Localization of the succinate receptor in the distal nephron and its signaling in polarized MDCK cells

Abstract

When the succinate receptor (SUCNR1) is activated in the afferent arterioles of the glomerulus it increases renin release and induces hypertension. To study its location in other nephron segments and its role in kidney function, we performed immunohistochemical analysis and found that SUCNR1 is located in the luminal membrane of macula densa cells of the juxtaglomerular apparatus in close proximity to renin-producing granular cells, the cortical thick ascending limb, and cortical and inner medullary collecting duct cells. In order to study its signaling, SUCNR1 was stably expressed in Madin-Darby Canine Kidney (MDCK) cells, where it localized to the apical membrane. Activation of the cells by succinate caused Gq and Gi-mediated intracellular calcium mobilization, transient phosphorylation of extracellular regulated kinase (ERK)1/2 and the release of arachidonic acid along with prostaglandins E2 and I2. Signaling was desensitized without receptor internalization but rapidly resensitized upon succinate removal. Immunohistochemical evidence of phosphorylated ERK1/2 was found in cortical collecting duct cells of wild type but not SUCNR1 knockout streptozotocin-induced diabetic mice, indicating in vivo relevance. Since urinary succinate concentrations in health and disease are in the activation range of the SUCNR1, this receptor can sense succinate in the luminal fluid. Our study suggests that changes in the luminal succinate concentration may regulate several aspects of renal function.

Succinate: a metabolic signal in inflammation

“…Human embryonic kidney (HEK) 293 cells stably expressing SUCNR1 were stimulated with succinate, resulting in inositol trisphosphate (IP3) accumulation, calcium mobilization, and extracellular signalregulated kinase (ERK) phosphorylation [27] similar to Gi and Gq signaling cascades. Indeed, the use of the respective inhibitors of Gq and Gi signaling, YM254890 and pertussis toxin, impaired succinate-induced responses [30]. Calcium-dependent NO and prostaglandin E2 (PGE2) production was also observed following stimulation of SUCNR1 with succinate.…”

Insight into SUCNR1 (GPR91) structure and function

“…SUCNR1 was originally described as being coupled to both G i and G q proteins in human embryonic kidney (HEK293) cells (He et al, 2004). The initial results were later confirmed by Robben et al, in polarized Madin Darby Canine Kidney (MDCK) cells (Robben et al, 2009). Several authors repeatedly confirmed G i activation by demonstrating a decrease of cAMP levels upon succinate binding to SUCNR1 (Fig.…”

“…Resolution of SUCNR1 crystal structure is expected to confirm the presence of the disulfide bonds and mutations of these residues should show if the bridges are critical for interactions with ligands. SUCNR1 has two N-glycosylation sites, at N 8 and N 168 in the N-terminus and in the second extracellular loop, respectively (Robben et al, 2009;Wittenberger et al, 2001). However the precise role of this post-translational modification remains unknown.…”

“…Alternative mechanisms may also exist because P2Y 1 shows a HRY motif where H 3.49 repels R 3.50 . This configuration leads to the stabilization of the C-terminus via hydrogen bond between R 3.50 and A 7.55 (Robben et al, 2009;. …”

“…Succinate binding to SUCNR1 triggers the activation of mitogen-activated pathway (MAP) kinases, especially extracellular signal-regulated kinases 1 and 2 (ERK1/2) in HEK293 cell line (Gilissen etal., 2015;He et al, 2004), MDCK (Robben et al, 2009), immature dendritic cells (iDC) (Rubic et al, 2008), retinal ganglion neuronal cell line (RGC) (Hu et al, 2013), TF-1 (human erythroleukaemia cell line) and cardiomyocytes (Aguiar et al, 2010) (Fig. 3).…”