Expression of the B splice variant of NBCe1 (SLC4A4) in the mouse kidney

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study’s purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

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Acid-Base Effects of Combined Renal Deletion of NBCe1-A and NBCe1-B

AJP Renal Physiology ◽

10.1152/ajprenal.00358.2021 ◽

2022 ◽

Author(s):

Hyun-Wook Lee ◽

Jill W. Verlander ◽

Gary E Shull ◽

Autumn N. Harris ◽

I. David Weiner

Keyword(s):

Proximal Tubule ◽

Splice Variant ◽

Molecular Mechanisms ◽

Ammonia Excretion ◽

Basal Diet ◽

Acid Base ◽

Outer Medulla ◽

Ammonia Metabolism ◽

Induced Changes ◽

Acid Loading

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deleting the A splice variant of the Na⁺-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex, but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The current studies determined the effects of combined renal deletion of NBCe1-A and NBCe1-B on systemic and proximal tubule ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. Since renal NBCe1-A and NBCe1-B expression is limited to the proximal tubule, Cre-positive mice had proximal tubule NBCe1-A/B deletion (PT-NBCe1-A/B KO). While on basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased expression of phosphate-dependent glutaminase (PDG) and phosphoenolpyruvate carboxykinase (PEPCK) and increased expression of glutamine synthetase (GS), an ammonia recycling enzyme, in PT in both the cortex and OM. Exogenous acid-loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in PDG, PEPCK, and GS expression in the proximal tubule in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to proximal tubule ammonia metabolism in the OM and thereby to systemic acid-base regulation.

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Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

Clinical Chemistry ◽

10.1373/clinchem.2006.073015 ◽

2007 ◽

Vol 53 (1) ◽

pp. 53-61 ◽

Cited By ~ 29

Author(s):

Eleni Mavrogiannou ◽

Areti Strati ◽

Aliki Stathopoulou ◽

Emily G Tsaroucha ◽

Loukas Kaklamanis ◽

...

Keyword(s):

Tumor Cell ◽

Real Time ◽

Cell Lines ◽

Splice Variant ◽

Splice Variants ◽

Telomerase Activity ◽

Tumor Cell Lines ◽

Rt Pcr ◽

Human Telomerase Reverse Transcriptase ◽

Tissue Samples

Abstract Background: We developed and validated a real-time reverse transcription (RT)–PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (α+β+, α−β+, α+β−, α−β−) in tumor cell lines and non–small cell lung cancer (NSCLC). Methods: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript–specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. Results: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The α+β− splice variant showed the highest expression and α−β+ and α−β− the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the α+β− variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by α+β+, with the α−β+ and α−β− splice variants having the lowest expression. In the NSCLC tumors, the α+β+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. Conclusions: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT α+β+ splice variant may be an independent negative prognostic factor for NSCLC patients.

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Distribution and oligomeric association of splice forms of Na+-K+-ATPase regulatory γ-subunit in rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00146.2001 ◽

2002 ◽

Vol 282 (3) ◽

pp. F393-F407 ◽

Cited By ~ 55

Author(s):

Elena Arystarkhova ◽

Randall K. Wetzel ◽

Kathleen J. Sweadner

Keyword(s):

Splice Variants ◽

Rat Kidney ◽

Thick Ascending Limb ◽

Outer Medulla ◽

Α Subunit ◽

Proximal Tubule Cells ◽

Γ Subunit ◽

Outer Stripe ◽

A Minor ◽

Splice Forms

Renal Na+-K+-ATPase is associated with the γ-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, γa and γb. Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the α-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na+-K+-ATPase conformation. In the cortex, the proportion of the γb-subunit was markedly lower, and the γa-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the γb-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na+-K+-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the γb-subunit. Because different mechanisms maintain and regulate Na+ homeostasis in different nephron segments, the splice forms of the γ-subunit may have evolved to control the renal Na+ pump through pump properties, gene expression, or both.

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Cloning and functional studies of splice variants of the α-subunit of the amiloride-sensitive Na+ channel

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.4.c1081 ◽

1998 ◽

Vol 274 (4) ◽

pp. C1081-C1089 ◽

Cited By ~ 15

Author(s):

J. Kevin Tucker ◽

Kaichiro Tamba ◽

Young-Jae Lee ◽

Li-Ling Shen ◽

David G. Warnock ◽

...

Keyword(s):

Amino Acid ◽

Splice Variants ◽

Extracellular Domain ◽

Na Channel ◽

Wild Type ◽

Rt Pcr ◽

Α Subunit ◽

Functional Studies ◽

Human Lung Cell Line ◽

Lung Cell Line

The α-subunit of the amiloride-sensitive epithelial Na+ channel (αENaC) is critical in forming an ion conductive pore in the membrane. We have identified the wild-type and three splice variants of the human αENaC (hαENaC) from the human lung cell line H441, using RT-PCR. These splice variants contain various structures in the extracellular domain, resulting in premature truncation (hαENaCx), 19-amino acid deletion (hαENaC−19), and 22-amino acid insertion (hαENaC+22). Wild-type hαENaC and splice variants were functionally characterized in Xenopus oocytes by coexpression with hENaC β- and γ-subunits. Unlike wild-type hαENaC, undetectable or substantially reduced amiloride-sensitive currents were observed in oocytes expressing these splice variants. Wild-type hαENaC was the most abundantly expressed hαENaC mRNA species in all tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and functional diversity of hαENaC and that alternative splicing may play a role in regulating hENaC activity.

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Fasting Induces the Expression of PGC-1α and ERR Isoforms in the Outer Stripe of the Outer Medulla (OSOM) of the Mouse Kidney

PLoS ONE ◽

10.1371/journal.pone.0026961 ◽

2011 ◽

Vol 6 (11) ◽

pp. e26961 ◽

Cited By ~ 11

Author(s):

Christina T. Teng ◽

Yin Li ◽

Pat Stockton ◽

Julie Foley

Keyword(s):

Mouse Kidney ◽

Outer Medulla ◽

Outer Stripe

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Methylation Regulates Expression of Novel Splice Variant and Wild Type Transcripts of IRF8.

Blood ◽

10.1182/blood.v116.21.3634.3634 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3634-3634

Author(s):

Era L. Pogosova-Agadjanyan ◽

Hana Lee ◽

Crystal K. Cummings ◽

Soheil Meshinchi ◽

Jerald P. Radich ◽

...

Keyword(s):

Cell Lines ◽

Promoter Methylation ◽

Splice Variant ◽

Splice Variants ◽

Leukemic Cell ◽

Wild Type ◽

Over Expression ◽

Demethylating Agents ◽

Leukemic Cell Lines ◽

Transcript Variants

Abstract Abstract 3634 Introduction: Interferon regulatory factor 8 (IRF8) is a transcription factor that plays a critical role in normal hematopoiesis. IRF8−/− transgenic mice develop a myeloproliferative syndrome that transforms to acute myeloid leukemia (AML). IRF8 expression varies dramatically in the blasts of AML patients. A number of biological processes, including epigenetic changes, mutations, or alternative splicing, may contribute to the variability of IRF8 expression in AML patients. We investigated the potential causes of the aberrant IRF8 expression in AML blasts. Wild-type transcript (IRF8-WT): The entire coding sequence of IRF8 was amplified using HiFi Taq polymerase and sequenced from 7 leukemic cell lines and 12 AML samples. We did not find any mutations associated with aberrant expression of IRF8. Splice Variants (IRF8-SVs): The initial studies examining IRF8 coding region suggested transcript deviation in the 5′ region. The GeneRacer kit was used to sequence 5′-capped mRNA. We identified 3 previously-undescribed transcript variants. In all 3 sequences, exon 1 was spliced out and replaced by nucleotides from the terminal end of intron 1 (Figure 1A). Some of the splice variants contained potential in-frame start codons. Expression of 5′ Splice Variants in Leukemic Cell Lines and AML Samples: We examined the expression levels of transcript variants in 12 leukemic cell lines, 246 AML samples, and hematopoietic subpopulations of cells from “healthy” adults. IRF8-SVs were expressed at significantly higher levels in some AML blasts than normal hematopoietic cells. In fact, AML cell lines with the highest IRF8 levels primarily expressed the IRF8-SVs rather than the IRF8-WT. Promoter Methylation: Interferon response element (pIRE) within the IRF8 promoter, which controls the expression of the gene, was examined by PCR after bisulfite conversion. Cell lines with very low IRF8 expression were often methylated at the pIRE locus. In addition, some cell lines with marked over-expression of IFR8-SVs also displayed methylated pIRE, suggesting that promoter methylation may also be playing a role in controlling the expression of the splice variants. pIRE methylation was also examined in a more limited number of AML samples, finding that blasts with low expression of IRF8 were methylated at this locus (Figure 1B). Reversal of Splice Variant Expression Pattern: Our previous studies suggested that methylation may play a role in controlling IRF8-SVs expression. Therefore, we examined the effect of demethylating agents on IRF8-SVs expression in U937 cells – a leukemic cell line with very high levels of IRF8-SVs. IRF8 transcripts (WT and SVs) were examined before and after exposure to therapeutic levels of 5-azacytidine (5-Aza). These studies suggested that 5-Aza exposure and subsequent demethylation of the pIRE locus was associated with restoration of the IRF8-WT and decreased IRF8-SVs expression (Figure 1C). This later data provides additional evidence that pIRE hypermethylation may regulate the expression of the novel IRF8-SVs. Conclusions: We have identified novel IRF8 transcript variants that are over-expressed in AML blasts. These novel IRF8-SVs introduce additional nucleotides at the 5′ region of the gene and may result in a new start codon. AML blasts with high levels of IRF8 transcript often over-express IRF8-SVs. Although the functional significance of these variants is unknown, methylation may play a role in regulating their expression, such that demethylating agents decrease IRF8-SVs and promote the IRF8-WT expression. Additional studies are planned to investigate the biology and clinical significance of IRF8-SVs. Initial studies suggests that over-expression of IRF8-SVs is associated with an inferior clinical outcome for adult AML patients. Disclosures: No relevant conflicts of interest to declare.

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Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A

AJP Renal Physiology ◽

10.1152/ajprenal.00015.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. F489-F501 ◽

Cited By ~ 3

Author(s):

Gunars Osis ◽

Kierstin L. Webster ◽

Autumn N. Harris ◽

Hyun-Wook Lee ◽

Chao Chen ◽

...

Keyword(s):

Metabolic Acidosis ◽

Proximal Tubule ◽

Wild Type ◽

Outer Medulla ◽

Extracellular Signals ◽

Citrate Metabolism ◽

Acid Loading ◽

Convoluted Tubules ◽

Proximal Tubule Segments

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

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Exploration of Potential Roles of a New LOXL2 Splicing Variant Using Network Knowledge in Esophageal Squamous Cell Carcinoma

The Scientific World JOURNAL ◽

10.1155/2014/431792 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 4

Author(s):

Bing-Li Wu ◽

Guo-Qing Lv ◽

Hai-Ying Zou ◽

Ze-Peng Du ◽

Jian-Yi Wu ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Esophageal Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Splice Variant ◽

Large Scale ◽

Molecular Mechanisms ◽

Splice Variants ◽

Lysyl Oxidase ◽

Wild Type

LOXL2 (lysyl oxidase-like 2), an enzyme that catalyzes oxidative deamination of lysine residue, is upregulated in esophageal squamous cell carcinoma (ESCC). A LOXL2 splice variant LOXL2-e13 and its wild type were overexpressed in ESCC cells followed by microarray analyses. In this study, we explored the potential role and molecular mechanism of LOXL2-e13 based on known protein-protein interactions (PPIs), following microarray analysis of KYSE150 ESCC cells overexpressing a LOXL2 splice variant, denoted by LOXL2-e13, or its wild-type counterpart. The differentially expressed genes (DEGs) of LOXL2-WT and LOXL2-e13 were applied to generate individual PPI subnetworks in which hundreds of DEGs interacted with thousands of other proteins. These two DEG groups were annotated by Functional Annotation Chart analysis in the DAVID bioinformatics database and compared. These results found many specific annotations indicating the potential specific role or mechanism for LOXL2-e13. The DEGs of LOXL2-e13, comparing to its wild type, were prioritized by the Random Walk with Restart algorithm. Several tumor-related genes such as ERO1L, ITGA3, and MAPK8 were found closest to LOXL2-e13. These results provide helpful information for subsequent experimental identification of the specific biological roles and molecular mechanisms of LOXL2-e13. Our study also provides a work flow to identify potential roles of splice variants with large scale data.

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NBCe1-A is required for the renal ammonia and K+ response to hypokalemia

AJP Renal Physiology ◽

10.1152/ajprenal.00481.2019 ◽

2020 ◽

Vol 318 (2) ◽

pp. F402-F421 ◽

Cited By ~ 1

Author(s):

Hyun-Wook Lee ◽

Autumn N. Harris ◽

Michael F. Romero ◽

Paul A. Welling ◽

Charles S. Wingo ◽

...

Keyword(s):

Metabolic Acidosis ◽

Proximal Tubule ◽

Splice Variant ◽

Ammonia Excretion ◽

Wild Type ◽

Outer Medulla ◽

Ammonia Metabolism ◽

Tissue Sections ◽

K Response

Hypokalemia increases ammonia excretion and decreases K+ excretion. The present study examined the role of the proximal tubule protein NBCe1-A in these responses. We studied mice with Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion [knockout (KO) mice] and their wild-type (WT) littermates were provided either K+ control or K+-free diet. We also used tissue sections to determine the effect of extracellular ammonia on NaCl cotransporter (NCC) phosphorylation. The K+-free diet significantly increased proximal tubule NBCe1-A and ammonia excretion in WT mice, and NBCe1-A deletion blunted the ammonia excretion response. NBCe1-A deletion inhibited the ammoniagenic/ammonia recycling enzyme response in the cortical proximal tubule (PT), where NBCe1-A is present in WT mice. In the outer medulla, where NBCe1-A is not present, the PT ammonia metabolism response was accentuated by NBCe1-A deletion. KO mice developed more severe hypokalemia and had greater urinary K+ excretion during the K+-free diet than did WT mice. This was associated with blunting of the hypokalemia-induced change in NCC phosphorylation. NBCe1-A KO mice have systemic metabolic acidosis, but experimentally induced metabolic acidosis did not alter NCC phosphorylation. Although KO mice have impaired ammonia metabolism, experiments in tissue sections showed that lack of ammonia does impair NCC phosphorylation. Finally, urinary aldosterone was greater in KO mice than in WT mice, but neither expression of epithelial Na+ channel α-, β-, and γ-subunits nor of H+-K+-ATPase α1- or α2-subunits correlated with changes in urinary K+. We conclude that NBCe1-A is critical for the effect of diet-induced hypokalemia to increase cortical proximal tubule ammonia generation and for the expected decrease in urinary K+ excretion.

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Time course of renal glutamate dehydrogenase induction during NH4Cl loading in rats

AJP Renal Physiology ◽

10.1152/ajprenal.1992.262.6.f999 ◽

1992 ◽

Vol 262 (6) ◽

pp. F999-F1006 ◽

Cited By ~ 4

Author(s):

P. A. Wright ◽

R. K. Packer ◽

A. Garcia-Perez ◽

M. A. Knepper

Keyword(s):

Glutamate Dehydrogenase ◽

Time Course ◽

Renal Cortex ◽

Ammonium Excretion ◽

Mrna Levels ◽

Outer Medulla ◽

Acid Intake ◽

Blood Ph ◽

Outer Stripe ◽

Acid Loading

To study mechanisms involved in renal glutamate dehydrogenase (GDH) regulation in response to systemic acid loading, we have measured blood pH, ammonium excretion, renal GDH mRNA levels, and GDH activity in rats. Acid intake (0.28 M NH4Cl in drinking water for 3 days) increased GDH mRNA levels in the renal cortex, but had no effect in the outer stripe of the outer medulla, inner stripe of the outer medulla, or the inner medulla. Rats were subjected to a step change in acid intake by alkali loading for 3 days (7.2 meq NaHCO3 per day in food slurry) and shifting to acid loading for up to 7 days (7.2 meq NH4Cl in food slurry). Ammonium excretion rose rapidly, increasing by 14-fold in the first 24-h period and 38-fold in the second 24-h period. Cortical GDH mRNA levels were increased relative to alkali-loaded values by 3.7-fold in 24 h, 4.3-fold in 4 days, but only 2.2-fold in 7 days. GDH activity was unchanged after 24 h of acid intake, but was significantly increased after 48 h. We concluded the following: 1) GDH mRNA is present in all regions of the kidney, but levels increase in response to acid loading only in the renal cortex; 2) GDH mRNA levels increase within 1 day after the initiation of acid loading, but the associated increase in functional enzyme activity takes 2 or more days; and 3) the large increases in ammonium excretion that occur in the first day after initiation of acid loading are not dependent on increased GDH activity.

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