Nephrotoxic antisense oligonucleotide SPC5001 induces kidney injury biomarkers in a proximal tubule-on-a-chip.

Antisense oligonucleotides (ASOs) are a promising therapeutic modality. However, failure to predict acute kidney injury induced by SPC5001 ASO observed in a clinical trial suggests the need for additional preclinical models to complement the preceding animal toxicity studies. To explore the utility of in vitro systems in this space, we evaluated the induction of nephrotoxicity and kidney injury biomarkers by SPC5001 in human renal proximal tubule epithelial cells (HRPTEC), cultured in 2D, and in a recently developed kidney proximal tubule-on-a-chip. 2D HRPTEC cultures were exposed to the nephrotoxic ASO SPC5001 or the safe control ASO 556089 (0.16-40 µM) for up to 72 h, targeting PCSK9 and MALAT1, respectively. Both ASOs induced a concentration-dependent downregulation of their respective mRNA targets but cytotoxicity (determined by LDH activity) was not observed at any concentration. Next, chip-cultured HRPTEC were exposed to SPC5001 (0.5 and 5 µM) and 556089 (1 and 10 µM) for 48 h to confirm downregulation of their respective target transcripts, with 74.1 ± 5.2% for SPC5001 (5 µM) and 79.4 ± 0.8% for 556089 (10 µM). During extended exposure for up to 20 consecutive days, only SPC5001 induced cytotoxicity (at the higher concentration; 5 µM), as evaluated by LDH in the perfusate medium. Moreover, perfusate levels of biomarkers KIM-1, NGAL, clusterin, osteopontin and VEGF increased 2.5 ± 0.2-fold, 3.9 ± 0.9-fold, 2.3 ± 0.6-fold, 3.9 ± 1.7-fold and 1.9 ± 0.4-fold respectively, in response to SPC5001, generating distinct time-dependent profiles. In conclusion, target downregulation, cytotoxicity and kidney injury biomarkers were induced by the clinically nephrotoxic ASO SPC5001, demonstrating the translational potential of this kidney on-a-chip.

A Multicompartment Human Kidney Proximal Tubule-on-a-Chip Replicates Cell Polarization-Dependent Cisplatin Toxicity.

Drug-induced kidney injury is a major clinical problem and causes drug attrition in the pharmaceutical industry. To better predict drug-induced kidney injury, kidney in vitro cultures with enhanced physiologic relevance are developed. To mimic the proximal tubule, the main site of adverse drug reactions in the kidney, human-derived renal proximal tubule epithelial cells (HRPTECs) were injected in one of the channels of dual-channel Nortis chips and perfused for 7 days. Tubes of HRPTECs demonstrated expression of tight junction protein 1 (zona occludens-1), lotus lectin, and primary cilia with localization at the apical membrane, indicating an intact proximal tubule brush border. Gene expression of cisplatin efflux transporters multidrug and toxin extrusion transporter (MATE) 1 (SLC47A1) and MATE2-k (SLC47A2) and megalin endocytosis receptor increased 19.9 ± 5.0-, 23.2 ± 8.4-, and 106 ± 33-fold, respectively, in chip cultures compared with 2-dimensional cultures. Moreover, organic cation transporter 2 (OCT2) (SLC22A2) was localized exclusively on the basolateral membrane. When infused from the basolateral compartment, cisplatin (25 µM, 72 hours) induced toxicity, which was evident as reduced cell number and reduced barrier integrity compared with vehicle-treated chip cultures. Coexposure with the OCT2 inhibitor cimetidine (1 mM) abolished cisplatin toxicity. In contrast, infusion of cisplatin from the apical compartment did not induce toxicity, which was in line with polarized localization of cisplatin uptake transport proteins, including OCT2. In conclusion, we developed a dual channel human kidney proximal tubule-on-a-chip with a polarized epithelium, restricting cisplatin sensitivity to the basolateral membrane and suggesting improved physiologic relevance over single-compartment models. Its implementation in drug discovery holds promise to improve future in vitro drug-induced kidney injury studies. SIGNIFICANCE STATEMENT: Human-derived kidney proximal tubule cells retained characteristics of epithelial polarization in vitro when cultured in the kidney-on-a-chip, and the dual-channel construction allowed for drug exposure using the physiologically relevant compartment. Therefore, cell polarization-dependent cisplatin toxicity could be replicated for the first time in a kidney proximal tubule-on-a-chip. The use of this physiologically relevant model in drug discovery has potential to aid identification of safe novel drugs and contribute to reducing attrition rates due to drug-induced kidney injury.

Emerging In Vitro Systems to Screen and Predict Drug-Induced Kidney Toxicity.

Drug attrition related to kidney toxicity remains a challenge in drug discovery and development. In vitro models established over the past 2 decades to supplement in vivo studies have improved the throughput capacity of toxicity evaluation, but usually suffer from low predictive value. To achieve a paradigm shift in the prediction of drug-induced kidney toxicity, two aspects are fundamental: increased physiological relevance of the kidney model, and use of appropriate toxicity end points. Recent studies have suggested that increasing the physiological relevance of kidney models can improve their sensitivity to drug-induced damage. Here, we discuss how advanced culture models, including modified cell lines, induced pluripotent stem cells, kidney organoid cultures, and microfluidic devices enhance in vivo similarity. To this end, culture models aim to increase the proximal tubule epithelial phenotype, reconstitute multiple tissue compartments and extracellular matrix, allow exposure to fluid shear stress, and enable interaction between multiple cell types. Applying computation-aided end points and novel biomarkers to advanced culture models will further improve sensitivity and clinical relevance of in vitro drug-induced toxicity prediction. Implemented at the right stage of drug discovery and development and coupled to high-content evaluation techniques, these models have the potential to reduce attrition and aid the selection of candidate drugs with an appropriate safety profile.

A novel multi-parametric high content screening assay in ciPTEC-OAT1 to predict drug-induced nephrotoxicity during drug discovery.

Drug-induced nephrotoxicity is a major concern in the clinic and hampers the use of available treatments as well as the development of innovative medicines. It is typically discovered late during drug development, which reflects a lack of in vitro nephrotoxicity assays available that can be employed readily in early drug discovery, to identify and hence steer away from the risk. Here, we report the development of a high content screening assay in ciPTEC-OAT1, a proximal tubular cell line that expresses several relevant renal transporters, using five fluorescent dyes to quantify cell health parameters. We used a validation set of 62 drugs, tested across a relevant concentration range compared to their exposure in humans, to develop a model that integrates multi-parametric data and drug exposure information, which identified most proximal tubular toxic drugs tested (sensitivity 75%) without any false positives (specificity 100%). Due to the relatively high throughput (straight-forward assay protocol, 96-well format, cost-effective) the assay is compatible with the needs in the early drug discovery setting to enable identification, quantification and subsequent mitigation of the risk for nephrotoxicity.

Retinoid-related orphan receptor γ (RORγ) adult induced knockout mice develop lymphoblastic lymphoma.

RORγ is a nuclear hormone receptor which controls polarization of naive CD4+ T-cells into proinflammatory Th17 cells. Pharmacological antagonism of RORγ has therapeutic potential for autoimmune diseases; however, this mechanism may potentially carry target-related safety risks, as mice deficient in Rorc, the gene encoding RORγ, develop T-cell lymphoma with 50% frequency. Due to the requirement of RORγ during development, the Rorc knockout (KO) animals lack secondary lymphoid organs and have a dysregulation in the generation of CD4+ and CD8+ T cells. We wanted to extend the evaluation of RORγ deficiency to address the question whether lymphomas, similar to those observed in the Rorc KO, would develop in an animal with an otherwise intact adult immune system. Accordingly, we designed a conditional RORγ knockout mouse (Rorc CKO) where the Rorc locus could be deleted in adult animals. Based on these studies we can confirm that these animals also develop lymphoma in a similar time frame as embryonic Rorc knockouts. This study also suggests that in animals where the gene deletion is incomplete, the thymus undergoes a rapid selection process replacing Rorc deficient cells with remnant thymocytes carrying a functional Rorc locus and that subsequently, these animals do not develop lymphoblastic lymphoma.

Long-term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes.

Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.

Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes.

Human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep) hold great potential as an unlimited cell source for toxicity testing in drug discovery research. However, little is known about mechanisms of compound toxicity in hiPSC-Hep. In this study, modified mRNA was used to reprogram foreskin fibroblasts into hiPSC that were differentiated into hiPSC-Hep. The hiPSC-Hep expressed characteristic hepatic proteins and exhibited cytochrome P450 (CYP) enzyme activities. Next, the hiPSC-Hep, primary cryopreserved human hepatocytes (cryo-hHep) and the hepatic cell lines HepaRG and Huh7 were treated with staurosporine and acetaminophen, and the toxic responses were compared. In addition, the expression of genes regulating and executing apoptosis was analyzed in the different cell types. Staurosporine, an inducer of apoptosis, decreased ATP levels and activated caspases 3 and 7 in all cell types, but to less extent in Huh7. Furthermore, a hierarchical clustering and a principal component analysis (PCA) of the expression of apoptosis-associated genes separated cryo-hHep from the other cell types, while an enrichment analysis of apoptotic pathways identified hiPSC-Hep as more similar to cryo-hHep than the hepatic cell lines. Finally, acetaminophen induced apoptosis in hiPSC-Hep, HepaRG and Huh7, while the compound initiated a direct necrotic response in cryo-hHep. Our results indicate that for studying compounds initiating apoptosis directly hiPSC-Hep may be a good alternative to cryo-hHep. Furthermore, for compounds with more complex mechanisms of toxicity involving metabolic activation, such as acetaminophen, our data suggest that the cause of cell death depends on a balance between factors controlling death signals and the drug-metabolizing capacity.

Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice.

RHO family proteins are important for the function of inflammatory cells. They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I). Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins. Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders. However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis. The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments. The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane. Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines. The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.

Targeting the protein prenyltransferases efficiently reduces tumor development in mice with K-RAS-induced lung cancer.

RAS and RHO proteins, which contribute to tumorigenesis and metastasis, undergo posttranslational modification with an isoprenyl lipid by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase-I (GGTase-I). Inhibitors of FTase and GGTase-I were developed to block RAS-induced malignancies, but their utility has been difficult to evaluate because of off-target effects, drug resistance, and toxicity. Moreover, the impact of FTase deficiency and combined FTase/GGTase-I deficiency has not been evaluated with genetic approaches. We found that inactivation of FTase eliminated farnesylation of HDJ2 and H-RAS, prevented H-RAS targeting to the plasma membrane, and blocked proliferation of primary and K-RAS(G12D)-expressing fibroblasts. FTase inactivation in mice with K-RAS-induced lung cancer reduced tumor growth and improved survival, similar to results obtained previously with inactivation of GGTase-I. Simultaneous inactivation of FTase and GGTase-I markedly reduced lung tumors and improved survival without apparent pulmonary toxicity. These data shed light on the biochemical and therapeutic importance of FTase and suggest that simultaneous inhibition of FTase and GGTase-I could be useful in cancer therapeutics.

Nf1 deficiency cooperates with oncogenic K-RAS to induce acute myeloid leukemia in mice.

Hyperactive RAS signaling is caused by mutations in RAS genes or a deficiency of the neurofibromatosis gene (NF1) and is common in myeloid malignancies. In mice, expression of oncogenic K-RAS or inactivation of Nf1 in hematopoietic cells results in myeloproliferative disorders (MPDs) that do not progress to acute myeloid leukemia (AML). Because NF1 is a RAS-GTPase-activating protein it has been proposed that NF1 deficiency is functionally equivalent to an oncogenic RAS. It is not clear, however, whether Nf1 deficiency would be redundant in K-RAS-induced MPD development or whether the 2 mutations would cooperate in leukemogenesis. Here, we show that the simultaneous inactivation of Nf1 and expression of K-RAS(G12D) in mouse hematopoietic cells results in AML that was fatal in primary mice within 4 weeks and transplantable to sublethally irradiated secondary recipients. The data point to a strong cooperation between Nf1 deficiency and oncogenic K-RAS.

Inactivating Icmt ameliorates K-RAS-induced myeloproliferative disease.

Hyperactive signaling through the RAS proteins is involved in the pathogenesis of many forms of cancer. The RAS proteins and many other intracellular signaling proteins are either farnesylated or geranylgeranylated at a carboxyl-terminal cysteine. That isoprenylcysteine is then carboxyl methylated by isoprenylcysteine carboxyl methyltransferase (ICMT). We previously showed that inactivation of Icmt mislocalizes the RAS proteins away from the plasma membrane and blocks RAS transformation of mouse fibroblasts, suggesting that ICMT could be a therapeutic target. However, nothing is known about the impact of inhibiting ICMT on the development of malignancies in vivo. In the current study, we tested the hypothesis that inactivation of Icmt would inhibit the development or progression of a K-RAS-induced myeloproliferative disease in mice. We found that inactivating Icmt reduced splenomegaly, the number of immature myeloid cells in peripheral blood, and tissue infiltration by myeloid cells. Moreover, in the absence of Icmt, the ability of K-RAS-expressing hematopoietic cells to form colonies in methylcellulose without exogenous growth factors was reduced dramatically. Finally, inactivating Icmt reduced lung tumor development and myeloproliferation phenotypes in a mouse model of K-RAS-induced cancer. We conclude that inactivation of Icmt ameliorates phenotypes of K-RAS-induced malignancies in vivo.

GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer.

Protein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA, RAC1, and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized, they have very different properties, and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study, we determined the effects of GGTase-I deficiency on cell viability and K-RAS-induced cancer development in mice. Inactivating the gene for the critical beta subunit of GGTase-I eliminated GGTase-I activity, disrupted the actin cytoskeleton, reduced cell migration, and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover, the absence of GGTase-I activity reduced lung tumor formation, eliminated myeloproliferative phenotypes, and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly, several cell types remained viable in the absence of GGTase-I, and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS-induced malignancies.

Rce1 deficiency accelerates the development of K-RAS-induced myeloproliferative disease.

The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the "C" of the carboxyl-terminal CaaX motif). After farnesylation, the 3 amino acids downstream from the farnesyl cysteine (the -aaX of the CaaX motif) are released by RAS-converting enzyme 1 (RCE1). We previously showed that inactivation of Rce1 in mouse fibroblasts mislocalizes RAS proteins away from the plasma membrane and inhibits RAS transformation. Therefore, we hypothesized that the inactivation of Rce1 might inhibit RAS transformation in vivo. To test this hypothesis, we used Cre/loxP recombination techniques to simultaneously inactivate Rce1 and activate a latent oncogenic K-RAS allele in hematopoietic cells in mice. Normally, activation of the oncogenic K-RAS allele in hematopoietic cells leads to rapidly progressing and lethal myeloproliferative disease. Contrary to our hypothesis, the inactivation of Rce1 actually increased peripheral leukocytosis, increased the release of immature hematopoietic cells into the circulation and the infiltration of cells into liver and spleen, and caused mice to die more rapidly. Moreover, in the absence of Rce1, splenocytes and bone marrow cells expressing oncogenic K-RAS yielded more and larger colonies when grown in methylcellulose. We conclude that the inactivation of Rce1 worsens the myeloproliferative disease caused by oncogenic K-RAS.