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The ENaC γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitroactivation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146mutation to 143QQQQ146(γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+(JNa) and K+(JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4male mice on a low Na+diet did not exhibit altered ENaC POor flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4mice was not altered. γQ4/Q4males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+diet. Blood Na+and K+were unchanged on a regular, low Na+, or high K+diet. These findings suggest that biochemical evidence of γ subunit cleavage should be used in isolation to evaluate ENaC activity. Further, factors independent of γ subunit cleavage modulate channel POand the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.

Downregulation of heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX2), and nitric oxide synthase-2 (NOS2) in the kidneys of Dahl rodents causes salt sensitivity, while restoring their expression aids in Na+excretion and blood pressure reduction. Loading cholesterol into collecting duct (CD) cells represses fluid shear stress (FSS)-mediated COX2 activity. Thus, we hypothesized that cholesterol represses flow-responsive genes necessary to effectuate Na+excretion. To this end, CD cells were used to test whether FSS induces these genes and if cholesterol loading represses them. Mice fed either 0% or 1% cholesterol diet were injected with saline, urine volume and electrolytes were measured, and renal gene expression determined. FSS-exposed CD cells demonstrated increases in HO-1 mRNA by 350-fold, COX2 by 25-fold, and NOS2 by 8-fold in sheared cells compared with static cells (P< 0.01). Immunoblot analysis of sheared cells showed increases in HO-1, COX2, and NOS2 protein, whereas conditioned media contained more HO-1 and PGE2than static cells. Cholesterol loading repressed the sheared mediated protein abundance of HO-1 and NOS2 as well as HO-1 and PGE2concentrations in media. In cholesterol-fed mice, urine volume was less at 6 h after injection of isotonic saline (P< 0.05). Urinary Na+concentration, urinary K+concentration, and osmolality were greater, whereas Na+excretion was less, at the 6-h urine collection time point in cholesterol-fed versus control mice (P< 0.05). Renal cortical and medullary HO-1 (P< 0.05) and NOS2 (P< 0.05) mRNA were repressed in cholesterol-fed compared with control mice. Cholesterol acts to repress flow induced natriuretic gene expression, and this effect, in vivo, may contribute to renal Na+avidity.

The proper function of the organs that make up the urinary tract (kidneys, ureters, bladder, and urethra) depends on their ability to sense and respond to mechanical forces, including shear stress and wall tension. However, we have limited understanding of the mechanosensors that function in these organs and the tissue sites in which these molecules are expressed. Possible candidates include stretch-activated PIEZO channels (PIEZO1 and PIEZO2), which have been implicated in mechanically regulated body functions including touch sensation, proprioception, lung inflation, and blood pressure regulation. Using reporter mice expressing a COOH-terminal fusion of Piezo1with the sequence for the tandem-dimer Tomatogene, we found that PIEZO1 is expressed in the kidneys, ureters, bladder, and urethra as well as organs in close proximity, including the prostate, seminal vesicles and ducts, ejaculatory ducts, and the vagina. We further found that PIEZO1 expression is not limited to one cell type; it is observed in the endothelial and parietal cells of the renal corpuscle, the basolateral surfaces of many of the epithelial cells that line the urinary tract, the interstitial cells of the bladder and ureters, and populations of smooth and striated muscle cells. We propose that in the urinary tract, PIEZO1 likely functions as a mechanosensor that triggers responses to wall tension.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50= 9 µM) at −120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu= 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

Within the CCD of the distal nephron of the rabbit, the BK (maxi K) channel mediates Ca2+- and/or stretch-dependent flow-induced K+secretion (FIKS) and contributes to K+adaptation in response to dietary K+loading. An unresolved question is whether BK channels in intercalated cells (ICs) and/or principal cells (PCs) in the CCD mediate these K+secretory processes. In support of a role for ICs in FIKS is the higher density of immunoreactive apical BKα (pore-forming subunit) and functional BK channel activity than detected in PCs, and an increase in IC BKα expression in response to a high-K+diet. PCs possess a single apical cilium which has been proposed to serve as a mechanosensor; direct manipulation of cilia leads to increases in cell Ca2+concentration, albeit of nonciliary origin. Immunoperfusion of isolated and fixed CCDs isolated from control K+-fed rabbits with channel subunit-specific antibodies revealed colocalization of immunodetectable BKα- and β1-subunits in cilia as well as on the apical membrane of cilia-expressing PCs. Ciliary BK channels were more easily detected in rabbits fed a low-K+vs. high-K+diet. Single-channel recordings of cilia revealed K+channels with conductance and kinetics typical of the BK channel. The observations that 1) FIKS was preserved but 2) the high-amplitude Ca2+peak elicited by flow was reduced in microperfused CCDs subject to pharmacological deciliation suggest that cilia BK channels do not contribute to K+secretion in this segment, but that cilia serve as modulators of cell signaling.

Dyslipidemia in chronic kidney disease (CKD) is usually characterized by hypertriglyceridemia. Here we studied postprandial lipemia in children and young adults to determine whether an increasing degree of CKD results in a proportional increase in triglyceride and chylomicron concentration. Secondary goals were to determine whether subnephrotic proteinuria, apolipoprotein (apo)C-III and insulin resistance modify the CKD effect. Eighteen fasting participants (mean age of 15 years, mean glomerular filtration rate (GFR) of 50 ml/min/1.73 m2) underwent a postprandial challenge with a high fat milkshake. Triglycerides, apoB-48, insulin, and other markers were measured before and 2, 4, 6, and 8 hours afterward. Response was assessed by the incremental area under the curve of triglycerides and of apoB-48. The primary hypothesis was tested by correlation to estimated GFR. Significantly, for every 10 ml/min/1.73 m2lower estimated GFR, the incremental area under the curve of triglycerides was 17% greater while that of apoB-48 was 16% greater. Univariate analyses also showed that the incremental area under the curve of triglycerides and apoB-48 were significantly associated with subnephrotic proteinuria, apoC-III, and insulin resistance. In multivariate analysis, CKD and insulin resistance were independently associated with increased area under the curve and were each linked to increased levels of apoC-III. Thus, postprandial triglyceride and chylomicron plasma excursions are increased in direct proportion to the degree of CKD. Independent effects are associated with subclinical insulin resistance and increased apoC-III is linked to both CKD and insulin resistance.

The majority of patients with obesity, insulin resistance, and metabolic syndrome have hypertension, but the mechanisms of hypertension are poorly understood. In these patients, impaired sodium excretion is critical for the genesis of Na+-sensitive hypertension, and prior studies have proposed a role for the epithelial Na+channel (ENaC) in this syndrome. We characterized high fat-fed mice as a model in which to study the contribution of ENaC-mediated Na+reabsorption in obesity and insulin resistance. High fat-fed mice demonstrated impaired Na+excretion and elevated blood pressure, which was significantly higher on a high-Na+diet compared with low fat-fed control mice. However, high fat-fed mice had no increase in ENaC activity as measured by Na+transport across microperfused cortical collecting ducts, electrolyte excretion, or blood pressure. In addition, we found no difference in endogenous urinary aldosterone excretion between groups on a normal or high-Na+diet. High fat-fed mice provide a model of metabolic syndrome, recapitulating obesity, insulin resistance, impaired natriuresis, and a Na+-sensitive elevation in blood pressure. Surprisingly, in contrast to previous studies, our data demonstrate that high fat feeding of mice impairs natriuresis and produces elevated blood pressure that is independent of ENaC activity and likely caused by increased Na+reabsorption upstream of the aldosterone-sensitive distal nephron.

Diuretics acting on specific nephron segments to inhibit Na+reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K+complicate their use to achieve target blood pressure. ROMK is an attractive diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide-sensitive Na+-K+-2Cl−cotransporter (NKCC2) and the amiloride- and benzamil-sensitive epithelial Na+channel (ENaC). The development of small-molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how inhibition of ROMK alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC alter fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another ROMK inhibitor, termed compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na+excretion when combined with HCTZ, amiloride, or benzamil, but not when coadministered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, compound A inhibited the kaliuretic response induced by bumetanide and HCTZ, an effect we attribute to inhibition of ROMK-mediated K+secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow-sensitive large-conductance Ca2+-activated K+channels (Slo1/β1). In conclusion, compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target.

Flow-induced K+secretion in the aldosterone-sensitive distal nephron is mediated by high-conductance Ca2+-activated K+(BK) channels. Familial hyperkalemic hypertension (pseudohypoaldosteronism type II) is an inherited form of hypertension with decreased K+secretion and increased Na+reabsorption. This disorder is linked to mutations in genes encoding with-no-lysine kinase 1 (WNK1), WNK4, and Kelch-like 3/Cullin 3, two components of an E3 ubiquitin ligase complex that degrades WNKs. We examined whether the full-length (or “long”) form of WNK1 (L-WNK1) affected the expression of BK α-subunits in HEK cells. Overexpression of L-WNK1 promoted a significant increase in BK α-subunit whole cell abundance and functional channel expression. BK α-subunit abundance also increased with coexpression of a kinase dead L-WNK1 mutant (K233M) and with kidney-specific WNK1 (KS-WNK1), suggesting that the catalytic activity of L-WNK1 was not required to increase BK expression. We examined whether dietary K+intake affected L-WNK1 expression in the aldosterone-sensitive distal nephron. We found a paucity of L-WNK1 labeling in cortical collecting ducts (CCDs) from rabbits on a low-K+diet but observed robust staining for L-WNK1 primarily in intercalated cells when rabbits were fed a high-K+diet. Our results and previous findings suggest that L-WNK1 exerts different effects on renal K+secretory channels, inhibiting renal outer medullary K+channels and activating BK channels. A high-K+diet induced an increase in L-WNK1 expression selectively in intercalated cells and may contribute to enhanced BK channel expression and K+secretion in CCDs.

The kidney adjusts K+excretion to match intake in part by regulation of the activity of apical K+secretory channels, including renal outer medullary K+(ROMK)-like K+channels, in the cortical collecting duct (CCD). ANG II inhibits ROMK channels via the ANG II type 1 receptor (AT1R) during dietary K+restriction. Because AT1Rs and ANG II type 2 receptors (AT2Rs) generally function in an antagonistic manner, we sought to characterize the regulation of ROMK channels by the AT2R. Patch-clamp experiments revealed that ANG II increased ROMK channel activity in CCDs isolated from high-K+(HK)-fed but not normal K+(NK)-fed rats. This response was blocked by PD-123319, an AT2R antagonist, but not by losartan, an AT1R antagonist, and was mimicked by the AT2R agonist CGP-42112. Nitric oxide (NO) synthase is present in CCD cells that express ROMK channels. Blockade of NO synthase with N-nitro-l-arginine methyl ester and free NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt completely abolished ANG II-stimulated ROMK channel activity. NO enhances the synthesis of cGMP, which inhibits phosphodiesterases (PDEs) that normally degrade cAMP; cAMP increases ROMK channel activity. Pretreatment of CCDs with IBMX, a broad-spectrum PDE inhibitor, or cilostamide, a PDE3 inhibitor, abolished the stimulatory effect of ANG II on ROMK channels. Furthermore, PKA inhibitor peptide, but not an activator of the exchange protein directly activated by cAMP (Epac), also prevented the stimulatory effect of ANG II. We conclude that ANG II acts at the AT2R to stimulate ROMK channel activity in CCDs from HK-fed rats, a response opposite to that mediated by the AT1R in dietary K+-restricted animals, via a NO/cGMP pathway linked to a cAMP-PKA pathway.

An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2signaling regulate cation transport in the CCD and, at least in other systems, are affected by biomechanical forces. We hypothesized that FSS and CS differentially affect MAPK signaling and PGE2release to modulate cation transport in the CCD. To validate that CS is a physiological force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5 ± 2.0 or 170 ± 32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dyne/cm2of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed an approximately twofold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared with unstretched controls. FSS induced whereas CS reduced PGE2release by ∼40%. In conclusion, FSS and CS differentially affect ERK and p38 activation and PGE2release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.

The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and nonvolatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here, we demonstrate that treatment of rabbit isolated, perfused collecting ducts with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibited V-ATPase-dependent H+secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. Compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3−-containing media. Moreover, AICAR treatment of clone C rabbit intercalated cells expressing the WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.

Fluid shear stress (FSS) is a critical regulator of cation transport in the collecting duct (CD). High-dietary sodium (Na) consumption increases urine flow, Na excretion, and prostaglandin E2(PGE2) excretion. We hypothesize that increases in FSS elicited by increasing tubular flow rate induce the release of PGE2from renal epithelial cells into the extracellular compartment and regulate ion transport. Media retrieved from CD cells exposed to physiologic levels of FSS reveal several fold higher concentration of PGE2compared with static controls. Treatment of CD cells with either cyclooxygenase-1 (COX-1) or COX-2 inhibitors during exposure to FSS limited the increase in PGE2concentration to an equal extent, suggesting COX-1 and COX-2 contribute equally to FSS-induced PGE2release. Cytosolic phospholipase A2 (cPLA2), the principal enzyme that generates the COX substrate arachidonic acid, is regulated by mitogen-activated protein-kinase-dependent phosphorylation and intracellular Ca2+concentration ([Ca2+]i), both signaling processes, of which, are activated by FSS. Inhibition of the ERK and p38 pathways reduced PGE2release by 53.3 ± 8.4 and 32.6 ± 11.3%, respectively, while antagonizing the JNK pathway had no effect. In addition, chelation of [Ca2+]ilimited the FSS-mediated increase in PGE2concentration by 47.5 ± 7.5% of that observed in untreated sheared cells. Sheared cells expressed greater phospho-cPLA2 protein abundance than static cells; however, COX-2 protein expression was unaffected (P= 0.064) by FSS. In microperfused CDs, COX inhibition enhanced flow-stimulated Na reabsorption and abolished flow-stimulated potassium (K) secretion, but did not affect ion transport at a slow flow rate, implicating that high tubular flow activates autocrine/paracrine PGE2release and, in turn, regulates flow-stimulated cation transport. In conclusion, FSS activates cPLA2 to generate PGE2that regulates flow-mediated Na and K transport in the native CD. We speculate that dietary sodium intake modulates tubular flow rate to regulate paracrine PGE2release and cation transport in the CD.

Epithelial Na+channel (ENaC)-mediated Na+absorption and BK channel-mediated K+secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca2+concentration ([Ca2+]i), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO3−reabsorption in the proximal tubule due to changes in both luminal Na+/H+exchanger 3 and H+-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol290: F289–F296, 2006), we sought to test the hypothesis that flow also regulates H+-ATPase activity in the CCD. H+-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH4Cl prepulse technique). H+-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pHi) recovery in the absence of luminal K+, bilateral Na+, and CO2/HCO3−, from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min−1·mm−1stimulated H+-ATPase activity, 2) flow-stimulated H+pumping was Ca2+dependent and required microtubule integrity, and 3) basal and flow-stimulated pHirecovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorusagglutinin as well as cells that did not. We conclude that luminal flow modulates H+-ATPase activity in the rabbit CCD and that H+-ATPases therein are present in both principal and intercalated cells.

Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion (JK) and Na absorption (JNa) at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal JK, JNa, or the flow-induced [Ca2+]itransient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce JK. Basolateral Cl removal reversibly inhibited FIKS but not basal JKor JNa. Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH4+at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH4+addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH4+entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.

The renal outer medullary potassium (K+) channel, ROMK (Kir1.1), is a putative drug target for a novel class of loop diuretic that would lower blood volume and pressure without causing hypokalemia. However, the lack of selective ROMK inhibitors has hindered efforts to assess its therapeutic potential. In a high-throughput screen for small-molecule modulators of ROMK, we previously identified a potent and moderately selective ROMK antagonist, 7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (VU590), that also inhibits Kir7.1. Because ROMK and Kir7.1 are coexpressed in the nephron, VU590 is not a good probe of ROMK function in the kidney. Here we describe the development of the structurally related inhibitor 2,2'-oxybis(methylene)bis(5-nitro-1H-benzo[d]imidazole) (VU591), which is as potent as VU590 but is selective for ROMK over Kir7.1 and more than 65 other potential off-targets. VU591 seems to block the intracellular pore of the channel. The development of VU591 may enable studies to explore the viability of ROMK as a diuretic target.

The role of mechanical forces in the regulation of glomerulotubular balance in the proximal tubule (PT) and Ca2+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+, HCO3−, and water reabsorption in the PT and Ca2+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 cell's 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.

The fine regulation of Na+and K+transport takes place in the cortical distal nephron. It is well established that K+secretion occurs through apical K+channels: the ROMK and the Ca2+- and voltage-dependent maxi-K. Previously, we identified the voltage-gated Kv1.3 channel in the inner medulla of the rat kidney (Escobar LI, Martínez-Téllez JC, Salas M, Castilla SA, Carrisoza R, Tapia D, Vázquez M, Bargas J, Bolívar JJ. Am J Physiol Cell Physiol286: C965–C974, 2004). To examine the role of Kv1.3 in the renal regulation of K+homeostasis, we characterized the effect of dietary K+on the molecular and functional expression of this channel. We performed real-time-PCR and immunoblot assays in kidneys from rats fed a control (CK; 1.2% wt/wt) or high-K+(HK; 10% wt/wt) diet for 5–15 days. Kv1.3 mRNA and protein expression did not change with HK in the whole kidney. However, dietary K+loading provoked a change in the cellular distribution of Kv1.3 from the cytoplasm to apical membranes. Immunolocalization of Kv1.3 detected the channel exclusively in the intercalated cells. We investigated whether Kv1.3 mediated K+transport in microperfused cortical collecting ducts (CCDs). The HK diet led to an increase in net K+transport from 7.4 ± 1.1 (CK) to 11.4 ± 1.0 (HK) pmol·min−1·mm−1. Luminal margatoxin, a specific blocker of Kv1.3, decreased net K+secretion in HK CCDs to 6.0 ± 1.6 pmol·min−1·mm−1. Our data provide the first evidence that Kv1.3 channels participate in K+secretion and that apical membrane localization of Kv1.3 is enhanced in the intercalated cells by dietary K+loading.

Flow-stimulated net K secretion (JK) in the cortical collecting duct (CCD) is mediated by an iberiotoxin (IBX)-sensitive BK channel, and requires an increase in intracellular Ca2+concentration ([Ca2+]i). The α-subunit of the reconstituted BK channel is phosphorylated by PKA and PKC. To test whether the BK channel in the native CCD is regulated by these kinases, JKand net Na absorption (JNa) were measured at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in the presence of mPKI, an inhibitor of PKA; calphostin C, which inhibits diacylglycerol binding proteins, including PKC; or bisindolylmaleimide (BIM) and Gö6976, inhibitors of classic and novel PKC isoforms, added to luminal (L) and/or basolateral (B) solutions. L but not B mPKI increased JKin CCDs perfused at a slow flow rate; a subsequent increase in flow rate augmented JKmodestly. B mPKI alone or with L inhibitor abolished flow stimulation of JK. Similarly, L calphostin C increased JKin CCDs perfused at slow flow rates, as did calphostin C in both L and B solutions. The observation that IBX inhibited the L mPKI- and calphostin C-mediated increases in JKat slow flow rates implicated the BK channel in this K flux, a notion suggested by patch-clamp analysis of principal cells. The kinase inhibited by calphostin C was not PKC as L and/or B BIM and Gö6976 failed to enhance JKat the slow flow rate. However, addition of these PKC inhibitors to the B solution alone or with L inhibitor blocked flow stimulation of JK. Interpretation of these results in light of the effects of these inhibitors on the flow-induced elevation of [Ca2+]isuggests that the principal cell apical BK channel is tonically inhibited by PKA and that flow stimulation of JKin the CCD is PKA and PKC dependent. The specific targets of the kinases remain to be identified.

Apical large-conductance Ca2+-activated K+(BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K+secretion. Dietary K+loading for 10–14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K+secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol289: F922–F932, 2005). To test whether this adaptation was mediated by a K+-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na+(LS) or high-Na+(HS) diet for 7–10 days to alter circulating levels of aldosterone but not serum K+concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, α-splice variants, β-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na+(JNa) and K+(JK) transport by microperfusion; kidneys were processed for immunolocalization of BK α-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na+and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK α-, β2-, and β4-subunit mRNA and localization of immunodetectable α-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ∼1 to 5 nl·min−1·mm−1, the increase in JNawas greater in LS vs. HS rabbits, yet the flow-stimulated increase in JKwas similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K+loading.

Mutations of cilia-expressed proteins are associated with an attenuated shear-induced increase in intracellular Ca2+concentration ([Ca2+]i) in renal epithelial cell lines derived from murine models of autosomal recessive polycystic kidney disease (ARPKD). We hypothesized that human ARPKD cyst-lining renal epithelial cells also exhibited dysregulated mechanosensation. To test this, conditionally immortalized cell lines derived from human fetal ARPKD cyst-lining (pool and clone 5E) cell lines with low levels of fibrocystin/polyductin expression and age-matched normal collecting tubule [human fetal collecting tubule (HFCT) pool and clone 2C] cell lines were grown in culture, loaded with a Ca2+indicator dye, and subjected to laminar shear. Clonal cell lines were derived from single cells present in pools of cells from cyst-lining and collecting tubules, microdissected from human kidney. Resting and peak [Ca2+]iwere similar between ARPKD 5E and pool, and HFCT 2C and pool; however, the flow-induced peak [Ca2+]iwas greater in ARPKD 5E (700 ± 87 nM, n= 21) than in HFCT 2C (315 ± 58 nM, n= 12; P< 0.01) cells. ARPKD 5E cells treated with Gd3+, an inhibitor of nonselective cation channels, inhibited but did not abolish the shear-induced [Ca2+]itransient. Cilia were ∼20% shorter in ARPKD than HFCT cells, but no difference in ciliary localization or total cellular expression of polycystin-2, a mechanosenory Gd3+-sensitive cation channel, was detected between ARPKD and HFCT cells. The intracellular Ca2+stores were similar between cells. In summary, human ARPKD cells exhibit an exaggerated Gd3+-sensitive mechano-induced Ca2+response compared with controls; whether this represents dysregulated polycystin-2 activity in ARPKD cells remains to be explored.

Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. Confocal microscopy and small interfering RNA (siRNA) were used to identify the source of the calcium and to dissect the requisite viral–cell interactions. Binding of HSV to human epithelial cells induced no calcium response, but shifting the cells to temperatures permissive for penetration triggered increases in plasma membrane calcium followed by a global release of intracellular calcium. Transfection with siRNA targeting the proteoglycan syndecan-2 blocked viral binding and abrogated any calcium response. Transfection with siRNA targeting nectin-1, a glycoprotein D receptor, also prevented both membrane and intracellular calcium responses. In contrast, the membrane response was preserved after transfection with siRNA targeting integrinαv, a novel glycoprotein H receptor. The membrane response, however, was not sufficient for viral entry, which required interactions with integrinαv and release of inositol-triphosphate receptor-dependent intracellular calcium stores. Thus, calcium plays a critical, complex role in HSV entry.

Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. Confocal microscopy and small interfering RNA (siRNA) were used to identify the source of the calcium and to dissect the requisite viral–cell interactions. Binding of HSV to human epithelial cells induced no calcium response, but shifting the cells to temperatures permissive for penetration triggered increases in plasma membrane calcium followed by a global release of intracellular calcium. Transfection with siRNA targeting the proteoglycan syndecan-2 blocked viral binding and abrogated any calcium response. Transfection with siRNA targeting nectin-1, a glycoprotein D receptor, also prevented both membrane and intracellular calcium responses. In contrast, the membrane response was preserved after transfection with siRNA targeting integrinαv, a novel glycoprotein H receptor. The membrane response, however, was not sufficient for viral entry, which required interactions with integrinαv and release of inositol-triphosphate receptor-dependent intracellular calcium stores. Thus, calcium plays a critical, complex role in HSV entry.

Apical low-conductance SK and high-conductance Ca2+-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion (JK) in the CCD is 1) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and 2) associated with an increase in [Ca2+]i, leading us to conclude that BK channels mediate flow-stimulated JK. To examine the Ca2+dependence and sources of Ca2+contributing to flow-stimulated JK, JKand net Na absorption (JNa) were measured at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in the absence of luminal Ca2+or after pretreatment with BAPTA-AM to chelate intracellular Ca2+, 2-aminoethoxydiphenyl borate (2-APB), to inhibit the inositol 1,4,5-trisphosphate (IP3) receptor or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated JNa(Morimoto et al. Am J Physiol Renal Physiol291: F663–F669, 2006), inhibited flow-stimulated JK. Increases in [Ca2+]istimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 μM colchicine (COL) to disrupt microtubule function or 5 μg/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated JKbut not JNa(Morimoto et al. Am J Physiol Renal Physiol291: F663–F669, 2006), although COL but not BFA also blocked the flow-induced [Ca2+]itransient. We thus speculate that BK channel-mediated, flow-stimulated JKrequires an increase in [Ca2+]idue, in part, to luminal Ca2+entry and ER Ca2+release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane.

Rates of Na+absorption in the distal nephron increase proportionally with the rates of tubular flow. We tested the hypothesis that the deformation or tension generated in the plasma membrane in response to flow activates the epithelial sodium channel (ENaC). We modified the physical properties of the membrane by changing the temperature and the content of cholesterol. Rates of net Na+absorption measured in cortical collecting ducts (CCDs) perfused at room temperature at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates were less than those measured at 37°C at the same flow rates, although increases in tubular fluid flow rates led to comparable relative increases in net Na+absorption at both temperatures. Xenopus laevisoocytes expressing ENaC responded to an increase in shear stress at 22–25°C with a discrete delay followed by a monoexponential increase in whole-cell Na+currents. We observed that temperature affected 1) basal currents, 2) delay times, 3) kinetics of activation, and 4) fold-increase in macroscopic currents in response to flow. The magnitude of the response to flow displayed biphasic behavior as a function of temperature, with a minimal value at 25°C. Steady-state fluorescence anisotropic measurements of purified plasma membranes did not show any obvious phase transition behavior over a temperature range from 8.3°C to 36.5°C. Modification of the content of membrane cholesterol did not affect the response to flow. Our results suggest that the flow-dependent activation of ENaC is not influenced by modifications in the intrinsic properties of the plasma membrane.

Thiazide and loop diuretics induce renal K+secretion, often leading to renal K+wasting and hypokalemia. This phenomenon has been proposed to reflect an increase in delivery to and reabsorption of Na+by the distal nephron, with a resultant increase in the driving force for passive K+efflux across the apical membrane. Recent studies suggest that cellular mechanisms that lead to enhanced rates of Na+reabsorption as well as K+secretion in response to increases tubular flow rates are more complex. Increases in tubular flow rates directly enhance the activity of apical membrane Na+channels and indirectly activate a class of K+channels, referred to as maxi-K, that are functionally inactive under low flow states. This review addresses the role of biomechanical forces, generated by variations in urinary flow rate and tubular fluid volume, in the regulation of transepithelial Na+and K+transport in the distal nephron. The question of why the distal nephron has evolved to include a component of flow-dependent K+secretion is also addressed.

Vectorial Na+absorption across the aldosterone-sensitive distal nephron plays a key role in the regulation of extracellular fluid volume and blood pressure. Within this nephron segment, Na+diffuses from the urinary fluid into principal cells through an apical, amiloride-sensitive, epithelial Na+channel (ENaC), which is considered to be the rate-limiting step for Na+absorption. We have reported that increases in tubular flow rate in microperfused rabbit cortical collecting ducts (CCDs) lead to increases in net Na+absorption and that increases in laminar shear stress activate ENaC expressed in oocytes by increasing channel open probability. We therefore examined whether flow stimulates net Na+absorption (JNa) in CCDs by increasing channel open probability or by increasing the number of channels at the apical membrane. Both baseline and flow-stimulated JNain CCDs were mediated by ENaC, as JNawas inhibited by benzamil. Flow-dependent increases in JNawere observed following treatment of tubules with reagents that altered membrane trafficking by disrupting microtubules (colchicine) or Golgi (brefeldin A). Furthermore, reducing luminal Ca2+concentration ([Ca2+]) or chelating intracellular [Ca2+] with BAPTA did not prevent the flow-dependent increase in JNa. Extracellular trypsin has been shown to activate ENaC by increasing channel open probability, and we observed that trypsin significantly enhanced JNawhen tubules were perfused at a slow flow rate. However, trypsin did not further enhance JNain CCDs perfused at fast flow rates. Similarly, the shear-induced increase in benzamil-sensitive JNain oocytes expressing protease resistance ENaC mutants was similar to that of controls. Our results suggest the rise in JNaaccompanying increases in luminal flow rates reflects an increase in channel open probability.

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the progressive dilatation of collecting ducts, the nephron segments responsible for the final renal regulation of sodium, potassium, acid-base, and water balance. Murine models of ARPKD possess mutations in genes encoding cilia-associated proteins, including Tg737in orpkmice. New findings implicate defects in structure/function of primary cilia as central to the development of polycystic kidney disease. Our group (Liu W, Xu S, Woda C, Kim P, Weinbaum S, and Satlin LM, Am J Physiol Renal Physiol285: F998–F1012, 2003) recently reported that increases in luminal flow rate in rabbit collecting ducts increase intracellular Ca2+concentration ([Ca2+]i) in cells therein. We thus hypothesized that fluid shear acting on the apical membrane or hydrodynamic bending moments acting on the cilium increase renal epithelial [Ca2+]i. To further explore this, we tested whether flow-induced [Ca2+]itransients in collecting ducts from mutant orpkmice, which possess structurally abnormal cilia, differ from those in controls. Isolated segments from 1- and 2-wk-old mice were microperfused in vitro and loaded with fura 2; [Ca2+]iwas measured by digital ratio fluorometry before and after the rate of luminal flow was increased. All collecting ducts responded to an increase in flow with an increase in [Ca2+]i, a response that appeared to be dependent on luminal Ca2+entry. However, the magnitude of the increase in [Ca2+]iin 2- but not 1-wk-old mutant orpkanimals was blunted. We speculate that this defect in mechano-induced Ca2+signaling in orpkmice leads to aberrant structure and function of the collecting duct in ARPKD.

The cortical collecting duct (CCD) is a final site for regulation of K+ homeostasis. CCD K+ secretion is determined by the electrochemical gradient and apical permeability to K+. Conducting secretory K+ (SK/ROMK) and maxi-K channels are present in the apical membrane of the CCD, the former in principal cells and the latter in both principal and intercalated cells. Whereas SK channels mediate baseline K+ secretion, maxi-K channels appear to participate in flow-stimulated K+ secretion. Chronic dietary K+ loading enhances the CCD K+ secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol 104: 693–710, 1994). Long-term exposure of Ambystoma tigrinum to elevated K+ increases renal K+ excretion due to an increase in apical maxi-K channel density in their CDs (Stoner and Viggiano, J Membr Biol 162: 107–116, 1998). The purpose of the present study was to test whether K+ adaptation in the mammalian CCD is associated with upregulation of maxi-K channel expression. New Zealand White rabbits were fed a low (LK), control (CK), or high (HK) K+ diet for 10–14 days. Real-time PCR quantitation of message encoding maxi-K α- and β2–4-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals (P < 0.05); β1-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of α-subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in α-intercalated cells in HK kidneys. In summary, K+ adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical α-subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane.

The cortical collecting duct (CCD) is a final site for regulation of K+homeostasis. CCD K+secretion is determined by the electrochemical gradient and apical permeability to K+. Conducting secretory K+(SK/ROMK) and maxi-K channels are present in the apical membrane of the CCD, the former in principal cells and the latter in both principal and intercalated cells. Whereas SK channels mediate baseline K+secretion, maxi-K channels appear to participate in flow-stimulated K+secretion. Chronic dietary K+loading enhances the CCD K+secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol104: 693–710, 1994). Long-term exposure of Ambystoma tigrinumto elevated K+increases renal K+excretion due to an increase in apical maxi-K channel density in their CDs (Stoner and Viggiano, J Membr Biol162: 107–116, 1998). The purpose of the present study was to test whether K+adaptation in the mammalian CCD is associated with upregulation of maxi-K channel expression. New Zealand White rabbits were fed a low (LK), control (CK), or high (HK) K+diet for 10–14 days. Real-time PCR quantitation of message encoding maxi-K α- and β2–4-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals (P< 0.05); β1-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of α-subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in α-intercalated cells in HK kidneys. In summary, K+adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical α-subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane.

Somatic growth is associated with an increase in total body Kcontent. Khomeostasis is regulated, in large part, by urinary Kexcretion. Within the adult kidney and specifically the cortical collecting duct, Ksecretion is accomplished by the passive diffusion of cell Kinto the urinary fluid down a favorable electrochemical gradient through Kselective channels. The purpose of this review is to summarize the results of recent studies that provide insight into how the cortical collecting duct is uniquely adapted for Kretention early in life.

An acute increase in tubular fluid flow rate in the microperfused cortical collecting duct (CCD), associated with a ∼20% increase in tubular diameter, leads to an increase in intracellular Ca2+concentration ([Ca2+]i)in both principal and intercalated cells (Woda CB, Leite M Jr, Rohatgi R, and Satlin LM. Am J Physiol Renal Physiol283: F437-F446, 2002). The apical cilium present in principal but not intercalated cells has been proposed to be a flow sensor. To determine whether flow across the cilium and/or epithelial stretch mediates the [Ca2+]iresponse, CCDs from New Zealand White rabbits were microperfused in vitro, split-open (to isolate the effect of flow across cilia), or occluded (to examine the effect of stretch and duration/magnitude of the flow impulse), and [Ca2+]iwas measured using fura 2. In perfused and occluded CCDs, a rapid (<1 s) but not slow (>3 min) increase in luminal flow rate and/or circumferential stretch led to an approximately threefold increase in [Ca2+]iin both principal and intercalated cells within ∼10 s. This response was mediated by external Ca2+entry and inositol 1,4,5-trisphosphate-mediated release of cell Ca2+stores. In split-open CCDs, an increase in superfusate flow led to an approximately twofold increase in [Ca2+]iin both cell types within ∼30 s. These experimental findings are interpreted using mathematical models to predict the fluid stress on the apical membranes of the CCD and the forces and torques on and deformation of the cilia. We conclude that rapid increases in luminal flow rate and circumferential stretch, leading to shear or hydrodynamic impulses at the cilium or apical membrane, lead to increases in [Ca2+]iin both principal and intercalated cells.

High urinary flow rates stimulate K secretion in the fully differentiated but not neonatal or weanling rabbit cortical collecting duct (CCD). Both small-conductance secretory K and high-conductance Ca2+/stretch-activated maxi-K channels have been identified in the apical membrane of the mature CCD by patch-clamp analysis. We reported that flow-stimulated net K secretion in the adult rabbit CCD is 1) blocked by TEA and charybdotoxin, inhibitors of intermediate- and high-conductance (maxi-K) Ca2+-activated K channels, and 2) associated with increases in net Na absorption and intracellular Ca2+concentration ([Ca2+]i). The present study examined whether the absence of flow-stimulated K secretion early in life is due to a 1) limited flow-induced rise in net Na absorption and/or [Ca2+]iand/or 2) paucity of apical maxi-K channels. An approximately sixfold increase in tubular fluid flow rate in CCDs isolated from 4-wk-old rabbits and microperfused in vitro led to an increase in net Na absorption and [Ca2+]i, similar in magnitude to the response observed in 6-wk-old tubules, but it failed to generate an increase in net K secretion. By 5 wk of age, there was a small, but significant, flow-stimulated rise in net K secretion that increased further by 6 wk of life. Luminal perfusion with iberiotoxin blocked the flow stimulation of net K secretion in the adult CCD, confirming the identity of the maxi-K channel in this response. Maxi-K channel α-subunit message was consistently detected in single CCDs from animals ≥4 wk of age by RT-PCR. Indirect immunofluorescence microscopy using antibodies directed against the α-subunit revealed apical labeling of intercalated cells in cryosections from animals ≥5 wk of age; principal cell labeling was generally intracellular and punctate. We speculate that the postnatal appearance of flow-dependent K secretion is determined by the transcriptional/translational regulation of expression of maxi-K channels. Furthermore, our studies suggest a novel function for intercalated cells in mediating flow-stimulated K secretion.

Nucleotide binding to purinergic P2 receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Whereas P2 receptors have been functionally identified at the basolateral membrane of the cortical collecting duct (CCD), a final regulatory site of urinary Na+, K+, and acid-base excretion, controversy exists as to whether apical purinoceptors exist in this segment. Nor has the distribution of receptor subtypes present on the unique cell populations that constitute Ca2+the CCD been established. To examine this, we measured nucleotide-induced changes in intracellular Ca2+concentration ([Ca2+]i) in fura 2-loaded rabbit CCDs microperfused in vitro. Resting [Ca2+]idid not differ between principal and intercalated cells, averaging ∼120 nM. An acute increase in tubular fluid flow rate, associated with a 20% increase in tubular diameter, led to increases in [Ca2+]iin both cell types. Luminal perfusion of 100 μM UTP or ATP-γ-S, in the absence of change in flow rate, caused a rapid and transient approximately fourfold increase in [Ca2+]iin both cell types (P< 0.05). Luminal suramin, a nonspecific P2 receptor antagonist, blocked the nucleotide- but not flow-induced [Ca2+]itransients. Luminal perfusion with a P2X (α,β-methylene-ATP), P2X7(benzoyl-benzoyl-ATP), P2Y1(2-methylthio-ATP), or P2Y4/P2Y6(UDP) receptor agonist had no effect on [Ca2+]i. The nucleotide-induced [Ca2+]itransients were inhibited by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl borate, thapsigargin, which depletes internal Ca2+stores, luminal perfusion with a Ca2+-free perfusate, or the L-type Ca2+channel blocker nifedipine. These results suggest that luminal nucleotides activate apical P2Y2receptors in the CCD via pathways that require both internal Ca2+mobilization and extracellular Ca2+entry. The flow-induced rise in [Ca2+]iis apparently not mediated by apical P2 purinergic receptor signaling.

Na+absorption in the renal cortical collecting duct (CCD) is mediated by apical epithelial Na+channels (ENaCs). The CCD is subject to continuous variations in intraluminal flow rate that we speculate alters hydrostatic pressure, membrane stretch, and shear stress. Although ENaCs share limited sequence homology with putative mechanosensitive ion channels in Caenorhabditis elegans,controversy exists as to whether ENaCs are regulated by biomechanical forces. We examined the effect of varying the rate of fluid flow on whole cell Na+currents (INa) in oocytes expressing mouse α,β,γ-ENaC (mENaC) and on net Na+absorption in microperfused rabbit CCDs. Oocytes injected with mENaC but not water responded to the initiation of superfusate flow (to 4–6 ml/min) with a reversible threefold stimulation of INawithout a change in reversal potential. The increase inINawas variable among oocytes. CCDs responded to a threefold increase in rate of luminal flow with a twofold increase in the rate of net Na+absorption. An increase in luminal viscosity achieved by addition of 5% dextran to the luminal perfusate did not alter the rate of net Na+absorption, suggesting that shear stress does not influence Na+transport in the CCD. In sum, our data suggest that flow stimulation of ENaC activity and Na+absorption is mediated by an increase in hydrostatic pressure and/or membrane stretch. We propose that intraluminal flow rate may be an important regulator of channel activity in the CCD.

K+secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+(SK) and a high-conductance Ca2+-activated K+(maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+channel mediating flow-dependent K+secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+channels. Net K+secretion and Na+absorption were measured at varying flow rates. In the absence of TEA, net K+secretion increased from 8.3 ± 1.0 to 23.4 ± 4.7 pmol · min−1· mm−1(P< 0.03) as the tubular flow rate was increased from 0.5 to 6 nl · min−1· mm−1. Flow stimulation of net K+secretion was blocked by luminal TEA (8.2 ± 1.2 vs. 9.9 ± 2.7 pmol · min−1· mm−1at 0.6 and 6 nl · min−1· mm−1flow rates, respectively) or charybdotoxin (6.8 ± 1.6 vs. 8.3 ± 1.6 pmol · min−1· mm−1at 1 and 4 nl · min−1· mm−1flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+secretion is mediated by a maxi-K channel, whereas baseline K+secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.

Within the renal cortical collecting duct (CCD), transepithelial Na+absorption and K+secretion are linked to basolateral Na+-K+-ATPase activity. Our purpose was to examine the developmental changes in basolateral Na+-K+-ATPase-mediated86rubidium (Rb) uptake, its inhibitor sensitivity and relationship to pump hydrolytic activity and Na+transport. Multiple CCDs (∼6 mm) from maturing rabbits were affixed to coverslips, preincubated at 37°C for 10 min (±1–2.5 mM ouabain or 10 or 100 μM Schering-28080, an inhibitor of H+-K+-ATPase), and then transferred to prewarmed incubation solution containing tracer amounts of86Rb (±inhibitors). After 1 min at 37°C, tubular samples were rinsed and permeabilized and isotope counts were measured to calculate basolateral Rb uptake. Ouabain-inhibitable Rb uptake, an index of basolateral Na+-K+pump activity, increased ∼3-fold during the 1st 8 wk of postnatal life (P< 0.03). The ∼2-fold increase in absolute rate of Rb uptake between 1 and 6 wk (2.64 ± 0.45 to 5.02 ± 0.32 pmol · min−1· mm−1) did not reach statistical significance. The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 ± 0.53 pmol · min−1· mm−1in adult animals (P < 0.03 vs. 6 wk). Schering-28080 failed to inhibit Rb uptake, implying that functional H+-K+-ATPase is absent at the basolateral membrane. Na+-K+-ATPase hydrolytic activity, determined by using a microassay that measured inorganic phosphate release from [γ-32P]ATP under maximum velocity (Vmax) conditions, also increased in the differentiating CCD (from 316.2 ± 44.4 pmol · h−1· mm−1at 2 wk to 555.9 ± 105.1 at 4 wk to 789.7 ± 145.0 at 6 wk;r= 1.0 by linear regression analysis; P < 0.005). The parallel ∼2.5-fold increases in Na+-K+-ATPase activity and ouabain-sensitive Rb uptake between 2- and 6-wk postnatal age suggest that the developmental increase in basolateral transport capacity is due predominantly to an increase in enzyme abundance. The signals mediating the developmental increase in Na+-K+-ATPase activity in the CCD remain to be defined.

The apical secretory K+(SK) channel in the principal cell represents the rate-limiting step for K+secretion across the cortical collecting duct (CCD). Patch clamp analysis of maturing rabbit principal cells identifies an increase in number of conducting SK channels after the 2nd week of life [L. M. Satlin and L. G. Palmer. Am. J. Physiol.272 (Renal Physiol.41): F397–F404, 1997], ∼1 wk after an increase in activity of the amiloride-sensitive epithelial Na+channel (ENaC) is detected. To correlate the postnatal increase in channel activity with developmental expression of ROMK, the molecular correlate of the SK channel, we used gene-specific probes to show a developmental increase in abundance of renal ROMK mRNA and a ROMK-specific antibody to examine the nephron distribution, localization, and abundance of this protein in developing rat kidney. Using antibodies directed against aquaporin-3 (AQP-3) and Tamm-Horsfall protein (THP), we confirmed that ROMK was expressed along the apical membranes of principal cells and thick ascending limbs of Henle (TALH) in adult kidney. Within the midcortex of the neonatal kidney, ROMK-positive segments revealed weak coincident staining with the TALH-specific antibody but did not colabel with an antibody directed against distal and connecting tubule (CNT)-specific kallikrein or the lectin Dolichos biflorusagglutinin (DBA), which labels proximal tubules and collecting ducts. In inner cortex and outer medulla of kidneys from 1-wk-old animals, ROMK protein was identified in medullary TALH (MTALH) and DBA-positive collecting ducts. By 3 wk of age, coincident ROMK and DBA expression was detected in midcortical and outer cortical CNTs and CCDs. Immunoblot analysis of plasma membrane-enriched fractions of maturing rat kidney revealed a developmental increase in a ∼40-kDa band, the expected size for ROMK. Immunolocalization of α-ENaC showed apical staining of a majority of cells in distal nephron segments after the 1st week of postnatal life. The β- and γ-ENaC subunit expression was routinely detected in a mostly cytoplasmic distribution immediately after birth, albeit in low abundance; γ-ENaC showed some apical polarization. These results suggest that the postnatal increases in a principal cell apical SK and Na+channel activity are mediated, at least in part, by increases in abundance of ROMK message and protein and ENaC subunit proteins.

Summary The studies outlined in this review suggest that the immaturity of distal nephron segments may hinder urinary excretion of potassium early in life. Among the factors that may limit potassium secretion by principal cells in the neonatal cortical collecting duct are an unfavorable electrochemical gradient (reduced Ki, Na+-K+-ATPase activity and/or Vte), limited membrane permeability to potassium and sodium, low tubular fluid flow rate, reduced luminal sodium concentration, or increased paracellular backleak. Alternatively, enhanced potassium absorption by other relatively well-differentiated distal nephron segments may contribute in part to a reduced net potassium excretory rate in the newborn.

The mature, fully differentiated cortical collecting duct plays a major role in the final renal regulation of Na+, K+ and H+ transport. To characterize the growth of this segment, we measured the outer diameter and the dry weight of cortical collecting ducts isolated from newborn, 1-month-old, and adult rabbits. During the 1st month of life no significant changes were observed; however, there was a 60% increase in both parameters after the 4th week of life. Growth-related accretion of K+ was demonstrated by showing tubular K+ content to increase by 60% with maturation. Concomitant with the increase in tubular size, total cell number per millimeter of tubular length rose by 30%. Approximately 50% of the observed increment in tubular size could be accounted for by cell hyperplasia, with the remaining increase resulting from cell hypertrophy. Hypertrophy of principal cells was confirmed by scanning electron microscopy, which demonstrated a doubling of the circumferential width without any change in longitudinal length. Hyperplasia was confirmed, using a fluorescent chromatin stain, by our finding of a mitotic frequency of 3/1000 cells in the neonatal mid-cortical collecting duct; the observed number of mitoses was 10-fold higher at the most cortical end (ampulla). The number of intercalated cells per millimeter of tubule length, identified by bright green fluorescence after cortical collecting ducts were stained with 6-carboxyfluorescein diacetate, was found to double during maturation, the increase being significant only after the 4th postnatal week. We conclude that maturation of the mid-cortical collecting duct results from both cellular hyperplasia and hypertrophy. It is unlikely that this segment plays a major role in regulating Na+, K+, and H+ transport in the neonatal kidney.

K-dependent H⁺ extrusion was investigated using fluorescence techniques in rabbit cortical collecting tubules (CCTs). Experiments were performed in split-open tubules from normal animals exposed to the intracellular pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). This preparation permitted the study of individual intercalated cells (Ics). In the Ics, partial recovery of pHi was observed in response to an acute acid load upon readdition of 5 mM K to the superfusate. This recovery was SCH 28080-inhibitable (10^–5 M) and ouabain-insensitive suggesting the process is mediated by a gastric-type H-K ATPase. To see if H-K ATPase plays a role in acid secretion its function was evaluated under chronic metabolic acidosis (CMA) conditions. CMA was induced by replacing drinking water with 75 mM NH4Cl in 5% sucrose for 10–14 days. The SCH 28080-inhibitable K-dependent pHi recovery rate was three-fold higher in CMA ICs compared to controls. To determine the location of the H-K ATPase, CCTs were microperfused and individual peanut lectin binding (PNA) ICs studied. K-dependent pHi recovery was measured in response to an NH4Cl pulse. An apical SCH 28080-inhibited K-dependent pHi recovery process was observed in control and CMA Ics. Taken together these data confirm the existence of a gastric-type H-K ATPase in ICs of rabbit CCT. Based on our findings the H-K ATPase is found on the apical side of the cell and is stimulated under conditions of CMA. J. Exp. Zool. 279:443–455, 1997. © 1997 Wiley-Liss, Inc.

Organic anion transporting polypeptide (oatp) is an integral membrane protein cloned from rat liver that mediates Na+-independent transport of organic anions such as sulfobromophthalein and taurocholic acid. Previous studies in rat hepatocytes suggested that organic anion uptake is associated with base exchange. To better characterize the mechanism of oatp-mediated organic anion uptake, we examined transport of taurocholate in a HeLa cell line stably transfected with oatp under the regulation of a zinc-inducible promoter (Shi, X., Bai, S., Ford, A. C., Burk, R. D., Jacquemin, E., Hagenbuch, B., Meier, P. J., and Wolkoff, A. W. (1995) J. Biol. Chem.270, 25591–25595). Whereas noninduced transfected cells showed virtually no uptake of [3H]taurocholate, taurocholate uptake by induced cells was Na+-independent and saturable (Km= 19.4 ± 3.3 μm; Vmax= 62.2 ± 1.4 pmol/min/mg protein; n= 3). To test whether organic anion transport is coupled to HCO3−extrusion, we compared the rates of taurocholate-dependent HCO3−efflux from alkali-loaded noninduced and induced cells. Monolayers grown on glass coverslips were loaded with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH (pHi) was measured by excitation ratio fluorometry. Noninduced and induced cells were alkalinized to an equivalent pHi(∼7.7) by transient exposure to a 50 mmHCO3−, Cl−-free solution. In the absence of extracellular Cl−and taurocholate, isohydric reduction of superfusate HCO3−concentration from 50 to 25 mmresulted in an insignificant change in pHiover time (dpHi/dt) in both groups. Addition of 25 μmtaurocholate to the superfusate led to a rapid fall in pHiin induced (−0.037 ± 0.011 pH units/min to pHiof 7.41 ± 0.14) but not in noninduced (0.003 ± 0.006 pH units/min to pHiof 7.61 ± 0.08) cells (p< 0.03). These data indicate that oatp-mediated taurocholate transport is Na+-independent, saturable, and accompanied by HCO3−exchange. We conclude that organic anion/base exchange is an important, potentially regulatable component of oatp function.