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 2B AR), attenuated glomerular fibrosis (glomerulosclerosis). We aimed to investigate the association between A 2B AR 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 2B AR attenuated some clinical signs of renal dysfunction and glomerulosclerosis, and decreased intraglomerular macrophage infiltration and MMT in DN rats.Cells 2020, 9, 1051 2 of 21 that affects close to 50% of patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) [3,7]. During DN, patients lose glomerular function. This is clinically manifested by the appearance of proteins in the urine (proteinuria; an albumin excretion rate ≥300 mg/24 h per gram of creatinine) and/or a reduced glomerular filtration rate (GFR; below 60 mL/min/1.73 m 2 ) [2]. In addition, people with DN who reach the stage of CKD [8] show an increase in the production of urine (polyuria), the appearance of glucose in the urine (glycosuria), and an increase in blood urea nitrogen (BUN) and serum creatinine [9]. Currently, management of DN patients involves the use of antihypertensive, antidyslipidemic, and antidiabetic agents, however these drugs have only shown a modest efficacy in slowing the evolution of the disease [10]. Regardless of the treatment used, the progression of DN leads to renal fibrosis [11], which irreversibly remodels the parenchyma tissue replacing it with extracellular matrix (ECM), thereby losing functionality [12]. Renal fibrosis predisposes patients to organ replacement therapies, such as hemodialysis and kidney transplantation, which means serious economic and societal costs for health systems [1,2,[13][14][15]. The pathophysiological events that trigger renal fibrosis are still unknown, however, this process is orchestrated by myofibroblasts, cells which have the ca...