Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder causing progressive renal cyst formation, increased kidney volume, and impaired function. The PCK rat is a preclinical model for investigating new treatments, requiring accurate quantification of total kidney volume (TKV), total cyst volume (TCV), and cyst count. We propose an automated segmentation pipeline of kidneys and cysts on µCT scans of excised rat kidneys, followed by automated cyst counting. For segmentation, a 3D U-Net ensemble was implemented using the nnU-Net framework with dual-channel input (raw µCT volumes, Sobel-filtered images). Models were trained on Dataset 1 subsets (D1, n = 5, 10, 15, 20), and evaluated on internal test set (n = 5) and independent external Dataset 2 (D2, n = 5). Segmentation achieved Dice Similarity Coefficients > 0.99 for kidney and > 0.98 for cysts on D1, with comparable performance on D2, exploring cross-scanner generalizability. For cyst counting, a morphological algorithm based on 3D distance transform and peak detection was optimized via genetic algorithm on 10 samples and evaluated on independent 10-sample set. The automated method achieved low variability, with operator-algorithm agreement exceeding inter-operator agreement across all metrics, drastically reducing processing time. The pipeline provides fast, accurate, and reproducible quantification of morphological biomarkers required for preclinical PKD research.

Acute kidney injury (AKI) is a serious condition marked by a rapid decline in renal function, often leading to long-term complications. Mesenchymal stromal cells (MSCs) and their derivatives, including conditioned medium (CM) and extracellular vesicles (EVs), show promise as regenerative therapies. However, the comparative efficacy of CM and EVs and the development of clinically translatable interventions remains underexplored. This study systematically compared the renoprotective effects of CM and EVs derived from human umbilical cord MSCs in a murine cisplatin-induced AKI model, using a therapeutically feasible dose. Both treatments improved renal function, reduced histological damage, preserved mitochondrial integrity, energy metabolism, and antioxidant response. Notably, EVs induced the greatest proliferative response in renal tubular cells. To further enhance the regenerative potential of EVs, we engineered MSCs to overexpress nicotinamide phosphoribosyltransferase (NAMPT), a metabolic enzyme that plays a key role in NAD+ biosynthesis. NAMPT-transfected MSCs released NAMPT-enriched EVs, which more effectively enhanced cell viability, reduced apoptosis, and protected mitochondria in cisplatin-damaged tubular cells in vitro compared to EV-GFP. In mice with AKI, NAMPT-enriched EVs improved renal function and repaired damage by enhancing renal NAMPT and NAD+ levels, promoting tubular cell regeneration. Mechanistically, the amelioration of mitochondrial function was related to increased PGC1α and SIRT3 and consequently SOD2 and ATP5i expression. These findings highlight the therapeutic potential of EVs, particularly NAMPT-enriched EVs, in renal repair, supporting their promise as a clinically translatable approach for promoting recovery from AKI and other kidney diseases.

Preclinical and clinical evidence suggested the potential benefits of treatment with acetylsalicylic acid (ASA) in mitigating COVID-19 severity. While available studies largely focused on the intracellular pathways through which ASA impairs viral replication or dampens host immunoresponse stimulated by SARS-CoV-2, whether ASA directly affects the interaction between the viral spike protein and its cellular receptor angiotensin converting enzyme 2 (ACE2) remains unexplored. This question is clinically relevant, as circulating spike S1 has been shown to persist in patients with acute and long COVID-19, where its interaction with the broadly expressed ACE2 drives systemic manifestations and tissue damage. Here, we demonstrate that pre-incubation of the SARS-CoV-2 spike subunit 1 (S1) with ASA dose-dependently impaired ACE2 binding on Vero cells. The functional relevance of this finding was confirmed in transgenic mice with human ACE2, in which intratracheal administration of ASA-treated S1 markedly reduced lung injury, fibrosis, and inflammation compared to untreated S1. Glycoproteomic profiling revealed that ASA altered the glycosylation landscape of S1, particularly N-glycosylation at N61 and O-glycosylation at S325. Site-directed mutagenesis of these two residues confirmed the critical role of their glycosylation in S1-ACE2 binding in vitro. Consistently, the glycosylation-insensitive S1 had limited effect in inducing lung injury, fibrosis, and inflammation in transgenic mice compared to WT S1, phenocopying the protective effects of ASA. These findings unveil a previously unrecognized antiviral activity of ASA, providing a molecular rationale for its repurposing as a low-cost, readily available intervention to prevent the progression from mild to severe COVID-19.

BACKGROUND:Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by progressive cyst development and renal dysfunction. While thyroid hormones (THs) are known to regulate key pathways in various kidney diseases, their role in ADPKD pathobiology and therapeutic potential remains unexplored. Here, we aimed to elucidate the role of THs in ADPKD and evaluate whether their pharmacological modulation could serve as a therapeutic strategy. METHODS:Patient-derived renal epithelial cells were used to engineer 3D polycystic tubules and to test the anti-cystogenic effects of THs and their analogs. The therapeutic efficacy of thyroxine (T4) in reducing cyst formation and delaying disease progression was assessed in vivo using PCK rats, an animal model of ADPKD. Lastly, serum THs levels were measured in 90 ADPKD patients enrolled in the REORIENTED clinical study and correlated with estimated glomerular filtration rate to explore their clinical relevance (Clinical Trial Gov NCT05646420). RESULTS:Mechanistically, thyroxine inhibits cyst growth by modulating proliferative, metabolic and ferroptotic pathways through αvβ3 integrin binding. In PCK rats, an animal model of ADPKD, T4 administration decreased kidney weight and significantly reduced macrocystic area (%, Vehicle 9.255 ± 2.654 vs. T4 1.945 ± 0.850, p < 0.05). Clinical data from ADPKD patients showed that altered TH serum levels correlate with disease severity: in the overall population of the study reverse triiodothyronine (rT3, a T3's metabolite) levels inversely correlate with renal function (R2 = 0.159, r = -0.397, p < 0.001), while free triiodothyronine (fT3) levels show a positive correlation (R2 = 0.110, r = 0.332, p < 0.01). CONCLUSIONS:This study reveals that THs contribute to ADPKD progression and identifies them as potential prognostic and therapeutic agents. By modulating multiple pathogenic pathways, THs may offer a novel, multi-targeted approach to reduce cyst growth and preserve renal function. These findings further support the development of personalized, hormone-based treatments and more refined stratification in the clinical management of ADPKD.

Severe COVID-19 is characterized by thrombo-inflammatory processes within the lung microvasculature. In pursuit of effective treatments, clinical studies explored mesenchymal stromal cells (MSCs) as a promising approach due to their anti-inflammatory, immunomodulatory, and regenerative properties, through their paracrine action.Here, we tested the conditioned medium (CM) derived from human umbilical cord (UC)-MSCs in acute lung injury induced by the spike protein subunit 1 (S1) in ACE2-humanized male mice. Injection of CM significantly limited S1-induced lung injury, edema, and fibrosis. This was associated with reduced vascular dysfunction, in terms of restored thrombomodulin levels and decreased von Willebrand (vWF) expression. By preserving endothelial glycocalyx, CM reduced complement C3 accumulation, favoring factor H binding on the lung microvasculature. Reduced oxidative stress, nuclear NF-κB p65 accumulation, and inflammatory cell infiltration were also observed in response to CM in S1-injected mice.In vitro, CM counteracted thrombo-inflammation by preserving thrombomodulin, as well as limiting vWF expression, due to endothelial glycocalyx recovery. CM reduced nuclear translocation of NF-κB p65 and its downstream targets, ICAM-1 and P-selectin, translating in decreased C3 deposits, platelet aggregation, and leukocyte adhesion on S1-challenged endothelial cells.Collectively, these data indicate that UC-MSC-derived secretome represents a promising therapy in COVID-19 due to its potent anti-thrombotic and anti-inflammatory effects on lung microcirculation.

Myocardial infarction (MI) is a leading cause of heart failure, with thyroid hormone (TH) signaling playing a key role in heart function and postinfarct recovery. Despite evidence of TH administration's safety in cardiac patients, inconsistent therapeutic outcomes and limited understanding of its mechanisms hinder clinical translation. This study aims to investigate the long-term effect of acute triiodothyronine (T3) administration following MI and to elucidate the mechanisms of its cardioprotective actions. To this end, two doses (40 μg/kg) of T3 were administered immediately after injury and 24 h later in a cryoinjury mouse model of left ventricle (LV) infarction. Remarkably T3 administration significantly reduced scar expansion. Echocardiographic analysis conducted 28 days post-injury revealed that T3 administration improved LV remodeling and prevented LV hypertrophy. At molecular level, T3 administration strongly reduced apoptosis in the peri-infarcted area, without inducing cardiac cell proliferation. Furthermore, T3 prevented the accumulation of long-chain acylcarnitines and the subsequent mitochondrial damage. These findings demonstrate that acute T3 treatment following MI improves long-term LV function and reduces LV remodeling by limiting apoptosis in the peri-infarct region and by preserving mitochondrial function and structural integrity.

In the original publication [...].

IntroductionIn autoimmune diseases, autoreactive B cells comprise only the 0.1-0.5% of total circulating B cells. However, current first-line treatments rely on non-specific and general suppression of the immune system, exposing patients to severe side effects. For this reason, identification of targeted therapies for autoimmune diseases is an unmet clinical need.MethodsHere, we designed a novel class of immunotherapeutic molecules, Bi-specific AutoAntigen-T cell Engagers (BiAATEs), as a potential approach for targeting the small subset of autoreactive B cells. To test this approach, we focused on a prototype autoimmune disease of the kidney, membranous nephropathy (MN), in which phospholipase A2 receptor (PLA2R) serves as primary nephritogenic antigen. Specifically, we developed a BiAATE consisting of the immunodominant Cysteine-Rich (CysR) domain of PLA2R and the single-chain variable fragment (scFv) of an antibody against the T cell antigen CD3, connected by a small flexible linker.ResultsBiAATE creates an immunological synapse between autoreactive B cells bearing an CysR-specific surface Ig+ and T cells. Ex vivo, the BiAATE successfully induced T cell-dependent depletion of PLA2R-specific B cells isolated form MN patients, sparing normal B cells. Systemic administration of BiAATE to mice transgenic for human CD3 reduced anti-PLA2R antibody levels following active immunization with PLA2R.DiscussionShould this approach be confirmed for other autoimmune diseases, BiAATEs could represent a promising off-the-shelf therapy for precision medicine in virtually all antibody-mediated autoimmune diseases for which the pathogenic autoantigen is known, leading to a paradigm shift in the treatment of these diseases.

Abstract Background and Aims Thyroid hormone (TH) signaling plays a crucial role in the regulation of the cell differentiation state, cell cycle and metabolism in various organs. Low TH levels are observed in most patients with chronic kidney disease, and correlate with cell de-differentiation and reactivation of the cell cycle. Despite this knowledge, possible alterations of TH signaling in Autosomal Dominant Polycystic Kidney Disease (ADPKD) and the capacity of TH to modulate anti-cystogenic and renoprotective pathways have not yet been studied. In this study we aimed to (i) investigate the role of TH signaling in the pathogenesis of ADPKD in patients and experimental models, and (ii) assess whether the pharmacological modulation of this pathway with TH (T3, T4) and TH analogs could arrest or reverse ADPKD progression. Method Ninety patients with a diagnosis of ADPKD based on renal ultrasonography findings or genetic testing were included in the study and divided into five subgroups of chronic kidney disease stages, according to the KDIGO classification. Serum TH was measured using ELISA and chemiluminescent immunoassays. The correlation between TH levels and renal function parameter (eGFR) in these patients was assessed using linear regression. 3D polycystic tubules were engineered from patient-derived cells as previously described (Benedetti et al. 2016). For the in vivo experiments, PCK rats—an animal model of ADPKD—were treated with T4 (10 μg/kg) or vehicle by gavage from 4 to 11 weeks of age. Age-matched SD rats were followed for the same length of time and used as controls. Results The analysis of patients’ data showed a strong linear correlation between the levels of FT3 and eGFR, and corresponding inverse correlation between rT3 (the inactivated form of T3) and eGFR. Treating patients-derived tubules with THs strongly inhibited cyst formation and growth. Interestingly, the results were validated in engineered polycystic tubules from three ADPKD patients with different mutations (missense, splice-site and stop-gain) in the PKD1 gene, further confirming that T4 plays an essential role in the cystogenesis and, presumably, progression of the disease in humans. Treating PCK rats—an animal model of ADPKD—with T4 resulted in a significant increase in T4 serum levels and a simultaneous reduction in the size of the macrocystic area compared to vehicle-treated PCK rats. Most importantly, T4 serum levels positively correlated with a reduction in both the size of the macrocystic area and loss of protein into the urine, indicating that TH signaling plays a role in the growth of cysts, and that exogenous T4 can slow down cystogenesis and improve renal function. Interestingly, our mechanistic studies revealed that T4 exerts its anti-cystogenic effects, at least partially, by binding to the membrane receptor αvβ3 and activating the MAPK cascade. Conclusion These results suggest that TH signaling plays a role in the progression of ADPKD and timely administration of T4, ideally at a relatively early stage of the disease, could delay progression or even reverse key clinical features of ADPKD.

Complement alternative pathway (AP) dysregulation drives C3 glomerulopathy (C3G), a rare renal disorder characterized by glomerular C3 deposition and glomerular damage, for which no effective treatments are available. Blockade of complement C3 is emerging as a viable therapeutic option. In an earlier study we showed that SLN500, a small interfering RNA targeting liver C3 synthesis, was able to limit AP dysregulation and glomerular C3d deposits in mice with partial factor H (FH) deficiency (Cfh+/- mice). Here, we assessed the pharmacological effects of SLN501 - an optimized SLN500 version - in mice with complete FH deficiency (Cfh-/- mice) that exhibit a more severe C3G phenotype. SLN501 effectively prevented liver C3 synthesis, thus limiting AP dysregulation, glomerular C3d deposits and the development of ultrastructural alterations. These data provide firm evidence of the use of siRNA-mediated liver C3 gene silencing as a potential therapy for treating C3G patients with either partial or complete FH loss of function.

Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease. Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely. Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.

Diabetes mellitus and alterations in thyroid hormone (TH) signaling are closely linked. Though the role of TH signaling in cell differentiation and growth is well known, it remains unclear whether its alterations contribute to the pathobiology of diabetic cells. Here, we aim to investigate whether the administration of exogenous T3 can counteract the cellular remodeling that occurs in diabetic cardiomyocytes, podocytes, and pancreatic beta cells. Treating diabetic rats with T3 prevents dedifferentiation, pathological growth, and ultrastructural alterations in podocytes and cardiomyocytes. In vitro, T3 reverses glucose-induced growth in human podo-cytes and cardiomyocytes, restores cardiomyocyte cytoarchitecture, and reverses pathological alterations in kidney and cardiac organoids. Finally, T3 treatment counteracts glucose-induced transdifferentiation, cell growth, and loss in pancreatic beta cells through TH receptor alpha1 activation. Our studies indicate that TH signaling activation substantially counteracts diabetes-induced pathological remodeling, and provide a potential therapeutic approach for the treatment of diabetes and its complications.

The spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can interact with endothelial cells. However, no studies demonstrated the direct effect of the spike protein subunit 1 (S1) in inducing lung vascular damage and the potential mechanisms contributing to lung injury. Here, we found that S1 injection in mice transgenic for human angiotensin converting enzyme 2 (ACE2) induced early loss of lung endothelial thromboresistance at 3 days, as revealed by thrombomodulin loss and von Willebrand factor (vWF) increase. In parallel, vascular and epithelial C3 deposits and enhanced C3a receptor (C3aR) expression were observed. These changes preceded diffuse alveolar damage and lung vascular fibrin(ogen)/platelets aggregates at 7 days, as well as inflammatory cell recruitment and fibrosis. Treatment with C3aR antagonist (C3aRa) inhibited lung C3 accumulation and C3a/C3aR activation, limiting vascular thrombo-inflammation and fibrosis. Our study demonstrates that S1 triggers vascular dysfunction and activates complement system, instrumental to lung thrombo-inflammatory injury. By extension, our data indicate C3aRa as a valuable therapeutic strategy to limit S1-dependent lung pathology.

Sirtuin 3 (SIRT3), the main deacetylase of mitochondria, modulates the acetylation levels of substrates governing metabolism and oxidative stress. In the kidney, we showed that SIRT3 affects the proper functioning of high energy-demanding cells, such as tubular cells and podocytes. Less is known about the role of SIRT3 in regulating endothelial cell function and its impact on the progression of kidney disease. Here, we found that whole body Sirt3 -deficient mice exhibited reduced renal capillary density, reflecting endothelial dysfunction, and VEGFA expression compared to wild-type mice. This was paralleled by activation of hypoxia signaling, upregulation of HIF-1α and Angiopietin-2, and oxidative stress increase. These alterations did not result in kidney disease. However, when Sirt3 -deficient mice were exposed to the nephrotoxic stimulus Adriamycin (ADR) they developed aggravated endothelial rarefaction, altered VEGFA signaling, and higher oxidative stress compared to wild-type mice receiving ADR. As a result, ADR-treated Sirt3 -deficient mice experienced a more severe injury with exacerbated albuminuria, podocyte loss and fibrotic lesions. These data suggest that SIRT3 is a crucial regulator of renal vascular homeostasis and its dysregulation is a predisposing factor for kidney disease. By extension, our findings indicate SIRT3 as a pharmacologic target in progressive renal disease whose treatments are still imperfect.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Peritubular capillary rarefaction is a recurrent aspect of progressive nephropathies. We previously found that peritubular capillary density was reduced in BTBR ob/ob mice with type 2 diabetic nephropathy. In this model, we searched for abnormalities in the ultrastructure of peritubular capillaries, with a specific focus on the endothelial glycocalyx, and evaluated the impact of treatment with an angiotensin-converting enzyme inhibitor (ACEi). Mice were intracardially perfused with lanthanum to visualise the glycocalyx. Transmission electron microscopy analysis revealed endothelial cell abnormalities and basement membrane thickening in the peritubular capillaries of BTBR ob/ob mice compared to wild-type mice. Remodelling and focal loss of glycocalyx was observed in lanthanum-stained diabetic kidneys, associated with a reduction in glycocalyx components, including sialic acids, as detected through specific lectins. ACEi treatment preserved the endothelial glycocalyx and attenuated the ultrastructural abnormalities of peritubular capillaries. In diabetic mice, peritubular capillary damage was associated with an enhanced tubular expression of heparanase, which degrades heparan sulfate residues of the glycocalyx. Heparanase was also detected in renal interstitial macrophages that expressed tumor necrosis factor-α. All these abnormalities were mitigated by ACEi. Our findings suggest that, in experimental diabetic nephropathy, preserving the endothelial glycocalyx is important in order to protect peritubular capillaries from damage and loss.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

A reduced nephron number at birth, due to critical gestational conditions, including maternal malnutrition, is associated with the risk of developing hypertension and chronic kidney disease in adulthood. No interventions are currently available to augment nephron number. We have recently shown that sirtuin 3 (SIRT3) has an important role in dictating proper nephron endowment. The present study explored whether SIRT3 stimulation, by means of supplementation with nicotinamide riboside (NR), a precursor of the SIRT3 co-substrate nicotinamide adenine dinucleotide (NAD+), was able to improve nephron number in a murine model of a low protein (LP) diet. Our findings show that reduced nephron number in newborn mice (day 1) born to mothers fed a LP diet was associated with impaired renal SIRT3 expression, which was restored through supplementation with NR. Glomerular podocyte density, as well as the rarefaction of renal capillaries, also improved through NR administration. In mechanistic terms, the restoration of SIRT3 expression through NR was mediated by the induction of proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α). Moreover, NR restored SIRT3 activity, as shown by the reduction of the acetylation of optic atrophy 1 (OPA1) and superoxide dismutase 2 (SOD2), which resulted in improved mitochondrial morphology and protection against oxidative damage in mice born to mothers fed the LP diet. Our results provide evidence that it is feasible to prevent nephron mass shortage at birth through SIRT3 boosting during nephrogenesis, thus providing a therapeutic option to possibly limit the long-term sequelae of reduced nephron number in adulthood.

Sirtuin 3 (SIRT3) is the primary mitochondrial deacetylase that controls the antioxidant pathway and energy metabolism. We previously found that renal Sirt3 expression and activity were reduced in mice with type 2 diabetic nephropathy associated with oxidative stress and mitochondrial abnormalities and that a specific SIRT3 activator improved renal damage. SIRT3 is modulated by diet, and to assess whether Sirt3 deficiency aggravates mitochondrial damage and accelerates kidney disease in response to nutrient overloads, wild-type (WT) and Sirt3−/− mice were fed a high-fat-diet (HFD) or standard diet for 8 months. Sirt3−/− mice on HFD exhibited earlier and more severe albuminuria compared to WT mice, accompanied by podocyte dysfunction and glomerular capillary rarefaction. Mesangial matrix expansion, tubular vacuolization and inflammation, associated with enhanced lipid accumulation, were more evident in Sirt3−/− mice. After HFD, kidneys from Sirt3−/− mice showed more oxidative stress than WT mice, mitochondria ultrastructural damage in tubular cells, and a reduction in mitochondrial mass and energy production. Our data demonstrate that Sirt3 deficiency renders mice more prone to developing oxidative stress and mitochondrial abnormalities in response to HFD, resulting in more severe kidney diseases, and this suggests that mitochondria protection may be a method to prevent HFD-induced renal injury.