Proximal tubule apical endocytosis is modulated by fluid shear stress via an mTOR-dependent pathway

Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na+/K+-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.

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

In the proximal tubule, axial flow (drag on brush-border microvilli) stimulates Na+ and HCO3− reabsorption by modulating both Na/H exchanger 3 (NHE3) and H-ATPase 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 HCO3− ( JHCO3) were measured. We examined the effect of high luminal Ca2+ (5 mM), 0 mM Ca2+, the Ca2+ 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+ reabsorption, high luminal Ca2+ decreased transport at low flow, but preserved the flow-induced increase; low luminal Ca2+ 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 HCO3− reabsorption, high luminal Ca2+ decreased transport at low flow and mildly diminished the flow-induced increase; low luminal Ca2+ 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 Ca2+ signaling as a critical step in transduction of microvillous drag to modulate Na+ and HCO3− transport.

Regulation of glomerulotubular balance. II. Impact of angiotensin II on flow-dependent transport

Underlying glomerulotubular balance (GTB) is the impact of axial flow to regulate Na+ and HCO3− transport by modulating Na+-H+ exchanger 3 (NHE3) and H-ATPase activity. It is not known whether the cascade of events following a change in flow relies on local angiotensin (ANG II) generation or receptor availability. Mouse tubules were microperfused in vitro at flows of 5 and 20 nl/min, and net fluid (Jv) and HCO3− (JHCO3) absorption and cell height were measured. Na+ (JNa) and Cl− (JCl) absorption and changes in microvillous torque were estimated. Raising flow increased Na+ and HCO3− reabsorption but did not change either Cl− transport or cell volume. Losartan reduced absolute Na+ and HCO3− absorption at both low and high flows but did not affect fractional flow-stimulated transport. Compared with controls, in AT1a knockout (KO) mouse tubules, 53% of flow-stimulated Na+ absorption was abolished, but flow-stimulated HCO3− absorption was retained at similar levels. The remaining flow-stimulated JHCO3 was eliminated by the H-ATPase inhibitor bafilomycin. Inhibition of the AT2 receptor by PD123319 increased both JNa and JHCO3 but did not affect flow-mediated fractional changes. NHE3 expression at the protein level was reduced in AT1a KO mice kidneys. We conclude that 1) although the AT1a receptor is necessary for flow to impact NHE3, the effect on H+-ATPase is independent of AT1a; 2) the small flow-mediated changes in cell volume suggest a coordinate flow effect on both luminal and basolateral transporters; and 3) there is no evidence of flow-dependent Cl− transport, and thus no evidence for convective paracellular Cl− transport in mouse tubules.

Regulation of glomerulotubular balance. I. Impact of dopamine on flow-dependent transport

In response to volume expansion, locally generated dopamine decreases proximal tubule reabsorption by reducing both Na/H-exchanger 3 (NHE3) and Na-K-ATPase activity. We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ and HCO3− reabsorption and have suggested that this observation underlies glomerulotubular balance. In the present work, we investigate the impact of dopamine on the sensitivity of reabsorptive fluxes to changes in luminal flow. Mouse proximal tubules were microperfused in vitro at low and high flow rates, and volume and HCO3− reabsorption (Jv and JHCO3) were measured, while Na+ and Cl− reabsorption (JNa and JCl) were estimated. Raising luminal flow increased Jv, JNa, and JHCO3 but did not change JCl. Luminal dopamine did not change Jv, JNa, and JHCO3 at low flow rates but completely abolished the increments of Na+ absorption by flow and partially inhibited the flow-stimulated HCO3− absorption. The remaining flow-stimulated HCO3− absorption was completely abolished by bafilomycin. The DA1 receptor blocker SCH23390 and the PKA inhibitor H89 blocked the effect of exogenous dopamine and produced a two to threefold increase in the sensitivity of proximal Na+ reabsorption to luminal flow rate. Under the variety of perfusion conditions, changes in cell volume were small and did not always parallel changes in Na+ transport. We conclude that 1) dopamine inhibits flow-stimulated NHE3 activity by activation of the DA1 receptor via a PKA-mediated mechanism; 2) dopamine has no effect on flow-stimulated H-ATPase activity; 3) there is no evidence of flow stimulation of Cl− reabsorption; and 4) the impact of dopamine is a coordinated modulation of both luminal and peritubular Na+ transporters.

Renal NMDA receptors independently stimulate proximal reabsorption and glomerular filtration

N-methyl-d-aspartate receptors (NMDA) are expressed in the kidney, where little is known of their functional role. Several series of micropuncture experiments were performed in hydropenic rats using the NMDA channel blocker, MK801, and the NMDA coagonist, l-glycine, to probe NMDA for effects on single-nephron glomerular filtration rate (SNGFR) and proximal reabsorption ( Jprox). During intravenous infusion of MK801 or l-glycine, Henle's loop was perfused to manipulate SNGFR via tubuloglomerular feedback (TGF), thereby facilitating analysis of glomerulotubular balance. To confirm local actions on the kidney, MK801 was delivered to the glomerulus by microperfusion past the macula densa and to the proximal tubule by microperfusion into the early S1 segment. By all measures, MK801 acted on the glomerulus to reduce SNGFR, and acted on the proximal tubule to suppress Jprox, while having no effect on the responsiveness of TGF. l-Glycine raised SNGFR, dampened the TGF response, and could not be proved to independently stimulate proximal reabsorption. NMDA exerts a tonic vasodilatory influence on the glomerulus and a proreabsorptive effect on the proximal tubule. These combined effects allow NMDA to modulate SNGFR with minimal impact on late proximal flow. The full effects of l-glycine infusion on proximal tubule and TGF response do not extrapolate from the response to NMDA blockade.