scholarly journals Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance

2008 ◽  
Vol 295 (4) ◽  
pp. F859-F866 ◽  
Author(s):  
Hayo Castrop ◽  
Jurgen Schnermann
Keyword(s):  
Functional Significance ◽  
Splice Variants ◽  
Single Gene ◽  
Macula Densa ◽  
Full Length ◽  
Salt Transport ◽  
Thick Ascending Limb ◽  
Differential Splicing ◽  
Splice Isoforms ◽  
Variable Exon

The renal Na-K-2Cl cotransporter (NKCC2, BSC1) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene ( Slc12a1), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations.

scholarly journals Salt-sensitive splice variant of nNOS expressed in the macula densa cells

2010 ◽  
Vol 298 (6) ◽  
pp. F1465-F1471 ◽  
Author(s):  
Deyin Lu ◽  
Yiling Fu ◽  
Arnaldo Lopez-Ruiz ◽  
Rui Zhang ◽  
Ramiro Juncos ◽  
...  
Keyword(s):  
Salt Intake ◽  
Splice Variants ◽  
Macula Densa ◽  
High Salt ◽  
Tubuloglomerular Feedback ◽  
No Production ◽  
Thick Ascending Limb ◽  
High Salt Diet ◽  
Salt Diet ◽  
High Salt Intake

Neuronal nitric oxide synthase (nNOS), which is abundantly expressed in the macula densa cells, attenuates tubuloglomerular feedback (TGF). We hypothesize that splice variants of nNOS are expressed in the macula densa, and nNOS-β is a salt-sensitive isoform that modulates TGF. Sprague-Dawley rats received a low-, normal-, or high-salt diet for 10 days and levels of the nNOS-α, nNOS-β, and nNOS-γ were measured in the macula densa cells isolated with laser capture microdissection. Three splice variants of nNOS, α-, β-, and γ-mRNAs, were detected in the macula densa cells. After 10 days of high-salt intake, nNOS-α decreased markedly, whereas nNOS-β increased two- to threefold in the macula densa measured with real-time PCR and in the renal cortex measured with Western blot. NO production in the macula densa was measured in the perfused thick ascending limb with an intact macula densa plaque with a fluorescent dye DAF-FM. When the tubular perfusate was switched from 10 to 80 mM NaCl, a maneuver to induce TGF, NO production by the macula densa was increased by 38 ± 3% in normal-salt rats and 52 ± 6% ( P < 0.05) in the high-salt group. We found 1) macula densa cells express nNOS-α, nNOS-β, and nNOS-γ, 2) a high-salt diet enhances nNOS-β, and 3) TGF-induced NO generation from macula densa is enhanced in high-salt diet possibly from nNOS-β. In conclusion, we found that the splice variants of nNOS expressed in macula densa cells were α-, β-, and γ-isoforms and propose that enhanced level of nNOS-β during high-salt intake may contribute to macula densa NO production and help attenuate TGF.

scholarly journals Effects of NKCC2 isoform regulation on NaCl transport in thick ascending limb and macula densa: a modeling study

2014 ◽  
Vol 307 (2) ◽  
pp. F137-F146 ◽  
Author(s):  
Aurélie Edwards ◽  
Hayo Castrop ◽  
Kamel Laghmani ◽  
Volker Vallon ◽  
Anita T. Layton
Keyword(s):  
Cell Model ◽  
Basolateral Membrane ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Thick Ascending Limb ◽  
Differential Splicing ◽  
Dietary Salt ◽  
Nacl Transport ◽  
Low Salt

This study aims to understand the extent to which modulation of the Na+-K+-2Cl− cotransporter NKCC2 differential splicing affects NaCl delivery to the macula densa. NaCl absorption by the thick ascending limb and macula densa cells is mediated by apical NKCC2. A recent study has indicated that differential splicing of NKCC2 is modulated by dietary salt (Schieβl IM, Rosenauer A, Kattler V, Minuth WW, Oppermann M, Castrop H. Am J Physiol Renal Physiol 305: F1139–F1148, 2013). Given the markedly different ion affinities of its splice variants, modulation of NKCC2 differential splicing is believed to impact NaCl reabsorption. To assess the validity of that hypothesis, we have developed a mathematical model of macula densa cell transport and incorporated that cell model into a previously applied model of the thick ascending limb (Weinstein AM, Krahn TA. Am J Physiol Renal Physiol 298: F525–F542, 2010). The macula densa model predicts a 27.4- and 13.1-mV depolarization of the basolateral membrane [as a surrogate for activation of tubuloglomerular feedback (TGF)] when luminal NaCl concentration is increased from 25 to 145 mM or luminal K+ concentration is increased from 1.5 to 3.5 mM, respectively, consistent with experimental measurements. Simulations indicate that with luminal solute concentrations consistent with in vivo conditions near the macula densa, NKCC2 operates near its equilibrium state. Results also suggest that modulation of NKCC2 differential splicing by low salt, which induces a shift from NKCC2-A to NKCC2-B primarily in the cortical thick ascending limb and macula densa cells, significantly enhances salt reabsorption in the thick limb and reduces Na+ and Cl− delivery to the macula densa by 3.7 and 12.5%, respectively. Simulation results also predict that the NKCC2 isoform shift hyperpolarizes the macula densa basolateral cell membrane, which, taken in isolation, may inhibit the release of the TGF signal. However, excessive early distal salt delivery and renal salt loss during a low-salt diet may be prevented by an asymmetric TGF response, which may be more sensitive to flow increases.

scholarly journals Bisbee: A proteomics validated analysis package for detecting differential splicing, identifying splice outliers, and predicting splice event protein effects

2020 ◽  
Author(s):  
Rebecca F. Halperin ◽  
Apurva Hegde ◽  
Jessica D. Lang ◽  
Elizabeth A. Raupach ◽  
Christophe Legendre ◽  
...  
Keyword(s):  
Statistical Approach ◽  
Splice Variants ◽  
Splice Event ◽  
Mass Spectrometry Data ◽  
Differential Splicing ◽  
Splice Isoforms ◽  
Independent Dataset ◽  
Normal Tissues ◽  
Analysis Package ◽  
Splicing Detection

AbstractHere we present a novel statistical approach to splicing outlier and differential splicing detection, implemented in a software package called Bisbee. We leverage Bisbee’s prediction of protein level effects to benchmark using matched RNAseq and mass spectrometry data from normal tissues. Bisbee exhibits improved sensitivity and specificity over existing approaches. We applied Bisbee to confirm a pathogenic splicing event in a rare disease and to identify tumor-specific splice isoforms associated with an oncogenic splice factor mutation. We also identified common tumor associated splice isoforms replicated in an independent dataset, demonstrating the utility of Bisbee in discovering disease relevant splice variants.

Single-molecule Real-time (SMRT) Isoform Sequencing (Iso-Seq) in Plants: The Status of the Bioinformatics Tools to Unravel the Transcriptome Complexity

2019 ◽  
Vol 14 (7) ◽  
pp. 566-573 ◽  
Author(s):  
Yubang Gao ◽  
Feihu Xi ◽  
Hangxiao zhang ◽  
Xuqing Liu ◽  
Huiyuan Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Splice Variants ◽  
Alternative Polyadenylation ◽  
Full Length ◽  
Rna Seq ◽  
Bioinformatics Pipeline ◽  
Splice Isoforms ◽  
Bioinformatics Tools ◽  
Genome Annotations

Background: The advent of the Single-Molecule Real-time (SMRT) Isoform Sequencing (Iso-Seq) has paved the way to obtain longer full-length transcripts. This method was found to be much superior in identifying full-length splice variants and other post-transcriptional events as compared to the Next Generation Sequencing (NGS)-based short read sequencing (RNA-Seq). Several different bioinformatics tools to analyze the Iso-Seq data have been developed and some of them are still being refined to address different aspects of transcriptome complexity. However, a comprehensive summary of the available tools and their utility is still lacking. Objective: Here, we summarized the existing Iso-Seq analysis tools and presented an integrated bioinformatics pipeline for Iso-Seq analysis, which overcomes the limitations of NGS and generates long contiguous Full-Length Non-Chimeric (FLNC) reads for the analysis of posttranscriptional events. Results: In this review, we summarized recent applications of Iso-Seq in plants, which include improved genome annotations, identification of novel genes and lncRNAs, identification of fulllength splice isoforms, detection of novel Alternative Splicing (AS) and Alternative Polyadenylation (APA) events. In addition, we also discussed the bioinformatics pipeline for comprehensive Iso-Seq data analysis, including how to reduce the error rate in the reads and how to identify and quantify post-transcriptional events. Furthermore, the visualization approach of Iso-Seq was discussed as well. Finally, we discussed methods to combine Iso-Seq data with RNA-Seq for transcriptome quantification. Conclusion: Overall, this review demonstrates that the Iso-Seq is pivotal for analyzing transcriptome complexity and this new method offers unprecedented opportunities to comprehensively understand transcripts diversity.

scholarly journals Development of an Androgen Receptor Inhibitor Targeting the N-Terminal Domain of Androgen Receptor for Treatment of Castration Resistant Prostate Cancer

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3488
Author(s):  
Fuqiang Ban ◽  
Eric Leblanc ◽  
Ayse Derya Cavga ◽  
Chia-Chi Flora Huang ◽  
Mark R. Flory ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Splice Variants ◽  
Full Length ◽  
Cancer Resistance ◽  
Castration Resistant Prostate Cancer ◽  
Terminal Domain ◽  
Castration Resistant ◽  
Coregulatory Proteins ◽  
Androgen Binding

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC.

scholarly journals Differential Expression of BARD1 Isoforms in Melanoma

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 320
Author(s):  
Lorissa I. McDougall ◽  
Ryan M. Powell ◽  
Magdalena Ratajska ◽  
Chi F. Lynch-Sutherland ◽  
Sultana Mehbuba Hossain ◽  
...  
Keyword(s):  
Long Range ◽  
Patient Outcomes ◽  
Splice Variants ◽  
Significant Proportion ◽  
Nanopore Sequencing ◽  
Rna Seq ◽  
Splice Isoforms ◽  
Tissue Samples ◽  
Multiple Transcripts ◽  
Mrna Variants

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies

1995 ◽  
Vol 269 (6) ◽  
pp. C1496-C1505 ◽  
Author(s):  
C. Lytle ◽  
J. C. Xu ◽  
D. Biemesderfer ◽  
B. Forbush
Keyword(s):  
Monoclonal Antibodies ◽  
Loop Diuretic ◽  
Chloride Secretion ◽  
Salt Transport ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Animal Cells ◽  
Immunoreactive Protein ◽  
Site Density ◽  
Carboxy Terminal

The Na-K-Cl cotransporter (NKCC) is present in most animal cells where it functions in cell volume homeostasis and epithelial salt transport. We developed six monoclonal antibodies (designated T4, T8, T9, T10, T12, and T14) against a fusion protein fragment encompassing the carboxy-terminal 310 amino acids of the human colonic NKCC. These T antibodies selectively recognized putative NKCC proteins in a diverse variety of animal tissues. Western blot analysis of membranes isolated from 23 types of cells identified single bands of immunoreactive protein ranging in mass from 146 to 205 kDa. The amount of immunoreactive protein detected in these cells correlated with loop diuretic binding site density. Proteins identified previously as Na-K-Cl cotransporters by loop diuretic photoaffinity labeling were mutually recognized by multiple T antibodies. Most of the T antibodies effectively immunoprecipitated the denatured form of the NKCC protein. Immunocytochemical studies on the rabbit parotid gland demonstrated that NKCC is restricted to the basolateral margin of the acinar cells and absent from the ducts, in accord with the central role of Na-K-Cl cotransport in chloride secretion. In the rabbit kidney, NKCC was localized to the apical membrane of thick ascending limb cells, consistent with its role in chloride reabsorption.

Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase

2007 ◽  
Vol 292 (6) ◽  
pp. F1867-F1872 ◽  
Author(s):  
Ruisheng Liu ◽  
Jeffrey L. Garvin ◽  
YiLin Ren ◽  
Patrick J. Pagano ◽  
Oscar A. Carretero
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Oxidase Inhibitor ◽  
Fluorescent Dye ◽  
Macula Densa ◽  
Superoxide Production ◽  
Tubuloglomerular Feedback ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Nacl Concentration

Superoxide (O2−) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O2− production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O2− production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O2− production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O2− production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O2−, we used tempol (10−4 M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O2−, we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O2− production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O2− production was blocked. To test whether depolarization of the macula densa induces O2− production, we artificially induced depolarization by adding valinomycin (10−6 M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O2− production. We conclude that increasing luminal NaCl induces O2− production during tubuloglomerular feedback. O2− generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.

scholarly journals Potential role of luminal potassium in tubuloglomerular feedback.

1997 ◽  
Vol 8 (12) ◽  
pp. 1831-1837 ◽  
Author(s):  
V Vallon ◽  
H Osswald ◽  
R C Blantz ◽  
S Thomson
Keyword(s):  
Potential Role ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Retrograde Perfusion ◽  
Luminal Membrane ◽  
K Concentration ◽  
Tubular Flow

Transport through the Na+-2Cl(-)-K+ cotransporter in the luminal membrane of macula densa cells is considered critical for tubuloglomerular feedback (TGF). Although various studies could support the importance of luminal Na+ and Cl-, the role of luminal K+ in TGF has not been thoroughly addressed. The study presented here examines this issue in nephrons with superficial glomeruli of anesthetized male Munich-Wistar-Frömter rats. Ambient Na+ concentration in early distal tubular fluid was approximately 22 mM, suggesting collection sites relatively close to the macula densa segment. First, it was found that ambient early distal tubular K+ concentration is approximately 1.3 mM, i.e., close to the K+ affinity of the Na+-2Cl(-)-K+ cotransporter in the thick ascending limb. Second, it was observed that a change in late proximal tubular flow rate, i.e., a maneuver that is known to induce a TGF response, significantly alters early distal tubular K+ concentration. Third, previous experiments failed to show an inhibition in TGF response during retrograde perfusion of the macula densa with K+-free solutions. Because of a potential K+ influx into the lumen between the perfusion site and the macula densa, however, the K+ channel blocker U37883A was added to the K+-free perfusate. TGF response was assessed as the fall in nephron filtration rate in response to retrograde perfusion of the macula densa segment from early distal tubular site. It was observed that luminal U37883A (100 microM) significantly attenuated TGF. Because adding 5 mM KCl to the perfusate restored TGF in the presence of U37883A and because the inhibitory action of U37883A on tubular K+ secretion was confirmed, the effect of U37883A on TGF was most likely caused by inhibition of K+ influx into the perfused segment, which decreased luminal K+ concentration at the macula densa. The present findings support a potential role for luminal K+ in TGF, which is in accordance with a transmission of the TGF signal across the macula densa via Na+-2Cl(-)-K+ cotransporter.

scholarly journals A short ERK5 isoform modulates nucleocytoplasmic shuttling of active ERK5 and associates with poor survival in breast cancer

2021 ◽  
Author(s):  
Mariska Miranda ◽  
Jodi M. Saunus ◽  
Seçkin Akgül ◽  
Mahdi Moradi Marjaneh ◽  
Jamie R. Kutasovic ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Splice Variants ◽  
Clinicopathological Features ◽  
Full Length ◽  
Nucleocytoplasmic Shuttling ◽  
Poor Survival ◽  
Mesenchymal Transition

AbstractBackgroundThe nucleocytoplasmic shuttling of ERK5 has gained recent attention as a regulator of its diverse roles in cancer progression but the exact mechanisms for this shuttling are still under investigation.MethodsUsing in vitro, in vivo and in silico studies, we investigated the roles of shorter ERK5 isoforms in regulating the nucleocytoplasmic shuttling of active phosphorylated-ERK5 (pERK5). Retrospective cohorts of primary and metastatic breast cancer cases were used to evaluate the association of the subcellular localization of pERK5 with clinicopathological features.ResultsExtranuclear localization of pERK5 was observed during cell migration in vitro and at the invasive fronts of metastatic tumors in vivo. The nuclear and extranuclear cell fractions contained different isoforms of pERK5, which are encoded by splice variants expressed in breast and other cancers in the TCGA data. One isoform, isoform-3, lacks the C-terminal transcriptional domain and the nuclear localization signal. The co-expression of isoform-3 and full-length ERK5 associated with high epithelial-to-mesenchymal transition (EMT) and poor patient survival. Experimentally, expressing isoform-3 with full-length ERK5 in breast cancer cells increased cell migration, drove EMT and led to tamoxifen resistance. In breast cancer patient samples, pERK5 showed variable subcellular localizations where its extranuclear localization associated with aggressive clinicopathological features, metastasis, and poor survival.ConclusionOur studies support a model of ERK5 nucleocytoplasmic shuttling driven by splice variants in an interplay between mesenchymal and epithelial states during metastasis. Using ERK5 as a biomarker and a therapeutic target should account for its splicing and context-dependent biological functions.Graphical AbstractERK5 isoform-3 expression deploys active ERK5 (pERK5) outside the nucleus to facilitate EMT and cell migration. In cells dominantly expressing isoform-1, pERK5 shuttles to the nucleus to drive cell expansion.

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