Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct

Abstract

Knepper MA. Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct. Am J Physiol Renal Physiol 295: F192-F201, 2008. First published April 16, 2008 doi:10.1152/ajprenal.00011.2008.-Collecting duct cells swell when exposed to arginine vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated, perfused rat inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomyosin). When a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradient and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl cotransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs from NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath caused significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry. cell volume; blebbistatin; cytoskeleton; kidney; inner medulla AN EARLY OBSERVATION made soon after introduction of the isolated, perfused tubule technique was that the cortical collecting duct (CCD) of the rabbit undergoes certain morphological changes upon exposure to vasopressin, including an increase in cell height, widening of the intercellular space, a change in cell shape with bulging of the center of the apical region into the lumen, and the formation of intracellular vacuoles (10). Similar observations have been made in isolated, perfused inner medullary collecting ducts (20). These changes were seen also in vivo (31) and are unique features of the collecting duct system.

Quantitative estimation of transmembrane ion transport in rat renal collecting duct principal cells

“…As the experiments were performed on principal cells from the open-end portion of the collecting duct fragments, where the single epithelial layer 16 Ilyaskin et al of cells was well-stirred, the simple "symmetrical" model could be used. The set of membrane osmolyte carriers and transporters taken into account in the model was chosen according to expression patterns in collecting duct cells Palmer 1987, 1989;Duc et al 1994;Bachmann et al 1999;Feraille and Doucet 2001;Muto 2001;Najjar et al 2005;Pisitkun et al 2006;Chou et al 2008;Uawithya et al 2008;Wakamatsu et al 2009). The transport of osmolytes and water depended on the area of the cell surface A, which was assumed to be constant and independent of cellular volume because of the low extensibility of the lipid bilayer.…”

“…There are multiple different pathways involved in the transport of solutes across the cell plasma membrane. Ion transport across principal cells is mediated by sodium (Duc et al 1994;Bachmann et al 1999;Loffing and Kaissling 2003;Frindt et al 2007), potassium (Frindt and Palmer 1987;Frindt and Palmer 1989;Muto 2001;Najjar et al 2005) and chloride (Sansom et al 1990;Todd-Turla et al 1996) channels, Na/K-pumps (Feraille and Doucet 2001;Wetzel and Sweadner 2001) and several types of ion transporters (Kuwahara et al 1991;Chou et al 2008). It is thus important to quantify the relative contribution of the various transporting mechanisms to the net transmembrane ion flux in order to understand the mechanism of the maintenance of the water-electrolyte balance in a principal cell.…”

Vasopressin and hyperosmolality regulate NKCC1 expression in rat OMCD

“…Although our data show that hyperosmolality by mannitol and raffinose also increased the NKCC1 mRNA expression, mannitol-induced hyperosmolality did not cause cell swelling by NKCC1 in their study. 27 They observed a very acute effect of NaCl or hyperosmolality on NKCC1 in isolated perfused IMCD. In contrast, we incubated OMCD for 60 min.…”

“…In contrast, an isolated tubule perfusion technique showed cell swelling of collecting ducts by exposure to vasopressin. 27 A larger increase in water permeability of the luminal membrane than that of the basolateral membrane has been speculated for the mechanism of cell swelling. Chou et al 27 reported that AVP-induced cell height increase is dependent on solute uptake by NKCC1, but is not dependent on apical water entry into an isolated perfused rat inner medullary collecting duct (IMCD).…”

“…27 A larger increase in water permeability of the luminal membrane than that of the basolateral membrane has been speculated for the mechanism of cell swelling. Chou et al 27 reported that AVP-induced cell height increase is dependent on solute uptake by NKCC1, but is not dependent on apical water entry into an isolated perfused rat inner medullary collecting duct (IMCD). Although our data show that hyperosmolality by mannitol and raffinose also increased the NKCC1 mRNA expression, mannitol-induced hyperosmolality did not cause cell swelling by NKCC1 in their study.…”