Author:Peter Haima, Ph.D1, and Martin Shaw2

1. Life-Force biomedical communication, The Netherlands2. Technical Support, EKF Diagnostics

TECOmedical Clinical & Technical Review

July 2014

Novel Liver and Kidney Biomarkers for Improved

Drug Safety TestingIn-vitro (2D and 3D cell culture), pre-clinical (animal) and human studies

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CONTENT

Summary 3

1 Introduction 3

2 LEAKAGE AND APOPTOSIS: biomarkers for drug induced liver injury 52.1 Leakage Biomarker: α-GST to detect drug induced hepatocyte injury 52.2 Apoptosis and total cell death biomarkers: ccK18 and K18 to detect drug induced hepatocyte injury 9

3 Biomarkers for drug induced kidney injury 133.1 Introduction 133.2 Summary 15

4 References 17

5 Application notes (protocols) 185.1 Alpha-GST Release as a Predictive Marker of Drug Induced Hepatotoxicity 18 Utilising 3D InSightTM Liver Microtissues

6 Overview of assays and intended use 21

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SUMMARYLiver and kidney toxicity are important causes of late stage drug failure and post-launch warning texts. Reasons for this include the shortcomings of current toxicology biomarkers that lack sufficient sensitivity and specificity. This document will present how the use of biomarkers with known origins and mechanisms of release can improve the detection and monitoring of potential toxicity, potentially saving time and costs in drug development and improving patient care.

Special emphasis in this review will be placed on the liver injury biomarker alpha Glutathione S-Transferase (alpha GST or αGST), for hepatocellular necrosis, and biomarkers caspase-cleaved keratin 18 (ccK18) and total soluble keratin 18 (K18; caspase-cleaved and uncleaved), providing insight into the mode of hepatocyte cell death: apoptosis or necrosis.

The renal biomarkers urinary alpha and pi GST will be presented and discussed as sensitive and specific biomarkers of proximal and distal renal tubular injury.

The above liver and kidney biomarkers enable the detection of adverse effects earlier and with greater sensitivity and specificity than traditional biomarkers of hepatic and renal injury. Examples will be given of their application and value in in-vitro, animal and human toxicology.

1 INTRODUCTIONToxicology testing is an important and expensive part of drug development. But in spite of the large sums spent and sophisticated technologies employed, many drugs are failing late in the development process, increasing the cost of drug development and slowing down the drug development process. Hepatotoxicity and nephrotoxicity are important causes of post-launch withdrawal of pharmaceuticals and an important cause of Adverse Drug Reactions (ADRs) in the clinic. ADRs rate among the leading causes of death in the Western world. Drug-induced liver injury (DILI) now ranks as a leading cause of liver failure and transplantation in western countries. Similarly, drug-induced kidney injury (DIKI) contributes to between 8-60% of all cases of acute renal failure in hospitalized patients, a serious problem considering the high morbidity and mortality associated with Acute Kidney Injury (AKI).

An important reason for compounds failing late during pre-clinical phase is that current tests used to detect and monitor renal and hepatic injury have shortcomings. For example, Aspartate Aminotransferase (AST) and Alanine amino transferase (ALT), common tests for hepatic injury, are well known to be insensitive and non-specific for the liver. Similarly, the commonest renal biomarker test, serum creatinine, is known to be a late, insensitive, indicator of renal glomerular function while most nephrotoxins affect the renal tubules. Therefore, European and USA initiative programs (the Innovative Medicines Initiative (IMI) SAFE-T consortium [1] and C-Path’s Predictive Safety Testing Consortium [2]) are currently developing and validating new safety biomarkers to: • Make better regulatory decisions in drug R&D and in a translational context• Detect, assess and monitor drug-induced kidney and liver injury in humans• Diagnose disease in clinical practiceUsing biomarkers with known sources and mechanisms of release can increase the specificity and sensitivity of organ damage testing. This can eliminate compounds with an increased risk of adverse reactions early from the drug discovery process and improve the monitoring of subjects with suspected liver or renal disease.

This review article will present information on a range of sensitive and specific biomarkers that offer this potential. The main focus will be on the cell leakage biomarkers alpha and pi GST for the early detection of liver or kidney injury and caspase-cleaved keratin 18, for detection of hepatocyte apoptosis. Examples of their use in in vitro (cell culture), in pre-clinical (animal) and human studies will be presented (see tables 1a and 1b).

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LiverBiomarker TissueSpecificity Indicativefor Species Cell culture LIVER(SERUM) HUMAN ANIMALαGST(Alpha Glutathione Hepatocytes Necrosis √ Rat √ S-Transferase) Mouse (2D & 3D)

πGST(Pi Glutathione Bileduct Necrosis √ - √S-Transferase)

ccK18 (Caspase - cleaved Hepatocytes Apoptosis √ Monkey √Keratin 18: M30 Elisa Bovine (2D & 3D)

K18

(cleaved and uncleaved Hepatocytes Necrosisand √ Monkey √Keratin 18: M65 Elisa) Apoptosis (2D & 3D)

KidneyBiomarker TissueSpecificity Indicativefor Responsetime Species Cell followinginjury* culture KIDNEY(URINE) HUMAN ANIMALαGST(Alpha Glutathione Proximaltubule Necrosis within4-6hours √ Rat √S-Transferase) (2D & 3D)

πGST(Pi Glutathione Distaltubule Necrosis within6hours √ - √S-Transferase)

KIM-1(Kidney Injury Proximaltubule Regeneration within12-24hours √ RatMolecule-1) afterinjury

L-FABP

(Liver Type Fatty Acid Proximaltubule Necrosis within4-6hours √ Binding Protein)

NGAL

(Neutrophil Gelatinase Distaltubule Regeneration within4-6hours √ Associated Lipocalin) afterinjury

CollagenIV Glomerulus Glomerular Chronicdeposition √ damage

* response times displayed are indications.

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2 LEAKAGE AND APOPTOSIS: biomarkers for drug induced liver injury

Detecting potential hepatotoxicity is important during drug development in order to reduce the risk of late stage failure of compounds and to reduce the risk of drugs showing unwanted hepatic effects entering clinical practice. Standard liver tests, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) show inadequate sensitivity and specificity and limited predictive value from a translational value and clinical outcome perspective [3]. During pre-clinical drug development, AST/ALT increases are commonly observed in the absence of evidence of hepatic injury and, conversely, sometimes they do not increase even when tissue injury is observed.

In clinical settings, liver biopsy is still the gold standard for the detection of liver damage, however it is invasive and is affected by sampling errors (e.g. inhomogeneous liver damage) and the risk of clinical complications. Utilising serum biomarkers linked to histological changes can reduce these problems.

International safety programs, IMI and PSTCTo mitigate the problems with standard liver tests, The European Innovative Medicines Initiative (IMI) is validating the liver injury biomarkers Alpha-GST, caspase-cleaved and total Keratin 18 for application in the regulatory decision-making process in clinical drug development and as potential clinical tests. C-Path’s Predictive Safety Testing Consortium, a program run in partnership with the Food and Drug Administration (FDA), is validating Alpha GST for similar reasons to the IMI. The first results obtained by the IMI SAFE-T consortium are very promising for ccK18, K18 and Alpha GST.

2.1 Leakage Biomarker: α-GST to detect drug induced hepatocyte injury Alpha glutathione S-transferase (Alpha GST) is a 56 kDa phase II intracellular detoxification enzyme, found in high concentrations in hepatocytes (2-5%). The protein behaves as an ideal leakage marker; in absence of liver injury, serum levels are very low but large quantities are rapidly released from injured hepatocytes. Moreover, its even distribution across the liver lobules makes it a sensitive indicator of even localised hepatocyte injury. After release, it is rapidly removed from the circulation with a half-life of only 60-90 minutes making it an excellent indicator of the kinetics of hepatotoxicity. The distribution of alpha GST in the liver is similar across all mammals utilised in toxicological testing, facilitating translational studies. Currently, the commonest indicators of hepatocyte injury are the transaminases and, in summary, it can be stated that compared with them, alpha GST is more specific, is present in the hepatocytes in greater quantities, is released more rapidly and is a more sensitive indicator of toxicokinetics. A brief comparison of the properties of alpha GST and transaminases as hepatic biomarkers is given in table 2.

Alpha GST has been studied as a biomarker of hepatocyte injury in many situations, including toxicology (In-vitro, pre-clinical and human studies), transplantation, surgical injury, hepatitis and clinical toxicology, especially paracetamol poisoning. Alpha GST has been proven to be an excellent biomarker in all these fields, providing rapid, sensitive and specific information regarding the onset, severity and resolution of hepatocyte injury. Over 150 articles have been published on alpha GST as a hepatocyte biomarker. This provides confidence in the results obtained.

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2.1.1 In vitro studies (2D and 3D cell cultures)Hepatocytes in cell culture express alpha GST and release it in response to toxins. The use of the combination of hepatocytes and alpha GST release has been widely validated. Exposure of hepatocytes to toxic levels of substances leads to the release of large quantities of alpha GST, while little is released from healthy cells (giving the marker an excellent Negative Predictive Value). As little as a few thousand cells per well are often sufficient to perform toxicological studies utilising the assay of Alpha GST.

For example, using hepG2 cells, McKim et al showed that the release of alpha GST from hepatocytes in culture was predictive of toxicity in rat and humans [4]. They showed that for Ketoconozole the LD50 (dose at which 50% of cells die) in cell culture, was closely related to the toxic levels in rat and human plasma.Similarly, Vickers et al showed that using precision cut liver slices, the release of alpha GST could indicate the relative toxicities of different cyclosporin analogues, while assaying both alpha and pi GST release could show differential effects on the hepatocytes and biliary cells [5].Lactate dehydrogenase (LDH) is commonly used to monitor in-vitro toxicology, however, the GSTs are more sensitive and due to their cellular specificity, allow different cell types to be monitored simultaneously and individually.

Figure 1 shows the release of different liver biomarkers from a culture of fresh human hepatocytes exposed to to the known hepatotoxin Erythromycin estolate. Note the much greater release of alpha GST compared to the other liver biomarkers.

Figure 1

Erythromycin Estolate Biomarker Release @ 24 hrs.( Abbott Laboratories 1995, Personal communication).

AlphaGST Transaminases(AST/ALT)

5%solubleliverprotein 0.7%solubleliverprotein• Largereleases• Easytoseechanges

MW56000Da MW147000Da• Easierrelease• 18xhighermolarconcentration

Shorthalf-life:60-90minutes Longhalf-life:AST/ALT,17/47hours

Clearandfastkinetics Slowkinetics

Table 2

Immunohistochemical Localisation of αGST in Hepatocytes. Reprinted from [41] with

permission from IOS Press.

 

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In vitro studies can be performed using two or three dimensional cell cultures. The advantage of 3D liver microtissues Is that they better mimic liver physiology by providing a liver-like cell structure with dense inter-hepatocyte contacts.In a recent study [6] (see application note at the end of this article) highly sensitive detection of alpha GST release from 3D rat and human InSightTM liver microtissues indicated Amaridone- and Diclofenac-induced hepatotoxicity with as little as 1000 cells/microtissue. Alpha GST as an in-vitro biomarker could be measured in the cell supernatant without killing the cells, enabling sequential sampling. Alpha GST release and reduced ATP production showed similar sensitivity to detect hepatotoxicity, but since the reduction in ATP production and the release of alpha GST reflect different pathological processes, their combination may provide additional valuable information. This was shown in this study where the combination of reduced ATP production and the release of alpha GST supported the hypothesis that the known toxicities of Amaridone and Diclofenac are mediated by effects on mitochondrial function.

2.1.2 Pre-clinical studies (animal models)Alpha GST (also known as Ya and Yc) has been shown in many studies to be a sensitive and specific biomarker of hepatotoxicity in rats. For example, in a study on the classic hepatotoxin, carbon tetrachloride (CCl4), alpha GST was shown to be a more sensitive biomarker than the transaminase Aspartate Transaminase (AST), regarding both dose and toxicokinetics [7] Furthermore, the release was more closely related to the dose than that of AST (Figure 2).

In a study on the drug valproic acid, elevations in serum alpha GST indicated hepatic effects in advance of changes in liver pathology [8]. In clinical use, valproic acid was shown to carry risks of hepatic side effects and the FDA issued a warning notice [9].

Figure 2

A) Sensitivity of Alpha GST – Dose Response: Hepatoxicity in the Rat.

B) Sensitivity of Alpha GST – Kinetics: Hepatotoxicity in the Rat (Reprinted from [41] with permission from IOS Press).

Sensitivity is very valuable, but specificity is also important. A major drawback of transaminases is that they are also expressed in muscles and the heart. In a study on the toxicity of caffeine, transaminases levels increased, but alpha GST remained normal. Histological examination, supported by the measurement of serum troponin, showed that the effect was on the heart and that the transaminases were not indicating hepatotoxicity. Alpha GST was more specific [10].

2.1.3 Human studiesAlpha GST has been shown to be a more discriminating indicator of hepatotoxicity than transaminases.For example, Eason et al found a four-dose escalating double blind study, that alpha GST rose more strongly than transaminases in subjects given the active substance, while it was more stable in the controls. Alpha GST was both more sensitive and specific. Furthermore, alpha GST more accurately showed toxicokinetics, falling faster after the study was terminated [11].In studies on the known hepatotoxin Paracetamol (Acetaminophen or APAP), alpha GST was shown to be more sensitive than transaminases. In subjects given high therapeutic or supra-therapeutic doses of Paracetamol, alpha GST rose when transaminases were normal [12]. The high sensitivity and specificity of alpha GST makes it unlikely that hepatotoxicity is occurring when levels are normal.

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2.1.4 Clinical HepatotoxicityAlpha GST is a very sensitive indicator of clinical hepatotoxicity. The assay of alpha GST has been widely used to study Paracetamol hepatotoxicity where it has been shown to be both a more sensitive and specific indicator of hepatic injury than transaminases. For example, Beckett and Hayes found that alpha GST rose faster and to a greater extent than transaminases, but then fell faster following successful therapy (Figure 3). Subjects presenting with serum alpha GST levels of over 10 μg/L went on to show liver damage [13]. Alpha GST provided prognostic information regarding severity of liver injury and therapeutic outcome.

Figure 3

Toxicokinetics - Paracetamol Overdose. Broken lines indicate biomarker behavior after initiation of therapy at 12 hours post overdose (adapted

from [13]).

The anti-inflammatory drug methotrexate is prescribed to treat inflammatory diseases. However, it is a known hepatotoxin and liver biopsy is recommended following a cumulative dose of 1.5 g or every six months. Methotrexate is taken weekly and in a study by Maxwell et al, it was shown that the day after the drug was taken, alpha GST was clearly elevated while other biomarkers were little changed (Figure 4).

Alpha GST was an early (predictive) biomarker of long-term liver injury [14].

Figure 4

Predictive Toxicology – Methotrexate

Hepatotoxicity (adapted from [14]).

To summarise, alpha GST is a sensitive and specific biomarker of hepatocyte injury and the ability to easily compare cross-species studies makes it particularly useful. By its application, hepatotoxic compounds may be detected earlier in the drug development process and subjects suffering from the effects of hepatotoxins followed more closely.

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2.2 Apoptosis and total cell death biomarkers: ccK18 and K18 to detect drug induced hepatocyte injury

2.2.1 ccK18 (M30) and K18 (M65) ELISAsHepatocyte cell death, specifically hepatocyte apoptosis, is considered to play a crucial role in the formation of liver fibrosis or liver cirrhosis. Numerous studies have demonstrated that hepatocyte apoptosis can be specifically assessed by means of caspases-cleaved fragments of Keratin 18 (ccK18), a major intermediate filament protein expressed in epithelial tissue and hepatocytes. The M30 Apoptosense® ELISA is based on the monoclonal antibody M30 CytoDEATH™. This antibody recognizes a neo-epitope on K18 which is exposed after caspase cleavage at residue Asp396. The M30 Aptosense® and CytoDeath® ELISAS (see Table 3) detects only caspase-cleaved fragments of K18 (ccK18; K18Asp396) but not the native protein. Antigen generation is strictly dependent on caspase activation (activity is inhibited by broad-spectrum caspase inhibitors). A number of different caspases are able to cleave K18 at Asp396, primarily caspase-3, -7 and -9; activity is, therefore, not dependent on the action of a specific caspase. M30 measurements will, therefore, represent epithelial and hepatocyte cell apoptosis.

The M65 and EpiDeath® ELISAs (for clinical and toxicology testing, respectively, see Table 3); utilizing the antibodies M5 and M6, measure total soluble K18 released from dead cells (caspase-cleaved and uncleaved). M65 measurements will, therefore, represent the total epithelial and hepatocyte cell death by any cause (apoptosis plus necrosis).

Figure 5

The principle of the M30 and M65 assays,

measuring apoptosis and total cell death

(necrosis + apoptosis) respectively.

Table 3

Specific application of the various M30 and M65 ELISAS.

Assay Description Application

M30Apoptosense®ELISA Apoptosis Clinicaluse:NASH/LiverDisease Toxicology:druginducedhepatoxicityM30CytoDeath™ Apoptosis Toxicology

M65®ELISA Apoptosis+Necrosis: Clinicaluse TotalCellDeath

M30Apoptosense®/M65®ELISA Apoptosis/TotalCellDeath ClinicaluseandToxicology: RatioofCellDeathModes

M65EpiDeath®ELISA Apoptosis&Necrosis: Toxicology:druginducedhepatoxicity TotalCellDeath

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Assaying ccK18 with the M30 ELISA provides an integrated measurement of apoptosis.The M30 ELISA has major advantages for screening and characterization of apoptotic cell responses. The assay quantifies the accumulation of an apoptosis-generated intracellular “death product” that is very stable in cells and after its release into culture media. Thus, any signal derived from cells that have already undergone apoptosis remains in the culture medium or blood, even after all apoptotic processes are completed and all cells are dead; and caspase activity is reduced. The M30 assay, therefore, provides an integrated measurement of apoptosis, where a single late time point is sufficient to quantify the cellular response to the apoptotic stimulus regardless of its kinetics. In contrast, enzymatic methods that measure the caspase activity in cells only at specific time points may return incorrect results (See illustration, Figure 6). This is especially true in circumstances when non-lethal caspase activation may be associated with differentiation or proliferation.

Fast induction of apoptosis Slow induction of apoptosis

Figure 6

M30 assay provides an integrated measurement of apoptosis.

Red lines: Signal in the M30 ELISA; Blue lines: signal of ongoing apoptosis (e. g. Annexin V).

Dotted lines: Example of fast induction of apoptosis.

Solid lines: Example of slow induction of apoptosis.

t1: Peak apoptotic rate for a rapidly acting stimulus. The signal following a treatment with a slower effect will still be low.

t2: Peak apoptotic rate following slower apoptosis induction. Following treatment with the more rapidly acting stimulus,

the apoptotic process has almost been completed and so the signal will be low.

t3: Late time point, when the apoptotic process is completed for both treatments; The signals of ongoing apoptosis are now very low.

However, the concentrations of the accumulated apoptotic product, measured by theM30 assay, are stable and indicate the total

extent of apoptosis, irrespective of its kinetics.

Combining M30 & M65 provides information on cell death mode trendsM30 measurements represent epithelial and hepatocyte cell apoptosis, M65 measurements represent the total epithelial and hepatocyte cell death by any cause (apoptosis plus necrosis). Combining the information from M30 and M65 measurements facilitates the determination of cell death mode trends.

Note: Conclusions on trending are only recommended for samples ≥ 300 U/L.

2.2.2 In vitro studies (2D and 3D cell cultures)Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate, thereby, losing their hepatocyte functions and metabolic competence. This has led to increasing interest into three-dimensional in-vitro models, using different extracellular matrix-based hydrogel or scaffolds methods, which allow for long-term culture of the human hepatoma cell line HepG2. HepG2 is a cell line with a very high cellular content of Keratin 18, which enables the sensitive measurement of ccK18 (M30) and K18 (M65) in HepG2 cell lysates and/or cell culture supernatants, respectively [16, 17].

   

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In a recent study [42] (see application note at the end of this article) ccK18 could be detected in human InSphero’s 3D InSight™ human liver microtissues co-cultured with Kupffer cells. Apoptosis was induced through 48 hour exposure to staurosporine and after removal of supernatant, cells were lysed using cytoskeleton solubilization buffer (CSB). The M30 Apoptosense® ELISA could detect an increase of ccK18 upon treatment of microtissues (2000 cells) with Staurosporine. These results demonstrate that M30 measurements can be used to monitor apoptosis in human liver microtissues.

In another study Shevchenko et al. [18] performed toxicity testing on cryopreserved differentiated HepaRG™ cells. HepaRG™ cells exhibit many characteristics of primary human hepatocytes, including morphology and expression of key metabolic enzymes, nuclear receptors, and drug transporters. Unlike HepG2 cells, HepaRG™ cells have high P450 activity and complete expression of all nuclear receptors. HepaRG™ were exposed to different compounds, including paracetamol, chloropromazine, rotenone, rosiglitazone and omeprazole. Toxicity and apoptosis were assayed from collected cell culture supernatants measuring K18 (M65 EpiDeath Elisa and ccK18 (M30 CytoDeath (CD) ELISA). M65 results were consistent with cell viability, whereas, M30 results indicated that apoptosis was induced at lower drug concentrations while necrosis was more prominent at higher ones (Figure 7).

The combined use of M30 and M65 measurements with the cell viability markers provided valuable information on viability, toxicity and apoptosis for each individual compound.

2.2.3 Clinical HepatotoxicityAdverse drug reactions, in particular drug-induced hepatotoxicity, represent a major challenge for clinicians and an impediment to safe drug development. Novel blood biomarkers that can provide information about pathways leading to hepatocyte death should aid earlier identification of potential hepatotoxins and accelerate drug development. New mechanism-based biomarkers such as ccK18 and total K18, which reflect particular cellular processes involved in DILI, should offer a clear rationale for patient stratification and novel therapeutic interventions.

Numerous studies have demonstrated that hepatocyte apoptosis or total hepatocyte cell death (apoptosis and necrosis) can be specifically assessed by means of caspases cleaved fragments of keratin 18 and total soluble K18.

Figure 7

Toxicity of Human HepaRGTM hepatocytes exposed to different

compounds. Toxicity and apoptosis were assayed from collected

supernatants measuring full-length cytokeratin 18(K18) and caspases

cleaved fragments of ccK18 and using M65 EpiDeath and M30 CytoDeath

ELISAS (adapted from [18]).

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The use of these immunoassays has been reported in clinical studies as biomarkers for the quantification of hepatocyte cell death modes during liver diseases such as non-alcoholic steatohepatitis (NASH) and hepatitis C infection.

Antoine et al. [19-22] evaluated Paracetamol overdose in large multi-centre patient cohort to determine the balance between apoptosis, necrosis and the inflammatory response during the time course of clinical Paracetamol. Their results showed that during the acute phase of Paracetamol hepatotoxicity, necrosis was the dominant form of cell death. At presentation, serum, ccK18 (apoptosis) and total K18 (necrosis plus apoptosis) values, all significantly correlated with later peak ALT activity values.

Circulating ccK18 levels were superior to serum ALT activity in identifying Acute Liver Injury (ALI) patients whose first blood sample was taken within 8 hours of overdose.

For assessing the safety of a new drug candidate it is of similar high importance that novel mechanism-based biomarkers such as ccK18 and K18 show no, or only a marginal increase (high specificity) in circumstances where false positive, elevations of standard liver enzymes ALT/AST may occur. As a model system Harrill et al [23] investigated the potential hepatotoxicity of heparin. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin, enoxaparin sodium, dalteparin sodium, or adomiparin sodium every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Minor elevations were observed in the levels of full-length keratin 18; but ccK18 fragments (apoptosis specific) were not detected suggesting that while heparins may cause mild hepatocyte necrosis, they do not induce apoptosis (Figure 8).

Figure 8

Effect of heparin on liver injury. Serum ccK18 and uncleaved K18 in subjects receiving injections of heparin. (adapted from [23]).

These studies clearly indicate that ccK18 and K18 hold potential as mechanism-based prognostic biomarkers during drug-induced liver injury.

2.3 SummaryBy utilising biomarkers specific for specific cell types and toxic mechanisms, hepatotoxicity can be detected earlier and with greater accuracy than with traditional biomarkers while providing important information as to toxic mechanisms.These biomarkers provide the opportunity to detect and eliminate potentially hepatotoxic compounds early from the development process and to improve the therapy of patients suffering from the effects of hepatotoxins.

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3 BIOMARKERS FOR DRUG INDUCED KIDNEY INJURY

3.1 IntroductionRenal diseases are an expanding problem. According to the European Kidney Health Alliance in 2012, 8% of Europeans have chronic kidney disease and renal dialysis may consume 2% of health budgets. This figure could double in five years [24].

Furthermore, drug-induced renal injury is the second most common cause of toxicity-related failure in drug development. Renal injury may be an under-recognised problem due to the ability of the kidneys to regenerate and their great reserve capacity [25]. The commonest test of renal function is serum creatinine. Creatinine is produced by the muscles at an approximately constant rate and mainly removed from the blood by glomerular filtration; therefore, the serum level is approximately inversely related to the glomerular filtration rate (GFR), the rate at which plasma is filtered by the glomeruli. However, serum creatinine is affected by many non-renal factors [26] and, furthermore, it is a very late biomarker as there must be a considerable loss of glomerular function before significant increases occur ([27] and see Figure 9). Furthermore even small increases in serum creatinine which apparently resolve are associated with increased long term morbidity and mortality and so should be prevented or avoided [43]. Better biomarkers can enable this.

Finally, most toxins affect the renal tubules and, even in cases of glomerular injury, changes in renal tubular biomarkers are important diagnostic indicators [28]. Because of the above, the measurement of biomarkers of renal tubular injury

and function are important in the understanding and monitoring of renal effects.

Figure 9

Response of novel renal injury biomarkers and serum creatine

following renal injury.

By measuring a selection of biomarkers with known origins, injury can be localised to distinct cell types. The absence of a biomarker from the urine also provides valuable information in that it shows where injury is not occurring and serves as a negative control. The

glutathione S-transferases have distinct distributions along the nephron [29]. They are found in high concentrations (4% of the soluble protein) in the cytosol and they are rapidly released into the urine in the event of injury to the tubular cells. Alpha GST is localised to the proximal convoluted tubule and the pi form in the distal tubules (Figure 10). In the rat, a mu form of GST (GSTYb1) has a similar distribution to pi GST in humans [30]. There are few biomarkers for distal tubular injury, although there is evidence that injury to it is associated with more progressive renal disease[44]. Pi GST is a well validated biomarker for distal tubule and so is especially valuable.

 

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Figure 10

Localization of alpha and Pi GST using immunohistochemistry

(Reprinted from [41] with permission from IOS Press).

The relationship between urinary GSTs and renal injury can be summarized as follows:· Elevated alpha GST – Acute proximal tubular injury· Elevated pi GST - Acute distal tubular injury· Both elevated - Global tubular injury

Urinary GSTs can be usefully combined with other biomarkers of renal injury and repair. For example McGuire et showed in a study on Hexachloro-1:3-Butadiene–induced acute nephrotoxicity in rats [40] how urinary alpha GST

combined with KIM-1 could define the time course and pathology of acute nephrotoxicity.Urinary alpha GST showed the time course of renal degeneration while the later induction and increase of KIM-1 indicate the time course of renal regeneration. Both biomarkers provided valuable complimentary information.

Figure 11

HCBD induced nephrotoxicity in rats, α-GST and KIM-1 responses post-dosing

(Schematic representation based on [40]).

Urinary GSTs have been studied as biomarkers of renal tubular injury in many situations, including toxicology (In-vitro, pre-clinical and human studies), environmental medicine, infection, diabetes, transplantation, acute renal failure, surgical injury and clinical toxicology, Alpha and pi GST have been proven to be excellent biomarkers in all these fields, providing rapid, sensitive and specific information regarding the site, severity and resolution of renal tubular injury (See [31] for a review).

Over 100 articles have been published on GSTs as renal tubular biomarkers including a large clinical validation, This provides confidence in the results obtained. Furthermore, urinary alpha GST was shown to be a valuable biomarker in the ILSI and IMI projects on nephrotoxicity biomarkers.

3.1.1 In vitro studies (cell culture)Histologically defined biomarkers, such as Alpha GST can provide sensitive and site-specific information on renal injury from cell cultures. For example, Sonee et al. [32] demonstrated the release of alpha GST from rat renal cell cultures in response to the known nephrotoxin, cisplatin. Similarly, Vickers et al., using cultured slices of human kidney, could demonstrate cell specificity in-vitro by the earlier release of alpha GST than pi GST in response to the proximal tubular toxin cisplatin [33]. Furthermore, the differential toxicity of different analogues of the known nephrotoxin, cyclosporin could also be shown by the relative release of alpha GST from human tissue slices (5).

Figure 12

Urinary α-GST, correlation with renal injury (Reprinted from [41]

with permission from IOS Press).

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3.1.2 Pre-clinical use (animal models)Animal studies offer the opportunity to obtain quantitative information between the release of biomarkers and histological changes. Urinary levels of biomarkers can be compared with the severity and time course of histological changes. For example, in a study in rats on the nephrotoxin compound A (a breakdown product of the anaesthetic Sevoflurane®), Kharasch et al. [34] showed that urinary alpha GST levels correlated closely with the extent of renal injury. Furthermore, even a few percent of necrotic renal tubules caused urinary alpha GST levels to increase several fold (Figure 12). In both human and animal studies, urinary alpha GST levels responded to the dose of toxin [34,35].

3.1.3 Human Studies Histological validation of renal injury is seldom possible in clinical trials; therefore, the use of histologically proven biomarkers is valuable in that they can provide a window to look into the kidney and a means of comparing animal and human responses. Published human studies include antibiotics, cyclosporin, contrast media, and anesthetics (See [31] for review). As a general rule, specific biomarkers are released in advance of changes in serum creatinine ([36], Figure 13).

Figure 13

Proximal tubular injury: aminoglycoside therapy

(adapted from [7]).

Combining biomarkers can also provide information as to the mechanism of renal injury. For example, Amphotericin B caused the release of alpha GST and pi GST in human subjects, while urinary N acetyl glucosaminamidase (NAG) rose little [37] This matches the toxicological mechanism of Amphotericin, which is to affect cell membranes, resulting in the release of cytosolic biomarkers, but not the lysosomal biomarker NAG (at the doses given) (Figure 14).

Figure 14

Amphoptericin toxicity (adapted from [36]).

Furthermore, only male volunteers showed increases in urinary GSTs, which agrees with the greater frequency of reactions in male subjects.

Specific biomarkers also proved the opportunity to compare the renal effect of different dosage regimes. For example, Ahlmén et al. demonstrated that the addition of a calcium blocker to cyclosporin reduced the release of alpha GST into the urine [38]. In humans, where biopsy is seldom possible in trials, the opportunity to localize injury to specific regions of the nephron is especially valuable.

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Figure 15

Distal tubular injury: Foscarnet therapy (adapted from [39]).

Most nephrotoxins affect the proximal tubule, but the distal tubular effects of the anti-fungal drug Foscarnet could be shown by the release of urinary pi GST in the absence of alpha GST increases [39] (Figure 15). Urinary Pi GST opens the opportunities to study toxic effects on new regions of the nephron. The assay of urinary GSTs provides unique insight into the site, severity and time course of nephrotoxic events.

3.2 SummaryBy utilising histologically defined renal injury biomarkers, nephrotoxicity can be detected earlier and with more sensitivity than when utilizing traditional biomarkers. Injury can be localised to specific regions of the renal tubule.

These biomarkers provide the opportunity to detect and eliminate potentially nephrotoxic compounds early from the development process and to reduce the exposure of patients to potentially damaging doses of nephrotoxic drugs.

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4 REFERENCES[1] http://www.imi-safe-t.eu/biomarker/drug-induced-injury/ consortium[2] http://c-path.org/ [3] Ozer J, et al. The current state of serum biomarkers of hepatotoxicity. Toxicology (245,3) 194-205 (2008).[4] McKim, Jr, JM et al. A new approach to in vitro toxicity screening based on multi-endpoint analysis provides information on mechanism and predicts in vivo toxicity. Poster presented at the 40th annual meeting of the Society of Toxicology, San Francisco, March 25-29, 2001.[5] Vickers AEM et al. Changes in Human Liver and Kidney Slice Function Related to Potential Side-Effects in the Presence of Biotransformation of Four Cyclosporin Derivatives GSA, IMM, OG, and PSC. In Vitro & Molecular Toxicology 11(2) (1998).[6] Alpha-GST Release as a Predictive Marker of Drug Induced Hepatotoxicity Utilising 3D InSightTM Liver Microtissues. Application note by Insphero (2013).[7] Clarke H. et al. Glutathione S-Transferase (αGST) release, an early indicator of carbon tetrachloride hepatotoxicity in the rat. Human and Experimental Toxicology (16) 154-157 (1997).[8] Tong V. et al. Valproic Acid I: Time Course of Lipid Peroxidation Biomarkers, Liver Toxicity, and Valproic Acid Metabolite Levels in Rats. Xiao Wei Teng, Thomas K. H. Chang, and Frank S. Abbott.Toxicological Sciences (86,2), 427–435 (2005).[9] http://www.fda.gov/Drugs/DrugSafety/ucm085729.htm[10] Coluccio D et al. A study to evaluate Biotrin rat alpha-glutathione S-transferases (alpha-GST) enzyme immunoassay (EIA) and its value to detect liver changes compared to standard liver enzymes.; American Association of Clinical Chemists. 52nd Annual Meeting, San Francisco, CA. USA. Clin Chem. (46) A140 (2000).[11] Eason S et al. Serum Glutathione S-Transferase Alpha (α-Gst): A Suitable Biomarker For Drug-Related Liver Injury?, 8th World congress on clinical pharmacology and therapeutics Brisbane, Australia, 1-6th August 2004.[12] Marco LA et al. Serum α-glutathione S-transferase (α GST) becomes elevated shortly after subtoxic acetaminophen overdose. Can J Emerg Med (3,3) 2002.[13] Beckett GJ et al. Glutathione S-Transferases: Biomedical Applications. Advances in Clinical Chemistry, (30) 281-380 (1993).[14] Maxwell PR et al. Serum alpha glutathione S-transferase as a biomarker of Methotrexate induced hepatotoxicity. Poster presented at the 43rd annual meeting of the Society of Toxicology, Baltimore USA, 21-25th March 2004.[15] Rees, GW et al Evaluation of an enzyme-immunoassay for serum α-glutathione S-transferase. Ann. Clin. Biochem (32) 575-583 (1995).[16] Andersson TB et al. The HepaRG cell line: a unique in vitro tool for understanding drug metabolism and toxicology in human. Expert Opin Drug Metab Toxicol (8) 909-20 (2012).[17] Szabo M et al. Comparison of human hepatoma HepaRG cells with human and rat hepatocytes in uptake transport assays in order to predict a risk of drug induced hepatotoxicity. PLoS One. 8:e59432 (2013).[18] Shevchenko V et al. Poster presented at at SOT’s 53rd Annual Meeting, March 24–27 (2014).[19] Antoine DJ et al. Mechanism-based bioanalysis and biomarkers for hepatic chemical stress. Xenobiotica. (39) 565-77 (2009).[20] Antoine DJ et al. Molecular forms of HMGB1 and keratin-18 as mechanistic biomarkers for mode of cell death and prognosis during clinical acetaminophen hepatotoxicity. J Hepatol (2012); 56:1070-9[21] Antoine DJ et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. J Hepatology (58) 777-87 (2013).[22] Antoine DJ et al. Stratification of paracetamol overdose patients using new toxicity biomarkers: current candidates and future challenges. Expert Rev Clin Pharmacol (7) 181-9 (2014).

[23] Harrill AH et al. The effects of heparins on the liver: application of mechanistic serum biomarkers in a randomized study in healthy volunteers. Clin Pharmacol Ther. (92) 214-20 (2012).[24] European Kidney Health Alliance. Policy Options for Kidney Health in Europe. www.ekha.eu (2012).[25] Hook JB et al. Toxic responses of the kidney. In The Basic Science of Poisons. 2nd Edition (Doll, J. and Klaasen, C.D. eds). Macmillan, New York. pp 310–329 (1980).[26] Price CP et al. Developments in the assessment of the glomerular filtration rate. Clin Chim Acta (297) 55–66 (2000).[27] Shemesh, O et al. Limitations of creatinine as a filtration marker in glomerulopathic patients. Kidney Int (28,5), 830–838 (1985).[28] D’Amico G et al. Urinary protein and enzyme excretion as markers of tubular damage. Curr Opin Nephrol Hypertens (12,6), 639–643 (2003).[29] Harrison DJ et al. Glutathione S-transferase isoenzymes in the human kidney: Basis for possible markers of renal injury. J Clin Pathol (42) 624–629 (1989).[30] Rozell B et al. Glutathione transferases of classes alpha, mu and pi show selective expression in different regions of rat kidney. Xenobiotica 23(8), 835–849 (1993)[31] Shaw M. Cell specific biomarkers in renal medicine and research The Urinary Proteome, Methods and Protocols. Ed Alex, J. Rai. Humana Press (2010).[32] Sonee M. et al. Evaluation of kidney toxicity biomarkers in human kidney cells. Presentation at the 46th Society of Toxicology meeting. Charlotte, NC, USA, 25–29 March 2007.[33] Vickers AEM et al. Kidney Slices of Human and Rat to Characterize Cisplatin-Induced Injury on Cellular Pathways and Morphology. Toxicologic Pathology. (32) 577–590 (2004).[34] Kharasch, E.D. et al. Role of renal cysteine conjugate β-lyase pathway in inhaled compound A nephrotoxicity in rats. Anesthesiology (88,6) 1624–1633 (1998).[35] Goldberg, ME et al. Dose of compound A, not Sevoflurane, determines changes in the biochemical markers of renal injury in volunteers. Anesth Analg (88) 437–445 (1999). [36] Bäckman, L et al. Glutathione transferase in the urine: A marker for post-transplant tubular lesions. Kidney Int (33) 571–577 (1998).[37] Pai, MP et al Assessment of effective renal plasma flow, enzymuria, and cytokine release in healthy volunteers receiving a single dose of Amphotericin B desoxycholate. Antimicrob Agents Chemother (49,9) 3784–3788 (2005).[38] Ahlmén J, et al. Decreased nephrotoxicity after the use of a microemulsion preparation of cyclosporin A compared to a conventional solution. Transplant Proc 27(6), 3432–3437 (1995).[39] Sundberg AGM, et al. Urinary pi-class glutathione transferase as an indicator of tubular damage in the human kidney. Nephron (67) 308–316 (1994)[40] Maguire DP et al. Correlation of histopathology, urinary biomarkers, and gene expression responses following hexachloro-1:3-butadiene-induced acute nephrotoxicity in male Hanover Wistar rats: a 28-day time course study. Toxicol Pathol. (41, 5) 779-94 (2013).[41] Shaw M. The use of histologically defined specific biomarkers in drug development with special reference to the glutathione S-transferases. Cancer Biomarkers 1(1) 69-74 (2005). [42] Caspase-cleaved Keratin 18 Release as a Predictive Marker of Drug Induced Hepatocyte Apoptosis Utilising 3D InSightTM

Liver Microtissues. Application note by Insphero (2014).[43] Nenov, VD et al. Multi-hit nature of chronic renal disease. Curr Opin Nephrol Hypertens 9, 85-97 (2000).[44] Branten, AJ et al. Urinary excretion of isoenzymes of glutathione S-transferase alpha and pi in patients with proteinuria. Reflection of the site of tubular injury. Nephron 85, 120-126 (2000).

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5 APPLICATION NOTES5.1 Alpha-GST Release as a Predictive Marker of Drug Induced Hepatotoxicity Utilising 3D InSightTM Liver Microtissues

Introduction and BackgroundDrug induced liver injury (DILI) is a major obstacle in the development of new pharmaceutical identities. Evaluation of potential DILI effects of novel compounds during pre-clinical phases is, therefore, a prerequisite for their safe entry into clinical trials. Hepatocytes, the major cell type in the liver, contain high amounts of metabolic enzymes, which are released upon cellular injury into the cell culture supernatant. Beside the commonly used markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), alpha-GST is increasingly being used as a liver injury marker. Over 150 articles have been published on its use in hepatotoxicity and in a wide range of clinical conditions. Alpha-GST provides advantages as a biomarker for hepatotoxicity due to its high cytosolic concentration in hepatocytes and its rapid release upon their injury1. Detection of alpha-GST release is therefore a specific and early indicator of hepatocyte injury. Mitochondrial injury leads to decreases in ATP production, making the assay of intracellular ATP a sensitive test for this pathological process. By combining the assay of intracellular ATP and alpha GST, one can potentially both detect hepatotoxicity and obtain information as to the potential toxic mechanism.

InSphero’s 3D InSightTM liver microtissues are co-culture spheroids that mimic liver physiology by providing a liver-like cell structure with dense inter-hepatocyte contacts. The unique production technology utilising hanging-drop culture enables excellent size-reproducibility and avoids the need for artificial extracellular-matrix components2. The culture of liver microtissues in 96-well format is possible for several weeks, making long-term toxicity testing possible. Since only 1000 hepatocytes/microtissue are necessary, large numbers of 3D insight microtissues can be produced from a single vial of primary cells. However, the low numbers of hepatocytes present means that very sensitive assays for monitoring changes in cellular status are required. In this study, the assay of alpha GST release, utilising the EKF Diagnostics alpha-GST ELISA kits, and CellTiter-Glo® Luminescent Cell ATP assay were compared as tests for hepatotoxicity utilising InSphero’s 3D InSightTM liver microtissues. As test substances, two compounds with known hepatotoxic risks were chosen, Amiodarone3 and Diclofenac5.

Key Features• Highlysensitivedetectionofreleasedalpha-GSTfrom3DInSightTM liver microtissues • Detectshepatotoxicityofratandhumanlivermicrotissueswithaslittleas1000cells/microtissue

Materials and MethodsASSAYS• HumanAlpha-GSTEIA (EKFDiagnostic,REF:BIO91RUO)• RatAlpha-GSTEIA (EKFDiagnostic,REF:BIO64RT)• CellTiter-Glo®LuminescentCellViabilityAssay (Promega,Cat:G7572)

3D LIVER MICROTISSUES• 3DInSight™HumanLiverMicrotissues (InSpheroAG,Cat:MT-02-002-04)• 3DInSight™RatLiverMicrotissues (InSpheroAG,Cat:MT-02-001-04)

MEDIA AND COMPOUNDS• 3DInSight™HumanLiverMaintenanceMedium InSpheroAG,Cat:CS-07-001-01)• 3DInSight™RatLiverMaintenanceMedium (InSpheroAG,Cat:CS-07-002-01)• Diclofenacsodiumsalt (Sigma,Cat:D6899)• Amiodaronehydrochloride (Sigma,Cat:A8423)

KeyFeatures• Highlysensitivedetectionofreleasedalpha-GST from3DInSightTMlivermicrotissues

• Detectshepatotoxicityofratandhumanliver microtissueswithaslittleas1000cells/microtissue

APPLICATIONNOTES

19

CELL CULTURE AND TEST SET-UPRat liver microtissues were treated with Amiodarone at the following concentrations: 500µM; 250µM; 125µM; 62.5µM; 31.25µM; 15.63µM; 7.81µM. Human liver microtissues were treated with Diclofenac at the following concentrations: 1000µM; 500µM; 250µM; 25µM; 62.5µM; 31.25µM; 15.63µM. Control cells were treated with DMSO in 70 µl maintenance medium per well. After five days incubation, the release of alpha-GST into the culture medium of the microtissues was quantified. Measurement of viability was performed with CellTiter-Glo®.

ALPHA GSTCell culture supernatants of at least 3 independent liver microtissues after compound incubation were frozen at -20°C until alpha-GST assessment. The alpha-GST assays were performed according to the manufacturer’s instruction whereby the cell culture supernatants were diluted 1:1 with sample diluent (50µl + 50µl) before incubation on the ELISA plates. The same procedure was applied for rat and human samples.

Table 1

Instrument settings for alpha-GST

readout on the Infinite M200 PRO.

ATP-ASSAYMeasurement of ATP-content of liver microtissues was performed with CellTiter-Glo® reagent (Promega, Madison, USA). The assay was performed according to manufacturer’s protocol with an increased incubation time of 20 minutes. Luminescence was quantified with the Tecan Infinite M200Pro (Tecan Group Ltd., Männedorf, Switzerland).

Results and DiscussionRAT LIVER MICROTISSUES AND AMIODARONEAmiodarone is known to cause hepatotoxicity by affecting mitochondrial function3. Cell toxicity was studied by simultaneously measuring the decrease of ATP concentration and the release of the cytosolic protein alpha GST. As is evident in Figure 1A+B, the treatment of rat liver microtissues with Amiodarone led to a dose-dependent decrease in ATP production and a concomitant release of alpha-GST into the either biomarker corresponded very closely (ATP: 55.26 µM alpha-GST: 52.36 µM) and were in agreement with previously published studies on cultures of rat hepatocytes in 2D-culture (IC50 of 38.3 µM) 4. In comparison with 2D cultivated HepG2 cells (IC50 of 78.9 µM) 4 the rat liver microtissues showed increased sensitivity towards this drug.

Figure 1

Dose response curve of rat liver microtissues treated over 5 days with Amiodarone. Measurement of intracellular ATP-content (A) and released

alpha-GST (B) from the same tissues (n=4).

Measurementparameter Instrumentsettings

Mode Absorbance

MeasurementWavelength 450nm

ReferenceWavelength 630nm

A B

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Human Liver Microtissues and DiclofenacDiclofenac is known to cause hepatotoxicity via mechanisms that involve mitochondrial injury5 It produced a concentration-dependent cytotoxic effect on the human liver microtissues, as assessed by CellTiter-Glo® (ATP production) and alpha-GST release (Figure 2A+B) with IC50 values of 91.3 µM (ATP) and 136.2 µM (alpha-GST). Compared with published data on 2D cultures (IC50 of primary human hepatocytes 331 µM, primary rat hepatocytes 392 µM and HepG2 399µM) 4,5 the 3D human liver microtissues were more sensitive than conventional standard liver in-vitro models. The toxicity of Diclofenac requires active phase I+II enzyme metabolism, and these enzyme systems are highly expressed in 3D liver models, but are severely down-regulated in other model systems. In this case, the observed difference between the calculated IC50 from ATP or alpha-GST assay may be an indication that decreases in ATP production and the release of alpha GST are reflecting different aspects of the pathological process. Possibly, the cells could survive a certain level of mitochondrial injury with a decrease in ATP production and retain viable but when a threshold is exceeded the cells die and alpha GST is released.

Figure 2

Dose response curve of human liver microtissues treated over 5 days with Diclofenac. Measurement of intracellular ATP-content (A) and released

alpha-GST (B) from the same tissues (n=3).

ConclusionIn summary, alpha-GST release assayed with the EKF Diagnostics alpha GST assays was shown to be a highly reliable and sensitive biomarker to detect hepatotoxicity utilising single liver microtissues. Alpha GST release showed similar sensitivity to the reduction in ATP production, however, since the reduction in ATP production and the release of alpha GST reflect different pathological processes, their combination may provide additional valuable information. This is demonstrated in this study where both Amaridone and Diclofenac are known to affect mitochondrial function.Since destruction of the microtisssues is not required for the assay of alpha GST in the supernatant, as opposed to the assay of intra-cellular ATP, its assay more conveniently enable the monitoring of microtissue viability over time. This can provide more detailed toxicokinetics and reduce the quantities of liver microtissues required. Thus, the combination of EKF diagnostic’s alpha-GST ELISA test kit with InSphero’s 3D InSight™ Liver Microtissues is a valuable combination well suited for the investigation of drug-induced hepatotoxicity in vitro.

References

A B

[1] Douglas, K., Mian, Z., Irshad, C. & Ping, W. Plasma alpha-Glutathione S-Transferase. Archives of Surgery 135, 198–203 (2000). [2] Messner, S., Agarkova, I., Moritz, W. & Kelm, J. M. Multi-cell type human liver microtissues for hepatotoxicity testing. Archives of Toxicology 87, 209–13 (2012).[3] Giri, S., Nieber, K. & Bader, A. Hepatotoxicity and hepatic metabolism of available drugs: current problems and possible solutions in preclinical stages. Expert Opinion on Drug

Metabolism & Toxicology 6, 895–917 (2010).[4] Wang, K., Shindoh, H., Inoue, T. & Horii, I. Advantages of in vitro cytotoxicity testing by using primary rat hepatocytes in comparison with established cell lines. The Journal of Toxicological Sciences 27, 229–37 (2002).[5] Bort, R., Ponsoda, X., Jover, R., Gómez-Lechón, M. J. & Castell, J. V Diclofenac toxicity to hepatocytes: a role for drug metabolism in cell toxicity. The Journal of Pharmacology and Experimental Therapeutics 288, 65–72 (1999).

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Species

Reference values

Cross reaction

Background

Intended use

Serum & Plasma

BIO9196 ELISA2.5 - 80 μg/l1.9 μg/l2 h100 μl (Suggested initial dilution 1:5)Serum & PlasmaCentrifuge within 3 hours from time of collection and transfer the sample from the original tube for storage. Samples can be stored at 20 – 25 °C for up to 48 hours, at 2 – 8 °C for up to one week or at -20 °C for >1 year. Repeated freeze thawing of samples should be avoided.

Human

0 - 12 μg/l

No cross-reactivity was observed with μGST at 500 μg/l or π-GST at 500 μg/l

α-GST is a member of the GST superfamily of small cytosolic proteins primarily involved in cellular detoxification reactions. α-GST is approximately 51kDa and can comprise upto 2 % of the cytosolic soluble protein content. As a result of its constitutive nature, α-GST is immediately released into the urine upon lysis of epithelial cells of the proximal tubule.

The α-GST EIA provides a method for the quantitative determination of alpha glutathione S-transferase (α-GST) in human serum and plasma. The α-GST EIA can also be used for the determination of αGST in human urine, to assess kidney injury.

α-GST is expressed in hepatocytes and its increase in blood is a sensitive indicator of acute hepatic injury. Increased αGST has been demonstrated to be indicative of hepatotoxicity, allograft rejection, viral hepatitis.

α-GST, Human serum & plasmaNovel Liver Injury Biomarkers

6 OVERVIEW OF ASSAYS AND INTENDED USE

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Species

Reference values

Cross reaction

Background

Intended use

BIO9196 ELISA2.5 - 80 μg/l1.9 μg/l2 h100 μlUrine (Suggested initial dilution 1:2) Overnight or 24 h urine is recommended.As soon as possible after collection sample should be diluted with urine stabilizing buffer (USB). Sample:USB 4:1. After the addition of USB, samples can be stored at 20-25 °C for up to 48 hours, at 2-8 °C for up to one week or at - 20 °C for >1 year.Repeated freeze thawing of samples should be avoided.

Human

0 - 29.0 µg/l (n = 120)

No cross-reactivity was observed with μ-GST at 500 μg/l or π-GST at 500 μg/l

Alpha Glutathione S-Transferase (α-GST) is a member of the GST superfamily of small cytosolic proteins primarily involved in cellular detoxification reactions. α-GST is approximately 51kDa and can comprise up to 2 % of the cytosolic soluble protein content. As a result of its constitutive nature, α-GST is immediately released into the urine upon lysis of epithelial cells of the proximal tubule.

The α-GST EIA provides a method for the quantitative determination of alpha glutathione S-transferase (α-GST) in human urine. The α-GST EIA can also be used for the determination of α-GST in human, serum and plasma, to assess liver injury.

Elevated urinary α-GST levels are an early indicator of acute kidney injury (AKI) onset in cases of nephrotoxicity, environmental toxicity, cardiothoracic surgery and in transplantation rejection. The release of π-GST has been shown to be associated with distal tubular damage, thus simultaneous measurement of both α-GST and π-GST allows discrimination between proximal and distal tubular damage. Low basal levels of α-GST are released in normal healthy individuals.

α-GST, Human urineKidney Injury Biomarker for proximal tubular damage

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α-GST, Rat serumNovel Liver Injury Biomarkers

Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample type

Sample preparation

Reference values

Species

Cross reaction

Background

Intended use

BIO64RT96 ELISA1.56 – 100 µg/l0.2 µg/l2.5 hours100 μlSerum (1:50)Cell Culture Supernatant (1:5 – 1:30 dependant on expected concentration)Sample preparation: For serum sample analysis, blood samples should be allowed to clot at room temperature for 2 hours or 2 – 8 ºC overnight. The sample is then centrifuged (3000 rpm/10 minutes) and the serum collected. Serum samples should be assayed immediately or may be stored at 2 – 8 ºC for 4 days. If necessary, the samples may be frozen at -20 ºC. Avoid repeated freezing and thawing. Do not store diluted samples. Samples can be stored at -20ºC for at least one month.

Cell Culture Supernatant:A hepatocyte cell number of 4 x 105 cells /ml cell culture medium is recommended.

Sprague-Dawley Rats 43 ± 56μg/l (mean ± 2SD) Normal ranges may vary between different rat strains. Therefore, it is important for each laboratory to assess a normal range for the rat strain used in the study.

Rat

Cross reaction: No significant cross-reactivity is observed with Yp or Yb1 isoforms.Cross reactivity with human, canine and porcine αGST is undetectable.

α-GST is a member of the GST superfamily of small cytosolic proteins primarily involved in cellular detoxification reactions. α-GST is approximately 51kDa and can comprise upto 2% of the cytosolic soluble protein content. α-GST is expressed in the cytosol of epithelial cells of the proximal tubule. As a result of its constitutive nature, α-GST is immediately released into the urine upon lysis of epithelial cells of the proximal tubule.In the liver, α-GST is expressed at high concentrations in the cytosol of hepatocytes throughout the liver parenchyma and is rapidly released upon cellular lysis making it a sensitive biomarker of acute hepatic injury

In the event of liver injury, α-GST is released more rapidly than transaminases and has a shorter half-life in the circulation. Therefore, α-GST levels more accurately indicate the onset and resolution of hepatocyte injury than transaminases. α-GST has been proven to be a superior indicator of hepatocyte injury in hepatotoxicity, transplantation and ischemia-reperfusion injury.

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α-GST, Rat urineKidney Injury Biomarker for proximal tubular damage

Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample type

Sample preparation

Reference values

Species

Cross reaction

Background

Intended use

BIO64RT96 ELISA1.56 – 100 µg/l0.2 µg/l2.5 hours100 μlUrine (Suggested initial dilution 1:5) Cell Culture Supernatant (1:5 – 1:30 dependent on expected concentration)As soon as possible after sample collection, add 100 μl of Rat Urine Stabilizing Buffer to 400 μl urine (4/5 dilution of patient sample), even if the samples are not to be stored. Do not store samples without the addition of Rat Urine Stabilizing Buffer. Rat Urine Stabilizing Buffer should be added within 12 hours of sample collection. After the addition of Rat Urine Stabilizing Buffer, samples can be stored at 2-8 ºC for at least 48 hours or at -20 ºC for at least a month.

Sprague-Dawley Rats: 46.9 ± 44.6 μg/l (mean ± 2SD) Wistar Rats: 23.0 ± 26.4 μg/l (mean ± 2SD)

Rat

No significant cross-reactivity is observed with Yp or Yb1 isoforms. Cross reactivity with human, canine and porcine α-GST is undetectable.

Alpha Glutathione S-Transferase (α-GST) is a member of the GST superfamily of small cytosolic proteins primarily involved in cellular detoxification reactions. α-GST is approximately 51kDa and can comprise up to 2% of the cytosolic soluble protein content. α-GST is expressed in the cytosol of epithelial cells of the proximal tubule. As a result of its constitutive nature, α-GST is immediately released into the urine upon lysis of epithelial cells of the proximal tubule.

The α-GST Rat EIA provides a method for the quantitative determination of alpha glutathione S-transferase (α-GST) in rat urine. Urinary α-GST levels correlate closely with the time course and the severity of renal injury. Urinary α-GST levels are more sensitive indicators of renal tubular injury than serum creatinine.

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Reference values

Species

Cross reaction

Background

Intended use

BIO8596 ELISA1.25 - 40 μg/l1.3 μg/l2.5 h100 μlUrine (Suggested initial dilution of 1:2)Samples should be mixed 1 part urine stabilizing buffer with 4 parts urine prior to storage. If the sample seems to contain blood, it must be centrifuged to remove the erythrocytes prior to adding the stabilizing buffer. (See IFU for full instructions).It is recommended that samples are assayed as soon as possible after collection.After the addition of USB, samples can be stored at 20-25 °C for up to 48 hours, at 2-8 °C for up to one week, or at -20 °C for >1 year.Repeated freeze-thaw cycles should be avoided.

0 – 30 µg/l (n=132)

Human

No cross-reactivity was observed with μ-GST at 500 μg/l or α-GST at 500 μg/l.

Pi Glutathione S-Transferase (π-GST) is a member of the GST superfamily of small cytosolic proteins primarily involved in cellular detoxification reactions. π-GST is approximately 47kDa and can comprise up to 2 % of the cytosolic soluble protein content. As a result of its constitutive nature, π-GST is immediately released into the urine upon lysis of epithelial cells of the distal tubule.

The π-GST EIA provides a method for the quantitative determination of π-GST in human urine.

π-GST is specifically expressed in epithelial cells of the distal tubules of the human kidney whereas α-GST expression is confined to cells of the proximal tubules. π-GST is readily released from epithelial cells upon cellular lysis making it a rapid and sensitive indicator of acute kidney injury (AKI) onset in cases of nephrotoxicity, environmental toxicity, cardiothoracic surgery and the intensive care setting. π-GST has also demonstrated clinical value in assessing transplant rejection and diabetes.π-GST has been demonstrated to be an excellent predictor of severe acute kidney injury leading to dialysis requirement in cardiothoracic surgery. The combination of sensitivity and specificity makes urinary π-GST the biomarker of choice when accurate diagnosis of renal acute distal tubular injury is required.

π-GST, Human urineNovel Liver Injury Biomarkers

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Species

Hook Effect

Intended use

*Note

1090096ELISA250 – 3000 U/L (units are defined against a synthetic peptide standard containing the M30 and M6 epitopes; 1 U/L = 1.24 pM)60 U/L 4.5 hours 25 µlCell lysates or culture supernatantsFresh samples are stable for up to maximum one day at 2 – 8 °C, for at least 9 months at -20 °C, and for at least two years when stored at -80 °C . Avoid repeated freeze-thawing .

Human, monkey and bovine K18 fragments

No high dose “hook effect” occur until 26000 U/L which is well above concentrations of K18Asp396-NE (M30)-reactive material observed in cell culture samples.

Quantitative measurement of the apoptotic cell death biomarker K18-Asp396-NE in cell culture ex¬periments. Can be used for cell lysates and/or culture supernatants. The assay only detects apoptosis in cells of epithelial origin that express K18. Cells should be of human, monkey or bovine origin.To be used to determine accumulation of caspase-cleaved K18 (ccK18) in cell cultures, providing an integra-tive measure of apoptosis. The K18-Asp396 neo-epitope is formed by caspase-3, -7 or -9 activation.

caspase-cleaved K18 = ccK18 previously Cytokeratin 18 (CK18/ccCK18)

M30 CytoDeath™ ELISA (PEVIVA®)Measurement of soluble caspase-cleaved fragments of the intermediate filament protein keratin 18 (K18) containing the M30 neo-epitope (K18-Asp396-NE). Released from human, monkey or bovine epithelial cells in cell culture.

27

Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample type

Sample preparation

Reference values

Species

Hook Effect

Intended use

*Note

10011 96 ELISA75 – 1000 U/l (units are defined against a synthetic peptide standard containing the M30 and M5 epitopes; 1 U/l = 1.24 pM)25 U/L4.5 hours25 µlHuman serum or plasma (EDTA, citrate, heparin plasma), K18Asp396-NE (M30)-reactive material released from apoptotic K18 positive human cells. The same type of material, i.e. serum or plasma collected by one method, should be used for a specific project .Fresh samples are stable for up to maximum one day at 2 – 8 °C, for at least 9 months at -20 °C, and for at least two years when stored at -80 °C. Avoid repeated freeze-thawing.

Healthy ≤ 150 U/LSlightly elevated 150 – 200 U/lElevated > 200 U/l CcK18 values were measured in 200 apparently normal Swedish blood donors. With the M30 Apoptosense® ELISA, male and female values showed a similar distribution and no age dependency. Based on the distribution the recommended cut-off value for elevated ccK18 has been set at > 200 U/L. In case of suspected fibrosis or fatty liver disease: ccK18 values between 150 and 200 U/L suggest an indication for mild fibrosis (for example NAFL); ccK18 values > 200 U/L are a strong indication for severe significant liver damage (for example NASH).

Human, non-human primates

No high dose “hook effect” occurs until 50,000 U/L which is well above concentrations of K18Asp396-NE (M30)-reactive material observed in cell culture samples.

The M30 neo-epitope (K18Asp396-NE) is a sensitive and integrative indicator specific for epithelial cell death involving caspase -3, -7 or -9 activation.

Quantitative measurement of the apoptotic cell death biomarker K18Asp396-NE (“M30 antigen”) released from dying human epithelial cell lines in vitro or in vivo. The cells or tissues should be of human epithelial origin (e.g. kidney, gut, colon, lung or liver) expressing K18.

caspase-cleaved K18 = ccK18 previously Cytokeratin 18 (CK18/ccCK18)

M30 Apoptosense® ELISA (PEVIVA®)Measurement of soluble human intermediate filament protein fragments of keratin 18 (K18)* that contain the M30 neo-epitope (K18Asp396-NE). Released from human epithelial cells.

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample type

Sample preparation

Reference values

Species

Hook Effect

Specificity

Intended use

*Note

1004096ELISA 67 – 5000 U/l (The units measured by the M65 EpiDeath® ELISA are defined against native antigen spiked into serum. Native antigen is calibrated against a recombinant protein standard.1 U/l = 1 .24 pM).25 U/l4.5 hours25 µl Human serum or plasma (EDTA, citrate, heparin plasma), K18-reactive material released from K18 positive human cells . Multiple freeze-thaw cycles of samples are well tolerated. The same type of material, i.e. serum or plasma collected by one method, should be used for a specific project. Cell culture supernatants from K18 positive (epithelial) cells or tissues.If the assay is to be performed the same day, the samples can be stored at 2 – 8 °C. Samples are stable for at least 9 months at -20 °C, and for at least two years when stored at -80 °C.

Healthy ≤ 150 U/LSlightly elevated 150 – 200 U/lElevated > 200 U/l

Total soluble K18 (uncleaved and cleaved Keratin 18 representing total cell death (necrosis and apoptosis) was measured in 200 apparently normal Swedish blood donors. Male and female values showed a similar distribution and no age dependency. Based on the distribution the recommended cut-off value for elevated K18 has been set at > 200 U/L.

Human, non-human primates

No high dose “hook effect” occur until 200,000 U/l which is well above concentrations of M65-reactive material observed in cell culture or serum/plasma samples.

The assay uses two monoclonal antibodies directed to epitopes in the 284 – 396 region of the K18 protein. Soluble full length K18 as well as K18 fragments and protein complexes that expose these epitopes will be detected by the assay. Quantitative measurement of total soluble keratin 18 (K18) released from dead cells (necrotic and apoptotic). The cells or tissues should be of human epithelial origin (e.g. kidney, gut, colon, lung or liver) expressing K18.

The M65 EpiDeath® ELISA can be combined with the M30 Apoptosense® ELISA (PEVIVA Prod. No. 10010) for determination of cell death mode (apoptosis versus necrosis). Death mode can be determined in vitro and in serum from cancer patients.

caspase-cleaved K18 = ccK18 previously Cytokeratin 18 (CK18/ccCK18)

M65 EpiDeath® ELISA (PEVIVA®)Measurement of total cell death (apoptosis + necrosis) of human intermediate filament protein keratin 18 (K18) released from human epithelial cells.

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Species

Background

Intended use

H-RENA-E-00196 ELISA0.31 - 10 ng/ml0.01 ng/ml3.5 hours50 μlUrine (Suggested initial dilution of 1:3)4 °C for up to four days or -20 °C for longer periods.Human

Kidney Injury Molecule-1 (KIM-1) is a type I trans-membrane structural glycoprotein expressed on epithelial cells of the renal proximal tubule. KIM-1 is undetectable in healthy tissue but its expression is highly induced in response to toxic and ischemic injury. The ectodomain of KIM-1 is shed into the urine in response to these injuries making it an early and specific biomarker for acute kidney injury (AKI).

The KIM-1 EIA provides a method for the quantitative determination of KIM-1 in human urine. KIM-1 has demonstrated excellent value as a pre-clinical tool for monitoring drug induced nephrotoxicity. In the clinical setting KIM-1 has been investigated as a marker of AKI onset in nephrotoxicity studies, cardiothoracic surgery and in the intensive care setting. KIM-1 has also been investigated as a biomarker of nephropathy status in chronic disorders such as diabetes.

KIM - 1, Human urineKidney Injury Biomarker for proximal tubular damage

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample type

Sample preparation

Species

Background

Intended use

R-RENA-E-00196 ELISA0.31 - 10 ng/ml0.31 ng/ml3.5 hours50 μlUrine (Suggested initial dilution of 1:3)

4 °C for up to four days or -20 °C for longer periods.

Rat

Kidney Injury Molecule-1 (KIM-1) is a type I trans-membrane structural glycoprotein expressed on epithelial cells of the renal proximal tubule. KIM-1 is undetectable in healthy tissue but its expression is highly induced in response to toxic and ischemic injury. The ectodomain of KIM-1 is shed into the urine in response to these injuries making it an early and specific biomarker for acute kidney injury (AKI).

The KIM-1 Rat EIA provides a method for the quantitative determination of KIM-1 in rat urine. Urinary KIM-1 is an early and specific biomarker for AKI and is an excellent tool in pre-clinical studies to monitor acute renal toxicity, by identifying adverse reactions from drugs and therapeutic agents in development. KIM-1 has been qualified by the Predictive Safety Testing Consortium (PSTC) for preclinical toxicity testing in drug development studies.

KIM - 1, Rat urineKidney Injury Biomarker for proximal tubular damage

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Species

Cross reaction

Background

Intended use

Z-00196 ELISA3 - 400 ng/ml3 ng/ml3 hours50 μlUrineSamples should be tested soon after collection, or store at -20 °C.Repeated freeze-thawing of samples should be avoided.

Human

No cross-reactivity (≤ 0.1 %) with I-FABP.

Liver Type Fatty Acid Binding Protein (L-FABP) is a small 14kDa cytosolic protein of the FABP family of proteins. These proteins are involved in the transport of long chain fatty acids to the mitochondria or peroxisome for β-oxidization. L-FABP is expressed in the cytoplasm of proximal tubular epithelial cells reflecting their high energy requirements met through fatty acid metabolism. Due to its small size, FABP leaks rapidly out of cells damaged by protein overload stress or ischaemia leading to a rapid rise in urinary levels.

The L-FABP EIA provides a method for the quantitative determination of L-FABP in human urine. L-FABP is rapidly released into the urine in response to injury and has demonstrated significant value as an early biomarker for the detection of acute kidney injury (AKI) onset in various clinical settings including:• CardiothoracicSurgery• Emergencyroomandintensivecare-unit• ContrastInducedNephropathy

L-FABP has also been investigated as a biomarker of disease progression in:• DiabeticNephropathy

L-FABP, Human urineKidney Injury Biomarker for proximal tubular damage

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample type

Sample preparation

Species

Cross reaction

Background

Intended use

BIO9096 ELISA0.4-100 ng/ml0.4 ng/ml3.5 hours100 μlUrine (Suggested initial dilution of 1:20)Plasma (Suggested initial dilution of 1:10)Cell cultureUrine:Collect urine using normal aseptic techniques. Centrifuge the urine to remove debris (1500xg at 4 °C for 15 min). Transfer urine to a fresh polypropylene tube.Plasma:Separate plasma from whole blood within 20 min of sampling (1500xg at 4 °C for 15 min). Remove plasma and transfer to fresh polypropylene tube. Recentrifuge the transferred plasma in order to avoid every contamination with white blood cells (1500xg at 4 °C for 15 min). Most reliable results are obtained if EDTA plasma is used.Storage:Store samples below -20 °C, preferably at -70 °C in polypropylene tubes. Use samples within 24 hours after thawing. Avoid multiple freeze-thaw cycles. Do not use hemolyzed, hyperlipemic, heat-treated or contaminated samples.

Human

Cross reactivity for other protein / peptides has not been tested Human Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a low molecular weight 25kDa glycoprotein and a member of the lipocalin superfamily, a family of small extracellular proteins that are characterized by the ability to bind small hydrophobic molecules. NGAL can also bind to specific cell surface receptors.NGAL has been identified as an iron-transporting protein during nephrogenesis, demonstrating a role for NGAL in renal organogenesis. NGAL is widely expressed in a variety of human tissues, including, kidney trachea, lungs, stomach and colon. In the kidney NGAL is thought to be expressed in the collecting duct and distal tubule.

The NGAL EIA provides a method for the quantitative determination of NGAL in human urine, plasma and cell culture supernatant. NGAL is rapidly upregulated and released in response to injury and has demonstrated value as an early biomarker for the detection of acute kidney injury (AKI) onset in various clinical settings including:• CardiothoracicSurgery• Emergencyroomandintensivecare-unit• ContrastInducedNephropathy

NGAL has also been investigated as a biomarker of renal injury in:• Acuteallograftrejection• Chronickidneydisease

Serum NGAL has been investigated as a biomarker of AKI onset in the critical care population.

NGAL, HumanKidney Injury Biomarker for proximal tubular damage

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Reference values

Species

Cross reaction

Interference

Intended use

BIO8296 ELISA15.6 – 1000 μg/l15.6 μg/l1 hour50 μlSerumSamples can be stored at 2 – 8 °C for one week.Samples may be stored at -20 °C for 12 months.Repeated freeze-thawing of samples should be avoided.

99 ± 23 μg/l (Mean ±1 SD, N= 180)

Human

Cross reactivity is less than 2 % with Collagen II and less than 0.5 % with other forms of collagen.

No significant interference has been observed in this assay with lipaemic, haemolytic or icteric samples.• Lipaemia: Lessthan10%interferenceupto1200Formazineturbidityunits.• Haemolysis: Lessthan10%interferenceupto3g/lhaemoglobin.• Icteri: Lessthan10%interferenceupto0.2g/lbilirubin.

• Chronicliverdiseasecomprisesanumberofprogressivedisorderswhichculminateinliver cirrhosis and which are characterized by excessive deposition of collagen.

• Collagen IV deposition increases in the liver with the progression of fibrosis and this increase is directly correlated in rising serum levels. Serum Collagen IV levels have been demonstrated to be an effective marker of disease progression and also therapeutic efficacy in several disorders such as hepatitis C patients and abstaining alcoholics.

Collagen IV, Human serumNovel Liver Injury Biomarkers

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Cat. No.Tests

MethodRange

SensitivityIncubation timeSample volume

Sample typeSample preparation

Reference values

Species

Cross reaction

Interference

Background

Intented use

BIO8396 ELISA0.8 – 50 μg/l0.8 μg/l25 hours50 μlUrineIf left standing Collagen IV can precipitate out of urine resulting in falsely low reported results. For this reason we recommend the use of Collagen IV sample collection tubes which ensure accurate reproducible measurement. After collection in this manner samples may be stored at 2-8 °C for one week or at -20 °C for nine months. Care should be taken to avoid freeze thaw cycles.

Reference values are dependent on the type of sample collection proceedure

Collagen IV (μg/g Creatinine) N

Early morning 30-39 years <4,0 122 urine > 40 years < 4,9 64

Spot urine < 21 years < 7,3 390

Human

Cross reactivity is less than 2 % with Collagen II and less than 0.5 % with other forms of collagen. No significant interference has been observed in this assay with creatinine, hemolytic or icteric samples.• Creatinine:Lessthan10%interferenceupto3g/linsample.• Haemolysis:Lessthan10%interferenceupto4.8g/lhaemoglobin.• Icterus:Lessthan10%interferenceupto0.2g/lbilirubin.

Collagen IV (Col IV) is a large extracellular protein and a major component of the mammalian extracellular matrix. It is also a key component of the glomerular basement membrane (BM) and tubular BM, and is considered to constitute their basic framework. Glomerular BM expansion is a hallmark of several chronic renal pathologies, with increased urinary Col IV being indicative of the extent of these morphological alterations.

This Coll IV EIA provides a method for the quantitative determination of Col IV in human urine. Col IV levels are elevated in a variety of renal pathologies and have been found to correlate with disease staging eg. diabetic and non-diabetic nephropathies, renal transplantation and acute rejection Increased Col IV has been demonstrated to correlate directly with the rate of renal functional decline in type 1 and type 2 diabetics with urinary Col IV levels being demonstrated to be a more sensitive marker of disease progression than urinary albumin levels.

Collagen IV, Human urineKidney Injury Biomarker for progression of renal fibrosis

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NotesNOTES

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