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The pore-forming a-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channelmediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney. NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+. [ABSTRACT FROM AUTHOR]

The epithelial Na+ channel (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 vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation 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/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, 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 not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo. NEW & NOTEWORTHY: The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets. [ABSTRACT FROM AUTHOR]

Cannabis and synthetic cannabinoid consumption are increasing worldwide. Cannabis contains numerous phytocannabinoids that act on the G protein-coupled cannabinoid receptor type 1 (CB1R) and cannabinoid receptor type 2 expressed throughout the body, including the kidney. Essentially every organ, including the kidney, produces endocannabinoids, which are endogenous ligands to these receptors. Cannabinoids acutely increase urine output in rodents and humans, thus potentially influencing total body water and electrolyte homeostasis. As the kidney collecting duct (CD) regulates total body water, acid/base, and electrolyte balance through specific functions of principal cells (PCs) and intercalated cells (ICs), we examined the cell-specific immunolocalization of CB1R in the mouse CD. Antibodies against either the C-terminus or N-terminus of CB1R consistently labeled aquaporin 2 (AQP2)-negative cells in the cortical and medullary CD and thus presumably ICs. Given the well-established role of ICs in urinary acidification, we used a clearance approach in mice that were acid loaded with 280 mM NH4Cl for 7 days and nonacid-loaded mice treated with the cannabinoid receptor agonist WIN55,212-2 (WIN) or a vehicle control. Although WIN had no effect on urinary acidification, these WIN-treated mice had less apical + subapical AQP2 expression in PCs compared with controls and developed acute diabetes insipidus associated with the excretion of large volumes of dilute urine. Mice maximally concentrated their urine when WIN and 1-desamino-8- d -arginine vasopressin [desmopressin (DDAVP)] were coadministered, consistent with central rather than nephrogenic diabetes insipidus. Although ICs express CB1R, the physiological role of CB1R in this cell type remains to be determined. NEW & NOTEWORTHY: The CB1R agonist WIN55,212-2 induces central diabetes insipidus in mice. This research integrates existing knowledge regarding the diuretic effects of cannabinoids and the influence of CB1R on vasopressin secretion while adding new mechanistic insights about total body water homeostasis. Our findings provide a deeper understanding about the potential clinical impact of cannabinoids on human physiology and may help identify targets for novel therapeutics to treat water and electrolyte disorders such as hyponatremia and volume overload. [ABSTRACT FROM AUTHOR]

Kidney organoids are three-dimensional structures generated from pluripotent stem cells (PSCs) that are capable of recapitulating the major structures of mammalian kidneys. As this technology is expected to be a promising tool for studying renal biology, drug discovery, and regenerative medicine, the functional capacity of kidney organoids has emerged as a critical question in the field. Kidney organoids produced using several protocols harbor key structures of native kidneys. Here, we review the current state, recent advances, and future challenges in the functional characterization of kidney organoids, strategies to accelerate and enhance kidney organoid functions, and access to PSC resources to advance organoid research. The strategies to construct physiologically relevant kidney organoids include the use of organ-on-a-chip technologies that integrate fluid circulation and improve organoid maturation. These approaches result in increased expression of the major tubular transporters and elements of mechanosensory signaling pathways suggestive of improved functionality. Nevertheless, continuous efforts remain crucial to create kidney tissue that more faithfully replicates physiological conditions for future applications in kidney regeneration medicine and their ethical use in patient care. NEW & NOTEWORTHY Kidney organoids are three-dimensional structures derived from stem cells, mimicking the major components of mammalian kidneys. Although they show great promise, their functional capacity has become a critical question. This review explores the advancements and challenges in evaluating and enhancing kidney organoid function, including the use of organ-on-chip technologies, multiomics data, and in vivo transplantation. Integrating these approaches to further enhance their physiological relevance will continue to advance disease modeling and regenerative medicine applications. [ABSTRACT FROM AUTHOR]

Kidney organoids cultured on adherent matrices in the presence of superfusate flow generate vascular networks and exhibit more mature podocyte and tubular compartments compared with static controls (Homan KA, Gupta N, Kroll KT, Kolesky DB, Skylar-Scott M, Miyoshi T, Mau D, Valerius MT, Ferrante T, Bonventre JV, Lewis JA, Morizane R. Nat Methods 16: 255-262, 2019; Takasato M, Er PX, Chiu HS, Maier B, Baillie GJ, Ferguson C, Parton RG, Wolvetang EJ, Roost MS, Chuva de Sousa Lopes SM, Little MH. Nature 526: 564-568, 2015.). However, their physiological function has yet to be systematically investigated. Here, we measured mechano-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in tubules isolated from organoids cultured for 21-64 days, microperfused in vitro or affixed to the base of a specimen chamber, and loaded with fura-2 to measure [Ca2+]i. A rapid >2.5-fold increase in [Ca2+]i from a baseline of 195.0 ± 22.1 nM (n = 9; P ≤ 0.001) was observed when microperfused tubules from organoids >40 days in culture were subjected to luminal flow. In contrast, no response was detected in tubules isolated from organoids <30 days in culture. Nonperfused tubules (41 days) subjected to a 10-fold increase in bath flow rate also exhibited a threefold increase in [Ca2+]i from baseline (P < 0.001). Mechanosensitive PIEZO1 channels contribute to the flowinduced [Ca2+]i response in mouse distal tubule (Carrisoza-Gaytan R, Dalghi MG, Apodaca GL, Kleyman TR, Satlin LM. The FASEB J 33: 824.25, 2019.). Immunodetectable apical and basolateral PIEZO1 was identified in tubular structures by 21 days in culture. Basolateral PIEZO1 appeared to be functional as basolateral exposure of nonperfused tubules to the PIEZO1 activator Yoda 1 increased [Ca2+]i (P ≤ 0.001) in segments from organoids cultured for >30 days, with peak [Ca2+]i increasing with advancing days in culture. These results are consistent with a maturational increase in number and/or activity of flow/stretch-sensitive Ca2+ channels, including PIEZO1, in tubules of static organoids in culture. [ABSTRACT FROM AUTHOR]

The role of mechanical forces in the regulation of glomerulotubular balance in the proximal tubule (PT) and [Ca.sup.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.sup.+], HC[O.sup.-.sub.3], and water reabsorption in the PT and [Ca.sup.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 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. proximal tubule; cortical collecting duct; microvilli; primary cilia; polycystic kidney disease; fluid flow in renal tubule doi: 10.1152/ajprenal.00453.2010

The fine regulation of [Na.sup.+] and [K.sup.+] transport takes place in the cortical distal nephron. It is well established that [K.sup.+] secretion occurs through apical [K.sup.+] channels: the ROMK and the [Ca.sup.2+]-and voltage-dependent maxi-K. Previously, we identified the voltage-gated Kvl.3 channel in the inner medulla of the rat kidney (Escobar LI, Martinez-Tellez JC, Salas M, Castilla SA, Carrisoza R, Tapia D, Vazquez M, Bargas J, Bolivar JJ. Am J Physiol Cell Physiol 286: C965-C974, 2004). To examine the role of Kv1.3 in the renal regulation of [K.sup.+] homeostasis, we characterized the effect of dietary [K.sup.+] 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.sup.+] (HK; 10% wt/wt) diet for 5-15 days. Kvl.3 mRNA and protein expression did not change with HK in the whole kidney. However, dietary [K.sup.+] loading provoked a change in the cellular distribution of Kvl.3 from the cytoplasm to apical membranes. Immunolocalization of Kvl.3 detected the channel exclusively in the intercalated cells. We investigated whether Kvl.3 mediated [K.sup.+] transport in microperfused cortical collecting ducts (CCDs). The HK diet led to an increase in net [K.sup.+] transport from 7.4 [+ or -] 1.1 (CK) to 11.4 [+ or -] 1.0 (HK) pmol x [min.sup.-1] x [mm .sup.-1.] Luminal margatoxin, a specific blocker of Kv1.3, decreased net [K.sup.+] secretion in HK CCDs to 6.0 [+ or -] 1.6 pmol x [min.sup.-1] [mm.sup.-1]. Our data provide the first evidence that Kvl.3 channels participate in [K.sup.+] secretion and that apical membrane localization of Kvl.3 is enhanced in the intercalated cells by dietary [K.sup.+] loading. potassium diet; potassium transport; distal nephron; intercalated cell; rat kidney; urine pH doi: 10.1152/ajprenal.00697.2009.

Flow-stimulated net K secretion ([J.sub.K]) in the cortical collecting duct (CCD) is mediated by an iberiotoxin (IBX)-sensitive BK channel, and requires an increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). The [alpha]-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, [J.sub.K] and net Na absorption ([J.sub.Na]) were measured at slow (~1) and fast (~5 nl x [min.sup.-1] x [mm.sup.-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 Go6976, inhibitors of classic and novel PKC isoforms, added to luminal (L) and/or basolateral (B) solutions. L but not B mPKI increased [J.sub.K] in CCDs perfused at a slow flow rate; a subsequent increase in flow rate augmented [J.sub.K] modestly. B mPKI alone or with L inhibitor abolished flow stimulation of [J.sub.K]. Similarly, L calphostin C increased [J.sub.K] in 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 [J.sub.K] at 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 Go6976 failed to enhance [J.sub.K] at 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 [[[Ca.sup.2+]].sub.i] suggests that the principal cell apical BK channel is tonically inhibited by PKA and that flow stimulation of JK in the CCD is PKA and PKC dependent. The specific targets of the kinases remain to be identified. K secretion; ROMK; mechanoregulation; in vitro microperfusion; laminar shear doi: 10.1152/ajprenal.90685.2008

Apical large-conductance [Ca.sup.2+]-activated [K.sup.+] (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated [K.sup.+] secretion. Dietary [K.sup.+] loading for 10-14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated [K.sup.+] 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 Physiol 289: F922-F932, 2005). To test whether this adaptation was mediated by a [K.sup.+]-induced increase in aldosterone, New Zealand White rabbits were fed a low-[Na.sup.+] (LS) or high-[Na.sup.+] (HS) diet for 7-10 days to alter circulating levels of aldosterone but not serum [K.sup.+] concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, s-splice variants, [beta]-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial [Na.sup.+] (JNa) and [K.sup.+] ([J.sub.K]) transport by microperfusion; kidneys were processed for immunolocalization of BK [alpha]-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary [Na.sup.+] and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK [alpha]-, [[beta].sub.2]-, and [[beta].sub.4]-subunit mRNA and localization of immunodetectable [alpha]-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ~1 to 5 nl x [min.sup.-1] x [mm.sup.-1], the increase in [J.sub.Na] was greater in LS vs. HS rabbits, yet the flow-stimulated increase in [J.sub.K] was 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.sup.+] loading. ROMK; epithelial sodium channel; mechanoregulation; laminar shear stress; epithelial cell

Mutations of cilia-expressed proteins are associated with an attenuated shear-induced increase in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.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 [Ca.sup.2+] 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 [[[Ca.sup.2+].sub.i] were similar between ARPKD 5E and pool, and HFCT 2C and pool; however, the flowinduced peak [[Ca.sup.2+].sub.i] was greater in ARPKD 5E (700 [+ or -] 87 nM, n = 21) than in HFCT 2C (315 [+ or -] 58 nM, n = 12; P < 0.01) cells. ARPKD 5E cells treated with [Gd.sup.3+] an inhibitor of nonselective cation channels, inhibited but did not abolish the shear-induced [[Ca.sup.2+].sub.i transient. Cilia were ~20% shorter in ARPKD than HFCT cells, but no difference in ciliary localization or total cellular expression of polycystin-2, a mechanosenory [Gd.sup.3+]-sensitive cation channel, was detected between ARPKD and HFCT cells. The intracellular [Ca.sup.2+] stores were similar between cells. In summary, human ARPKD cells exhibit an exaggerated [Gd.sup.3+]-sensitive mechano-induced [Ca.sup.2+] response compared with controls; whether this represents dysregulated polycystin-2 activity in ARPKD cells remains to be explored. calcium; ion channel; transport; laminar shear stress

Apical low-conductance SK and high-conductance [Ca.sup.2+]-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion ([J.sub.K]) in the CCD is 1) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and 2) associated with an increase in [[Ca.sup.2+].sub.i], leading us to conclude that BK channels mediate flow-stimulated [J.sub.K]. To examine the [Ca.sup.2+] dependence and sources of [Ca.sup.2+] contributing to flow-stimulated [J.sub.K], [J.sub.K] and net Na absorption ([J.sub.Na]) were measured at slow (~1) and fast (~5 nl x [min.sup.-1 x m[m.sup.-1]) flow rates in rabbit CCDs microperfused in the absence of luminal [Ca.sup.2+] or after pretreatment with BAPTA-AM to chelate intracellular [Ca.sup.2+], 2-aminoethoxydiphenyl borate (2-APB), to inhibit the inositol 1,4,5-trisphosphate (I[P.sub.3]) receptor or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated [J.sub.Na] (Morimoto et al. Am J Physiol Renal Physiol 291 : F663-F669, 2006), inhibited flow-stimulated [J.sub.K]. Increases in [[[Ca.sup.2+]].sub.i] stimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 [micro]M colchicine (COL) to disrupt microtubule function or 5 [micro]g/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated [J.sub.K] but not [J.sub.Na] (Morimoto et al. Am J Physiol Renal Physiol 291: F663-F669, 2006), although COL but not BFA also blocked the flow-induced [[[Ca.sup.2+]].sub.i] transient. We thus speculate that BK channel-mediated, flow-stimulated [J.sub.K] requires an increase in [[[Ca.sup.2+]].sub.i] due, in part, to luminal [Ca.sup.2+] entry and ER [Ca.sup.2+] release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. maxi-K channel; BK channel; SK channel; mechanoregulation; ENaC doi:10.1152/ajprenal.00057.2007

Rates of [Na.sup.+] 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.sup.+] absorption measured in cortical collecting ducts (CCDs) perfused at room temperature at slow (~1) and fast (~5 nl x [min.sup.-1] x [mm.sup.-1]) flow rates were less than those measured at 37[degrees]C at the same flow rates, although increases in tubular fluid flow rates led to comparable relative increases in net [Na.sup.+] absorption at both temperatures. Xenopus laevis oocytes expressing ENaC responded to an increase in shear stress at 22-25[degrees]C with a discrete delay followed by a monoexponential increase in whole-cell [Na.sup.+] 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[degrees]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[degrees]C to 36.5[degrees]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. shear stress; mechanosensitive; mechanoregulation; anisotropy doi:10.1152/ajprenal.00455.2006

Thiazide and loop diuretics induce renal [K.sup.+] secretion, often leading to renal [K.sup.+] wasting and hypokalemia. This phenomenon has been proposed to reflect an increase in delivery to and reabsorption of [Na.sup.+] by the distal nephron, with a resultant increase in the driving force for passive [K.sup.+] efflux across the apical membrane. Recent studies suggest that cellular mechanisms that lead to enhanced rates of [Na.sup.+] reabsorption as well as [K.sup.+] secretion in response to increases tubular flow rates are more complex. Increases in tubular flow rates directly enhance the activity of apical membrane [Na.sup.+] channels and indirectly activate a class of [K.sup.+] 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.sup.+] and [K.sup.+] transport in the distal nephron. The question of why the distal nephron has evolved to include a component of flow-dependent [K.sup.+] secretion is also addressed. epithelial [Na.sup.+] channel; ROMK; cortical collecting duct; shear stress; SK channel

Vectorial [Na.sup.+] 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.sup.+] diffuses from the urinary fluid into principal cells through an apical, amiloride-sensitive, epithelial [Na.sup.+] channel (ENaC), which is considered to be the rate-limiting step for [Na.sup.+] absorption. We have reported that increases in tubular flow rate in microperfused rabbit cortical collecting ducts (CCDs) lead to increases in net [Na.sup.+] 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.sup.+] absorption ([J.sub.Na]) in CCDs by increasing channel open probability or by increasing the number of channels at the apical membrane. Both baseline and flow-stimulated [J.sub.Na], in CCDs were mediated by ENaC, as [J.sub.Na], was inhibited by benzamil. Flow-dependent increases in [J.sub.Na] were observed following treatment of tubules with reagents that altered membrane trafficking by disrupting microtubules (colchicine) or Golgi (brefeldin A). Furthermore, reducing luminal [Ca.sup.2+] concentration ([Ca.sup.2+]) or chelating intracellular [[Ca.sup.2+]] with BAPTA did not prevent the flow-dependent increase in [J.sub.Na]. Extracellular trypsin has been shown to activate ENaC by increasing channel open probability, and we observed that trypsin significantly enhanced [J.sub.Na] when tubules were perfused at a slow flow rate. However, trypsin did not further enhance [J.sub.Na] in CCDs perfused at fast flow rates. Similarly, the shear-induced increase in benzamil-sensitive [J.sub.Na] in oocytes expressing protease resistance ENaC mutants was similar to that of controls. Our results suggest the rise in [J.sub.Na] accompanying increases in luminal flow rates reflects an increase in channel open probability. epithelial sodium channel; in vitro microperfusion; protein trafficking; mechanoregulation; laminar shear; principal cell doi:10.1152/ajprenal.00514.2005

The Tg[737.sup.orpk] autosomal recessive polycystic kidney disease (ARPKD) mouse carries a hypomorphic mutation in the Tg737 gene. Because of the absence of its protein product Polaris, the nonmotile primary monocilium central to the luminal membrane of ductal epithelia, such as the cortical collecting duct (CCD) principal cell (PC), is malformed. Although the functions of the renal monocilium remain elusive, primary monocilia or flagella on neurons act as sensory organelles. Thus we hypothesized that the PC monocilium functions as a cellular sensor. To test this hypothesis, we assessed the contribution of Polaris and cilium structure and function to renal epithelial ion transport electrophysiology. Properties of Tg[737.sup.orpk] mutant CCD PC clones were compared with clones genetically rescued with wild-type Tg737 cDNA. All cells were grown as polarized cell monolayers with similarly high transepithelial resistance on permeable filter supports. Three- to fourfold elevated transepithelial voltage ([V.sub.te]) and short-circuit current ([I.sub.sc]) were measured in mutant orpk monolayers vs. rescued controls. Pharmacological and cell biological examination of this enhanced electrical end point in mutant monolayers revealed that epithelial [Na.sup.+] channels (ENaCs) were upregulated. Amiloride, ENaC-selective amiloride analogs (benzamil and phenamil), and protease inhibitors (aprotinin and leupeptin) attenuated heightened [V.sub.te] and [I.sub.sc]. Higher concentrations of additional amiloride analogs (ethylisopropylamiloride and dimethylamiloride) also revealed inhibition of [V.sub.te]. Cell culture requirements and manipulations were also consistent with heightened ENaC expression and function. Together, these data suggest that ENaC expression and/or function are upregulated in the luminal membrane of mutant, cilium-deficient orpk CCD PC monolayers vs. cilium-competent controls. When the genetic lesion causes loss or malformation of the monocilium, ENaC-driven [Na.sup.+] hyperabsorption may explain the rapid emergence of severe hypertension in a majority of patients with ARPKD. cilia; hypertension; ion transport; epithelial cells

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 Tg737 in orpk mice. 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 Physiol 285: F998-F1012, 2003) recently reported that increases in luminal flow rate in rabbit collecting ducts increase intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.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 [[[Ca.sup.2+]].sub.i]. To further explore this, we tested whether flow-induced [[[Ca.sup.2+]].sub.i] transients in collecting ducts from mutant orpk mice, 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; [[[Ca.sup.2+]].sub.i] was 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 [[[Ca.sup.2+]].sub.i], a response that appeared to be dependent on luminal [Ca.sup.2+] entry. However, the magnitude of the increase in [[[Ca.sup.2+]].sub.i] in 2- but not 1-wk-old mutant orpk animals was blunted. We speculate that this defect in mechano-induced [Ca.sup.2+] signaling in orpk mice leads to aberrant structure and function of the collecting duct in ARPKD. autosomal recessive polycystic kidney disease; calcium signaling; principal cell; intercalated cell; differentiation

The cortical collecting duct (CCD) is a final site for regulation of [K.sup.+] homeostasis. CCD [K.sup.+] secretion is determined by the electrochemical gradient and apical permeability to [K.sup.+]. Conducting secretory [K.sup.+] (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.sup.+] secretion, maxi-K channels appear to participate in flow-stimulated [K.sup.+] secretion. Chronic dietary [K.sup.+] loading enhances the CCD [K.sup.+] secretory capacity due, in part, to an increase in SK channel density (Palmer et al., J Gen Physiol 104: 693-710, 1994). Longterm exposure of Ambystoma tigrinum to elevated [K.sup.+] increases renal [K.sup.+] 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.sup.+] 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.sup.+] diet for 10-14 days. Real-time PCR quantitation of message encoding maxi-K [alpha]- and [[beta].sub.2-4]-subunits in single CCDs from HK animals was greater than that detected in CK and LK animals (P < 0.05); [[beta].sub.1]-subunit was not detected in any CCD sample but was present in whole kidney. Indirect immunofluorescence microscopy revealed a predominantly intracellular distribution of [alpha]-subunits in LK kidneys. In contrast, robust apical labeling was detected primarily in [alpha]-intercalated cells in HK kidneys. In summary, [K.sup.+] adaptation is associated with an increase in steady-state abundance of maxi-K channel subunit-specific mRNAs and immunodetectable apical [alpha]-subunit, the latter observation consistent with redistribution from an intracellular pool to the plasma membrane. potassium adaptation; intercalated cell; principal cell; in vitro micropeffusion; [H.sup.+]-[K.sup.+]-ATPase

Liu, Wen, Shiyun Xu, Craig Woda, Paul Kim, Sheldon Weinbaum, and Lisa M. Satlin. Effect of flow and stretch on the [[[Ca.sup.2.sup.+]].sub.i] response of principal and intercalated cells in cortical collecting duct. Am J Physiol Renal Physiol 285: F998-F1012, 2003. First published July 1, 2003; 10.1152/ajprenal.00067.2003.--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 [Ca.sup.2.sup.+]concentration ([[[Ca.sup.2.sup.+]].sub.i]) in both principal and intercalated cells (Woda CB, Leite M Jr, Rohatgi R, and Satlin LM. Am J Physiol Renal Physiol 283: 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 [[[Ca.sup.2.sup.+]].sub.i] response, 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 [[[Ca.sup.2.sup.+]].sub.i] was measured using fura 2. In perfused and occluded CCDs, a rapid (3 min) increase in luminal flow rate and/or circumferential stretch led to an approximately threefold increase in [[[Ca.sup.2.sup.+]].sub.i] in both principal and intercalated cells within ~10 s. This response was mediated by external [Ca.sup.2.sup.+] entry and inositol 1,4,5-trisphosphate-mediated release of cell [Ca.sup.2.sup.+] stores. In split-open CCDs, an increase in superfusate flow led to an approximately twofold increase in [[[Ca.sup.2.sup.+]].sub.i] in 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 [[[Ca.sup.2.sup.+]].sub.i] in both principal and intercalated cells. cilium; cytoskeletal deformation; fluid shear stress; mechanotransduction; fura 2; intracellular calcium concentration

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 [Ca.sup.2+]/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) [Ca.sup.2+]-activated K channels, and 2) associated with increases in net Na absorption and intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]]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 [[Ca.sup.2+]]i and/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 [[Ca.sup.2+]]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 [alpha]-subunit message was consistently detected in single CCDs from animals [greater than or equal to] 4 wk of age by RT-PCR. Indirect immunofluorescence microscopy using antibodies directed against the [alpha]-subunit revealed apical labeling of intercalated cells in cryosections from animals [greater than or equal to] 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. maxi-K channel; iberiotoxin; in vitro microperfusion; intracellular calcium concentration; mechanoregulation; development

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.sup.+], [K.sup.+], 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 [Ca.sup.2+] the CCD been established. To examine this, we measured nucleotide-induced changes in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) in fura 2-loaded rabbit CCDs microperfused in vitro. Resting [[[Ca.sup.2+]].sub.i] did 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 [[[Ca.sup.2+]].sub.i] in both cell types. Luminal perfusion of 100 [micro]M UTP or ATP-[gamma]-S, in the absence of change in flow rate, caused a rapid and transient approximately fourfold increase in [[[Ca.sup.2+]].sub.i] in both cell types (P < 0.05). Luminal suramin, a nonspecific P2 receptor antagonist, blocked the nucleotide- but not flow-induced [[[Ca.sup.2+]].sub.i] transients. Luminal perfusion with a P2X ([alpha],[beta]-methylene-ATP), P2[X.sub.7] (benzoyl-benzoyl-ATP), P2[Y.sub.1] (2-methylthio-ATP), or P2[Y.sub.4]/P2[Y.sub.6] (UDP) receptor agonist had no effect on [[[Ca.sup.2+]].sub.i]. The nucleotide-induced [[[Ca.sup.2+]].sub.i] transients were inhibited by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl borate, thapsigargin, which depletes internal [Ca.sup.2+] stores, luminal perfusion with a [Ca.sup.2+]-free perfusate, or the L-type [Ca.sup.2+] channel blocker nifedipine. These results suggest that luminal nucleotides activate apical P2[Y.sub.2] receptors in the CCD via pathways that require both internal [Ca.sup.2+] mobilization and extracellular [Ca.sup.2+] entry. The flow-induced rise in [[[Ca.sup.2+]].sub.i] is apparently not mediated by apical P2 purinergic receptor signaling. microperfusion; fura 2; purinergic receptor; principal cell; intercalated cell; intracellular calcium concentration

The expression of carbonic anhydrase IV in carbonic anhydrase II-deficient mice was analyzed using Western blot analysis. Results of the research indicated that there was a higher expression of carbonic anhydrase IV (CA IV) immunoreactive protein in renal membranes in males than females. Furthermore, the membrane-bound CA IV activity in mice was upregulated in microsomal membranes.

The effects of low apical K+ permeability on the low rate of transepithelial K+ secretion were analyzed in perfused cortical collecting ducts (CCDs) isolated from newborn rabbits. Patch-clamp analysis of perfused rabbit CCDs indicated the presence of a maturation-associated increase in the activity of he predominant K+-permeable channel. Furthermore, the increased numbers of conducting apical K+-secretory channels in CCD contributed to the postnatal surge in net K+ secretion.

The role of the H-K-adenosinetriphosphatase (H-K-ATPase) in the intercalated cells of neonatal rabbit cortical collecting duct (CCD) was analyzed with fluorescent hydrogen-ion concentration-sensitive dyes. Analysis of neonatal rabbit CCDs and fully differentiated rhodamine peanut lectin (PNA)-differentiated intercalated cells indicated the presence of H-K-ATPases in the early stages of development. Furthermore, H-K-ATPase activity in neonatal rabbit CCDs were inhibited by renal cytostolic proteins.

The function of renal H-K-adenosine triphosphatase (ATPase) in relation to acidosis-induced net hydrogen secretion was investigated. Measurements of the rate of K-dependent intracellular pH recovery in split open rabbit cortical collecting duct (CCD) intercalated cells (ICs) with chronic metabolic acidosis (CMA) using the single-cell fluorescence technique showed a higher K-dependent pH recovery rate in CMA ICs than in control ICs. It was concluded that CMA enhanced the functional activity of H-K-ATPase.

The effect of the low rate of sodium absorption observed during early postnatal life on the differentiation of rabbit cortical collecting ducts (CCD) was investigated using the patch-clamp technique in microperfused CCDs. Data on conductance, number and open probability of apical sodium channels in principal cells revealed a rate-limiting step at the apical Na+ channel. Both number and open probability increased immediately after birth when amiloride was added, suggesting sensitivity of the Na+ channels.

A study of the site of H+ secretion in the kidney by investigating isolated perfused mesonephric collecting tubules using pH-sensitive fluorescent dyes and microcalorimetric techniques reveals that the secretion of H+ in the collecting tubules is twice that in the mature outer medullary collecting duct (OMCD) of the metanephric kidney. While ouabain does not block H+ secretion, Cl- depletion from the bathing solution and luminally applied H+-ATPase blocker NEM inhibit H+ secretion, indicating that the transporters that facilitate H+ secretion in the mesonephric collecting tubule are similar to the ones in OMCD. pH measurements and staining of the collecting tubules with 6-carboxy fluorescein diacetate confirm this finding.

A monoclonal antibody, B63 antigen, controls bicarbonate transport in peanut lectin-positive intercalated cells (ICC). B63 antigen is either the apical anion exchanger of beta-ICC or an associated modulatory protein. B63 antigen is present in tissues that involve bicarbonate secretion such as the pancreas, salivary glands and the small-intestine, and reacts with the apical membrane of peanut lectin agglutin-positive cells of the kidney cortex.

An analysis of adult rabbit cortical collecting direct (CCD), a major region for potassium release, involves a study of the potassium transport ability of this region during postnatal maturation and reveals that the low secretory ability of the CCD is partially responsible for low rates of potassium excretion typical of newborn kidneys. Functional immaturity of the paracellular pathway in the neonatal area may further restrict the production of transepithelial cation gradients. Functional growth of apical potassium channels in the CCD succeeds maturation of luminal sodium channels or basolateral Na(+)-K(+) pump.

Large-conductance K+ (BK) channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high-K+ diet. In cell culture, the long isoform of with-no-lysine kinase 1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high-K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high-K+ diet. Mice with IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high-K+ (5% K+, as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical þ subapical α-subunit expression and BK channel-dependent whole cell currents compared with controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high-K+ diet have higher blood K+ concentration and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high-K+ diet. [ABSTRACT FROM AUTHOR]

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 PGE2 than static cells. Cholesterol loading repressed the sheared mediated protein abundance of HO-1 and NOS2 as well as HO-1 and PGE2 concentrations 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. [ABSTRACT FROM AUTHOR]

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 Piezo1 with the sequence for the tandem-dimer Tomato gene, 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. [ABSTRACT FROM AUTHOR]

[Na.sup.+] absorption in the renal cortical collecting duct (CCD) is mediated by apical epithelial [Na.sup.+] 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.sup.+] currents ([I.sub.Na]) in oocytes expressing mouse [Alpha],[Beta],[Gamma]-ENaC (mENaC) and on net [Na.sup.+] 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 [I.sub.Na] without a change in reversal potential. The increase in [I.sub.Na] was variable among oocytes. CCDs responded to a threefold increase in rate of luminal flow with a twofold increase in the rate of net [Na.sup.+] absorption. An increase in luminal viscosity achieved by addition of 5% dextran to the luminal perfusate did not alter the rate of net [Na.sup.+] absorption, suggesting that shear stress does not influence [Na.sup.+] transport in the CCD. In sum, our data suggest that flow stimulation of ENaC activity and [Na.sup.+] 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. epithelial sodium channel; ROMK; collecting duct; transepithelial transport; oocyte

Flow-dependent [K.sup.+] secretion in the cortical collecting duct is mediated by a maxi-K channel. Am J Physiol Renal Physiol 49: F786-F793, 2001.--[K.sup.+] secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory [K.sup.+] (SK) and a high-conductance [Ca.sup.2+]-activated [K.sup.+] (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline [K.sup.+] secretion. The role of the stretch- and [Ca.sup.2+]-activated maxi-K channel is still uncertain. The purpose of this study was to identify the [K.sup.+] channel mediating flow-dependent [K.sup.+] 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.sup.+] channels. Net [K.sup.+] secretion and [Na.sup.+] absorption were measured at varying flow rates. In the absence of TEA, net [K.sup.+] secretion increased from 8.3 [+ or -] 1.0 to 23.4 [+ or -] 4.7 pmol [multiplied by] [min.sup.-1] [multiplied by] [mm.sup.-1] (P [is less than] 0.03) as the tubular flow rate was increased from 0.5 to 6 nl [multiplied by] [min.sup.-1] [multiplied by] [mm.sup.-1]. Flow stimulation of net [K.sup.+] secretion was blocked by luminal TEA (8.2 [+ or -] 1.2 vs. 9.9 [+ or -] 2.7 pmol [multiplied by] [min.sup.-1] [mm.sup.-1] at 0.6 and 6 nl [multiplied by] [min.sup.-1] [multiplied by] [mm.sup.-1] flow rates, respectively) or charybdotoxin (6.8 [+ or -] 1.6 vs. 8.3 [+ or -] 1.6 pmol [multiplied by] [min.sup.-1] [multiplied by] [mm.sup.-1] at 1 and 4 nl [multiplied by] [min.sup.-1] [multiplied by] [mm.sup.-1] flow rates, respectively) but not by apamin. These results suggest that flow-dependent [K.sup.+] secretion is mediated by a maxi-K channel, whereas baseline [K.sup.+] secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel. principal cell; intercalated cell; ROMK; epithelial sodium channels; charybdotoxin; apamin

Constantinescu, Alexandru R., Jerome C. Lane, John Mak, Beth Zavilowitz, and Lisa M. Satlin. [Na.sup.+]-[K.sup.+]-ATPase-mediated basolateral rubidium uptake in the maturing rabbit cortical collecting duct. Am J Physiol Renal Physiol 279: F1161-F1168, 2000.--Within the renal cortical collecting duct (CCD), transepithelial [Na.sup.+] absorption and [K.sup.+] secretion are linked to basolateral [Na.sup.+]-[K.sup.+]-ATPase activity. Our purpose was to examine the developmental changes in basolateral [Na.sup.+]-[K.sup.+]-ATPase-mediated [sup.86]rubidium (Rb) uptake, its inhibitor sensitivity and relationship to pump hydrolytic activity and [Na.sup.+] transport. Multiple CCDs (~6 mm) from maturing rabbits were affixed to coverslips, preincubated at 37 [degrees] C for 10 min ([+ or -] 1-2.5 mM ouabain or 10 or 100 [micro]M Schering-28080, an inhibitor of [H.sup.+] -[K.sup.+] -ATPase), and then transferred to prewarmed incubation solution containing tracer amounts of [sup.86]Rb ([+ or -] inhibitors). After 1 min at 37 [degrees] 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.sup.+]-[K.sup.+] pump activity, increased ~3-fold during the 1st 8 wk of postnatal life (P [is less than] 0.03). The ~2-fold increase in absolute rate of Rb uptake between 1 and 6 wk (2.64 [+ or -] 0.45 to 5.02 [+ or -] 0.32 pmol [multiplied by] [min.sup.-1] [multiplied by] [mm.sup.-1]) did not reach statistical significance. The rate of basolateral Rb uptake increased further after the 6th wk of life to 7.29 [+ or -] 0.53 pmol [multiplied by] [min.sup.-1] [multiplied by] [mm.sup.-1] in adult animals (P [is less than] 0.03 vs. 6 wk). Schering-28080 failed to inhibit Rb uptake, implying that functional [H.sup.+]-[K.sup.+]-ATPase is absent at the basolateral membrane. [Na.sup.+]-[K.sup.+]-ATPase hydrolytic activity, determined by using a microassay that measured inorganic phosphate release from [[Gamma]-[sup.32]P]ATP under maximum velocity ([V.sub.max]) conditions, also increased in the differentiating CCD (from 316.2 [+ or -] 44.4 pmol [multiplied by] [h.sup.-1] [multiplied by] [mm.sup.-1] at 2 wk to 555.9 [+ or -] 105.1 at 4 wk to 789.7 [+ or -] 145.0 at 6 wk; r = 1.0 by linear regression analysis; P [is less than] 0.005). The parallel ~2.5-fold increases in [Na.sup.+]-[K.sup.+]-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.sup.+]-[K.sup.+]-ATPase activity in the CCD remain to be defined. ontogeny; transepithelial sodium transport; ouabain; Schering-28080; microassay

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 BK' 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 subunitspecific antibodies revealed colocalization of immunodetectable BK'- and '1-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. [ABSTRACT FROM AUTHOR]