Diabetic nephropathy (DN) is considered the main cause of kidney disease in which myofibroblasts lead to renal fibrosis. Macrophages were recently identified as the major source of myofibroblasts in a process known as macrophage–myofibroblast transition (MMT). Adenosine levels increase during DN and in vivo administration of MRS1754, an antagonist of the A_2B adenosine receptor (A_2BAR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A_2BAR and MMT in glomerulosclerosis during DN. Kidneys/glomeruli of non-diabetic, diabetic, and MRS1754-treated diabetic (DM+MRS1754) rats were processed for histopathologic, transcriptomic, flow cytometry, and cellular in vitro analyses. Macrophages were used for in vitro cell migration/transmigration assays and MMT studies. In vivo MRS1754 treatment attenuated the clinical and histopathological signs of glomerulosclerosis in DN rats. Transcriptomic analysis demonstrated a decrease in chemokine-chemoattractants/cell-adhesion genes of monocytes/macrophages in DM+MRS1754 glomeruli. The number of intraglomerular infiltrated macrophages and MMT cells increased in diabetic rats. This was reverted by MRS1754 treatment. In vitro cell migration/transmigration decreased in macrophages treated with MRS1754. Human macrophages cultured with adenosine and/or TGF-β induced MMT, a process which was reduced by MRS1754. We concluded that pharmacologic blockade of A_2BAR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease, which remains incurable. The progression of DN is associated with progressive and irreversible renal fibrosis and also high levels of adenosine. Our aim was to evaluate the effects of ADORA3 antagonism on renal injury in streptozotocin-induced diabetic rats. An ADORA3 antagonist that was administered in diabetic rats greatly inhibited the levels of inflammatory interleukins IL-1β and IL-18, meanwhile when adenosine deaminase was administered, there was a non-selective attenuation of the inflammatory mediators IL-1β, IL-18, IL-6, and induction of IL-10. The ADORA3 antagonist attenuated the high glucose-induced activation of caspase 1 in HK2 cells in vitro. Additionally, ADORA3 antagonisms blocked the increase in caspase 1 and the nuclear localization of NFκB in the renal tubular epithelium of diabetic rats, both events that are involved in regulating the production and activation of IL-1β and IL-18. The effects of the A3 receptor antagonist resulted in the attenuation of kidney injury, as evidenced by decreased levels of the pro-fibrotic marker α-SMA at histological levels and the restoration of proteinuria in diabetic rats. We conclude that ADORA3 antagonism represents a potential therapeutic target that mechanistically works through the selective blockade of the NLRP3 inflammasome.

Background: In type I diabetes mellitus (T1DM) pancreatic β cells are destroyed. Treatment entails exogenous insulin administration and strict diet control, yet optimal glycemic control is hardly attainable. Islet transplant could be an alternative in patients with poor glycemic control, but inefficient islet purification and autoimmune response of patients is still a challenge. For these reasons, it is necessary to explore new cellular sources and immunological isolation methods oriented to develop T1DM cell-based therapies. Aims: We postulate human adipose-derived stem cell (hASC) as an adequate source to generate pancreatic islet cells in vitro, and to produce islet-like structures. Furthermore, we propose microencapsulation of these aggregates as an immunological isolation strategy. Methods: hASC obtained from lipoaspirated fat tissue from human donors were differentiated in vitro to insulin (Ins) and glucagon (Gcg) producing cells. Then, insulin producing cells (IPC) and glucagon producing cells (GPC) were cocultured in low adhesion conditions to form cellular aggregates, and later encapsulated in a sodium alginate polymer. Expression of pancreatic lineage markers and secretion of insulin or glucagon in vitro were analyzed. Results: The results show that multipotent hASC efficiently differentiate to IPC and GPC, and express pancreatic markers, including insulin or glucagon hormones which they secrete upon stimulation (fivefold for insulin in IPC, and fourfold for glucagon, compared to undifferentiated cells). In turn, calculation of the Feret diameter and area of cellular aggregates revealed mean diameters of ~ 80 µm, and 65% of the aggregates reached 4000 µm² at 72 h of formation. IPC/GPC aggregates were then microencapsulated in sodium-alginate polymer microgels, which were found to be more stable when stabilized with Ba²⁺, yielding average diameters of ~ 300 µm. Interestingly, Ba²⁺-microencapsulated aggregates respond to high external glucose with insulin secretion. Conclusions: The IPC/GPC differentiation process from hASC, followed by the generation of cellular aggregates that are later microencapsulated, could represent a possible treatment for T1DM.

Studies have emphasized alleviating fibrogenesis through interference with adenosine signaling in experimental diabetic nephropathy. We found that the in vivo antagonism of the adenosine A_2B receptor (A_2BAR) using MRS1754 in diabetic rats impedes the diabetes-induced glomerular expression of the mesenchymal-like transformation markers Snail and α-SMA, while the loss of the epithelial podocyte-specific proteins nephrin and ZO-1 was prevented. Furthermore, the production of MCP-1, CCL3, TGF-β, and the transcript levels of inflammatory mediators was reduced by A_2BAR antagonism. Using human podocytes in vitro, we demonstrated that A_2BAR antagonism affected the TGF-β-induced activation of SMAD2/-3, as evidenced by the attenuated phosphorylation of SMAD2/-3 and decreased SMAD3 occupancy at target gene promoters following the MRS1754 treatment. Moreover, the non-canonical activation of p65-NF-κB, the primary inflammatory signaling pathway downstream of TGF-β, and the expression of Snail were also reduced by MRS1754. We conclude that an A_2BAR blockade interferes with the pathogenic TGF-β signaling cascade responsible for the phenotypical transformation of podocytes, thereby alleviating diabetic glomerulopathy.

Accurate chromosome segregation and ribosomal gene expression silencing are essential for maintaining genome integrity, and disruptions in these processes are key for oncogenesis and cancer progression. Here, we demonstrate a novel role for the transcriptional co-repressor SKI in regulating rDNA and pericentromeric heterochromatin (PCH) silencing in human cells. We found that SKI localizes to the rDNA promoter on acrocentric chromosomes and is crucial for maintaining H3K9 trimethylation (H3K9me3) and repressing 45S rRNA gene expression. SKI is also associated with BSR and HSATII satellites within PCH, where is necessary for H3K9 methylation and recruitment of SUV39H1 and HP1α, key players for heterochromatin silencing and centromere function. Consequently, SKI deficiency disrupted centromere integrity and resulted in aberrant chromosome segregation, micronuclei formation, and chromosome instability. The identification of SKI as a key participant in the epigenetic-mediated silencing of pericentromeric and ribosomal DNA provides a fundamental insight, paving the way for new research into the intricate relationship between transcriptional regulation and genome instability during cancer progression, and opening novel opportunities for therapeutic intervention.

Purpose: Molecular mechanisms underlying COVID-19 susceptibility and severity are still poorly understood. The presence of genetic risk factors associated with ethnic background has been suggested, highlighting non-European ancestry as a risk factor for hospitalization in the United States. However, the representation of non-European populations in genomic case-control and cohort studies remains insufficient, and Latin American populations have been significantly understudied. Addressing this gap, we established The Chilean COVID-19 Biorepository, a multicentric endeavor comprising high-quality biological samples and associated data collected throughout Chile under stringent biobanking standards that ensure high quality, reproducibility, and interoperability. Participants: The Chilean COVID-19 Biorepository was established by a network of nine nodes distributed in five macro-zones nationwide. The study enrolled adult participants living in Chile who had tested positive for SARS-CoV-2 infection and provided broad written informed consent. Blood samples were collected with EDTA and processed to store blood, plasma, buffy-coat, and DNA. Quality control measures, such as Standard Preanalytical Code (SPREC), incident reporting, DNA concentration, and absorbance ratio (260/280), were implemented to ensure the reliability and quality of the collected samples. Sociodemographic data, habits, clinical information, use of medications, and preexisting pathologies were registered. A weekly iterative workflow was implemented to ensure the quality and integrity of specimens and data. Findings to date: Between October 2020 and February 2021, 2262 participants were recruited, pseudonymized, and categorized by disease severity into six categories, from asymptomatic to lethal. Notably, the Biorepository exhibited high compliance rates (>90%) across all quality control assessed items, reflecting high adherence to biobanking standards. A noteworthy feature of this cohort is the self-identification of 279 participants (12.3%) into thirteen different ethnic groups. Amerindian ancestry from genome-wide genetic data was 44.0%[SD15.5%] and increased to 61.2%[SD19.5%] when considering participants who identified as Native South Americans. As a data-contributor partner of the COVID-19 Host Genetics Initiative, the Chilean COVID-19 Biorepository has contributed to the publication of a second updated genome-wide association study, further enhancing our knowledge of the role of host genetics in susceptibility and severity to SARS-CoV-2. Future plans: The Chilean COVID-19 Biorepository, under the leadership of Latin American researchers from a Latin American country, substantially adds to the integration of Latin American populations in the global collections landscape. Just as ocurred with the COVID-19 Host Genetics Initiative, we expect that this repository will attract global network collaborations for comparative studies on the effects of COVID-19 across diverse populations, including exploring potential genetic advantages or disadvantages in the context of SARS-CoV-2 infection. Researchers involved in establishing this biorepository are currently associated within a collaborative initiative known as COVID-19 Genomics Network (C19-GenoNet), aimed to accelerate the identification of genetic factors in both hosts and pathogens that influence the short and long-term outcomes of SARS-CoV-2 infection. The broad informed consent utilized enables longitudinal cohort follow-up, thereby allowing for investigating the long-term consequences of SARS-CoV-2 infection, particularly concerning long-COVID. Thus, participants of this cohort were re-contacted to assess the development of long-COVID through a survey-based approach. The re-contact and recruitment procedures yielded a high response rate (82.11%), demonstrating strong participant engagement. In this case as well, this cohort has been leveraged by collaboration with the COVID-19 Host Genetics Initiative for the forthcoming publication of a genome-wide association study on long-COVID. The concerted endeavors invested in this Chilean initiative have led to the establishment and consolidation of C19-GenoNet as both a research network and a biobanking network. A comprehensive catalog of the C19-GenoNet biobank network has been created and is accessible online at https://redcovid.uchile.cl/ . STRENGHT AND LIMITATIONS OF THIS STUDY: This study is one of the largest cohorts of COVID-19 patients with associated Biobank reported so far in Latin America. The study’s design and rigorous weekly monitoring ensured effective collection of high-quality simples and maximized the quality and completeness of data, with the ability to re-contact participants in case of problematic information. There were no control or reliable information about the time between the infection and the sampling, which may hamper the comparison of some parameters among cases due to transcriptional dynamics after SARS-CoV-2 infection. The study is based on a self-reported survey, which may represent a bias when analyzing specific clinical phenotypes.