PROBLEMS OF SPECIFICITY IN THE USE OF A STRONTIUM CAPTURE TECHNIQUE FOR THE CYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE IN MAMMALIAN RENAL TUBULES

The p-nitrophenyl phosphate-strontium procedure for the localization of the phosphatase component of Na-K-activated adenosine triphosphatase was evaluated using rat renal cortex as a test tissue. The results obtained by light microscopy were unexpected in that reaction product was found only on the brush borders of proximal tubule cells; this reaction was ouabain-resistant, K-independent and partially Mg-dependent, but could be completely inhibited by l-tetramisole. Electron microscopy showed that a reaction was also present on the cytoplasmic surfaces of the lateral and basal plasma membranes of the proximal and distal tubule cells. That seen in the distal tubule was sensitive to ouabain but not to l-tetramisole, whereas that in the proximal tubule showed a mixture of ouabain-sensitive and l-tetramisole-sensitive components. It is concluded that the procedure as originally described is not specific, demonstrating alkaline phosphatase as well as Na-K-adenosine triphosphatase, but that this problem may be overcome by the use of an alkaline phosphatase inhibitor.

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The characterisation and uptake of paraquat in cultured baboon kidney proximal tubule cells (bPTC)

Human & Experimental Toxicology ◽

10.1191/096032701672136818 ◽

2001 ◽

Vol 20 (2) ◽

pp. 90-99 ◽

Cited By ~ 10

Author(s):

R Machaalani ◽

V Lazzaro ◽

G G Duggin

Keyword(s):

Proximal Tubule ◽

Mdck Cells ◽

Distal Tubule ◽

Genetic Mutation ◽

Immunoperoxidase Staining ◽

Renal Tubules ◽

Trypan Blue Exclusion ◽

Proximal Tubule Cells ◽

Morphological Studies ◽

Tubule Cells

A primary culture of baboon proximal tubule cells (bPTC) was prepared and characterised using LLC-PK1 cells of proximal tubule origin and MDCK cells of distal tubule origin, as positive and negative references, respectively. The proximal tubular origin of the bPTC was determined by morphological studies, immunoperoxidase staining and the expression of proximal tubule markers alkaline phosphatase and gammaglutamyltransferase. The hypothesis that paraquat (PQ) is transported by the bPTC was investigated. The cytotoxic threshold for PQ in these cells was determined and compared to the LLC-PK1 and MDCK cells. Furthermore, this study investigated the transport of the monovalent cation tetraethyl ammonium (TEA) and the polyvalent cation cimetidine in the bPTC and demonstrated their effect on the cellular uptake of PQ. The cytotoxic threshold of PQ in the bPTC, determined by cellular viability studies using the method of Trypan blue exclusion, is 0.05 mM at 2 h incubation. The LC50 after 24 his 76, 61 and 455 pM for the bPTC, LLC-PK1 and MDCK cells, respectively. This indicates that proximal tubule cells are more susceptible to PQ toxicity compared to distal tubule cells, which is consistent with clinical PQ toxicity where renal damage is found predominantly in the proximal renal tubules. The cations PQ and cimetidine were actively transported by the bPTC. The uptake of PQ (0.05 mM) commenced after 15 min whereas cimetidine (0.5 mM) uptake was evident after 2 min. Furthermore, cimetidine was shown to compete with PQ for uptake in the bPTC. Coincubating PQ (0.05 mM) and cimetidine (0.5 mM) for 60 min resulted in an approximate 50% decrease in PQ uptake. The cation TEA was not transported by the bPTC suggesting either a genetic mutation or complete absence of the transporter for TEA in the cells. The results suggest that PQ may be transported by the same cation transporter as cimetidine and not TEA, indicating PQ uptake in the bPTC to be via a polyvalent organic cation transporter.

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TRANSPORT ADENOSINE TRIPHOSPHATASE CYTOCHEMISTRY II. CYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE ACTIVITY IN THE SECRETORY EPITHELIUM OF THE AVIAN SALT GLAND

Journal of Histochemistry & Cytochemistry ◽

10.1177/20.1.23 ◽

1972 ◽

Vol 20 (1) ◽

pp. 23-38 ◽

Cited By ~ 168

Author(s):

STEPHEN A. ERNST

Keyword(s):

Phosphatase Activity ◽

Adenosine Triphosphatase ◽

Plasma Membranes ◽

Salt Gland ◽

Ultrastructural Localization ◽

Defined Medium ◽

Secretory Cells ◽

Cytochemical Localization ◽

Fixed Tissue ◽

Nitrophenyl Phosphate

A cytochemical procedure is described for the ultrastructural localization of K-dependent, ouabain-sensitive nitrophenyl phosphatase activity in avian salt gland. Cryostat sections (50 µ) of paraformaldehyde-fixed tissue were incubated in a kinetically defined medium containing: 5 mM p-nitrophenyl phosphate, 10 mM MgCl2, 10 mM KCl, 100 mM Tris-HCl buffer (pH 8.5 or 9.0) and 20 mM SrCl2 to precipitate hydrolyzed phosphate. After incubation at room temperature, the sections were treated with Pb(NO3)2 to convert SrPi to PbPi precipitates for visualization in the electron microscope. Reaction product was localized on the cytoplasmic side of the secretory cell lateral and basal plasma membranes. Little, if any, reaction product was associated with the apical surfaces of the secretory cells or with endothelial surfaces of capillaries. Appropriate control experiments indicated that deposition of reaction product was dependent on Mg and K and was sensitive to ouabain. Furthermore, nonenzymatic hydrolysis of nitrophenyl phosphate did not occur in the medium, and deposition of artifactually produced precipitates did not resemble deposition of enzymatically produced precipitates. The relationship of this localization to transport adenosine triphosphatase cytochemistry is discussed, and the physiologic implications of the localization for tracing the route of active Na transport in the salt gland are considered.

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Aluminium deposition in liver and kidney following acute intravenous administration of aluminium chloride or citrate in conscious rats

Human & Experimental Toxicology ◽

10.1177/096032719501401002 ◽

1995 ◽

Vol 14 (10) ◽

pp. 787-794 ◽

Cited By ~ 20

Author(s):

AJ Spencer ◽

JA Wood ◽

HC Saunders ◽

MS Freeman ◽

CJ Lote

Keyword(s):

Proximal Tubule ◽

Intravenous Administration ◽

Distal Tubule ◽

Dry Weight ◽

Aluminium Chloride ◽

Proximal Tubule Cells ◽

Al Content ◽

Liver And Kidney ◽

Wet Weight ◽

Tubule Cells

1 Plasma, urinary, liver and kidney cell aluminium (Al) levels were monitored in the rat, 1h after intravenous administration of 29630 nmol (800 μg) Al as either Al chloride or as Al citrate (Al chloride plus excess sodium citrate). Al levels were measured in plasma, urine and liver by atomic absorption spectroscopy (AAS). Liver and kidney Al content was measured at the cellular and subcellular level by electron probe X-ray microanalysis (EPXMA). 2 Urinary excretion of Al was significantly higher ( P < 0.01), when Al was given as the citrate than as the chloride. After 1h, plasma Al levels were significantly lower in the Al citrate group than the Al chloride group (59 ± 3.7 vs 877 ± 214 nmol ml-1, respectively; P< 0.01). 3 Al concentrations were significantly higher in the livers of rats receiving Al chloride (818 ± 252 nmol g-1 wet weight; P < 0.05), than in either control or Al citrate groups (122 ± 41 and 107 ± 26 nmol g-1 wet weight, respectively). Al concentrations derived from EPXMA measurements were in agreement with AAS values for the three groups, with significantly higher Al concentra tions in the Al chloride group (1.7 ± 0.4 nmol mg-1 dry weight; P < 0.05) than in the control or Al citrate groups, where Al was not detectable. EPXMA analysis showed that Al was distributed in all liver organelles analysed (cytoplasm, mitochondria, nucleus, ER) and was not preferentially taken up by any one organelle in Al chloride treated rats. 4 Significant amounts of Al were found in cytoplasm and mitochondria of proximal tubule cells of rats given Al citrate (0.64 ± 0.15 and 0.80 ± 0.11 nmol mg-1 dry weight, respectively), but not in nuclei or lysosomes of these cells. Al levels were not detectable in control kid neys, in proximal tubule cells after Al chloride adminis tration or distal tubule cells after either Al treatment.

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Antenatal betamethasone attenuates the angiotensin-(1–7)-Mas receptor-nitric oxide axis in isolated proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00593.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. F1056-F1062 ◽

Cited By ~ 6

Author(s):

Yixin Su ◽

Jianli Bi ◽

Victor M. Pulgar ◽

Mark C. Chappell ◽

James C. Rose

Keyword(s):

Nitric Oxide ◽

Proximal Tubule ◽

Cell Model ◽

Primary Cultures ◽

Specific Effect ◽

Renal Tubules ◽

Proximal Tubule Cells ◽

Mas Receptor ◽

Tubule Cells

We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to angiotensin-(1–7) [Ang-(1–7)]. The present study determined the Na+ uptake and nitric oxide (NO) response to low-dose Ang-(1–7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as percentage of basal uptake or area under the curve for Na+ or percentage of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433 ± 28 vs. 330 ± 26%; P < 0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255 ± 16 vs. 255 ± 14%; P > 0.05). Ang-(1–7) significantly inhibited Na+ uptake in RPTC from vehicle male (214 ± 11%) and from both vehicle (190 ± 14%) and Beta (209 ± 11%) females but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1–7) in male but not female RPTC. Both the Na+ and NO responses to Ang-(1–7) were blocked by Mas receptor antagonist d-Ala7-Ang-(1–7). We conclude that the tubular Ang-(1–7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.

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Monoclonal antibodies to human erythrocyte membrane Ca++-Mg++ adenosine triphosphatase pump recognize an epitope in the basolateral membrane of human kidney distal tubule cells.

Journal of Clinical Investigation ◽

10.1172/jci113196 ◽

1987 ◽

Vol 80 (5) ◽

pp. 1225-1231 ◽

Cited By ~ 83

Author(s):

J L Borke ◽

J Minami ◽

A Verma ◽

J T Penniston ◽

R Kumar

Keyword(s):

Monoclonal Antibodies ◽

Erythrocyte Membrane ◽

Human Erythrocyte ◽

Adenosine Triphosphatase ◽

Basolateral Membrane ◽

Human Kidney ◽

Distal Tubule ◽

Human Erythrocyte Membrane ◽

Tubule Cells

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Structural and functional analysis of Na+/Ca2+ exchange in distal convoluted tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.1996.271.3.f560 ◽

1996 ◽

Vol 271 (3) ◽

pp. F560-F570 ◽

Cited By ~ 6

Author(s):

K. E. White ◽

F. A. Gesek ◽

P. A. Friedman

Keyword(s):

Plasma Membranes ◽

Calcium Absorption ◽

Single Cells ◽

Primary Cultures ◽

Distal Tubule ◽

Variable Regions ◽

Tubule Cells ◽

Conserved Region ◽

Single Transcript ◽

And Function

Renal distal convoluted tubules (DCT) are a major site of hormone-regulated, active calcium absorption. Calcium exit across basolateral plasma membranes is thought to be mediated by Na+/Ca2+ exchange and a Ca(2+)-ATPase. In this report the presence and function of Na+/Ca2+ exchangers in DCT cells were assessed. cDNAs encoding a conserved region and the variable regions of three alternatively spliced isoforms of the Na+/Ca2+ exchanger, NACA2, NACA3, and NACA6, were isolated in a ratio of 7:12:1 using homology-based reverse transcription-polymerase chain reaction (RT-PCR) with RNA from an immortalized mouse DCT cell line. Northern blots probed with a 32P-labeled PCR product from a conserved region of the exchanger were positive for a single transcript of 7 kb in primary cultures of distal tubule cells (cortical ascending limb + DCT cells), consistent with the reported size of the exchanger in other tissues. Na+/Ca2+ exchange was assessed by measuring sodium-dependent changes of intracellular calcium ([Ca2+]i), in single cells. In the presence of an outward Na+ gradient, [Ca2+]i increased by 240%. Collapsing the Na+ gradient with monensin inhibited the rise of [Ca2+]i. Removal of extracellular Ca2+ or the addition of an Na+ ionophore inhibited the rise of [Ca2+]i. The intracellular Na+ concentration decreased upon removal of extracellular Na+ in parallel with the rise of [Ca2+]i. Western analysis performed on membranes prepared from DCT cells or primary cultures of distal tubule cells with a polyclonal antibody revealed bands at approximately 125 and 85 kDa, consistent with reported sizes for exchanger protein. These findings show that Na+/Ca2+ exchanger transcripts, protein, and activity are present in DCT cells and that Na(+)-dependent Ca2+ efflux may be mediated by NACA2, NACA3, and NACA6.

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Gentamicin suppresses endotoxin-driven TNF-α production in human and mouse proximal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00333.2007 ◽

2007 ◽

Vol 293 (4) ◽

pp. F1373-F1380 ◽

Cited By ~ 24

Author(s):

Richard A. Zager ◽

Ali C. M. Johnson ◽

Adam Geballe

Keyword(s):

Protein Synthesis ◽

Proximal Tubule ◽

Renal Tubules ◽

Mrna Accumulation ◽

Chemokine Production ◽

Proximal Tubule Cells ◽

Gram Negative ◽

Essentially Normal ◽

Tnf Α ◽

Tubule Cells

Gentamicin is a mainstay in treating gram-negative sepsis. However, it also may potentiate endotoxin (LPS)-driven plasma TNF-α increases. Because gentamicin accumulates in renal tubules, this study addressed whether gentamicin directly alters LPS-driven tubular cell TNF-α production. HK-2 proximal tubular cells were incubated for 18 h with gentamicin (10–2,000 μg/ml). Subsequent LPS-mediated TNF-α increases (at 3 or 24 h; protein/mRNA) were determined. Gentamicin effects on overall protein synthesis ([35S]methionine incorporation), monocyte chemoattractant protein-1 (MCP-1) levels, and LPS-stimulated TNF-α generation by isolated mouse proximal tubules also were assessed. Finally, because gentamicin undergoes partial biliary excretion, its potential influence on gut TNF-α/MCP-1 mRNAs was probed. Gentamicin caused striking, dose-dependent inhibition of LPS-driven TNF-α production (up to 80% in HK-2 cells/isolated tubules). Surprisingly, this occurred despite increased TNF-α mRNA accumulation. Comparable changes in MCP-1 were observed. These changes were observed at clinically relevant gentamicin concentrations and despite essentially normal overall protein synthetic rates. Streptomycin also suppressed LPS-driven TNF-α increases, suggesting an aminoglycoside drug class effect. Gentamicin doubled basal TNF-α mRNA in cecum and in small intestine after LPS. Gentamicin can suppress LPS-driven TNF-α production in proximal tubule cells, likely by inhibiting its translation. Overall preservation of protein synthesis and comparable MCP-1 suppression suggest a semiselective blockade within the LPS inflammatory mediator cascade. These results, coupled with increases in gut TNF-α/MCP-1 mRNAs, imply that gentamicin may exert protean, countervailing actions on systemic cytokine/chemokine production during gram-negative sepsis.

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The significance of inhibitor-resistant alkaline phosphatase in the cytochemical demonstration of transport adenosine triphosphatase.

Journal of Histochemistry & Cytochemistry ◽

10.1177/23.8.169303 ◽

1975 ◽

Vol 23 (8) ◽

pp. 571-574 ◽

Cited By ~ 11

Author(s):

J A Firth ◽

B Y Marland

Keyword(s):

Alkaline Phosphatase ◽

Adenosine Triphosphatase ◽

Renal Cortex ◽

Sodium Potassium ◽

Cytochemical Demonstration ◽

Nitrophenyl Phosphate ◽

Atpase Cytochemistry ◽

Strontium Ions ◽

Dependent Transport ◽

Hydrolysis Of

The hydrolysis of disodium p-nitrophenyl phosphate at pH 9.0 by slices of formaldehydee-fixed rat renal cortex was investigated by colorimetric estimation of the nitrophenol liberated. It was found that three types of activity could be identified on the basis of their responses to inhibitors and cations: (a) alkaline phosphatase sensitive to inhibition by L-tetramisole; (b) potassium-dependent phosphatase, probably identifiable with the phosphatase component of sodium-potassium-dependent transport adenosine triphosphatase (?Na-K-ATPase); and (c) alkaline phosphatase insensitive to L-tetramisole. It was found that in the presence of strontium ions, as used in Na-K-ATPase cytochemistry, the activities of the second and third types of enzyme were approximately equal. The implications of these findings for the cytochemical demonstration of Na-K-ATPase are discussed.

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Steroidogenic acute regulatory-related lipid transfer domain protein 5 localization and regulation in renal tubules

AJP Renal Physiology ◽

10.1152/ajprenal.90433.2008 ◽

2009 ◽

Vol 297 (2) ◽

pp. F380-F388 ◽

Cited By ~ 16

Author(s):

Yu-Chyu Chen ◽

Renate K. Meier ◽

Shirong Zheng ◽

Syed J. Khundmiri ◽

Michael T. Tseng ◽

...

Keyword(s):

Er Stress ◽

Proximal Tubule ◽

Mouse Kidney ◽

Cell Periphery ◽

Type I ◽

Renal Tubules ◽

Lipid Transfer ◽

Proximal Tubule Cells ◽

Cholesterol Levels ◽

Tubule Cells

STARD5 is a cytosolic sterol transport protein that is predominantly expressed in liver and kidney. This study provides the first report on STARD5 protein expression and distribution in mouse kidney. Immunohistochemical analysis of C57BL/6J mouse kidney sections revealed that STARD5 is expressed in tubular cells within the renal cortex and medullar regions with no detectable staining within the glomeruli. Within the epithelial cells of proximal renal tubules, STARD5 is present in the cytoplasm with high staining intensity along the apical brush-border membrane. Transmission electronmicroscopy of a renal proximal tubule revealed STARD5 is abundant at the basal domain of the microvilli and localizes mainly in the rough endoplasmic reticulum (ER) with undetectable staining in the Golgi apparatus and mitochondria. Confocal microscopy of STARD5 distribution in HK-2 human proximal tubule cells showed a diffuse punctuate pattern that is distinct from the early endosome marker EEA1 but similar to the ER membrane marker GRP78. Treatment of HK-2 cells with inducers of ER stress increased STARD5 mRNA expression and resulted in redistribution of STARD5 protein to the perinuclear and cell periphery regions. Since recent reports show elevated ER stress response gene expression and increased lipid levels in kidneys from diabetic rodent models, we tested STARD5 and cholesterol levels in kidneys from the OVE26 type I diabetic mouse model. Stard5 mRNA and protein levels are increased 2.8- and 1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVB control kidneys. Renal free cholesterol levels are 44% elevated in the OVE26 mice. Together, our data support STARD5 functioning in kidney, specifically within proximal tubule cells, and suggest a role in ER-associated cholesterol transport.

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