The Urothelium: Life in a Liquid Environment.

The urothelium, which lines the renal pelvis, ureters, urinary bladder, and proximal urethra, forms a high-resistance but adaptable barrier that surveils its mechanochemical environment and communicates changes to underlying tissues including afferent nerve fibers and the smooth muscle. The goal of this review is to summarize new insights into urothelial biology and function that have occurred in the past decade. After familiarizing the reader with key aspects of urothelial histology, we describe new insights into urothelial development and regeneration. This is followed by an extended discussion of urothelial barrier function, including information about the roles of the glycocalyx, ion and water transport, tight junctions, and the cellular and tissue shape changes and other adaptations that accompany expansion and contraction of the lower urinary tract. We also explore evidence that the urothelium can alter the water and solute composition of urine during normal physiology and in response to overdistension. We complete the review by providing an overview of our current knowledge about the urothelial environment, discussing the sensor and transducer functions of the urothelium, exploring the role of circadian rhythms in urothelial gene expression, and describing novel research tools that are likely to further advance our understanding of urothelial biology.

Antinociceptive effect of the calcitonin gene-related peptide receptor antagonist BIBN4096BS in mice with bacterial cystitis.

Patients with urinary tract infections (UTIs) suffer from urinary frequency, urgency, dysuria, and suprapubic pain, but the mechanisms by which bladder afferents sense the presence of uropathogens and encode this information is not well understood. Calcitonin gene-related peptide (CGRP) is a 37-mer neuropeptide found in a subset of bladder afferents that terminate primarily in the lamina propria. Here, we report that the CGRP receptor antagonist BIBN4096BS lessens lower urinary tract symptoms and prevents the development of pelvic allodynia in mice inoculated with uropathogenic Escherichia coli (UPEC) without altering urine bacterial loads or the host immune response to the infection. These findings indicate that CGRP facilitates the processing of noxious/inflammatory stimuli during UPEC infection. Using fluorescent in situ hybridization, we identified a population of suburothelial fibroblasts in the lamina propria, a region where afferent fibers containing CGRP terminate, that expresses the canonical CGRP receptor components Calcrl and Ramp1. We propose that these fibroblasts, in conjunction with CGRP+ afferents, form a circuit that senses substances released during the infection and transmit this noxious information to the central nervous system.NEW & NOTEWORTHY Afferent C fibers release neuropeptides including calcitonin gene-related peptide (CGRP). Here, we show that the specific CGRP receptor antagonist, BIBN409BS, ameliorates lower urinary tract symptoms and pelvic allodynia in mice inoculated with uropathogenic E. coli. Using fluorescent in situ hybridization, we identified a population of suburothelial fibroblasts in the lamina propria that expresses the canonical CGRP receptor. Our findings indicate that CGRP contributes to the transmission of nociceptive information arising from the bladder.

Studies of ultrastructure, gene expression, and marker analysis reveal that mouse bladder PDGFRA+ interstitial cells are fibroblasts.

Fibroblasts are crucial to normal and abnormal organ and tissue biology, yet we lack basic insights into the fibroblasts that populate the bladder wall. Candidates may include bladder interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells), which express the fibroblast-associated marker PDGFRA along with VIM and CD34 but whose form and function remain enigmatic. By applying the latest insights in fibroblast transcriptomics, coupled with studies of gene expression, ultrastructure, and marker analysis, we observe the following: 1) that mouse bladder PDGFRA+ cells exhibit all of the ultrastructural hallmarks of fibroblasts including spindle shape, lack of basement membrane, abundant endoplasmic reticulum and Golgi, and formation of homotypic cell-cell contacts (but not heterotypic ones); 2) that they express multiple canonical fibroblast markers (including Col1a2, CD34, LY6A, and PDGFRA) along with the universal fibroblast genes Col15a1 and Pi16 but they do not express Kit; and 3) that PDGFRA+ fibroblasts include suburothelial ones (which express ACTA2, CAR3, LY6A, MYH10, TNC, VIM, Col1a2, and Col15a1), outer lamina propria ones (which express CD34, LY6A, PI16, VIM, Col1a2, Col15a1, and Pi16), intermuscular ones (which express CD34, VIM, Col1a2, Col15a1, and Pi16), and serosal ones (which express CD34, PI16, VIM, Col1a2, Col15a1, and Pi16). Collectively, our study revealed that the ultrastructure of PDFRA+ interstitial cells combined with their expression of multiple canonical and universal fibroblast-associated gene products indicates that they are fibroblasts. We further propose that there are four regionally distinct populations of fibroblasts in the bladder wall, which likely contribute to bladder function and dysfunction.NEW & NOTEWORTHY We currently lack basic insights into the fibroblasts that populate the bladder wall. By exploring the ultrastructure of mouse bladder connective tissue cells, combined with analyses of their gene and protein expression, our study revealed that PDGRA+ interstitial cells (also referred to as myofibroblasts, telocytes, and interstitial cells of Cajal-like cells) are fibroblasts and that the bladder wall contains multiple, regionally distinct populations of these cells.

Bladder infection with uropathogenic Escherichia coli increases the excitability of afferent neurons.

Urinary tract infections (UTIs) cause bladder hyperactivity and pelvic pain, but the underlying causes of these symptoms remain unknown. We investigated whether afferent sensitization contributes to the bladder overactivity and pain observed in mice suffering from experimentally induced bacterial cystitis. Inoculation of mouse bladders with the uropathogenic Escherichia coli strain UTI89 caused pelvic allodynia, increased voiding frequency, and prompted an acute inflammatory process marked by leukocytic infiltration and edema of the mucosa. Compared with controls, isolated bladder sensory neurons from UTI-treated mice exhibited a depolarized resting membrane potential, lower action potential threshold and rheobase, and increased firing in response to suprathreshold stimulation. To determine whether bacterial virulence factors can contribute to the sensitization of bladder afferents, neurons isolated from naïve mice were incubated with supernatants collected from bacterial cultures with or depleted of lipopolysaccharide (LPS). Supernatants containing LPS prompted the sensitization of bladder sensory neurons with both tetrodotoxin (TTX)-resistant and TTX-sensitive action potentials. However, bladder sensory neurons with TTX-sensitive action potentials were not affected by bacterial supernatants depleted of LPS. Unexpectedly, ultrapure LPS increased the excitability only of bladder sensory neurons with TTX-resistant action potentials, but the supplementation of supernatants depleted of LPS with ultrapure LPS resulted in the sensitization of both population of bladder sensory neurons. In summary, the results of our study indicate that multiple virulence factors released from UTI89 act on bladder sensory neurons to prompt their sensitization. These sensitized bladder sensory neurons mediate, at least in part, the bladder hyperactivity and pelvic pain seen in mice inoculated with UTI89.NEW & NOTEWORTHY Urinary tract infection (UTI) produced by uropathogenic Escherichia coli (UPEC) promotes sensitization of bladder afferent sensory neurons with tetrodotoxin-resistant and tetrodotoxin-sensitive action potentials. Lipopolysaccharide and other virulence factors produced by UPEC contribute to the sensitization of bladder afferents in UTI. In conclusion, sensitized afferents contribute to the voiding symptoms and pelvic pain present in mice bladder inoculated with UPEC.

Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel.

The paraoxonase (PON) family comprises three highly conserved members: PON1, PON2, and PON3. They are orthologs of Caenorhabditis elegans MEC-6, an endoplasmic reticulum-resident chaperone that has a critical role in proper assembly and surface expression of the touch-sensing degenerin channel in nematodes. We have shown recently that MEC-6 and PON2 negatively regulate functional expression of the epithelial Na+ channel (ENaC), suggesting that the chaperone function is conserved within this family. We hypothesized that other PON family members also modulate ion channel expression. Pon3 is specifically expressed in the aldosterone-sensitive distal tubules in the mouse kidney. We found here that knocking down endogenous Pon3 in mouse cortical collecting duct cells enhanced Na+ transport, which was associated with increased γENaC abundance. We further examined Pon3 regulation of ENaC in two heterologous expression systems, Fisher rat thyroid cells and Xenopus oocytes. Pon3 coimmunoprecipitated with each of the three ENaC subunits in Fisher rat thyroid cells. As a result of this interaction, the whole-cell and surface abundance of ENaC α and γ subunits was reduced by Pon3. When expressed in oocytes, Pon3 inhibited ENaC-mediated amiloride-sensitive Na+ currents, in part by reducing the surface expression of ENaC. In contrast, Pon3 did not alter the response of ENaC to chymotrypsin-mediated proteolytic activation or [2-(trimethylammonium)ethyl]methanethiosulfonate-induced activation of αßS518Cγ, suggesting that Pon3 does not affect channel open probability. Together, our results suggest that PON3 regulates ENaC expression by inhibiting its biogenesis and/or trafficking.

Acid-sensing ion channels modulate bladder nociception.

Sensitization of neuronal pathways and persistent afferent drive are major contributors to somatic and visceral pain. However, the underlying mechanisms that govern whether afferent signaling will give rise to sensitization and pain are not fully understood. In the present report, we investigated the contribution of acid-sensing ion channels (ASICs) to bladder nociception in a model of chemical cystitis induced by cyclophosphamide (CYP). We found that the administration of CYP to mice lacking ASIC3, a subunit primarily expressed in sensory neurons, generates pelvic allodynia at a time point at which only modest changes in pelvic sensitivity are apparent in wild-type mice. The differences in mechanical pelvic sensitivity between wild-type and Asic3 knockout mice treated with CYP were ascribed to sensitized bladder C nociceptors. Deletion of Asic3 from bladder sensory neurons abolished their ability to discharge action potentials in response to extracellular acidification. Collectively, the results of our study support the notion that protons and their cognate ASIC receptors are part of a mechanism that operates at the nerve terminals to control nociceptor excitability and sensitization.NEW & NOTEWORTHY Our study indicates that protons and their cognate acid-sensing ion channel receptors are part of a mechanism that operates at bladder afferent terminals to control their function and that the loss of this regulatory mechanism results in hyperactivation of nociceptive pathways and the development of pain in the setting of chemical-induced cystitis.

Murine epithelial sodium (Na+) channel regulation by biliary factors.

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone-sensitive distal nephron, distal colon, and biliary epithelium. Numerous factors regulate ENaC activity, including extracellular ligands, post-translational modifications, and membrane-resident lipids. However, ENaC regulation by bile acids and conjugated bilirubin, metabolites that are abundant in the biliary tree and intestinal tract and are sometimes elevated in the urine of individuals with advanced liver disease, remains poorly understood. Here, using a Xenopus oocyte-based system to express and functionally study ENaC, we found that, depending on the bile acid used, bile acids both activate and inhibit mouse ENaC. Whether bile acids were activating or inhibiting was contingent on the position and orientation of specific bile acid moieties. For example, a hydroxyl group at the 12-position and facing the hydrophilic side (12α-OH) was activating. Taurine-conjugated bile acids, which have reduced membrane permeability, affected ENaC activity more strongly than did their more membrane-permeant unconjugated counterparts, suggesting that bile acids regulate ENaC extracellularly. Bile acid-dependent activation was enhanced by amino acid substitutions in ENaC that depress open probability and was precluded by proteolytic cleavage that increases open probability, consistent with an effect of bile acids on ENaC open probability. Bile acids also regulated ENaC in a cortical collecting duct cell line, mirroring the results in Xenopus oocytes. We also show that bilirubin conjugates activate ENaC. These results indicate that ENaC responds to compounds abundant in bile and that their ability to regulate this channel depends on the presence of specific functional groups.

Acid-sensing ion channels in sensory signaling.

Acid-sensing ion channels (ASICs) are cation-permeable channels that in the periphery are primarily expressed in sensory neurons that innervate tissues and organs. Soon after the cloning of the ASIC subunits, almost 20 yr ago, investigators began to use genetically modified mice to assess the role of these channels in physiological processes. These studies provide critical insights about the participation of ASICs in sensory processes, including mechanotransduction, chemoreception, and nociception. Here, we provide an extensive assessment of these findings and discuss the current gaps in knowledge with regard to the functions of ASICs in the peripheral nervous system.

Expression and distribution of PIEZO1 in the mouse urinary tract.

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.

Molecular determinants of afferent sensitization in a rat model of cystitis with urothelial barrier dysfunction.

The internal surface of the urinary bladder is covered by the urothelium, a stratified epithelium that forms an impermeable barrier to urinary solutes. Increased urothelial permeability is thought to contribute to symptom generation in several forms of cystitis by sensitizing bladder afferents. In this report we investigate the physiological mechanisms that mediate bladder afferent hyperexcitability in a rat model of cystitis induced by overexpression in the urothelium of claudin-2 (Cldn2), a tight junction-associated protein upregulated in bladder biopsies from patients with interstitial cystitis/bladder pain syndrome. Patch-clamp studies showed that overexpression of Cldn2 in the urothelium sensitizes a population of isolectin GS-IB4-negative [IB4(-)] bladder sensory neurons with tetrodotoxin-sensitive (TTX-S) action potentials. Gene expression analysis revealed a significant increase in mRNA levels of the delayed-rectifier voltage-gated K+ channel (Kv)2.2 and the accessory subunit Kv9.1 in this population of bladder sensory neurons. Consistent with this finding, Kv2/Kv9.1 channel activity was greater in IB4(-) bladder sensory neurons from rats overexpressing Cldn2 in the urothelium than in control counterparts. Likewise, current density of TTX-S voltage-gated Na+ (Nav) channels was greater in sensitized neurons than in control counterparts. Significantly, guangxitoxin-1E (GxTX-1E), a selective blocker of Kv2 channels, blunted the repetitive firing of sensitized IB4(-) sensory neurons. In summary, our studies indicate that an increase in the activity of TTX-S Nav and Kv2/Kv9.1 channels mediates repetitive firing of sensitized bladder sensory neurons in rats with increased urothelial permeability.NEW & NOTEWORTHY Hyperexcitability of sensitized bladder sensory neurons in a rat model of interstitial cystitis/bladder pain syndrome (IC/BPS) results from increased activity of tetrodotoxin-sensitive voltage-gated Na+ and delayed-rectifier voltage-gated K+ (Kv)2/Kv9.1 channels. Of major significance, our studies indicate that Kv2/Kv9.1 channels play a major role in symptom generation in this model of IC/BPS by maintaining the sustained firing of the sensitized bladder sensory neurons.

Renal sensory nerves increase sympathetic nerve activity and blood pressure in 2-kidney 1-clip hypertensive mice.

Renal denervation lowers arterial blood pressure (ABP) in multiple clinical trials and some experimental models of hypertension. These antihypertensive effects have been attributed to the removal of renal afferent nerves. The purpose of the present study was to define the function, anatomy, and contribution of mouse renal sensory neurons to a renal nerve-dependent model of hypertension. First, electrical stimulation of mouse renal afferent nerves produced frequency-dependent increases in ABP that were eliminated by ganglionic blockade. Stimulus-triggered averaging revealed renal afferent stimulation significantly increased splanchnic, renal, and lumbar sympathetic nerve activity (SNA). Second, kidney injection of wheat germ agglutinin into male C57Bl6 mice (12-14 wk; Jackson Laboratories) produced ipsilateral labeling in the T11-L2 dorsal root ganglia. Next, 2-kidney 1-clip (2K1C) hypertension was produced in male C57Bl6 mice (12-14 wk; Jackson Laboratories) by placement of a 0.5-mm length of polytetrafluoroethylene tubing around the left renal artery. 2K1C mice displayed an elevated ABP measured via telemetry and a greater fall in mean ABP to ganglionic blockade at day 14 or 21 vs. day 0. Renal afferent discharge was significantly higher in 2K1C-clipped vs. 2K1C-unclipped or sham kidneys. In addition, 2K1C-clipped vs. 2K1C-unclipped or sham kidneys had lower renal mass and higher mRNA levels of several proinflammatory cytokines. Finally, both ipsilateral renal denervation (10% phenol) or selective denervation of renal afferent nerves (periaxonal application of 33 mM capsaicin) at time of clipping resulted in lower ABP of 2K1C mice at day 14 or 21. These findings suggest mouse renal sensory neurons are activated to increase SNA and ABP in 2K1C hypertension. NEW & NOTEWORTHY This study documents the function, anatomy, and contribution of mouse renal sensory nerves to neurogenic hypertension produced by renal stenosis. Activation of renal afferents increased sympathetic nerve activity and blood pressure. Renal afferent activity was elevated in hypertensive mice, and renal afferent denervation lowered blood pressure. Clinically, patients with renal stenosis have been excluded from clinical trials for renal denervation, but this study highlights the potential therapeutic efficacy to target renal nerves in these patients.

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel.

The epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (α, ß, and γ). We previously reported that cytoplasmic residues on the ß (ßCys-43 and ßCys-557) and γ (γCys-33 and γCys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, γ subunit palmitoylation had a dominant role over ß subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when γ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking ß subunit palmitoylation sites. ß subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking ß and γ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCDcl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCDcl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na+ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.

ASIC3 fine-tunes bladder sensory signaling.

Acid-sensing ion channels (ASICs) are trimeric proton-activated, cation-selective neuronal channels that are considered to play important roles in mechanosensation and nociception. Here we investigated the role of ASIC3, a subunit primarily expressed in sensory neurons, in bladder sensory signaling and function. We found that extracellular acidification evokes a transient increase in current, consistent with the kinetics of activation and desensitization of ASICs, in ~25% of the bladder sensory neurons harvested from both wild-type (WT) and ASIC3 knockout (KO) mice. The absence of ASIC3 increased the magnitude of the peak evoked by extracellular acidification and reduced the rate of decay of the ASIC-like currents. These findings suggest that ASICs are assembled as heteromers and that the absence of ASIC3 alters the composition of these channels in bladder sensory neurons. Consistent with the notion that ASIC3 serves as a proton sensor, 59% of the bladder sensory neurons harvested from WT, but none from ASIC3 KO mice, fired action potentials in response to extracellular acidification. Studies of bladder function revealed that ASIC3 deletion reduces voiding volume and the pressure required to trigger micturition. In summary, our findings indicate that ASIC3 plays a role in the control of bladder function by modulating the response of afferents to filling.

The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization.

Acid-sensing ion channels (ASICs) are cation-selective proton-gated channels expressed in neurons that participate in diverse physiological processes, including nociception, synaptic plasticity, learning, and memory. ASIC subunits contain intracellular N and C termini, two transmembrane domains that constitute the pore, and a large extracellular loop with defined domains termed the finger, ß-ball, thumb, palm, and knuckle. Here we examined the contribution of the finger, ß-ball, and thumb domains to activation and desensitization through the analysis of chimeras and the assessment of the effect of covalent modification of introduced Cys at the domain-domain interfaces. Our studies with ASIC1a-ASIC2a chimeras showed that swapping the thumb domain between subunits results in faster channel desensitization. Likewise, the covalent modification of Cys residues at selected positions in the ß-ball-thumb interface accelerates the desensitization of the mutant channels. Studies of accessibility with thiol-reactive reagents revealed that the ß-ball and thumb domains reside apart in the resting state but that they become closer to each other in response to extracellular acidification. We propose that the thumb domain moves upon continuous exposure to an acidic extracellular milieu, assisting with the closing of the pore during channel desensitization.

Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia.

The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin and cubilin. Changes in GFR and the accompanying fluid shear stress (FSS) modulate acute changes in PT ion transport thought to be mediated by microvillar bending. We found that FSS also affects apical endocytosis in PT cells. Exposure of immortalized PT cell lines to physiologically relevant levels of FSS led to dramatically increased internalization of the megalin-cubilin ligand albumin as well as the fluid phase marker dextran. FSS-stimulated apical endocytosis was initiated between 15 and 30 min postinduction of FSS, occurred via a clathrin- and dynamin-dependent pathway, and was rapidly reversed upon removing the FSS. Exposure to FSS also caused a rapid elevation in intracellular Ca(2+) [Ca(2+)]i, which was not observed in deciliated cells, upon treatment with BAPTA-AM, or upon inclusion of apyrase in the perfusion medium. Strikingly, deciliation, BAPTA-AM, and apyrase also blocked the flow-dependent increase in endocytosis. Moreover, addition of ATP bypassed the need for FSS in enhancing endocytic capacity. Our studies suggest that increased [Ca(2+)]i and purinergic signaling in response to FSS-dependent ciliary bending triggers a rapid and reversible increase in apical endocytosis that contributes to the efficient retrieval of filtered proteins in the PT.

An unexpected journey: conceptual evolution of mechanoregulated potassium transport in the distal nephron.

Flow-induced K secretion (FIKS) in the aldosterone-sensitive distal nephron (ASDN) is mediated by large-conductance, Ca(2+)/stretch-activated BK channels composed of pore-forming α-subunits (BKα) and accessory ß-subunits. This channel also plays a critical role in the renal adaptation to dietary K loading. Within the ASDN, the cortical collecting duct (CCD) is a major site for the final renal regulation of K homeostasis. Principal cells in the ASDN possess a single apical cilium whereas the surfaces of adjacent intercalated cells, devoid of cilia, are decorated with abundant microvilli and microplicae. Increases in tubular (urinary) flow rate, induced by volume expansion, diuretics, or a high K diet, subject CCD cells to hydrodynamic forces (fluid shear stress, circumferential stretch, and drag/torque on apical cilia and presumably microvilli/microplicae) that are transduced into increases in principal (PC) and intercalated (IC) cell cytoplasmic Ca(2+) concentration that activate apical voltage-, stretch- and Ca(2+)-activated BK channels, which mediate FIKS. This review summarizes studies by ourselves and others that have led to the evolving picture that the BK channel is localized in a macromolecular complex at the apical membrane, composed of mechanosensitive apical Ca(2+) channels and a variety of kinases/phosphatases as well as other signaling molecules anchored to the cytoskeleton, and that an increase in tubular fluid flow rate leads to IC- and PC-specific responses determined, in large part, by the cell-specific composition of the BK channels.

Intracellular Na+ regulates epithelial Na+ channel maturation.

Epithelial Na(+) channel (ENaC) function is regulated by the intracellular Na(+) concentration ([Na(+)]i) through a process known as Na(+) feedback inhibition. Although this process is known to decrease the expression of proteolytically processed active channels on the cell surface, it is unknown how [Na(+)]i alters ENaC cleavage. We show here that [Na(+)]i regulates the posttranslational processing of ENaC subunits during channel biogenesis. At times when [Na(+)]i is low, ENaC subunits develop mature N-glycans and are processed by proteases. Conversely, glycan maturation and sensitivity to proteolysis are reduced when [Na(+)]i is relatively high. Surface channels with immature N-glycans were not processed by endogenous channel activating proteases, nor were they sensitive to cleavage by exogenous trypsin. Biotin chase experiments revealed that the immature surface channels were not converted into mature cleaved channels following a reduction in [Na(+)]i. The hypothesis that [Na(+)]i regulates ENaC maturation within the biosynthetic pathways is further supported by the finding that Brefeldin A prevented the accumulation of processed surface channels following a reduction in [Na(+)]i. Therefore, increased [Na(+)]i interferes with ENaC N-glycan maturation and prevents the channel from entering a state that allows proteolytic processing.

Cell-specific regulation of L-WNK1 by dietary K.

Flow-induced K(+) secretion in the aldosterone-sensitive distal nephron is mediated by high-conductance Ca(2+)-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.

Increased urothelial paracellular transport promotes cystitis.

Changes in the urothelial barrier are observed in patients with cystitis, but whether this leads to inflammation or occurs in response to it is currently unknown. To determine whether urothelial barrier dysfunction is sufficient to promote cystitis, we employed in situ adenoviral transduction to selectively overexpress the pore-forming tight junction-associated protein claudin-2 (CLDN-2). As expected, the expression of CLDN-2 in the umbrella cells increased the permeability of the paracellular route toward ions, but not to large organic molecules. In vivo studies of bladder function revealed higher intravesical basal pressures, reduced compliance, and increased voiding frequency in rats transduced with CLDN-2 vs. controls transduced with green fluorescent protein. While the integrity of the urothelial barrier was preserved in the rats transduced with CLDN-2, we found that the expression of this protein in the umbrella cells initiated an inflammatory process in the urinary bladder characterized by edema and the presence of a lymphocytic infiltrate. Taken together, these results are consistent with the notion that urothelial barrier dysfunction may be sufficient to trigger bladder inflammation and to alter bladder function.