Robyn L. Fisher

Kidney slices of human and rat to characterize cisplatin-induced injury on cellular pathways and morphology

Kidney slices represent an in vitro model that has the cellular complexity of in vivo tissue to provide insights into mechanisms of organ injury, as shown in this study with the model nephrotoxicant cisplatin. Cell pathways altered by cisplatin exposure are assessed by gene expression analysis, cell function, and morphology in human and rat kidney slices in comparison to rat kidney from an in vivo study. The acute nephrosis of the tubular epithelium induced by cisplatin in vivo was reproduced in both human and rat kidney slices, while the glomerulus appeared resistant even at high concentrations. Kidney gene expression changes of in vivo and in vitro samples were indicative of transcription, DNA damage, cell cycle, proliferation, and apoptosis that are in agreement with the mechanism of cisplatin causing DNA damage, growth arrest, and apoptosis; while genes indicative of protein damage, the disruption of transport and calcium homeostasis, cellular metabolism, and oxidative stress are pathways linked with cisplatin binding to various cellular proteins and macromolecules. Both concentration and time-dependent gene expression changes evident in the in vitro model preceded a change in tissue morphology. Functional assays confirming cell dysfunction and increased apoptosis revealed the rat kidney to be more sensitive to the effects of cisplatin than human kidney as demonstrated by significant decreases in slice ATP and GSH levels, significant increases in caspase 9 and 3 activity, p53 protein levels, and increased DNA laddering. The regional markers of proximal and distal tubular injury, alpha- and pi-glutathione S-transferases, were shown for the human kidney slices to be significantly increased by cisplatin. In this study, cisplatin-induced nephrotoxicity was demonstrated morphologically in rat and human kidney slices, and the associated gene expression and functional changes characterized the cellular pathways involved.

Precision-cut organ slices to investigate target organ injury.

Drug-induced organ injury is a multifaceted process, involving numerous cell types and mediators, and remains a significant safety issue in pharmaceutical development and clinical therapy. Organ slices, an in vitro model representing the multicellular, structural and functional features of in vivo tissue, is a promising model for elucidating mechanisms of drug-induced organ injury and for characterising species susceptibilities. Time- and concentration-dependent drug-induced effects on organ slice gene expression, function and morphology are providing insight into the molecular and biochemical pathways leading to organ dysfunction, an altered morphology and the induction of repair pathways. Human organ slice studies are valuable for bridging the extrapolation of animal-derived data and for identifying mechanisms relevant for humans. The liver is the major organ used in organ slice studies; however, the utility of extrahepatic-derived slices, as well as cocultures for investigating multiple organ involvement in tissue injury is increasing. Organ slice investigations can further our understanding of the cell types and cell interactions involved in drug-induced injury and the consequences of drug-induced off-target effects for identifying compound liabilities that will impact safety.

Cryopreservation of pig and human liver slices.

The ability to cryopreserve human liver slices would greatly enhance the opportunities to test potentially hepatotoxic drugs and environmental contaminants as well as the metabolism of these compounds. This study focused on trying to cryopreserve pig and human liver slices. Since the acquisition of human liver tissue is unpredictable and scarce, an animal model was sought to predict problems associated with cryopreservation of human tissue. The pig liver was chosen because of its anatomical and physiological resemblance to human liver. The human liver tissues that did become available were obtained through the Arizona Organ Bank and the National Disease Research Interchange and from surgical liver resections. An in vitro culture system that employed precision-cut liver slices was used in this study. Different types and concentrations of cryoprotectants, cooling rates, and culture media were all tried in an attempt to cryopreserve pig and human liver slices. The viabilities of fresh and cryopreserved liver slices were evaluated using slice K+ retention and protein synthesis. Pig liver slices following cryopreservation retained between 80 and 85% of intracellular K+ content and protein synthesis as compared to controls using 1.4 M Me2SO, a 12 degrees C/min cooling rate, and a rapid rewarming rate of direct submersion of the slice into 37 degrees C fetal calf serum. Human liver slices following cryopreservation retained between 54 and 89% of intracellular K+ content and protein synthesis as compared to controls using the same protocol as for pigs, except that lower cooling rates were giving better results. The large variation seen in cryopreserved human liver slices was due to the length of warm and cold ischemia to which the tissue was exposed before arriving at the laboratory. This study indicated that pig and human liver slices can be cryopreserved and used for future toxicological and metabolic studies.

Repair pathways evident in human liver organ slices

The extension of human liver slice culture viability for several days broadens the potential of this ex vivo model for characterizing pathways of organ injury and repair, and allows for the multiple dosing of compounds. Extended viability is demonstrated by continued synthesis of GSH and ATP, and maintenance of intracellular K(+) levels. Gene expression profiling revealed the activation of regeneration pathways via increased expression of collagens (I, IV, and V), laminins, ninjurin, growth factors (EGF, epiregulin, and TGF-β1), matrix metalloproteinase-7, and insulin like growth factor 5. Collagen IV protein levels began to increase by day 4 of culture. Some markers of hepatic stellate cells, detected by RT-PCR, were up-regulated (HSP47, αSMA, pro-collagen 1a1, PDGF receptor, thrombospondin-2) with time in culture, while other markers exhibited no change or were down-regulated (αB-crystallin, synaptophysin), suggesting that the induction of regenerative pathways may in part be the role of the stellate cells as well as resident fibroblasts. Complimentary to the gene expression was evidence of regeneration in the human liver slices, as evaluated by histopathology. Improvements in organ acquisition, organ slice preparation and culture methods demonstrates that organ slice viability, integrity and morphology can be extended reproducibly for several days in culture which allows for the investigation of injury and repair processes.

Protein Arylation Precedes Acetaminophen Toxicity in a Dynamic Organ Slice Culture of Mouse Kidney

Acetaminophen (APAP) is an analgesic and antipyretic agent which may cause hepatotoxicity and nephrotoxicity with overdose in man and laboratory animals. In vivo studies suggest that in situ activation of APAP contributes to the development of nephrotoxicity. Associated with target organ toxicity is selective arylation of proteins, with a 58-kDa acetaminophen binding protein (58-ABP) being the most prominent cytosolic target. In this study a mouse kidney slice model was developed to further evaluate the contribution of in situ activation of APAP to the development of nephrotoxicity and to determine the selectivity of protein arylation. Precision cut kidney slices from male CD-1 mice were incubated with selected concentrations of APAP (0-25 mM) for 2 to 24 hr. APAP caused a dose- and time-dependent decrease in nonprotein sulfhydryls (NPSH), ATP content, and K+ retention. Preceding toxicity was arylation of cytosolic proteins, the most prominent one being the 58-ABP. The association of 58-ABP arylation with APAP toxicity in this mouse kidney slice model is consistent with earlier, in vivo results and demonstrates the importance of in situ activation of APAP for the development of nephrotoxicity. Precision cut renal slices and dynamic organ culture are a good model for further mechanistic studies of APAP-induced renal toxicity.

Preparation and culture of precision-cut organ slices from human and animal

1.u2002Human and animal precision-cut organ slices are being widely used to obtain drug metabolism and toxicity profiles in vitro. These data are then used to predict what might be seen in human patients. The accuracy of this prediction and extrapolation of the findings based on human or animal in vitro systems to the findings that occur in vivo is dependent on both the quality of the tissue itself and the quality of the in vitro system. 2.u2002The quality of human organs used in research is dependent on procurement methods, warm ischaemia time, preservation solutions, cold ischaemia time, and donor-specific factors. It is important to confirm that the organs being used are highly viable and fully functional before using them in scientific studies. 3.u2002The optimal preparation and incubation of organ slices is also essential in maintaining slice viability and function. It is important to prepare the slices in a cold preservation solution, to prepare the slices at a correct thickness, and to incubate the slices in a system where the slice rotates in out of the oxygen atmosphere and medium. 4.u2002Meeting the criteria outlined here will lead to successful organ slice cultures for investigating drug-induced mechanisms and organ-specific toxicity.

Thyroid organotypic rat and human cultures used to investigate drug effects on thyroid function, hormone synthesis and release pathways.

Drug induced thyroid effects were evaluated in organotypic models utilizing either a rat thyroid lobe or human thyroid slices to compare rodent and human response. An inhibition of thyroid peroxidase (TPO) function led to a perturbation in the expression of key genes in thyroid hormone synthesis and release pathways. The clinically used thiourea drugs, methimazole (MMI) and 6-n-propyl-2-thioruacil (PTU), were used to evaluate thyroid drug response in these models. Inhibition of TPO occurred early as shown in rat thyroid lobes (2 h) and was sustained in both rat (24-48 h) and human (24 h) with ≥ 10 μM MMI. Thyroid from rats treated with single doses of MMI (30-1000 mg/kg) exhibited sustained TPO inhibition at 48 h. The MMI in vivo thyroid concentrations were comparable to the culture concentrations (~15-84 μM), thus demonstrating a close correlation between in vivo and ex vivo thyroid effects. A compensatory response to TPO inhibition was demonstrated in the rat thyroid lobe with significant up-regulation of genes involved in the pathway of thyroid hormone synthesis (Tpo, Dio1, Slc5a5, Tg, Tshr) and the megalin release pathway (Lrp2) by 24h with MMI (≥ 10 μM) and PTU (100 μM). Similarly, thyroid from the rat in vivo study exhibited an up-regulation of Dio1, Slc5a5, Lrp2, and Tshr. In human thyroid slices, there were few gene expression changes (Slc5a5, ~2-fold) and only at higher MMI concentrations (≥ 1500 μM, 24h). Extended exposure (48 h) resulted in up-regulation of Tpo, Dio1 and Lrp2, along with Slc5a5 and Tshr. In summary, TPO was inhibited by similar MMI concentrations in rat and human tissue, however an increased sensitivity to drug treatment in rat is indicated by the up-regulation of thyroid hormone synthesis and release gene pathways at concentrations found not to affect human tissue.

Evaluation of drug-induced injury and human response in precision-cut tissue slices

1.u2002Drug induced organ injury is multifaceted, encompassing a spectrum of cell types and numerous networks reflecting cell-cell and cell-matrix interactions. Characterization of drug induced side effects and human response can be addressed in organ slice models. 2.u2002The application of human tissue to various organ slice models including liver, intestine, kidney, liver-blood co-cultures and thyroid enhances our ability to focus on the clinical relevance of side effects identified in animal studies for human, and to evaluate potential biomarkers of the side effects. Dose–response relationships can help discern drug concentrations which alter organ function or affect morphology, to identify drug concentrationswhich could pose a risk for humans. 3.u2002Insight into pathways of organ injury, by incorporating gene and protein expression profiling, with functional measurements and morphology, aid to define species differences and sensitivity. 4.u2002Human organ slice studies are valuable for bridging the extrapolation of animal derived data and for identifying mechanisms relevant for humans, thereby expanding the scope of translational research for drug safety assessment.

Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues.

A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (>or=1000 microM at 48 h) and human tissues (>or=1000 microM at 48 h, >or=750 microM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.

Isoproterenol effects evaluated in heart slices of human and rat in comparison to rat heart in vivo.

Human response to isoproterenol induced cardiac injury was evaluated by gene and protein pathway changes in human heart slices, and compared to rat heart slices and rat heart in vivo. Isoproterenol (10 and 100μM) altered human and rat heart slice markers of oxidative stress (ATP and GSH) at 24h. In this in vivo rat study (0.5mg/kg), serum troponin concentrations increased with lesion severity, minimal to mild necrosis at 24 and 48h. In the rat and the human heart, isoproterenol altered pathways for apoptosis/necrosis, stress/energy, inflammation, and remodeling/fibrosis. The rat and human heart slices were in an apoptotic phase, while the in vivo rat heart exhibited necrosis histologically and further progression of tissue remodeling. In human heart slices genes for several heat shock 70kD members were altered, indicative of stress to mitigate apoptosis. The stress response included alterations in energy utilization, fatty acid processing, and the up-regulation of inducible nitric oxide synthase, a marker of increased oxidative stress in both species. Inflammation markers linked with remodeling included IL-1α, Il-1β, IL-6 and TNFα in both species. Tissue remodeling changes in both species included increases in the TIMP proteins, inhibitors of matrix degradation, the gene/protein of IL-4 linked with cardiac fibrosis, and the gene Ccl7 a chemokine that induces collagen synthesis, and Reg3b a growth factor for cardiac repair. This study demonstrates that the initial human heart slice response to isoproterenol cardiac injury results in apoptosis, stress/energy status, inflammation and tissue remodeling at concentrations similar to that in rat heart slices.