MAFLD and CKD: An Updated Narrative Review

Accumulating evidence now indicates that non-alcoholic fatty liver disease (NAFLD), which is the most common chronic liver disease observed in clinical practice worldwide, is independently associated with an increased risk of incident chronic kidney disease (CKD). Given that NAFLD is linked to insulin resistance, obesity and type 2 diabetes mellitus, an international panel of experts have recently proposed a name change from NAFLD to metabolic associated fatty liver disease (MAFLD). Since the diagnostic criteria for NAFLD and MAFLD are different, observational studies assessing the potential concordance (or even superiority) of MAFLD, compared with NAFLD, in detecting patients at increased risk of hepatic and extra-hepatic complications (including CKD) are required. Hence, in the last two years, some observational studies have investigated the potential relationship between MAFLD and CKD. The result is that, at present, evidence regarding the concordance or even superiority of MAFLD, compared with NAFLD, in detecting patients at higher risk of CKD is still preliminary, although some data indicate that MAFLD identifies patients with CKD as accurately as NAFLD. In this narrative review, we will discuss: (a) the epidemiological evidence assessing the association between NAFLD and risk of incident CKD, (b) the epidemiological data investigating the association between MAFLD and risk of CKD and (c) the biological mechanisms underlying the association between NAFLD/MAFLD and CKD.     

Genetic and Diet-Induced Animal Models for Non-Alcoholic Fatty Liver Disease (NAFLD) Research

A rapidly increasing incidence of non-alcoholic fatty liver disease (NAFLD) is noted worldwide due to the adoption of western-type lifestyles and eating habits. This makes the understanding of the molecular mechanisms that drive the pathogenesis of this chronic disease and the development of newly approved treatments of utmost necessity. Animal models are indispensable tools for achieving these ends. Although the ideal mouse model for human NAFLD does not exist yet, several models have arisen with the combination of dietary interventions, genetic manipulations and/or administration of chemical substances. Herein, we present the most common mouse models used in the research of NAFLD, either for the whole disease spectrum or for a particular disease stage (e.g., non-alcoholic steatohepatitis). We also discuss the advantages and disadvantages of each model, along with the challenges facing the researchers who aim to develop and use animal models for translational research in NAFLD. Based on these characteristics and the specific study aims/needs, researchers should select the most appropriate model with caution when translating results from animal to human.     

Autophagy Dysregulation in Metabolic Associated Fatty Liver Disease: A New Therapeutic Target

Metabolic associated fatty liver disease (MAFLD) is one of the most common causes of chronic liver disease worldwide. To date, there is no FDA-approved treatment, so there is an urgent need to determine its pathophysiology and underlying molecular mechanisms. Autophagy is a lysosomal degradation pathway that removes damaged organelles and misfolded proteins after cell injury through endoplasmic reticulum stress or starvation, which inhibits apoptosis and promotes cell survival. Recent studies have shown that autophagy plays an important role in removing lipid droplets from hepatocytes. Autophagy has also been reported to inhibit the production of pro-inflammatory cytokines and provide energy for the hepatic stellate cells activation during liver fibrosis. Thyroid hormone, irisin, melatonin, hydrogen sulfide, sulforaphane, DA-1241, vacuole membrane protein 1, nuclear factor erythroid 2-related factor 2, sodium-glucose co-transporter type-2 inhibitors, immunity-related GTPase M, and autophagy-related gene 7 have been reported to ameliorate MAFLD via autophagic induction. Lipid receptor CD36, SARS-CoV-2 Spike protein and leucine aminopeptidase 3 play a negative role in the autophagic function. This review summarizes recent advances in the role of autophagy in MAFLD. Autophagy modulates major pathological changes, including hepatic lipid metabolism, inflammation, and fibrosis, suggesting the potential of modulating autophagy for the treatment of MAFLD.     

Role of B Lymphocytes in the Pathogenesis of NAFLD: A 2022 Update

Non-alcoholic fatty liver disease and its related complications are becoming one of the most important health problems globally. The liver functions as both a metabolic and an immune organ. The crosstalk between hepatocytes and intrahepatic immune cells plays a key role in coordinating a dual function of the liver in terms of the protection of the host from antigenic overload as a result of receiving nutrients and gut microbiota antigenic stimulation via facilitating immunologic tolerance. B cells are the most abundant lymphocytes in the liver. The crucial role of intrahepatic B cells in energy metabolism under different immune conditions is now emerging in the literature. The accumulating evidence has demonstrated that the antibodies and cytokines produced by B cells in the microenvironment play key and distinct roles in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Herein, we have aimed to consolidate and update the current knowledge about the pathophysiological roles of B cells as well as the underlying mechanisms in energy metabolism. Understanding how B cells can exacerbate and suppress liver damage by exploiting the antibodies and cytokines they produce will be of great importance for designing B-cell targeting therapies to treat various liver diseases.     

The Effects of Probiotics,Prebiotics and Synbiotics in Non-Alcoholic Fat Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH): A Systematic Review

Modifications in the microbiota caused by environmental and genetic reasons can unbalance the intestinal homeostasis, deregulating the host’s metabolism and immune system, intensifying the risk factors for the development and aggravation of non-alcoholic fat liver disease (NAFLD). The use of probiotics, prebiotics and synbiotics have been considered a potential and promising strategy to regulate the gut microbiota and produce beneficial effects in patients with liver conditions. For this reason, this review aimed to evaluate the effectiveness of probiotics, prebiotics, and symbiotics in patients with NAFLD and NASH. Pubmed, Embase, and Cochrane databases were consulted, and PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines were followed. The clinical trials used in this study demonstrated that gut microbiota interventions could improve a wide range of markers of inflammation, glycemia, insulin resistance, dyslipidemia, obesity, liver injury (decrease of hepatic enzymes and steatosis and fibrosis). Although microbiota modulators do not play a healing role, they can work as an important adjunct therapy in pathological processes involving NAFLD and its spectrums, either by improving the intestinal barrier or by preventing the formation of toxic metabolites for the liver or by acting on the immune system.     

Plasma Levels of Homocysteine and Cysteine Increased in Pediatric NAFLD and Strongly Correlated with Severity of Liver Damage

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from simple triglyceride accumulation in the hepatocytes to hepatic steatosis with inflammation, ballooning and fibrosis. It has been demonstrated that the pathogenesis of NAFLD involves increased oxidative stress, with consumption of the major cellular antioxidant, glutathione (GSH). Liver has a fundamental role in sulfur compound metabolism, although the data reported on plasma thiols status in NAFLD are conflicting. We recruited 63 NAFLD patients, and we analyzed all plasma thiols, such as homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly) and GSH, by high-performance liquid chromatography (HPLC) with fluorescence detection. Hcy, Cys and CysGly plasma levels increased in NAFLD patients (p < 0.0001); whereas GSH levels were decreased in NAFLD patients when compared to controls (p < 0.0001). On the contrary, patients with steatohepatitis exhibited lower levels of Hcy and Cys than subjects without. Furthermore, a positive correlation was found between Hcy and Cys and the presence of fibrosis in children with NAFLD. Taken together, these data demonstrated a defective hepatic sulfur metabolism in children with NAFLD, and that high levels of Hcy and Cys probably correlates with a pattern of more severe histological liver damage, due to mechanisms that require further studies.     

Lessons from Mouse Models of High-Fat Diet-Induced NAFLD

Nonalcoholic fatty liver disease (NAFLD) encompasses a clinicopathologic spectrum of diseases ranging from isolated hepatic steatosis to nonalcoholic steatohepatitis (NASH), the more aggressive form of fatty liver disease that may progress to cirrhosis and cirrhosis-related complications, including hepatocellular carcinoma. The prevalence of NAFLD, including NASH, is also increasing in parallel with the growing epidemics of obesity and diabetes. However, the causal relationships between obesity and/or diabetes and NASH or liver tumorigenesis have not yet been clearly elucidated. Animal models of NAFLD/NASH provide crucial information, not only for elucidating the pathogenesis of NAFLD/NASH, but also for examining therapeutic effects of various agents. A high-fat diet is widely used to produce hepatic steatosis and NASH in experimental animals. Several studies, including our own, have shown that long-term high-fat diet loading, which can induce obesity and insulin resistance, can also induce NASH and liver tumorigenesis in C57BL/6J mice. In this article, we discuss the pathophysiology of and treatment strategies for NAFLD and subsequent NAFLD-related complications such as NASH and liver tumorigenesis, mainly based on lessons learned from mouse models of high-fat diet-induced NAFLD/NASH.     

Long-Term Adverse Effect of Liver Stiffness on Glycaemic Control in Type 2 Diabetic Patients with Nonalcoholic Fatty Liver Disease: A Pilot Study

Currently, there are limited data regarding the long-term effect of liver stiffness on glycaemic control in patients with type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD). We prospectively followed an outpatient sample of 61 consecutive postmenopausal women with T2DM and NAFLD who had baseline data on liver ultrasonography and Fibroscan^®-assessed liver stiffness measurement (LSM) in 2017 and who underwent follow-up in 2022. Haemoglobin A1c (HbA1c) was measured both at baseline and follow-up. At baseline, 52 patients had NAFLD (hepatic steatosis) alone, and 9 had NAFLD with coexisting clinically significant fibrosis (defined as LSM ≥ 7 kPa on Fibroscan^®). At follow-up, 16 patients had a worsening of glycaemic control (arbitrarily defined as HbA1c increase ≥ 0.5% from baseline). The prevalence of NAFLD and coexisting clinically significant fibrosis at baseline was at least three times greater among patients who developed worse glycaemic control at follow-up, compared with those who did not (31.3% vs. 8.9%; p = 0.030). In logistic regression analysis, the presence of NAFLD and clinically significant fibrosis was associated with an approximately 4.5-fold increased likelihood of developing worse glycaemic control at follow-up (odds ratio 4.66, 95% confidence interval 1.07–20.3; p = 0.041), even after adjustment for baseline confounding factors, such as age, body mass index, haemoglobin A1c (or HOMA-estimated insulin resistance) and use of some glucose-lowering agents that may positively affect NAFLD and liver fibrosis. In conclusion, our results suggest that the presence of Fibroscan^®-assessed significant fibrosis was associated with a higher risk of developing worse glycaemic control in postmenopausal women with T2DM and NAFLD.     

Meta-Omic Platforms to Assist in the Understanding of NAFLD Gut Microbiota Alterations: Tools and Applications

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide as a result of the increasing prevalence of obesity, starting from early life stages. It is characterized by a spectrum of liver diseases ranging from simple fatty liver (NAFL) to steatohepatitis (NASH), with a possible progression to fibrosis, thus increasing liver-related morbidity and mortality. NAFLD development is driven by the co-action of several risk factors, including obesity and metabolic syndrome, which may be both genetically induced and diet-related. Recently, particular attention has been paid to the gut-liver axis, which may play a physio-pathological role in the onset and progression of the disease. The gut microbiota is intended to act as a bioreactor that can guarantee autonomous metabolic and immunological functions and that can drive functional strategies within the environment of the body in response to external stimuli. The complexity of the gut microbiota suggests that it behaves as an organ. Therefore, the concept of the gut-liver axis must be complemented with the gut-microbiota-liver network due to the high intricacy of the microbiota components and metabolic activities; these activities form the active diet-driven power plant of the host. Such complexity can only be revealed using systems biology, which can integrate clinical phenomics and gut microbiota data.     

Nonalcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis: Current Issues and Future Perspectives in Preclinical and Clinical Research

Nonalcoholic fatty liver disease (NAFLD) is a continuum of liver abnormalities often starting as simple steatosis and to potentially progress into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. Because of its increasing prevalence, NAFLD is becoming a major public health concern, in parallel with a worldwide increase in the recurrence rate of diabetes and metabolic syndrome. It has been estimated that NASH cirrhosis may surpass viral hepatitis C and become the leading indication for liver transplantation in the next decades. The broadening of the knowledge about NASH pathogenesis and progression is of pivotal importance for the discovery of new targeted and more effective therapies; aim of this review is to offer a comprehensive and updated overview on NAFLD and NASH pathogenesis, the most recommended treatments, drugs under development and new drug targets. The most relevant in vitro and in vivo models of NAFLD and NASH will be also reviewed, as well as the main molecular pathways involved in NAFLD and NASH development.     

Assessment of Ultra-Early-Stage Liver Fibrosis in Human Non-Alcoholic Fatty Liver Disease by Second-Harmonic Generation Microscopy

Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients.     

Molecular Mechanisms and New Treatment Strategies for Non-Alcoholic Steatohepatitis (NASH)

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD), in which most patients exhibit non-progressive, non-alcoholic fatty liver (NAFL) attributable to simple steatosis. Multiple hits, including genetic differences, fat accumulation, insulin resistance and intestinal microbiota changes, account for the progression of NASH. NAFLD is strongly associated with obesity, which induces adipokine secretion, endoplasmic reticulum (ER) and oxidative stress at the cellular level, which in turn induces hepatic steatosis, inflammation and fibrosis. Among these factors, gut microbiota are acknowledged as having an important role in initiating this multifactorial disease. Oxidative stress is considered to be a key contributor in the progression from NAFL to NASH. Macrophage infiltration is apparent in NAFL and NASH, while T-cell infiltration is apparent in NASH. Although several clinical trials have shown that antioxidative therapy with vitamin E can effectively control hepatitis pathology in the short term, the long-term effects remain obscure and have often proved to be ineffective in many other diseases. Several long-term antioxidant protocols have failed to reduce mortality. New treatment modalities that incorporate current understanding of NAFLD molecular pathogenesis must be considered.     

The Emerging Role of Extracellular Vesicles and Autophagy Machinery in NASH—Future Horizons in NASH Management

Non-alcoholic fatty liver disease (NAFLD) is considered the most frequent chronic hepatic disease in the general population, while it is the first cause of liver transplantation in the US. NAFLD patients will subsequently develop non-alcoholic steatohepatitis (NASH), which is characterized by aberrant hepatocellular inflammation with or without the presence of fibrosis. The lack of specific biomarkers and therapeutic strategies makes non-alcoholic steatohepatitis (NASH) management a difficult task for clinicians. Extracellular vesicles (EVs) constitute a heterogenic population of vesicles produced by inward or outward plasma-membrane budding. There is an emerging connection between autophagy EVs production, via an unconventional non-degradative procedure. Alterations in the amount of the secreted EVs and the cargo they carry are also involved in the disease progression and development of NASH. Autophagy constitutes a multistep lysosomal degradative pathway that reassures cell homeostasis and survival under stressful conditions, such as oxygen and energy deprivation. It prevents cellular damage by eliminating defected proteins or nοn-functional intracellular organelles. At the same time, it reassures the optimal conditions for the cells via a different mechanism that includes the removal of cargo via the secretion of EVs. Similarly, autophagy machinery is also associated with the pathogenetic mechanism of NAFLD, while it has a significant implication for the progression of the disease and the development of NASH. In this review, we will shed light on the interplay between autophagy and EVs in NASH, the emerging connection of EVs production with the autophagy pathway, and their possible manipulation for developing future therapeutic strategies for NASH.     

Fibrogenic Pathways in Metabolic Dysfunction Associated Fatty Liver Disease (MAFLD)

The prevalence of nonalcoholic fatty liver disease (NAFLD), recently also re-defined as metabolic dysfunction associated fatty liver disease (MAFLD), is rapidly increasing, affecting ~25% of the world population. MALFD/NAFLD represents a spectrum of liver pathologies including the more benign hepatic steatosis and the more advanced non-alcoholic steatohepatitis (NASH). NASH is associated with enhanced risk for liver fibrosis and progression to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) activation underlies NASH-related fibrosis. Here, we discuss the profibrogenic pathways, which lead to HSC activation and fibrogenesis, with a particular focus on the intercellular hepatocyte–HSC and macrophage–HSC crosstalk.     

The Alterations of Mitochondrial Function during NAFLD Progression—An Independent Effect of Mitochondrial ROS Production

The progression of non-alcoholic fatty liver (NAFL) into non-alcoholic steatohepatitis implicates multiple mechanisms, chief of which is mitochondrial dysfunction. However, the sequence of events underlying mitochondrial failure are still poorly clarified. In this work, male C57BL/6J mice were fed with a high-fat plus high-sucrose diet for 16, 20, 22, and 24 weeks to induce NAFL. Up to the 20th week, an early mitochondrial remodeling with increased OXPHOS subunits levels and higher mitochondrial respiration occurred. Interestingly, a progressive loss of mitochondrial respiration along “Western diet” feeding was identified, accompanied by higher susceptibility to mitochondrial permeability transition pore opening. Importantly, our findings prove that mitochondrial alterations and subsequent impairment are independent of an excessive mitochondrial reactive oxygen species (ROS) generation, which was found to be progressively diminished along with disease progression. Instead, increased peroxisomal abundance and peroxisomal fatty acid oxidation-related pathway suggest that peroxisomes may contribute to hepatic ROS generation and oxidative damage, which may accelerate hepatic injury and disease progression. We show here for the first time the sequential events of mitochondrial alterations involved in non-alcoholic fatty liver disease (NAFLD) progression and demonstrate that mitochondrial ROS are not one of the first hits that cause NAFLD progression.     

Roles of Macrophages in Advanced Liver Fibrosis,Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c⁺/Ly6C⁻ and CD11c⁻/Ly6C⁺ cells. CD11c⁺ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c⁺ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C⁺ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c⁺/Ly6C⁻ cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c⁻/Ly6C⁺ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.     

Current Options and Future Directions for NAFLD and NASH Treatment

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, with a broad spectrum ranging from simple steatosis to advanced stage of nonalcoholic steatohepatitis (NASH). Although there are many undergoing clinical trials for NAFLD treatment, there is no currently approved treatment. NAFLD accounts as a major causing factor for the development of hepatocellular carcinoma (HCC), and its incidence rises accompanying the prevalence of obesity and diabetes. Reprogramming of antidiabetic and anti-obesity medicine is a major treatment option for NAFLD and NASH. Liver inflammation and cellular death, with or without fibrosis account for the progression of NAFLD to NASH. Therefore, molecules and signaling pathways involved in hepatic inflammation, fibrosis, and cell death are critically important targets for the therapy of NAFLD and NASH. In addition, the avoidance of aberrant infiltration of inflammatory cytokines by treating with CCR antagonists also provides a therapeutic option. Currently, there is an increasing number of pre-clinical and clinical trials undergoing to evaluate the effects of antidiabetic and anti-obesity drugs, antibiotics, pan-caspase inhibitors, CCR2/5 antagonists, and others on NAFLD, NASH, and liver fibrosis. Non-invasive serum diagnostic markers are developed for fulfilling the need of diagnostic testing in a large amount of NAFLD cases. Overall, a better understanding of the underlying mechanism of the pathogenesis of NAFLD is helpful to choose an optimized treatment.     

Evolving Concepts in the Pathogenesis of NASH: Beyond Steatosis and Inflammation

Non-alcoholic steatohepatitis (NASH) is characterised by hepatic steatosis and inflammation and, in some patients, progressive fibrosis leading to cirrhosis. An understanding of the pathogenesis of NASH is still evolving but current evidence suggests multiple metabolic factors critically disrupt homeostasis and induce an inflammatory cascade and ensuing fibrosis. The mechanisms underlying these changes and the complex inter-cellular interactions that mediate fibrogenesis are yet to be fully elucidated. Lipotoxicity, in the setting of excess free fatty acids, obesity, and insulin resistance, appears to be the central driver of cellular injury via oxidative stress. Hepatocyte apoptosis and/or senescence contribute to activation of the inflammasome via a variety of intra- and inter-cellular signalling mechanisms leading to fibrosis. Current evidence suggests that periportal components, including the ductular reaction and expansion of the hepatic progenitor cell compartment, may be involved and that the Th17 response may mediate disease progression. This review aims to provide an overview of the pathogenesis of NASH and summarises the evidence pertaining to key mechanisms implicated in the transition from steatosis and inflammation to fibrosis. Currently there are limited treatments for NASH although an increasing understanding of its pathogenesis will likely improve the development and use of interventions in the future.