Drug-Induced Nephrotoxicity: Clinical Impact and Preclinical in Vitro

Review pubs.acs.org/molecularpharmaceutics

Drug-Induced Nephrotoxicity: Clinical Impact and Preclinical in Vitro Models Ho Yee Tiong,† Peng Huang,‡ Sijing Xiong,‡ Yao Li,‡ Anantharaman Vathsala,*,† and Daniele Zink*,‡ †

Yong Loo Lin School of Medicine, National University Health System, 1E Kent Ridge Road, NUHS Tower Block, Singapore 119228, Singapore ‡ Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore ABSTRACT: The kidney is a major target for drug-induced toxicity. Druginduced nephrotoxicity remains a major problem in the clinical setting, where the use of nephrotoxic drugs is often unavoidable. This leads frequently to acute kidney injury, and current problems are discussed. One strategy to avoid such problems would be the development of drugs with decreased nephrotoxic potential. However, the prediction of nephrotoxicity during preclinical drug development is difficult and nephrotoxicity is typically detected only late. Also, the nephrotoxic potential of newly approved drugs is often underestimated. Regulatory approved or validated in vitro models for the prediction of nephrotoxicity are currently not available. Here, we will review current approaches on the development of such models. This includes a discussion of three-dimensional and microfluidic models and recently developed stem cell based approaches. Most in vitro models have been tested with a limited number of compounds and are of unclear predictivity. However, some studies have tested larger numbers of compounds and the predictivity of the respective in vitro model had been determined. The results showed that high predictivity can be obtained by using primary or stem cell derived human renal cells in combination with appropriate end points. KEYWORDS: drug-induced nephrotoxicity, nephrotoxicant, in vitro model, predictivity, renal proximal tubular cell, stem cell

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population rises to between 30% to 60%.8 Globally, the incidence of AKI has steadily increased between 1988 and 2003.6 At least part of this increase appears to be due to increased prescription of drugs that can cause AKI. For example, in the United Kingdom, a 5.1% increment in practice level prescription of angiotensin converting enzyme inhibitors or angiotensin receptor-II antagonist drugs over a 4 year period since 2007 was shown to account for a 14.8% increase in AKI hospital admissions.9 Drug or toxicant induced AKI accounts for about 20% of hospital- and community-acquired AKI.3,10 The main feature of AKI is a sudden decrease in kidney function over a variable period of time between hours to days, resulting in the accumulation of creatinine, urea, and other toxins.8 Other associated features of AKI are sodium and water retention and the development of metabolic acidosis and hyperkalemia. The Kidney Disease: Improving Global Outcomes (KDIGO) clinical guidelines workgroup in 201211 defined AKI as an increase in the serum creatinine level of 0.3 mg/dL (26.5 μmol/ L) or more within 48 h; a serum creatinine level that has increased by at least 1.5 times the baseline value in the first 7

INTRODUCTION Besides the liver, the kidney is a major target for drug-induced toxic effects. Kidneys receive nearly 25% of cardiac output and, as one of the major organs of excretion, are naturally exposed to a greater proportion of circulating drugs and chemicals.1 Renal toxicity has been reported for various agents including heavy metals, chemicals, fungal toxins, and a large number of drugs.2,3 The use of nephrotoxic drugs frequently leads to acute kidney injury (AKI). AKI is associated with increased morbidity and mortality. One strategy to avoid these problems would be the development of drugs with decreased nephrotoxic potential. However, it is difficult to predict nephrotoxicity during preclinical drug development, and the nephrotoxic potential of newly developed drugs is often underestimated.4,5 Such problems lead to substantially increased costs for the pharmaceutical industry. A major problem is the current lack of regulatory approved or validated in vitro models for the prediction of nephrotoxicity. Here, we will review the problems with drug-induced nephrotoxicity in the clinical setting. We will then give a comprehensive overview on the development of in vitro models for the prediction of drug-induced nephrotoxicity, with a focus on recent approaches.

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Special Issue: Engineered Biomimetic Tissue Platforms for in Vitro Drug Evaluation

CLINICAL IMPACT Drug-Induced Acute Kidney Injury. AKI has been estimated to account for 1% of hospital admissions in the United States and to develop in 5% to 7% of hospitalized patients.6,7 The incidence in the intensive care unit (ICU) © XXXX American Chemical Society

Received: November 28, 2013 Revised: February 5, 2014 Accepted: February 6, 2014

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dx.doi.org/10.1021/mp400720w | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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AKI19 in a dose dependent manner. Despite forced diuresis, dose reduction, and case selection, the incidence of cisplatin induced AKI remains high between 4% to 23%.22 Renoprotective agents have been tried but have unclear utility.22 Other well-known agents which cause similar clinical problems (although the cellular injury mechanisms are different) in the treatment of oncology patients include bevacizumab, gemcitabine, interferon, ifosfamide, pemetrexed, and methotrexate. Drug-Induced AKI in Sepsis. Sepsis is a systemic response to infection and is a common cause of multiorgan failure including AKI. During sepsis, hypotension, vascular endothelial dysfunction, and regional hypoperfusion within the kidneys are common features. Such renal insufficiency and volume depletion, which are exaggerated during sepsis, are risk factors for drug-induced nephrotoxicity by the same antibiotics needed to treat the infection. Antibiotics used in critically ill septic patients that also have potential to cause AKI include the aminoglycosides (gentamicin, amikacin), vancomycin, and amphotericin B (antifungal).23 Even a brief empiric course with aminoglycosides has a reported rate of AKI between 10% and 20%.23 This is due to accumulation of aminoglycosides, which are taken up by PTC by the megalin/cubilin endocytotic receptor complex, to levels vastly exceeding serum concentration. This in turn results in problems with protein synthesis and folding, disrupted protein sorting, increased lysosomal permeability, proteolysis and mitochondrial dysfunction, and probably other damages resulting in cell death.24 Vancomycin is important for the therapy of serious Staphylococcus aureus infections, especially methicillin-resistant Staphylococcus aureus infections. Initial reported incidence of up to 43% of AKI was attributed to impurities in the original formulation.25 With the modern purification, nephrotoxicity is still around the range of 5% to 7% after 4 to 17 days of its use, even when recommended target trough levels are used.25 While the mechanism of vancomycin-induced AKI is not clearly known,25 the condition was reversible in the majority of cases with short-term dialysis required in 3% of patients. Drug-Induced AKI in Transplantation. Kidney transplantation is the best treatment for patients with end stage kidney disease, and despite improved short-term outcomes from better immunosuppressant drugs, 3% to 5% of allografts are still lost yearly to chronic allograft nephropathy/calcineurin inhibitor nephrotoxicity. Cyclosporine and tacrolimus are calcineurin inhibitors in use for many years and are the basis of many immunosuppressive regimens because of their clinical success. However, standard recommended doses are associated with nephrotoxicity. Ten years after transplantation, all recipients of kidney and pancreas transplants on cyclosporinebased immunosuppression had varying degrees of chronic allograft nephropathy on protocol kidney biopsies due to calcineurin inhibitor nephrotoxicity.26 Nephrotoxicity was thought to be due to effects on larger renal vessels causing regional ischemia. While there are other mechanisms of renal injury due to these drugs, calcineurin inhibitors appear to be also toxic to renal cells, causing apoptosis.27 There is evidence that lower doses of calcineurin inhibitor use may reduce this chronic nephrotoxicity and reduce episodes of acute or chronic nephrotoxicity but increase the risk of rejection.28 The clinical dilemma is the uncertainty of whether episodes of AKI in kidney transplant dysfunction are due to cyclosporine toxicity (exacerbated by too high drug levels) or due to kidney transplant rejection (too low drug levels).

days; or a urine volume of less than 0.5 mL per kg body weight/hour for 6 h.11 This definition retains the earlier RIFLE (risk, injury, failure, loss of kidney function, and end-stage kidney disease)12 and AKIN (acute kidney injury network)13 staging criteria with the ability to prognosticate patients based on serum creatinine and urine output. In spite of progress in medical research in this area, AKI is still associated with high morbidity and mortality. In critically ill patients, AKI that requires dialysis is associated with a mortality of 40% to 70%.7,14 Statistics have not shown significant improvement despite advances in supportive therapy.8 Supportive measures include intensive care monitoring with the use of invasive central lines and urinary catheters to maintain careful fluid balance, reduction or removal of exposure to nephrotoxicants, drugs to induce renal diuresis and correct electrolyte imbalance, and if necessary dialysis in the form of hemofiltration. AKI with its complications exerts a direct effect on other organs and systems, and it contributes to multiorgan failure in critically ill patients. Several studies indicate that the development of AKI in any setting is a major risk factor for nonrenal complications and is an independent contributor to overall mortality.15 In survivors of the acute insult, damaged renal structures are regenerated and recellularized. However, maladaptive repair mechanisms following AKI appear to predispose to chronic kidney disease (CKD).15 As many as 50% of patients are estimated to have residual structural and functional renal defects that may predispose to subsequent episodes of renal failure or progression to end stage kidney disease.15 In addition to its clinical sequelae, AKI prolongs hospital stays and significantly increases hospital expenses with an annual estimated cost of $8 billion USD.16 In the U.K., the cost to the NHS of AKI was estimated to be between 434 million and 620 million pounds. Nephrotoxicants exert toxic effects on the kidney by one or several mechanisms, including altered systemic and local hemodynamics, direct toxic effects on renal cells, inflammation, and crystal nephropathy.2,3 Acute tubular necrosis is the most common cause of AKI and follows ischemic or nephrotoxic injury to the tubules. The epithelial cells of the renal proximal tubule (PTC) are a major target for nephrotoxicants due to their roles in glomerular filtrate concentration and drug transport and metabolism.17,18 Several drugs are widely used despite known evidence of kidney injury, PTC toxicity, and other systemic toxicity. These include aminoglycosides, vancomycin, cisplatin, and iodinated radiographic contrast agents. Several clinical scenarios particularly render the kidneys vulnerable to drug-induced nephrotoxicity and subsequent AKI. These are briefly summarized in the following paragraphs. Drug-Induced AKI in Oncology. Despite improvements in chemotherapeutics for various malignancies, nephrotoxicity remains a complication and sometimes limits life-saving therapy. Not all patients exposed to nephrotoxic chemotherapeutic agents develop AKI, as different factors enhance patient risk for nephrotoxicity. These include tumor related kidney effects, innate drug toxicity, patient factors, and renal drug handling.19 Cisplatin is the archetypal nephrotoxicant and is a platinum compound that is effective therapy for many carcinomas, sarcomas, and lymphomas. Cisplatin enters PTC through the organic cation transporter 2 (OCT2) and the copper transporter Ctr1.20 After entering the cells it causes cell injury by damaging nuclear and mitochondrial DNA. This leads to apoptosis and necrosis of tubular cells20,21 and causes clinical B



Review

Drug-Induced AKI in Diagnostic Imaging. Radiocontrast agents are widely used for contrast-enhanced cross sectional imaging, as well as in interventional radiological procedures such as angiography. Contrast-induced AKI is reported to account for 11%−12% of all cases of inpatient AKI, making it a very common cause of hospital acquired AKI.29 In coronary angiography, the incidence rate of contrast-induced AKI was as high as 14.5% with inpatient mortality rate of 7.1% in those not requiring dialysis and 35.7% in those requiring. It has been defined specifically as a rise of >0.5 mg/dL in serum creatinine or 25% increase, assessed within 48−72 h after administration of the contrast medium.29 It has been reported that it causes injury due to direct tubular cell toxicity associated with the formation of reactive oxygen species as well as by inducing regional ischemia.2,3 Other Implications on Diagnosis and Prediction of Drug-Induced AKI. As described above, the current gold standard for detection of AKI relies on the laboratory measurement of increased serum creatinine and/or plasma urea, products of nitrogen metabolism. Indeed, measurements of serum creatinine and urine output, an even less sensitive measure of renal function, constitute current diagnostic criteria for AKI.11−13 Unfortunately, laboratory measurements of urea and creatinine are insensitive markers of decreased glomerular filtration rate (GFR) and are measurably abnormal only after nearly 50% of GFR has been lost.8 Further, their levels are affected by nutrition and food intake, fluid resuscitation, muscle mass, age, gender, muscle injury, use of steroids, the presence of blood in the gastrointestinal tract, and other factors, rendering them suboptimal markers for detection of early AKI. Delay in detection of AKI due to the insensitivity of creatinine and urea as adequate and early biomarkers leads to loss of a therapeutic opportunity. There is thus an urgent clinical need for more sensitive biomarkers to detect AKI at an earlier stage. As such, there has been extensive research into developments of novel serum and urine biomarkers with high sensitivity and specificity for diagnosing renal cellular injury (see, for instance, refs 30−37 from the extensive body of literature on this topic). Such potential novel biomarkers include clusterin, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL, also called lipocalin 2), which are upregulated in injured PTC.30,31,34−37 It remains to be seen if such potential novel biomarkers meet the current clinical needs.32 Nevertheless, such biomarkers hold promise for improving safety during drug development.33 Apart from early detection of AKI, there is also a clear need to prevent AKI by developing therapeutic drugs with reduced nephrotoxic potential.

milestones.htm) are currently not active in the evaluation of respective methods (a summary of current validated and accepted alternative methods is provided by AltTox: http:// www.alttox.org/ttrc/validation-ra/validated-ra-methods.html). The European Centre for the Validation of Alternative Test Methods (ECVAM, competence inherited by the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM)) has funded one prevalidation study, which was performed with two animal cell lines and 15 compounds and published in 2002.38 This study used transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) flux as end points. There were no validation studies on this or other models for in vitro nephrotoxicology. What are the difficulties in developing in vitro assays for the prediction of drug-induced nephrotoxicity? Research studies on this topic that were published in this millennium are listed in Table 1. Most of these studies were retrieved from PubMed by using search terms like “nephrotoxicity”, “in vitro”, “proximal tubule cells”, and combinations of these terms as well as “in vitro nephrotoxicology”, “nephrotoxicity and 3D”, “nephrotoxicity and chip”, and “nephrotoxicity and microfluidics”. Except for two studies on three-dimensional (3D) models using hydrogel-embedded isolated murine renal proximal tubules (PT),39,40 only cell culture based models were selected. The search focused on research articles describing new cell models, 3D or microfluidic models, and models where larger numbers of compounds have been tested to determine predictivity. Not all studies focusing mainly on mechanistic aspects, where only 1 or few compounds had been tested, were included in Table 1. Interestingly, only 3 studies were retrieved from PubMed by using the search term “in vitro nephrotoxicology”. In this case, the database suggested instead the search term “in vitro neurotoxicology” with 596 items. Such findings strengthen the notion that research on in vitro models for nephrotoxicology is generally underrepresented compared to other organ systems. Table 1 reveals one of the main problems in the development of in vitro models for the prediction of drug-induced nephrotoxicity. Most of the studies have been performed with a very limited number of compounds, which does not allow determination of the predictivity of the model. Only 5 studies have been performed with >10 compounds, and in only 3 cases have major performance metrics such as sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) been determined. Many studies that investigated 2 mM) of gentamicin60,61 and tobramycin.60 Human Primary Renal Proximal Tubular Cells. Together, the findings summarized above suggest that HK-2 cells have limitations with respect to applications in in vitro nephrotoxicology, and especially the uptake of drugs is compromised. This is in accordance with the finding that compared to human primary renal proximal tubular cells60 (HPTC; this acronym will be used here, also with respect to studies where different acronyms have been used for primary human PTC) and stem cell derived HPTC-like cells the sensitivity and overall predictivity were markedly reduced when HK-2 cells were used60,62. In these studies aiming at the development of predictive models, a comprehensive comparison of different PTC cell types was performed and 41 well-characterized compounds were screened. These included 22 compounds that are known to be toxic for human PTC in vivo, 11 nephrotoxicants that are not toxic for human PTC in vivo, and 8 compounds that are not nephrotoxic in humans. Best in terms of predicting PTC toxicity in humans were HPTC (mean area under the curve (AUC) value of the receiver operating characteristic (ROC) curves of 0.85), followed by human embryonic stem cell (hESC) derived HPTC-like cells (AUC value of 0.80; the AUC values obtained for HK-2 and Lewis lung cancer-porcine kidney 1 (LLC-PK1) cells were 0.71 and 0.73, respectively). In the following, HPTC and hESC-derived HPTC-like cells will be addressed in more detail. Human primary PTC (HPTC) have been applied in various studies on in vitro nephrotoxicology (Table 1). It has been shown that many PTC-specific drug transporters are expressed in monolayer cultures of HPTC, at least during early passages. In a study investigating cultures of HPTC from different donors up to day 5, OAT1−4 were detected, and expression levels of OAT1 and OAT3 were highest.63 Expression of OCT2 was low and variable among cultures from different donors, and the main organic cation transporter was OCTN2 (organic cation/ carnitine transporter 2). MRPs were detectable as well as Pglycoprotein. Functional studies confirmed expression of these diverse arrays of transporters for major classes of important drugs, and also showed functionality of the peptide transporter 2 (PEPT2).63 These results were in agreement with another study investigating monolayer cultures of human primary proximal and distal tubule/collecting duct cells after 10 days in culture.64 The results revealed expression of the Na+/phosphate cotransporter NaPiIIa, the sodium-dependent glucose cotrans-

Figure 1. Drug transporter activities of HPTC. HPTC were detached from monolayer cultures and suspended in indicator-free vehicle (DMSO)-containing cell culture medium, or medium containing inhibitors for specific drug transporters. The following inhibitors were added (affected transporters are indicated in parentheses): 20 μM verapamil (MDR1), 50 μM MK-57 (MRP1/2), 100 μM novobiocin (BCRP), and 50 μM tetrapentylammonium (OCT2). After incubation for 5 min eFluxx-ID Gold dye solution (eFluxx-ID Gold multidrug resistance assay kit, Enzo Life Sciences, Farmingdale, NY, USA) was added for measuring the activities of ATP-binding cassette (ABC) efflux transporters (MDR1, MRP1/2, and BCRP). For measuring the activity of the uptake transporter OCT2, 25 μM ASP+ (4-(4(dimethylamino)styryl)-N-methylpyridinium iodide) was added. After incubation with the fluorescent substrates for 30 min, propidium iodide was added to monitor cell viability, and cells were analyzed immediately by fluorescence-activated cell sorting. The diagrams show the fluorescence intensities (n = 3) of the intracellular eFluxx-ID Gold dye (A, B, and C) or of ASP+ (D; vehicle controls, white; inhibitortreated samples, gray). On each panel it is indicated which transporter was inhibited. Inhibition of efflux transporters increases intracellular fluorescence, whereas inhibition of the uptake transporter OCT2 decreases intracellular fluorescence. Data were analyzed by calculating MDR activity factor (MAF) values.107 Samples with MAF values >25 are considered as being positive for transporter activity. This was the case for all results shown here. F



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Figure 2. Morphology of hESC-derived HPTC-like cells in vitro. (A, B) Differentiated simple epithelium generated on TCPS. Tight junctions were detected by immunostaining of zonula occludens-1 (ZO-1) (red; cell nuclei, blue). The boxed region (A) is shown enlarged in panel B. (C, D) The apical brush border was imaged by scanning electron microscopy. (E) Overview of tubule-like structures generated on Matrigel (cell nuclei, blue). (F) On Matrigel HPTC-like cells generate cords consisting of rows of single cells (arrowheads) and multicellular tubule-like structures (arrows). The tubule-like structures marked with arrows are shown enlarged in the insets (right). Scale bars: (A, B) 200 μm, (C) 3 μm, (D) 4 μm, (E) 500 μm, and (F) 200 μm. Reprinted with permission from ref 67. Copyright 2013 The Authors.

involved in drug metabolism. These include γ-glutamyl transferase (GGT), γ-glutamylcysteine synthetase, glutathione S-transferases (GSTA, GSTP, and GSTT), glutathione (GSH) disulfide reductase, and GSH peroxidase.65,66 The cytochrome P450 (CYP) enzymes found to be expressed are CYP1B1, CYP3A4, CYP3A5, CYP4A11, and possibly CYP2D6.65,66 Three sulfotransferases and three UDP-glucuronosyltransferases were expressed as well.66 Interdonor variability as well as some decrease of enzymatic activities over time was observed, although enzymatic activities remained detectable. Altogether, the results summarized above show that monolayers of HPTC express a broad variety of transporters and drug metabolizing enzymes and possess significant capacity for transport and metabolism of many classes of drugs. However, the disadvantages of this cell model are obvious and include interdonor variability, functional changes during passaging, and low expression levels of some transporters and enzymes. Other major problems are cell source limitations and the limited proliferative capacity. Human Proximal Tubular Cells Immortalized with Human Telomerase Reverse Transcriptase. Currently, two major strategies are used to address these problems: one strategy is “mild” immortalization of HPTC with human telomerase reverse transcriptase (hTERT). Alternatively, stem cell based approaches are attractive. Recently, the first protocol was

established for the generation of HPTC-like cells from human embryonic stem cells.67 Both approaches will be described in the following paragraphs. An excellent review of other renal cell models and non-hTERT-based immortalization strategies is provided in ref 68. The first HPTC-derived cell line that was immortalized by overexpression of hTERT was established by Wieser et al. (2008)69 and is called RPTEC/TERT1. These cells display morphological and functional characteristics of HPTC and express the megalin/cubilin transport system. Recently a transcriptomic and metabolomic analysis of HPTC and RPTEC/TERT1 monolayers that matured over a time period of 16 days revealed major changes after confluency was reached.70 As characteristic features of HPTC, such as cell−cell junctions, can only be expressed in the confluent state, it is probably preferable to work with confluent cultures. RPTEC/ TERT1 cells have been applied in several recent mechanistic studies using limited numbers of compounds, which included a detailed transcriptomic, proteomic, and metabolomics analysis of cyclosporine A-induced changes.71−74 Part of this work was funded by the Predict-IV project (European Union’s seventh Framework Programme (FP7/2007−2013)). The Predict-IV project addressed the development of preclinical in vitro models for the prediction of drug-induced toxicity. In the course of this project, it was also tested whether G



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transplants, high VIM expression in the tubular epithelium is predictive of graft dysfunction.80 Other typical markers that are upregulated in vivo after PTC damage are the paired box gene 2 (PAX-2), NGAL, KIM-1, and clusterin.31,32,34,35,79,81,82 These markers are typically also upregulated and expressed in in vitro cultures of primary rat and mouse PTC and HPTC that have not been exposed to any toxicants.60,76,78,81 The fact that primary cultures of rodent PTC and HPTC upregulate a whole panel of typical injury markers strongly suggests that the primary cells are functionally in an injury response state. In vivo, cells expressing injury markers transiently adopt a proliferative and dedifferentiated phenotype.77,81−83 In vitro, isolated cells have proliferative capacity, and the typically observed downregulation of differentiation markers and differentiated functions in vitro is probably not just “dedifferentiation”, but a normal part of an injury response state of these cells. A highly interesting gene expression microarray analysis on primary rat PTC showed that these cells upregulated typical stress and injury markers, while characteristic differentiation markers were downregulated.76 Such differentiation markers included drug and other membrane transporters, as well as genes for enzymes catalyzing phase I and phase II reactions and enzymes involved in oxidative stress management (typically such transporters and enzymes continued to be expressed, but at lower levels compared to the in vivo situation). However, other genes, including those coding for extracellular matrix (ECM) proteins and the basolateral glucose transporter GLUT1, were strongly upregulated. This suggested that the cells not only displayed an injury response but also adapted to the specific conditions of the in vitro environment.76 We observed comparable gene expression patterns with expression of injury markers, upregulation of the basolateral glucose transporter GLUT5 and relatively low expression levels of some drug transporters in HPTC.60 Probably it is not surprising that primary PTC show a typical injury response, including changes in gene expression patterns and the proliferative capacity, given the fact that these cells are obtained by organ disruption and isolation procedures which they would never experience under normal conditions in vivo. Also other adaptive changes are not unexpected as the cultivation conditions are usually very different from the normal in vivo environment. This involves not only the absence of a 3D architecture or microfluidic shear stress but also the absence of the typical chemical microenvironment with a complex ECM and different fluid compositions at the apical and basal sides. Also mechanical substrate properties need to be considered, and surprisingly HPTC as well as primary human endothelial cells perform best on stiff substrates in vitro.84 This surprising finding is currently further addressed, but it might reflect the altered functional state of the cells or the requirement of mechanical stress in the absence of shear stress. Finally, also the large variety of cues from neighboring and more distant cells in the body is absent in vitro, and the importance of this influence was recently demonstrated in an in vitro coculture system of HPTC and primary human endothelial cells.85 Cell States and Conditions in Vivo and in Vitro. Given all of these complex features, it will be challenging to develop really “biomimetic” systems, and addressing only one aspect by putting the cells, for instance, in a 3D gel or by applying shear stress will be not sufficient. It is also not clear how a respective improvement in the functional state should be determined, but given the complex functional changes, measuring only the

the nephrotoxicity of compounds could be predicted by using models employing HPTC or RPTEC/TERT1. End point was the expression of the potential novel AKI biomarkers clusterin, KIM-1, and NGAL (also called lipocalin 2). No upregulation of these potential biomarkers was observed after treatment with nephrotoxicants (Predict IV, third and fourth Annual Report, http://www.predict-iv.toxi.uni-wuerzburg.de/periodic_reports/ ). Also the establishment of transport assays was not successful with these two cell types. It was concluded that the culture conditions were not amenable (Predict IV, fifth Annual Report). In these studies, cells were kept in static monolayer cultures on tissue culture plastic (TCPS) or Transwell filter supports. However, many studies showing expression and proper functioning of drug transporters and drug metabolizing enzymes in HPTC were performed with comparable monolayer cultures. An alternative explanation for at least some of these negative results of the Predict-IV project could be the end points used. This will be further discussed below. Pluripotent Stem Cell Derived Cells. As mentioned above, another approach to develop an unlimited cell source is to use stem cell derived renal cells. A protocol has been developed for the differentiation of human induced pluripotent stem cells (hiPSC) into podocyte-like cells,75 and recently a method for the generation of stem cell derived HPTC-like cells in vitro has been published.67 Currently the method is based on hESCs. It involves cultivation of the hESCs on an extracellular matrix in renal epithelial cell medium in the presence of defined concentrations of bone morphogenetic protein (BMP)2 and BMP7. In vitro hESC-derived HPTC-like cells display characteristic morphological features of HPTC such as the formation of a differentiated simple polarized epithelium with tight junctions and an apical brush border, and formation of tubule-like structures when cultivated on a gel (Figure 2). The cells display characteristic functional features of HPTC including pH-dependent ammoniagenesis, water transport, GGT activity, and response to parathyroid hormone. Functional features are maintained in bioreactors under shear stress. Further, HPTC-like cells generate tubular structures in implants in vivo and integrate into renal epithelia in ex vivo organ cultures.67 Gene and protein expression patterns were strikingly similar to those of HPTC, although MEG and OAT3 were expressed at lower levels in HPTC-like cells.67 This could provide an explanation why the sensitivity of HPTC-like cells was lower compared to HPTC when the predictive performance was tested in vitro with a set of 41 compounds that are either toxic or nontoxic for PTC in humans.62 However, the sensitivity of HPTC-like cells was higher than the sensitivity of HK-2 or LLC-PK1 cells, and also the overall predictive performance of the in vitro model was improved when HPTC-like cells were employed (compared to HK-2 or LLC-PK-1 cells62). Together, the results suggested that hESC-derived HPTC-like cells are suitable for applications in in vitro models for the prediction of PTC toxicity in humans. Currently the approach is adapted to hiPSC. Expression of Stress and Injury Markers in Vitro. The striking similarities between HPTC and HPTC-like cells with respect to gene and protein expression patterns included expression of high levels of vimentin (VIM).67 Expression of VIM in primary cultures of rat PTC76,77 or HPTC60,67,78 has been consistently observed in vitro, in proliferating as well as in confluent cultures. In vivo, VIM is normally not expressed in PTC, but is upregulated after injury.77,79 In human kidney H



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One of the best investigated potential novel biomarkers for AKI is NGAL. NGAL expression is not restricted to the kidney, and NGAL can be expressed in a wide variety of cell types.90,91 Upregulation of NGAL occurs in the course of many benign and malignant human diseases and is associated with inflammation or infection.90,91 NGAL is upregulated by a wide variety of stimuli associated with tissue damage and is involved in inflammatory and immune responses. The nuclear factor-κB (NF-κB) is important for NGAL regulation, and the NGAL promoter has an NF-κB binding site.91−93 NGAL shares all of these features (expression in a wide variety of cell types, upregulation in many diseases and in response to tissue damage, regulation of inflammatory, and immune responses) with the proinflammatory interleukins (IL) IL-6 and IL-8, which are also target genes of NF-κB.94−97 As in the case of NGAL, activation of NF-κB and upregulation of proinflammatory interleukins occurs in PTC in vivo in response to nephrotoxicant-induced injury.98−100 In fact, also the proinflammatory interleukins IL-6, IL-8, and IL-18 had been proposed as potential novel biomarkers for the detection of nephrotoxicant-induced kidney injury.91 Given these findings we tested upregulation IL-6, IL-8, and IL-18 in comparison to NGAL, KIM-1, and VIM in response to gentamicin and CdCl2 in HPTC in vitro. Significant and consistent upregulation of IL-6 and IL-8 was observed, whereas less clear responses were obtained with NGAL, KIM-1, VIM, and IL-18.60 Further, by using IL-6 and IL-8 expression as end points in in vitro models based on HPTC or hESC-derived HPTC-like cells, the PTC toxicity of compounds could be predicted with high accuracy60,62. In these studies, a test set of 41 compounds was used. In contrast, when otherwise the same conditions and cell types were used, the predictivity was low when cell viability, lactate dehydrogenase (LDH) leakage, GSH depletion, or ATP depletion was used as end point. The results obtained with respect to ATP depletion were in agreement with a recent study on organ-specific toxicity using 621 compounds. In this study, ATP depletion was used as end point, and low sensitivities together with high false-negative rates were observed.41 So far, the results of the various studies suggested that IL-6 and IL-8 expression could be suitable end points for in vitro models for the prediction of PTC toxicity. Usual points of criticism with respect to these end points are that they are not cell type specific and are triggered by a wide variety of stressors. This is correct. But is this a problem? With regard to this question it is important to consider which kinds of specificities end points display and what the respective requirements are in in vitro and in vivo. With respect to the specificity of end points it is important to distinguish between different types of specificity (Table 2): (i) cell type specificity, (ii) mechanism of injury specificity, and (iii) cellular response/pathway specificity. Cell type specificity is important in vivo for diagnostic purposes in order to determine the site of injury. This is, for instance, a problem with respect to NGAL, which is expressed in many cell types in the human body. Plasma levels of this and other potential novel biomarkers for AKI rather correlate with the severity of the disease than with true kidney damage.32 However, for in vitro models usually only one cell type is selected and therefore the cell type specificity of the end point does not play a role. What is important is that the end point reliably indicates damage of the cell type selected, and whether this occurs in a cell type specific way depends on the cell type specific differentiation

improvement of some cellular functions will not be very informative. At the level of marker expression we suggest to determine at least the expression of injury markers (VIM, NGAL, KIM-1, PAX-2, and clusterin), glucose transporters (GLUT1 and GLUT5), drug transporters, drug metabolizing enzymes, and stem cell markers (CD133, CD24; see below) in addition to other differentiation markers to assess whether the cells were switched back under particular culture conditions into a state similar to the normal cell state in vivo. In principle, this appears to be possible. An increasing number of studies provides strong evidence that in vivo the regeneration of injured tubules is not accomplished by a resident stem cell population or precursor cells, but rather by previously fully differentiated surviving PTC that only transiently dedifferentiate and proliferate during repair.79,81−83,86,87 These cells express the typical injury markers (VIM, NGAL, KIM-1, Pax-2, and clusterin) as well as stem cell markers (CD133 and CD24). As PTC expressing such markers appear to adopt again the fully differentiated state after injury repair in vivo, it should be principally possible to obtain fully differentiated PTC from cells expressing such markers also in vitro. Altogether, the results summarized above show that PTC display a high degree of plasticity, which appears to be linked to their regenerative potential in vivo. Further, the results suggest that currently no in vitro model exists where PTC are in a normal in vivo like state, and it will be probably a long way to develop such models. As long as this has not been achieved, it cannot be expected that PTC respond in vitro in the same way as in vivo. Most critical in this regard appear to be dose− response relationships, and typically high doses of PTC-specific toxicants need to be applied in vitro in order to obtain a response.44,60,61,88 This was predicted, based on the observed downregulation of drug metabolizing enzymes and transporters in vitro76 (as mentioned, most of these genes remain to be expressed, but at lower levels). As it will be a tough nut to develop fully biomimetic models and achieve normal cell behavior in vitro, it would be currently most straightforward to carefully characterize PTC under in vitro conditions and to determine how they respond differently from their in vivo counterparts. This would allow to interpret in vitro results properly and to predict in vivo responses correctly, also in the presence of functional differences. End Points. One recent example for problems that may be associated with a limited understanding of the functional cell state in vitro are the difficulties with identifying appropriate end points and the failure of getting consistent results with potential novel biomarkers for AKI in vitro. As outlined above, such potential novel biomarkers, including KIM-1, NGAL, and clusterin, are upregulated in PTC in vivo after kidney injury. This has been also observed with respect to AKI induced by various nephrotoxic drugs.30,31,33,35,36,89 Therefore, it was thought that upregulation of KIM-1, NGAL, and clusterin could be suitable end points for in vitro assays for the prediction of drug-induced nephrotoxicity. However, consistent upregulation in vitro was not observed after exposure to nephrotoxicants (Table 1), also not in a recently developed 3D model44 or in vitro models based on HPTC or RPTEC/TERT1 cells (ref 60 and Predict IV, third and fourth Annual Report, http://www. predict-iv.toxi.uni-wuerzburg.de/periodic_reports/).These results might be explained by the fact that these biomarkers are already upregulated in vitro in the absence of nephrotoxicants, in contrast to the in vivo situation. I



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status (e.g., drug transporter expression), but not on the cell type specificity of the end point. Further, end points can be specific for the mechanism of injury. In this respect it is worth mentioning that the potential novel biomarkers for AKI give no mechanistic insights. Their upregulation in vivo indicates in the best case that a particular renal cell type has been damaged, but not how. In vitro, the specificity for a particular injury mechanism inversely correlates with predictivity if a library of compounds is screened that injure cells by various uncharacterized mechanisms (Figure 3). Therefore, end points that are specific for a particular injury mechanism are very useful for mechanistic studies, but not for predictive models with major applications in early preclinical screening. In addition, end points can be specific for particular cellular responses/pathways that are triggered by toxicant-induced injury. In this respect it should be noted that cellular responses and respective pathways are often highly conserved and not specific for a particular cell type or mechanism. The same applies to respective end points, and this is illustrated in Figure 4. Apoptosis and necrosis, both resulting in cell death, can both be triggered by different drugs (cisplatin and acetaminophen) in different cell types (PTC or hepatocytes) by different mechanisms, depending on the cell type specific expression patterns of drug metabolizing enzymes and transporters. Cell

Table 2. End Points Have Different Types of Specificity with Different Relevance in Vivo and in Vitroa specificity cell type injury mechanism cellular response/ pathway

in vivo

in vitro

important for determining the site of injury and correct diagnosis no insight provided by classical diagnostic markers for AKI and potential novel biomarkers often highly conserved and not specific for cell type and injury mechanism

not important, typically only one cell type selected specificity correlates inversely with predictivity often highly conserved and not specific for cell type and injury mechanism

a End points display three different types of specificity: (i) cell type specificity, (ii) mechanism of injury specificity, and (iii) cellular response/pathway specificity. The cell type specificity of the end point is usually important in vivo for diagnostic purposes in order to determine the site of injury. In contrast to the many cell types present in vivo, typically only one cell type is selected for in vitro models. In in vitro models the cell type specificity of the response is determined by the differentiation status of the selected cell type, and not by the end point. In vivo, classical diagnostic markers for AKI (blood urea nitrogen and serum creatinine) and potential novel biomarkers do not provide insight into the injury mechanism. In vitro, the specificity of the end point for a particular injury mechanism inversely correlates with its predictivity when compounds are screened that injure cells by various mechanisms (Figure 3). Cellular responses and pathways triggered by cellular stress and injury are often highly conserved and specific neither for the cell type nor for the mechanism of injury (Figure 4).

Figure 3. The injury mechanism specificity of end points inversely correlates with predictivity. The figure shows the typical 96-well plate design for high-throughput screening. Blue bars with increasing thickness on the left indicate increasing compound concentrations. The left-hand plate shows the distribution of potentially nephrotoxic compounds on the plate. Four compounds would be PTC-specific toxicants (A, B, C, and D, red), that would injure this cell type by mechanisms a, b, c, and d. This information would be unknown in a typical preclinical screen. The right-hand plates show typical test results (concentration-dependent positive results green and orange). If an end point specific for mechanism “a” would be used (upper right-hand plate), the information would be obtained that this is the injury mechanism in the case of positive test results. However, in the example shown here only one of four PTC-specific nephrotoxicants would be detected, and the sensitivity would be only 25%. In contrast, if an end point would be used that is generally triggered by cell stress or injury, no information on the injury mechanism would be obtained. However, all compounds that damage PTC and induce therefore cell stress or injury could be detected, and the sensitivity would be 100%. J



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Microfluidic and 3D Models. Generally it is assumed that 2D and static cultivation conditions are artificial and therefore results could be improved by using 3D and microfluidic models. The question of how a good performance of in vitro models can be obtained requires a differentiated discussion, and also the properties of the different tissues and cell types must be taken into account. For instance, PTC form simple (single cell layer) epithelia in vivo. This is in contrast to other cell types, such as hepatocytes, which are arranged into true 3D structures in vivo. Further, the discussion above suggests that some of the negative results obtained, so far, were probably due to the selection of inappropriate cell types and end points and not due to the use of inappropriate culture conditions. A clear disadvantage of current microfluidic and 3D models is their incompatibility with high throughput screening. This problem might be solved in the future by advanced imaging techniques.46,47 In addition, 3D gels compromise the controlled application of compounds and probably also the detection of cellular responses.44 However, although it is the classical approach to use 3D gels for the generation of kidney tubules in vitro,101,102 3D gels can be avoided. Recently it was shown that large and functional human renal tubules can be generated in a gel-free environment on the TCPS surface (Figure 5).103 In this model, the kidney tubules are freely accessible for compound application and imaging. It was shown that nanoparticles can be applied in controlled manners to such gel-free tubules, and their uptake and transport were investigated by high-resolution imaging in a study on nanotoxicology.104 Two different 3D models based on hydrogel cultures have been employed for nephrotoxicology.39,40,44 One model used freshly isolated murine PT embedded in a modified hyaluronic acid based hydrogel.39,40 Cell viability dropped about 2-fold within the first 2 weeks of culture. This murine model would require the use of animals, and results are expected to be affected by interspecies variability. Due to the requirement for fresh tissue it will be difficult to adapt it to human material. The model was characterized by immunostaining, reverse-transcriptase polymerase chain reaction (RT-PCR), and assays for enzymatic activities and gluconeogenesis. After treatment with cisplatin several metabolites of this drug were detected.40 In order to assess the effects of toxicants, the 3D constructs were exposed for 3 days to doxorubicin, colchicine, 4-aminophenol, and cisplatin (negative control: untreated samples). The end point measured was cytokine release. Consistently negative results were obtained with tumor necrosis factor alpha (TNFalpha) secretion as end point. All four drugs increased the release of IL-6 and monocyte chemoattractant protein-1 (MCP-1). RANTES (regulated on activation, normal T cell expressed and secreted) and macrophage inflammatory protein1alpha (MIP-1alpha) were increased by three of the four compounds (4-aminophenol was negative), and IL-1beta was increased by cisplatin and doxorubicin.40 Thus, with the exception of TNFalpha, consistently positive results were obtained with cisplatin and doxorubicin. Doxorubicin displays profound cardio- and hepatotoxicity in humans. Renal side effects are rare and mainly due to glomerular damage.105 The same four drugs were also applied in another study, in which the performance of this 3D model was compared to the performance of monolayer cultures.39 Major differences were found. Whereas the 3D model employed murine PT, the immortalized cell lines were of porcine (LLC-PK1) and human origin (HEK293). As mentioned above, HEK293 cells appear to be of neuronal origin.54

Figure 4. Cellular pathways and responses are often highly conserved, and respective end points are not cell type specific and not informative with respect to the injury mechanism. Necrosis and apoptosis result in cell death, and measurement of these end points is widely applied. Necrosis and apoptosis are triggered in PTC by cisplatin20,21 and by acetaminophen in hepatocytes.108−111 Hepatotoxicity is typically associated with overdoses of acetaminophen, but renal insufficiency is rare.112 Cisplatin has dose-limiting nephrotoxicity due to the relatively high degree of accumulation in the kidney.113 The basolateral transporters OCT2 and Ctr1 mediate the uptake of cisplatin by PTC.20 After uptake, cisplatin damages nuclear (blue) and mitochondrial DNA (red arrows indicate damage).20 Acetaminophen is metabolized in hepatocytes by CYP2E1 (pink dots).114 The metabolite depletes GSH pools and leads to oxidative damage of mitochondria (light-brown ovals).110,114 CYP2E1 is not expressed in PTC.65,66

viability and apoptosis/necrosis are widely used end points in in vitro nephrotoxicology (Table 1). Together, the considerations outlined above suggest that the differentiation status of the selected cell type is important in in vitro assays for the prediction of nephrotoxicity, but not the cell type specificity of the end point. Further, end points that are specific for a particular injury mechanism are useful for mechanistic studies, but not for predictive models for early preclinical screening. Here, it would be probably the most efficient strategy to select an end point that reacts to cellular stress or injury induced by a broad variety of toxicants (and mechanisms) in order to obtain high predictivity (Figure 3). If some of the compounds that were predicted to be toxic in this way should still be of interest, such selected compounds can be further characterized. Such a screening strategy would be probably more efficient than multiplexing different mechanismspecific end points in order to obtain good predictivity in early screens. End points currently used in in vitro nephrotoxicology that indicate cellular stress or injury induced by a wide range of toxicants and mechanisms are lactate production88 and IL-6/IL8 expression.60,62 K



Review

over time in 2D cultures of NKi-2 cells, which were used for comparison, but not in 3D cultures. This was explained by potential adsorption of KIM-1 to the 3D matrix. In terms of NGAL expression there was not a big difference between 2D and 3D cultures. LD50 values were calculated based on the results on LDH release (the term LD50 values was used in the study, but probably these were EC50 values). With respect to cisplatin there were no significant differences between 2D and 3D cultures. Significant differences were found with respect to gentamicin and doxorubicin, and in this case 3D cultures were more sensitive.44 As outlined above, cisplatin is a well-known nephrotoxicant, whereas renal side effects of doxorubicin are rare and usually not due to PTC damage. A microfluidic model was recently described, where commercial HPTC were grown in a microfluidic chip on a porous polyester membrane coated with collagen IV.45 HPTC differentiation was found to be compromised on such membranes in comparison to TCPS.84,106 After growing to confluence, cells were exposed to fluid shear stress of 0.2 dyn/ cm2 for 3 days.45 Cell morphology, marker expression, albumin uptake, and glucose transport were improved under fluidic conditions. The toxic effects of cisplatin were determined by measuring LDH release and apoptosis. Significant changes in LDH release and the percentages of apoptotic cells were observed under static and fluidic conditions, and the toxic effects were more pronounced under static conditions. Amelioration of cisplatin toxicity was observed in the presence of the OCT2 inhibitor cimetidine, and these effects were more pronounced under fluidic conditions. Improved cell recovery during a period of four days was observed under fluidic conditions when cisplatin was washed out after day 1 of exposure.45

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Figure 5. Large and functional human renal tubules generated by HPTC in a gel-free system. Panel A shows a tubule on the TCPS surface of the culture dish. Human renal tubules generated in this gelfree system are in the size range of native human renal proximal tubules. Due to the large size of the tubule several microscopic images were stitched together. (B) The fluorescent organic anion Lucifer Yellow (green) was transported into the tubular lumen after addition to the culture medium surrounding the tubule. The arrowhead marks the tubular epithelium (cell nuclei, blue). Lucifer Yellow specific fluorescence in the cytoplasm was relatively low. Together with the substantial enrichment in the lumen this indicates efficient transepithelial transport of the organic anion. Panel C shows a larger part of the tubule displayed enlarged in B. Scale bars: (A) 1 mm and (B, C) 100 μm. The panels were reproduced with slight modifications from ref 103 and are reprinted with permission. Copyright 2011 The Authors Journal of Cellular and Molecular Medicine and Copyright 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

CONCLUSION In order to develop in vitro models for the prediction of druginduced nephrotoxicity, it will be inevitable to test larger numbers of compounds and to determine the predictive performance of the evaluated in vitro model. Further, it would be important to carefully select the cell types and end points used, and the current results suggest that both have a major impact on the predictive performance. With respect to PTC, currently no cell type is available that shows in vitro a similar performance as in vivo, and this appears to be due to an injury response of the cells. Whether this can be ameliorated by using biomimetic models remains to be shown. As long as this has not been achieved, it would be important to carefully characterize the differences between the renal cell states in vivo and in vitro in order to understand the different requirements, in particular with respect to the dosing of compounds. Nevertheless, even in the presence of different cell states in vivo and in vitro, high predictivity can be obtained, at least with respect to the prediction of PT toxicity.

Another 3D model44 used NKi-2 cells, which are hTERT immortalized human renal cortical cells. These cells displayed some features of PTC. However, NKi-2 cells responded to antidiuretic hormone, were not responsive to parathyroid hormone, and expressed very low levels of GGT.44 These features are not typical for PTC. After embedment into a mixture of Matrigel and rat tail collagen, tubule-like structures were formed by branching morphogenesis. With this 3D model the response to three drugs (cisplatin, gentamicin, and doxorubicin) during 3 days and 14 days of exposure was tested. Cell viability and LDH release were determined, as well as secretion of KIM-1 and NGAL. KIM-1 increased significantly

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AUTHOR INFORMATION

Corresponding Authors

*A.V.: phone, +65 67726124; fax, +65 67794112; e-mail, [email protected]. *D.Z.: phone, +65 6824 7107; fax, +65 6478 9080; e-mail, [email protected]. Notes

The authors declare no competing financial interest. L



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ACKNOWLEDGMENTS This work was supported by a grant from the Joint Council Office (Agency for Science, Technology and Research (A*STAR)) Development Program and the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, A*STAR, Singapore).

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NOTE ADDED IN PROOF While this article was processed, four other articles were published that address the differentiation of human pluripotent stem cells into renal precursor structures.129−132 Goal of these approaches was step-wise recapitulation of embryonic development. Major applications of the renal precursors obtained, which often contained a mix of different and not very mature renal cell types, may be in regenerative medicine. Any applications have not been demonstrated, so far. Whether the precursors obtained may be suitable for drug safety testing is unclear. In contrast, ref 62 describes the successful development of an in vitro model for the prediction of PTC toxicity that employs HPTC-like cells derived from human pluripotent stem cells. The protocol used for the generation of HPTC-like cells (ref 67) did not involve step-wise recapitulation of embryonic development.

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Drug-Induced Nephrotoxicity: Clinical Impact and Preclinical in Vitro

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