A Quantitative Approach to Screen for Nephrotoxic Compounds In Vitro.

Nephrotoxicity due to drugs and environmental chemicals accounts for significant patient mortality and morbidity, but there is no high throughput in vitro method for predictive nephrotoxicity assessment. We show that primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells rendering them desirable to use in such in vitro systems. To identify a reliable biomarker of nephrotoxicity, we conducted multiplexed gene expression profiling of HPTECs after exposure to six different concentrations of nine human nephrotoxicants. Only overexpression of the gene encoding heme oxygenase-1 (HO-1) significantly correlated with increasing dose for six of the compounds, and significant HO-1 protein deregulation was confirmed with each of the nine nephrotoxicants. Translatability of HO-1 increase across species and platforms was demonstrated by computationally mining two large rat toxicogenomic databases for kidney tubular toxicity and by observing a significant increase in HO-1 after toxicity using an ex vivo three-dimensional microphysiologic system (kidney-on-a-chip). The predictive potential of HO-1 was tested using an additional panel of 39 mechanistically distinct nephrotoxic compounds. Although HO-1 performed better (area under the curve receiver-operator characteristic curve [AUC-ROC]=0.89) than traditional endpoints of cell viability (AUC-ROC for ATP=0.78; AUC-ROC for cell count=0.88), the combination of HO-1 and cell count further improved the predictive ability (AUC-ROC=0.92). We also developed and optimized a homogenous time-resolved fluorescence assay to allow high throughput quantitative screening of nephrotoxic compounds using HO-1 as a sensitive biomarker. This cell-based approach may facilitate rapid assessment of potential nephrotoxic therapeutics and environmental chemicals.

ASL mRNA-LNP Therapeutic for the Treatment of Argininosuccinic Aciduria Enables Survival Benefit in a Mouse Model.

Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5' and 3' untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.

The enhanced value of combining conventional and "omics" analyses in early assessment of drug-induced hepatobiliary injury.

The InnoMed PredTox consortium was formed to evaluate whether conventional preclinical safety assessment can be significantly enhanced by incorporation of molecular profiling ("omics") technologies. In short-term toxicological studies in rats, transcriptomics, proteomics and metabolomics data were collected and analyzed in relation to routine clinical chemistry and histopathology. Four of the sixteen hepato- and/or nephrotoxicants given to rats for 1, 3, or 14days at two dose levels induced similar histopathological effects. These were characterized by bile duct necrosis and hyperplasia and/or increased bilirubin and cholestasis, in addition to hepatocyte necrosis and regeneration, hepatocyte hypertrophy, and hepatic inflammation. Combined analysis of liver transcriptomics data from these studies revealed common gene expression changes which allowed the development of a potential sequence of events on a mechanistic level in accordance with classical endpoint observations. This included genes implicated in early stress responses, regenerative processes, inflammation with inflammatory cell immigration, fibrotic processes, and cholestasis encompassing deregulation of certain membrane transporters. Furthermore, a preliminary classification analysis using transcriptomics data suggested that prediction of cholestasis may be possible based on gene expression changes seen at earlier time-points. Targeted bile acid analysis, based on LC-MS metabonomics data demonstrating increased levels of conjugated or unconjugated bile acids in response to individual compounds, did not provide earlier detection of toxicity as compared to conventional parameters, but may allow distinction of different types of hepatobiliary toxicity. Overall, liver transcriptomics data delivered mechanistic and molecular details in addition to the classical endpoint observations which were further enhanced by targeted bile acid analysis using LC/MS metabonomics.

Modulation of key regulators of mitosis linked to chromosomal instability is an early event in ochratoxin A carcinogenicity.

Ochratoxin A (OTA) is a potent renal carcinogen, but little is known regarding the mechanism of OTA carcinogenicity. Early histopathological alterations induced by OTA in rat kidney include single cell death, stimulation of cell proliferation and prominent karyomegaly indicative of blocked nuclear division during mitosis. Based on these observations, it has been suggested that disruption of mitosis by OTA may be the principal cause of cell death and subsequent trigger for cell proliferation to compensate for cell loss. To gain further insight into the molecular mechanism of OTA toxicity, we used targeted quantitative real-time polymerase chain reaction arrays to investigate the expression of genes involved in cell cycle control and mitosis in kidneys of male F344 rats treated with 0, 21, 70 and 210 microg/kg body wt OTA for up to 90 days. Treatment with OTA resulted in overexpression of key regulators of mitosis, including the mitotic protein kinases Polo-like kinase 1, Aurora B and cyclin-dependent kinase 1 (Cdk1Cdc2), several cyclins and cyclin-dependent kinase inhibitors, topoisomerase II and survivin. Immunohistochemical analysis confirmed upregulation of Cdk1, p21(WAF1/CIP1), topoisomerase II and survivin in S3 proximal tubule cells, from which OTA-induced tumors in rats arise, and demonstrated increased phosphorylation of histone H3, a target of Aurora B. Importantly, many of the genes found to be deregulated in response to OTA have been linked to chromosomal instability and malignant transformation, supporting the hypothesis that aberrant mitosis, resulting in blocked or asymmetric cell division, accompanied by an increased risk of aneuploidy acquisition, may play a critical role in OTA carcinogenicity.

A High-Throughput Screening Assay to Identify Kidney Toxic Compounds.

Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc.

Managing incidental findings and disclosure of results in a paediatric research cohort - the LIFE Child Study cohort.

OBJECTIVE: To assess the frequency of incidental findings (IFs) in the population-based "Leipzig Research Centre for Civilization Diseases (LIFE) Child Study" within 1 year. METHODS: From July 2011 to June 2012, 969 children participated in the study. The IFs were analysed with respect to age, gender, type of examination and clinical action taken. RESULTS: The IFs were detected in 63 participants (6.5%), including five children who presented with two IFs simultaneously. Eleven children received a new, hence previously unknown, clinical diagnosis. Alternatively, 18 IFs could not be confirmed or were of a transient and self-limiting condition. The frequency of IFs varied widely depending on the type of examination, but did not differ by gender. CONCLUSION: Although IFs were common events, there was no finding with a profound clinical impact on the subject's life. Our current IF management protocol may be useful in creating management plans for other cohort studies.

Performance of novel kidney biomarkers in preclinical toxicity studies.

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

Comparative analysis of novel noninvasive renal biomarkers and metabonomic changes in a rat model of gentamicin nephrotoxicity.

Although early detection of toxicant induced kidney injury during drug development and chemical safety testing is still limited by the lack of sensitive and reliable biomarkers of nephrotoxicity, omics technologies have brought enormous opportunities for improved detection of toxicity and biomarker discovery. Thus, transcription profiling has led to the identification of several candidate kidney biomarkers such as kidney injury molecule (Kim-1), clusterin, lipocalin-2, and tissue inhibitor of metalloproteinase 1 (Timp-1), and metabonomic analysis of urine is increasingly used to indicate biochemical perturbations due to renal toxicity. This study was designed to assess the value of a combined (1)H-NMR and gas chromatography-mass spectrometry (GC-MS) metabonomics approach and a set of novel urinary protein markers for early detection of nephrotoxicity following treatment of male Wistar rats with gentamicin (60 and 120 mg/kg bw, s.c.) for 7 days. Time- and dose-dependent separation of gentamicin-treated animals from controls was observed by principal component analysis of (1)H-NMR and GC-MS data. The major metabolic alterations responsible for group separation were linked to the gut microflora, thus related to the pharmacology of the drug, and increased glucose in urine of gentamicin-treated animals, consistent with damage to the S(1) and S(2) proximal tubules, the primary sites for glucose reabsorption. Altered excretion of urinary protein biomarkers Kim-1 and lipocalin-2, but not Timp-1 and clusterin, was detected before marked changes in clinical chemistry parameters were evident. The early increase in urine, which correlated with enhanced gene and protein expression at the site of injury, provides further support for lipocalin-2 and Kim-1 as sensitive, noninvasive biomarkers of nephrotoxicity.