Regulation of transepithelial phosphate transport by PTH in chicken proximal tubule epithelium

Abstract

2001.-The effect of parathyroid hormone (PTH) and activation of protein kinase C (PKC) and protein kinase A (PKA) on transepithelial Pi transport was examined in monolayers of chick proximal tubule cells in primary culture (PTCs). Acute exposure of the PTCs to PTH (10 Ϫ9 M, basolateral side) significantly decreased the net reabsorption of Pi by ϳ66%. There was no effect after the addition of PTH to the luminal side. Activation of PKC by phorbol 12-myristate 13-acetate (PMA; 0.1 M) dramatically decreased net P i reabsorption by ϳ60%. Bisindolylmaleimide I (BIM; 1 M), a highly selective PKC inhibitor, prevented PMA-induced inhibition. Activation of adenylate cyclase/PKA by forskolin (10 M) mimicked the effect of PTH by significantly reducing net P i reabsorption by one-half. Addition of H-89 (10 M), a potent inhibitor of PKA, abolished forskolin-induced inhibition. PTH inhibition was blocked by either BIM or H-89. Tissue electrophysiology remained stable after all treatments. There was a decreased immunoreactivity of the luminal Na ϩ -Pi cotransporter NaPi-IIa after PTH treatment. These data indicate that PTH inhibition of Pi reabsorption in this in vitro system is mediated by PKC and PKA. primary cultures; reabsorptive flux; secretory flux; luminal sodium-inorganic phosphate cotransporter NaPi-IIa; protein kinase C; protein kinase A THE STUDY OF RENAL FUNCTION in birds and other nonmammalian vertebrates has provided valuable insights into the regulation of renal phosphate (P i ) excretion. In mammals, filtration and reabsorption provide sufficient control of P i excretion; however, in birds, which generally have lower and more variable glomerular filtration rates (46), the capacity for net tubular P i secretion has been reported (3). P i can be excreted by the avian kidney in quantities severalfold higher than the filtered load (21). Because avian kidneys possess this additional level of control for renal P i handling, comparative studies of its regulation should further our understanding of P i homeostasis.In both the avian and mammalian renal proximal tubule, P i reabsorption occurs via secondary, Na ϩ -dependent electroneutral transport across the luminal membrane [brush-border membrane (BBM)] (15, 34) coupled to an undefined exit process in the basolateral membrane (BLM) (1). In both mammals and birds, parathyroid hormone (PTH) has been found to be the major regulator of this process (28,24). PTH decreases BBM Na ϩ -P i cotransport activity in both mammals and birds and, in the latter, additionally stimulates net P i secretion (1, 34), which likely occurs through a unique Na ϩ -independent, voltage-and K ϩ -dependent transporter in the BBM (1, 35).Studies with the opossum kidney cell line (OK cells) have shown that PTH exerts its effects through activation of both protein kinase A (PKA) and protein kinase C (PKC) (7, 32), resulting in inhibition of BBM Na ϩ -P i cotransport. Other studies indicated the phosphaturic effect of PTH involves endocytic retrieval followed by lysosomal degradation of the BBM Na ϩ -P...

PTH stimulates a Cl−-dependent and EIPA-sensitive current in chick proximal tubule cells in culture

“…All monolayers grown under these conditions and tested with normal Cl Ϫ transport buffer consistently displayed low PDs and a modest TER, ranging from 0.6 to 1.9 mV and 63 to 150 ⍀ ⅐ cm 2 , respectively. There were some significant differences in TER among groups in Table 1, mostly compared with the higher TER seen in the monolayers tested at the lowest Cl consistently displayed a glucose-stimulated increment in I sc , attributable to a Na ϩ -glucose luminal cotransporter and characteristic of the vertebrate PT (13,15,45).…”

“…Nevertheless, it is interesting to consider several avian PT transport systems that are affected by PTH. In birds, PTH is known to stimulate a secretory component of P i transport (52) in addition to its inhibition of reabsorptive transport (13,36). PTH was also shown to increase urate clearance in birds, although the mechanism of this response was not determined (23).…”

“…The avian PT also possesses specific systems for secretory transport (basolateral to apical) of both urate and P i , although there is little known about these systems (3,7,13,36,52). Such secretory processes may have evolved in part to compensate for the lower filtration rates found in nonmammalian nephrons (7,36).…”

“…Such secretory processes may have evolved in part to compensate for the lower filtration rates found in nonmammalian nephrons (7,36). Thus PTH in birds is thought to increase P i excretion by both inhibition of a reabsorptive flux, as in mammals, and by stimulation of a secretory flux (13,36,52).…”

PTH Stimulated Growth and Decreased Col‐X Deposition are Phosphotidylinositol‐3,4,5 Triphosphate Kinase and Mitogen Activating Protein Kinase Dependent in Avian Sterna

“…Therefore, the range of MAPK-I doses tested were above and below 20 lM with more latitude on the high side to compensate for anticipated difficulty with matrix penetration. Application of the PKA inhibitor, H89, in other investigations ranged from .1 to 30 lM (Akino et al, 2000;Dudas et al, 2002;Zakany et al, 2002). We used the range of 1 to 10 lM for dose response testing.…”