Acute-on-chronic liver failure (ACLF), a syndrome observed among patients with decompensated liver cirrhosis is characterised by an intense systemic inflammatory response, multiple organ system failure and high 28-day mortality. The loss of kidney function is the most common extra-hepatic organ failure; acute kidney injury (AKI) being reported in up to 92% of patients with ACLF (Napoleone et al 2022). The intense systemic inflammation occurring in ACLF, triggered by bacteria or endotoxins such as lipopolysaccharide (LPS) due to gut leakage, has been shown to be involved in the pathogenesis of organ dysfunction and/or failure. The FDA approved antiparasitic drug nitazoxanide (NTZ) presents hepatoprotective and anti-inflammatory properties, making it a promising therapeutic approach for the treatment of ACLF. Our aim was to assess the efficacy of NTZ to alleviate kidney injury and inflammation and restore renal function in preclinical models of ACLF. ACLF was triggered through an acute injection of LPS in rats with advanced liver injury induced by either bile duct ligation (BDL) or carbon tetrachloride (CCl4). NTZ (100 mg/kg) or vehicle were orally administered concomitantly or one hour before LPS injection. Serum and tissues were collected 3 or 24 hours post-LPS in BDL and CCl4 rats, respectively. To evaluate renal function, serum levels of cystatin C, creatinine and urea were measured. Renal transcriptome was assessed using RNA sequencing and pairwise comparisons were performed to identify differentially expressed genes. In the CCl4 + LPS-induced ACLF model, NTZ alleviated hepatocellular injury by reducing LPS-induced rise of AST (−100%, p = 0.03) and GGT (−99%, p = 0.014) at 24 h post-LPS. Compared to fibrotic rats (CCl4 alone), CCl4+LPS-induced ACLF rats presented alterations of kidney function as shown by elevated levels of serum creatinine (113 µM vs 50 µM) and serum urea (127 mg/dL vs 47 mg/dL), which were reduced upon NTZ administration (−99% creatinine, p = 0.03 and −96% urea, p = 0.0005). LPS injection in rats with BDL-induced liver fibrosis led to systemic inflammation as shown by increased serum pro-inflammatory cytokines levels at 3 h post-LPS, which were significantly reduced upon NTZ treatment: −99% for IL6 (p = 0.001) and −94% for TNF alpha (p = 0.006). Compared to BDL alone rats, BDL+LPS-induced ACLF rats also presented renal dysfunction as shown by elevated levels of cystatin-C (4.6 µg/mL vs 2 µg/mL, Fig. A). This renal impairment was associated with a significant remodeling of the renal transcriptome (Fig. B), with differentially expressed genes mainly related to innate immune response and pro-inflammatory signaling pathways. Treatment of rats with NTZ restored the LPS-induced level of cystatin C by 85% (p = 0.004) and the inflammatory gene signature in the kidney (Figure), with expression level of genes such as lipocalin 2 (LCN2), a marker of renal injury, being reduced. Interestingly, serum and renal levels of Ripk3, a marker of necroptotic cell death, was also alleviated by NTZ treatment. A single oral administration of NTZ rapidly alleviates kidney inflammation and injury and prevents alteration of renal function in pre-clinical models of ACLF. By protecting the major extrahepatic organ failing in ACLF, in addition to improvement of other organ functions, these findings further support the investigation of NTZ therapy as a promising approach for ACLF treatment.

Background & Aims: Robust performance of non-invasive tests (NITs) across ages is critical to assess liver disease among patients with metabolic dysfunction-associated liver disease (MASLD). We evaluated the impact of age on the performance of NIS2+(TM) vs. other NITs. Methods: An analysis cohort (N = 1,926) with biopsy-proven MASLD was selected among individuals screened for the phase III RESOLVE-IT clinical trial and divided into <= 45, 46-55, 56-64, and >= 65 years groups. To avoid potential confounding effects, a well-balanced cohort (n = 708; n = 177/age group) was obtained by applying a propensity score-matching algorithm to the analysis cohort. Baseline values of biomarkers and NITs were compared across age groups using one-way ANOVA, and the impact of age and histology were compared through three-way ANOVA. The impact of age on NIT performance for the detection of at-risk metabolic dysfunction-associated steatohepatitis (MASH; MASLD activity score [MAS] >= 4 and fibrosis stage [F] >= 2) was also evaluated. Results: Age did not affect the distributions of NIS2+(TM) and APRI (aspartate aminotransferase-to-platelet ratio index), but significantly (p <0.0001) impacted those of NFS (NAFLD fibrosis score), FIB-4 (Fibrosis-4 index), and Enhanced Liver Fibrosis (ELF (TM)) score. NIS2+(TM) was the only NIT on which fibrosis and MAS exerted a moderate to large effect. While the impact of fibrosis on APRI was moderate, that of MAS was low. The impact of age on FIB-4 and NFS was larger than that of fibrosis. NIS2+(TM) exhibited the highest AUROC values for detecting at-risk MASH across age groups, with stable performances irrespective of cut-offs. Conclusions: NIS2+(TM) was not significantly impacted by age and was sensitive to both fibrosis and MAS grade, demonstrating a robust performance to rule in/out at-risk MASH with fixed cut-offs. Impact and Implications: While metabolic dysfunction-associated steatotic liver disease (MASLD) can affect individuals of all ages, patient age could represent an important confounding factor when interpreting non-invasive test (NIT) results, highlighting the need for reliable and efficient NITs that are not impacted by age and that could be interpreted with fixed cut-offs, irrespective of patient age. We report the impact of age on different well-established NITs - among those tested, only two panels, NIS2+(TM) and APRI, were not impacted by age and can be used and interpreted independently of patient age. NIS2+(TM) was also sensitive to both fibrosis and MAS, further confirming its efficiency for the detection of the composite endpoint of at-risk MASH and its potential as a valuable candidate for large-scale implementation in clinical practice and clinical trials. (c) 2024 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY -NC -ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).