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.

Induction of the adenosine receptor A_2B (A_2BAR) expression in diabetic glomeruli correlates with an increased abundance of its endogenous ligand adenosine and the progression of kidney dysfunction. Remarkably, A_2BAR antagonism protects from proteinuria in experimental diabetic nephropathy. We found that A_2BAR antagonism preserves the arrangement of podocytes on the glomerular filtration barrier, reduces diabetes-induced focal adhesion kinase (FAK) activation, and attenuates podocyte foot processes effacement. In spreading assays using human podocytes in vitro, adenosine enhanced the rate of cell body expansion on laminin-coated glass and promoted peripheral pY397-FAK subcellular distribution, while selective A_2BAR antagonism impeded these effects and attenuated the migratory capability of podocytes. Increased phosphorylation of the Myosin2A light chain accompanied the effects of adenosine. Furthermore, when the A_2BAR was stimulated, the cells expanded more broadly and more staining of pS19 myosin was detected which co-localized with actin cables, suggesting increased contractility potential in cells planted onto a matrix with a stiffness similar to of the glomerular basement membrane. We conclude that A_2BAR is involved in adhesion dynamics and contractile actin bundle formation, leading to podocyte foot processes effacement. The antagonism of this receptor may be an alternative to the intervention of glomerular barrier deterioration and proteinuria in the diabetic kidney disease.

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.

The monoamine oxidase (MAO) gene family encodes for enzymes that perform the oxidative deamination of monoamines, a process required to degrade norepinephrine, serotonin, dopamine, and other amines. While mammalian MAO enzymes, MAO A and MAO B, have been extensively studied, the molecular properties of the other family members are only partly uncovered. This study aims to explore the evolution of MAOs, emphasizing understanding of the MAO gene repertoire among vertebrates. Our analyses show that the duplication that gave rise to MAO A and MAO B occurred in the ancestor of tetrapods, between 408 and 352 million years ago. Nontetrapod jawed vertebrates possess the ancestral preduplicative condition of MAO A/B. Our results also identified a new family member, MAO F, in nontetrapod jawed vertebrates. Thus, most jawed vertebrates possess a repertoire of two MAO genes, MAO A and MAO B in tetrapods and MAO A/B and MAO F in nontetrapod jawed vertebrates, representing different MAO gene lineages. Jawless vertebrates possess the ancestral condition of a single copy gene, MAO A/B/F. Enzymatic assays conducted on the MAO recombinant enzymes of the Indo-Pacific tarpon show that both proteins, MAO A/B and MAO F, have enzymatic and molecular properties more similar to human MAO A, with the former featuring a strikingly higher activity rate when compared with all other MAO enzymes. Our analyses underscore the importance of scanning the tree of life for new gene lineages to understand phenotypic diversity and gain detailed insights into their function.

Nanobodies (VHHs or single-domain antibodies) are powerful affinity reagents, but their routine use is often limited by production constraints and by the lack of a conserved Fc region for secondary detection. We describe a simplified strategy in which functional GST–nanobody fusion proteins are expressed directly in the cytoplasm of Escherichia coli Origami^TM 2 (DE3), a strain that supports disulfide bond formation through trxB/gor mutations. Using well-characterized nanobodies against GFP (Lag2) and mCherry (C11), we designed N-terminal GST fusions and confirmed by AlphaFold3-based modeling that both constructs preserve the GST fold and the VHH (Variable domain of the Heavy-chain antibody of Heavy-chain-only antibodies) β-sandwich with defined CDR loops and a predicted intradomain disulfide bond. Following IPTG induction and purification by glutathione affinity and size-exclusion chromatography, we obtained soluble GST-nb-GFP and GST-nb-mCherry at ~8–12 mg/L. Isothermal titration calorimetry showed nanomolar binding to their antigens (K_d ~123 nM for GFP and ~199 nM for mCherry). Consistent with conformational epitope recognition, GST-nanobodies were reactive in native-state dot blots but not in denaturing Western blots under the conditions tested. The GST moiety enabled indirect immunofluorescence via anti-GST antibodies, yielding specific labeling of GFP- or mCherry-tagged TGN38 in HeLa and H4 cells. Finally, we demonstrate “GST-nanobody pulldown” as a robust method for affinity capture from cell lysates. Together, this platform provides a low-cost, versatile route to functional nanobody reagents without requiring tag removal, and complements other nanobody designs (e.g., VHH-Fc fusions) in an application-dependent manner.