Whole exome data prioritization unveils the hidden weight of Mendelian causes of male infertility. A report from the first Italian cohort.

Almost 40% of infertile men cases are classified as idiopathic when tested negative to the current diagnostic routine based on the screening of karyotype, Y chromosome microdeletions and CFTR mutations in men with azoospermia or oligozoospermia. Rare monogenic forms of infertility are not routinely evaluated. In this study we aim to investigate the unknown potential genetic causes in couples with pure male idiopathic infertility by applying variant prioritization to whole exome sequencing (WES) in a cohort of 99 idiopathic Italian patients. The ad-hoc manually curated gene library prioritizes genes already known to be associated with more common and rare syndromic and non-syndromic male infertility forms. Twelve monogenic cases (12.1%) were identified in the whole cohort of patients. Of these, three patients had variants related to mild androgen insensitivity syndrome, two in genes related to hypogonadotropic hypogonadism, and six in genes related to spermatogenic failure, while one patient is mutant in PKD1. These results suggest that NGS combined with our manually curated pipeline for variant prioritization and classification can uncover a considerable number of Mendelian causes of infertility even in a small cohort of patients.

Ductular reaction-associated neutrophils promote biliary epithelium proliferation in chronic liver disease.

BACKGROUND & AIMS: Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response. METHODS: The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion. RESULTS: In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion. CONCLUSIONS: Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response. IMPACT AND IMPLICATIONS: Our results indicate that neutrophils are highly plastic and can have an extended lifespan. Moreover, we identify a new role of neutrophils as triggers of expansion of the biliary epithelium. Overall, the results of this study indicate that ductular reaction-associated neutrophils (or DRANs) are new players in the maladaptive tissue-healing response in chronic liver injury and may be a potential target for therapeutic interventions to reduce ductular reaction expansion and promote tissue repair in advanced liver disease.

Ductular reaction promotes intrahepatic angiogenesis through Slit2-Roundabout 1 signaling.

BACKGROUND AND AIMS: Ductular reaction (DR) expands in chronic liver diseases and correlates with disease severity. Besides its potential role in liver regeneration, DR plays a role in the wound-healing response of the liver, promoting periductular fibrosis and inflammatory cell recruitment. However, there is no information regarding its role in intrahepatic angiogenesis. In the current study we investigated the potential contribution of DR cells to hepatic vascular remodeling during chronic liver disease. APPROACH AND RESULTS: In mouse models of liver injury, DR cells express genes involved in angiogenesis. Among angiogenesis-related genes, the expression of Slit2 and its receptor Roundabout 1 (Robo1) was localized in DR cells and neoangiogenic vessels, respectively. The angiogenic role of the Slit2-Robo1 pathway in chronic liver disease was confirmed in ROBO1/2-/+ mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which displayed reduced intrahepatic neovascular density compared to wild-type mice. However, ROBO1/2 deficiency did not affect angiogenesis in partial hepatectomy. In patients with advanced alcohol-associated disease, angiogenesis was associated with DR, and up-regulation of SLIT2-ROBO1 correlated with DR and disease severity. In vitro, human liver-derived organoids produced SLIT2 and induced tube formation of endothelial cells. CONCLUSIONS: Overall, our data indicate that DR expansion promotes angiogenesis through the Slit2-Robo1 pathway and recognize DR cells as key players in the liver wound-healing response.

NK cells eliminate Epstein-Barr virus bound to B cells through a specific antibody-mediated uptake.

Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.

The multifaceted role of cathepsins in liver disease.

Proteases are the most abundant enzyme gene family in vertebrates and they execute essential functions in all living organisms. Their main role is to hydrolase the peptide bond within proteins, a process also called proteolysis. Contrary to the conventional paradigm, proteases are not only random catalytic devices, but can perform highly selective and targeted cleavage of specific substrates, finely modulating multiple essential cellular processes. Lysosomal protease cathepsins comprise 3 families of proteases that preferentially act within acidic cellular compartments, but they can also be found in other cellular locations. They can operate alone or as part of signalling cascades and regulatory circuits, playing important roles in apoptosis, extracellular matrix remodelling, hepatic stellate cell activation, autophagy and metastasis, contributing to the initiation, development and progression of liver disease. In this review, we comprehensively summarise current knowledge on the role of lysosomal cathepsins in liver disease, with a particular emphasis on liver fibrosis, non-alcoholic fatty liver disease and hepatocellular carcinoma.

Cathepsins in the Pathophysiology of Mucopolysaccharidoses: New Perspectives for Therapy.

Cathepsins (CTSs) are ubiquitously expressed proteases normally found in the endolysosomal compartment where they mediate protein degradation and turnover. However, CTSs are also found in the cytoplasm, nucleus, and extracellular matrix where they actively participate in cell signaling, protein processing, and trafficking through the plasma and nuclear membranes and between intracellular organelles. Dysregulation in CTS expression and/or activity disrupts cellular homeostasis, thus contributing to many human diseases, including inflammatory and cardiovascular diseases, neurodegenerative disorders, diabetes, obesity, cancer, kidney dysfunction, and others. This review aimed to highlight the involvement of CTSs in inherited lysosomal storage disorders, with a primary focus to the emerging evidence on the role of CTSs in the pathophysiology of Mucopolysaccharidoses (MPSs). These latter diseases are characterized by severe neurological, skeletal and cardiovascular phenotypes, and no effective cure exists to date. The advance in the knowledge of the molecular mechanisms underlying the activity of CTSs in MPSs may open a new challenge for the development of novel therapeutic approaches for the cure of such intractable diseases.

Heparan sulfate in chronic kidney diseases: Exploring the role of 3-O-sulfation.

One of the main feature of chronic kidney disease is the development of renal fibrosis. Heparan Sulfate (HS) is involved in disease development by modifying the function of growth factors and cytokines and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. Using a mouse model of renal fibrosis, we found that total HS 2-O-sulfation was increased in damaged kidneys, whilst, tubular staining of HS 3-O-sulfation was decreased. The expression of HS modifying enzymes significantly correlated with the development of fibrosis with HS3ST1 demonstrating the strongest correlation. The pro-fibrotic factors TGFß1 and TGFß2/IL1ß significantly downregulated HS3ST1 expression in both renal epithelial cells and renal fibroblasts. To determine the implication of HS3ST1 in growth factor binding and signalling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signalling.

Chronic psychosocial defeat differently affects lipid metabolism in liver and white adipose tissue and induces hepatic oxidative stress in mice fed a high-fat diet.

It is widely accepted that chronic stress may alter the homeostatic mechanisms of body weight control. In this study, we followed the metabolic changes occurring in mice when chronic stress caused by psychosocial defeat (CPD) is associated with ad libitum exposure to a palatable high-fat diet (HFD). In this model, CPD mice consumed more HFD than unstressed (Un) mice without gaining body weight. We focused on metabolic processes involved in weight control, such as de novo lipogenesis (DNL), fatty acid ß-oxidation (FAO), and thermogenesis. The activity and expression of DNL enzymes were reduced in the liver and white adipose tissue of mice consuming the HFD. Such effects were particularly evident in stressed mice. In both CPD and Un mice, HFD consumption increased the hepatic expression of the mitochondrial FAO enzyme carnitine palmitoyltransferase-1. In the liver of mice consuming the HFD, stress exposure prevented accumulation of triacylglycerols; however, accumulation of triacylglycerols was observed in Un mice under the same dietary regimen. In brown adipose tissue, stress increased the expression of uncoupling protein-1, which is involved in energy dissipation, both in HFD and control diet-fed mice. We consider increased FAO and energy dissipation responsible for the antiobesity effect seen in CPD/HFD mice. However, CPD associated with HFD induced hepatic oxidative stress.-Giudetti, A. M., Testini, M., Vergara, D., Priore, P., Damiano, F., Gallelli, C. A., Romano, A., Villani, R., Cassano, T., Siculella, L., Gnoni, G. V., Moles, A., Coccurello, R., Gaetani, S. Chronic psychosocial defeat differently affects lipid metabolism in liver and white adipose tissue and induces hepatic oxidative stress in mice fed a high-fat diet.

Lysosomal protease deficiency or substrate overload induces an oxidative-stress mediated STAT3-dependent pathway of lysosomal homeostasis.

Diverse cellular processes depend on the lysosomal protease system but how cells regulate lysosomal proteolytic capacity is only partly understood. We show here that cells can respond to protease/substrate imbalance in this compartment by de novo expression of multiple lysosomal hydrolases. This response, exemplified here either by loss of asparagine endopeptidase (AEP) or other lysosomal cysteine proteases, or by increased endocytic substrate load, is not dependent on the transcription factor EB (TFEB) but rather is triggered by STAT3 activation downstream of lysosomal oxidative stress. Similar lysosomal adaptations are seen in mice and cells expressing a constitutively active form of STAT3. Our results reveal how cells can increase lysosomal protease capacity under 'fed' rather than 'starved' conditions that activate the TFEB system. In addition, STAT3 activation due to lysosomal stress likely explains the hyperproliferative kidney disease and splenomegaly observed in AEP-deficient mice.

Mitochondrial-Lysosomal Axis in Acetaminophen Hepatotoxicity.

Acetaminophen (APAP) toxicity is the most common cause of acute liver failure and a major indication for liver transplantion in the United States and Europe. Although significant progress has been made in understanding the molecular mechanisms underlying APAP hepatotoxicity, there is still an urgent need to find novel and effective therapies against APAP-induced acute liver failure. Hepatic APAP metabolism results in the production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which under physiological conditions is cleared by its conjugation with glutathione (GSH) to prevent its targeting to mitochondria. APAP overdose or GSH limitation leads to mitochondrial NAPQI-protein adducts formation, resulting in oxidative stress, mitochondrial dysfunction, and necrotic cell death. As mitochondria are a major target of APAP hepatotoxicity, mitochondrial quality control and clearance of dysfunctional mitochondria through mitophagy, emerges as an important strategy to limit oxidative stress and the engagement of molecular events leading to cell death. Recent evidence has indicated a lysosomal-mitochondrial cross-talk that regulates APAP hepatotoxicity. Moreover, as lysosomal function is essential for mitophagy, impairment in the fusion of lysosomes with autophagosomes-containing mitochondria may compromise the clearance of dysfunctional mitochondria, resulting in exacerbated APAP hepatotoxicity. This review centers on the role of mitochondria in APAP hepatotoxicity and how the mitochondrial/lysosomal axis can influence APAP-induced liver failure.

Increased intake of energy-dense diet and negative energy balance in a mouse model of chronic psychosocial defeat.

PURPOSE: Chronic exposure to stress may represent a risk factor for developing metabolic and eating disorders, mostly driven by the overconsumption of easily accessible energy-dense palatable food, although the mechanisms involved remain still unclear. In this study, we used an ethologically oriented murine model of chronic stress caused by chronic psychosocial defeat (CPD) to investigate the effects of unrestricted access to a palatable high fat diet (HFD) on food intake, body weight, energy homeostasis, and expression of different brain neuropeptides. Our aim was to shed light on the mechanisms responsible for body weight and body composition changes due to chronic social stress. METHODS: In our model of subordinate (defeated), mice (CPD) cohabitated in constant sensory contact with dominants, being forced to interact on daily basis, and were offered ad libitum access either to an HFD or to a control diet (CD). Control mice (of the same strain as CPD mice) were housed in pairs and left unstressed in their home cage (UN). In all these mice, we evaluated body weight, different adipose depots, energy metabolism, caloric intake, and neuropeptide expression. RESULTS: CPD mice increased the intake of HFD and reduced body weight in the presence of enhanced lipid oxidation. Resting energy expenditure and interscapular brown adipose tissue (iBAT) were increased in CPD mice, whereas epididymal adipose tissue increased only in HFD-fed unstressed mice. Propiomelanocortin mRNA levels in hypothalamic arcuate nucleus increased only in HFD-fed unstressed mice. Oxytocin mRNA levels in the paraventricular nucleus and neuropeptide Y mRNA levels within the arcuate were increased only in CD-fed CPD mice. In the arcuate, CART was increased in HFD-fed UN mice and in CD-fed CPD mice, while HFD intake suppressed CART increase in defeated animals. In the basolateral amygdala, CART expression was increased only in CPD animals on HFD. CONCLUSIONS: CPD appears to uncouple the intake of HFD from energy homeostasis causing higher HFD intake, larger iBAT accumulation, increased energy expenditure and lipid oxidation, and lower body weight. Overall, the present study confirms the notion that the chronic activation of the stress response can be associated with metabolic disorders, altered energy homeostasis, and changes of orexigenic and anorexigenic signaling. These changes might be relevant to better understand the etiology of stress-induced obesity and eating disorders and might represent a valid therapeutic approach for the development of new therapies in this field.

The NF-κB1 is a key regulator of acute but not chronic renal injury.

The NF-κB family of transcription factors is important for many cellular functions, in particular initiation and propagation of inflammatory and immune responses. However, recent data has suggested that different subunits of the NF-κB family can suppress the inflammatory response. NF-κB1, from the locus nfκb1, can inhibit transcription, acting as a brake to the recognised pro-inflammatory activity of other NF-κB subunits. We tested the function of NF-κB1 in an acute (nephrotoxic serum (NTS) nephritis) and a chronic (unilateral ureteric obstruction (UUO)) model of renal injury using NF-κB1 (nfκb1-/-) knockout mice. Deficiency in NF-κB1 increased the severity of glomerular injury in NTS-induced nephritis and was associated with greater proteinuria and persistent pro-inflammatory gene expression. Induction of disease in bone marrow chimeric mice demonstrated that the absence of NF-κB1 in either bone marrow or glomerular cells increased the severity of injury. Early after UUO (day 3) there was more severe histological injury in the nfκb1-/- mice but by day 10, disease severity was equivalent in wild type and nfκb1-/- mice. In conclusion, NF-κB1 modifies acute inflammatory renal injury but does not influence chronic fibrotic injury.

A sketch of known and novel MYCN-associated miRNA networks in neuroblastoma.

Neuroblastoma (NB) originates from neural crest-derived precursors and represents the most common childhood extracranial solid tumour. MicroRNAs (miRNAs), a class of small non-coding RNAs that participate in a wide variety of biological processes by regulating gene expression, appear to play an essential role within the NB context. High-throughput next generation sequencing (NGS) was applied to study the miRNA transcriptome in a cohort of NB tumours with and without MYCN-amplification (MNA and MNnA, respectively) and in dorsal root ganglia (DRG), as a control. Out of the 128 miRNAs differentially expressed in the NB vs. DRG comparison, 47 were expressed at higher levels, while 81 were expressed at lower levels in the NB tumours. We also found that 23 miRNAs were differentially expressed in NB with or without MYCN-amplification, with 17 miRNAs being upregulated and 6 being downregulated in the MNA subtypes. Functional annotation analysis of the target genes of these differentially expressed miRNAs demonstrated that many mRNAs were involved in cancer-related pathways, such as DNA-repair and apoptosis as well as FGFR and EGFR signalling. In particular, we found that miR-628-3p negatively affects MYCN gene expression. Furthermore, we identified a novel miRNA candidate with variable expression in MNA vs. MNnA tumours, whose putative target genes are implicated in the mTOR pathway. The present study provides further insight into the molecular mechanisms that correlate miRNA dysregulation to NB development and progression.

A Review of Research-Grade Human Induced Pluripotent Stem Cells Qualification and Biobanking Processes.

Human induced pluripotent stem cell (hiPSC) biobanks are invaluable resources for basic and clinical research, since they provide a sustainable supply of accessible cell lines that meet high quality and safety standards. hiPSCs are particularly useful for understanding disease mechanisms, creating cell models for drug development, and generating novel clinical therapies. For clinical applications and drug discovery, it is fundamental that the acquired pluripotent cell lines never touch animal-derived products nor xenogeneic reagents (Good Manufacturing Practice-grade); whereas for research grade, it is sufficient to operate under Good Laboratory Practice conditions. However, regardless of the end use, it is important that every step in the whole process, starting from the original cells throughout expansion and manipulation, must be performed and recorded rigorously. Here, we describe our biobanking management system that is applied specifically to human pluripotent stem cells.

The Multifaceted Role of the Lysosomal Protease Cathepsins in Kidney Disease.

Kidney disease is worldwide the 12th leading cause of death affecting 8-16% of the entire population. Kidney disease encompasses acute (short-lasting episode) and chronic (developing over years) pathologies both leading to renal failure. Since specific treatments for acute or chronic kidney disease are limited, more than 2 million people a year require dialysis or kidney transplantation. Several recent evidences identified lysosomal proteases cathepsins as key players in kidney pathophysiology. Cathepsins, originally found in the lysosomes, exert important functions also in the cytosol and nucleus of cells as well as in the extracellular space, thus participating in a wide range of physiological and pathological processes. Based on their catalytic active site residue, the 15 human cathepsins identified up to now are classified in three different families: serine (cathepsins A and G), aspartate (cathepsins D and E), or cysteine (cathepsins B, C, F, H, K, L, O, S, V, X, and W) proteases. Specifically in the kidney, cathepsins B, D, L and S have been shown to regulate extracellular matrix homeostasis, autophagy, apoptosis, glomerular permeability, endothelial function, and inflammation. Dysregulation of their expression/activity has been associated to the onset and progression of kidney disease. This review summarizes most of the recent findings that highlight the critical role of cathepsins in kidney disease development and progression. A better understanding of the signaling pathways governed by cathepsins in kidney physiopathology may yield novel selective biomarkers or therapeutic targets for developing specific treatments against kidney disease.

Cysteine cathepsins control hepatic NF-κB-dependent inflammation via sirtuin-1 regulation.

Sirtuin-1 (SIRT1) regulates hepatic metabolism but its contribution to NF-κB-dependent inflammation has been overlooked. Cysteine cathepsins (Cathepsin B or S, CTSB/S) execute specific functions in physiological processes, such as protein degradation, having SIRT1 as a substrate. We investigated the roles of CTSB/S and SIRT1 in the regulation of hepatic inflammation using primary parenchymal and non-parenchymal hepatic cell types and cell lines. In all cells analyzed, CTSB/S inhibition reduces nuclear p65-NF-κB and κB-dependent gene expression after LPS or TNF through enhanced SIRT1 expression. Accordingly, SIRT1 silencing was sufficient to enhance inflammatory gene expression. Importantly, in a dietary mouse model of non-alcoholic steatohepatitis, or in healthy and fibrotic mice after LPS challenge, cathepsins as well as NF-κB-dependent gene expression are activated. Consistent with the prominent role of cathepsin/SIRT1, cysteine cathepsin inhibition limits NF-κB-dependent hepatic inflammation through the regulation of SIRT1 in all in vivo settings, providing a novel anti-inflammatory therapeutic target in liver disease.

Lysosomal protease cathepsin D; a new driver of apoptosis during acute kidney injury.

Acute kidney injury (AKI) is an abrupt reduction in kidney function caused by different pathological processes. It is associated with a significant morbidity and mortality in the acute phase and an increased risk of developing End Stage Renal Disease. Despite the progress in the management of the disease, mortality rates in the last five decades remain unchanged at around 50%. Therefore there is an urgent need to find new therapeutic strategies to treat AKI. Lysosomal proteases, particularly Cathepsin D (CtsD), play multiple roles in apoptosis however, their role in AKI is still unknown. Here we describe a novel role for CtsD in AKI. CtsD expression was upregulated in damaged tubular cells in nephrotoxic and ischemia reperfusion (IRI) induced AKI. CtsD inhibition using Pepstatin A led to an improvement in kidney function, a reduction in apoptosis and a decrease in tubular cell damage in kidneys with nephrotoxic or IRI induced AKI. Pepstatin A treatment slowed interstitial fibrosis progression following IRI induced AKI. Renal transplant biopsies with acute tubular necrosis demonstrated high levels of CtsD in damaged tubular cells. These results support a role for CtsD in apoptosis during AKI opening new avenues for the treatment of AKI by targeting lysosomal proteases.

Histone Modifications in a Mouse Model of Early Adversities and Panic Disorder: Role for Asic1 and Neurodevelopmental Genes.

Hyperventilation following transient, CO2-induced acidosis is ubiquitous in mammals and heritable. In humans, respiratory and emotional hypersensitivity to CO2 marks separation anxiety and panic disorders, and is enhanced by early-life adversities. Mice exposed to the repeated cross-fostering paradigm (RCF) of interference with maternal environment show heightened separation anxiety and hyperventilation to 6% CO2-enriched air. Gene-environment interactions affect CO2 hypersensitivity in both humans and mice. We therefore hypothesised that epigenetic modifications and increased expression of genes involved in pH-detection could explain these relationships. Medullae oblongata of RCF- and normally-reared female outbred mice were assessed by ChIP-seq for H3Ac, H3K4me3, H3K27me3 histone modifications, and by SAGE for differential gene expression. Integration of multiple experiments by network analysis revealed an active component of 148 genes pointing to the mTOR signalling pathway and nociception. Among these genes, Asic1 showed heightened mRNA expression, coherent with RCF-mice's respiratory hypersensitivity to CO2 and altered nociception. Functional enrichment and mRNA transcript analyses yielded a consistent picture of enhancement for several genes affecting chemoception, neurodevelopment, and emotionality. Particularly, results with Asic1 support recent human findings with panic and CO2 responses, and provide new perspectives on how early adversities and genes interplay to affect key components of panic and related disorders.

Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease.

During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.

Hedonic sensitivity to natural rewards is affected by prenatal stress in a sex-dependent manner.

Palatable food is a strong activator of the reward circuitry and may cause addictive behavior leading to eating disorders. How early life events and sex interact in shaping hedonic sensitivity to palatable food is largely unknown. We used prenatally restraint stressed (PRS) rats, which show abnormalities in the reward system and anxious/depressive-like behavior. Some of the hallmarks of PRS rats are known to be sex-dependent. We report that PRS enhanced and reduced milk chocolate-induced conditioned place preference in males and females, respectively. Male PRS rats also show increases in plasma dihydrotestosterone (DHT) levels and dopamine (DA) levels in the nucleus accumbens (NAc), and reductions in 5-hydroxytryptamine (5-HT) levels in the NAc and prefrontal cortex (PFC). In male rats, systemic treatment with the DHT-lowering drug finasteride reduced both milk chocolate preference and NAc DA levels. Female PRS rats showed lower plasma estradiol (E2 ) levels and lower DA levels in the NAc, and 5-HT levels in the NAc and PFC. E2 supplementation reversed the reduction in milk chocolate preference and PFC 5-HT levels. In the hypothalamus, PRS increased ERα and ERß estrogen receptor and CARTP (cocaine-and-amphetamine receptor transcript peptide) mRNA levels in males, and 5-HT2C receptor mRNA levels in females. Changes were corrected by treatments with finasteride and E2 , respectively. These new findings show that early life stress has a profound impact on hedonic sensitivity to high-palatable food via long-lasting changes in gonadal hormones. This paves the way to the development of hormonal strategies aimed at correcting abnormalities in the response to natural rewards.