V-ATPase B1 Research Articles

V-ATPase B2 promotes microglial phagocytosis of myelin debris by inactivating the MAPK signaling pathway

Microglial phagocytosis of myelin debris is a crucial process for promoting myelin regeneration in conditions such as multiple sclerosis (MS). Vacuolar-ATPase B2 (V-ATPase B2) has been implicated in various cellular processes, but its role in microglial phagocytosis and its potential impact on MS-related responses remain unclear. In this study, we employed BV-2 murine microglial cells to investigate the influence of V-ATPase B2 on the phagocytosis of myelin debris by microglia. The results revealed that V-ATPase B2 expression increased in response to myelin debris exposure. Overexpression of V-ATPase B2 significantly enhanced BV-2 phagocytosis of myelin debris. Additionally, V-ATPase B2 overexpression shifted microglial polarization towards an anti-inflammatory M2 phenotype, coupled with decreased lysosomal pH and enhanced lysosome degradation capacity. Moreover, endoplasmic reticulum (ER) stress inhibitor, 4-PBA, reversed the effects of V-ATPase B2 silencing on ER stress, M2 polarization, and lysosomal degradation of BV-2 cells. The MAPK pathway was inhibited upon V-ATPase B2 overexpression, contributing to heightened myelin debris clearance by BV-2 cells. Notably, MAPK pathway inhibition partially attenuated the inhibitory effects of V-ATPase B2 knockdown on myelin debris clearance. In conclusion, our findings reveal a pivotal role for V-ATPase B2 in promoting microglial phagocytosis of myelin debris by regulating microglial polarization and lysosomal function via the MAPK signaling pathway, suggesting that targeting V-ATPase B2 may hold therapeutic potential for enhancing myelin debris clearance and modulating microglial responses in MS and related neuroinflammatory disorders.

Overexpression of V-ATPase B2 attenuates lung injury/fibrosis by stabilizing lysosomal membrane permeabilization and increasing collagen degradation

Excessive oxidative stress causes lysosomal membrane permeabilization (LMP), which leads to cell death. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H+ into the cytosol and thus maintaining intracellular pH. Previously, we reported that V-ATPase B2 subunit expression is upregulated in the TiO2-exposed lung epithelium. We investigated the role of the lysosomal V-ATPase B2 subunit in oxidative stress-induced alveolar epithelial cell death and in an experimental lung injury/fibrosis model. Overexpression of V-ATPase B2 increased lysosomal pH and lysosomal activities in the cells. In the presence of H2O2, overexpression of V-ATPase B2 increased survival, and silencing of V-ATPase B2 dramatically increased cell death. Overexpression of V-ATPase B2 diminished H2O2-triggered LMP, as evidenced by a reduction in acridine orange staining and leakage of cathepsin D from the lysosome to the cytoplasm. In addition, V-ATPase B2-overexpressing macrophages exhibited significantly enhanced uptake and degradation of collagen. V-ATPase B2-overexpressing transgenic mice showed significant inhibition of the bleomycin-induced increases in lung inflammation and fibrosis. We conclude that V-ATPase B2 is critical for maintaining lysosomal activities against excessive oxidative stress by stabilizing LMP. Our findings reveal a previously unknown role of this V-ATPase subunit in a lung injury and fibrosis model.

Impaired V-ATPase leads to increased lysosomal pH, results in disrupted lysosomal degradation and autophagic flux blockage, contributes to fluoride-induced developmental neurotoxicity

Fluoride is capable of inducing developmental neurotoxicity, yet its mechanisms remain elusive. We aimed to explore the possible role and mechanism of autophagic flux blockage caused by abnormal lysosomal pH in fluoride-induced developmental neurotoxicity, focusing on the role of V-ATPase in regulating the neuronal lysosomal pH. Using Sprague-Dawley rats exposed to sodium fluoride (NaF) from gestation through delivery until the neonatal offspring reached six months of age as an in vivo model. The results showed that NaF impaired the cognitive abilities of the offspring rats. In addition, NaF reduced V-ATPase expression, diminished lysosomal degradation capacity and blocked autophagic flux, and increased apoptosis in the hippocampus of offspring. Consistently, these results were validated in SH-SY5Y cells incubated with NaF. Moreover, NaF increased the SH-SY5Y lysosomal pH. Mechanistically, V-ATPase B2 overexpression and ATP effectively restored V-ATPase expression, reducing NaF-induced lysosomal alkalinization while increasing lysosomal degradation capacity. Notably, those above pharmacological and molecular interventions diminished NaF-induced apoptosis by restoring autophagic flux. Collectively, the present findings suggested that NaF impairs the lysosomal pH raised by V-ATPase. This leads to reduced lysosomal degradation capacity and triggers autophagic flux blockage and apoptosis, thus contributing to neuronal death. Therefore, V-ATPase might be a promising indicator of developmental fluoride neurotoxicity.

Investigating the Transitional Cell Type in the Kidney Collecting Duct

The kidney cortical collecting duct (CCD) has long been thought to contain two distinct cell types, principal cells (PC) and intercalated cells (IC), which facilitate 4-5% of sodium and 10% of water reabsorption in the kidney. Recent single cell RNA-Seq data has identified a distinct transitional cell expressing both PC and IC markers and a number of distinct markers. In this study, the presence of CCD cell types was investigated in mice and rats under the disease model of the syndrome of apparent mineralocorticoid excess (SAME, conferred by a mutation of the Hsd11b2gene, expressed by PCs) and also in two CCD derived cell lines. Inactivation of HSD11B2in humans results in a severe, and often fatal, phenotype in early neonatal life, whilst milder mutations, with residual enzyme activity cause hypertension, sodium retention and hypokalemia in adulthood. The Hsd11b2 gene knockout (KO) model of SAME in mice and rats was studied in the context of CCD cell plasticity and it was hypothesised to reduce the proportion of PCs within the CCD. Cells in the CCD were counted in WT and Hsd11b2 KO animals following immunofluorescent staining for PC- and IC-specific markers (Aqp2 and V-ATPase B1, respectively) in paraffin-embedded kidney sections. ImageJ and GraphPadPrism were used to analyse data, which are expressed as mean ± SEM. Significance was assessed using Tukey's multiple comparisons test, p<0.0001. PC proportion was significantly reduced in KO mice CCDs compared to controls at 4 weeks (52.1 ± 1.9 % vs. 73.0 ± 1.9 %) and 17 weeks (54.1 ± 1.8 % vs. 72.2 ± 1.3 %). A significant increase in the number of cells expressing both PC and IC markers was observed between WT and KO at 4 weeks (8.4 ± 1.2 % vs.25.9 ± 2.0 %) and at 17 weeks (6.5 ± 0.8 % vs. 22.1 ± 2.0 %). Similarly, in the rat KO a significant reduction in the proportion of PCs was noted compared to controls at 5 weeks (60.69 ± 2.51 % vs. 73.30 ± 1.84 %) and 24 weeks (38.40 ± 2.07 % vs. 66.80 ± 1.94 %). This study shows CCD cell plasticity in models of SAME, but also provides further evidence for a PC+/IC+cell in the kidney. The mCCDcl1­ and mpkCCDcl4­ cell lines are typically used as models of PC function. Immunofluorescent staining using antibodies against PC, IC and transitional cell markers (Aqp2, V-ATPase B1 and Parm1, respectively) was carried out on fixed, polarised cells and images analysed using ImageJ software. It was found that both cell line lines contained cells that co-expressed PC and IC markers, similar to the cell type seen in mouse and rat kidneys. Furthermore, a large proportion of cells in both cell lines expressed the transitional cell marker, Parm1. These results provide further support for the presence of a transitional cell type in the CCD, especially in pathophysiological states. The observation of similar cells in CCD cell lines also indicates their use as in vitro models for investigating the role and plasticity of CCD cell types.

Knock‐out of Adam10 in mCCD cl1 cells gives insight into the effects of Notch pathway disruption for kidney collecting duct cell plasticity

The kidney cortical collecting duct (CCD) is responsible for the fine tuning of sodium, volume, and acid-base homeostasis, and comprises of principal and intercalated cells (PCs and ICs) as well as a newly discovered ‘intermediate’ cell type (1). Plasticity between PCs and ICs has been reported previously in mice in which genes for factors implicated in the Notch pathway, such as Adam10, have been knocked out (KO) (2). In vivo, disruption of the Notch pathway leads to a decreasing PC:IC ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs and a response to physiological concentrations of hormones such as aldosterone; it also has been shown to express both PC and IC markers, a phenotype transmitted through single cell cloning (3). These characteristics make mCCDcl1 cells a useful platform for the study of plasticity in the CCD. Adam10 was knocked out in mCCDcl1 cells using CRISPR-Cas9 technology and independent clones were isolated and expanded for study. Despite mCCDcl1 cells being polyploid, our strategy enabled all alleles for each gene to be successfully targeted. Clones were cultured on Transwell filters over 10 days and their electrophysiological properties measured (trans-epithelial resistance Rte, voltage Vte, calculated short-circuit current Isc). Both Adam10 KO clones showed a slightly reduced baseline Rte compared to control (0.90±0.19 kΩ.cm2 vs 1.12±0.18 kΩ.cm2) but a complete reduction in baseline Isc (0.3±0.1 μA/cm2 vs -8.1±0.4 μA/cm2 for control) as well as aldosterone response, decreased by 95% (fold change 0.19±0.03 down 3.70±0.11 for control). Single-cell RNA sequencing principal component analysis shows different clustering of KO clones compared to WT. Potentially explaining the loss of sodium transport function, a significant drop in expression for the gamma subunit of the epithelial sodium channel (γ-ENaC) (between ~40% and ~60% depending on the cluster) was detected in the KO clones, as well as a decrease in the expression of 11ßHSD2 (between ~20% and ~60%). Immunostaining in KO clones confirmed the decrease in expression of γ-ENaC and showed an altered distribution and localisation of γ-ENaC, Aqp2, and V-ATPase B1 compared to WT mCCDcl1, overall suggesting a loss of polarisation. These data suggest that genomic modification of mCCDcl1 cells provides an effective means to investigate the mechanisms through which CCD cells mediate their plasticity and can identify new pathways involved in regulating cell phenotype as well as specific effects of pathway disruption on collecting duct cells functions. (1) Park et al.; Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease; Science; 360: 758-763, 2018. (2) Guo et al.; Adam10 Mediates the Choice between Principal Cells and Intercalated Cells in the Kidney; JASN; 26: 149-159, 2015. (3) Assmus et al.; mCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells; AJP-renal; 314 (5): 820-831, 2018.

NBCe2 (Slc4a5) Is Expressed in the Renal Connecting Tubules and Cortical Collecting Ducts and Mediates Base Extrusion.

Arterial hypertension, is a common disorder with multiple and variable etiologies. Single nucleotide polymorphism analyses have detected an association between variants in the gene encoding the electrogenic Na+:HCO3– cotransporter NBCe2 (Slc4a5), and salt-sensitive hypertension. Mice with genetic deletion of NBCe2 are hypertensive, and the cause of the blood pressure (BP) increase is believed to arise from a lack of renal NBCe2 function. The exact cellular expression of NBCe2 in the kidney tubular system is, however, not determined. Here, we find NBCe2 to be expressed predominantly in isolated connecting tubules (CNT) and cortical collecting ducts (CD) by RT-PCR. In isolated renal CNT and CCD, genetic deletion of NBCe2 leads to decreased net base extrusion. To determine the role of renal NBCe2 in the development of hypertension, we generated CNT and intercalated cell NBCe2 knockout mice by crossing an Slc4a5 lox mouse with mice expressing cre recombinase under the V-ATPase B1 subunit promotor. Although the mice displayed changes in the expression of renal membrane transporters, we did not detect hypertension in these mice by tail cuff recordings. In conclusion, while global NBCe2 deletion certainly causes hypertension this study cannot confirm the role of renal NBCe2 expression in blood pressure regulation.

Sex-dependent differences in water homeostasis in wild-type and V-ATPase B1-subunit deficient mice

Men tend to dehydrate more than women after prolonged exercise, possibly due to lower water intake and higher perspiration rate. Women are prone to exercise-associated hyponatremia, primarily attributed to the higher water consumption causing hypervolemia. Since aquaporin-2 (AQP2) water channels in the kidney collecting duct (CD) principal cells (PCs) are involved in maintaining water balance, we investigated their role in sex-dependent water homeostasis in wild-type (WT) C57BL/6 mice. Because CD intercalated cells (ICs) may also be involved in water balance, we also assessed the urine concentrating ability of V-ATPase B1 subunit-deficient (Atp6v1b1-/-) mice. Upon 12-hour water deprivation, urine osmolality increased by 59% in WT female mice and by only 28% in males. This difference was abolished in Atp6v1b1-/- mice, in which dehydration induced a ~30% increase in urine osmolarity in both sexes. AQP2 levels were highest in WT females; female Atp6v1b1-/- mice had substantially lower AQP2 expression than WT females, comparable to the low AQP2 levels seen in both Atp6v1b1-/- and WT males. After dehydration, AQP2 relocates towards the PC apical pole, especially in the inner stripe and inner medulla, and to a greater extent in WT females than in WT males. This apparent sex-dependent concentrating advantage was absent in Atp6v1b1-/- females, whose reduced AQP2 apical relocation was similar to WT males. Accordingly, female mice concentrate urine better than males upon dehydration due to increased AQP2 expression and mobilization. Moreover, our data support the involvement of ICs in water homeostasis, at least partly mediated by V-ATPase, in a sex-dependent manner.

Development of a 3D in vitro Model of the Kidney Cortical Collecting Duct

The cortical collecting duct (CCD) of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH and osmolarity and is composed of two distinct cells types, principal cells (PCs) and intercalated cells (ICs). Each cell type is detectable in the kidney by the localization of specific transport proteins such as Aqp2 and ENaC in principal cells and V‐ATPase B1 in intercalated cells. The inter‐relationship between PCs and ICs is complex and yet to be fully elucidated. The ratio of the number of PCs to ICs (typically 70:30 in healthy mice) has been shown to be influenced by multiple factors involved in Notch signalling [1]. A transitional cell expressing both PC and IC markers, was also recently described. mCCDcl1 cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. However, previous studies have shown plasticity of mCCDcl1 cells, a direct lineage relationship between principal and intercalated cells, and evidence consistent with mCCDcl1 cells being precursor cells [2]. These characteristics make mCCDcl1 cells a useful tool in the study of collecting duct physiology and cell differentiation. However, classic (2D) cell culture techniques have limitations for the development of an optimal in vitro model of the collecting duct.In this study, a 3D model of collecting duct was developed using polyHIPEs, an emulsion‐based polymer materials offering a highly interconnected micro‐porosity network [3]. 3D‐printed polyHIPEs structures were designed and integrated in a PDMS‐free fluidic device and offered a neutral support structure for mCCDcl1 cells, allowing cell attachment and polarization. Cells were cultured on the 3D scaffold for up to 6 weeks, showing an improved lifespan compared to classic cell culture, and displayed a continuous monolayer. Cell thickness was determined using confocal microscopy and increased from 7.5 ± 1.1 μm in 2D culture to 20.2 ± 2.5 μm in 3D culture. Immunocytochemistry of sodium channels (α‐ENaC) showed a highly polarized monolayer and increased expression in 3D cultures with its fluorescence mean intensity up to 31.64 ± 2.69 %, from 23.74 ± 2.19 % on a 2D culture. ENaC expression was confirmed by q‐PCR, which showed a 2.82 ± 0.15 fold increase in cells on the 3D scaffold, and V‐ATPase B1 expression increased by a factor of 23.77 ± 8.20. Amiloride sensitive sodium transport was observed using a fluorescent sodium‐binding dye and demonstrates that 3D polyHIPEs scaffolds may enable quantitative visualisation of ion transport in cultured collecting duct cells.The 3D model developed here is a useful tool for elucidating the mechanisms involved in collecting duct plasticity, to study drug toxicity, and for studying the cellular mechanisms underlying genetic diseases such as the syndrome of apparent mineralocorticoid excess (SAME).Support or Funding InformationBHF (British Heart Foundation)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells.

The cortical collecting duct of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH, and osmolarity and is composed of two distinct cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney by the localization of specific transport proteins such as aquaporin 2 (Aqp2) and epithelial sodium channel (ENaC) in principal cells and V-ATPase B1 and connexin 30 (Cx30) in intercalated cells. mCCDcl1 cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. In this study, the mCCDcl1 parental cell line and three sublines cloned from isolated single cells (Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance, transepithelial voltage, equivalent short circuit current and expression of the cell-specific markers Aqp2, ENaC, V-ATPaseB1, and Cx30. The parental mCCDcl1 cell line presented amiloride-sensitive electrogenic sodium transport indicative of principal cell function; however, immunocytochemistry and RT-PCR showed that some cells expressed the intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive for both intercalated and principal cell-specific markers. The vertical transmission of both principal and intercalated cell characteristics via single cell cloning reveals the plasticity of mCCDcl1 cells and a direct lineage relationship between these two physiologically important cell types and is consistent with mCCDcl1 cells being precursor cells.

Development and Diseases of the Collecting Duct System.

The collecting duct of the mammalian kidney is important for the regulation of extracellular volume, osmolarity, and pH. There are two major structurally and functionally distinct cell types: principal cells and intercalated cells. The former regulates Na+ and water homeostasis, while the latter participates in acid-base homeostasis. In vivo lineage tracing using Cre recombinase or its derivatives such as CreGFP and CreERT2 is a powerful new technique to identify stem/progenitor cells in their native environment and to decipher the origins of the tissue that they give rise to. Recent studies using this technique in mice have revealed multiple renal progenitor cell populations that differentiate into various nephron segments and collecting duct. In particular, emerging evidence suggests that like principal cells, most of intercalated cells originate from the progenitor cells expressing water channel Aquaporin 2. Mutations or malfunctions of the channels, pumps, and transporters expressed in the collecting duct system cause various human diseases. For example, gain-of-function mutations in ENaC cause Liddle's syndrome, while loss-of-function mutations in ENaC lead to Pseudohypoaldosteronism type 1. Mutations in either AE1 or V-ATPase B1 result in distal renal tubular acidosis. Patients with disrupted AQP2 or AVPR2 develop nephrogenic diabetes insipidus. A better understanding of the function and development of the collecting duct system may facilitate the discovery of new therapeutic strategies for treating kidney disease.

Corrigendum: Interaction between V-ATPase B2 and (Pro) renin Receptors in Promoting the progression of Renal Tubulointerstitial Fibrosis.

Scientific Reports 6: Article number: 25035; published online: 28 April 2016; updated: 10 June 2016 The Acknowledgements section in this Article is incomplete. “Thanks very much for the contributions on statistical work by Dr Qian Zhou”. should read: “Thanks very much for the contributions on statistical work by Dr Qian Zhou.

Interaction between V-ATPase B2 and (Pro) renin Receptors in Promoting the progression of Renal Tubulointerstitial Fibrosis.

To investigate the levels of (Pro) renin receptor [(P) RR], α-smooth muscle actin (α-SMA), fibronectin (FN), and vacuolar H+-ATPase (V-ATPase) subunits (B2, E, and c) in rat unilateral ureteral obstruction (UUO) models and rat proximal tubular epithelial cells (NRK-52E) treated with prorenin to elucidate the role of V-ATPase in these processes by activating the (P) RR. UUO significantly upregulated (P) RR, V-ATPase subunits, α-SMA and FN expression in tubulointerstitium or tubular epithelial cells. A marked colocalization of (P) RR and the B2 subunit was also observed. Prorenin treatment upregulated α-SMA, FN, (P) RR, and V-ATPase subunits and activity in NRK52E cell in a dose- and time-dependent manner. The V-ATPase inhibitor bafilomycin A1 partially blocked prorenin-induced (P) RR, FN, and α-SMA expression. Co-immunoprecipitate and immunofluorescence results demonstrated that the V-ATPase B2 subunit bound to the (P) RR, which was upregulated after prorenin stimulation. Either siRNA-mediated (P) RR or B2 subunit knockdown partially reduced V-ATPase activity and attenuated prorenin-induced FN and α-SMA expression. From the data we can assume that activation of (P) RR and V-ATPase may play an important role in tubulointerstitial fibrosis with possible involvement of interaction of V-ATPase B2 subunit and (P)RR.

Dot1l deficiency leads to increased intercalated cells and upregulation of V-ATPase B1 in mice

The collecting duct in the mammalian kidney consists of principal cells (PCs) and intercalated cells (ICs), which regulate electrolyte/fluid and acid/base balance, respectively. The epigenetic regulators of PC and IC differentiation remain obscure. We previously used Aqp2 and V-ATPase B1B2 to label PCs and ICs, respectively. We found that mice with histone H3 K79 methyltransferase Dot1l disrupted in Aqp2-expressing cells (Dot1lAC) vs. Dot1lf/f possessed ~20% more ICs coupled with a similar decrease in PCs. Here, we performed multiple double immunofluorescence staining using various PC and IC markers and confirmed that this finding. Both α-IC and β-IC populations were significantly expanded in Dot1lAC vs. Dot1lf/f. These changes are associated with significantly upregulated V-ATPase B1 and B2, but not Aqp2, AE1, and Pendrin. Chromatin immunoprecipitation assay unveiled a significant reduction of Dot1l and H3K79 di-methylation bound at the Atp6v1b1 5′ flanking region. Overexpression of Dot1a significantly downregulated a stably-transfected luciferase reporter driven by the Atp6v1b1 promoter in IMCD3 cells. This downregulation was impaired, but not completely abolished when a methyltransferase-dead mutant was overexpressed. Taken together, our data suggest that Dot1l is a new epigenetic regulator of PC and IC differentiation and Atp6v1b1 is a new transcriptional target of Dot1l.

Abstract 601: A Biased AT1 Receptor Agonist Stimulates COX2 Expression In Intercalated Cells

We recently discovered a pathway by which angiotensin (Ang) II, acting via angiotensin type 1 receptors (AT1R), triggers expression of cyclo-oxygenase (COX)-2 in intercalated cells (IC) of the collecting duct (CD), generating vasodilator prostaglandins, and attenuating the development of hypertension. We found that AT1R-dependent activation of COX2 could be recapitulated in isolated medullary collecting ducts (IMCDs) and in the C11 clone of the Madin-Darby canine kidney (MDCK) cell line permanently transfected with the AT1AR. This cell line has many features resembling ICs including expression of V-ATPase B1. The robust increase in COX2 expression in C11 cells after AngII was blunted after p38 inhibition (5.1*±0.8 vs. 1.4*±0.4; p=0.014), while PKC and MEK1 inhibition had negligible effects. Ang II activation of AT1R triggers both canonical G-protein and β-arrestin pathways and each pathway may have distinct physiological consequences. In IMCDs, we found that stimulation of COX2 by Ang II requires β-arrestin2, as this response was abrogated in IMCDs from mice lacking β-arrestin2. We next tested the capacity for an AT1R β-arrestin-biased ligand, TRV120023, to activate COX2 in C11 cells. These biased ligands block G-protein signalling like conventional AT1R blockers (ARBs), while stimulating β-arrestin-dependent pathways. TRV120023 caused significant stimulation of COX2 at concentrations of 0.1μM-10μM, indicating that activation of AT1R-linked β-arrestin pathways alone is sufficient to stimulate COX2. Exposure of the IC cell line by TRV also triggered phosphorylation of ERK1/2. The p38 inhibitor SB203580 attenuated TRV-dependent stimulation of ERK1/2 and reduced COX2 expression. In summary, activation of β-arrestin with TRV120023 is sufficient to induce COX2 expression via a pathway involving ERK1/2 and p38 MAPK. This suggests that β-arrestin-biased AT1R agonists might have added beneficial effects compared to conventional ARBs by blocking detrimental G-protein-dependent pathways while preserving beneficial β-arrestin-dependent pathways, such as enhanced expression of COX2.

Loss of the V-ATPase B1 subunit isoform expressed in non-neuronal cells of the mouse olfactory epithelium impairs olfactory function.

The vacuolar proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also regulates transmembrane proton (H+) secretion, which is necessary for an array of physiological functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells. Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite sex). We conclude that V-ATPase-mediated H+ secretion in the olfactory epithelium is required for optimal olfactory function.

ATP secretion in the male reproductive tract: essential role of CFTR

Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations.

V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells

The ubiquitous vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na(+)-independent pH recovery was significantly faster after an NH(4)Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-β1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Vacuolar H(+)-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC(50) of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.

HIGHLIGHTS FROM THE LITERATURE

HIGHLIGHTS FROM THE LITERATURE

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.