P0968THE INFLUENCE OF ALPHA KLOTHO ON NPP1 ACTIVITY IN CULTURED RAT PODOCYTES

Abstract

Abstract Background and Aims Kidney is the principal contributor to circulating αKlotho (αKL), and simultaneously, constitutes the major organ where circulating αKlotho is taken up from the circulation. Klotho is mainly expressed in proximal renal tubule and in distal tubular epithelium. In the kidney soluble αKlotho acts from the urinary luminal side as an autocrine or paracrine enzyme to regulate transporters and ion channels. Recently, it has been demonstrated that the glomerulus is another source of αKlotho production. NPP1 is the best characterized nucleotide pyrophosphatases/phosphodiesterases (NPP) family member, encoded by the ENPP1 gene, and is a type II transmembrane glycoprotein that forms disulfide-bonded homodimers in the plasma membrane and in the mineral-depositing matrix vesicles. The major function of NPP1 is generation of PPi from adenosine triphosphate (ATP). Recently, we demonstrated that extracellular nucleotides stimulate an increase in glomerular albumin permeability, in part due to glomerular NO production and actin reorganization in podocytes . Furthermore, both αKlotho and NPP1 regulate insulin-dependent signaling. Thus, a key aim of this study is to fully examine the role of αKlotho on NPP1 function and its role in the protection of glomerular filtration barrier and podocyte function in diabetes. Method We measured glomerular permeability to albumin (Palb) in a single isolated rat glomerulus based on the video-microscopy method and permeability to albumin across the podocyte monolayer. Expression of αKlotho and NPP1 was confirmed in podocytes using Western blotting and immunofluorescence. Ecto-nucleotide pyrophosphatase/phosphodiesterase activity in podocytes was assessed using p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as a substrate. The ATP concentration was measured using luciferin-luciferase reaction. Results Since NPP1 and Klotho regulate insulin-dependent signaling we investigated the role of Klotho on NPP1 dimerization and NPPs activity. Podocyte treatment withαKlotho (0.5 nM, 30 min) in non-reducing conditions (maleimide, 100 mM) increased the disulfide bond formation of NPP1 (Fig. 1A). The percentage of dimerized (260 kDa) NPP1 increased approximately by 45%. This effect was abolished in the presence of ATP (100 µM, 30 min). As a positive control, we used hydrogen peroxide (100 μM, 5 min) which induces oxidative disulfide bond formation (Fig. 1A). We also demonstrated time-dependent decrease of NPPs activity in the presence of αKL (Fig. 1B). Klotho also induces an increase of extracellular ATP concentration by 76% (from 3.19±0.01 to 5.62±0.43, n=6, P<0.05). Moreover, we confirmed the role of soluble αKlotho in ATP-evoked increases of glomerular and podocyte albumin permeability. Conclusion Summarizing, we have clearly demonstrated an interaction between αKL and NPP1 in podocytes. Moreover, we observed the protective role of αKlotho in podocyte permeability to albumin.