Regulation of glomerulotubular balance. III. Implication of cytosolic calcium in flow-dependent proximal tubule transport

Abstract

In the proximal tubule, axial flow (drag on brush-border microvilli) stimulates Na ϩ and HCO 3 Ϫ reabsorption by modulating both Na/H exchanger 3 (NHE3) and HATPase activity, a process critical to glomerulotubular balance. We have also demonstrated that blocking the angiotensin II receptor decreases baseline transport, but preserves the flow effect; dopamine leaves baseline fluxes intact, but abrogates the flow effect. In the current work, we provide evidence implicating cytosolic calcium in flow-dependent transport. Mouse proximal tubules were microperfused in vitro at perfusion rates of 5 and 20 nl/min, and reabsorption of fluid (Jv) and HCO 3 Ϫ (JHCO3) were measured. We examined the effect of high luminal Ca 2ϩ (5 mM), 0 mM Ca 2ϩ , the Ca 2ϩ chelator BAPTA-AM, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB), and the Ca-ATPase inhibitor thapsigargin. In control tubules, increasing perfusion rate from 5 to 20 nl/min increased Jv by 62% and JHCO3 by 104%. With respect to Na ϩ reabsorption, high luminal Ca 2ϩ decreased transport at low flow, but preserved the flow-induced increase; low luminal Ca 2ϩ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect; thapsigargin decreased baseline flow, leaving the flow effect intact. With respect to HCO 3 Ϫ reabsorption, high luminal Ca 2ϩ decreased transport at low flow and mildly diminished the flow-induced increase; low luminal Ca 2ϩ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect. These data implicate IP3 receptor-mediated intracellular Ca 2ϩ signaling as a critical step in transduction of microvillous drag to modulate Na ϩ and HCO 3 Ϫ transport.calcium signals; tubule transport; flow-dependent; sodium bicarbonate; IP 3 receptor; extracellular Ca 2ϩ GLOMERULOTUBULAR BALANCE (GTB) was first established in the rat kidney via micropuncture (18), where variation in glomerular filtration (over a range of flows from 15 to 60 nl/min) was accompanied by a constant fractional proximal reabsorption. We have studied the mechanism of axial flow-induced changes in Na ϩ and HCO 3 Ϫ absorption by microperfusion of mouse proximal tubules in vitro under low and high flow rates. From these studies we have demonstrated that the underlying mechanism of GTB is the flow regulation of both Na/H exchanger 3 (NHE3) and H-ATPase activities. This modulation is torque dependent (bending moment at the apical membrane due to fluid flow) and requires the intact actin cytoskeleton (2). In the studies of mouse proximal tubule cells, we have shown that fluid shear stress stimulates NHE3 and H-ATPase trafficking to the apical and Na-K-ATPase to the basolateral membrane surfaces (6); this observation is supported by the mathematical model that shows both apical and basolateral transporters are regulated by flow (25). Regulatory mechanisms of the GTB, including the role of dopamine and ANG II receptors and the role of cAMP-PKA have been examined. From these st...

Regulation of glomerulotubular balance: flow-activated proximal tubule function

“…These results are consistent with the hypothesis that increased flow causes redistribution of NHE3 within the apical membrane, as found in proximal tubule cells [18]; this activation is essential for flow-stimulated Na + and HCO 3 − absorption. Finally, we have demonstrated that IP3 receptor-mediated intracellular Ca 2+ signaling plays a critical role in transduction of microvillus torque to increase Na + and HCO 3 − absorption [16]. High luminal Ca 2+ downregulates Na + and HCO 3 − absorption in the proximal tubule, and increasing Ca 2+ influx has no impact on flow-stimulated proximal tubule transport [16].…”

“…Finally, we have demonstrated that IP3 receptor-mediated intracellular Ca 2+ signaling plays a critical role in transduction of microvillus torque to increase Na + and HCO 3 − absorption [16]. High luminal Ca 2+ downregulates Na + and HCO 3 − absorption in the proximal tubule, and increasing Ca 2+ influx has no impact on flow-stimulated proximal tubule transport [16]. These results indicate that the role of Ca 2+ signals in flow regulation of Na + and HCO 3 − absorption in proximal tubule is different from its role in flow regulation of K secretion in the collecting duct [52].…”

“…It has been suggested that in the proximal tubule, cytosolic Ca 2+ functions as a negative feedback signal at the luminal membrane to blunt Na + entry [21]. For those reasons, the effect of low and high luminal Ca 2+ , the Ca 2+ chelator BAPTA-AM, the IP3 receptor antagonist 2-APB, and the Ca-ATPase inhibitor thapsigargin on flow-stimulated Na + and HCO 3 − absorption were examined to investigate the role of calcium signals in flow-activated proximal tubule transport [16]. As shown in Fig.…”

“…This observation is captured by a mathematical model that shows that both apical and basolateral transporters are regulated by flow [50]. To understand the regulatory mechanisms of GTB, the following three major signaling transduction pathways have been examined: angiotensin II (Ang II), dopamine, and calcium signals [15, 16, 47]. It has been demonstrated that the AT1 receptor is important to maximize the NHE3 activity activated by flow; however, it is not critical for the flow-stimulated HCO 3 − transport, which still exists when the inhibitors are present or when the AT1a receptor is knocked out [15].…”

“…Thus, the tubule sensitivity changes in microvillus torque are dependent upon transporter availability on the membrane surface [18]. It was also demonstrated that changes in extracellular Ca 2+ did not alter flow signals, but inositol 1,4,5-trisphosphate (IP3) receptor-mediated intracellular Ca 2+ signaling is a critical step in transduction of microvillus drag to modulate Na + and HCO 3 − transport [16]. This recent work has been a start in a program to define the underlying cellular mechanisms and regulation of GTB.…”