycoprotein was demonstrated to be functional, but limited as compared to freshly isolated PTEC.38 Thus, HK-2 cell line is of limited value for testing compounds for kidney repair. The optimal model available in terms of completeness in PTE characteristics is probably freshly isolated human PTEC from renal tissue.22,39,40 The expression of metabolic enzymes (e.g., CYP1B1, CYP3A4/5, glutathione-S-transferases (GSTs), and UGTs) and functionality of transporters (e.g., OATs, OCT2, BCRP, Pgp, MRPs) were demonstrated for up to 5 days. Importantly, primary PTEC cultured on semi-permeable membranes formed a tight monolayer, allowing bi-directional flux assays to study renal clearance. However, the functionality of primary cells was highly variable between donors and, moreover, expression levels of specific transporters decreased within days upon seeding. These shortcomings make primary cells unsuitable for long-term exposures, which are required to study kidney repair. Maintained functionality, at least for 2 weeks and preferably up to 1 month, is pivotal when considering the use of these cells in cell-aided devices or for kidney regeneration. Immortalization techniques can provide biotechnologists the in vitro tools to extend the expression and functionality of primary human PTEC. Maintained proliferation of immortalized and subcloned cell lines is also an advantage for the reproducibility of in vitro testing. The introduction of oncogenes into a human derived cell line is often used as immortalization technique and stimulates proliferation. This technique was successful for the immortalization of PTEC transfected with simian virus 40 T (SV40T)41 or using HPV16 E6/E7 in the HK2 cell line as mentioned earlier.37 Both transfections led to unlimited availability of cells, although the uncontrolled proliferation capacity led to undesired phenotypical changes as well. Importantly, transfections using oncogenes implies a potential hazardous situation when used in a clinical environment, and needs careful evaluation before future

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clinical applications can be considered. To limit the risk of oncogene expression, conditionally immortalization techniques have been developed. Only specific defined conditions allow cells to proliferate, induced by oncogene expression. The addition of chemical compounds (such as doxycyline) and application of physical parameters (such as temperature) are then used to trigger proliferation. Most valuable achievements were made using a temperature sensitive vector SV40tsA58, which was applied to obtain conditionally immortalized PTEC (ciPTEC).42 When culturing at a permissive temperature of 33 C, the cells could be maintained for up to 50 passages and were characterized at 39 C for membrane transporter activity (succinate; phosphate; Na/KATPase) and maintained proximal tubular characteristics (such as apical microvilli). In addition to SV40tsA58, cells can be transfected with human telomerase (hTERT) that limits replicative senescence by maintaining telomere length and further improves the unlimited availability of human cells with intact characteristics.43 Using human fibroblasts and endothelial cells, the combination of SV40tsA58 and hTERT was demonstrated to result in functionally intact immortalized cell lines, whereas the immortalization of either one of the vectors was not sufficient.43 Double transfection with SV40tsA58 and hTERT was applied in human proximal tubule cells derived from urine.44 These well-characterized ciPTEC have maintained drug transporter functionality (OCT2; Pgp; BCRP, MRPs), megalin-mediated endocytosis, and metabolic enzyme activity.44 46 The transporter activity was demonstrated to be intact for more than 45 passages, indicating that the availability of these cells theoretically is sufficient for the development of a bioartificial kidney or a kidney-on-a-chip. Furthermore, upon gentamicin-induced nephrotoxicity, kidney regeneration was studied, which was stimulated by mesenchymal stem cell-conditioned medium.47 On the other hand, as for all renal cells in culture,48 the expression of OATs rapidly decreased upon culturing and could not be restored by conditional immortalization. ciPTEC isolated from freshly derived tissue exhibited similar epithelial characteristics as compared to ciPTEC developed from cells exfoliated in urine, although expression of ECM proteins was higher in tissue derived cells.49 This expression allows monolayer formation on semi-permeable membranes without additional collagen coating, which is required for cells derived from urine. Strikingly, single transfection with hTERT was demonstrated to maintain proximal tubule epithelial characteristics in renal PTEC expressing hTERT (RPTEC/TERT), including expression of aminopeptidase N, tight junction formation, and receptor-mediated endocytosis.50 Both Pgp and OCT2 activity was demonstrated in RPTEC/TERT, as well as expression of other relevant drug transporters.51 Again, functionality of OAT proteins could not be demonstrated, but was recently effectively introduced through viral transduction.111 To overcome safety issues introduced by transfections with oncogenes, pluripotent stem cell derived cells are an emerging and attractive approach to obtain sufficient cells to build a kidney-on-a-chip. Renal progenitor cells isolated from adult kidneys using the CD133 positive fraction could be differentiated further into endothelial cells and integrated in tubules of injured kidney, indicating their multipotent phenotype.52 Human induced pluripotent stem cells (iPSCs)53 offers a new avenue to potentially generate functionally mature renal cells through directed differentiation into kidney lineages, although the technology is still in its infancy.54 Human embryonic stem cells, on the other hand, could be differentiated into PTEC by culturing in conditioned medium with specific supplements and kidney developmental factors, including bone morphogenetic proteins 2 and 7 (BMP2/7).55 Next to expression of relevant PTEC proteins, response to parathyroid hormone demonstrated the differentiation into a PTE phenotype. So far, availability of human derived stem cells is limited and the use of embryonic stem cells hampered by ethical issues. The iPSC technology is promising and improving culturing conditions in the future could allow for an unlimited source of human differentiated PTEC that will find their applications in the field of regenerative medicine. Moreover, with this technology patient-derived cells can be obtained, even from urine samples, allowing disease modeling, for which its application in 3D engineered renal models would offer excellent drug-screening platforms as well.56,57

82.3 ENGINEERED RENAL MODELS: FROM 1D TO 3D PTEC and other renal cells have important characteristics such as cobblestone morphology, expression and activity of membrane transporters and metabolizing enzymes, which can often be improved when incorporated in a 3D model. Flow and signaling between multiple cell types in the kidney are other relevant factors to consider in a 3D kidney model. Examples that are successfully used in the development of a 3D kidney model are summarized in Table 82.1 and will be discussed further, with an emphasis on the cross-talk between various cell types, physiological conditions, and the 3D architectural design.

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TABLE 82.1

Summary of Literature Search on Three 3D Kidney Model Approaches Discussed in This Chapter Multiple channels

Flow Materials

Permeable Polarized membrane monolayer

Endpoint read-outs

Summary

Refs

HK-2 and MDCK

No

No

PEG or PES

Yes

N/A

N/A

Fiber testing for development of BAK

58

Primary human renal PT cells

Yes

Yes

PES/PVP fiber coated with LDOPA and collagen IV

Yes

Yes

N/A

Performance renal PT in fluidic device for development of BAK

59

Yes

PES fiber

Yes

Yes

N/A

Fiber testing for development of BAK

60,61

Yes

Fiber of PES, PVP and NMP coated with fibrin in PDMS body

Yes

Yes

N/A

Development of BAK

62

Human proximal No epithelial cells (HKC8) and human dermal fibroblasts (WS-1)

No

Collagen I

No

N/A

RANTES, IL-6, MCP-1, IL-8

Cisplatin-induced nephrotoxicity in coculture

63

NKi-2 cells No immortilzed human renal cortical cells (via hTERT)

No

Gel mixture of Matrigel and rat tail collagen I

No

KIM1, NGAL

Development 3D model 64 for nephrotoxicity in 2D and 3D kidney model

NKi-2 cells No immortilzed human renal cortical cells (via hTERT)

No

Mixture of Matrigel and rat tail collagen I in Transwell

No

N/A

KIM1, NGAL, MCP-1, TNFα, IL-6, IL-8

Effects of Shiga Toxin type 2 in 2D and 3D kidney model

Murine proximal tubules

No

No

Hyaluronic acid- N/A based hydrogel

Yes

KIM1, ALP, CYP, ILDevelopment of 3D 1β, IL-6, RANTES, organoid model MIP-1α, MCP-1, TNFα, γ-GGT

35

Murine proximal tubules, HEK293 and LLC-PK1

No

No

Hyaluronic acid- N/A based hydrogel

Yes

KIM1, NAG, CYP, TNFα, NGAL, HO, VIM, Spp1, CLU

Nephrotoxicity screening in 2D cell cultures (HEK293 and LLC-PK1) and 3D PT model

36

Murine proximal tubules

No

No

Hyaluronic acid- N/A based hydrogel

Yes

KIM1, TNFα, MIP-2, IL-1β, IL-2, IL-6, IL-10, MCP-1, MIP-1α, MIP1β, INFγ, RANTES, TNFα

Nephrotoxicity of nanoparticles in 3D kidney model

66

Cell culture(s) HOLLOW FIBER

ciPTEC Human RPTEC

Yes

GEL-BASED CULTURES

65

MICROFLUIDIC-BASED Primary rat IMCD cells (3D) and MDCK (2D)

Yes

Yes

PMDS (channels), polyester (membrane)

Yes

Yes

N/A

Analysis renal PT cells on microfluidic device compared to 2D cell culture

67

MDCK

Yes

Yes

PMDS

No

No

Gene and protein expression

Analysis cell function and proliferation of MDCK under fluidic

68

(Continued)

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TABLE 82.1

(Continued)

Cell culture(s)

Multiple channels

Flow Materials

Permeable Polarized membrane monolayer

Endpoint read-outs

Summary

Refs

conditions compared to static RPTEC

Yes

Yes

Glass (channels), Yes polycarbonate (membrane) coated with Matrigel

N/A

N/A

Optimizing conditions for renal PT on fluidic device

69

Human PT cel line (HK2)

No

Yes

PMDS with glass coating

No

Yes, Na1/ K1-ATPase n HK2

N/A

Formation of kidney stones

70

Primary rat IMCD cells

Yes

Yes

PMDS (channels), polyester (membrane)

Yes

Uncertain due to relatively short duration of time

N/A

Influence of fluid shear stress on cell function and proliferation

71

Primary RPTEC and Yes HK-2

Yes

Poly-carbonate Yes (membrane) and PDMS (channels)

Yes

N/A

Optimizing porous membrane for renal microfluidic device

72

Primary HUVEC, MDCK and NIH3T3

Yes

Yes

Hydrogel mixture of rat collagen I and alginate

Yes

N/A

N/A

Passive diffusion in coculture in microfluidic device containing multiple channels

73

MDCK

Yes

Yes

PDMS

No

No

Gene and protein expression only

Ifosfamide nephrotoxicity under static and fluidic conditions

74

MDCK, HepG2/ C3a and HepaRG

Yes

Yes

PMDS

No

No

Gene expression and intracellular Ca21 release

Metabolism-induced ifosfamide nephrotoxicity

75

Primary human Yes kidney PT epithelial cells

Yes

PMDS (channels), polyester (membrane)

Yes

Yes

KIM1, LDH, P-gp activity

Nephrotoxicity study in 76 2D cell culture and 3D model

ALP, alkaline phosphatase; BAK, bioartificial kidney; ciPTEC, conditionally immortilized proximal tubular epithelial cells; CLU, clusterin; CYP, cytochrome P450; HO-1, heme oxygenase-1; IL, interleukin; IMCD, inner medullary collecting duct; INFγ, interferon γ; KIM1, kidney injury molecule-1; LCC-PK1, porcine renal proximal tubule epithelial cells; LDH, lactase hydrogenase; MCP-1, monocyte chemotactic protein-1; MIP-1α, macrophage inflammatory protein α; NAG, lysozomal enzyme; NGAL, neutrophil gelatinase-associated lipcalin, NMP, N-methyl-2-2pyrrolidone; PDMS, polydimethylxiloxane; PEG, polyethylene glycol; Pgp, P-glycoprotein; PES, polyethersulfone; PT, kidney proximal tubules; PT, kidney proximal tubular epithelial cells; PVP, polyvinylpyrrolidone; RPTECs, renal proximal tubule epithelial cells; SMIE, submandibular immortalized epithelial cells; Spp1, osteopontin; TNFα, tumor necrosis factor α; VIM, vimentin; γ-GGT, γ-glutamyl-transerase.

Morphologic characteristics of renal epithelial cells include a columnar shape with a cobblestone formation and a folded, brush-border, apical membrane. Cells spread mainly in horizontal direction in conventional 2D cell culture resulting in flattened cells compared to 3D cell culture.76,77 Design of pharmacological studies based on 2D culture could, therefore, be biased. In addition, a tight monolayer is essential for enhanced expression of gap junction proteins and proper barrier function. This also advances cross-talk between different cell types. Ideally, cells from different renal segments (glomerulus, proximal tubules and distal) and other cell types, such as vascular endothelial cells, should be incorporated in one system. In a close-proximity co-culture setting, signaling between microvascular endothelial cells and PTEC has been demonstrated.78 Vascular growth factor, cytokines and ECM proteins influenced expression and functional phenotype of PTEC, resulting in enhanced expression of catalytic enzymes and cytokines in this co-culture. In addition, co-culturing enhanced barrier function.78

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(A)

(C)

(B)

(D)

FIGURE 82.2 3-Dimensional models for studying kidney injury and repair processes. Cells grown on hollow fiber permeable membranes (A) allowing implementation of flow and apical and basolateral compartements. Hollow fiber models allow for studying the functional role of PTEC in the clearance of metabolic wastes and are components of bioartificial kidney device developments. Gel-based cultures (B) embedded in ECM-like gel, preserving apical and basolateral phenotype and facilitating diffusion. Gel-based models allow for studying branching and drug toxicity screenings. Microfluidic membrane-based devices (C) consist of multiple channels separated by a permeable membrane seeded with PTEC. Apical and basolateral compartments together with flow are implemented in this model. Transcellular transport is facilitated by PTEC on permeable membranes. Microfluidic models allow for studying drug toxicity and kidney diseases. Microfluidic membrane-free device (D) consist of multiple channels separated by cells seeded on ECM-like structures (e.g., gel supports). Apical and basolateral compartments and flow are incorporated in this model, as well as the ability to study membrane-free transcellular transport.

This underlines the importance of the incorporation of multiple cell types next to PTEC in the optimization of a 3D kidney model. In native kidneys, cells are embedded in the ECM, which is key in inter- and intracellular signaling, regeneration, and support. The ECM forms a depot for gasses, nutrients, and effector molecules, such as growth factors and cytokines, indicating a role in tissue maintenance. The ECM consists of the basement membrane (BM) and the stromal matrix (SM). The BM is a sheet-like scaffold and comprises fibronectin, proteoglycans, laminin, and collagen IV. The SM is composed of collagen I, proteoglycans, and glycosaminoglycans, which form fibrous structures providing the major structural support of the ECM. The cell-ECM adhesion and signaling is mediated by integrins, which are transmembrane receptors located in the PTEC plasma membrane.79 As the ECM has a pivotal role in cell adhesion, structure, and function, it is essential to incorporate its components in a 3D model. Current advances in 3D kidney models have incorporated ECM components in hollow fiber devices, gel systems, and membrane-based microfluidic devices (Fig. 82.2). The rigidity of the ECM also influences mechanical stress in the 3D culture. Under physiological circumstances PTEC experience pressure due to ultrafiltration by the glomerulus,80 leading to shear stress. The importance of shear stress or flow in cell cultures was underscored by the demonstration of primary cilium expressed in most epithelial tissues. The solitary organelle functions as a flow sensor, both in vitro and in vivo, for which polycystin-1 and polycystin-2 (PC1, PKD1 and PC2, PKD2) are mediating the mechanosensation.81 As a consequence, cultured PTEC expressing functional primary cilia are affected when a shear stress is applied by induction of apical flow. This physiological condition is used in hollow fiber systems and microfluidics, further contributing to improvements in the in vitro systems. In a study by Gao et al. a dual channel microfluidic device was described. Channels were separated with human PTEC cultured on a polycarbonate membrane for the development of a bioartificial renal tubule.69 Cell adhesion and proliferation were tested using different types of synthetic or natural ECM components. It was nicely shown that the ECM components, including gelatin, laminin, and collagen I, positively affect cell adhesion and proliferation.69 As earlier mentioned, PTEC form a tight and polarized monolayer consisting of an apical and basolateral membrane. Implementing compartmentalization via a fiber, a gel, or a permeable membrane creates such a polarized system. This multicompartmentalization enables the study of transcellular transport, via diffusion or facilitated by membrane transporters, thereby mimicking processes of diffusion, active secretion and (active and

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TABLE 82.2

Comparison Summary Table of Three 3D Kidney Model Approaches Discussed in this Chapter

Hollow fiber

Gel-based cultures

Membrane-based microfluidics

Fibers coated with renal cells, flow in channels

Renal cells in gel forming branch-like structures or renal tissue encapsulated in hydrogel

Multiple channels separated with membrane, coated with renal cells

Advantages Renal flow can be incorporated

Extensive protocols available

Renal flow can be incorporated Long-term studies possible

Limitations

Time-consuming Difficult to be used for HTS

Necrotic centers (spheroid-like structures) Limited abilities to implement fluidic conditions

Membrane porosity Pumps are often required to maintain flow

References

58,59,62,70

35,36,63 66,73,82 84

67 69,71,72,74 76,85,86

Definition

Long-term studies possible

passive) reabsorption. Three commonly used approaches for 3D renal models suitable for pharmacological studies in renal diseases are being discussed and an overview of various studies is presented in Table 82.1. Obviously, the three technologies described each have their possibilities and restraints. A comparison of these aspects is presented in Table 82.2.

82.3.1 Hollow Fiber Membranes for 3D Renal Cultures Cells cultured within or on hollow fiber membranes can be used for bioartifical kidney or renal tubule assist device (RAD) developments,8,58 60,62 mimicking tubular structures able to remove waste products that accumulate during uremia. Such cell-aided devices could potentially improve current dialysis treatments, although their clinical applications are still far from common practice. Seeding PTEC in a permeable hollow fiber allows the formation of an apical side facing inwards and basolateral side facing outwards (Fig. 82.2A) or vice versa when cells are cultured on the fibers. This increases membrane surface area and stimulates establishment of a barrier function. Increased curvature of hollow fibers improved PTEC function, likely due to imitation of the curvature in native renal tubules and increasing mechanical stress.58 Oo et al. developed a device with human primary PTEC using a 3,4-dihydroxy-L-phenylalanine (DOPA) and collagen IV double coating on a hollow polyethersylfone (PES)/polyvinylpyrrolidone (PVP) fiber membrane.59 Cells were cultured in this bioreactor for up to 7 days and perfusion was started at day three and maintained until day seven. Filtration of the bioreactor was tested, and expression levels of renal-specific markers proved to be sustained. Expression levels of transport proteins (Na/KATPase), brush border enzymes (CD13 and GGT) and the tight junction protein, ZO-1, was significantly upregulated compared to static conditions.59 Jansen and colleagues used a similar double coating strategy with human ciPTEC cultured on hollow PES fibers, after optimization of the coating in 2D,61 and demonstrated active OCT2mediated transport.60 Ng et al. developed a comparable model where human primary PTECs were cultured inside a hollow fiber on a fibrin coating.62 Biomarker read-out from perfusates of hollow fibers upon exposure to drugs or nephrotoxicants is feasible, but has, as of yet, not been described. However, production of a hollow fiber bioreactor is time demanding and expensive, and developing this method for drug screening on a large scale would be unsuitable. It is, therefore, not surprising that hollow fibers find their application mainly in bioartificial kidney developments.

82.3.2 Gel-Based 3D Kidney Models Kidney tubular tissue or cells encapsulated by a gel has been applied mainly in drug toxicity screenings in high-throughput settings, using renal stress biomarkers as end-point indicators. Although diverse gel types are available now a days, ranging from synthetic gels to cell-derived gels, only the latter gels found their application in 3D kidney models. Synthetic gels currently fail to mimic the complex function of the ECM and may unexpectedly interact with cells not naturally occurring.87 Although some studies showed that rat kidney cells and human primary PTEC maintained in vivo-like characteristics in these synthetic gels, including polarity, epithelial phenotype, and function.88,89 Natural polymers, such as alginate, or cell-derived gels, containing a collagen mixture or hyaluronic acid, have been more often successfully used to engineer 3D kidney models. The frequently applied Matrigels consists of a mixture of collagens derived from Engelbreth-Holm-Swarm mouse sarcoma cells

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(e.g., BD Biosciences and Sigma-Aldrich). While its application demonstrated successful 3D modeling, compositions of Matrigel change from batch-to-batch, resulting in variable outcomes.90 Gel-based 3D kidney models have been used for various applications, ranging from developmental studies64,65 to functional testing of whole mouse proximal tubules embedded in gel.35,36,66 These models proved suitable for drug toxicity screening using endpoint markers for cellular and renal stress.35,36,64 66,73 Other methods, such as 3D spheroids, have been applied for pharmacological studies as well as for renal epithelial disorder modeling.82,91 Viability and phenotype of kidney cells in gels can be maintained for prolonged periods of time (for up to 2 weeks or longer), allowing long-term testing of compounds.35,36,64 66,73 Gel-based systems are also compatible with co-culturing for disease modeling, as demonstrated by a co-culture of human dermal fibroblasts (WS-1) and human PTEC (HKC8) in a gel of collagen type I study cisplatin-induced renal fibrosis.63 DesRochers et al. have developed a spheroid-like method using immortalized human renal cortical cells (NKi2) seeded in a 50:50 gel mixture of Matrigel and rat tail collagen I in semi-permeable Transwells plates.64 After 2 weeks, formation of 3D branch-like structures of PTEC was demonstrated and cultures maintained viable for up to 8 weeks. Expression of several transport proteins (a.o. OAT1, OAT4, MRP2, MRP4 and megalin) was shown and long-term effects of commonly used drugs could be studied. To assess cell integrity, clinically relevant endpoints of nephrotoxicity were used, such as KIM1 and NGAL, upon exposure to several drugs, and confirmed the model’s validity. Overall, compared to 2D cell cultures, this 3D model was more sensitive to nephrotoxic drugs.64 Recently, cytotoxicity of Shiga Toxin type 2, a bacterial toxin produced by an Escherichia coli strain, was studied in this same model.65 Eminently, enhanced secretion of KIM1 and IL-8 compared to 2D cell cultures was observed, again validating the use of this 3D model.65 Another approach effectively applied in a gel-based 3D kidney model is freshly isolated proximal tubules encapsulated in a gel containing the natural polymer hyaluronic acid.35,36,66 Astashkina et al. developed this model using murine proximal tubules embedded in a hydrogel, containing the natural polymer hyaluronic acid.35 This model has been used for nephrotoxicity screenings under static conditions.35,36,66 Cells preserved well in this gel for up to 6 weeks without any significant phenotypical changes.35 Furthermore, cell viability and expression of OAT1, megalin and aquaporin-1 (AQP1) were maintained for up to 6 weeks.35 Upon exposure to nephrotoxic drugs, such as cisplatin or doxorubicin, levels of cytokines and KIM1 increased, indicating tubulotoxicity.35,36 More recently, the 3D proximal tubule model has been used to test nephrotoxicity of nanoparticles as well.66 Furthermore, for studying regenerative medicine, studying differentiation and branching of kidney cells in gelmatrices could be possible applications. However, as replacement therapy this method would not be suitable. The inability for significant up scaling of the 3D model in culture is a hurdle limiting its application for regenerative medicine. In addition, formation of hypoxic centers inducing necrosis is a risk for gel-based methods, especially for spheroid-like methods.83,84 Moreover, another significant limitation of this model is the lack of flow, especially since fluidic conditions appeared to be an important factor for expression and activity of several essential proteins. But these limitations can be addressed by microfluidic systems, currently widely under development.

82.3.3 Microfluidic Systems for 3D Kidney Modeling Microfluidic devices of 3D kidney models described contain two or more channels separated by permeable membranes or a gel, which are usually coated with ECM components before seeding cells (Fig. 82.2C). Implementation of multiple channels and flow allows for studying transcellular transport under fluidic conditions. The first described microfluidic 3D kidney models focused on proof-of-concept of cell growth using MDCK cells68 or primary rat inner medullary collecting duct (IMCD) cells.67 In addition, Wei et al. described a polydimethyl siloxane (PDMS) microfluidic system, channels coated with glass covered fibronectin containing HK-2 cells.70 In this model, formation of calcium phosphate stones in PTEC was studied. The feasibility of transfections within such a system was demonstrated by overexpression of green fluorescence protein (GFP), allowing investigation of the role of a specific transporter or ion channel in kidney stone formation.70 Positive effects of fluidic conditions on viability, expression of drug transporters, and sensitivity to nephrotoxic compounds were demonstrated in microfluidic 3D kidney models in comparison to static conditions.67 69,71,76 Furthermore, these models have been used for optimizing coating,69 membrane design,72 and drug screenings.74 76 Microfluidics meet the requirements of co-culturing, which stimulates signaling between diverse cell types. This was successfully demonstrated studying kidney fibrosis63 and mechanisms of renal transport.73 Mu et al. created a co-culture of primary human umbilical vascular endothelial cells (HUVEC) and MDCK cells in

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separated channels to study passive renal diffusion.73 Channels were constructed using a hydrogel mixture of collagen type I and alginate and diffusion was assessed using two CellTrackers dyes (green and red).73 Choucha-Snouber et al. developed a microfluidic biochip, containing multiple channels.68 Briefly, MDCK cells were seeded in channels on PDMS membranes and allowed to adhere for 24 hours. Experiments were then conducted under fluidic conditions for another 72 hours.68,74,75 Expression levels of several transporters were determined and activation of (oxidative) stress pathways, such as the Nrf-2 pathway, were compared to conventional 2D cell cultures under static conditions.68 Furthermore, this model was used in a similar set-up to assess ifosfamide-induced nephrotoxicity.74 Although treatment had no effect on MDCK cells’ viability under static conditions, an inflammatory response was induced upon ifosfamide exposure when biochips were used, again indicating that flow and 3D environment increase the sensitivity to toxicants. In addition, this model was combined with a 3D liver model using HepG2/C3a and HepaRG cells to study toxic effects of ifosfamide metabolites.75 The release of intracellular Ca21 by the MDCK cells upon exposure was demonstrative of an inter-organ response. Although the model is an excellent tool to study nephrotoxicity under fluidic conditions, the lack of compartmentalization, dividing apical from basal side, limits this system for replacement therapy. Recently, Jang et al. published an advanced biomimetic model of the kidney, consisting of a microfluidic device containing two channels separated by a PDMS membrane.76 This membrane was coated with collagen IV and seeded with primary human PTEC, and allowed to reach a confluent monolayer within 3 days. Expression of ZO-1, Na/K-ATPase, and AQP1 clearly improved under fluidic conditions. Moreover, morphology of renal proximal tubular epithelial cells, such as cilia presence and cell height, was more in vivo-like under fluidic conditions. Functionally, uptake of albumin and glucose was demonstrated, as well as activity of P-gp and brush border membrane alkaline phosphatase activity. P-gp activity, demonstrated using the calcein-AM assay,92 was inhibited following verapamil administration further confirming epithelial function. In addition, this kidney-ona-chip model demonstrated susceptibility for cisplatin-induced toxicity, using lactate dehydrogenase (LDH) release and apoptosis as endpoint readouts. Toxicity was less severe after co-treatment with the OCT2-inhibitor cimetidine under fluidic conditions, confirming earlier interaction studies performed under static conditions.93 95 In agreement with clinical observations,96 PTEC recovered from cisplatin-induced toxicity when culturing cells under drug-free conditions for 4 days after cisplatin exposure at the first day. These examples demonstrate the broad perspectives of the microfluidics system, however there are also some limitations. The most often applied membrane in microfluidics is PDMS, but membrane porosity is a serious problem and can affect compound concentrations due to adsorption. For example, adsorption of the fluorescent dye tetramethylrhodamine85 and the drug fluoxetine86 to PDMS have been described. But, it was demonstrated that coating the membrane with parylene could limit this adsorption.97 In addition, mimicking renal flow adds an extra dimension but also extra complexity. Flow is often implemented using pumps and tubings, making this system to some extent complicated as culture set-ups need to be adjusted. Fluidic conditions can also be created by other methods, such as leveling through mechanical constructions. OrganoPlatess (Mimetas BV, Leiden, The Netherlands) is an example of a microfluidic titer plate where flow is created by leveling.98 The plate consists of two or three channels separated by a phaseguide, which is used to establish a gel support. Channels can function both as gel or medium perfusion channel. These microtiter plates were shown to be suitable platforms for 3D culturing of HepG2 liver cells and human neuroepithelial stem cells (hNESCs) and was compatible with fluorescent assays, opening possibilities to use this platform in high-throughput screening (HTS).98,99 Developing a 3D kidney model for HTS requires techniques for fast and validated end-point readouts. For regeneration, one could think of cell- and biomarkers-based assays correlated with cell proliferation, cell viability, monolayer integrity, and functional assays, such as transport activity. End-point readouts following exposure to nephrotoxic drugs have been described widely for 3D kidney models.35,36,64 66,76 The kidney-on-a-chip model by Jang et al. could be compatible with HTS and it was demonstrated that this biochip was a better biomimetic method compared to the Transwell cell cultures under static conditions due to an improved epithelial phenotype.76 Several other 3D organoid models in microfluid systems have been described, and pioneering work led to development of a highly sophisticated model of the lungs on a chip.100 This model includes co-cultures of human alveolar epithelial cells and human pulmonary microvascular endothelial cells in two separate channels cultured on an ECM-coated PDMS membrane. Incorporating two vacuum channels alongside the microfluidic channel mimicked mechanical stress on the cells due to expansion of the alveoli. In this lung-on-a-chip, mechanical strain intensified silica nanoparticle-induced toxic response in alveolar epithelial cells due to increased uptake of the nanoparticles.100 It was proposed that employing such mechanical stress in addition to flow advances tissue performance of other organ systems as well, including the kidney-on-a-chip.101

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82.4 FUTURE PROSPECTS IN REGENERATIVE NEPHROPHARMACOLOGY Current therapy in CKD is dialysis and kidney transplantation, but both options have significant shortcomings. Dialysis treatment fails to arrest disease progression and has not made sufficient technological improvement the past forty years to ameliorate the decline in quality of life of patients with end-stage kidney disease. With respect to organ transplantation, the shortage in available kidneys along with the necessity of permanent immunosuppressive therapy after transplantation, which presents recipient patients with substantial morbidity, still form major clinical impediments. Moreover, the prevalence of CKD continues emphasizing the need for alternatives for which the emerging field of regenerative medicine holds great promise. Many advances in this field have been made recently but will no doubt expand further in future. As discussed in this chapter, developments in organ-on-chip technologies let miniature organs grow in the lab, using human or patient-derived cells. But also other techniques, such as native organ scaffolds102 or 3D printing techniques for development of microfluidic devices might form platforms in regenerative nephropharmacology.103 Furthermore, mini-organs, recently developed by Davies’ group, could evolve in more sophisticated models to fill the gap between in vitro and in vivo in understanding functional kidney development and repair.104 107 A significant amount of 3D organoid systems developed for disease modeling have been described, recently reviewed by Benam et al.9 A 3D organoid model involves bio-mimetic development of a certain organ for multiple purposes, ranging from organogenesis to toxicity screenings. Overall benefit is that a 3D culture better represents organ function on intercellular level improving its predicting value. More recently, advances were made in development of bioscaffolds. Researchers from the Wake Forrest Institute for Regenerative Medicine (NC, USA), and others, showed that decellularized kidneys from animals and human could be used as 3D biological scaffolds. These scaffolds can be used as templates for functional kidney reconstruction after recellularization with autologous stem cells or differentiated cells, and, eventually, be used as a transplantable organ.102,108 110 In addition, these recellularized organs can provide a drug discovery platform to study cells behavior in their natural environment, which is superior to studies with cells cultured in 2D platforms. Most recently, advances in 3D printing technology have allowed the creation of cellular 3D bioprinted kidney tissue, consisting of polarized human RPTEC and a living interstitial layer comprised of renal fibroblasts and endothelial cells. The tissue appeared to form E-cadherin positive junctions between the epithelial cells, microvascular structures, and maintained layered architecture for at least 2 weeks in vitro. Furthermore, expression of metabolism enzymes was found. Still, microfabrication of the complex vascular and tubular network of a native kidney facilitating its physiological function is a challenge that not only the 3D printing technology, but basically all technologies described in this chapter, are facing. In conclusion, innovative technologies in 3D kidney bioengineering, including bioartificial kidney development, gel-based organoid systems, and microfluidics, each have strengths and weaknesses in studying pharmacological and therapeutic approaches in regenerative nephrology. The need for alternatives in renal replacement therapy advances expanding these developments, and requires multidisciplinary methods in which engineers, biologists, pharmacologists, and clinicians cooperate. Such interdisciplinary approach may, eventually, lead to a proper understanding of the kidney’s regeneration and repair processes, which increases the possibility of development of clinically applicable treatment strategies in kidney disease.

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