The effect of 2,3,4-trimethylpentane on the ultrastructure of proximal tubular cells in primary cell culture

1989 ◽  
Vol 47 ◽  
pp. 1090-1091
Author(s):  
D.R. Mattie ◽  
J.J. Maslanka ◽  
N.J. Del Raso ◽  
M.R. Chase
Keyword(s):  
Proximal Tubule ◽  
Male Rat ◽  
Proximal Tubular Cells ◽  
Thin Sections ◽  
Proximal Tubule Cells ◽  
Tubular Cells ◽  
Transmission Electron ◽  
Cacodylate Buffer ◽  
Proximal Tubular ◽  
Tubule Cells

To aid in the assessment of the risk of Air Force personnel working with hydrocarbon fuels and compounds, an attempt was made to further characterize the nephropathy that results from exposure to hydrocarbons. The purpose of this study was to isolate and establish purified primary cultures of male rat proximal tubular cells suitable for experimental exposure to sublethal concentrations of solubilized 2,3, 4-trimethylpentane (TMP), a model hydrocarbon. Experiments were conducted to evaluate the cytotoxicity and metabolism of solubilized TMP in media containing or lacking the protein albumin.Proximal tubule cells in primary suspension culture were exposed to one of the following levels of TMP: 7.9, 12.0, 15.7, 19.1 or 25.5 mM. After 4 hours of exposure, pelleted cells were fixed for transmission electron microscopy by resuspension in 2% glutaraldehyde and 2.5% paraformaldehyde in 0.1M cacodylate buffer at pH 7.4. After a minimum fixation of at least 24 hours, the cells were post-fixed with 2% osmium tetroxide in 0,1M cacodylate buffer at pH 7.4. Cells were processed into Polybed 812 plastic capsules. Sections one micron thick, were cut in order to verify that cells were intact and suitable for thin sectioning. Thin sections (60- 90 nm) were cut on an ultramicrotome using a diamond knife. Thin sections, stained with uranyl acetate and lead citrate, were examined with a transmission electron microscope at 60 kV. Photographs of representative proximal tubule cells were taken at three levels of magnification.

scholarly journals Immunotoxin SS1P is rapidly removed by proximal tubule cells of kidney, whose damage contributes to albumin loss in urine

2020 ◽  
Vol 117 (11) ◽  
pp. 6086-6091 ◽  
Author(s):  
Xui-Fen Liu ◽  
Junxia Wei ◽  
Qi Zhou ◽  
Bruce A. Molitoris ◽  
Ruben Sandoval ◽  
...  
Keyword(s):  
Half Life ◽  
Proximal Tubular Cells ◽  
Proximal Tubule Cells ◽  
Tubular Cells ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Protein Toxin ◽  
Mouse Imaging ◽  
Proximal Tubular ◽  
Recombinant Immunotoxins

Recombinant immunotoxins (RITs) are chimeric proteins composed of an Fv and a protein toxin being developed for cancer treatment. The Fv brings the toxin to the cancer cell, but most of the RITs do not reach the tumor and are removed by other organs. To identify cells responsible for RIT removal, and the pathway by which RITs reach these cells, we studied SS1P, a 63-kDa RIT that targets mesothelin-expressing tumors and has a short serum half-life. The major organs that remove RIT were identified by live mouse imaging of RIT labeled with FNIR-Z-759. Cells responsible for SS1P removal were identified by immunohistochemistry and intravital two-photon microscopy of kidneys of rats. The primary organ of SS1P removal is kidney followed by liver. In the kidney, SS1P passes through the glomerulus, is taken up by proximal tubular cells, and transferred to lysosomes. In the liver, macrophages are involved in removal. The short half-life of SS1P is due to its very rapid filtration by the kidney followed by degradation in proximal tubular cells of the kidney. In mice treated with SS1P, proximal tubular cells are damaged and albumin in the urine is increased. SS1P uptake by kidney is reduced by coadministration ofl-lysine. Our data suggests thatl-lysine administration to humans might prevent SS1P-mediated kidney damage, reduce albumin loss in urine, and alleviate capillary leak syndrome.

scholarly journals Regulation of Megalin Expression in Cultured Proximal Tubule Cells by Angiotensin II Type 1A Receptor- and Insulin-Mediated Signaling Cross Talk

Endocrinology ◽  
2009 ◽  
Vol 150 (2) ◽  
pp. 871-878 ◽  
Author(s):  
Michihiro Hosojima ◽  
Hiroyoshi Sato ◽  
Keiko Yamamoto ◽  
Ryohei Kaseda ◽  
Taeko Soma ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Cross Talk ◽  
Proximal Tubule Cells ◽  
Opossum Kidney ◽  
Receptor Associated Protein ◽  
Ok Cells ◽  
Proximal Tubular ◽  
Tubule Cells ◽  
Proximal Tubular Function

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT1AR)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT1AR but are deficient in endogenous angiotensin II receptors (AT1AR-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalin’s endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT1AR-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT1AR-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT1AR-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT1AR- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs. Angiotensin II type 1A receptor- and insulin-mediated competitive signaling cross-talk regulates the expression of megalin, a multi-ligand endocytic receptor, in cultured proximal tubule cells.

scholarly journals Long-term protein exposure reduces albumin binding and uptake in proximal tubule-derived opossum kidney cells.

1998 ◽  
Vol 9 (6) ◽  
pp. 960-968 ◽  
Author(s):  
M Gekle ◽  
S Mildenberger ◽  
R Freudinger ◽  
S Silbernagl
Keyword(s):  
Amino Acids ◽  
Proximal Tubule ◽  
Uptake Rate ◽  
Kidney Cells ◽  
Proximal Tubular Cells ◽  
Opossum Kidney ◽  
Tubular Cells ◽  
Opossum Kidney Cells ◽  
Proximal Tubular ◽  
Protein Reabsorption

To avoid renal loss of large amounts of proteins, filtered proteins are reabsorbed by endocytosis along the proximal tubule. However, although protein reabsorption is a task of proximal tubular cells, it is also a threat because it may cause cell injury. This study determines whether exposure to bovine serum albumin (BSA) leads to regulatory changes in endocytosis of FITC-BSA in proximal tubule-derived opossum kidney cells. Preincubation with BSA led to a decrease of FITC-BSA endocytosis with an IC50 value of 0.58 g/L. Specific binding of FITC-BSA to the apical membrane was also reduced (IC50 = 0.69 g/L). Kinetic analyses revealed that maximal uptake rate and maximal binding capacity were decreased with no change in affinity. Similar effects were observed after preincubation with equimolar amounts of other proteins (lactalbumin, transferrin, and conalbumin), but not after preincubation with dextran. The effect of preincubation with BSA could be mimicked by preincubation with some amino acids. Preincubation with L-Ala, L-Gln, or NH4Cl, but not with L-Leu, L-Glu, or L-Asp, reduced FITC-BSA endocytosis and binding. Preincubation with BSA, but not with dextran, reduced protein degradation and increased ammonia production, vesicular pH, as well as the rate of lactate dehydrogenase release. Apical fluid-phase endocytosis and apical uptake of neutral amino acids were not reduced. It is concluded that proximal tubular cells reduce the uptake rate for proteins, but not for other substrates, in response to increased protein load. This reduction is achieved by reducing the number of apical binding sites, partially in response to increased ammoniagenesis with deranged vesicular pH and enzyme activities. Thus, increased protein filtration could result in reduced protein reabsorption, thereby enhancing proteinuria.

Intracisternal microtubules in proximal tubule cells of duck kidney: A serendipitous finding

1987 ◽  
Vol 45 ◽  
pp. 898-899
Author(s):  
M.K. Bhatnagar ◽  
S.M. Snelgrove-Hobson ◽  
P.V.V. Prasada Rao
Keyword(s):  
Electron Microscope ◽  
Proximal Tubule ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Kidney Cortex ◽  
Lead Citrate ◽  
Cell Organelles ◽  
Thin Sections ◽  
Proximal Tubule Cells ◽  
Tubule Cells

Cytpplasmic microtubules, ubiquitous cell organelles, lie free in the cytosol without being compartmented off by membranes. In rare instances, however, intracisternal microtubules (ICM) have been reported in ganglionic neurons of aging dogs and in the proximal tubule cells (PTC) of rabbit kidneys. We report here the presence of ICM in the PTC of the kidneys of Peking ducks (Anas platyrhynhos).A total of 106 Peking ducks (24 males and 24 females of 9 months, 48 females of 15 months, 6 males of 30 months and 2 males and 2 females of 48 months of age) were anesthetized and exsanguinated. Pieces of kidney cortex were fixed in 4% glutaraldehyde in 0.5M phosphate buffer (pH 7.3) at 29°C for four hours. Samples were post fixed in 2% osmium tetroxide for two hours. One micron sections of Epon-embedded cortices were stained with toluidine blue and thin sections (70-70 nm) contrasted with uranyl acetate and lead citrate were examined with a JEOL 100-S electron microscope at 80 kV.

A1 adenosine receptor knockout mice are protected against acute radiocontrast nephropathy in vivo

2006 ◽  
Vol 290 (6) ◽  
pp. F1367-F1375 ◽  
Author(s):  
H. Thomas Lee ◽  
Michael Jan ◽  
Soo Chan Bae ◽  
Jin Deok Joo ◽  
Farida R. Goubaeva ◽  
...  
Keyword(s):  
Renal Failure ◽  
Acute Renal Failure ◽  
Proximal Tubule ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Proximal Tubule Cells ◽  
Proximal Tubular ◽  
Tubule Cells

The role of renal A1 adenosine receptors (A1AR) in the pathogenesis of radiocontrast nephropathy is controversial. We aimed to further elucidate the role of A1AR in the pathogenesis of radiocontrast nephropathy and determine whether renal proximal tubule A1AR contribute to the radiocontrast nephropathy. To induce radiocontrast nephropathy, A1AR wild-type (WT) or knockout (KO) mice were injected with a nonionic radiocontrast (iohexol, 1.5–3 g iodine/kg). Some A1WT mice were pretreated with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a selective A1AR antagonist) before iohexol injection. A1AR contribute to the pathogenesis of radiocontrast nephropathy in vivo as the A1WT mice developed significantly worse acute renal failure, more renal cortex vacuolization, and had lower survival 24 h after iohexol treatment compared with the A1KO mice. DPCPX pretreatment also protected the A1WT mice against radiocontrast-induced acute renal failure. No differences in renal cortical apoptosis or inflammation were observed between A1WT and A1KO mice. To determine whether the proximal tubular A1AR mediate the direct renal cytotoxicity of radiocontrast, we treated proximal tubules in culture with iohexol with or without 2-chloro- N6-cyclopentyladenosine (a selective A1AR agonist) or DPCPX pretreatment. We also subjected cultured proximal tubule cells overexpressing A1AR or lacking A1AR to radiocontrast injury. Iohexol caused a direct dose-dependent reduction in proximal tubule cell viability as well as proliferation. Neither the A1AR agonist nor the antagonist treatment affected proximal tubule viability or proliferation. Moreover, overexpression or lack of A1AR failed to impact the iohexol toxicity on proximal tubule cells. Therefore, we conclude that radiocontrast causes acute renal failure via mechanisms dependent on A1AR; however, renal proximal tubule A1AR do not contribute to the direct tubular toxicity of radiocontrast.

scholarly journals Angiotensin II stimulates vesicular H+-ATPase in rat proximal tubular cells

1998 ◽  
Vol 95 (16) ◽  
pp. 9665-9668 ◽  
Author(s):  
Carsten A. Wagner ◽  
Gerhard Giebisch ◽  
Florian Lang ◽  
John P. Geibel
Keyword(s):  
Angiotensin Ii ◽  
Atpase Activity ◽  
Proximal Tubule ◽  
Brush Border ◽  
Channel Blocker ◽  
Proximal Tubular Cells ◽  
Proton Extrusion ◽  
Tubular Cells ◽  
Ion Secretion ◽  
Proximal Tubular

Two mechanisms of H+ ion secretion in the proximal tubule that mediate bicarbonate reabsorption have been identified: the brush border Na/H exchanger and electrogenic H+ ion secretion. Angiotensin II (AII) has been shown to be a regulator of the luminal Na+/H+ exchanger and the basolateral Na+/HCO3− cotransporter. In the present study, we examined the effects of AII on H+-ATPase activity in isolated proximal tubule fragments. H+-ATPase activity was assessed by monitoring intracellular pH after Na+ removal from the bath. In addition, we investigated the effects on pH recovery of the proton pump inhibitor bafilomycin A1, removal of Cl−, and of colchicine. pH was continuously measured with the pH-sensitive fluorescent dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recovery of cell pH was observed in the absence of external Na+ and was significantly accelerated by AII. The AII-stimulated pH recovery was completely abolished by bafilomycin A1, by removal of Cl−, by NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate; a potent Cl− channel blocker], and by colchicine. We conclude from these studies that AII stimulates proton extrusion via H+-ATPase by a Cl−-dependent process involving brush border insertion of vesicles. This process may contribute to up-regulation of HCO3− reabsorption along the proximal tubule when tubules are exposed to AII.

Transepithelial fluxes of adenosine and 2′-deoxyadenosine across human renal proximal tubule cells: roles of nucleoside transporters hENT1, hENT2, and hCNT3

2009 ◽  
Vol 296 (6) ◽  
pp. F1439-F1451 ◽  
Author(s):  
Adam N. Elwi ◽  
Vijaya L. Damaraju ◽  
Michelle L. Kuzma ◽  
Delores A. Mowles ◽  
Stephen A. Baldwin ◽  
...  
Keyword(s):  
Cell Surface ◽  
Proximal Tubule ◽  
Apical Cell ◽  
Nucleoside Transporters ◽  
Proximal Tubule Cells ◽  
Protein Levels ◽  
Anti Cancer ◽  
Proximal Tubular ◽  
Tubule Cells ◽  
Renal Proximal Tubule Cells

This study examined the roles of human nucleoside transporters (hNTs) in mediating transepithelial fluxes of adenosine, 2′-deoxyadenosine, and three purine nucleoside anti-cancer drugs across polarized monolayers of human renal proximal tubule cells (hRPTCs), which were shown in previous studies to have human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) activities ( 11 ). Early passage hRPTCs were cultured on transwell inserts under conditions that induced formation of polarized monolayers with experimentally accessible apical and basolateral domains. Polarized hRPTC cultures were monitored for inhibitor sensitivities and sodium-dependence of the following: 1) transepithelial fluxes of radiolabeled adenosine, 2′-deoxyadenosine, fludarabine (9-β-d-arabinosyl-2-fluoroadenine), cladribine (2-chloro-2′-deoxyadenosine), and clofarabine (2-chloro-2′-fluoro-deoxy-9-β-d-arabinofuranosyladenine); 2) mediated uptake of radiolabeled adenosine, 2′-deoxyadenosine, fludarabine, cladribine, and clofarabine from either apical or basolateral surfaces; and 3) relative apical cell surface hCNT3 protein levels. Transepithelial fluxes of adenosine were mediated from apical-to-basolateral sides by apical hCNT3 and basolateral hENT2, whereas transepithelial fluxes of 2′-deoxyadenosine were mediated from basolateral-to-apical sides by apical hENT1 and basolateral human organic anion transporters (hOATs). The transepithelial fluxes of adenosine, hCNT3-mediated cellular uptake of adenosine, and relative apical cell surface hCNT3 protein levels correlated positively in polarized hRPTCs. The purine nucleoside anti-cancer drugs fludarabine, cladribine, and clofarabine, like adenosine exhibited apical-to-basolateral fluxes. Collectively, this evidence suggested that apical hCNT3 and basolateral hENT2 are involved in proximal tubular reabsorption of adenosine and some nucleoside drugs and that apical hENT1 and basolateral hOATs are involved in proximal tubular secretion of 2′-deoxyadenosine.

20-Hydroxyeicosatetraenoic acid is formed in response to EGF and is a mitogen in rat proximal tubule

1995 ◽  
Vol 269 (6) ◽  
pp. F806-F816 ◽  
Author(s):  
F. Lin ◽  
A. Rios ◽  
J. R. Falck ◽  
Y. Belosludtsev ◽  
M. L. Schwartzman
Keyword(s):  
Arachidonic Acid ◽  
Proximal Tubule ◽  
Cell Lines ◽  
Primary Cultures ◽  
Cell Number ◽  
Brdu Incorporation ◽  
Maximal Effect ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular

Hydroxyeicosatetraenoic acid (20-HETE) is a major cytochrome P-450-arachidonic acid metabolite in the rat kidney, and its synthesis along the nephron is specifically localized to the proximal tubule, where receptor density for epidermal growth factor (EGF) is the highest. EGF stimulated endogenous 20-HETE formation in a concentration and time-dependent manner, i.e., from 1.6 to 2.6 +/- 0.3 and 3.0 +/- 0.6 pmol 20-HETE.mg-1.min-1 at 10(-8) and 10(-7) M EGF, respectively. The effect of 20-HETE on proximal tubular cell proliferation was examined using primary cultures of rat proximal tubular cells and proximal tubular-derived cell lines, LLC-PK1 and opossum kidney OK. In both cell lines, 20-HETE increased thymidine incorporation into DNA with maximal effect at 10(-9) M. Addition of 20-HETE to serum-deprived LLC-PK1 or OK cells for 48 h caused a concentration-dependent increase in cell number with maximal effect at 10(-9) M. This effect was specific, as structurally similar eicosanoids such as 20-COOH-arachidonic acid, 19(R)-HETE, and 19(S)-HETE did not increase cell number. In 4-day primary cultures of proximal tubular cells, EGF (10(-9) M) and 20-HETE (10(-9) M) increased bromodeoxyuridine (BrdU) incorporation by 40 and 28%, respectively. Addition of both resulted in a twofold increase in BrdU incorporation. Although 20-HETE synthesis in cultured cells is greatly diminished with time, significant picomolar concentrations can be obtained in 4-day cultures. Addition of 17-octadecynoic acid (17-ODYA), an inhibitor of 20-HETE synthesis, significantly inhibited EGF-stimulated BrdU incorporation. The demonstrations that EGF stimulates proximal tubular 20-HETE production and that the latter is a potent mitogen to these cells suggests that 20-HETE may act as a mediator of the EGF effect on cellular growth in the proximal tubule.

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