Calcium-sensing receptor regulation of PTH-dependent calcium absorption by mouse cortical ascending limbs

Abstract

Resting Ca2+ absorption by cortical thick ascending limbs (CALs) is passive and proceeds through the paracellular pathway. In contrast, parathyroid hormone (PTH) stimulates active, transcellular Ca2+ absorption ( J Ca). The Ca2+-sensing receptor (CaSR) is expressed on serosal membranes of CALs. In the present study, we tested the hypothesis that activation of the CAL CaSR indirectly inhibits passive Ca2+ transport and directly suppresses PTH-induced cellular J Ca. To test this theory, we measured J Ca and Na absorption ( J Na) by single perfused mouse CALs. Net absorption was measured microfluorimetrically in samples collected from tubules perfused and bathed in symmetrical HEPES-buffered solutions or those in which luminal Na+ was reduced from 150 to 50 mM. We first confirmed that Gd3+ activated the CaSR by measuring intracellular Ca2+ concentration ([Ca2+]i) in CALs loaded with fura 2. On stepwise addition of Gd3+ to the bath, [Ca2+]i increased, with a half-maximal rise at 30 μM Gd3+. J Ca and transepithelial voltage ( V e,) were measured in symmetrical Na+-containing solutions. PTH increased J Ca by 100%, and 30 μM Gd3+inhibited this effect. V e was unchanged by either PTH or Gd3+. Similarly, NPS R-467, an organic CaSR agonist, inhibited PTH-stimulated J Ca without altering V e. Neither PTH nor Gd3+affected J Na. Addition of bumetanide to the luminal perfusate abolished J Na and V e. These results show that CaSR activation directly inhibited PTH-induced transcellular J Caand that cellular Ca2+ and Na+ transport can be dissociated. To test the effect of CaSR activation on passive paracellular Ca2+ transport, J Ca was measured under asymmetrical Na conditions, in which passive Ca2+ transport dominates transepithelial absorption. PTH stimulated J Ca by 24% and was suppressed by Gd3+. In this setting, Gd3+ reduced V e by 32%, indicating that CaSR activation inhibited both transcellular and paracellular Ca2+transport. We conclude that the CaSR regulates both active transcellular and passive paracellular Ca2+ reabsorption but has no effect on J Na by CALs.