Adeno-associated virus in the liver: natural history and consequences in tumour development.

OBJECTIVE: Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%-80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. This study aimed to characterise the natural history of AAV infection in the liver and its consequence in tumour development. DESIGN: Viral DNA was quantified in tumour and non-tumour liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analysed using a DNAse/TaqMan-based assay and quantitative RT-PCR. In silico analyses using viral capture data explored viral variants and new clonal insertions. RESULTS: AAV DNA was detected in 21% of the patients, including 8% of the tumour tissues, equally distributed in two major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumour tissues, frequently associated with viral RNA expression and human herpesvirus type 6, the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B and GLI1/INHBE. AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localisation of the integration site. CONCLUSION: We provided an integrated analysis of the wild-type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. Clonal AAV insertions were positive selected during HCC development on non-cirrhotic liver challenging the notion of AAV as a non-pathogenic virus.

tRNA fragmentation and protein translation dynamics in the course of kidney injury.

Cells under stressful microenvironmental conditions initiate integrated molecular circuitries that aim at reducing general protein translation rates while redirecting protein synthesis toward a selective set of stress-response proteins. The consequence of the activation of this dynamic system is a reduction of the energy expenditure of the cell, and a metabolic rewiring that shapes adaptation under stress, which will, in fine, promote cell survival. In general, the translation initiation step is the prime target of translation reduction, with 2 molcular modules inhibiting translation initiation: the mechanistic target of Rapamycin complex 1, and the stress related kinases eIF2 kinases, which are all involved in the cellular responses to kidney injuries. tRNA (tRNA) dynamics and fragmentation have recently gained a considerable weight in the field of the non-coding RNA biology, and emerge as an important system for protein translation modulation under cellular stress. More precisely, stress-induced tRNA (tiRNA), the cleavage products of the ribonuclease angiogenin, are generated under various stress conditions, including oxidative stress and endoplasmic reticulum stress, and contribute to protein translation reprogramming in mammal cells. Current clinical and experimental evidence indicates that the angiogenin-tRNA fragmentation system is initiated under renal insults, and is involved in the tissue adaptation upon kidney injury. In addition, this system represents a potential source for minimally-invasive or non invasive biomarkers of early kidney injury. Besides RNA interference, tRNA fragments are likely involved in other fundamental cellular functions, including inflammation, and a better understanding of the molecular basis of tRNA functions will drive discoveries on the fundamental role of non coding RNA biology, as exemplified by microRNA, in the regulation of kidney homeostasis.

Urinary Angiogenin Reflects the Magnitude of Kidney Injury at the Infrahistologic Level.

The ribonuclease angiogenin is a component of the mammalian stress response that is secreted by renal epithelial cells on activation of the inositol-requiring enzyme 1α (IRE1α)-active spliced X-box binding protein 1 (sXBP1) axis and instrumental to the adaptation to AKI associated with endoplasmic reticulum stress. To determine whether the amount of angiogenin in urine of individuals with a kidney injury reflects the magnitude of the lesions and provides information on the risk of organ failure, we examined individuals referred for a kidney injury and determined the biochemical characteristics of urinary angiogenin and its diagnostic and prognostic values. Urinary angiogenin did not correlate with the urinary concentrations of high molecular weight proteins and correlated only weakly with low molecular weight proteins, suggestive of tubular production. In a cohort of 242 kidney transplant recipients with acute allograft dysfunction, higher urinary angiogenin concentrations at the time of the biopsy associated with worse renal function and higher proteinuria but did not correlate with histologic lesions as defined in the Banff classification. Kidney transplant recipients with urinary angiogenin amounts in the highest 50% had a risk of graft failure 3.59 times as high (95% confidence interval, 1.12 to 15.94) as that of patients with amounts in the lowest 50%. Finally, the amount of urinary angiogenin reflected the activity of the IRE1α-XBP1 axis in allografts. Our approach identified urinary angiogenin as a noninvasive indicator of the extent of tissue damage, independent of the histologic lesions, and a risk predictor of kidney allograft failure.

A Novel Extrinsic Pathway for the Unfolded Protein Response in the Kidney.

The ribonuclease angiogenin is a component of the mammalian stress response, and functions in both cell-autonomous and non-cell-autonomous ways to promote tissue adaptation to injury. We recently showed that angiogenin regulates tissue homeostasis during AKI associated with endoplasmic reticulum (ER) stress through the production of transfer RNA fragments that interfere with translation initiation and thereby alleviate ER stress. However, whether the paracrine signaling mediated by angiogenin secretion is a genuine component of the ER stress response to kidney injury is unknown. Here, we explored the molecular mechanisms by which angiogenin is secreted upon ER stress, and determined how it modulates the inflammatory microenvironment. In cultured renal epithelial cells, ER stress specifically induced angiogenin secretion under the selective control of inositol-requiring enzyme 1α, a key activator of the unfolded protein response. The transcription factors spliced X-box-binding protein 1 and p65, which are activated by inositol-requiring enzyme 1α upon ER stress, each bound the angiogenin promoter and controlled the amount of angiogenin secreted. Furthermore, p65 promoted angiogenin transcription in an ER stress-dependent manner. Similar to secretion of the ER stress-induced proinflammatory cytokine IL-6, secretion of angiogenin required the ER-Golgi pathway. Notably, incubation of human macrophages with angiogenin promoted macrophage reprogramming toward an activated and proinflammatory phenotype. In patients, angiogenin expression increased upon renal inflammation, and the urinary concentration of angiogenin correlated with the extent of immune-mediated kidney injury. Collectively, our data identify angiogenin as a mediator of the ER stress-dependent inflammatory response and as a potential noninvasive biomarker of AKI.

Angiogenin Mediates Cell-Autonomous Translational Control under Endoplasmic Reticulum Stress and Attenuates Kidney Injury.

Endoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but the molecular bases underlying the biologic outcomes on the evolution of renal disease remain mostly unknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but its contribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediated contribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression was significantly higher in samples from injured human kidneys than in samples from normal human kidneys, and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renal epithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcription factor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules and inhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramatically greater in ANG knockout mice (Ang(-/-)) mice than in wild-type mice. These results indicate that ANG is a critical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism.

High prevalence of and potential mechanisms for chronic kidney disease in patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a genetic disorder of the synthesis of heme caused by a deficiency in hydroxymethylbilane synthase (HMBS), leading to the overproduction of the porphyrin precursors δ-aminolevulinic acid and porphobilinogen. The aim of this study is to describe the clinical and biological characteristics, the renal pathology, and the cellular mechanisms of chronic kidney disease associated with AIP. A total of 415 patients with HMBS deficiency followed up in the French Porphyria Center were enrolled in 2003 in a population-based study. A follow-up study was conducted in 2013, assessing patients for clinical, biological, and histological parameters. In vitro models were used to determine whether porphyrin precursors promote tubular and endothelial cytotoxicity. Chronic kidney disease occurred in up to 59% of the symptomatic AIP patients, with a decline in the glomerular filtration rate of ~1 ml/min per 1.73 m(2) annually. Proteinuria was absent in the vast majority of the cases. The renal pathology was a chronic tubulointerstitial nephropathy, associated with a fibrous intimal hyperplasia and focal cortical atrophy. Our experimental data provide evidence that porphyrin precursors promote endoplasmic reticulum stress, apoptosis, and epithelial phenotypic changes in proximal tubular cells. In conclusion, the diagnosis of chronic kidney disease associated with AIP should be considered in cases of chronic tubulointerstitial nephropathy and/or focal cortical atrophy with severe proliferative arteriosclerosis.

Tryptophan depletion and the kinase GCN2 mediate IFN-γ-induced autophagy.

IFN-γ is a master regulator of the immune responses that occur in the transplanted kidney, acting both on the immune system and on the graft itself. The cellular responses to IFN-γ are complex, and emerging evidence suggests that IFN-γ may regulate autophagic functions. Conversely, autophagy modulates innate and adaptive immune functions in various contexts. In this study, we identify a novel mechanism by which IFN-γ activates autophagy in human kidney epithelial cells and provide new insights into how autophagy regulates immune functions in response to IFN-γ. Our results indicate that IFN-γ promotes tryptophan depletion, activates the eIF2α kinase general control nonderepressible-2 (GCN2), and leads to an increase in the autophagic flux. Further, tryptophan supplementation and RNA interference directed against GCN2 inhibited IFN-γ-induced autophagy. This process is of functional relevance because autophagy regulates the secretion of inflammatory cytokines and growth factors by human kidney epithelial cells in response to IFN-γ. These findings assign to IFN-γ a novel function in the regulation of autophagy, which, in turn, modulates IFN-γ-induced secretion of inflammatory cytokines.

Cyclin A2/E1 activation defines a hepatocellular carcinoma subclass with a rearrangement signature of replication stress.

Cyclins A2 and E1 regulate the cell cycle by promoting S phase entry and progression. Here, we identify a hepatocellular carcinoma (HCC) subgroup exhibiting cyclin activation through various mechanisms including hepatitis B virus (HBV) and adeno-associated virus type 2 (AAV2) insertions, enhancer hijacking and recurrent CCNA2 fusions. Cyclin A2 or E1 alterations define a homogenous entity of aggressive HCC, mostly developed in non-cirrhotic patients, characterized by a transcriptional activation of E2F and ATR pathways and a high frequency of RB1 and PTEN inactivation. Cyclin-driven HCC display a unique signature of structural rearrangements with hundreds of tandem duplications and templated insertions frequently activating TERT promoter. These rearrangements, strongly enriched in early-replicated active chromatin regions, are consistent with a break-induced replication mechanism. Pan-cancer analysis reveals a similar signature in BRCA1-mutated breast and ovarian cancers. Together, this analysis reveals a new poor prognosis HCC entity and a rearrangement signature related to replication stress.