Journal of Clinical Investigation

The loss of lysosomal cystine transporter cystinosin (CTNS) disrupts kidney proximal tubule (PT) function, causing cystinosis - a prototypical lysosomal storage disorder characterized by cystine accumulation and metabolic dyshomeostasis. Cystine storage disrupts lysosomal nutrient sensing and downstream mTORC1 signalling, driving loss of PT differentiation and proximal tubulopathy. Here, using cross-species disease models, differentiated cellular systems, lysosome-based assays, and transcriptomics profiling, we demonstrate that sodium-glucose co-transporter 2 (SGLT2) inhibitors (empagliflozin or dapagliflozin) ameliorate proximal tubulopathy in cystinosis. In CTNS-deficient PT cells, SGLT2 inhibition restores lysosome proteolysis, autophagic flux, metabolic homeostasis, and epithelial differentiation and function, independently of cystine clearance. Mechanistically, SGLT2 inhibition reduces the assembly of the v-ATPase-Ragulator-Rag scaffolding complex at lysosomes, thereby decoupling cystine storage from pathological mTORC1 activation. These effects reprogram PT metabolic trajectories and differentiation states, mitigating proximal tubulopathy across zebrafish and rodent models of cystinosis. Together, these findings define a lysosome-metabolism crosstalk that links glucose handling to mTORC1 regulation and provide a rationale for repurposing SGLT2 inhibitors as a disease-modifying therapy for cystinosis and related lysosome-driven PT disorders.

Cystinuria is a rare inherited disease characterized by increased urinary cystine levels resulting in the formation of cystine stones in the urinary tract. Mutations in the genes encoding the cystine transporter complex, SLC3A1 and SLC7A9, are the primary drivers of the disease. Current mouse models used to study cystinuria rely on gene deficiency or spontaneous mutations in mice that do not accurately reflect the pathogenic mutations found in humans. To overcome this limitation, we generated novel Slc7a9G105R mice carrying the most common pathogenic single-point mutation in the SLC7A9 gene. Both male and female Slc7a9G105R mice developed a cystinuria phenotype by 9 weeks of age, characterized by substantial cystine stone formation and increased urinary cystine, lysine, arginine, and ornithine. Slc7a9G105R mice displayed distinct serum and urinary metabolite profiles mapped to dibasic amino acid pathways. Fecal metagenomics revealed that Slc7a9G105R mice had a heterogeneous microbiota with altered functional pathways, including increased L-cysteine biosynthesis. Depletion of the microbiota with antibiotics did not impact cystine stone burden but reduced urinary tract inflammation. Prophylactic or therapeutic dietary supplementation with alpha-lipoic acid reduced stone burden and inflammation, but it also caused damage to the urothelium. Untargeted metabolomics analysis following alpha-lipoic acid supplementation identified metabolites that can increase cystine solubility, reduce inflammation, and damage epithelial cells. Correlation analysis revealed novel serum biomarkers of stone burden, including blood urea nitrogen, 2-hydroxybutyric acid, 2-amino-2-thiazoline-4-carboxylic acid, and indole-3-acetylglycine. Collectively, the Slc7a9G105R mutant mouse model offers a precise, rapid-onset, and translational platform for investigating cystinuria pathogenesis and evaluating potential therapeutic strategies. Translational statementThe development of a novel knock-in mouse model carrying the most common pathogenic point mutation in the human SLC7A9 gene provides a clinically relevant and translationally valuable platform for investigating cystinuria pathogenesis and testing emerging therapies. This model represents the closest possible approximation of human SLC7A9-mediated cystinuria, enabling rigorous preclinical evaluation of small molecules and gene therapies. It has also facilitated the identification of candidate biomarkers for cystine stone burden and treatment response, which are urgently needed to improve disease monitoring and clinical decision-making. The next critical step is to validate these biomarkers in human cystinuria cohorts to support their clinical translation.

Chemotherapy-induced neuropathic pain (CINP) is a frequent and debilitating adverse effect of anti-tumor therapies, for which current treatments are largely non-specific and offer limited efficacy. Identifying molecular mechanisms that drive CINP may enable the development of targeted therapeutic strategies. Here, we demonstrate that paclitaxel-induced mechanical pain hypersensitivity in mice occurs independently of classical Nav1.8+ nociceptors but critically depends on TrkB+ sensory neurons. Transcriptomic analysis of TrkB+ sensory neurons revealed selective expression of Scn5a, which encodes the voltage-gated sodium channel Nav1.5, a channel classically associated with cardiac excitability. Importantly, SCN5A expression was also detected in human primary sensory neurons, indicating potential translational relevance. Functional studies further showed that Scn5a knockdown, using small interfering RNA, significantly attenuates paclitaxel-induced mechanical pain hypersensitivity. Together, these findings identify TrkB+ sensory neurons as key drivers of CINP and reveal Nav1.5 as a previously unrecognized contributor to chemotherapy-induced neuropathic pain. Targeting Nav1.5 in TrkB+ sensory neurons may therefore represent a novel therapeutic strategy for the treatment of CINP.

ObjectiveAutoimmune diseases (ADs) markedly elevate venous thromboembolism (VTE) risk, yet the shared genetic architecture and tissue-specific regulatory mechanisms of this "Autoimmune-Thrombotic Axis" remain poorly defined. We aimed to characterize the genomic landscape of immunothrombosis to identify causal links and therapeutic targets. Approach and ResultsWe integrated large-scale GWAS data for VTE and 16 ADs using a multi-omics framework, including pleiotropy scanning, local genetic correlation, and summary-based Mendelian randomization (SMR). We identified 21 Immunothrombotic Shared Loci (ISLs) and 274 pleiotropic genes enriched in complement and coagulation cascades. Mendelian randomization (MR) analysis revealed a robust causal effect of genetically predicted systemic lupus erythematosus (SLE) on VTE risk (OR = 1.018, 95% CI: 1.008-1.029, P = 0.0003). Mechanistically, IL6R and PLCL1 emerged as central mediators with distinct tissue-specific regulatory partitioning. Colocalization confirmed that shared genetic susceptibility is primarily driven by expression in arterial tissues (aorta and coronary) rather than exclusively in immune cells. Furthermore, the lead SNP rs4129267 was identified as a potential predictor for VTE in rheumatoid arthritis patients, and drug prioritization nominated TNF inhibitors as promising candidates for mitigating thrombotic burden. ConclusionThis study establishes the first genomic atlas of the autoimmune-thrombotic axis, demonstrating that vasculature-specific gene regulation drives immunothrombosis. These findings provide a biological basis for VTE risk stratification and suggest that genotype-guided therapy may optimize vascular outcomes in AD patients.

Severe hemolysis is a life-threatening condition with limited therapeutic options. Although haptoglobin and hemopexin sequester hemoglobin and heme, these protective systems are rapidly saturated during acute hemolysis, leading to the accumulation of cytotoxic free heme. In this study, we identify scavenger receptor BI (SR-BI) as a critical mediator of free heme clearance. SR-BI binds heme and facilitates its hepatic uptake under pathological conditions. Mice lacking hepatic SR-BI exhibit impaired heme clearance and increased susceptibility to heme- and hemolysis-induced lethality. Pharmacological upregulation of hepatic SR-BI via imatinib or adenoviral delivery confers protection against heme toxicity. Using a humanized model of sickle cell disease (SCD), we further demonstrate that sickle hepatopathy significantly reduces hepatic SR-BI expression compared to non-SCD littermates, potentially increasing vulnerability to heme-induced injury. Notably, adenoviral-mediated SR-BI upregulation rescues SCD mice from heme toxicity. These findings reveal a previously unrecognized mechanism of heme detoxification via hepatic SR-BI and identify a promising therapeutic target for hemolytic disorders. One-Sentence SummaryIdentification of scavenger receptor BI as a targetable scavenger of heme in hemolysis

Mixed granulocytic asthma (MGA) is a severe Th2-low endotype, characterized by high Th17/neutrophilic burden and exacerbated airway remodeling. Both features confer resistance to inhaled corticosteroids, and typical asthma treatments. Thus, MGA is an enormous public health burden. Gaps in knowledge include how Th17 cells induce pathological tissue remodeling, and how Th17 differentiation occurs in response to allergens. We generated a Th2-low murine model of asthma that recapitulates major features of human MGA namely, heightened airway reactivity to methacholine, Th17/neutrophilic inflammation, airway remodeling, and resistance to corticosteroid treatment. Two specific biomarkers enriched in human MGA, the TNF superfamily member 14 (aka LIGHT), and the mitochondrial oxidative phosphorylation (OXPHOS) pathway, are upregulated in this model. We show OXPHOS promotes the metabolic reprograming of Th17 cells, to produce LIGHT that controls airway remodeling. Mechanistically, OXPHOS regulates ROR{gamma}t expression and the subsequent transcriptional network to program survival and differentiation of Th17 cells, whereas LIGHT drives airway remodeling by activating the MMP9-dependent TGF{beta} pathway. Additionally, OXPHOS+Th17 cells promote the expression of osteopontin necessary for fibroblast activation. LIGHT antagonistic blockade reduces airway remodeling, whereas OXPHOS chemical inhibition reduces Th17 cells and neutrophilia. Importantly, the dual blockade of LIGHT and OXPHOS reverses all features of MGA and reciprocally increase the numbers of Treg cells. Thus, the dual blockade of LIGHT and OXPHOS constitutes a promising target for clinical interventions in human MGA, possibly extending to other Th17-driven fibrotic diseases.

Plant-based dietary strategies may offer a tractable approach to mitigating microbiome disruption and improving outcomes in patients undergoing autologous hematopoietic cell transplantation (auto-HCT) for multiple myeloma, a population in whom intestinal dysbiosis has been linked to infectious complications and inferior survival. We conducted a single-arm study to test the feasibility and biological activity of a high-fiber, plant-based, whole-food meal delivery intervention during the peri-transplant period. Adults with multiple myeloma (n = 22) received fully prepared, plant-based meals for 5 weeks spanning conditioning, neutropenia, and early recovery, with the goal of supporting consumption of nutrient-dense, high-fiber foods despite transplant-related symptoms that often limit oral intake. The primary endpoints were feasibility and tolerability, defined by successful enrollment, adherence to study procedures, and patient-reported intake of study meals; diet was quantified using prospective food diaries and 24-hour dietary recall surveys. Secondary endpoints included changes in gut microbiome composition and function assessed by shotgun metagenomic sequencing and stool short-chain fatty acid (SCFA) measurements. The intervention was feasible and generally well tolerated, with all participants consuming at least some proportion of delivered meals and with adherence sufficient to support planned dietary and correlative analyses. Greater intake of study meals was associated with more pronounced shifts in gut microbial communities, including enrichment of SCFA-producing taxa and compositional changes consistent with a fiber-responsive microbiome. Stool SCFA concentrations increased from baseline to the end of the intervention, suggesting a functional impact of the dietary strategy on microbial metabolite production during the peri-transplant period. These findings demonstrate that a plant-based meal delivery intervention is implementable during auto-HCT and suggest dose-dependent modulation of the gut microbiome and its metabolic output. Larger randomized trials are warranted to determine whether microbiome-targeted nutrition can reduce transplant-related toxicities, enhance immune recovery, and improve disease control in multiple myeloma. The trial is registered at ClinicalTrials.gov (NCT06559709).

Allogeneic hematopoietic stem cell transplantation (HSCT) is a life-saving treatment for hematologic malignancies, but long-term survivors present with lower muscle mass and functional capacity. In adult HSCT survivors 10-20 years after treatment, single nucleus RNA sequencing uncovered elevated XRRA1 expression levels in all muscle nuclei populations, which was retained in primary muscle stem cell cultures. HSCT survivors were characterized in vivo by impaired neuromuscular innervation that associated with muscle weakness, and lower muscle stem cell neurotrophic action. Despite these impairments, the molecular and physiological responses to heavy resistance training (HReT) were preserved in HSCT survivors, as demonstrated in a pre-registered clinical trial (ClinicalTrials.gov: NCT04922970). After 12 weeks of HReT, gains in muscle mass and strength were similar in HSCT survivors and healthy controls. In addition, we observed that [~]9% of muscle-resident immune cells persist into adulthood and that bone marrow derived cells do not adopt alternative cell fates in muscle tissue, resolving long-standing questions in human muscle biology. Together, these findings uncover molecular mechanisms of HSCT sequelae in muscle nuclei and muscle stem cells, which, importantly, can at least partly be overcome by mechanical loading. Given the growing population of HSCT survivors and the multitude of benefits of HReT for all organ systems, our findings support the importance of HReT in this population to promote healthspan.

Patients with advanced non-small cell lung cancer (NSCLC) and mutations in epidermal growth factor receptor (EGFR) benefit from EGFR tyrosine kinase inhibitors (TKIs). Osimertinib, a third-generation EGFR TKI, is standard first-line therapy for EGFR-mutated NSCLC, but most patients develop resistance to it. Here, we demonstrate that increased formation of primary cilia, microtubule-based sensory organelles, is associated with osimertinib-refractory NSCLC progression. EGFR-mutated, osimertinib-resistant human NSCLC cells had increased cilia formation and acetylation of -tubulin and reduced histone deacetylase 6 (HDAC6) activity compared to their osimertinib-sensitive counterparts. HDAC6 inhibition increases cilia formation in osimertinib-sensitive NSCLC cells, and overexpression of exogenous HDAC6 sensitized osimertinib-resistant NSCLC cells to osimertinibs anti-proliferative effects. Because intraflagellar transport (IFT) proteins are essential for primary cilium formation and function, we knocked down IFT88 in osimertinib-resistant NSCLC cells, which reversed osimertinib resistance in orthotopic and subcutaneous mouse models of lung cancer. Mechanistically, increased sodium influx during osimertinib-induced inhibition of EGFR signalling promotes cilia formation through sustained HDAC6 inactivity and greater -tubulin acetylation. Inhibition of sodium influx with dibutyryl-cAMP decreased cilium formation, increased sensitivity to osimertinib, and reduced tumor progression in mice bearing osimertinib-resistant lung tumors. Collectively, our findings suggest that enhanced primary cilium formation mediates EGFR TKI resistance and that targeted inhibition of ciliogenesis may prevent or overcome osimertinib resistance.

BackgroundPeripheral artery disease (PAD) and its severe form, chronic limb-threatening ischemia (CLTI), significantly impair blood flow to the lower extremities, affecting millions of adults globally. Intramuscular adipose tissue (IMAT) and fibrosis accumulation distinguish patients with CLTI from those with mild PAD, suggesting a role in CLTI pathobiology. However, the functional consequences of IMAT in CLTI remain unclear. MethodsWe compared gastrocnemius muscle samples from patients with PAD/CLTI, intermittent claudication, and non-PAD individuals. We analyzed bulk RNA sequencing, proteomic, lipidomic, and single-cell/nucleus RNA sequencing datasets. Additionally, we used murine models of hindlimb ischemia (HLI) with genetic manipulation of Ppar{gamma}, a key adipogenic transcription factor, specifically in fibro-adipogenic progenitor cells (FAPs), the cellular source of IMAT, to modulate IMAT formation and assessed the impact on limb function and pathology. ResultsPatients with CLTI exhibited significantly elevated expression of adipogenic genes and proteins in muscle specimens when compared to non-PAD controls. Murine models showed that increasing IMAT formation significantly worsened ischemic limb muscle strength and work output. In contrast, preventing IMAT formation significantly improved ischemic limb muscle strength and work output. These findings were consistent across both male and female mice, although females had greater tendency to form IMAT compared with male mice. ConclusionsIMAT accumulation is a key determinant of limb function in PAD/CLTI. Our studies demonstrate that targeting IMAT formation could improve limb function in mice with experimental PAD. Together, these findings suggest that developing strategies to limit or reduce IMAT may improve limb function and walking performance in patients with PAD/CLTI, providing a novel therapeutic avenue to address a critical unmet need. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIIntramuscular adipose tissue accumulation (IMAT) distinguishes patients with chronic limb-threatening ischemia from those with milder peripheral artery disease or those without PAD and directly impairs ischemic limb muscle function. C_LIO_LIGenetic gain- and loss-of-function mouse models demonstrate that increasing IMAT worsens, while preventing IMAT formation improves, ischemic limb strength and performance independent of perfusion. C_LIO_LIAdipogenic signatures in human calf muscle negatively correlates with muscle strength and disease severity, identifying IMAT as a functional biomarker and modifiable target in PAD/CLTI. C_LI What are the clinical implications?O_LIIMAT accumulation represents an underappreciated, non-vascular mechanism contributing to leg dysfunction in PAD/CLTI. C_LIO_LITherapies aimed at limiting or reversing IMAT formation may improve leg strength and walking performance in patients with PAD/CLTI, addressing a critical unmet clinical need. C_LIO_LIIdentifying and targeting cellular pathways regulating IMAT formation from fibro-adipogenic progenitors may complement vascular interventions to enhance functional recovery after revascularization. C_LI

Vincristine-induced peripheral neuropathy (VIPN) is a frequent and dose-limiting complication of cancer therapy, yet the upstream mechanisms coupling vascular activation to neuroinflammation remain poorly defined. Here we identify E-selectin as a critical orchestrator of vincristine-induced neuropathy. Systematic interrogation of endothelial adhesion molecules in a murine model of VIPN revealed that blockade of E-selectin, but not ICAM-1, PECAM-1 or P-selectin, completely prevented mechanical hypersensitivity and markedly reduced F4/80 immune cell accumulation in dorsal root ganglia and peripheral nerves. Genetic deletion of E-selectin conferred equivalent protection, despite the absence of structural loss of intraepidermal or myelinated fibres, indicating a predominantly functional neuroimmune pathology. Spatial transcriptomics demonstrated that vincristine induces a conserved stress and neuroinflammation-associated transcriptional programme in dorsal root ganglia, with immune and stromal populations acting as dominant signalling hubs. Genetic or pharmacological perturbation of E-selectin did not abolish injury-associated pathways but redistributed cell-cell communication networks, reducing immune-cell dominance and reshaping interferon and metabolic signalling states without inducing Sele expression. Mechanistically, E-selectin exerted non-canonical effects beyond endothelial adhesion. Local E-selectin administration was sufficient to induce macrophage-dependent mechanical hypersensitivity that was abolished in Fut4/7-deficient mice and following phagocyte depletion. In macrophages, E-selectin enhanced vincristine-driven NF-{kappa}B activation, NLRP3 inflammasome assembly and IL-1{beta} release. Together, these findings position E-selectin as an upstream regulator of IL-1{beta}-dependent neuroinflammation in VIPN and identify selective targeting of E-selectin-mediated immune-neuron interactions as a therapeutic strategy for chemotherapy-induced neuropathy.

Metabolic dysfunction does not necessarily correlate with adiposity. Metabolically healthy obese individuals and insulin-resistant lean individuals represent a fundamental paradox that implicates immune cell intrinsic mechanisms in the pathogenesis of type 2 diabetes. Here, we identify myeloid Gi signaling as a previously unrecognized determinant of whole-body glucose homeostasis. Single-cell transcriptomic analysis of adipose tissue macrophages from obese mice and humans reveals marked alteration in Gnai isoform, suggesting that myeloid Gi signaling is functionally engaged during metabolic disease. Using complementary myeloid-specific rodent models of Gi inhibition (pertussis toxin) and chemogenetic Gi activation (DREADD), we demonstrate that inhibition of Gi signaling improves glucose tolerance and enhances insulin sensitivity under both regular chow and high-fat diet conditions, independent of body weight and energy expenditure. Whereas acute Gi activation in lean mice modestly enhances glucose disposal, the same intervention during diet-induced obesity markedly impairs systemic glucose homeostasis, revealing context-dependent pathway function. Mechanistically, Gi inhibition amplifies macrophage cAMP-CREB signaling to drive IL-6 production, engaging STAT3- and AMPK-dependent pathways in adipose tissue and skeletal muscle to support insulin action. Conversely, Gi activation engages a previously uncharacterized G{beta}{gamma}-mTOR/AKT-JNK cascade, driving IL-1{beta} secretion that directly impairs insulin signaling in adipocytes and myotubes. Pharmacological IL-6 receptor blockade abolishes the metabolic benefits of Gi inhibition, whereas IL-1 receptor antagonism fully rescues Gi activation-induced metabolic dysfunction, establishing these cytokines as obligate downstream effectors. This signaling architecture is conserved in human macrophages, and ATAC-seq profiling reveals chromatin remodeling at cAMP-CREB and IL-6 regulatory pathway loci, consistent with the observed transcriptional reprogramming. Together, these findings establish myeloid Gi signaling as a weight-independent immunometabolic switch that couples opposing cytokine programs to systemic insulin sensitivity and identify this pathway as a therapeutic target in obesity-associated metabolic disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/713834v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@136b8faorg.highwire.dtl.DTLVardef@1aa654forg.highwire.dtl.DTLVardef@1e12f9forg.highwire.dtl.DTLVardef@fd7bf8_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

STUDY QUESTIONAre pathogenic variants in Homeodomain-interacting protein kinase (HIPK4) associated with sperm head abnormalities causing male infertility? SUMMARY ANSWERHIPK4 is a novel candidate gene associated with sperm head defects and human male infertility. WHAT IS KNOWN ALREADYNumerous genes causing male infertility due to Multiple Morphological Abnormalities of the sperm flagella (MMAF) have been described but the genetic basis of sperm head defects is less well understood. STUDY DESIGN, SIZE, DURATIONFour infertile brothers displaying varying degrees of quantitatively and/or qualitatively impaired spermatogenesis, their parents, and their fertile brother were included in the study. Further, the Male Reproductive Genomics (MERGE) cohort comprising exome/genome sequencing data of >3,300 men was queried. PARTICIPANTS/MATERIALS, SETTING, METHODSWe performed exome sequencing in all five brothers and their parents. To characterise the sperm phenotype, standard semen analysis, immunofluorescence staining, and transmission-electron microscopy (TEM) were carried out. Further, we evaluated the impact of the HIPK4 variant in cell culture experiments using HEK293T cells. MAIN RESULTS AND THE ROLE OF CHANCEAnalysing the exome data, we could not identify a common genetic cause in all four affected brothers. However, one of the affected brothers was compound heterozygous for two loss-of-function variants in DNAH17 (c.1076_1077dup p.(Lys360*) and c.7752+2T>A p.?) associated with markedly reduced sperm motility and MMAF. The variants pathogenicity was further validated by TEM of flagellar cross-sections revealing an outer dynein arm defect and axonemal disruption. On the contrary, his three infertile brothers were homozygous for the start-loss variant c.1A>G in HIPK4. This gene is expressed during spermiogenesis and is reportedly involved in sperm head shaping in mice. Heterologous expression of (partial) HIPK4 variant cDNA elucidated the alternative use of an in frame start codon located 35 amino acids downstream, resulting in an N-terminally truncated protein p.(Met1_Glu35del). The truncated HIPK4 protein lacks parts of its kinase domain and shows reduced protein stability. In line with published mouse models, all three brothers displayed 100% abnormal sperm head morphology with variable defects. Importantly, one brother affected by HIPK4 variants fathered a child after successful intracytoplasmic sperm injection demonstrating that it is a treatment option for HIPK4-related teratozoospermia. No further men from the MERGE cohort were affected by biallelic HIPK4 variants. Taken together, HIPK4 is an autosomal-recessive candidate gene associated with sperm head defects and male infertility. LARGE SCALE DATAThe reported variants in DNAH17 and HIPK4 were submitted to ClinVar. LIMITATIONS, REASONS FOR CAUTIONIndependent replication is required to assess the phenotypic spectrum and the reproductive outcome associated with biallelic HIPK4 variants and to formally establish the gene-disease relationship for male infertility. WIDER IMPLICATIONS OF THE FINDINGSThis study raises awareness of the significant genetic heterogeneity of male infertility. The described family highlights that distinct genetic causes may underlie a seemingly similar phenotype. Exome sequencing of families is helpful to efficiently disentangle individual causes among affected family members. STUDY FUNDING/COMPETING INTEREST(S)N.N., J.R., H.O., S.L., C.F., and F.T. were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Clinical Research Unit Male Germ Cells (CRU326, project number 329621271). R.T.W., N.N., J.R., H.O., and F.T. were supported by the Federal Ministry of Research, Technology and Space (BMFTR) as part of the project ReproTrack.MS (grant 01GR2303). S.A.K. was supported by the DFG Clinician Scientist programme CareerS Munster (project number 493624047). A.S.G. was supported by the Medical Faculty Munster via an Innovative Medical Research (IMF) grant (GA-122104).

Claudin-4 (CLDN4) is a key determinant of paracellular ion transport in the distal nephron, where it contributes to chloride permeability and transepithelial resistance. Although CLDN4 knockout mice exhibit hypercalciuria, the epithelial mechanism linking CLDN4 to calcium permeability and kidney stone disease remains unclear. We examined the molecular and functional effects of a kidney stone-associated CLDN4 variant P74L which was identified in two unrelated individuals with nephrolithiasis from the Bern Kidney Stone Registry. Using doxycycline-inducible epithelial cell models expressing human wild-type (WT) or mutant CLDN4, we show that the P74L variant displayed reduced protein stability, impaired junctional incorporation, and decreased surface expression. In contrast to WT CLDN4, whose overexpression increased transepithelial electrical resistance and restricted paracellular sodium, chloride, and calcium permeability, P74L CLDN4 failed to confer these effects. Expression of P74L CLDN4 was associated with reduced CLDN3 and CLDN7 messenger abundance without significant changes in CLDN8 or transcriptional regulation of other distal calcium (and other ion) transport genes. Together, these findings identify CLDN4 P74L as a loss-of-function variant that increases epithelial calcium permeability, possibly leading to increased calcium back-flux in the distal nephron relevant to nephrolithiasis.

People with chronic hyperglycemia are more susceptible to fungal skin infections, but the mechanisms underlying their worse clinical outcomes remain unclear. Using both in vivo and in vitro models, we explored how hyperglycemia influences skin antifungal defenses and how GLP1 agonists might restore host defense in diabetic conditions. Hyperglycemic mice showed increased susceptibility to Candida albicans skin infections, with larger lesions and higher fungal loads at all time points tested. Histology revealed larger abscesses, more extensive myeloid cell infiltration, and poorer control of fungal invasion, associated with increased chemoattractant production on day 1 post-infection. Despite heightened inflammatory responses, macrophages and keratinocytes exposed to high glucose exhibit markedly impaired fungal ingestion. RNAseq analysis of C. albicans-infected dermal macrophages cultured in high glucose showed enrichment of genes related to antimicrobial effectors and the C-type lectin receptor pathway, including Clec7a (Dectin-1), while suppressing downstream signaling pathways required for effective phagocytosis. Pharmacologic blockade or genetic deletion of Dectin-1 restored fungal uptake under high-glucose conditions and improved host defense in vivo. Mechanistically, Dectin-1 signaling in hyperglycemia promoted increased prostaglandin E2 (PGE2) production via induction of microsomal Prostaglandin E Synthase-1 (mPGES-1), and inhibition of PGE2 synthesis rescued deficient phagocytic function. Finally, treatment with the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide reduced lesion size, fungal burden, inflammation, and tissue damage in diabetic mice, linking metabolic control to restoration of innate immune function. These findings identify maladaptive innate immune sensing as a key mechanism underlying susceptibility to fungal infection in diabetes and reveal how metabolic stress converts antifungal recognition pathways into drivers of inflammatory dysfunction.

Metabolic-dysfunction associated steatotic liver disease (MASLD) is the most common chronic liver disease. Fracture risk is increased among people with MASLD, however, the genetic contribution to risk is undetermined. PNPLA3I148M is a common SNP which accounts for most MASLD heritability and increases MASLD morbidity and mortality. However, PNPLA3I148M impact on bone is unexplored. To bridge this gap, we used a validated murine model of MASLD (DIAMOND mice) which received human PNPLA3 transgenes via adeno-associated vector serotype 8 (AAV8) and assessed bone morphology, cellularity, and transcriptomics. PNPLA3I148M was expressed in bone and associated with bone loss, decreased bone formation, increased bone resorption, and increased bone marrow adiposity. PNPLA3I148M reprogrammed the transcriptome in bone, enriching expression of pathways associated with fatty acid metabolism and hampering bone turnover. Notably, these findings occurred in the absence of MASLD. These findings suggest PNPLA3I148M possesses an intrinsic deleterious skeletal role.

The glomerulus, a unique capillary network in the nephron, filters an entire blood volume approximately 300 times a day. Specialized epithelial cells known as podocytes form a critical component of the glomerular filtration barrier, and diseases linked to podocyte injury include minimal change disease, focal segmental glomerulosclerosis, and diabetic kidney disease. Because podocytes are terminally differentiated, their ability to respond to external stress is critical. The unfolded protein response (UPR), a cellular stress pathway, is associated with glomerular injury, although the role of the UPR in glomerular injury is undefined. The UPR is initially protective, leading to upregulation of molecular chaperones, a class of proteins that promote protein folding and are required to survive oxidative and ischemic injury. An unresolved UPR, however, leads to apoptosis. We previously found that one molecular chaperone, GRP170, provides protection against acute kidney injury since GRP170 depletion led to UPR induction and widespread kidney injury. Here we generated a new podocyte specific GRP170 knock out mouse (GRP170Pd-/-). Surprisingly, GRP170Pd-/- mice were born healthy, and podocyte development appeared normal. Within a month, however, the knockout mice exhibited profound glomerular injury manifesting as proteinuria, hypoalbuminemia, hyperlipidemia, and kidney injury. Concomitant with glomerular injury, we observed increased expression of the pro-apoptotic UPR target, CHOP, in podocytes. Together, our new model not only defines a protective role for GRP170 against glomerular injury but also provides a new model to test the therapeutic potential of small molecule UPR modulators to treat glomerular injury.

Rhabdomyolysis is the acute breakdown of skeletal muscle resulting from failure of cellular homeostasis in response to metabolic stress. Recurrent forms are frequently linked to inherited defects affecting energy metabolism or calcium handling. Ryanodine receptor type 3 (RyR3) is an intracellular calcium release channel, expressed in skeletal muscle, that contributes to the fine-tuning of calcium signaling. Although variants in other calcium-handling proteins have been implicated in rhabdomyolysis, the role of RyR3 has not been established. In this study, we report rare compound heterozygous missense variants in RYR3 identified in two unrelated individuals with severe, fever-triggered recurrent rhabdomyolysis. Muscle biopsies revealed mild structural changes with triadic disorganization, mitochondrial alterations, lipid accumulation, and autophagic material, while overall muscle architecture was largely preserved. Structural modeling supports the pathogenicity of the variants, and calcium flux analysis demonstrated significantly reduced ryanodine receptor-mediated calcium release in patient-derived myoblasts. Functional analyses showed that RyR3 deficiency impaired starvation-induced autophagy, characterized by defective autophagosome formation and reduced autophagic flux, and increased susceptibility to metabolic stress. Mitochondrial bioenergetic profiling revealed reduced oxidative phosphorylation capacity and decreased membrane potential under stress conditions, consistent with compromised mitochondrial adaptation. In zebrafish, ryr3 knockdown resulted in structural and functional muscle abnormalities, including reduced myotome area and decreased locomotor activity, associated with impaired autophagic flux. This study establishes a novel association between recessive RYR3 variants and recurrent rhabdomyolysis and identifies RyR3 as a critical regulator of skeletal muscle stress adaptation through calcium-dependent control of autophagy and mitochondrial homeostasis. More broadly, our findings further highlight autophagy as a central determinant of muscle resilience in the context of rhabdomyolysis.

Increased literature support the pathogenetic role of dysfunctional energetic metabolism in the setup and progression of organ damage and failure. Genetic diseases often offer the possibility to investigate pathogenetic mechanisms. In particular, excessive cardiac damage is the most frequent cause of mortality in Fabry disease (FD), a genetic condition caused by deficient -galactosidase A (GLA) activity, leading to globotriaosylceramide (Gb3) accumulation. Beyond Gb3 storage, metabolic alterations and mitochondrial dysfunction, supported by in vitro evidence or studies in other tissues, may contribute to FD cardiomyopathy. This study investigated, for the first time, the mechanisms of mitochondrial involvement in FD, its role in determining cardiac manifestations, and its potential as a therapeutic target. We used a humanized FD mouse model (R301Q-Tg/GLA knockout), along with derived embryonic fibroblasts and neonatal and adult cardiomyocytes, to assess mitochondrial function across the lifespan. FD cells showed impaired mitophagy, reduced mitochondrial respiration, and increased reactive oxygen species production. Importantly, this mitochondrial dysfunction exacerbated the lysosomal deficit in FD cells, forming a vicious cycle. In cardiomyocytes, these alterations progressed with age, leading to the accumulation of dysfunctional mitochondria, energetic failure, and, in adult hearts, terminal mitochondrial damage and apoptosis. These events ultimately result in cardiac remodeling and dysfunction, including hypertrophy and diastolic impairment. Indeed, L-arginine supplementation, which promotes NO/PGC-1-dependent mitochondrial rescue, prevented the development of cardiac abnormalities in FD mice. Our findings identify early mitochondrial dysfunction as a key driver of FD cardiomyopathy and support mitochondrial targeting, including L-arginine supplementation, as a promising adjuvant therapeutic strategy. The mechanistic link between lysosomal dysfunction, altered mitochondrial turnover, and energetic collapse emerges as a key targetable pathway in organ damage, extending beyond FD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/718770v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@2a5c4borg.highwire.dtl.DTLVardef@1117767org.highwire.dtl.DTLVardef@1b634c5org.highwire.dtl.DTLVardef@1429b6c_HPS_FORMAT_FIGEXP M_FIG C_FIG Cardiac manifestations vs mitochondrial alterations in Fabry disease: the visible tip and the hidden base of the icebergCardiac manifestations in hR301Q Tg/KO mice become evident from 9 months of age. However, mitochondrial homeostasis is perturbed much earlier (neonatal to young stages), with impaired mitophagy, reduced mitochondrial respiration and membrane potential, increased ROS production and PGC-1 downregulation. At later stages, from 6 months of age, mitochondrial dysfunction progresses and begins to impact cellular energetics, as indicated by reduced ETC expression and the onset of energetic deficit (ATP reduction). The resulting energetic collapse, together with progressive mitochondrial leakage, leads to cardiomyocyte hypertrophy, apoptosis, and dysfunction, which become detectable from 9 months of age, when clinical signs emerge. These findings support a mechanistic model in which 1) lysosomal incompetence due to GLA deficit is the initiating event inducing impairment of mitophagy; 2) Unsuccessful mitophagy, induces downregulation of PGC-1a-dependent mitogenesis; 3) exhausted mitochondria accumulate, inducing energetic collapse (able to exacerbate lysosomal dysfunction and further perturb mitophagy in a vitious cycle); 4) ultimate mitochondrial leakage induces Cytochrome C release and apoptosis activation. This cascade of molecular events is responsible for clinical manifestations, and mitochondrial targeting prevents cardiac organ damage. Significance statementFabry disease is a rare genetic disorder in which cardiac complications are a major cause of death, yet underlying mechanisms remain unclear. Here, we identify mitochondrial dysfunction as an early pathogenic event associated with impaired mitophagy, whereby defective mitochondrial quality control both results from and exacerbates lysosomal dysfunction, creating a self-reinforcing cycle that drives disease progression. Using a humanized model, we demonstrate that mitochondrial dysfunction is a key determinant of cardiac phenotype in vivo, driving energetic failure, oxidative stress, and cardiac damage. Importantly, L-arginine treatment restores mitochondrial function and prevents cardiac abnormalities. Our findings define a broadly relevant pathogenic axis linking lysosomal dysfunction, mitophagy failure, and mitochondrial impairment, that lead to impaired energetic metabolism and consequent cardiac hypertrophy, independently from GB3 accumulation. The implications of our study go beyond Fabry disease and support the therapeutic targeting of cellular energy homeostasis to prevent and treat organ damage and failure in chronic diseases. IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org

Obesity is a major driver of hepatic steatosis, yet the molecular link between excess adiposity and hepatocellular lipid accumulation remains incompletely defined. Here, we identify circulating fatty acid-binding protein 4 (FABP4) as a key mediator of adipocyte-hepatocyte lipid crosstalk in obesity. Analyses of human liver specimens and mouse models reveal aberrant accumulation of FABP4 protein--but not transcript--in hepatocytes during steatosis, indicating an extrinsic source. Genetic deletion of FABP4, specifically in adipocytes, protects against high fat diet-induced hepatic steatosis without altering obesity or systemic lipid levels. Mechanistically, circulating FABP4 directly binds to hepatocytes, facilitating free fatty acid uptake. Furthermore, we developed a high-affinity humanized monoclonal antibody that selectively neutralizes circulating FABP4, blocks hepatocyte binding, suppresses fatty acid uptake, and markedly attenuates hepatic steatosis in multiple obese mouse models. These findings establish circulating FABP4 as a pathogenic lipid chaperone and a promising therapeutic target for obesity-associated hepatic steatosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/701451v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@200778org.highwire.dtl.DTLVardef@ca7204org.highwire.dtl.DTLVardef@1037674org.highwire.dtl.DTLVardef@55c680_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHepatocytic accumulation of extrinsic FABP4 links adiposity to liver lipid deposition. C_LIO_LISpecific deletion of FABP4 in adipocytes prevents hepatic steatosis without affecting systemic lipid levels or obesity. C_LIO_LICirculating FABP4 derived from adipocytes directly binds hepatocytes to facilitate free fatty acid transfer. C_LIO_LIBlocking circulating FABP4 with a high-affinity anti-FABP4 monoclonal antibody inhibits hepatocyte lipid uptake and attenuates steatosis in multiple obese mouse models. C_LI