Strategies used by nurses regarding underreporting of rural work accidents due to pesticide use.

OBJECTIVE: To learn the strategies used regarding underreporting of pesticide use in rural areas. METHODS: A qualitative study was carried out in eight primary healthcare units in rural areas and two emergency care units in a municipality in southern Brazil. Data collection took place in 2023 through interviews. Twenty professional nurses participated. The data was submitted to content analysis. RESULTS: The strategies identified were lifelong and continuing education for the professionals who carry out the notification, active search and training of workers who deal directly with this type of substance, computerizing the notification by filling in the forms online, and carrying out research on the subject. FINAL CONSIDERATIONS: Nurses play an important role in reporting occupational accidents caused by the use of pesticides, improving occupational safety in rural areas.

The spatially resolved transcriptome signatures of glomeruli in chronic kidney disease.

Here, we used digital spatial profiling (DSP) to describe the glomerular transcriptomic signatures that may characterize the complex molecular mechanisms underlying progressive kidney disease in Alport syndrome, focal segmental glomerulosclerosis, and membranous nephropathy. Our results revealed significant transcriptional heterogeneity among diseased glomeruli, and this analysis showed that histologically similar glomeruli manifested different transcriptional profiles. Using glomerular pathology scores to establish an axis of progression, we identified molecular pathways with progressively decreased expression in response to increasing pathology scores, including signal recognition particle-dependent cotranslational protein targeting to membrane and selenocysteine synthesis pathways. We also identified a distinct signature of upregulated and downregulated genes common to all the diseases investigated when compared with nondiseased tissue from nephrectomies. These analyses using DSP at the single-glomerulus level could help to increase insight into the pathophysiology of kidney disease and possibly the identification of biomarkers of disease progression in glomerulopathies.

Urine-derived podocytes from steroid resistant nephrotic syndrome patients as a model for renal-progenitor derived extracellular vesicles effect and drug screening.

Background: Personalized disease models are crucial for assessing the specific response of diseased cells to drugs, particularly novel biological therapeutics. Extracellular vesicles (EVs), nanosized vesicles released by cells for intercellular communication, have gained therapeutic interest due to their ability to reprogram target cells. We here utilized urinary podocytes obtained from children affected by steroid-resistant nephrotic syndrome with characterized genetic mutations as a model to test the therapeutic potential of EVs derived from kidney progenitor cells. Methods: EVs were isolated from kidney progenitor cells (nKPCs) derived from the urine of a preterm neonate. Three lines of urinary podocytes obtained from nephrotic patients' urine and a line of Alport patient podocytes were characterized and used to assess albumin permeability in response to various drugs or to nKPC-EVs. RNA sequencing was conducted to identify commonly modulated pathways. Results: Podocytes appeared unresponsive to pharmacological treatments, except for a podocyte line demonstrating responsiveness, in alignment with the patient's clinical response at 48 months. At variance, treatment with the nKPC-EVs was able to significantly reduce permeability in all the steroid-resistant patients-derived podocytes as well as in the line of Alport-derived podocytes. RNA sequencing of nKPC-EV-treated podocytes revealed the common upregulation of two genes (small ubiquitin-related modifier 1 (SUMO1) and Sentrin-specific protease 2 (SENP2)) involved in the SUMOylation pathway, a process recently demonstrated to play a role in slit diaphragm stabilization. Gene ontology analysis on podocyte expression profile highlighted cell-to-cell adhesion as the primary upregulated biological activity in treated podocytes. Conclusions: nKPCs emerge as a promising non-invasive source of EVs with potential therapeutic effects on podocyte dysfunction. Furthermore, our findings suggest the possibility of establishing a non-invasive in vitro model for screening regenerative compounds on patient-derived podocytes.

C3aR-initiated signaling is a critical mechanism of podocyte injury in membranous nephropathy.

The deposition of antipodocyte autoantibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.

Strategies used by nurses regarding underreporting of rural work accidents due to pesticide use / Estrategias utilizadas por los enfermeros en relación con el subregistro de accidentes laborales rurales causados por el uso de plaguicidas / Estratégias utilizadas por enfermeiros sobre subnotificações de acidentes de trabalho rural por uso de agrotóxicos

ABSTRACT Objective: To learn the strategies used regarding underreporting of pesticide use in rural areas. Methods: A qualitative study was carried out in eight primary healthcare units in rural areas and two emergency care units in a municipality in southern Brazil. Data collection took place in 2023 through interviews. Twenty professional nurses participated. The data was submitted to content analysis. Results: The strategies identified were lifelong and continuing education for the professionals who carry out the notification, active search and training of workers who deal directly with this type of substance, computerizing the notification by filling in the forms online, and carrying out research on the subject. Final considerations: Nurses play an important role in reporting occupational accidents caused by the use of pesticides, improving occupational safety in rural areas.

RESUMEN Objetivo: Conocer las estrategias utilizadas para el subregistro del uso de plaguicidas en áreas rurales. Métodos: Estudio cualitativo realizado en ocho unidades de atención primaria en áreas rurales y dos unidades de atención de emergencia en un municipio del sur de Brasil. La recopilación de datos se realizó en 2023 mediante entrevistas. Participaron 20 profesionales de enfermería. Los datos fueron sometidos a análisis de contenido. Resultados: Las estrategias identificadas fueron la educación continua y permanente de los profesionales que realizan la notificación, la búsqueda activa y la capacitación de los trabajadores que tratan directamente con este tipo de sustancia, la informatización de la notificación mediante el llenado de los formularios en línea y la realización de investigaciones sobre el tema. Consideraciones finales: Los enfermeros desempeñan un papel importante en la notificación de accidentes laborales causados por el uso de plaguicidas, mejorando la seguridad laboral en las zonas rurales.

RESUMO Objetivo: Conhecer as estratégias utilizadas acerca das subnotificações por uso de agrotóxicos no meio rural. Método: Estudo qualitativo realizado em oito unidades básicas de regiões rurais e duas unidades de pronto atendimento de um município do sul do Brasil. A coleta de dados deu-se em 2023, por entrevistas. Participaram 20 profissionais enfermeiros. Os dados foram submetidos à análise de conteúdo. Resultados: Apontaram como estratégias a realização da educação permanente e continuada dos profissionais que realizam a notificação, a busca ativa e a capacitação dos trabalhadores que lidam diretamente com esse tipo de substância, a informatização da notificação por meio do preenchimento das fichas online e a realização de pesquisas acerca da temática. Considerações Finais: Os enfermeiros desempenham um papel importante na notificação de acidentes de trabalho pelo uso de agrotóxicos, melhorando a segurança ocupacional no meio rural.

Extracellular vesicles in kidney development and pediatric kidney diseases.

Extracellular vesicles (EVs) are membranous cargo particles that mediate intercellular communication. They are heterogeneous in size and mechanism of release, and found in all biological fluids. Since EV content is in relation to the originating cell type and to its physiopathological conditions, EVs are under study to understand organ physiology and pathology. In addition, EV surface cargo, or corona, can be influenced by the microenvironment, leading to the concept that EV-associated molecules can represent useful biomarkers for diseases. Recent studies also focus on the use of natural, engineered, or synthetic EVs for therapeutic purposes. This review highlights the role of EVs in kidney development, pediatric kidney diseases, including inherited disorders, and kidney transplantation. Although few studies exist, they have promising results and may guide researchers in this field. Main limitations, including the influence of age on EV analyses, are also discussed.

Mitochondria Transplantation Mitigates Damage in an In Vitro Model of Renal Tubular Injury and in an Ex Vivo Model of DCD Renal Transplantation.

OBJECTIVES: To test whether mitochondrial transplantation (MITO) mitigates damage in 2 models of acute kidney injury (AKI). BACKGROUND: MITO is a process where exogenous isolated mitochondria are taken up by cells. As virtually any morbid clinical condition is characterized by mitochondrial distress, MITO may find a role as a treatment modality in numerous clinical scenarios including AKI. METHODS: For the in vitro experiments, human proximal tubular cells were damaged and then treated with mitochondria or placebo. For the ex vivo experiments, we developed a non-survival ex vivo porcine model mimicking the donation after cardiac death renal transplantation scenario. One kidney was treated with mitochondria, although the mate organ received placebo, before being perfused at room temperature for 24 hours. Perfusate samples were collected at different time points and analyzed with Raman spectroscopy. Biopsies taken at baseline and 24 hours were analyzed with standard pathology, immunohistochemistry, and RNA sequencing analysis. RESULTS: In vitro, cells treated with MITO showed higher proliferative capacity and adenosine 5'-triphosphate production, preservation of physiological polarization of the organelles and lower toxicity and reactive oxygen species production. Ex vivo, kidneys treated with MITO shed fewer molecular species, indicating stability. In these kidneys, pathology showed less damage whereas RNAseq analysis showed modulation of genes and pathways most consistent with mitochondrial biogenesis and energy metabolism and downregulation of genes involved in neutrophil recruitment, including IL1A, CXCL8, and PIK3R1. CONCLUSIONS: MITO mitigates AKI both in vitro and ex vivo.

Identification and Characterization of the Wilms Tumor Cancer Stem Cell.

A nephrogenic progenitor cell (NP) with cancer stem cell characteristics driving Wilms tumor (WT) using spatial transcriptomics, bulk and single cell RNA sequencing, and complementary in vitro and transplantation experiments is identified and characterized. NP from WT samples with NP from the developing human kidney is compared. Cells expressing SIX2 and CITED1 fulfill cancer stem cell criteria by reliably recapitulating WT in transplantation studies. It is shown that self-renewal versus differentiation in SIX2+CITED1+ cells is regulated by the interplay between integrins ITGß1 and ITGß4. The spatial transcriptomic analysis defines gene expression maps of SIX2+CITED1+ cells in WT samples and identifies the interactive gene networks involved in WT development. These studies define SIX2+CITED1+ cells as the nephrogenic-like cancer stem cells of WT and points to the renal developmental transcriptome changes as a possible driver in regulating WT formation and progression.

Differential detection workflows for multi-sample single-cell RNA-seq data.

In single-cell transcriptomics, differential gene expression (DE) analyses typically focus on testing differences in the average expression of genes between cell types or conditions of interest. Single-cell transcriptomics, however, also has the promise to prioritise genes for which the expression differ in other aspects of the distribution. Here we develop a workflow for assessing differential detection (DD), which tests for differences in the average fraction of samples or cells in which a gene is detected. After benchmarking eight different DD data analysis strategies, we provide a unified workflow for jointly assessing DE and DD. Using simulations and two case studies, we show that DE and DD analysis provide complementary information, both in terms of the individual genes they report and in the functional interpretation of those genes.

Regenerative medicine technologies applied to transplant medicine. An update.

Regenerative medicine (RM) is changing how we think and practice transplant medicine. In regenerative medicine, the aim is to develop and employ methods to regenerate, restore or replace damaged/diseased tissues or organs. Regenerative medicine investigates using tools such as novel technologies or techniques, extracellular vesicles, cell-based therapies, and tissue-engineered constructs to design effective patient-specific treatments. This review illustrates current advancements in regenerative medicine that may pertain to transplant medicine. We highlight progress made and various tools designed and employed specifically for each tissue or organ, such as the kidney, heart, liver, lung, vasculature, gastrointestinal tract, and pancreas. By combing both fields of transplant and regenerative medicine, we can harbor a successful collaboration that would be beneficial and efficacious for the repair and design of de novo engineered whole organs for transplantations.

Immune responses to SARS-CoV-2 in dialysis and kidney transplantation.

Despite progressive improvements in the management of patients with coronavirus disease 2019 (COVID-19), individuals with end-stage kidney disease (ESKD) are still at high risk of infection-related complications. Although the risk of infection in these patients is comparable to that of the general population, their lower rate of response to vaccination is a matter of concern. When prevention strategies fail, infection is often severe. Comorbidities affecting patients on maintenance dialysis and kidney transplant recipients clearly account for the increased risk of severe COVID-19, while the role of uremia and chronic immunosuppression is less clear. Immune monitoring studies have identified differences in the innate and adaptive immune response against the virus that could contribute to the increased disease severity. In particular, individuals on dialysis show signs of T cell exhaustion that may impair antiviral response. Similar to kidney transplant recipients, antibody production in these patients occurs, but with delayed kinetics compared with the general population, leaving them more exposed to viral expansion during the early phases of infection. Overall, unique features of the immune response during COVID-19 in individuals with ESKD may occur with severe comorbidities affecting these individuals in explaining their poor outcomes.

The spatially resolved transcriptional profile of acute T cell-mediated rejection in a kidney allograft.

Analysis of the transcriptional profile of graft biopsies represents a promising strategy to study T cell-mediated-rejection (TCMR), also known as acute cellular rejection. However, bulk RNA sequencing of graft biopsies may not capture the focal nature of acute rejection. Herein, we used the whole exome GeoMX Digital Space Profiling platform to study five tubular and three glomerular regions of interest in the kidney graft biopsy from a patient with a chronic-active TCMR episode and in analogous areas from two different normal kidney control biopsies. All kidney sections were from paraffin blocks. Overall, inflammatory genes were significantly upregulated in the tubular areas of the TCMR biopsy and showed an enrichment for gene-ontology terms associated with T-cell activation, differentiation, and proliferation. Enrichment analysis of the 100 genes with the highest coefficient of variation across the TCMR tubular regions of interest revealed that these highly variable genes are involved in kidney development and injury and interestingly do not associate with the 2019 Banff classification pathology scores within the individual regions of interest. Spatial transcriptomics allowed us to unravel a previously unappreciated variability across different areas of the TCMR biopsy related to the graft response to the alloimmune attack, rather than to the immune cells. Thus, our approach has the potential to decipher clinically relevant, new pathogenic mechanisms, and therapeutic targets in acute cellular rejection and other kidney diseases with a focal nature.

Effect of disease progression on the podocyte cell cycle in Alport Syndrome.

Progression of glomerulosclerosis is associated with loss of podocytes with subsequent glomerular tuft instability. It is thought that a diminished number of podocytes may be able to preserve tuft stability through cell hypertrophy associated with cell cycle reentry. At the same time, reentry into the cell cycle risks podocyte detachment if podocytes cross the G1/S checkpoint and undergo abortive cytokinesis. In order to study cell cycle dynamics during chronic kidney disease (CKD) development, we used a FUCCI model (fluorescence ubiquitination-based cell cycle indicator) of mice with X-linked Alport Syndrome. This model exhibits progressive CKD and expresses fluorescent reporters of cell cycle stage exclusively in podocytes. With the development of CKD, an increasing fraction of podocytes in vivo were found to be in G1 or later cell cycle stages. Podocytes in G1 and G2 were hypertrophic. Heterozygous female mice, with milder manifestations of CKD, showed G1 fraction numbers intermediate between wild-type and male Alport mice. Proteomic analysis of podocytes in different cell cycle phases showed differences in cytoskeleton reorganization and metabolic processes between G0 and G1 in disease. Additionally, in vitro experiments confirmed that damaged podocytes reentered the cell cycle comparable to podocytes in vivo. Importantly, we confirmed the upregulation of PDlim2, a highly expressed protein in podocytes in G1, in a patient with Alport Syndrome, confirming our proteomics data in the human setting. Thus, our data showed that in the Alport model of progressive CKD, podocyte cell cycle distribution is altered, suggesting that cell cycle manipulation approaches may have a role in the treatment of various progressive glomerular diseases characterized by podocytopenia.

Generation of a Glomerular Filtration Barrier on a Glomerulus-on-a-Chip Platform.

Despite an enormous investment of clinical and financial resources, chronic kidney disease (CKD) remains a global health threat. The lack of reliable in vitro systems that can efficiently mimic the renal and glomerular environment has hampered our ability to successfully develop novel and more renal specific drugs. Even though some success in generating in vitro tubule analogues and kidney organoids has been described, a major challenge remains for the in vitro assembly of the filtration unit of the kidney, the glomerulus. We have recently developed a novel glomerulus-on-a-chip system that mimics the characteristic and functionality of the glomerular filtration barrier, including its response to injury. This system recapitulates the functions and structure of the in vivo glomerulus, including permselectivity; indeed, we have confirmed free diffusion of insulin as well as impermeability to physiological concentrations of albumin. Exposure to nephrotoxic agents like puromycin aminonucleoside leads to a significant increase in albumin leakage. When exposed to sera from patients with anti-podocyte autoantibodies, the chip shows albumin leakage to an extent proportional to in vivo clinical data, phenomenon not observed with sera from either healthy controls, confirming functional response to injury. We describe here the detailed procedure to obtain a glomerulus-on-a-chip system that replicates both phenotypically and functionally the in vivo glomerular microenvironment.

Intravital imaging reveals glomerular capillary distension and endothelial and immune cell activation early in Alport syndrome.

Alport syndrome (AS) is a genetic disorder caused by mutations in type IV collagen that lead to defective glomerular basement membrane, glomerular filtration barrier (GFB) damage, and progressive chronic kidney disease. While the genetic basis of AS is well known, the molecular and cellular mechanistic details of disease pathogenesis have been elusive, hindering the development of mechanism-based therapies. Here, we performed intravital multiphoton imaging of the local kidney tissue microenvironment in a X-linked AS mouse model to directly visualize the major drivers of AS pathology. Severely distended glomerular capillaries and aneurysms were found accompanied by numerous microthrombi, increased glomerular endothelial surface layer (glycocalyx) and immune cell homing, GFB albumin leakage, glomerulosclerosis, and interstitial fibrosis by 5 months of age, with an intermediate phenotype at 2 months. Renal histology in mouse or patient tissues largely failed to detect capillary aberrations. Treatment of AS mice with hyaluronidase or the ACE inhibitor enalapril reduced the excess glomerular endothelial glycocalyx and blocked immune cell homing and GFB albumin leakage. This study identified central roles of glomerular mechanical forces and endothelial and immune cell activation early in AS, which could be therapeutically targeted to reduce mechanical strain and local tissue inflammation and improve kidney function.

Emerging Technologies to Study the Glomerular Filtration Barrier.

Kidney disease is characterized by loss of glomerular function with clinical manifestation of proteinuria. Identifying the cellular and molecular changes that lead to loss of protein in the urine is challenging due to the complexity of the filtration barrier, constituted by podocytes, glomerular endothelial cells, and glomerular basement membrane. In this review, we will discuss how technologies like single cell RNA sequencing and bioinformatics-based spatial transcriptomics, as well as in vitro systems like kidney organoids and the glomerulus-on-a-chip, have contributed to our understanding of glomerular pathophysiology. Knowledge gained from these studies will contribute toward the development of personalized therapeutic approaches for patients affected by proteinuric diseases.