Alan M. Weinstein

Shear-induced reorganization of renal proximal tubule cell actin cytoskeleton and apical junctional complexes

In this study, we demonstrate that fluid shear stress (FSS)-induced actin cytoskeletal reorganization and junctional formation in renal epithelial cells are nearly completely opposite the corresponding changes in vascular endothelial cells (ECs) [Thi MM et al. (2004) Proc Natl Acad Sci USA 101:16483–16488]. Mouse proximal tubule cells (PTCs) were subjected to 5 h of FSS (1 dyn/cm2) to investigate the dynamic responses of the cytoskeletal distribution of filamentous actin (F-actin), ZO-1, E-cadherin, vinculin, and paxillin to FSS. Immunofluorescence analysis revealed that FSS caused basal stress fiber disruption, more densely distributed peripheral actin bands (DPABs), and the formation of both tight junctions (TJs) and adherens junctions (AJs). A dramatic reinforcement of vinculin staining was found at the cell borders as well as the cell interior. These responses were abrogated by the actin-disrupting drug, cytochalasin D. To interpret these results, we propose a “junctional buttressing” model for PTCs in which FSS enables the DPABs, TJs, and AJs to become more tightly connected. In contrast, in the “bumper-car” model for ECs, all junctional connections were severely disrupted by FSS. This “junctional buttressing” model explains why a FSS of only 1/10 of that used in the EC study can cause a similarly dramatic, cytoskeletal response in these tall, cuboidal epithelial cells; and why junctional buttressing between adjacent cells may benefit renal epithelium in maximizing flow-activated, brush border-dependent, transcellular salt and water reabsorption.

Mechanotransduction in the renal tubule

The role of mechanical forces in the regulation of glomerulotubular balance in the proximal tubule (PT) and Ca(2+) signaling in the distal nephron was first recognized a decade ago, when it was proposed that the microvilli in the PT and the primary cilium in the cortical collecting duct (CCD) acted as sensors of local tubular flow. In this review, we present a summary of the theoretical models and experiments that have been conducted to elucidate the structure and function of these unique apical structures in the modulation of Na(+), HCO(3)(-), and water reabsorption in the PT and Ca(2+) signaling in the CCD. We also contrast the mechanotransduction mechanisms in renal epithelium with those in other cells in which fluid shear stresses have been recognized to play a key role in initiating intracellular signaling, most notably endothelial cells, hair cells in the inner ear, and bone cells. In each case, small hydrodynamic forces need to be greatly amplified before they can be sensed by the cells intracellular cytoskeleton to enable the cell to regulate its membrane transporters or stretch-activated ion channels in maintaining homeostasis in response to changing flow conditions.

A mathematical model of the inner medullary collecting duct of the rat: pathways for Na and K transport

A mathematical model of the inner medullary collecting duct (IMCD) of the rat has been developed representing Na+, K+, Cl-, HCO3-, CO2, H2CO3, phosphate, ammonia, and urea. Novel model features include: finite rates of hydration of CO2, a kinetic representation of the H-K-ATPase within the luminal cell membrane, cellular osmolytes that are regulated in defense of cell volume, and the repeated coalescing of IMCD tubule segments to yield the ducts of Bellini. Model transport is such that when entering Na+ is 4% of filtered Na+, approximately 75% of this load is reabsorbed. This requirement renders the area-specific transport rate for Na+ comparable to that for proximal tubule. With respect to the luminal membrane, there is experimental evidence for both NaCl cotransport and an Na+ channel in parallel. The experimental constraints that transepithelial potential difference is small and that the fractional apical resistance is greater than 85% mandate that more than 75% of luminal Na+ entry be electrically silent. When Na+ delivery is limited, an NaCl cotransporter can be effective at reducing luminal Na+ concentration to the observed low urinary values. Given the rate of transcellular Na+ reabsorption, there is necessarily a high rate of peritubular K+ recycling; also, given the lower bound on luminal membrane Cl- reabsorption, substantial peritubular Cl- flux must be present. Thus, if realistic limits on cell membrane electrical resistance are observed, then this model predicts a requirement for peritubular electroneutral KCl exit.A mathematical model of the inner medullary collecting duct (IMCD) of the rat has been developed representing Na+, K+, Cl-,[Formula: see text] CO2, H2CO3, phosphate, ammonia, and urea. Novel model features include: finite rates of hydration of CO2, a kinetic representation of the H-K-ATPase within the luminal cell membrane, cellular osmolytes that are regulated in defense of cell volume, and the repeated coalescing of IMCD tubule segments to yield the ducts of Bellini. Model transport is such that when entering Na+ is 4% of filtered Na+, approximately 75% of this load is reabsorbed. This requirement renders the area-specific transport rate for Na+ comparable to that for proximal tubule. With respect to the luminal membrane, there is experimental evidence for both NaCl cotransport and an Na+ channel in parallel. The experimental constraints that transepithelial potential difference is small and that the fractional apical resistance is greater than 85% mandate that more than 75% of luminal Na+ entry be electrically silent. When Na+delivery is limited, an NaCl cotransporter can be effective at reducing luminal Na+ concentration to the observed low urinary values. Given the rate of transcellular Na+ reabsorption, there is necessarily a high rate of peritubular K+recycling; also, given the lower bound on luminal membrane Cl- reabsorption, substantial peritubular Cl- flux must be present. Thus, if realistic limits on cell membrane electrical resistance are observed, then this model predicts a requirement for peritubular electroneutral KCl exit.

Cortical distal nephron Cl− transport in volume homeostasis and blood pressure regulation

Renal intercalated cells mediate the secretion or absorption of Cl(-) and OH(-)/H(+) equivalents in the connecting segment (CNT) and cortical collecting duct (CCD). In so doing, they regulate acid-base balance, vascular volume, and blood pressure. Cl(-) absorption is either electrogenic and amiloride-sensitive or electroneutral and thiazide-sensitive. However, which Cl(-) transporter(s) are targeted by these diuretics is debated. While epithelial Na(+) channel (ENaC) does not transport Cl(-), it modulates Cl(-) transport probably by generating a lumen-negative voltage, which drives Cl(-) flux across tight junctions. In addition, recent evidence indicates that ENaC inhibition increases electrogenic Cl(-) secretion via a type A intercalated cells. During ENaC blockade, Cl(-) is taken up across the basolateral membrane through the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and then secreted across the apical membrane through a conductive pathway (a Cl(-) channel or an electrogenic exchanger). The mechanism of this apical Cl(-) secretion is unresolved. In contrast, thiazide diuretics inhibit electroneutral Cl(-) absorption mediated by a Na(+)-dependent Cl(-)/HCO3(-) exchanger. The relative contribution of the thiazide and the amiloride-sensitive components of Cl(-) absorption varies between studies and probably depends on the treatment model employed. Cl(-) absorption increases markedly with angiotensin and aldosterone administration, largely by upregulating the Na(+)-independent Cl(-)/HCO3(-) exchanger pendrin. In the absence of pendrin [Slc26a4((-/-)) or pendrin null mice], aldosterone-stimulated Cl(-) absorption is significantly reduced, which attenuates the pressor response to this steroid hormone. Pendrin also modulates aldosterone-induced changes in ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function, at least in part, by altering luminal HCO3(-). This review summarizes mechanisms of Cl(-) transport in CNT and CCD and how these transporters contribute to the regulation of extracellular volume and blood pressure.

Shear stress-induced changes of membrane transporter localization and expression in mouse proximal tubule cells

Our previous studies of microperfused single proximal tubule showed that flow-dependent Na+ and HCO3− reabsorption is due to a modulation of both NHE3 and vacuolar H+-ATPase (V-ATPase) activity. An intact actin cytoskeleton was indicated to provide a structural framework for proximal tubule cells to transmit mechanical forces and subsequently modulate cellular functions. In this study, we have used mouse proximal tubule (MPT) cells as a model to study the role of fluid shear stress (FSS) on apical NHE3 and V-ATPase and basolateral Na/K-ATPase trafficking and expression. Our hypothesis is that FSS stimulates both apical and basolateral transporter expression and trafficking, which subsequently mediates salt and volume reabsorption. We exposed MPT cells to 0.2 dynes/cm2 FSS for 3 h and performed confocal microscopy and Western blot analysis to compare the localization and expression of both apical and basolateral transporters in control cells and cells subjected to FSS. Our findings show that FSS leads to an increment in the amount of protein expression, and a translocation of apical NHE3 and V-ATPase from the intracellular compartment to the apical plasma membrane and Na/K-ATPase to the basolateral membrane. Disrupting actin by cytochalasin D blocks the FSS-induced changes in NHE3 and Na/K-ATPase, but not V-ATPase. In contrast, FSS-induced V-ATPase redistribution and expression are largely inhibited by colchicine, an agent that blocks microtubule polymerization. Our findings suggest that the actin cytoskeleton plays an important role in FSS-induced NHE3 and Na/K-ATPase trafficking, and an intact microtubule network is critical in FSS-induced modulation of V-ATPase in proximal tubule cells.

A mathematical model of rat ascending Henle limb. III. Tubular function

K+ plays a catalytic role in AHL Na+ reabsorption via Na+-K+-2Cl- cotransporter (NKCC2), recycling across luminal K+ channels, so that luminal K+ is not depleted. Based on models of the ascending Henle limb (AHL) epithelium, it has been hypothesized that NH4+ may also catalyze luminal Na+ uptake. This hypothesis requires that luminal NH4+ not be depleted, implying replenishment via either direct secretion of NH4+, or NH3 in parallel with a proton. In the present work, epithelial models of rat medullary and cortical AHL (Weinstein AM, Krahn TA. Am J Physiol Renal Physiol 298: F000-F000, 2009) are configured as tubules and examined in simulations of function in vitro and in vivo to assess the feasibility of a catalytic role for NH4+ in Na+ reabsorption. Modulation of Na+ transport is also examined by peritubular K+ concentration and by Bartter-type transport defects in NKCC2 (type 1), in luminal membrane K+ channels (type 2), and in peritubular Cl- channels (type 3). It is found that a catalytic role for NH4+, which is significant in magnitude (relative to K+), is quantitatively realistic, in terms of uptake via NKCC2, and in terms of luminal membrane ammonia backflux. Simulation of a 90% NKCC2 defect is predicted to double distal Na+ delivery; it is also predicted to increase distal acid delivery (principally as NH4+). With doubling of medullary K+, the model predicts a 30% increase in distal Na+ delivery, but in this case there is a decrease in AHL acidification. This effect of peritubular K+ on proton secretion appears to be akin to type 3 Bartters pathophysiology, in which there is decreased peritubular HCO3- exit, cytosolic alkalinization, and a consequent decrease in luminal proton secretion by NHE3. One consequence of overlapping and redundant roles for K+ and NH4+, is a blunted impact of luminal membrane K+ permeability on overall Na+ reabsorption, so that type 2 Bartter pathophysiology is not well captured by the model.

A mathematical model of rat ascending Henle limb. II. Epithelial function

A mathematical model of ascending Henle limb (AHL) epithelium has been fashioned using kinetic representations of Na+-K+-2Cl- cotransporter (NKCC2), KCC4, and type 3 Na+/H+ exchanger (NHE3), with transporter densities selected to yield the reabsorptive Na+ flux expected for rat tubules in vivo. Of necessity, this model predicts fluxes that are higher than those measured in vitro. The kinetics of the NKCC and KCC are such that Na+ reabsorption by the model tubule is responsive to variation in luminal NaCl concentration over the range of 30 to 130 mM, with only minor changes in cell volume. Peritubular KCC accounts for about half the reabsorptive Cl- flux, with the remainder via peritubular Cl- channels. Transcellular Na+ flux is turned off by increasing peritubular KCl, which produces increased cytosolic Cl- and thus inhibits NKCC2 transport. In the presence of physiological concentrations of ammonia, there is a large acid challenge to the cell, due primarily to NH4+ entry via NKCC2, with diffusive NH3 exit to both lumen and peritubular solutions. When NHE3 density is adjusted to compensate this acid challenge, the model predicts luminal membrane proton secretion that is greater than the HCO3(-)-reabsorptive fluxes measured in vitro. The model also predicts luminal membrane ammonia cycling, with uptake via NKCC2 or K+ channel, and secretion either as NH4+ by NHE3 or as diffusive NH3 flux in parallel with a secreted proton. If such luminal ammonia cycling occurs in vivo, it could act in concert with luminal K+ cycling to facilitate AHL Na+ reabsorption via NKCC2. With physiological ammonia, peritubular KCl also blunts NHE3 activity by inhibiting NH4+ uptake on the Na-K-ATPase, and alkalinizing the cell.

ENaC inhibition stimulates Cl− secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na(+) channel (ENaC) reduces Cl(-) absorption. Since ENaC does not transport Cl(-), the purpose of this study was to determine how ENaC modulates Cl(-) absorption. Thus, Cl(-) absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl(-) absorption or transepithelial voltage (V(T)). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na(+)-K(+)-ATPase inhibitor to the bath reduced Cl(-) absorption by ∼66-75% and nearly obliterated lumen-negative V(T). In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1a(loxlox)). Whereas benzamil-sensitive Cl(-) absorption did not depend on CFTR, application of a Na(+)-K(+)-2Cl(-) cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na(+)-K(+)-2Cl(-) cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl(-) absorption, although the benzamil-sensitive component of V(T) was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl(-) absorption is benzamil sensitive, whereas thiazide-sensitive Cl(-) absorption is undetectable. Second, benzamil-sensitive Cl(-) absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V(T). Finally, benzamil-sensitive Cl(-) flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.

Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis.

Mutations in SLC4A4, the gene encoding the electrogenic Na(+)-HCO3(-) cotransporter NBCe1, cause severe proximal renal tubular acidosis (pRTA), growth retardation, decreased IQ, and eye and teeth abnormalities. Among the known NBCe1 mutations, the disease-causing mechanism of the T485S (NBCe1-A numbering) mutation is intriguing because the substituted amino acid, serine, is structurally and chemically similar to threonine. In this study, we performed intracellular pH and whole cell patch-clamp measurements to investigate the base transport and electrogenic properties of NBCe1-A-T485S in mammalian HEK 293 cells. Our results demonstrated that Ser substitution of Thr485 decreased base transport by ~50%, and importantly, converted NBCe1-A from an electrogenic to an electroneutral transporter. Aqueous accessibility analysis using sulfhydryl reactive reagents indicated that Thr485 likely resides in an NBCe1-A ion interaction site. This critical location is also supported by the finding that G486R (a pRTA causing mutation) alters the position of Thr485 in NBCe1-A thereby impairing its transport function. By using NO3(-) as a surrogate ion for CO3(2-), our result indicated that NBCe1-A mediates electrogenic Na(+)-CO3(2-) cotransport when functioning with a 1:2 charge transport stoichiometry. In contrast, electroneutral NBCe1-T485S is unable to transport NO3(-), compatible with the hypothesis that it mediates Na(+)-HCO3(-) cotransport. In patients, NBCe1-A-T485S is predicted to transport Na(+)-HCO3(-) in the reverse direction from blood into proximal tubule cells thereby impairing transepithelial HCO3(-) absorption, possibly representing a new pathogenic mechanism for generating human pRTA.

Potassium excretion during antinatriuresis: perspective from a distal nephron model

Renal excretion of Na(+) and K(+) must be regulated independently within the distal nephron, but is complicated by the fact that changing excretion of one solute requires adjustments in the transport of both. It is long known that hypovolemia increases Na(+) reabsorption while impairing K(+) excretion, even when distal Na(+) delivery is little changed. Renewed interest in this micropuncture observation came with identification of the molecular defects underlying familial hyperkalemic hypertension (FHH), which also increases distal Na(+) reabsorption and impairs K(+) excretion. In this work, a mathematical model of the distal nephron (Weinstein AM. Am J Physiol Renal Physiol 295: F1353-F1364, 2008), including the distal convoluted tubule (DCT), connecting segment (CNT), and collecting duct (CD), is used to examine renal K(+) excretion during antinatriuresis. Within the model, Na(+) avidity is represented as the modulation of DCT NaCl reabsorption, and the K(+) secretion signal is an aldosterone-like effect on principal cells of the CNT and CD. The first model prediction is that changes in DCT NaCl reabsorption are not mediated by NaCl cotransporter density alone, but require additional adjustments of both peritubular Na-K-ATPase and KCl cotransport. A second observation is that the CNT response to increased DCT Na(+) reabsorption should not only stabilize CD K(+) delivery but also compensate for the compromise of K(+) excretion downstream, as low Na(+) delivery increases CD K(+) reabsorption. Such anticipatory regulation is seen with the aldosterone response of hypovolemia, while the FHH phenotype manifests enhanced DCT NaCl transport but a blunted aldosterone effect. The model emphasizes the need for two distinct signals to the distal nephron, regulating Na(+) excretion and K(+) excretion, in contrast to a single switch apportioning NaCl reabsorption and Na(+)-for-K(+) exchange.