Abstract
The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H+-ATPases and alternative functions in H+-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H+-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H+-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H+-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H+-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H+-ATPases. In mice treated with NH4Cl, NaHCO3, KHCO3, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H+-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH4Cl or NaHCO3 treated mice demonstrated always colocalization of PRR/Atp6ap2 with H+-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H+-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H+-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H+-ATPase activity in intercalated cells.
Highlights
Therenin receptor (P)RR is a protein spanning the membrane once and with a large extracellular domain
The distribution ofrenin receptor/Atp6ap2 mRNA was examined in mouse kidney using hand-dissected nephron segments by semi-quantitative RT-qPCR. (P)RR/Atp6ap2 mRNA was detected in the glomerulus and all other segments, with highest levels in the connecting tubule/ cortical collecting duct (CNT/collecting ducts (CCDs)) and outer medullary collecting duct (OMCD) (Fig 1)
The high mRNA abundance of (P)RR/Atp6ap2 in CNT/CCD and OMCD was paralleled by high mRNA levels of the B1 (Atp6v1b1) H+-ATPase subunit which is selectively enriched in intercalated cells [40,41]
Summary
The (pro)renin receptor (P)RR is a protein spanning the membrane once and with a large extracellular domain. The activity of plasma membrane-associated H+-ATPases is regulated by various hormones and factors including angiotensin II, aldosterone, acidosis or alkalosis [7] Some of these effects are mediated by intracellular signaling cascades involving cAMP/PKA, PKC, ERK1/2 or AMPK [10,11,12,13,14]. In various model organisms such as Drosophila or Xenopus laevis larvae, the (P)RR/ Atp6ap is critical for fundamental cellular processes such as endocytic retrieval of proteins and Wnt signaling [22,23,24] Whether these functions of the (P)RR/Atp6ap are related to its possible role as accessory subunit of the H+-ATPase or due to other functions has not been fully elucidated. The main questions addressed in this manuscript are 1) the localization of (P)RR/Atp6ap protein along the murine nephron and its colocalization with plasma membrane associated H+-ATPases, 2) the coregulation of (P)RR/Atp6ap and two major H+-ATPase subunits on mRNA and protein level, and 3) to test whether acute application of prorenin could regulate native plasma membrane H+-ATPase in intercalated cells in freshly isolated murine collecting ducts
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