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Ionising radiation (IR) is a recognised carcinogen responsible for cancer development in patients previously treated using radiotherapy, and in individuals exposed as a result of accidents at nuclear energy plants. However, the mutational signatures induced by distinct types and doses of radiation are unknown. Here, we analyse the genetic architecture of mammary tumours, lymphomas and sarcomas induced by high (56Fe-ions) or low (gamma) energy radiation in mice carrying Trp53 loss of function alleles. In mammary tumours, high-energy radiation is associated with induction of focal structural variants, leading to genomic instability and Met amplification. Gamma-radiation is linked to large-scale structural variants and a point mutation signature associated with oxidative stress. The genomic architecture of carcinomas, sarcomas and lymphomas arising in the same animals are significantly different. Our study illustrates the complex interactions between radiation quality, germline Trp53 deficiency and tissue/cell of origin in shaping the genomic landscape of IR-induced tumours.

ObjectiveThe Collaborative Cross (CC) is a mouse population model with diverse and reproducible genetic backgrounds used to identify novel disease models and genes that contribute to human disease. Since spontaneous tumour susceptibility in CC mice remains unexplored, we assessed tumour incidence and spectrum.DesignWe monitored 293 mice from 18 CC strains for tumour development. Genetic association analysis and RNA sequencing were used to identify susceptibility loci and candidate genes. We analysed genomes of patients with gastric cancer to evaluate the relevance of genes identified in the CC mouse model and measured the expression levels of ISG15 by immunohistochemical staining using a gastric adenocarcinoma tissue microarray. Association of gene expression with overall survival (OS) was assessed by Kaplan-Meier analysis.ResultsCC mice displayed a wide range in the incidence and types of spontaneous tumours. More than 40% of CC036 mice developed gastric tumours within 1 year. Genetic association analysis identified Nfκb1 as a candidate susceptibility gene, while RNA sequencing analysis of non-tumour gastric tissues from CC036 mice showed significantly higher expression of inflammatory response genes. In human gastric cancers, the majority of human orthologues of the 166 mouse genes were preferentially altered by amplification or deletion and were significantly associated with OS. Higher expression of the CC036 inflammatory response gene signature is associated with poor OS. Finally, ISG15 protein is elevated in gastric adenocarcinomas and correlated with shortened patient OS.ConclusionsCC strains exhibit tremendous variation in tumour susceptibility, and we present CC036 as a spontaneous laboratory mouse model for studying human gastric tumourigenesis.

Variations in oral bacterial communities have been linked to oral cancer suggesting that the oral microbiome is an etiological factor that can influence oral cancer development. The 4-nitroquinoline 1-oxide (4-NQO)-induced murine oral and esophageal cancer model is frequently used to assess the effects of preventive and/or therapeutic agents. We used this model to assess the impact of the microbiome on tumorigenesis using axenic (germ-free) and conventionally housed mice. Increased toxicity was observed in germ-free mice, however, no difference in tumor incidence, multiplicity, and size was observed. Transcriptional profiling of liver tissue from germ-free and conventionally housed mice identified 254 differentially expressed genes including ten cytochrome p450 enzymes, the largest family of phase-1 drug metabolizing enzymes in the liver. Gene ontology revealed that differentially expressed genes were enriched for liver steatosis, inflammation, and oxidative stress in livers of germ-free mice. Our observations emphasize the importance of the microbiome in mediating chemical toxicity at least in part by altering host gene expression. Studies on the role of the microbiome in chemical-induced cancer using germ-free animal models should consider the potential difference in dose due to the microbiome-mediated changes in host metabolizing capacity, which might influence the ability to draw conclusions especially for tumor induction models that are dose dependent.

BackgroundDespite careful clinical examination, procurement biopsy and assessment on hypothermic machine perfusion, a significant number of potentially useable deceased donor kidneys will be discarded because they are deemed unsuitable for transplantation. Ex vivo normothermic perfusion (EVNP) may be useful as a means to further assess high-risk kidneys to determine suitability for transplantation.MethodsFrom June 2014 to October 2015, 7 kidneys (mean donor age, 54.3 years and Kidney Donor Profile Index, 79%) that were initially procured with the intention to transplant were discarded based on a combination of clinical findings, suboptimal biopsies, long cold ischemia time (CIT) and/or poor hypothermic perfusion parameters. They were subsequently placed on EVNP using oxygenated packed red blood cells and supplemental nutrition for a period of 3 hours. Continuous hemodynamic and functional parameters were assessed.ResultsAfter a mean CIT of 43.7 hours, all 7 kidneys appeared viable on EVNP with progressively increasing renal blood flow over the 3-hour period of perfusion. Five of the 7 kidneys had excellent macroscopic appearance, rapid increase in blood flow to 200 to 250 mL/min, urine output of 40 to 260 mL/h and increasing creatinine clearance.ConclusionsFavorable perfusion characteristics and immediate function after a 3-hour course of EVNP suggests that high-risk kidneys subjected to long CIT may have been considered for transplantation. The combined use of ex vivo hypothermic and normothermic perfusion may be a useful strategy to more adequately assess and preserve high-risk kidneys deemed unsuitable for transplantation. A clinical trial will be necessary to validate the usefulness of this approach.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Hepatic steatosis develops after liver transplantation (LT) in 30% of adults, and nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in nontransplanted children. However, posttransplant steatosis has been minimally studied in pediatric LT recipients. We explored the prevalence, persistence, and association with chronic liver damage of hepatic steatosis in these children. In this single-center study of pediatric patients transplanted 1988-2015 (n = 318), 31% of those with any posttransplant biopsy (n = 271) had ≥ 1 biopsy with steatosis. Median time from transplant to first biopsy with steatosis was 0.8 months (interquartile range [IQR], 0.3-6.5 months) and to last biopsy with steatosis was 5.5 months (IQR, 1.0-24.5 months); 85% of patients with steatosis also had for-cause biopsies without steatosis. All available for-cause biopsies were re-evaluated (n = 104). Of 9 biopsies that could be interpreted as nonalcoholic steatohepatitis (NASH)/borderline NASH, with steatosis plus inflammation or ballooning, 8 also had features of cholestasis or rejection. Among 70 patients with surveillance biopsies 3.6-20.0 years after transplant, only 1 overweight adolescent had a biopsy with NAFLD (grade 1 steatosis, mild inflammation, no ballooning or fibrosis)-despite a 30% prevalence of overweight/obesity in the cohort and 27% with steatosis on previous for-cause biopsy. Steatosis on preceding for-cause biopsy was not associated with portal (P = 0.49) or perivenular fibrosis (P = 0.85) on surveillance biopsy. Hepatic steatosis commonly develops early after transplant in children and adolescents, but it rarely persists. Biopsies that did have steatosis with NASH characteristics were all for-cause, mostly in patients with NAFLD risk factors and/or confounding causes of liver damage. Prospective studies that follow children into adulthood will be needed to evaluate if and when hepatic steatosis presents a longterm risk for pediatric LT recipients. Liver Transplantation 23 957-967 2017 AASLD.

NUCKS1 is a 27 kD vertebrate-specific protein, with a role in the DNA damage response. Here, we show that after 4 Gy total-body X-irradiation, Trp53+/- Nucks1+/- mice more rapidly developed tumors, particularly thymic lymphoma (TL), than Trp53+/- mice. TLs in both cohorts showed loss of heterozygosity (LOH) of the Trp53+ allele in essentially all cases. In contrast, LOH of the Nucks1+ allele was rare. Nucks1 expression correlated well with Nucks1 gene dosage in normal thymi, but was increased in the majority of TLs from Trp53+/- Nucks1+/- mice, suggesting that elevated Nucks1 message may be associated with progression towards malignancy in vivo. Trp53+/- Nucks1+/- mice frequently succumbed to CD4- CD8- TLs harboring translocations involving Igh but not Tcra/d, indicating TLs in Trp53+/- Nucks1+/- mice mostly originated prior to the double positive stage and at earlier lineage than TLs in Trp53+/- mice. Monoclonal rearrangements at Tcrb were more prevalent in TLs from Trp53+/- Nucks1+/- mice, as was infiltration of primary TL cells to distant organs (liver, kidney and spleen). We propose that, in the context of Trp53 deficiency, wild type levels of Nucks1 are required to suppress radiation-induced TL, likely through the role of the NUCKS1 protein in the DNA damage response.

The tumor suppressor TP53 can initiate a plethora of anti-proliferative effects to maintain genomic integrity under conditions of genotoxic stress. The N-terminal proline-rich domain (PRD) of TP53 is important in the regulation of TP53 activity and stability. A common polymorphism at codon 72 in this region has been associated with altered cancer risk in humans. The Trp53ΔP mouse, which carries a germline homozygous deletion of a region of the PRD, does not develop spontaneous tumors in a mixed 129/Sv and C57BL/6 genetic background, but is highly susceptible to a broad range of tumor types following total body exposure to 4 Gy gamma (γ) radiation. This contrasts with the tumor spectrum in Trp53 null (-/-) mice, which mainly develop thymic lymphomas and osteosarcomas. Analysis of genomic instability in tissues and cells from Trp53ΔP mice demonstrated elevated basal levels of aneuploidy, but this is not sufficient to drive spontaneous tumorigenesis, which requires an additional DNA damage stimulus. Levels of genomic instability did not increase significantly in Trp53ΔP mice following irradiation exposure, suggesting that other radiation effects including tissue inflammation, altered metabolism or autophagy, may play an important role. The Trp53ΔP mouse is a novel model to dissect the mechanisms of tumor development induced by radiation exposure. © 2015 Wiley Periodicals, Inc.

BackgroundAA amyloidosis due to subcutaneous injection of drugs of abuse has been described in the USA, but all the existing literature is from more than 20 years ago. There is more recent literature from Europe. We have observed a high incidence of AA amyloidosis in the county hospital in San Francisco.DesignHere, we describe 24 patients who had kidney biopsy-proven AA amyloidosis from our hospital from 1998 to 2013. All the patients were thought to have AA amyloidosis from skin popping of illicit drugs after having exhausted the intravenous route. These patients with biopsy-proven AA amyloidosis were analysed further.ResultsAll patients were found to have hepatitis C infection, hypertension was not common, most had advanced kidney failure, and acidosis was common as was tubulointerstitial involvement on the kidney biopsy. Other organ involvement included hepatomegaly and splenomegaly in a number of patients; direct myocardial involvement was not seen, but pulmonary hypertension, history of deep vein thrombosis and pulmonary embolism were common. The prognosis of these patients was poor. The mortality rate approached 50% 1 year after biopsy, and most of the patient needed dialysis shortly after diagnosis. Cessation of drug use seemed beneficial but rarely achievable.ConclusionAA amyloidosis from skin popping is common in San Francisco. Most patients with renal involvement end up on dialysis, and mortality rates are exceedingly high.

Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies.

Oxidative stress has been proposed to be a unifying cause for diabetic nephropathy and a target for novel therapies. Here we apply a new endogenous reduction-oxidation (redox) sensor, hyperpolarized (HP) (13)C dehydroascorbate (DHA), in conjunction with MRI to noninvasively interrogate the renal redox capacity in a mouse diabetes model. The diabetic mice demonstrate an early decrease in renal redox capacity, as shown by the lower in vivo HP (13)C DHA reduction to the antioxidant vitamin C (VitC), prior to histological evidence of nephropathy. This correlates with lower tissue reduced glutathione (GSH) concentration and higher NADPH oxidase 4 (Nox4) expression, consistent with increased superoxide generation and oxidative stress. ACE inhibition restores the HP (13)C DHA reduction to VitC with concomitant normalization of GSH concentration and Nox4 expression in diabetic mice. HP (13)C DHA enables rapid in vivo assessment of altered redox capacity in diabetic renal injury and after successful treatment.

Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras (Kras(LA2)). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the Kras(LA2) model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the Kras(LA2) model. By contrast, the Kras(LA2) tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C > T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.

Exposure to levamisole-adulterated cocaine can induce a distinct clinical syndrome characterized by retiform purpura and/or agranulocytosis accompanied by an unusual constellation of serologic abnormalities including antiphospholipid antibodies, lupus anticoagulants, and very high titers of antineutrophil cytoplasmic antibodies. Two recent case reports suggest that levamisole-adulterated cocaine may also lead to renal disease in the form of pauci-immune glomerulonephritis. To explore this possibility, we reviewed cases of pauci-immune glomerulonephritis between 2010 and 2012 at an inner city safety net hospital where the prevalence of levamisole in the cocaine supply is known to be high. We identified 3 female patients and 1 male patient who had biopsy-proven pauci-immune glomerulonephritis, used cocaine, and had serologic abnormalities characteristic of levamisole-induced autoimmunity. Each also had some other form of clinical disease known to be associated with levamisole, either neutropenia or cutaneous manifestations. One patient had diffuse alveolar hemorrhage. Three of the 4 patients were treated with short courses of prednisone and cyclophosphamide, 2 of whom experienced stable long-term improvement in their renal function despite ongoing cocaine use. The remaining 2 patients developed end-stage renal disease and became dialysis-dependent. This report supports emerging concern of more wide spread organ toxicity associated with the use of levamisole-adulterated cocaine.

The Aurora-A kinase gene is frequently amplified and/or overexpressed in a variety of human cancers, leading to major efforts to develop therapeutic agents targeting this pathway. Here, we show that Aurora-A is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7 in a process that is regulated by GSK3β. Using a series of truncated Aurora-A proteins and site-directed mutagenesis, we identified distinct FBXW7 and GSK3β-binding sites in Aurora-A. Mutation of critical residues in either site substantially disrupts degradation of Aurora-A. Furthermore, we show that loss of Pten results in the stabilization of Aurora-A by attenuating FBXW7-dependent degradation of Aurora-A through the AKT/GSK3β pathway. Moreover, radiation-induced tumor latency is significantly shortened in Fbxw7(+/-)Pten(+/-) mice as compared with either Fbxw7(+/-) or Pten(+/-) mice, indicating that Fbxw7 and Pten appear to cooperate in suppressing tumorigenesis. Our results establish a novel posttranslational regulatory network in which the Pten and Fbxw7 pathways appear to converge on the regulation of Aurora-A level.