Axial flow modulates proximal tubule NHE3 and H-ATPase activities by changing microvillus bending moments.
We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ absorption (Du Z, Duan Y, Yan Q, Weinstein AM, Weinbaum S, and Wang T. Proc Natl Acad Sci USA 101: 13068-13073, 2004). It was hypothesized that brush-border microvilli function as a sensor to detect and amplify luminal hydrodynamic forces and transmit them to the actin cytoskeleton. In the present study we examine whether 1) flow-dependent HCO3- transport is proportional to flow-dependent variations in microvillous torque (bending moment); 2) both luminal membrane Na(+)/H+ exchange (NHE3) and H(+)-ATPase activity are modulated by axial flow; and 3) paracellular permeabilities contribute to the flux perturbations. HCO3- absorption is examined by microperfusion of mouse S2 proximal tubules in vitro, with varying perfusion rates, and in the presence of the Na/H-exchange inhibitor EIPA, the H(+)-ATPase inhibitor bafilomycin, and the actin cytoskeleton inhibitor cytochalasin D. Paracellular permeability changes are assessed with measurements of epithelial HCO3- permeability and transepithelial potential difference (PD). It is found that 1) an increase in perfusion rate enhances HCO3- absorption and microvillous torque, and the fractional changes of each are nearly identical; 2) inhibition of NHE3 by EIPA, or H(+)-ATPase by bafilomycin, produced only partial inhibition of flow-stimulated bicarbonate transport; 3) disruption of the actin cytoskeleton by cytochalasin D blocked the increment of HCO3- absorption by high flow; and 4) HCO3- permeability and transepithelial PD are not modulated by flow. We conclude that flow-dependent modulation of proximal tubule HCO3- reabsorption is due to changes in both NHE3 and H(+)-ATPase activity within the luminal cell membrane and this requires an intact actin cytoskeleton. Paracellular permeability changes do not contribute to this flow dependence. Perfusion-absorption balance in the proximal tubule is a direct effect of flow-induced torque on brush-border microvilli to regulate luminal cell membrane transporter activity.
Du
Zhaopeng
Z
Department of Cellular and Molecular Physiology, Yale School of Medicine, 333 Cedar St., PO Box 208026, New Haven, CT 06520-8026, USA.
Yan
Qingshang
Q
Duan
Yi
Y
Weinbaum
Sheldon
S
Weinstein
Alan M
AM
Wang
Tong
T
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008871:Microvilli / Q000502:physiology; D010477:Perfusion; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D026941:Sodium Channel Blockers / Q000494:pharmacology; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D019415:Torque
D001639:Bicarbonates; D018942:Macrolides; C000613386:Slc9a3 protein, mouse; D026941:Sodium Channel Blockers; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D015638:Cytochalasin D; D000584:Amiloride; D012964:Sodium; D006180:Proton-Translocating ATPases; C039614:ethylisopropylamiloride