Cellular localization of adenine receptors in the rat kidney and their functional significance in the inner medullary collecting duct

Abstract

The Gi-coupled adenine receptor (AdeR) binds adenine with high affinity and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in the renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex and outer medulla compared with the inner medulla. Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid COOH-terminal sequence of rat AdeR, which we generated, detected two bands between ∼30 and 40 kDa (molecular mass of native protein: 37 kDa) in the cortex, outer medulla, and inner medulla. These bands were ablated by preadsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature, including the glomerular arterioles, and less intense labeling in the cells of the collecting duct system. Confocal immunofluorescence imaging colocalized AdeR with aquaporin-2 protein to the apical plasma membrane in the collecting duct. Functionally, adenine (10 μM) significantly decreased (P < 0.01) 1-deamino-8-d-arginine vasopressin (10 nM)-induced cAMP production in ex vivo preparations of inner medullary collecting ducts, which was reversed by PSB-08162 (20 μM, P < 0.01), a selective antagonist of AdeR. Thus, we demonstrated the expression of AdeR in the renal vasculature and collecting ducts and its functional relevance. This study may open a new avenue for the exploration of autocrine/paracrine regulation of renal vascular and tubular functions by the nucleobase adenine in health and disease.

Impaired natriuretic response to high-NaCl diet plus aldosterone infusion in mice overexpressing human CD39, an ectonucleotidase (NTPDase1)

“…Both primary antibodies are known to work on paraffin-embedded tissue sections. Formalin-fixed and paraffin-embedded kidneys from untreated mice were processed as described previously (8) except for the following modification with respect to antigen retrieval. Deparaffinized and rehydrated sections were microwaved in citrate-based antigen retrieval solution (Antigen Unmasking Solution, H-3300; Vector Laboratories) just until boiling (2-3 min), and then cooled in deionized water.…”

“…Deparaffinized and rehydrated sections were microwaved in citrate-based antigen retrieval solution (Antigen Unmasking Solution, H-3300; Vector Laboratories) just until boiling (2-3 min), and then cooled in deionized water. Sections were then rinsed with PBS, blocked with BSA (mouse cd39) or BSA plus goat serum (hCD39), and processed according to the previously published method (8). For proper visualization of labeling for human CD39 in certain structures of the kidney, kidneys were also processed by snapfreezing.…”

Mas and Its Related G Protein–Coupled Receptors, Mrgprs

“…However, the localization of rMrgprA expression in the vasculature and in the collecting duct of the kidney was recently reported in detail, and an inhibition of vasopressin signaling by rMrgprA was postulated (Kishore et al, 2013). One group has systematically analyzed the expression of all Mrgprs in mouse intestine (Avula et al, 2011(Avula et al, , 2013.…”

“…The binding of adenine to its receptors is very specific, because even small molecular alterations markedly reduce affinity of the ligand for rMrgprA and mMrgprA10 (Gorzalka et al, 2005;von Kügelgen et al, 2008;Borrmann et al, 2009). A first small-molecule antagonist for rMrgprA has also been developed (Kishore et al, 2013).…”

“…Accordingly, it has been shown that hMRGPRX1 can form dimers with the d-opioid receptor, and binding of hMRGPRX1 ligands to this heterodimer inhibits d-opioid signaling (Breit et al, 2006). rMrgprX1 activation modulates m-opioid receptor coupling in rat DRG neurons, in which they are coexpressed (Wang et al, 2013a), and activation of rMrgprA inhibits the signaling of coexpressed vasopressin V2 receptors in the collecting duct of the kidney (Kishore et al, 2013), but in both cases a direct interaction of the receptors was not analyzed. Moreover, Mas was found to bind the Ang II AT1 receptor (Kostenis et al, 2005;Santos et al, 2007).…”