The Neurotoxic Effect of Environmental Temperature Variation in Adult Zebrafish (Danio rerio).

Neurotoxicity consists of the altered functionality of the nervous system caused by exposure to chemical agents or altered chemical-physical parameters. The neurotoxic effect can be evaluated from the molecular to the behavioural level. The zebrafish Danio rerio is a model organism used in many research fields, including ecotoxicology and neurotoxicology. Recent studies by our research group have demonstrated that the exposure of adult zebrafish to low (18 °C) or high (34 °C) temperatures alters their brain proteome and fish behaviour compared to control (26 °C). These results showed that thermal variation alters the functionality of the nervous system, suggesting a temperature-induced neurotoxic effect. To demonstrate that temperature variation can be counted among the factors that generate neurotoxicity, eight different protein datasets, previously published by our research group, were subjected to new analyses using an integrated proteomic approach by means of the Ingenuity Pathway Analysis (IPA) software (Release December 2022). The datasets consist of brain proteome analyses of wild type adult zebrafish kept at three different temperatures (18 °C, 26 °C, and 34 °C) for 4 days (acute) or 21 days (chronic treatment), and of BDNF+/- and BDNF-/- zebrafish kept at 26 °C or 34 °C for 21 days. The results (a) demonstrate that thermal alterations generate an effect that can be defined as neurotoxic (p value ≤ 0.05, activation Z score ≤ -2 or ≥2), (b) identify 16 proteins that can be used as hallmarks of the neurotoxic processes common to all the treatments applied and (c) provide three protein panels (p value ≤ 0.05) related to 18 °C, 34 °C, and BDNF depletion that can be linked to anxiety-like or boldness behaviour upon these treatments.

Kidney stone matrix proteins: Role in stone formation.

Stone formation is induced by an increased level of urine crystallization promoters and reduced levels of its inhibitors. Crystallization inhibitors include citrate, magnesium, zinc, and organic compounds such as glycosaminoglycans. In the urine, there are various proteins, such as uromodulin (Tamm-Horsfall protein), calgranulin, osteopontin, bikunin, and nephrocalcin, that are present in the stone matrix. The presence of several carboxyl groups in these macromolecules reduces calcium oxalate monohydrate crystal adhesion to the urinary epithelium and could potentially protect against lithiasis. Proteins are the most abundant component of kidney stone matrix, and their presence may reflect the process of stone formation. Many recent studies have explored the proteomics of urinary stones. Among the stone matrix proteins, the most frequently identified were uromodulin, S100 proteins (calgranulins A and B), osteopontin, and several other proteins typically engaged in inflammation and immune response. The normal level and structure of these macromolecules may constitute protection against calcium salt formation. Paradoxically, most of them may act as both promoters and inhibitors depending on circumstances. Many of these proteins have other functions in modulating oxidative stress, immune function, and inflammation that could also influence stone formation. Yet, the role of these kidney stone matrix proteins needs to be established through more studies comparing urinary stone proteomics between stone formers and non-stone formers.

Mass spectrometry-based proteomic strategy for ecchymotic skin examination in forensic pathology.

Mass spectrometry (MS)-based proteomics has recently attracted the attention from forensic pathologists. This work is the first report of the development of a shotgun bottom-up proteomic approach based on rapid protein extraction and nano-liquid chromatography/high-resolution mass spectrometry applied to full-thickness human skin for the differential analysis of normal and ecchymotic tissues to identify new biomarkers for bruise characterization and dating. We identified around 2000 proteins from each pooled extract. The method showed excellent precision on independent replicates, with Pearson correlation coefficients always higher than 95%. Glycophorin A, a known biomarker of vital wounds from immunochemical studies, was identified only in ecchymotic tissues, as confirmed by Western blotting analysis. This finding suggests that this protein can be used as a MS-detectable biomarker of wound vitality. By focusing on skin samples from individuals with known wound dating, besides Glycophorin A, other proteins differentially expressed in ecchymotic samples and dependant on wound age were identified, although further analysis on larger datasets are needed to validate these findings. This study paves the way for an in-depth investigation of the potential of MS-based techniques for wound examination in forensic pathology, overcoming the limitations of immunochemical assays.

Nuevas estrategias terapéuticas para tratar la hiperfosfatemia en la IRC: foco en la absorción intestinal / New therapeutic strategies to treat hyperphosphatemia in CRF: focus on intestinal absorption

RESUMEN El manejo de la hiperfosfatemia de los pacientes con insuficiencia renal crónica en diálisis permanece como un desafío. A pesar de utilizar un enfoque multifacético que incluye la restricción dietética, la remoción de fósforo por la diálisis y el uso de quelantes de fósforo, esta estrategia múltiple no logra reducir los niveles de fósforo en más de 2 mg/dl. El control de fósforo de los pacientes en diálisis es fundamental en razón de la relación monotónica entre los niveles séricos de fosfato y el incremento del riesgo cardiovascular. Por lo tanto, hay una necesidad de explorar nuevas estrategias para reducir los niveles séricos de fosfato a niveles normales. Recientes avances en nuestra compresión de los mecanismos que subyacen a la homeostasis del fósforo sugieren que el transporte gastrointestinal del fósforo podría ser un objetivo. Recientemente se han desarrollado inhibidores de los cotransportadores sodio fosfato del intestino y se ha revalorizado el uso de la nicotinamida, en su formulación de liberación prolongada, que también actuaria por ese mecanismo. También se han drogas como el tenapanor, que inhibiendo el intercambiador sodio/hidrogeno isoforma 3 del enterocito, disminuyen la absorción paracelular de fósforo.

ABSTRACT Management of hyperphosphatemia in patients with chronic renal failure on dialysis remains challenging. Despite using a multifaceted approach that includes dietary restriction, phosphorus removal by dialysis, and phosphate binders, these multiple strategies fail to reduce phosphorus levels by more than 2 mg/dL. Phosphorus control in dialysis patients is essential due to the monotonic relationship between serum phosphate levels and increased cardiovascular risk. Therefore, there is a need to explore new strategies to reduce serum phosphate levels to normal levels. Recent advances in understanding the mechanisms underlying phosphorus homeostasis suggest that the gastrointestinal transport of phosphorus could be a target. Inhibitors of intestinal sodium phosphate cotransporters recently developed, and using of nicotinamide, in its prolonged release formulation, which would also act by this mechanism, has been revalued. There have also been drugs such as tenapanor, which, by inhibiting the isoform three sodium/hydrogen exchanger of the enterocyte, decreases the paracellular absorption of phosphorus.

Role of prolyl hydroxylase/HIF-1 signaling in vascular calcification.

Morbidity and mortality of chronic kidney disease (CKD) patients are largely associated with vascular calcification, an actively regulated process in which vascular smooth muscle cells (VSMCs) change into cells similar to osteocytes/chondrocytes, known as trans-differentiation. Cellular and systemic response to low oxygen (hypoxia) is regulated by the prolyl hydroxylase/hypoxia-inducible factor (HIF)-1 pathway. Recent studies highlighted that hypoxia-mediated activation of HIF-1 induces trans-differentiation of VSMCs into bone-forming type through an increase in osteo-/chondrogenic genes. Inhibition of the HIF-1 pathway abolished osteochondrogenic differentiation of VSMCs. Hypoxia strongly enhanced elevated phosphate-induced VSMC osteogenic trans-differentiation and calcification. HIF-1 was shown to be essential for phosphate enhanced VSMC calcification. O2-dependent degradation HIF-1 is triggered by the prolyl hydroxylase domain proteins (PHD). Prolyl hydroxylase inhibitors, daprodustat and roxadustat, increase high phosphate-induced VC in VSMCs, stabilizing HIF-1α and activating the HIF-1 pathway in these cells. Whether the use of these PHD inhibitors to treat anemia in CKD patients will favor the development and progression of vascular calcification remains to be explored.

Phosphoproteomic mapping of CCR5 and ACKR2 signaling properties.

ACKR2 is an atypical chemokine receptor which is structurally uncoupled from G proteins and is unable to activate signaling pathways used by conventional chemokine receptors to promote cell migration. Nonetheless, ACKR2 regulates inflammatory and immune responses by shaping chemokine gradients in tissues via scavenging inflammatory chemokines. To investigate the signaling pathways downstream to ACKR2, a quantitative SILAC-based phosphoproteomic analysis coupled with a systems biology approach with network analysis, was carried out on a HEK293 cell model expressing either ACKR2 or its conventional counterpart CCR5. The model was stimulated with the common agonist CCL3L1 for short (3 min) and long (30 min) durations. As expected, many of the identified proteins are known to participate in conventional signal transduction pathways and in the regulation of cytoskeleton dynamics. However, our analyses revealed unique phosphorylation and network signatures, suggesting roles for ACKR2 other than its scavenger activity. In conclusion, the mapping of phosphorylation events at a holistic level indicated that conventional and atypical chemokine receptors differ in signaling properties. This provides an unprecedented level of detail in chemokine receptor signaling and identifying potential targets for the regulation of ACKR2 and CCR5 function.

Brain Proteome and Behavioural Analysis in Wild Type, BDNF+/- and BDNF-/- Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures.

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 °C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF+/- and knock out BDNF-/-) were kept for 21 days at 26 °C or 34 °C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment.

Role of claudins in idiopathic hypercalciuria and renal lithiasis.

Paracellular transport in the kidney is mediated by a family of proteins located in the tight junctions called claudins which confers its ionic selectivity. Claudin-2 is highly expressed in the proximal tubule and descending limb of Henle and mediate paracellular reabsorption of sodium and calcium cations. In the thick ascending limb of Henle (TALH) calcium is reabsorbed by a paracellular channel formed by Claudin-16 and-19. Claudin-16 mediates cationic permeability while Claudin-19 increases the cationic selectivity of Claudin-16 by blocking anionic permeability. On the other hand, Claudin 14, that is also located in TALH, inhibits the paracellular permeability of Claudin-16 to calcium. Recent wide genomic association analysis studies have detected four common synonymous variants (genetic polymorphisms of a single nucleotide, SNPs) at the locus of Claudin-14 gene that were significantly associated with the presence of renal lithiasis. Another study of wide genomic association and nephrolithiasis was carried out in the general population but including chromosome X, where claudin-2 gene is located. They detected nine SNPs that had a significant association with renal lithiasis risk. A greater knowledge of the paracellular pathway controlled by claudins and its regulation will allow us to develop future new treatments for idiopathic hypercalciuria and renal lithiasis.

Hyponatremia, Inflammation at Admission, and Mortality in Hospitalized COVID-19 Patients: A Prospective Cohort Study.

Background: Systemic inflammation has been associated with severe coronavirus disease 2019 (COVID-19) disease and mortality. Hyponatremia can result from inflammation due to non-osmotic stimuli for vasopressin production. Methods: We prospectively studied 799 patients hospitalized with COVID-19 between March 7 and November 7, 2020, at Hospital Posadas in Buenos Aires, Argentina in order to evaluate the association between hyponatremia, inflammation, and its impact on clinical outcomes. Admission biochemistries, high-sensitivity C-reactive protein (hsCRP), ferritin, patient demographics, and outcome data were recorded. Outcomes (within 30 days after symptoms) evaluated included ICU admission, mechanical ventilation, dialysis-requiring acute kidney injury (AKI), and in-hospital mortality. Length of hospital stay (in days) were evaluated using comprehensive data from the EHR. Results: Hyponatremia (median Na = 133 mmol/L) was present on admission in 366 (45.8%). Hyponatremic patients had higher hsCRP (median 10.3 [IR 4.8-18.4] mg/dl vs. 6.6 [IR 1.6-14.0] mg/dl, p < 0.01) and ferritin levels (median 649 [IQR 492-1,168] ng/dl vs. 393 [IQR 156-1,440] ng/dl, p = 0.02) than normonatremic patients. Hyponatremia was associated with higher odds of an abnormal hsCRP (unadjusted OR 5.03, 95%CI: 2.52-10.03), and remained significant after adjustment for potential confounders (adjusted OR 4.70 [95%CI: 2.33-9.49], p < 0.01). Hyponatremic patients had increased mortality on unadjusted (HR 3.05, 95%CI: 2.14-4.34) and adjusted (HR 2.76, 95%CI:1.88-4.06) in Cox proportional hazard models. Crude 30-day survival was lower for patients with hyponatremia at admission (mean [SD] survival 22.1 [0.70] days) compared with patients who were normonatremic (mean [SD] survival 27.2 [0.40] days, p < 0.01). Conclusion: Mild hyponatremia on admission is common, is associated with systemic inflammation and is an independent risk factor for hospital mortality. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT04493268.

Sarcopenia in hemodialysis patients from Buenos Aires, Argentina.

OBJECTIVES: Sarcopenia is the loss of skeletal muscle mass and function that occurs with aging that can lead to greater morbidity and mortality. Chronic kidney disease and hemodialysis (HD) favors the development of sarcopenia. We studied the prevalence of sarcopenia and its components using European Working Group on Sarcopenia in Elderly People 2 proposed criteria and risk factors for its development in HD patients. METHODS: In 100 adult HD patients, we evaluated: hand grip strength (HGS), muscle mass by dual energy X-ray absorptiometry and physical performance (gait-speed and sit-stand test). RESULTS: Sixty patients were male and 40 were female; mean age 55.6 years. Prevalence of sarcopenia was 16% (11.1% in males and 25% in females; P = 0.05); 7% had severe sarcopenia. Prevalence of low HGS was 33% in males and 28% in females; low muscle mass was 30% in males but 70% in females and low physical performance 23% in males and 45% in females. Falls were reported by 23 patients. Patients with lower HGS had a higher prevalence of falls in the last year (40% two or more falls; P = 0.03). Only females with sarcopenia had lower bone mineral content. Neither age, body mass index, time on dialysis, or prevalence of diabetes predicted sarcopenia. CONCLUSIONS: A significant proportion of dialysis patients had sarcopenia, more frequent in females. Low HGS was associated with a higher prevalence of falls. Only females with sarcopenia had lower bone mineral content.

High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma.

Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. In this study, the substrate profile of plasma proteases was evaluated using two nano-LC-ESI-MS/MS methods. Multiplex substrate profiling by mass spectrometry (MSP-MS) quantifies plasma protease activity in vitro using a global and unbiased library of synthetic peptide reporter substrates, and shotgun peptidomics quantifies protein degradation products that have been generated in vivo by proteases. The two approaches gave complementary results since they both highlight key peptidase activities in plasma including amino- and carboxypeptidases with different substrate specificity profiles. These assays provide a significant advantage over traditional approaches, such as fluorogenic peptide reporter substrates, because they can detect active plasma proteases in a global and unbiased manner, in comparison to detecting select proteases using specific reporter substrates. We discovered that plasma proteins are cleaved by endoproteases and these peptide products are subsequently degraded by amino- and carboxypeptidases. The exopeptidases are more active and stable in plasma and therefore were found to be the most active proteases in the in vitro assay. The protocols presented here set the groundwork for studies to evaluate changes in plasma proteolytic activity in shock.

Proteomic Analysis Reveals a Mitochondrial Remodeling of ßTC3 Cells in Response to Nanotopography.

Recently, using cluster-assembled zirconia substrates with tailored roughness produced by supersonic cluster beam deposition, we demonstrated that ß cells can sense nanoscale features of the substrate and can translate these stimuli into a mechanotransductive pathway capable of preserveing ß-cell differentiation and function in vitro in long-term cultures of human islets. Using the same proteomic approach, we now focused on the mitochondrial fraction of ßTC3 cells grown on the same zirconia substrates and characterized the morphological and proteomic modifications induced by the nanostructure. The results suggest that, in ßTC3 cells, mitochondria are perturbed by the nanotopography and activate a program involving metabolism modification and modulation of their interplay with other organelles. Data were confirmed in INS1E, a different ß-cell model. The change induced by the nanostructure can be pro-survival and prime mitochondria for a metabolic switch to match the new cell needs.

Acciones óseas de las hormonas de la neurohipófisis / Bone actions of neuro hypophyseal hormones

La oxitocina (OXT) como la arginina-vasopresina (AVP) son dos hormonas primitivas secretadas por la hipófisis posterior. Sus receptores están mucho más ampliamente distribuidos en el organismo de lo que se pensaba originalmente, incluido el hueso. En los estudios preclínicos, la OXT ha mostrado ser anabólica para el hueso, promoviendo la osteogénesis sobre la adipogénesis y favoreciendo la actividad osteoblástica sobre la osteoclástica. Tanto los osteoblastos como los osteoclastos tienen receptores para la OXT, y los efectos de los estrógenos sobre la masa ósea en ratones está mediada por lo menos en parte por la OXT. El mecanismo preciso por el cual la activación de los receptores de oxitocina (OXTR) se traduce en un incremento de la formación ósea permanece poco claro. La AVP también podría afectar el esqueleto en forma directa. Dos de los receptores de la AVP, V1a y V2 están expresados en osteoblastos y osteoclastos. La inyección de AVP en ratones de tipo salvaje aumenta la formación osteoclastos que producen resorción y reduce los osteoblastos formadores de hueso. En forma opuesta, la exposición de precursores osteoblásticos a antagonistas de los receptores V1a o V2, incrementan la osteoblastogénesis, como también lo hace la deleción genética del receptor V1a. (AU)

Both oxytocin (OXT) and argininevasopressin (AVP) are primitive hormones secreted by the posterior pituitary gland. OXT receptors are much more widely distributed in the body than originally thought, including in bone. In preclinical studies, OXT has been shown to be anabolic for bone, promoting osteogenesis over adipogenesis and favoring osteoblastic over osteoclastic activity. Both osteoblasts and osteoclasts have receptors for OXT, and the effects of estrogen on bone mass in mice is mediated at least in part by OXT. The precise mechanism by which the activation of oxytocin receptors (OXTRs) results in an increase in bone formation remains unclear. AVP could also have direct actions on the skeleton. The two AVP receptors, V1a and V2, are expressed in osteoblasts and osteoclasts. Injection of AVP in wild-type mice increases the formation of osteoclasts increasing bone resorption, and reduces bone-forming osteoblasts. On the contrary, the exposure of osteoblastic precursors to V1a and V2 antagonists increase osteoblastogenesis, the same as the genetic deletion of the V1a receptor. (AU)

Chronicity of Uncorrected Hyponatremia and Clinical Outcomes in Older Patients Undergoing Hip Fracture Repair.

Background: Chronic hyponatremia is a risk factor for hip fracture but remains uncorrected in most patients. This study evaluated if preoperative chronicity of uncorrected hyponatremia influences outcomes after hip fracture repair. Materials and Methods: Evaluated were older patients hospitalized for hip fracture repair between 2007 and 2012 with plasma sodium measured at admission and ≥1 preadmission outpatient measurement. Patients were classified as being normonatremic (NN; plasma sodium 135-145 mmol/L), chronic prolonged hyponatremia (CPH; ≥2 consecutive plasma sodium values <135 mmol/L over >90 days), or recent hyponatremia (one plasma sodium <135 mmol/L within 30 days before admission with previously normal plasma sodium). Length of hospital stay, in-hospital death, post-operative complications, 30-day readmission, and long-term mortality were the evaluated outcomes. Multivariable Cox regression was used to evaluate the association of hyponatremia status with outcomes. Results: Among 1,571 eligible patients, 76.7% were NN, 14% had CPH, and 9.1% had RH. Compared with NN patients, CHN patients were older and had more prior heart failure, alcoholism, and anticonvulsant drug use. In multivariable analyses, neither CPH or RH was associated with hospital length of stay, in-hospital or 30-day death, or 30-day readmission, while RH was associated with post-operative sepsis [adjusted odds ratio (aOR) 1.84, 95% CI: 1.01-3.35). Only CPH was independently associated with long-term all-cause death (OR 1.53, 95% CI: 1.12-2.09). Conclusions: Hyponatremia affects nearly 25% of patients undergoing hip fracture repair. Preoperative chronic untreated hyponatremia is associated with increased post-operative mortality following surgical repair of a hip fracture in older patients. Future studies should evaluate if correction of hyponatremia could decrease long-term mortality after hip fracture repair.