SO039
BETA3-ADRENORECEPTOR AS A NEW PLAYER IN SYMPATHETIC REGULATION OF THE ACID-BASE HOMEOSTASIS IN THE KIDNEY

Sympathetic nervous system has a key role in the regulation of renal function. We previously showed that, in mouse, the β₃-adrenoreceptor (β₃-AR) localizes in most of the nephron segments and its selective agonism promotes a potent antidiuretic effect. Here, we evaluate the expression of β₃-AR in kidney intercalated cells (ICs) which are associated with the regulation of acid-base homeostasis in distal segments of the kidney tubule. The aim of this study is to investigate whether the β₃-AR might play a role in the acid-base homeostasis operated by the kidney.

The β₃-AR expression in mouse kidney ICs was investigated by confocal microscopy. Wild type (wt) and β₃-AR knock-out (ko) mice were used to study the role of β₃-AR in the renal acid-base homeostasis. Mouse urine were collected using metabolic cages and pH was measured. Immunofluorescence and Western blotting experiments were performed to evaluate the localization and the abundance of renal H⁺-ATPase in wt and β₃-AR ko mice. Kidney cells expressing human β₃-AR (M1-β₃-AR) and endogenous H⁺-ATPase were used to investigate the possible effects of β₃-AR stimulation on the activity/localization of H⁺-ATPase. Live imaging experiments using the pH-sensitive dye BCECF were carried out to determine intracellular pH and assess H⁺-ATPase activity.  Immunofluorescence experiments were performed to reveal the effects of β₃-AR activation on H⁺-ATPase localization.

Co-localization study of β₃-AR with either H⁺-ATPase or Cl⁻/HCO₃₋ exchanger pendrin in mouse kidney showed that β₃-AR is expressed in H⁺-secreting type A, in HCO₃^— secreting type B, and in non-A non-B ICs. The urine pH of β₃-AR ko mice was significantly higher compared with wt mice. In line with these results, localization and expression analysis showed that renal H⁺-ATPase significantly decreased in β₃-AR ko mice compared to wt mice, supporting the idea that H⁺ secretion is partially blunted in these animals. Of note, exposure of M1-β₃-AR cells to a selective β₃-AR agonist induced a 2.5 fold increase of H⁺-ATPase activity compared to resting cells and this effect was prevented by a selective β₃-AR antagonist or by the H⁺-ATPase inhibitor bafilomycin. Moreover, β₃-AR agonism enhanced H⁺-ATPase apical expression in M1-β₃-AR cells. In addition, the PKA inhibitor H89 abolished the stimulatory effect of β₃-AR agonism, demonstrating the involvement of the cAMP/PKA pathway.

The present data suggest that modulation of H⁺-ATPase activity in renal ICs by endogenous β₃-AR agonists may play an addition physiological role in hormonal control of renal acid-base homeostasis.