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Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

[Ca.sup.2+]-activated BK channels in renal intercalated cells (ICs) mediate luminal flow-induced [K.sup.+] secretion (FIKS), but how ICs sense increased flow remains uncertain. We examined whether PIEZO1, a mechanosensitive [Ca.sup.2+]-permeable channel expressed in the basolateral membranes of ICs, is required for FIKS. In isolated cortical collecting ducts (CCDs), the mechanosensitive cation-selective channel inhibitor GsMTx4 dampened flow-induced increases in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]), whereas the PIEZO1 activator Yoda1 increased [[Ca.sup.2+].sub.i] and BK channel activity. CCDs from mice fed a high-[K.sup.+] (HK) diet exhibited a greater Yoda1-dependent increase in [[Ca.sup.2+].sub.i] than CCDs from mice fed a control [K.sup.+] diet. ICs in CCDs isolated from mice with a targeted gene deletion of Piezo1 in ICs (IC-Piezo1-KO) exhibited a blunted [[Ca.sup.2+].sub.i] response to Yoda1 or increased flow, with an associated loss of FIKS in CCDs. Male IC-Piezo1-KO mice selectively exhibited an increased blood [[K.sup.+]] in response to an oral [K.sup.+] bolus and blunted urinary [K.sup.+] excretion following a volume challenge. Whole-cell expression of BK[alpha] subunit was reduced in ICs of IC-Piezo1-KO mice fed an HK diet. We conclude that PIEZO1 mediates flow-induced basolateral [Ca.sup.2+] entry into ICs, is upregulated in the CCD in response to an HK diet, and is necessary for FIKS., Introduction The kidney is subject to variations in tubular (urinary) flow rate that regulate various physiologic functions, including [Na.sup.+] absorption and [K.sup.+] secretion in the aldosterone-sensitive distal nephron (ASDN), composed [...]

K+ secretion in the distal nephron has a critical role in K+ homeostasis and is the primary route by which K+ is lost from the body. Renal K+ secretion is enhanced by increases in dietary K+ intake and by increases in tubular flow rate in the distal nephron. This review addresses new and important insights regarding the mechanisms underlying flow‐induced K+ secretion (FIKS). While basal K+ secretion in the distal nephron is mediated by renal outer medullary K+ (ROMK) channels in principal cells (PCs), FIKS is mediated by large conductance, Ca2+/stretch activated K+ (BK) channels in intercalated cells (ICs), a distinct cell type. BK channel activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), and both PCs and ICs exhibit increases in [Ca2+]i in response to increases in tubular fluid flow rate, associated with an increase in tubular diameter. PIEZO1, a mechanosensitive, nonselective cation channel, is expressed in the basolateral membranes of PCs and ICs, where it functions as a mechanosensor. The loss of flow‐induced [Ca2+]i transients in ICs and BK channel‐mediated FIKS in microperfused collecting ducts isolated from mice with IC‐specific deletion of Piezo1 in the CCD underscores the importance of PIEZO1 in the renal regulation of K+ transport. [ABSTRACT FROM AUTHOR]

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