Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule

Abstract

-We previously demonstrated that uroguanylin (UGN) significantly inhibits Na ϩ /H ϩ exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK1). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H ϩ secretion in rat renal proximal segments, through a H ϩ -sensitive microelectrode. UGN (1 M) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK1 cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK1 cells. Finally, treatment of LLC-PK1 cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides are intricately involved in the maintenance of salt and water homeostasis. NHE3; uroguanylin; renal microperfusion; bicarbonate reabsorption; cGMP; cAMP IN THE PAST DECADES, IT HAS been postulated that Sta-like guanylin peptides participate in a control system regulating salt balance in response to oral salt intake, linking intestine and kidney in the process of salt and water homeostasis (8,19,22). These peptides are known as endogenous agonists for the Escherichia coli heatstable toxin receptor, guanylate cyclase C (GC-C), uroguanylin (UGN) being its most potent member (12,18,20,27). UGN is a 16 amino acid peptide (27), expressed throughout the intestinal tract, secreted by enterochromaffin cells as prouroguanylin that undergoes postsecretory proteolytic cleavage, generating the active form (15,28,41). Recently, Qian and colleagues (44) demonstrated that the conversion of prouroguanylin to UGN occurs within the lumen of renal tubules.Renal effects of UGN include natriuresis, kaliuresis, diuresis, and increased excretion of cGMP (17,26). In a previous work using stationary in vivo microperfusion, our group demonstrated that UGN perfusion stimulates potassium secretion through Maxi-K channels in connecting and cortical collecting ducts (2). Furthermore, this study demonstrated that UGN perfusion also leads to an inhibition of bicarbonate reabsorption in proximal tubules by reducing the activity of the Na ϩ /H ϩ exchanger, NHE3 isoform (2). In renal proximal tubule cells, NHE...

Changes in the activity and expression of protein phosphatase‐1 accompany the differential regulation of NHE3 before and after the onset of hypertension in spontaneously hypertensive rats

“…Previous studies have shown that the natriuretic peptide uroguanylin, which activates the guanylate cyclase C receptor/cGMP pathway, also inhibits NHE3 activity by increasing NHE3 phosphorylation at both the 552 and 605 PKA consensus sites and by decreasing the surface expression of NHE3 in proximal tubule cells (Lessa et al . ). Protein phosphatases are targets of NO (Pfeilschifter et al .…”

From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

“…Fonteles et al (68) have demonstrated that chronic ingestion of NaCl for 10 days, at a concentration of 2% in drinking water, causes the kidney tissues to respond to a sub-threshold uroguanylin dose during ex vivo renal perfusion, suggesting a sensitization of kidney receptors to this hormone. In a very recent publication, Lessa et al (69) showed that uroguanylin reduces sodium-proton transport activity (NHE 3 ) in rat renal proximal tubules in a mechanism mediated by both cGMP/PK6 and cAMP/PKA signaling pathways with a parallel reduction in NHE 3 surface expression (Figure 2B). Baba et al (70) demonstrated that uroguanylin, at concentrations that have no effect on normal rats, significantly increases urinary sodium excretion in nephrotic rats, which could be important when treating clinical conditions involving sodium retention.…”

Uroguanylin: a new actor in the energy balance movie

“…It was initially proposed that the dietary ingestion of salt induces an increase in the secretion of proUGN by EC . proUGN is converted to UGN in the intestine and the renal tubules , leading to an increase of Na + in the urine (Greenberg et al 1997, Nakazato et al 1998, Lessa et al 2012) and a decrease in intestinal Na + absorption (Joo et al 1998, Toriano et al 2011. As part of this entero-renal axis, UGN signaling acts by regulating Na + homeostasis.…”