INTRODUCTION:Podocyte injury and mesangial activation are hallmarks of a wide variety of glomerular diseases. Although podocyte damage is thought to be an early event that causes subsequent mesangial activation, the mechanism connecting these two events remains poorly understood. Since the protein fibrillin-1 (FBN1) may be involved in glomerular injury in several kidney diseases, we tested whether FBN1 expression in injured podocytes trigger mesangial cell activation and proliferation. METHODS:FBN1 expression was assessed in various models of glomerular diseases. Mice with podocyte-specific knockout of FBN1 were generated to evaluate the role of FBN1 in glomerular disease. The interaction between FBN1 and epidermal growth factor receptor (EGFR) was studied by molecular docking, cellular thermal shift assay, and co-immunoprecipitation. RESULTS:Podocyte FBN1 was upregulated in various animal models of glomerular diseases and in patients with proteinuric kidney disorders. FBN1 stimulated mesangial cell activation and proliferation both in vitro and in glomerular mini-organ cultures. Overexpression of FBN1 promoted mesangial cell activation and aggravated glomerulosclerosis in Adriamycin (ADR) nephropathy. Conversely, podocyte-specific knockout of the FBN1 gene attenuated mesangial activation and glomerulosclerosis in mice with ADR nephropathy or streptozotocin-induced diabetes. RNA sequencing, molecular docking simulation and protein interactions analyses demonstrated that FBN1 could directly bind to EGFR in mesangial cells, which resulted in a sequential phosphorylation of EGFR, phosphoinositide 3-kinase (PI3K) and AKT kinase. Compared to EGF, FBN1 triggered a sustained activation of EGFR, PI3K and AKT in comparable doses, but not extracellular signal-regulated kinase-1 and -2. Pharmacologic inhibition of EGFR, PI3K or AKT abolished FBN1-induced mesangial cell activation in vitro. Furthermore, inhibition of EGFR activation by erlotinib ameliorated FBN1-induced mesangial activation and glomerulosclerosis in vivo. CONCLUSIONS:These findings illustrate that extracellular matrix protein FBN1 is a previously unrecognized ligand for EGFR and induces a sustained activation of EGFR/PI3K/AKT signaling, which leads to mesangial cell activation and glomerulosclerosis.

BACKGRUOUND:Diabetic kidney disease (DKD) often progresses to end-stage renal disease more rapidly than nondiabetic kidney disease because of persistent hyperglycemia and early activation of multiple pathogenic pathways. Early detection of DKD is crucial for identifying subtle kidney damage before clinical symptoms appear. METHODS:This study combined human serum proteomics with public single-cell RNA sequencing and spatial transcriptomics data from diabetic kidneys to identify key biomarkers for DKD diagnosis. These biomarkers were validated in multiple organs of db/db mice at early and advanced stages. In a discovery cohort, sera from 173 healthy adults and 444 patients with type 2 diabetes mellitus (T2DM), with or without kidney disease, were analyzed using metabolomics and enzyme-linked immunosorbent assay (ELISA). Multiple machine learning algorithms were developed to integrate synergistic biomarkers and serum metabolites for early DKD detection, with results validated in 435 participants from four independent clinical cohorts. RESULTS:Metalloproteinase-7 (MMP-7) and tenascin C (TNC) were elevated in human diabetic kidneys at the single-cell and spatial levels. Proteomics indicated upregulation of serum amyloid A1 (SAA1) and TNC in the serum of patients with DKD. In db/db mice, all three biomarkers increased in multiple organs by 18 weeks of age. In sera from patients with DKD, MMP-7 and TNC levels were consistently elevated across cohorts. The new algorithms combining MMP-7, SAA1, and TNC enhanced early-stage DKD detection, with approximately 13% improvements in accuracy when serum metabolites were included to distinguish progression from early to advanced DKD stages. CONCLUSION:Integrating synergistic biomarkers with serum metabolomics enhances the early detection of DKD, potentially improving outcomes by slowing disease progression in patients with T2DM.

The fibrotic kidney microenvironment is shaped by cellular crosstalk, extracellular matrix (ECM) remodelling, metabolic reprogramming and spatial heterogeneity. While late-stage ECM changes dominate fibrosis, the role of early-activated matrix proteins remains unclear. Here we show that ECM1 is an early regulator of kidney remodelling. Global Ecm1 knockout mice develop spontaneous fibrosis and early death, whereas ECM1 levels markedly increase in biofluids during chronic kidney disease. Targeting Ecm1 through AAV9-mediated knockdown or fibroblast-specific deletion substantially reduces renal fibrosis. Mechanistically, Ecm1 deletion disrupts the integrin α2β1-RhoC axis, suppressing YAP activity. Reduced YAP nuclear translocation and diminished YAP-TEAD4 complex formation relieve TEAD4-mediated repression of Pgc1a, enhancing mitochondrial oxidative phosphorylation (OXPHOS) and promoting repair. Spatial transcriptomics and proteomics confirm this mechano-metabolic pathway, revealing mitochondrial reprogramming in tubules that counteracts fibrotic progression. Notably, YAP inactivation in fibroblasts limits aberrant activation without impairing their OXPHOS. This selective ECM-mitochondrial crosstalk uncovers a mechano-metabolic pathway in which mitochondrial shifts drive defence against kidney fibrosis.

Cisplatin is a commonly used chemotherapy agent for treating various solid tumors, but its clinical application is limited by nephrotoxicity. While the potential for cisplatin to cause chronic kidney disease (CKD) following repeated administration has been underexplored, effective therapeutic strategies for cisplatin-induced CKD are lacking. We found that cisplatin-induced CKD is characterized by renal dysfunction and inflammation, along with intestinal barrier impairment. 16S rRNA and metabolomics revealed that cisplatin disrupts the gut microbiome and raises levels of tryptophan metabolites- Indole-3-propionic acid (IPA). Notably, oral administration of IPA reproduced similar harmful effects in cisplatin-induced CKD. Integrated analyses of the microbiome, metabolomics, Raman spectroscopy and DESI-MSI indicated that IPA supplementation exacerbates the production of uremic toxins linked to tryptophan metabolism and promotes the growth of pathogenic bacteria. Our findings demonstrates that IPA exacerbates renal inflammation and fibrosis by regulating AHR/NF-κB signaling pathways, altering intestinal microbiome composition, and disrupting tryptophan metabolism.

Kidney fibrosis is the common outcome of chronic kidney disease (CKD). It often instigates in the focal sites by forming the fibrogenic niche after injury. In this study, using single-cell RNA sequencing (scRNA-seq) and a spatial transcriptomic (ST) approach, the cellular heterogeneity, spatial organization, and molecular interactions are delineated in the fibrotic kidney. Through analyses of the scRNA-seq and ST data from normal and fibrotic kidneys in mice subjected to unilateral ischemia-reperfusion injury, a tenascin C (TNC)-enriched, proinflammatory, and profibrotic microenvironment is identified that facilitated macrophage activation and promoted renal inflammation and fibrosis. Both TNC-enriched decellularized kidney tissue scaffold and exogenous TNC protein promoted bone marrow-derived macrophages activation though Toll-like receptor 4 (TLR4)/NF-κB signaling. Either pharmacological inhibition of TLR4 signaling or genetic knockout of its gene alleviated renal inflammation and fibrosis by inhibiting macrophage activation in vivo. Finally, chimeric mice that received bone marrow transplantation from TLR4-deficient donors are protected against kidney inflammation and fibrosis. These results suggest that TNC plays a crucial role in orchestrating the formation of a proinflammatory and profibrotic niche that promotes renal inflammation and fibrosis by activating macrophages via TLR4/NF-κB signaling. The findings underscore the complex interplay among fibroblasts, extracellular microenvironment, and macrophages that drive kidney fibrosis.

Wnt/β-catenin signaling plays a pivotal role in the pathogenesis of chronic kidney diseases (CKD), which is associated with macrophage activation and polarization. However, the relative contribution of macrophage-derived Wnts in the evolution of CKD is poorly understood. Here we demonstrate a critical role of Wnts secreted by macrophages in regulating renal inflammation and fibrosis after various injuries. In mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO), macrophages were activated and polarized to M1 and M2 subtypes, which coincided with the activation of Wnt/β-catenin signaling. In vitro, multiple Wnts were induced in primary cultured bone marrow-derived macrophages (BMDMs) after polarization. Conversely, Wnt proteins also stimulated the activation and polarization of BMDMs to M1 and M2 subtype. Blockade of Wnt secretion from macrophages in mice with myeloid-specific ablation of Wntless (Wls), a cargo receptor that is obligatory for Wnt trafficking and secretion, blunted macrophage infiltration and activation and inhibited the expression of inflammatory cytokines. Inhibition of Wnt secretion by macrophages also abolished β-catenin activation in tubular epithelium, repressed myofibroblast activation and reduced kidney fibrosis after either obstructive or ischemic injury. Furthermore, conditioned medium from Wls-deficient BMDMs exhibited less potency to stimulate fibroblast proliferation and activation, compared to the controls. These results underscore an indispensable role of macrophage-derived Wnts in promoting renal inflammation, fibroblasts activation and kidney fibrosis.

N6-methyladenosine (m6A) methylation plays a crucial role in various biological processes and the pathogenesis of human diseases. However, its role and mechanism in kidney fibrosis remain elusive. In this study, we show that the overall level of m6A methylated RNA was upregulated and the m6A methyltransferase METTL3 was induced in kidney tubular epithelial cells in mouse models and human kidney biopsies of chronic kidney disease (CKD). Proximal tubule-specific knockout of METTL3 in mice protected kidneys against developing fibrotic lesions after injury. Conversely, overexpression of METTL3 aggravated kidney fibrosis in vivo. Through bioinformatics analysis and experimental validation, we identified β-catenin mRNA as a major target of METTL3-mediated m6A modification, which could be recognized by a specific m6A reader, the insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3). METTL3 stabilized β-catenin mRNA, increased β-catenin protein and induced its downstream profibrotic genes, whereas either knockdown of IGF2BP3 or inhibiting β-catenin signaling abolished its effects. Collectively, these results indicate that METTL3 promotes kidney fibrosis by stimulating the m6A modification of β-catenin mRNA, leading to its stabilization and its downstream profibrotic genes expression. Our findings suggest that targeting METTL3/IGF2BP3/β-catenin pathway may be a novel strategy for the treatment of fibrotic CKD.

Alcohol abuse is one of the major public health problems in the world and is associated with various health conditions. However, little is known about the effect of alcohol consumption on acute kidney injury (AKI). In this study, we demonstrate that chronic and binge alcohol feeding with a Lieber-DeCarli diet containing 5 % ethanol for 10 days, followed by a single dose of 31.5 % ethanol by gavage, aggravated AKI after ischemia-reperfusion injury (IRI) in female, but not in male, mice. Kidney dysfunction, histopathology and tubular cell apoptosis were more severe in EtOH-fed female mice after IRI, compared to pair-fed controls. RNA sequencing and experimental validation uncovered that activation of integrin β1 and its downstream c-Jun NH2-terminal kinase (JNK) aggravated AKI in EtOH-fed mice. Knockdown of integrin β1 inhibited JNK phosphorylation and alleviated AKI in EtOH-fed mice, whereas activation of integrin β1 by agonist antibody increased JNK phosphorylation, worsened renal histological injury and tubular cell apoptosis, and aggravated kidney dysfunction. In vitro, activation of integrin β1 increased JNK phosphorylation and induced tubular epithelial cell apoptosis. The detrimental effect of EtOH feeding was primarily mediated by acetaldehyde, as its levels were increased in the blood, liver and kidney of female mice fed with ethanol. Acetaldehyde per se activated integrin β1/JNK signaling and induced tubular cell apoptosis in vitro. These findings suggest that alcohol consumption increases vulnerability to AKI in female mice, which is probably mediated by acetaldehyde/integrin β1/JNK signaling cascade.

The smoothened (Smo) receptor facilitates hedgehog signaling between kidney fibroblasts and tubules during acute kidney injury (AKI). Tubule-derived hedgehog is protective in AKI, but the role of fibroblast-selective Smo is unclear. Here, we report that Smo-specific ablation in fibroblasts reduced tubular cell apoptosis and inflammation, enhanced perivascular mesenchymal cell activities, and preserved kidney function after AKI. Global proteomics of these kidneys identified extracellular matrix proteins, and nidogen-1 glycoprotein in particular, as key response markers to AKI. Intriguingly, Smo was bound to nidogen-1 in cells, suggesting that loss of Smo could affect nidogen-1 accessibility. Phosphoproteomics revealed that the ‘AKI protector’ Wnt signaling pathway was activated in these kidneys. Mechanistically, nidogen-1 interacted with integrin β1 to induce Wnt in tubules to mitigate AKI. Altogether, our results support that fibroblast-selective Smo dictates AKI fate through cell-matrix interactions, including nidogen-1, and offers a robust resource and path to further dissect AKI pathogenesis.

Acute kidney injury (AKI) is a refractory clinical syndrome with limited effective treatments. Amid AKI, activation of the extracellular signal-regulated kinase (ERK) cascade plays a critical role in promoting kidney repair and regeneration. However, a mature ERK agonist in treating kidney disease remains lacking. This study identified limonin, a member of the class of compounds known as furanolactones, as a natural ERK2 activator. Employing a multidisciplinary approach, we systemically dissected how limonin mitigates AKI. Compared to vehicles, pretreatment of limonin significantly preserved kidney functions after ischemic AKI. We revealed that ERK2 is a significant protein linked to the limonin's active binding sites through structural analysis. The molecular docking study showed a high binding affinity between limonin and ERK2, which was confirmed by the cellular thermal shift assay and microscale thermophoresis. Mechanistically, we further validated that limonin promoted tubular cell proliferation and reduced cell apoptosis after AKI by activating ERK signaling pathway in vivo. In vitro and ex vivo, blockade of ERK abolished limonin's capacity of preventing tubular cell death under hypoxia stress. Our results indicated that limonin is a novel ERK2 activator with strong translational potential in preventing or mitigating AKI.

BACKGROUND:Acute liver injury (ALI) is a common side effect of cisplatin treatment in the clinic and can lead to liver failure if not treated promptly. Previous studies have revealed that Limonin, a critical bioactive substance in citrus fruits, can protect multiple organs from various medical conditions. However, whether Limonin could ameliorate cisplatin-induced ALI remains unclear.METHODS:In vivo and in vitro models were induced by cisplatin in the present study. Non-targeted metabolomics was employed to analyze the metabolic changes in the liver after ALI. In addition, molecular docking was utilized to predict the potential targets of Limonin.RESULTS:Limonin attenuated hepatic histopathological injury by reducing hepatocyte apoptosis, lipid peroxidation, and inflammation in cisplatin-challenged mice. Employing metabolomics, we revealed that Limonin mediated the balance of various disturbed metabolic pathways in the liver after cisplatin-induced ALI. Integrating public data mining, molecular docking studies, and in vitro experiments demonstrated that Limonin suppressed the expression and activity of its direct target, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), in the liver, thus reducing the production of corticosterone (CORT), a key metabolite promoted hepatocyte apoptosis.CONCLUSIONS:Limonin improves the liver metabolic microenvironment by inhibiting 11β-HSD1 to protect against cisplatin-induced ALI.

Wnt/β-catenin is a developmental signaling pathway that plays a crucial role in driving kidney fibrosis after injury. Activation of β-catenin is presumed to be regulated through the posttranslational protein modification. Little is known about whether β-catenin is also subjected to regulation at the posttranscriptional mRNA level. Here, we report that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) plays a pivotal role in regulating β-catenin. IGF2BP3 was upregulated in renal tubular epithelium of various animal models and patients with chronic kidney disease. IGF2BP3 not only was a direct downstream target of Wnt/β-catenin but also was obligatory for transducing Wnt signal. In vitro, overexpression of IGF2BP3 in kidney tubular cells induced fibrotic responses, whereas knockdown of endogenous IGF2BP3 prevented the expression of injury and fibrosis markers in tubular cells after Wnt3a stimulation. In vivo, exogenous IGF2BP3 promoted β-catenin activation and aggravated kidney fibrosis, while knockdown of IGF2BP3 ameliorated renal fibrotic lesions after obstructive injury. RNA immunoprecipitation and mRNA stability assays revealed that IGF2BP3 directly bound to β-catenin mRNA and stabilized it against degradation. Furthermore, knockdown of IGF2BP3 in tubular cells accelerated β-catenin mRNA degradation in vitro. These studies demonstrate that IGF2BP3 promotes β-catenin signaling and drives kidney fibrosis, which may be mediated through stabilizing β-catenin mRNA. Our findings uncover a previously underappreciated dimension of the complex regulation of Wnt/β-catenin signaling and suggest a potential target for therapeutic intervention of fibrotic kidney diseases.

Background: Renal infiltration of inflammatory cells including macrophages is a crucial event in kidney fibrogenesis. However, how macrophage regulates fibroblast activation in the fibrotic kidney remains elusive. In this study, we show that macrophages promoted fibroblast activation by assembling a vitronectin (Vtn)-enriched, extracellular microenvironment. Methods: We prepared decellularized kidney tissue scaffold (KTS) from normal and fibrotic kidney after unilateral ischemia-reperfusion injury (UIRI) and carried out an unbiased quantitative proteomics analysis. NRK-49F cells were seeded on macrophage-derived extracellular matrix (ECM) scaffold. Genetic Vtn knockout (Vtn-/-) mice and chronic kidney disease (CKD) model with overexpression of Vtn were used to corroborate a role of Vtn/integrin αvβ5/Src in kidney fibrosis. Results: Vtn was identified as one of the most upregulated proteins in the decellularized kidney tissue scaffold from fibrotic kidney by mass spectrometry. Furthermore, Vtn was upregulated in the kidney of mouse models of CKD and primarily expressed and secreted by activated macrophages. Urinary Vtn levels were elevated in CKD patients and inversely correlated with kidney function. Genetic ablation or knockdown of Vtn protected mice from developing kidney fibrosis after injury. Conversely, overexpression of Vtn exacerbated renal fibrotic lesions and aggravated renal insufficiency. We found that macrophage-derived, Vtn-enriched extracellular matrix scaffold promoted fibroblast activation and proliferation. In vitro, Vtn triggered fibroblast activation by stimulating integrin αvβ5 and Src kinase signaling. Either blockade of αvβ5 with neutralizing antibody or pharmacological inhibition of Src by Saracatinib abolished Vtn-induced fibroblast activation. Moreover, Saracatinib dose-dependently ameliorated Vtn-induced kidney fibrosis in vivo. These results demonstrate that macrophage induces fibroblast activation by assembling a Vtn-enriched extracellular microenvironment, which triggers integrin αvβ5 and Src kinase signaling. Conclusion: Our findings uncover a novel mechanism by which macrophages contribute to kidney fibrosis via assembling a Vtn-enriched extracellular niche and suggest that disrupting fibrogenic microenvironment could be a therapeutic strategy for fibrotic CKD.