Localization of NBC-1 variants in human kidney and renal cell carcinoma

doi:10.1016/j.bbrc.2003.09.147

Biochemical and Biophysical Research Communications

Volume 310, Issue 4, 31 October 2003, Pages 1213-1218

https://doi.org/10.1016/j.bbrc.2003.09.147 Get rights and content

Abstract

The Na⁺–HCO₃⁻ cotransporter (NBC-1) plays a major role in bicarbonate absorption from proximal tubules. However, which NBC-1 variant mediates proximal bicarbonate absorption has not been definitely determined. Moreover, the localization of this cotransporter in human kidney and renal cell carcinoma (RCC) tissues has not been clarified. To clarify these issues, immunohistochemical analysis was performed using the specific antibodies against kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters. In Western blot analysis the expression of kNBC-1 but not of pNBC-1 was detected in both normal human kidney and RCC tissues. In immunofluorescence analysis on normal renal tissues the anti-kNBC-1 antibody strongly and exclusively labeled the basolateral membranes of proximal tubules, which was confirmed by electron microscopic observation. In RCC cells, the anti-kNBC-1 antibody labeled both plasma membranes and intracellular organelles. The labeling by anti-pNBC-1 antibody was not detected in both normal kidney and RCC tissues. These results indicate that kNBC-1 is the dominant variant that mediates bicarbonate absorption from human renal proximal tubules. They also suggest that NBC-1 may have distinct roles in cancer cells.

Section snippets

Materials and methods

Tissues. Human kidneys were obtained with full informed consent from three patients (age: 60, 71, and 80 years old) who underwent the total nephrectomy for RCC. The histological analysis confirmed that all these tumors were of common type, clear cell subtype. Both histologically normal tissues and cancer tissues were immediately frozen in liquid nitrogen for Western blot analysis or processed for immunohistochemical analysis.

Western blot analysis. Western blot analysis was performed as

Western blot analysis

To determine the dominant NBC-1 variant(s) expressed in human kidney and RCC tissues, we first performed Western blot using the variant-specific antibodies. As shown in Fig. 1, the anti-kNBC-1 antibody yielded a clear band of ∼145 kDa from the normal renal tissues. In all three RCC tissues, a similar band was also detected by the anti-kNBC-1 antibody. By contrast, the anti-pNBC-1 antibody failed to yield a clear band from both normal kidney and RCC tissues (data not shown), though this antibody

Discussion

The anti-peptide antibodies used in the present study were raised against the N-terminal regions of human kNBC-1 and pNBC-1. The specificity of these antibodies was confirmed by the expression study on ECV304 cells [11]. Another NBC-1-related isoform, rb2NBC-1, has the novel C-terminal region, but its N-terminal region is identical to that of pNBC-1 [5]. Therefore rb2NBC-1 is expected to be detected by the anti-pNBC-1 antibody, though its human counterpart has not been cloned so far.

Acknowledgements

This study was in part supported by Grants 12671024 and 14571013 from the Ministry of Education, Science and Culture of Japan. Anti-NBC-1 antibodies were supplied by Kumamoto Immunochemical Laboratory (Kumamoto, Japan).

References (25)

C.E. Burnham et al.
Cloning and functional expression of a human kidney Na⁺:HCO₃⁻ cotransporter
J. Biol. Chem.
(1997)
N. Abuladze et al.
Molecular cloning, chromosomal localization, tissue distribution, and functional expression of the human pancreatic sodium bicarbonate cotransporter
J. Biol. Chem.
(1998)
N. Abuladze et al.
Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3
Gene
(2000)
M.F. Romero et al.
Expression cloning and characterization of a renal electrogenic Na⁺/HCO₃⁻ cotransporter
Nature
(1997)
M.O. Bevensee et al.
An electrogenic Na⁺–HCO₃⁻ cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain
Am. J. Physiol. Cell Physiol.
(2000)
M.F. Romero et al.
Electrogenic Na⁺/HCO₃⁻ cotransporters: cloning and physiology
Annu. Rev. Physiol.
(1999)
M.F. Romero et al.
Cloning and functional expression of rNBC, an electrogenic Na⁺–HCO₃⁻ cotransporter from rat kidney
Am. J. Physiol.
(1998)
I. Choi et al.
Cloning and characterization of a human electrogenic Na⁺–HCO₃⁻ cotransporter isoform (hhNBC)
Am. J. Physiol.
(1999)
T. Igarashi et al.
Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities
Nat. Genet.
(1999)
T. Igarashi et al.
Unraveling the molecular pathogenesis of isolated proximal renal tubular acidosis
J. Am. Soc. Nephrol.
(2002)

T. Usui et al.

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

J. Clin. Invest.

(2001)

T. Igarashi et al.

Novel nonsense mutation in the Na⁺/HCO₃⁻ cotransporter gene (SLC4A4) in a patient with permanent isolated proximal renal tubular acidosis and bilateral glaucoma

J. Am. Soc. Nephrol.

(2001)

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Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion
2011, Journal of Biological Chemistry
Citation Excerpt :
NBC1 has two variants: kNBC1 from kidney and pNBC1 from pancreas. We found that the NBC1 expressed in TG neurons is the pancreatic type, which has been reported in pancreas (36, 37) and salivary glands (38, 39). We further confirmed the role of NHE1 and pNBC1 in pHi recovery by treating TG neurons with siRNA.
The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca²⁺-induced acidification and subsequent recovery of intracellular pH (pH_i) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μm) and capsaicin (1 μm) increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) with a following decrease in pH_i. The recovery of [Ca²⁺]_i to the prestimulus level was inhibited by LaCl₃ (1 mm) and o-vanadate (10 mm), a plasma membrane Ca²⁺/ATPase (PMCA) inhibitor. Removal of extracellular Ca²⁺ also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na⁺/H⁺ exchanger type 1 (NHE1), pancreatic Na⁺-HCO₃⁻ cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1–3 were detected by RT-PCR. pH_i recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH_i. Application of the NHE1 inhibitor 5′-(N-ethyl-N-isopropyl) amiloride (5 μm) or the pNBC1 inhibitor 4′,4′-di-isothiocyanostilbene-2′,2′-sulfonic acid (500 μm) delayed pH_i recovery and decreased AP frequency. Simultaneous application of 5′-(N-ethyl-N-isopropyl) amiloride and 4′,4′-di-isothiocyanostilbene-2′,2′-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca²⁺]_i in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH_i recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.
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Increased expression of NBC1 has been reported in renal cell carcinoma. It is postulated that NBC1 may be involved in creating a microenvironment that may enhance tumor spread and aggressiveness.110 NBC3 and NBC4 also play a role of clinical importance.
The role of the Na⁺-coupled HCO₃⁻ transporter (NBC) family is indispensable in acid-base homeostasis. Almost all tissues express a member of the NBC family. NBC has been studied extensively in the kidney and plays a role in proximal tubule HCO₃⁻ reabsorption. Although the exact function of this transporter family on other tissues is not very clear, the ubiquitous expression of NBC family suggests a role in cell pH regulation. Altered NBC activity caused by mutations of the gene responsible for NBC protein expression results in pathophysiologic conditions. Mutations of NBC resulting in important clinical disorders have been reported extensively on one member of the NBC family, the kidney NBC (NBC1). These mutations have led to several structural studies to understand the mechanism of the abnormal NBC1 activity.
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¹: These authors equally contributed to this study.

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Localization of NBC-1 variants in human kidney and renal cell carcinoma

Abstract

Section snippets

Materials and methods

Western blot analysis

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

Gene

Expression cloning and characterization of a renal electrogenic Na+/HCO3− cotransporter

Nature

An electrogenic Na+–HCO3− cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain

Am. J. Physiol. Cell Physiol.

Electrogenic Na+/HCO3− cotransporters: cloning and physiology

Annu. Rev. Physiol.

Cloning and functional expression of rNBC, an electrogenic Na+–HCO3− cotransporter from rat kidney

Am. J. Physiol.

Cloning and characterization of a human electrogenic Na+–HCO3− cotransporter isoform (hhNBC)

Am. J. Physiol.

Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities

Nat. Genet.

Unraveling the molecular pathogenesis of isolated proximal renal tubular acidosis

J. Am. Soc. Nephrol.

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

J. Clin. Invest.

Novel nonsense mutation in the Na+/HCO3− cotransporter gene (SLC4A4) in a patient with permanent isolated proximal renal tubular acidosis and bilateral glaucoma

J. Am. Soc. Nephrol.

Expression cloning and characterization of a renal electrogenic Na⁺/HCO₃⁻ cotransporter

An electrogenic Na⁺–HCO₃⁻ cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain

Electrogenic Na⁺/HCO₃⁻ cotransporters: cloning and physiology

Cloning and functional expression of rNBC, an electrogenic Na⁺–HCO₃⁻ cotransporter from rat kidney

Cloning and characterization of a human electrogenic Na⁺–HCO₃⁻ cotransporter isoform (hhNBC)

Novel nonsense mutation in the Na⁺/HCO₃⁻ cotransporter gene (SLC4A4) in a patient with permanent isolated proximal renal tubular acidosis and bilateral glaucoma