scholarly journals Telomerase immortalization of principal cells from mouse collecting duct

2010 ◽  
Vol 299 (6) ◽  
pp. F1507-F1514 ◽  
Author(s):  
Stacy L. Steele ◽  
Yongren Wu ◽  
Robert J. Kolb ◽  
Monika Gooz ◽  
Courtney J. Haycraft ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Collecting Duct ◽  
Whole Cell ◽  
Renal Epithelial Cells ◽  
Principal Cells ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Well Differentiated

Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.

Whole cell sodium conductance of principal cells freshly isolated from rat cortical collecting duct

1995 ◽  
Vol 269 (3) ◽  
pp. C791-C796 ◽  
Author(s):  
J. K. Bubien
Keyword(s):  
Patch Clamp ◽  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cyclic Monophosphate ◽  
Principal Cells ◽  
Clamp Technique ◽  
Duct Cells ◽  
Collecting Duct Cells

Cortical collecting duct fragments were manually dissected from 6-wk-old Sprague-Dawley rats. The fragments were enzymatically digested (collagenase A) into single cells, washed, and resuspended in serum-free RPMI 1640. Individual cells were examined electrophysiologically using the whole cell patch-clamp technique. Two morphologically distinct cell types were present in the cell suspension. Small round cells that had a capacitance of 7 pF and larger oval cells with a capacitance of 29 pF were consistently observed. Whole cell electrophysiological examination revealed that the small round cells had virtually no plasma membrane ionic conductance, whereas both inward and outward currents were observed in the larger oval-type cells. Also, superfusion of 250 pM arginine vasopressin specifically increased the inward conductance of only the larger cells. The effect could be completely inhibited by 2 microM amiloride or 100 mumol of the Rp diastereomer of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a specific adenosine 3',5'-cyclic monophosphate inhibitor). These findings are consistent with the hypothesis that the larger cells are principal cells and the smaller cells are intercalated cells and directly demonstrate that an amiloride-sensitive whole cell conductance is readily observable in freshly isolated cortical collecting duct cells. Thus the whole cell configuration of the patch-clamp technique appears to be well suited for assessing cellular mechanisms that regulate the ionic conductances of cortical collecting duct cells.

Whole cell and unitary amiloride-sensitive sodium currents in M-1 mouse cortical collecting duct cells

1996 ◽  
Vol 270 (4) ◽  
pp. C998-C1010 ◽  
Author(s):  
M. L. Chalfant ◽  
T. G. O'Brien ◽  
M. M. Civan
Keyword(s):  
Collecting Duct ◽  
Cell Permeability ◽  
Na Channel ◽  
Na Channels ◽  
Cortical Collecting Duct ◽  
Ionic Selectivity ◽  
Whole Cell ◽  
Duct Cells ◽  
Excised Patches ◽  
Collecting Duct Cells

Amiloride-sensitive whole cell currents have been reported in M-1 mouse cortical collecting duct cells (Korbmacher et al., J. Gen. Physiol. 102: 761-793, 1993). We have confirmed that amiloride inhibits the whole cell currents but not necessarily the measured whole cell currents. Anomalous responses were eliminated by removing external Na+ and/or introducing paraepithelial shunts. The amiloride-sensitive whole cell currents displayed Goldman rectification. The ionic selectivity sequence of the amiloride-sensitive conductance was Li+ > Na+ >> K+. Growth of M-1 cells on permeable supports increased the amiloride-sensitive whole cell permeability, compared with cells grown on plastic. Single amiloride-sensitive channels were observed, which conformed to the highly selective low-conductance amiloride-sensitive class [Na(5)] of epithelial Na+ channels. Hypotonic pretreatment markedly slowed run-down of channel activity. The gating of the M-1 Na+ channel in excised patches was complex. Open- and closed-state dwell-time distributions from patches that display one operative channel were best described with two or more exponential terms each. We conclude that 1) study of M-1 whole cell Na+ currents is facilitated by reducing the transepithelial potential to zero, 2) these M-1 currents reflect the operation of Na(5) channels, and 3) the Na+ channels display complex kinetics, involving > or = 2 open and > or = 2 closed states.

Expression of mRNA for natriuretic peptide receptor subtypes in bovine kidney

1994 ◽  
Vol 267 (2) ◽  
pp. F318-F324 ◽  
Author(s):  
T. Yamamoto ◽  
L. Feng ◽  
T. Mizuno ◽  
S. Hirose ◽  
K. Kawasaki ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Natriuretic Peptide ◽  
Collecting Duct ◽  
Biologically Active ◽  
Ribonuclease Protection Assay ◽  
Receptor Subtypes ◽  
Total Rna ◽  
Duct Cells ◽  
Glomerular Epithelial Cells ◽  
Collecting Duct Cells

The localization of mRNA for atrial natriuretic peptide (ANP) receptor subtypes (A, B, C) in the kidney was examined. Quantitative analysis of the ribonuclease protection assay showed that the numbers of type A receptor (ANPRA) mRNA were 6.9 x 10(7) in the glomeruli and 10.4 x 10(7) molecules/micrograms of total RNA in the inner medulla, and that of type C receptor (ANPRC) mRNA was 21.7 x 10(7) molecules/micrograms of total RNA in the glomeruli. The type B receptor (ANPRB) mRNA was present in smaller numbers (4.5-4.9 x 10(6) molecules/micrograms of total RNA) evenly throughout the kidney fractions. In situ hybridization demonstrated both ANPRA and ANPRC mRNA selectively in the glomerular epithelial cells and ANPRA mRNA in the collecting duct cells of the inner medulla. ANPRC was also localized on the foot processes of glomerular epithelial cells by immunohistochemistry using a specific antibody against the receptor. These results indicate that ANPRA is the major biologically active receptor for the ANP family of hormones in the kidney and is present selectively on the glomerular epithelial cells and inner medullary collecting duct cells. These cells are presumed to play a role in the regulation of glomerular filtration rate and sodium excretion induced by the family of ANP.

Changes in cellular composition of kidney collecting duct cells in rats with lithium-induced NDI

2004 ◽  
Vol 286 (4) ◽  
pp. C952-C964 ◽  
Author(s):  
Birgitte Mønster Christensen ◽  
David Marples ◽  
Young-Hee Kim ◽  
Weidong Wang ◽  
Jørgen Frøkiær ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Lithium Treatment ◽  
Intercalated Cells ◽  
Cellular Organization ◽  
Principal Cells ◽  
Duct Cells ◽  
Collecting Ducts ◽  
Collecting Duct Cells ◽  
Principal And Intercalated Cells ◽  
Double Immunolabeling

Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H+]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H+]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 ± 3.4 vs. 62 ± 1.8% of controls ( P < 0.05; n = 4) and in inner medullary collecting ducts, 58 ± 1.6 vs. 81 ± 1.3% of controls ( P < 0.05; n = 4). In parallel, a significant increase in the fraction of intercalated ([H+]ATPase-positive) cells was shown. Urine output, whole kidney AQP-2 expression, cellular organization, and the fractions of principal and intercalated cells in cortex and inner medulla returned to control levels after 4 wk on a lithium-free diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus.

Stanniocalcin-1 secretion and receptor regulation in kidney cells

2008 ◽  
Vol 294 (4) ◽  
pp. F788-F794 ◽  
Author(s):  
Olga Sazonova ◽  
Kathi A. James ◽  
Christopher R. McCudden ◽  
Daniel Segal ◽  
Asghar Talebian ◽  
...  
Keyword(s):  
Cell Lines ◽  
Collecting Duct ◽  
Signal Transduction Pathway ◽  
Distal Tubule ◽  
Receptor Regulation ◽  
Thick Ascending Limb ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Medullary Collecting Duct ◽  
Pkc Activation

Kidney collecting duct principal cells are the main source of stanniocalcin-1 (STC-1) production and secretion. From there, the hormone targets thick ascending limb and distal convoluted tubule cells, as well as collecting duct cells. More specifically, STC-1 targets their mitochondria to exert putative antiapoptotic effects. Two distal tubule cell lines serve as models of STC-1 production and/or mechanism of action. Madin-Darby canine kidney-1 (MDCK-1) cells mimic collecting duct cells in their synthesis of STC-1 ligand and receptor, whereas inner medullary collecting duct-3 (IMCD-3) cells respond to additions of STC-1 by increasing their respiration rate. In the present study, MDCK cell STC-1 secretion was examined under normal and hypertonic conditions, vectorally, and in response to hormones and signal transduction pathway activators/inhibitors. STC-1 receptor regulation was monitored in both cell lines in response to changing ligand concentration. The results showed that NaCl-induced hypertonicity had concentration-dependent stimulatory effects on STC-1 secretion, as did the PKC activator TPA. Calcium and ionomycin were inhibitory, whereas calcium receptor agonists had no effect. Angiotensin II, aldosterone, atrial natriuretic factor, antidiuretic hormone, and forskolin also had no effects. Moreover, STC-1 secretion exhibited no vectoral preference. STC-1 receptors were insensitive to homologous downregulation in both cell lines. In contrast, they were upregulated when STC-1 secretion was inhibited by calcium. The findings suggest that hypertonicity-induced STC-1 secretion is regulated through PKC activation and that high intracellular calcium levels are a potent inhibitor of release. More intriguingly, the results suggest that the receptor may not accompany STC-1 in its passage to the mitochondria.

scholarly journals Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney

2020 ◽  
Vol 31 (6) ◽  
pp. 1212-1225 ◽  
Author(s):  
Ann M. Laszczyk ◽  
Atsuko Y. Higashi ◽  
Sanjeevkumar R. Patel ◽  
Craig N. Johnson ◽  
Abdul Soofi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Target Genes ◽  
Collecting Duct ◽  
Expression Patterns ◽  
Renal Medulla ◽  
Urine Concentration ◽  
Renal Epithelia ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Collecting Tubules

BackgroundAs the glomerular filtrate passes through the nephron and into the renal medulla, electrolytes, water, and urea are reabsorbed through the concerted actions of solute carrier channels and aquaporins at various positions along the nephron and in the outer and inner medulla. Proliferating stem cells expressing the nuclear transcription factor Pax2 give rise to renal epithelial cells. Pax2 expression ends once the epithelial cells differentiate into mature proximal and distal tubules, whereas expression of the related Pax8 protein continues. The collecting tubules and renal medulla are derived from Pax2-positive ureteric bud epithelia that continue to express Pax2 and Pax8 in adult kidneys. Despite the crucial role of Pax2 in renal development, functions for Pax2 or Pax8 in adult renal epithelia have not been established.MethodsTo examine the roles of Pax2 and Pax8 in the adult mouse kidney, we deleted either Pax2, Pax8, or both genes in adult mice and examined the resulting phenotypes and changes in gene expression patterns. We also explored the mechanism of Pax8-mediated activation of potential target genes in inner medullary collecting duct cells.ResultsMice with induced deletions of both Pax2 and Pax8 exhibit severe polyuria that can be attributed to significant changes in the expression of solute carriers, such as the urea transporters encoded by Slc14a2, as well as aquaporins within the inner and outer medulla. Furthermore, Pax8 expression is induced by high-salt levels in collecting duct cells and activates the Slc14a2 gene by recruiting a histone methyltransferase complex to the promoter.ConclusionsThese data reveal novel functions for Pax proteins in adult renal epithelia that are essential for retaining water and concentrating urine.

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