Search Results (127)

Organ shortage is a major challenge in transplantation, prompting the use of extended criteria donor grafts. These require improved preservation techniques and reliable methods to assess graft function. This study aimed to evaluate changes in the kidney metabolome following three preservation methods: normothermic ex vivo kidney perfusion (NEVKP), hypothermic machine perfusion (HMP) and static cold storage (SCS) in porcine autotransplant models. A chemical biopsy allowed minimally invasive sampling of metabolites, which were analyzed using liquid chromatography coupled with high-resolution mass spectrometry. The results highlighted metabolites affected by ischemia and oxidative stress in donor kidneys, as well as changes specific to each preservation method. Differences were observed immediately after transplantation and reperfusion and several days post-surgery. NEVKP was associated with the activation of physiological anti-oxidative and anti-inflammatory mechanisms, suggesting potential protective effects. However, some metabolites had dual roles, which may influence future graft treatment designs. HMP and SCS, while reducing energy demand in cells, also limit physiological repair mechanisms. These findings provide a basis for improving graft assessment and organ preservation, with chemical biopsy serving as both a tool for discovery and a potential diagnostic method for monitoring graft quality. Full article

The enzyme transglutaminase 2 (TG2) has an open conformation with transamidase activity which crosslinks matrix proteins contributing to fibrosis development. LDN-27219 promotes the closed conformation of TG2, which can enhance vasodilation, but its effects in renal tissue are unknown. We investigated whether LDN-27219 treatment affects albuminuria and markers of renal fibrosis as well as ex vivo vasodilatation. Male C57BL/6 mice (n = 48) underwent unilateral nephrectomy plus insertion of a deoxycorticosterone acetate pellet (DOCA group) or nephrectomy only (sham group). Both groups were randomized to intraperitoneal treatment with either LDN-27219 (8 mg/kg twice daily) or vehicle for 2 weeks. Urine albumin excretion was evaluated by metabolic cages. Kidney tissue fibrosis markers were assessed by qPCR and Western blotting, while the TG2 conformational state was evaluated using native gel electrophoresis. Collagen staining was performed using Picrosirius red and quantified under circularly polarized light. Mesenteric arteries were mounted in wire myographs for evaluation of vasorelaxation. DOCA mouse developed significant albuminuria (p < 0.001 vs. sham), but neither TG2 mRNA nor protein expression was upregulated in the kidney. However, the relative amount of TG2 in the closed conformation was higher in DOCA mice. LDN-27219 did not affect albuminuria, but LDN-27219-treated DOCA mice showed less urine production and less collagen staining than vehicle-treated DOCA mice. LDN-27219 did not affect TG2 mRNA or TG2 protein expression or mRNA of fibrosis markers. LDN-27219-treated mice had enhanced vasorelaxation to the nitric oxide donor sodium nitroprusside. In conclusion, LDN-27219 treatment in the one-kidney DOCA–salt model did not affect renal TG2 mRNA and protein expression or albuminuria but still exerted beneficial effects in terms of reduced kidney fibrosis and urine production in addition to enhanced vasodilatation. Full article

Background: Vascular imaging is essential for clinical practice, research, and the diagnosis and management of vascular diseases. Super-resolution ultrasound (SRUS) imaging is an emerging high-resolution imaging technique with broad applications in soft tissue vascular imaging. However, the impact of biological and clinical variables on its imaging accuracy is currently unknown. This study investigates these factors in an animal model and compares SRUS with contrast-enhanced µCT. Methods: Kidney scans from 29 Zucker rats (Zucker Diabetic Fatty and Zucker Lean) were retrospectively analysed. The left kidney was imaged in vivo using SRUS during microbubble infusion, then filled with Microfil and excised for ex vivo µCT. SRUS parameters and clinical variables were analysed, and SRUS scans were co-registered with µCT to compare vascular density measurements. Results: Mean arterial blood pressure and anaesthesia time showed significant linear relationships with SRUS microbubble detection and vascular track reconstruction. The anaesthesia time was also strongly correlated with vascular density measurement. Visualisation and velocity estimations of renal arteries were limited with SRUS. Ultrasound signal attenuation had significant impacts, particularly in cortical far-field imaging. Despite differences between kidney regions, the vascular density distribution did not differ considerably between SRUS and µCT datasets for whole-kidney imaging. Conclusions: This study outlines key factors SRUS users must consider for optimal technique use. Careful region selection and control of clinical variables ensure more reliable and comparable images. Further research is necessary to translate these findings from a rat model into clinical application. Full article

A precision genome edit in the bovine CD46 gene (A₈₂LPTFS₈₇) dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned heifer. However, pathogen evolution threatens the long-term efficacy of such interventions. Here, our aim is two-fold: first, to determine whether BVDV can adapt in vitro to use the edited CD46 receptor to infect Madin–Darby bovine kidney (MDBK) cells, and second, to evaluate the ex vivo infectivity of culture-adapted viruses in cells from the CD46-edited heifer. Serial passage of BVDV on CD46-edited MDBK cells selected for virus variants capable of CD46-independent infection. Virus genome sequencing revealed mutations in the viral E^RNS gene predicted to enhance HS-mediated entry. HS adaptation was confirmed by inhibiting virus infection with heparin or Heparinase I/III treatment. A naturally occurring HS-adapted field isolate from a persistently infected calf showed similar results. However, when tested on primary cells from the CD46-edited heifer, HS-adapted viruses showed reduced infectivity in skin fibroblasts, monocytes, and lymphocytes in a manner that correlated with HS expression. Thus, although BVDV can adapt to use HS as an alternative entry receptor, HS adaptation does not overcome the protection conferred by the CD46 edit in all relevant cell types. Full article

Gut-derived uremic toxins (UTs) contribute to cardiovascular disorders like atherosclerosis and cardiomyopathy in patients with chronic kidney disease (CKD), causing increased cardiovascular morbidity and mortality. The intermediate steps between higher concentrations of gut-derived UTs and organ damage caused by UTs are still insufficiently understood. Glycosaminoglycans (GAGs) as components of the extracellular matrix are known to interact with various ligands such as growth factors or receptors, thereby influencing (patho)physiological processes. We previously found that the UT inorganic phosphate (Pi) induces the synthesis and sulphation of the GAGs heparan sulphate and chondroitin sulphate in the rat vascular smooth muscle cell (VSMC) line A7r5 and in the human endothelial cell (EC) line EA.Hy926. The aim of this study was to investigate if other organic UTs modulate GAGs in vascular cells as well. We treated ex vivo cultures of rat aortic rings as well as primary rat VSMCs and human ECs with the UTs Pi, indoxylsulphate (IS), p-cresylsulphate (pCS), trimethylamine N-oxide (TMAO), and urea, and analyzed the samples by histological staining, qPCR, western blot, HPLC, and colorimetric assays. The UT treatment of aortic rings and cells increased contents of sulphated GAGs and hyaluronic acid. UT-treated cells contained higher amounts of 4S- and 6S-sulphated GAGs compared to controls. This was accompanied by altered expressions of genes and proteins relevant for GAG metabolism. Mechanistically, the effects of the UTs on GAGs involve the activation of the PI3K/Akt pathway and of the transcription factor NF-κB. In conclusion, the UT-induced remodeling of the cardiovascular matrix by upregulation of sulphated GAGs and hyaluronic acid in aortic tissue and vascular cells might be a missing link between gut-derived UT and pathophysiological alterations in the cardiovascular system in the sense of a gut–matrix axis. Full article

Background: Hypertension (HTN) is recognized as a major risk factor for cardiovascular disease, chronic kidney disease, and peripheral artery disease. Valsartan (VAL), an angiotensin receptor blocker drug for hypertension, has been limited due to its poor solubility and poor absorption from the GIT, which leads to low oral bioavailability. Objectives/Method: In the present research, firstly, VAL-loaded nanoliposomes were formulated and optimized using the Box–Behnken design (BBD). Optimized VAL-nanoliposomes were physically characterized and their fate was examined by scanning and transmission microscopy, DSC, FTIR, XRD, and ex vivo studies using rat skin. In vitro studies using human keratinocyte (HaCaT) cells showed a decrease in cell viability as the liposome concentration increased. Secondly, the formulation of VAL-loaded nanoliposomes was integrated into dissolvable microneedles (DMNs) to deliver the VAL transdermally, crossing the skin barrier for better systemic delivery. Results: The optimized nanoliposomes showed a vesicle size of 150.23 (0.47) nm, a ZP of −23.37 (0.50) mV, and an EE% of 94.72 (0.44)%. The DMNs were fabricated using a ratio of biodegradable polymers, sodium alginate (SA), and hydroxypropyl methylcellulose (HPMC). The resulting VAL-LP-DMNs exhibited sharp pyramidal microneedles, adequate mechanical properties, effective skin insertion capability, and rapid dissolution of the microneedles in rat skin. In the ex vivo analysis, the transdermal flux of VAL was significantly (5.36 (0.39) μg/cm²/h) improved by VAL-LP-DMNs. The enhancement ratio of the VAL-LP-DMNs was 1.85. In conclusion, liposomes combined with DMNs have shown high potential and bright prospects as carriers for the transdermal delivery of VAL. Conclusions: These DMNs can be explored in studies focused on in vivo evaluations to confirm their safety, pharmacokinetics profile, and pharmacodynamic efficacy. Full article

We report two conjugates of gem-diethyl pyrroline nitroxide radicals with D-mannosamine as potential metabolic organic radical contrast agents, mORCAs, circumventing the need for biorthogonal reactions. In-cell EPR spectroscopy, using Jurkat cells and analogous conjugate, based on a pyrrolidine nitroxide radical, shows an efficient incorporation of highly immobilized nitroxides, with a correlation time of τ_cor = 20 ns. In vivo MRI experiments in mice show that the paramagnetic nitroxide radical shortens the T₁ and T₂ relaxation times of protons in water located in the kidney and brain by only up to ~10% after 3 d. Ex vivo EPR spectroscopic analyses indicate that the contrast agents in mouse tissues are primarily localized in the kidney, lung, liver, heart, and blood, which primarily contain immobilized nitroxide radicals with τ_cor = 4–9 ns. The spin concentrations in tissues remain low (1–3 nmol g⁻¹) at 24 h after the third mORCA injection, approximately one to two orders of magnitude lower than those of ORCAFluor and BASP-ORCA (measured at ~24 h post-injection). These low spin concentrations explain the small proton T₁ and T₂ relaxation changes observed in in vivo MRI. Full article

This study aimed to evaluate the effects of autologous dendritic cell (DC) immunotherapy on clinical outcomes (glomerular filtration rate/GFR and urine creatinine albumin ratio/UACR) and endothelial dysfunction (ICAM, VCAM, VEGF) in patients with diabetic kidney disease (DKD). Endothelial dysfunction induced by inflammation is one of the key factors in the pathogenesis of DKD. In this one-group pretest–posttest quasi-experimental study, 69 subjects with DKD were administered a single dose of autologous DC immunotherapy ex vivo. UACR was measured at baseline and at weeks 1, 2, 3, and 4, while ICAM, VCAM, VEGF, and GFR were measured at baseline and at week 4 post-immunotherapy. The results showed a significant reduction in median UACR from 250 (IQR 71–668) mg/g at baseline to 164 (IQR 49–576) mg/g at week 4 (p < 0.05). GFR did not show any significant changes after immunotherapy. HbA1c (B = −33.270, p = 0.021) and baseline UACR (B = −0.185, p < 0.001) were identified as significant predictors of UACR change. Although there were no significant changes in ICAM, VCAM, and VEGF, subgroup analysis revealed a decrease in VCAM in macroalbuminuria patients and an increase in those with good glycemic control, suggesting differing endothelial responses. In conclusion, autologous DC immunotherapy effectively reduced UACR in DKD patients, and significant VCAM changes were found in macroalbuminuria and good glycemic control subjects. Further research is needed to understand the mechanisms behind UACR reduction and the long-term impact of this therapy. Full article

Radiofrequency ablation (RFA) is a minimally invasive procedure that utilizes localized heat to treat tumors by inducing localized tissue thermal damage. The present study aimed to evaluate the temperature evolution and spatial distribution, ablation size, and reproducibility of ablation zones in ex vivo liver, kidney, and lung using a commercial device, i.e., Dophi™ R150E RFA system (Surgnova, Beijing, China), and to compare the results with the manufacturer’s specifications. Optical fibers embedding arrays of fiber Bragg grating (FBG) sensors, characterized by 0.1 °C accuracy and 1.2 mm spatial resolution, were employed for thermometry during the procedures. Experiments were conducted for all the organs in two different configurations: single-electrode (200 W for 12 min) and double-electrode (200 W for 9 min). Results demonstrated consistent and reproducible ablation zones across all organ types, with variations in temperature distribution and ablation size influenced by tissue characteristics and RFA settings. Higher temperatures were achieved in the liver; conversely, the lung exhibited the smallest ablation zone and the lowest maximum temperatures. The study found that using two electrodes for 9 min produced larger, more rounded ablation areas compared to a single electrode for 12 min. Our findings support the efficacy of the RFA system and highlight the need for tailored RFA parameters based on organ type and tumor properties. This research provides insights into the characterization of RFA systems for optimizing RFA techniques and underscores the importance of accurate thermometry and precise procedural planning to enhance clinical outcomes. Full article

Background/Objectives: The prevalence of cardiovascular-kidney-metabolic (CKM) syndrome is increasing rapidly, and cardiovascular complications pose significant risks in individuals with kidney disease and metabolic dysfunction. Understanding the mechanisms of CKM disorders is crucial, as is the discovery of novel preventive treatments. This study aimed to examine the therapeutic effects of a specially formulated nitric oxide-enhancing food additive in a mouse model of CKM syndrome induced by unilateral nephrectomy (UNX) in combination with chronic Western diet (WD) feeding. Methods: C57BL/6J mice underwent UNX and were fed a WD high in salt, sugar, and fat for 12 weeks, compared to sham-operated mice on standard chow. One group of UNX+WD mice received Flexovital (FLX), a food additive containing extracts of Rhodiola rosea and beetroot, and the amino acids L-arginine and L-citrulline. CKM parameters were assessed both in vivo and ex vivo alongside histological and biochemical analyses. Results: The UNX+WD mice showed an increase in body fat mass, the fat/lean mass ratio, and adipocyte area, of which most were significantly reduced by FLX. Elevated fasting glucose levels were also reduced by FLX, which tended towards improving glucose clearance. Elevated arterial blood pressure and endothelial dysfunction in UNX+WD mice were significantly reduced by FLX. FLX improved GFR and reduced glomerular and tubular injuries in UNX+WD mice. Additionally, FLX increased the P/O ratios of oxidative phosphorylation in the isolated renal mitochondria of UNX+WD mice. Conclusions: In this model of CKM syndrome, FLX effectively prevented the onset and progression of CKM dysfunctions induced by UNX+WD, as well as the associated organ injuries. These promising results highlight the need for validation in upcoming human trials. Full article

Background/Objectives: Dual-modality probes, combining positron emission tomography (PET) with fluorescence imaging (FI) capabilities in a single molecule, are of high relevance for the accurate staging and guided resection of tumours. We herein present a pair of candidates targeting the cholecystokinin-2 receptor (CCK2R), namely [⁶⁸Ga]Ga-CyTMG and [⁶⁸Ga]Ga-CyFMG. In these probes, the SulfoCy5.5 fluorophore and two units of a CCK2R-binding motif are coupled to the chelator acting as a core scaffold, triazacyclononane-phosphinic acid (TRAP), and Fusarinine C (FSC), respectively. Using this approach, we investigated the influence of these chelators on the final properties. Methods: The synthetic strategy to both precursors was based on the stoichiometric conjugation of the components via click chemistry. The characterization in vitro included the evaluation of the CCK2R affinity and internalization in A431-CCK2R cells. Ex vivo biodistribution as well as PET and FI studies were performed in xenografted mice. Results: ⁶⁸Ga labelling was accomplished with high radiochemical yield and purity for both precursors. A CCK2R affinity in the subnanomolar range of the conjugates and a receptor-specific uptake of the radioligands in cells were observed. In A431-CCK2R/A431-mock xenografted mice, the investigated compounds showed specific accumulation in the tumours and reduced off-target uptake compared to a previously developed compound. Higher accumulation and prolonged retention in the kidneys were observed for [⁶⁸Ga]Ga-CyTMG when compared to [⁶⁸Ga]Ga-CyFMG. Conclusions: Despite the promising targeting properties observed, further probe optimization is required to achieve enhanced imaging contrast at early timepoints. Additionally, the results indicate a distinct influence of the chelators in terms of renal accumulation and retention. Full article

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the −405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD. Full article

Background/Objectives: Urinary tract infections (UTIs) are infections that involve the urethra, bladder, and, in much more severe cases, even kidneys. These infections represent one of the most common diseases worldwide. Various pathogens are responsible for this condition, the most common being Escherichia coli (E. coli). Bromelain is a proteolytic complex obtained from the stem and stalk of Ananas comosus (L.) Merr. showing several beneficial activities. In addition to bromelain, N-acetylcysteine (NAC) has also been used. Methods: The purpose of this experiment was to evaluate the antibacterial, anti-motility, and anti-biofilm effects of a new polyenzymatic complex (DIF17BRO^®) in combination with NAC (the Formulation) on various strains of E. coli isolated from patients with UTIs. Subsequently, the anti-inflammatory and antioxidant effects of the Formulation were studied in an ex vivo model of cystitis, using bladder samples from mice exposed to E. coli lipopolysaccharide (LPS). Results: Our results showed that the Formulation significantly affects the capability of bacteria to form biofilm and reduces the bacteria amount in the mature biofilm. Moreover, it combines the interesting properties of NAC and a polyenzyme plant complex based on bromelain in a right dose to affect the E. coli adhesion capability. Finally, the Formulation exhibited protective effects, as confirmed by the inhibitory activities on multiple inflammatory and oxidative stress-related pathways on bladder specimens exposed to LPS. Conclusions: This blend of active compounds could represent a promising and versatile approach to use to overcome the limitations associated with conventional therapies. Full article

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies. Full article

Cardiovascular calcification, characterized by hydroxyapatite deposition in the arterial wall and heart valves, is associated with high cardiovascular morbidity and mortality. Cardiovascular calcification is a hallmark of aging but is frequently seen in association with chronic diseases, such as chronic kidney disease (CKD), diabetes, dyslipidemia, and hypertension in the younger population as well. Currently, there is no therapeutic approach to prevent or cure cardiovascular calcification. The pathophysiology of cardiovascular calcification is highly complex and involves osteogenic differentiation of various cell types of the cardiovascular system, such as vascular smooth muscle cells and valve interstitial cells. In vitro cellular and ex vivo tissue culture models are simple and useful tools in cardiovascular calcification research. These models contributed largely to the discoveries of the numerous calcification inducers, inhibitors, and molecular mechanisms. In this review, we provide an overview of the in vitro cell culture and the ex vivo tissue culture models applied in the research of cardiovascular calcification. Full article