Kidney Effects by Alternative Classes of Medicines in Patients and Relationship to Effects in Nonclinical Toxicity Studies.

Drug-induced kidney injury has historically been associated with renal tubule injury related to small molecule pharmaceuticals such as nonsteroidal anti-inflammatory drugs, antineoplastic agents, or antibiotics, but as a greater number of alternative classes of medicines such as biotherapeutics, molecular-targeted antineoplastic drugs, chimeric antigen receptor T-cell therapies, antibody-drug conjugates, oligonucleotide therapies, or other immunomodulatory drugs come to market, the presentation of drug-induced nephrotoxicity is changing. This review article describes the potential rare clinical events in drug-induced kidney injury that might be noted with these new therapies and their potential impact on patients. Potential pathogenic mechanisms related to immunogenicity, immune complex formation, and stimulation of downstream proinflammatory pathways with some of these alternative medicine classes have resulted in the potential for glomerulonephritis, acute interstitial nephritis, renal vasculitis, and other immune-mediated renal disorders in humans. This contrasts with nonclinical toxicity studies, where biologic therapies more often result in vasculitis and glomerulonephritis associated with antidrug antibodies and immunomodulatory pharmacology, and which are not always predictive of clinical effects. While nonclinical antidrug antibody-related renal disease is generally not clinically relevant, other immune-mediated nephrotoxicities associated with immunomodulatory drugs may be predictive of clinical adverse events. Fortunately, these conditions are still rare and account for a small percentage of serious adverse events in kidneys of patients.

A Review of Specific Biomarkers of Chronic Renal Injury and Their Potential Application in Nonclinical Safety Assessment Studies.

A host of novel renal biomarkers have been developed over the past few decades which have enhanced monitoring of renal disease and drug-induced kidney injury in both preclinical studies and in humans. Since chronic kidney disease (CKD) and acute kidney injury (AKI) share similar underlying mechanisms and the tubulointerstitial compartment has a functional role in the progression of CKD, urinary biomarkers of AKI may provide predictive information in chronic renal disease. Numerous studies have explored whether the recent AKI biomarkers could improve upon the standard clinical biomarkers, estimated glomerular filtration rate (eGFR), and urinary albumin to creatinine ratio, for predicting outcomes in CKD patients. This review is an introduction to alternative assays that can be utilized in chronic (>3 months duration) nonclinical safety studies to provide information on renal dysfunction and to demonstrate specific situations where these assays could be utilized in nonclinical drug development. Novel biomarkers such as symmetrical dimethyl arginine, dickkopf homolog 3, and cystatin C predict chronic renal injury in animals, act as surrogates for GFR, and may predict changes in GFR in patients over time, ultimately providing a bridge from preclinical to clinical renal monitoring.

Carcinogenicity Assessment of Daprodustat (GSK1278863), a Hypoxia-Inducible Factor (HIF)-Prolyl Hydroxylase Inhibitor.

Daprodustat (GSK1278863) is a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor in development for treatment of anemia of chronic kidney disease. Daprodustat's biological activity simulates components of the natural response to hypoxia; inhibition of PHDs results in HIF stabilization and modulation of HIF-controlled gene products, including erythropoietin. The carcinogenic potential of daprodustat was evaluated in 2-year carcinogenicity studies in Sprague-Dawley rats and CD-1 mice, where once-daily doses were administered. The mouse study also included evaluation of daprodustat's 3 major circulating human metabolites. There were no neoplastic findings that were considered treatment related in either study. Exaggerated pharmacology resulted in significantly increased red cell mass and subsequent multiorgan congestion and secondary non-neoplastic effects in both species, similar to those observed in chronic toxicity studies. In rats, these included aortic thrombosis and an exacerbation of spontaneous rodent cardiomyopathy, which contributed to a statistically significant decrease in survival in high-dose males (group terminated in week 94). Survival was not impacted in mice at any dose. Systemic exposures (area under the plasma concentration-time curve) to daprodustat at the high doses in rats and mice exceed predicted maximal human clinical exposure by ≥143-fold. These results suggest that daprodustat and metabolites do not pose a carcinogenic risk at clinical doses.

Intrarenal Renin-Angiotensin System Involvement in the Pathogenesis of Chronic Progressive Nephropathy-Bridging the Informational Gap Between Disciplines.

Chronic progressive nephropathy (CPN) is the most commonly encountered spontaneous background finding in laboratory rodents. Various theories on its pathogenesis have been proposed, but there is a paucity of data regarding specific mechanisms or physiologic pathways involved in early CPN development. The current CPN mechanism of action for tumorigenesis is largely based on its associated increase in tubular cell proliferation without regard to preceding subcellular degenerative changes. Combing through the published literature from multiple biology disciplines provided insight into the preceding cellular events. Mechanistic pathways involved in the progressive age-related decline in rodent kidney function and several key inflexion points have been identified. These critical pathway factors were then connected using data from renal models from multiple rodent strains, other species, and mechanistic work in humans to form a cohesive picture of pathways and protein interactions. Abundant data linked similar renal pathologies to local events involving hypoxia (hypoxia-inducible factor 1α), altered intrarenal renin-angiotensin system (RAS), oxidative stress (nitric oxide), and pro-inflammatory pathways (transforming growth factor ß), with positive feedback loops and downstream effectors amplifying the injury and promoting scarring. Intrarenal RAS alterations seem to be central to all these events and may be critical to CPN development and progression.

Species Differences in Renal Development and Associated Developmental Nephrotoxicity.

The developing kidney is sensitive to both morphological and functional disturbances during the gestational and postnatal phases of growth and differentiation. Exposure to drugs or chemicals during these critical windows of renal development can result in aplasia, dysplasia, polycystic kidney disease, hydronephrosis, or other features characteristic of nephrotoxicity, including tubule dilation, necrosis, or mineralization. Functional effects can occur without associated morphological abnormalities. Differences in the timing of nephrogenesis and morphologic renal development among species help to explain specific phenotypes of various gestational and postnatal teratogens and nephrotoxins. Functional maturation follows anatomical maturation, but important differences in maximally achieved glomerular filtration rate, concentrating ability and acid-base equilibrium between species makes comparison of these timings critical for accurate and consistent translation of laboratory animal toxicity data to the human clinical experience. Species and age dependent differences in the maturation of kidney transporters, renal xenobiotic metabolism and renal blood flow can have a profound effect on the toxicity profiles of agents and marked differences in the tolerability based on age. Advances in the understanding of the genetics of inherited renal diseases and the underlying cellular and molecular pathogenesis of renal developmental anomalies has helped provide mechanistic understanding of many teratogenic and perinatal nephrotoxic agents. Investigative studies have provided important translational and mechanistic information for assessing human pediatric nephrotoxic potential. Birth Defects Research 109:1243-1256, 2017. © 2017 Wiley Periodicals, Inc.

Drug-induced Physeal Abnormalities in Preclinical Toxicity Studies.

Most toxic physeal changes are characterized microscopically by altered chondrocyte development, proliferation, or maturation in the growth plate and eventually result in disordered appositional bone growth. Many therapeutic drugs directly or indirectly target proteins involved in chondrocytic differentiation and maturation pathways, so toxic physeal injury has become increasingly common in preclinical toxicologic pathology. While physeal dysplasia has been associated with several different drug classes including bisphosphonates, vascular endothelial growth factor receptor inhibitors, fibroblast growth factor receptor inhibitors, transforming growth factor beta receptor inhibitors, and vascular targeting agents, physeal changes often share similar morphologic features including thickening and disorganization of the hypertrophic layer, increased numbers of hypertrophic chondrocytes, altered mineralization of endochondral ossification, and/or increased thickness of subphyseal bone. Knowledge of genetic and nutritional diseases affecting bone growth has been important in helping to determine which specific target drugs may be affecting that could result in toxic physeal lesions. A pathophysiologic mechanism for most physeal toxicants has been determined in detail using a variety of investigative techniques. However, due to the signaling cross talk and the tight regulation required for chondrocyte maturation in the physis, several growth factor pathways are likely to be affected simultaneously with pharmacologic disruption of physeal homeostasis and inhibition of one factor necessary for chondrocyte function often affects others.

Regulatory Forum Opinion Piece*: Dispelling Confusing Pathology Terminology: Recognition and Interpretation of Selected Rodent Renal Tubule Lesions.

Renal tubule lesions often prove troublesome for toxicologic pathologists because of the diverse nature and interrelated cell types within the kidney and the presence of spontaneous lesions with overlapping morphologies similar to those induced by renal toxicants. Although there are a number of guidance documents available citing straightforward diagnostic criteria of tubule lesions for the pathologist to refer to, most are presented without further advice on the when to or to the why and the why not of diagnosing one lesion over another. Documents presenting diagnostic perspectives and recommendations derived from an author's experience are limited since guidance documents are generally based on descriptive observations. In this Regulatory Forum opinion piece, the authors attempt to dispel confusing renal tubule lesion terminology in laboratory animal species by suggesting histological advice on the recognition and interpretation of these complex entities.

Proliferative and nonproliferative lesions of the rat and mouse urinary system.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the urinary tract of rats and mice. The standardized nomenclature of urinary tract lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for urinary tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Differences in effects on myocardium and mitochondria by angiogenic inhibitors suggest separate mechanisms of cardiotoxicity.

Several multikinase angiogenesis inhibitors demonstrate mitochondrial and/or cardiovascular toxicity, suggesting an on-target pharmacologic effect. To evaluate whether cardiotoxicity is directly related to vascular endothelial growth factor receptor inhibition, we investigated the effects of sunitinib, sorafenib, and pazopanib on myocardial function and structure. We used a rat model to assess myocardial effects of the inhibitors concurrently exposed to the cardiac stressor dobutamine. Echocardiographic abnormalities including premature ventricular contractions, decreases in heart rate, circumferential strain, and radial and circumferential strain rates were noted with sorafenib, but not with sunitinib or pazopanib. Ultrastructural analysis of ventricular cardiomyocytes by transmission electron microscopy revealed mitochondrial swelling, dense deposits, and matrix cavitation in rats given sunitinib and disrupted mitochondrial cristae in rats given sorafenib, but there were no effects with pazopanib. Effects on neonatal rat cardiomyocyte cultures were assessed, which identified decreases in mitochondrial membrane potential with sunitinib treatment, but not with sorafenib or pazopanib. Intracellular adenosine triphosphate depletion was observed with sunitinib and sorafenib, but not pazopanib. Our results show that cardiotoxicity is not necessarily related to a pharmacologic classwide effect of vascular endothelial growth factor receptor inhibition, and the rat myocardial structural and functional changes identified in this study may be instead a result of inhibition of other kinase pathways, the mechanism of which may be associated with mitochondrial toxicity.

Role of p38 in regulation of hematopoiesis: effect of p38 inhibition on cytokine production and transcription factor activity in human bone marrow stromal cells.

This study was designed to evaluate effects of specific p38 MAP kinase inhibition on gene and protein expression of essential hematopoietic cytokines in primary human bone marrow stromal cells (HBMSC) and to identify downstream transcription factors (TF) regulated by the p38 MAP kinase signalling pathway. In vitro effects of p38 inhibitors (p38i) on cytokine regulation were compared to inhibitors of other major signalling pathways including PI3 kinase, JNK, MEK-1, NF-kappaB or protein kinase C (PKC). HBMSC were pre-treated with p38i (SB-203580) for 1 h and then stimulated with 200 ng/ml lipopolysaccharide (LPS). Supernatants and RNA were collected 6 h post LPS treatment for quantitative protein and mRNA analyses by ELISA and real-time RT-PCR, respectively, for interleukin-6 (IL-6), interleukin-11 (IL-11), granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and Activin A. Effects of the inhibitors of PI3 kinase (LY294002), JNK (synthetic inhibitory peptide), MEK-1 (PD90859), NF-kappaB (pyrrolidinedithiocarbamate (PDTC)) and protein kinase C (calphostin C) on HBMSC expression hematopoietic cytokines were evaluated and compared. SB-203580 caused dose-dependent decreases in cytokine protein expression and decreased IL-6 and IL-11 mRNA expression. Of the pathway inhibitors examined, only NF-kappaB elicited similar effects on cytokine protein and mRNA expression. p38-regulated transcription factor activity was assessed using a DNA/Protein array. Several TFs linked to cytokine regulation were modulated by SB-203580, with 10 of 21 p38-regulated TFs identified have not been previously linked to downstream p38 signalling. These observations in cultured HBMSC have illustrated the involvement of cytokine proteins, mRNA and TF activities and may improve the current understanding of the in vivo p38i suppression of erythropoiesis. In addition, these results suggest that IL-6, IL-11, GM-CSF, G-CSF and Activin A are similarly regulated by p38 and NF-kappaB and that the MEK1, JNK and PKC pathways appear to play a more limited role in modulating cytokine expression in HBMSC.

Effects of p38 MAP kinase inhibitors on the differentiation and maturation of erythroid progenitors.

In rodents, p38 MAP kinase inhibitors (p38is) induce bone marrow hypocellularity and reduce reticulocyte and erythrocyte counts. To identify target cell populations affected, a differentiating primary liquid erythroid culture system using sca-1(+)cells from mouse bone marrow was developed and challenged with p38is SB-203580, SB-226882, and SB-267030. Drug-related alterations in genes involved at different stages of erythropoiesis, cell-surface antigen expression (CSAE), burst-forming unit erythroid (BFU-E) colony formation, and cellular morphology (CM), growth (CG), and viability were evaluated. CSAE, CM, and decreases in BFU-E formation indicated delayed maturation, while CG and viability were unaffected. Terminal differentiation was delayed until day 14 versus day 7 in controls. CSAE demonstrated higher percentages of sca-1(+)cells after day 2 and reduced percentages of ter119(+) cells after day 7 in all treated cultures. Real-time reverse transcriptase polymerase chain reaction revealed a transient delay in expression of genes involved at early, intermediate, and late stages of erythropoiesis, followed by rebound expression at later time points. Results demonstrate p38is do not irreversibly inhibit erythrogenesis but induce a potency-dependent, transient delay in erythropoietic activity. The delay in activity is suggestive of effects on sca-1(+)bone marrow cells caused by alterations in expression of genes related to erythroid commitment and differentiation resulting in delayed maturation.

Tumor-specific efficacy of transforming growth factor-beta RI inhibition in Eker rats.

PURPOSE: Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions. EXPERIMENTAL DESIGN: We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency. RESULTS: Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals. CONCLUSION: Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

Seroprevalence and comparison of isolates of endometriotropic bovine herpesvirus-4.

Sixty-eight cases of suppurative, ulcerative endometritis associated with Bovine Herpesvirus-4 (BHV-4) in postparturient dairy cows (62 Holsteins and 6 Jerseys, mean age 4.2 years) were confirmed by a combination of histopathology, fluorescent antibody assays, electron microscopic evaluation of uterus, and polymerase chain reaction (PCR). All cases occurred in the 3- to 28-day postpartum period, and histologic lesions among various cows were consistent when compared with postpartum interval. The endometrial lining epithelium was necrotic and ulcerated from 3 to 7 days postpartum, with only mild inflammation in the lamina propria and submucosa. From 1 to 4 weeks postpartum, the ulcers were confluent to diffuse. Epithelium was replaced by fibrinonecrotic, suppurative mats, resulting in severe bacterial pyometra by day 24. Seroprevalence to BHV-4 in one dairy with a history of 18 mortality cases was 36% (107 of 296). In a random sample of 8 cows from this herd, none had serologic titers in blood sampled 2 weeks prepartum, but 3 of 8 seroconverted with significant titers of 1:8 to 1:16 at 2 weeks postpartum. By 10 weeks postpartum, all 8 cows returned to negative serologic status. Two of 6 cats from the premises also had positive titers. Random serum samples taken from 480 dairy cattle at sale barns indicated 76 (16%) were positive by serum neutralization. Clinical signs, postparturient timing, and histologic lesions were very similar to those previously reported in Belgium with BHV-4. But sequence analysis of PCR products of the glycoprotein B region of 4 separate field isolates of endometriotropic BHV-4 suggests these field isolates were more closely related to the North American nonvirulent strain DN-599 than to the endometriotropic European strain V.

Bacterial pathogens isolated from cultured bullfrogs (Rana castesbeiana).

A commercial bullfrog (Rana castesbeiana) operation in south Georgia had multiple epizootics of systemic bacterial infections over a 3-year period, 1998-2000. A number of potential pathogens (Aeromonas hydrophila, Chryseobacterium (Flavobacterium) meningosepticum, Chryseobacterium (Flavobacterium) indolgenes, Edwardsiella tarda, Citrobacterfreundii, Pseudomonas spp., and (Streptococcus iniae) were isolated from various tissues. Clinically, frogs demonstrated acute onset of torticolis, stupor, and indifference to stimuli. Cutaneous hyperemia, subcutaneous and muscular hemorrhage, and peripheral edema were consistent gross findings. Histologically, clusters of lymphocytes, monocytes, and occasional acidophiles with scattered granulomas occurred in liver, kidney, and spleen. This is the first report of S. inae and C. meningosepticum as potential disease agents in R. castesbeiana. These findings suggest that a variety of bacteria may be associated with redleg and that culture results must be obtained for accurate diagnosis.