Prostaglandin I2 Receptor Agonism Preserves β-Cell Function and Attenuates Albuminuria Through Nephrin-Dependent Mechanisms

Abstract

Discovery of common pathways that mediate both pancreatic b-cell function and end-organ function offers the opportunity to develop therapies that modulate glucose homeostasis and separately slow the development of diabetes complications. Here, we investigated the in vitro and in vivo effects of pharmacological agonism of the prostaglandin I 2 (IP) receptor in pancreatic b-cells and in glomerular podocytes. The IP receptor agonist MRE-269 increased intracellular 39,59-cyclic adenosine monophosphate (cAMP), augmented glucose-stimulated insulin secretion (GSIS), and increased viability in MIN6 b-cells. Its prodrug form, selexipag, augmented GSIS and preserved islet b-cell mass in diabetic mice. Determining that this preservation of b-cell function is mediated through cAMP/protein kinase A (PKA)/ nephrin-dependent pathways, we found that PKA inhibition, nephrin knockdown, or targeted mutation of phosphorylated nephrin tyrosine residues 1176 and 1193 abrogated the actions of MRE-269 in MIN6 cells. Because nephrin is important to glomerular permselectivity, we next set out to determine whether IP receptor agonism similarly affects nephrin phosphorylation in podocytes. Expression of the IP receptor in podocytes was confirmed in cultured cells by immunoblotting and quantitative real-time PCR and in mouse kidneys by immunogold electron microscopy, and its agonism 1) increased cAMP, 2) activated PKA, 3) phosphorylated nephrin, and 4) attenuated albumin transcytosis. Finally, treatment of diabetic endothelial nitric oxide synthase knockout mice with selexipag augmented renal nephrin phosphorylation and attenuated albuminuria development independently of glucose change. Collectively, these observations describe a pharmacological strategy that posttranslationally modifies nephrin and the effects of this strategy in the pancreas and in the kidney.

EP4 inhibition attenuates the development of diabetic and non-diabetic experimental kidney disease

“…Firstly, we had earlier shown that glomerular injury in STZ-eNOS −/− mice predominantly occurs within podocytes 18 (which our histological survey revealed are the major glomerular cell-type that expresses EP4). Secondly, the magnitude and pace of albuminuria development in STZ-eNOS −/− mice occurring within two to three weeks of diabetes induction 18, 31 , allows the relatively rapid in vivo screening of compounds for possible anti-albuminuric actions 31 . Whereas the EP4 inhibitor ONO-AE3-208 caused an approximate 50% reduction in urinary albumin excretion in STZ-eNOS −/− mice, the broadspectrum COX inhibitor indomethacin made no difference.…”

“…It is possible, for example, that with COX inhibition, concurrent blockade of the synthesis of other prostanoids (e.g. prostaglandin I 2 31 ) or concurrent prevention of the activation of other prostanoid receptors (e.g. EP2 32 ) negates the apparently beneficial effects observed with selective PGE 2 -EP4 targeting.…”

“…The magnitude of mesangial matrix deposition in the diabetic mouse studies or glomerulosclerosis in the SNx rat study was determined on periodic acid-Schiff stained kidney sections using a semi-quantitative scoring method on approximately 50 glomerular profiles per kidney section as previously described 31, 45 .…”

Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

“…The magnitude of mesangial matrix accumulation was estimated using a semiquantitative scoring system on approximately 50 glomeruli in periodic acid-Schiff-stained (PAS-stained) kidney sections from 3 to 8 mice per group for adriamycin-and vehicle-treated mice and 9 to 12 mice per group for db/db and db/m mice, as previously described and by investigators blinded to the study groups (68).…”

Histone H3 Serine 10 Phosphorylation Facilitates Endothelial Activation in Diabetic Kidney Disease

“…Experiments were conducted in conditionally immortalized human podocytes (provided by M. Saleem, University of Bristol, Bristol, U.K.) (7) and in primary cultured human renal GECs (hGECs) (ScienCell Research Laboratories, Carlsbad, CA) (8,9). hGECs were cultured under control conditions (5.6 mmol/L glucose) or with the addition of 19.4 mmol/L glucose (final concentration 25 mmol/L, high glucose) or 19.4 mmol/L mannitol for 16 h. To generate human podocyte-conditioned medium, differentiated human podocytes were incubated under control conditions (5.6 mmol/L glucose [hpod_CM]) or high-glucose conditions (25 mmol/L [hpod_HGCM]) for 48 h. The Human Cytokine 41-Plex Discovery Assay was performed by Eve Technologies (Calgary, Alberta, Canada).…”