Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat

2000 ◽  
Vol 98 (4) ◽  
pp. 409 ◽  
Author(s):  
Leong L. NG ◽  
Sonja JENNINGS ◽  
Joan E. DAVIES ◽  
Paulene A. QUINN
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Spontaneously Hypertensive Rat ◽  
Temperature Sensitive ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Spontaneously Hypertensive ◽  
Hypertensive Rat

Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat

2000 ◽  
Vol 98 (4) ◽  
pp. 409-418 ◽  
Author(s):  
Leong L. NG ◽  
Sonja JENNINGS ◽  
Joan E. DAVIES ◽  
Paulene A. QUINN
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Western Blotting ◽  
Spontaneously Hypertensive Rat ◽  
Proximal Tubule Cell ◽  
Permissive Temperature ◽  
Proximal Tubule Cells ◽  
Spontaneously Hypertensive ◽  
Hypertensive Rat ◽  
Tubule Cells

Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na+/H+ exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar–Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 °C, but growth is abolished at the restrictive temperature of 39 °C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE-694-sensitive H+ efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0±2.8 and 39.9±5.7 mmol·min-1·l-1 respectively; P < 0.001; n = 6). H+ efflux due to non-Na+-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of 32P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers.

Increased expression of the SA gene in the kidney of the spontaneously hypertensive rat is localized to the proximal tubule

1994 ◽  
Vol 12 (12) ◽  
pp. 1347???1352 ◽  
Author(s):  
Hasmukh R. Patel ◽  
Amrik S. Thiara ◽  
Kevin P. West ◽  
David Lodwick ◽  
Nilesh J. Samani
Keyword(s):  
Proximal Tubule ◽  
Spontaneously Hypertensive Rat ◽  
Spontaneously Hypertensive ◽  
Hypertensive Rat

A morphological and functional comparison of proximal tubule cell lines established from human urine and kidney tissue

2014 ◽  
Vol 323 (1) ◽  
pp. 87-99 ◽  
Author(s):  
J. Jansen ◽  
C.M.S. Schophuizen ◽  
M.J. Wilmer ◽  
S.H.M. Lahham ◽  
H.A.M. Mutsaers ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Human Urine ◽  
Kidney Tissue ◽  
Proximal Tubule Cell ◽  
Tubule Cell

scholarly journals Molecular Dissection of Ca2+ Efflux in Immortalized Proximal Tubule Cells

1997 ◽  
Vol 109 (2) ◽  
pp. 217-228 ◽  
Author(s):  
Kenneth E. White ◽  
Frank A. Gesek ◽  
Teresa Nesbitt ◽  
Marc K. Drezner ◽  
Peter A. Friedman
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Cell Lines ◽  
Calcium Absorption ◽  
Northern Analysis ◽  
Proximal Tubule Cell ◽  
Rt Pcr ◽  
Tubule Cell ◽  
Western Analysis ◽  
Product Identity

Plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger participate in regulating cell function by maintaining proper intracellular Ca2+ concentrations ([Ca2+]i). In renal epithelial cells these proteins have been additionally implicated in cellular calcium absorption. The purpose of the present studies was to determine the Ca2+ extrusion mechanisms in cells derived from the proximal tubule. Homology-based RT-PCR was used to amplify PMCA transcripts from RNA isolated from mouse cell lines originating from the S1, S2, and S3 proximal tubule segments. S1, S2, and S3 cells exhibited only PMCA1 and PMCA4 products. PCR product identity was confirmed by sequence analysis. Northern analysis of proximal tubule cell RNAs revealed appropriate transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4, but were negative for PMCA2 and PMCA3. Western analysis with a monoclonal antibody to PMCA showed that all proximal cell lines expressed a reacting plasma membrane protein of 140 kD, the reported PMCA molecular mass. Na+/Ca2+ exchanger (NCX1) mRNA expression, analyzed by RT-PCR, protein expression by Western analysis, and functional exchange activity were uniformly absent from all proximal tubule cell lines. These observations support the idea that immortalized cells derived from the proximal tubule express PMCA1 and PMCA4, which may serve as the primary mechanism of cellular Ca2+ efflux.

Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin-angiotensin system

1995 ◽  
Vol 268 (3) ◽  
pp. F435-F446 ◽  
Author(s):  
S. S. Tang ◽  
F. Jung ◽  
D. Diamant ◽  
D. Brown ◽  
D. Bachinsky ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Renin Angiotensin System ◽  
Temperature Sensitive ◽  
Proximal Tubule Cell ◽  
Permissive Temperature ◽  
Ang Ii ◽  
Tubule Cell ◽  
Angiotensin System ◽  
Rat Proximal Tubule

Immortalized rat proximal tubule cell (IRPTC) lines should be useful for investigation of proximal tubule (PT) regulation and function but previously have been unavailable. We now report the establishment and characterization of an immortalized transformed, temperature-sensitive IRPTC cell line containing renin-angiotensin system (RAS) components. Primary PT cells prepared from male Wistar rats (4-5 wk old) after collagenase digestion, sieving, and Percoll gradient were cultured on collagen-coated T-75 flasks in Dulbecco's modified Eagle's medium containing 5% fetal calf serum. Subconfluent PT cells were transfected with the temperature-sensitive SV40 mutant viruses (tsA SV40) by direct exposure. After 7-8 wk, several clones were obtained, from which one has been characterized and designated as line 3-2. This cell line appears stable up to 45 passages. Clonal cells transformed with this virus exhibit a transformed phenotype at a permissive temperature of 34 degrees C and grow in multiple layers. When the cells are subsequently placed at a nonpermissive temperature of 41 degrees C, they return to morphology similar to that of untransformed cells of the same lineage. At either 34 degrees C or 41 degrees C, this cell line expresses a variety of PT markers including alkaline phosphatase, cytokeratin, carbonic anhydrase, and glucose transporter isoform 2 (GLUT2), while not expressing factor VIII. Uniquely, these cells also appear to express PT proteins gp330 and CHIP28, markers which are usually lost in cultured cells. Furthermore, the cell line expresses protein and mRNA components of RAS, including angiotensinogen, angiotensin converting enzyme, and renin. The IRPTC cell line expresses few angiotensin II (ANG II) receptors at 34 degrees C, the permissive temperature. However, at the nonpermissive temperature, 41 degrees C, IRPTC expresses ANG II receptor (dissociation constant of 0.7 nM; maximum binding capacity of 265 fmol/mg protein). ANG II (10(-8) M) induced a transient rise in cytoplasmic Ca2+ concentration, which was nearly abolished with losartan but not PD-123319, suggesting this finding is AT1 receptor mediated. This cell line should provide an excellent model of PT and should make it possible to study the cell and molecular biology of the RAS, as well as other regulatory systems of the PT.

scholarly journals Cofilin Interacts with ClC-5 and Regulates Albumin Uptake in Proximal Tubule Cell Lines

2003 ◽  
Vol 278 (41) ◽  
pp. 40169-40176 ◽  
Author(s):  
Deanne H. Hryciw ◽  
Yinghong Wang ◽  
Olivier Devuyst ◽  
Carol A. Pollock ◽  
Philip Poronnik ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cell Lines ◽  
Proximal Tubule Cell ◽  
Tubule Cell
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