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Results for “Kidney Tubules, Proximal”

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At least 19 recordsLinked to original sources

PubMed1990

Structural requirements for protection by small amino acids against hypoxic injury in kidney proximal tubules.

Kidney proximal tubules are resistant to hypoxic injury if glycine or L-alanine is present in their incubation medium. Protection does not depend on the concentration or turnover of ATP in cells. We have investigated structure-function relationships that govern this protective activity. Among more than 45 amino acids and analogs examined, only glycine, L-alanine, D-alanine, beta-alanine, and the neuronal glycine binding site agonist, 1-aminocyclopropane-1-carboxylic acid, were active. The protective effect could not be explained by amino acid metabolism. Ultrastructural features in protected cells were preserved to a degree which suggested that processes responsible for degradation during hypoxia were retarded. These results are consistent with stringent requirements of amino acid molecular structure for protection against hypoxia, and suggest the involvement of highly specific, acceptor-ligand effects on a process critical for maintaining cellular integrity.

Adenosine Triphosphate↗

PubMed1980

Testosterone-mediated sexual dimorphism of mitochondria and lysosomes in mouse kidney proximal tubules.

In kidney proximal tubules of male mice the mitochondria are larger and more electron-lucent, autophagic vacuoles and lysosomes (predominantly myeloid bodies) more numerous and voluminous, and exocytosed intraluminal myeloid bodies more common than in females. Males also have higher kidney activities of mitochondrial cytochrome c oxidase and lysosomal hydrolases, and excrete larger quantities of hydrolases and protein in the urine. Orchiectomy evokes the feminine pattern whereas testosterone administration induces the male pattern. Endogenous testosterone modulates mitochondrial structure and function and enhances the activity of the lysosomal-vacuolar system in proximal tubule cells.

Animals↗

PubMedDate not supplied

Characterization of rPEPT2-mediated Gly-Sar transport parameters in the rat kidney proximal tubule cell line SKPT-0193 cl.2 cultured in basic growth media.

The rat proximal kidney tubule cell line SKPT-0193 cl.2 (SKPT) expresses the di-/tripeptide transporter PEPT2 (rPEPT2) and has been used to study PEPT2-mediated transport. Traditionally, SKPT cells have been cultured in growth media supplemented with epidermal growth factor (EGF), apotransferrin, dexamethasone, and insulin. It was recently demonstrated that omission of EGF from the culture media caused a drastic increase in the expression of rPEPT2. The hypothesis was therefore that the SKPT cell line might be able to differentiate and express rPEPT2 in the absence of the four agonists traditionally added. The aim of the study was thus to characterize Gly-Sar transport parameters in SKPT cells cultured in basic growth media (conventional media without added agonists). Morphology was studied using confocal laser scanning microscopy (CLSM) and immunohistochemistry. Monolayer integrity was evaluated using transepithelial electrical resistance (TEER) measurements and [(3)H]-mannitol permeabilities. Di-/tripeptide transporter activity was studied using [(14)C]-glycylsarcosine ([(14)C]-Gly-Sar). SKPT cells grown in basic media for 4 days formed confluent monolayers with a TEER of 5.03 +/- 0.33 kOmega.cm(2) (n = 5). Apical Gly-Sar uptake peaked after 3-6 days in culture. Uptake at day 4 was 5.89 +/- 0.30 pmol.cm(-2).min(-1) (n = 3). Di-/tripeptide uptake displayed an optimum at approximately pH 6. Affinity values for cephalexin, kyotorphin, and delta-aminolevulinic acid were comparable to those obtained in other PEPT2-expressing model systems. It can be concluded that SKPT cells grown in the absence of the agonists traditionally added to the culture media retain all necessary properties for PEPT2-mediated peptide uptake studies. Furthermore, the absence of the agonists might facilitate studies of hormonal regulation of PEPT2 expression and transport activity.

Aminolevulinic Acid↗

PubMed1997

Symmetric pH dependence of buffering power in giant fused cells from frog kidney proximal tubule.

This study measures the intrinsic buffering power (beta(i)) of giant fused cells from the proximal kidney tubule of the frog (Rana ridibunda) as a function of intracellular pH (pHi). We monitored pHi and transmembrane potential difference during acid or alkaline cell loading, achieved by removal of NH4Cl-containing solutions or CO2-HCO3(-)-equilibrated solutions, respectively, in the absence of extracellular Na+. Data were well fit by the equation for a single, monoprotic buffer with a maximum beta(i) at a pHi of 7.39 +/- 0.06 and a total buffer concentration of 30.7 +/- 1.6 mM (means +/- SD). From pHi measurements obtained during CO2-HCO3- exposure, we also calculated the buffering power afforded by the CO2-HCO3- pair, and we show its increasing contribution to total buffering power at increasing PCO2 and pHi. To our knowledge, this is the first report of a cell type in which intrinsic cell buffers can be adequately approximated as a single monoprotic buffer with a negative logarithm of apparent dissociation constant in the normal physiological range and essentially symmetric dependence on pHi in both acid and alkaline ranges.

Ammonium Chloride↗

PubMed1993

Apical expression of an antigen common to rabbit yolk sac endoderm and kidney proximal tubule epithelium.

The tissue distribution, molecular weight, and biochemical nature of an antigen detected by a mouse monoclonal antibody designated 283D3 and raised against rabbit visceral yolk sac endodermal cells, has been investigated. The antigen is located on the luminal side of apical tubules and large sub-apical vesicles in rabbit yolk sac endoderm and proximal kidney tubule epithelial cells. It is expressed in a similar polarised fashion in epithelial cells lining the epididymis. Western blotting showed the antigen to comprise proteins of molecular weight 330-380 kDa. The antigen has been affinity purified from yolk sac and kidney and is predominantly protein in nature with a small percentage of N-linked carbohydrate. In terms of tissue distribution and molecular weight it has close similarity to Heymann nephritis antigen but differs in not being confined to coated pits. Its function is not known, but the association with endocytic elements implies a possible role in non-specific protein absorption.

Animals↗

PubMed1997

Receptor-mediated endocytosis in kidney proximal tubules: recent advances and hypothesis.

Preparation of kidney proximal tubules in suspension allows the study of receptor-mediated endocytosis, protein reabsorption, and traffic of endosomal vesicles. The study of tubular protein transport in vitro coupled with that of the function of endosomal preparation offers a unique opportunity to investigate a receptor-mediated endocytosis pathway under physiological and pathological conditions. We assume that receptor-mediated endocytosis of albumin in kidney proximal tubules in situ and in vitro can be regulated, on the one hand, by the components of the acidification machinery (V-type H+-ATPase, Cl(-)-channel and Na+/H+-exchanger), giving rise to formation and dissipation of a proton gradient in endosomal vesicles, and, on the other hand, by small GTPases of the ADP-ribosylation factor (Arf)-family. In this paper we thus analyze the recent advances of the studies of cellular and molecular mechanisms underlying the identification, localization, and function of the acidification machinery (V-type H+-ATPase, Cl(-)-channel) as well as Arf-family small GTPases and phospholipase D in the endocytotic pathway of kidney proximal tubules. Also, we explore the possible functional interaction between the acidification machinery and Arf-family small GTPases. Finally, we propose the hypothesis of the regulation of translocation of Arf-family small GTPases by an endosomal acidification process and its role during receptor-mediated endocytosis in kidney proximal tubules. The results of this study will not only enhance our understanding of the receptor-mediated endocytosis pathway in kidney proximal tubules under physiological conditions but will also have important implications with respect to the functional consequences under some pathological circumstances. Furthermore, it may suggest novel targets and approaches in the prevention and treatment of various diseases (cystic fibrosis, Dent's disease, diabetes and autosomal dominant polycystic kidney disease).

ADP-Ribosylation Factors↗

PubMed2005

Accumulation of nonesterified fatty acids causes the sustained energetic deficit in kidney proximal tubules after hypoxia-reoxygenation.

Kidney proximal tubules exhibit decreased ATP and reduced, but not absent, mitochondrial membrane potential (Deltapsi(m)) during reoxygenation after severe hypoxia. This energetic deficit, which plays a pivotal role in overall cellular recovery, cannot be explained by loss of mitochondrial membrane integrity, decreased electron transport, or compromised F1F0-ATPase and adenine nucleotide translocase activities. Addition of oleate to permeabilized tubules produced concentration-dependent decreases of Deltapsi(m) measured by safranin O uptake (threshold for oleate = 0.25 microM, 1.6 nmol/mg protein; maximal effect = 4 microM, 26 nmol/mg) that were reversed by delipidated BSA (dBSA). Cell nonesterified fatty acid (NEFA) levels increased from <1 to 17.4 nmol/mg protein during 60- min hypoxia and remained elevated at 7.6 nmol/mg after 60 min reoxygenation, at which time ATP had recovered to only 10% of control values. Safranin O uptake in reoxygenated tubules, which was decreased 85% after 60-min hypoxia, was normalized by dBSA, which improved ATP synthesis as well. dBSA also almost completely normalized Deltapsi(m) when the duration of hypoxia was increased to 120 min. In intact tubules, the protective substrate combination of alpha-ketoglutarate + malate (alpha-KG/MAL) increased ATP three- to fourfold, limited NEFA accumulation during hypoxia by 50%, and lowered NEFA during reoxygenation. Notably, dBSA also improved ATP recovery when added to intact tubules during reoxygenation and was additive to the effect of alpha-KG/MAL. We conclude that NEFA overload is the primary cause of energetic failure of reoxygenated proximal tubules and lowering NEFA substantially contributes to the benefit from supplementation with alpha-KG/MAL.

Adenosine Triphosphate↗

PubMed1983

Does water drag solutes through kidney proximal tubule?

Coupling of salt and water movements across kidney proximal tubules was studied in the presence of an induced transepithelial osmotic water flux. Convoluted proximal tubules from rabbit kidney were perfused in vitro with a control solution, with or without 50 mM/l of mannitol or raffinose in the both. Osmolalities of the perfused and collected fluids as well as the net water flux Jv were measured in each experiment. The net solute flux Js was calculated from the difference between the amount of total solutes delivered and collected at each end of the tubule. No apparent net transepithelial solute movements were detected in the presence of an osmotic water flux when active solute transport was inhibited either by an external to of 26 degrees C or by ouabain in the bath. The water flux observed was similar to that calculated assuming that only water crossed the epithelium, and no streaming potential was measured, whether or not active transport was blocked. It is concluded that the osmotic water flux through kidney proximal tubule does not drag a significant amount of solutes, probably because of the absence of convective solvent flux. This suggests the existence of different pathways for water and salt movement.

Animals↗

PubMed2001

Utilization of amino acids in growing kidney proximal tubule cell cultures.

The growth of rat kidney proximal tubule cells was monitored continuously by the cellular incorporation of [methyl-(14)C] thymidine using scintillating microplates. The radioisotope had no effect on cell proliferation over a 5 day period, neither was it extensively converted to thymine. Leibovitz L-15 medium supplemented with bicarbonate proved a good growth medium and its high levels of carbohydrates and amino acids facilitated the appearance of intermediates in the cells' metabolism of additional radioactive amino acids. Kidney proximal tubule cells had a greater potential to process amino acids than BHK-21 cells. The utilization of amino acids by proximal tubule cells differed from that of other organs. The amino acids could be classified into three classes. Members of the first type were only used for protein synthesis (arginine, lysine, histidine and tyrosine). The second class of amino acids yielded only one or two metabolites (leucine and isoleucine), while the last type gave more than two metabolites (alanine, aspartate, glycine, methionine, proline and valine).

Amino Acids↗

PubMed1978

A network thermodynamic model of salt and water flow across the kidney proximal tubule.

This network thermodynamic model of kidney proximal tubule epithelium treats coupled salt and water flow across each component membrane of the epithelium. We investigate the effects of various relative internal parameter values on the concentration of transepithelial flow, the concentrations in the cell and interspace, and the distribution of flows between cellular and paracellular routes. Best fit is obtaine if the apical and basolateral membrane reflection coefficients (or) are equal. The measured transepithelial filtration coefficient, Lp, is a function not only of the component Lps but also of the internal concentrations, or's, and permeabilities. For the given system topology (i.e., connectedness), parameters of component membranes must be within a narrow range to be consistent with experimental results. The dependence of the concentration of transported fluid on the balance between the solute pump rate and the transepithelial volume flow driving force is shown. This has implications for the effects of peritubular or lumen oncotic pressure on salt and water flow. With Appendix B of this paper and a user's guide for a circuit-simulation package (e.g., SPICE or PCAP) the reader can perform similar network analyses of transport models himself.

Biological Transport↗

PubMed1999

Expression of the MRP2 gene-encoded conjugate export pump in human kidney proximal tubules and in renal cell carcinoma.

Human kidney proximal tubule epithelia express the ATP-dependent export pump for anionic conjugates encoded by the MRP2 (cMRP/cMOAT) gene (symbol ABCC2). MRP2, the apical isoform of the multidrug resistance protein, is an integral membrane glycoprotein with a molecular mass of approximately 190 kD that was originally cloned from liver and localized to the canalicular (apical) membrane domain of hepatocytes. In this study, MRP2 was detected in human kidney cortex by reverse transcription-PCR followed by sequencing of a 826-bp cDNA fragment and by immunoblotting using two different antibodies. Human MRP2 was localized to the apical brush-border membrane domain of proximal tubules by double and triple immunofluorescence microscopy including laser scanning microscopy. The expression of MRP2 in renal cell carcinoma was studied by reverse transcription-PCR and immunoblotting in samples from patients undergoing tumor-nephrectomy without prior chemotherapy. Clear-cell carcinomas, originating from the proximal tubule epithelium, expressed MRP2 in 95% (18 of 19) of cases. Immunofluorescence microscopy of MRP2 in clear-cell carcinoma showed a lack of a distinct apical-to-basolateral tumor cell polarity and an additional localization of MRP2 on intracellular membranes. MRP2, the first cloned ATP-dependent export pump for anionic conjugates detected in human kidney, may be involved in renal excretion of various anionic endogenous substances, xenobiotics, and cytotoxic drugs. This conjugate-transporting ATPase encoded by the MRP2 gene has a similar substrate specificity as the multidrug resistance protein MRP1, and may contribute to the multidrug resistance of renal clear-cell carcinomas.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1993

A protein with anion exchange properties found in the kidney proximal tubule.

One important mechanism for reabsorption of chloride in the kidney proximal tubule involves anion exchange of chloride for a base. Anion exchange transport systems in general demonstrate sensitivity to inhibition by disulfonic stilbenes, probenecid, furosemide, and the arginyl amino group modifier phenylglyoxal. Using disulfonic stilbene affinity chromatography, we have identified and partially purified a protein with anion exchanger properties in luminal membrane vesicles isolated from rabbit kidney cortex. This protein has a molecular weight of 162 kD. The binding of the 162 kD protein to the stilbene affinity matrix is inhibited by disulfonic stilbenes, probenecid, furosemide, and phenylglyoxal. Reconstitution of the proteins eluted from the affinity matrix into liposomes demonstrates anion exchange activity as assayed by radiolabeled chloride influx. Deletion of the 162 kD protein from the eluted mixture by probenecid diminishes the anion exchanger activity in the reconstituted liposomes. Further purification of the disulfonic stilbene column eluant by Econo-Pac Q ion exchange chromatography resulted in significant enrichment in 162 kD protein abundance and also anion exchange activity in reconstituted liposomes. The results of the above experiments strongly suggest that the 162 kD protein is an anion exchanger. Insight into the functional and molecular characteristics of this protein should provide important information about the mechanism(s) of chloride reabsorption in the kidney proximal tubule.

Animals↗

PubMed1997

Membrane stress causes inhibition of water channels in brush border membrane vesicles from kidney proximal tubule.

Brush border membrane vesicles (BBMV) from rabbit kidney proximal tubule cells, prepared with different internal solute concentrations (cellobiose buffer 13, 18 or 85 mosM) developed an hydrostatic pressure difference across the membrane of 18.7 mosM, that causes a membrane tension close to 5 x 10(-5) N cm-1. When subjected to several hypertonic osmotic shocks an initial delay of osmotic shrinkage (a lag time), corresponding to a very small change in initial volume was apparent. This initial osmotic response, which is significantly retarded, was correlated with the initial period of elevated membrane tension, suggesting that the water permeability coefficient is inhibited by membrane stress. We speculate that this inhibition may serve to regulate cell volume in the proximal tubule.

Animals↗

PubMed2005

Primary kidney proximal tubule cells.

Primary rabbit kidney epithelial cell cultures can be obtained that express renal proximal tubule functions. Toward these ends, renal proximal tubules are purified from the rabbit kidney by the method of Brendel and Meezan. To summarize, each kidney is perfused with iron oxide, which becomes associated with glomeruli. The renal cortex is sliced and homogenized to liberate nephron segments. Renal proximal tubules and glomeruli are purified by sieving. The glomeruli, covered with iron oxide, are removed using a magnet. After a brief collagenase treatment (to disrupt basement membrane), the tubules are plated in hormonally defined serum-free medium supplemented with 5 micro g/mL bovine insulin, 5 microg/mL human transferrin, and 5 x 10(-8) M hydrocortisone. After 5-6 d of incubation, confluent monolayers are obtained that possess multicellular domes, indicative of their capacity for transepithelial solute transport.

Animals↗

PubMed1994

Nephrotoxicity of CdCl2 and Cd-metallothionein in cultured rat kidney proximal tubules and LLC-PK1 cells.

Nephrotoxicity is the major adverse effect produced by chronic exposure to cadmium (Cd). This injury is thought to be caused by the Cd-metallothionein complex (CdMT). In intact animals, CdMT is more efficiently taken up by the proximal tubules than CdCl2 and results in more renal damage. However, the mechanism(s) by which CdMT produces renal injury is not yet understood completely. Therefore, we used cultured renal proximal tubular cells to study the nephrotoxicity of CdMT and CdCl2. Rat kidney proximal tubules were isolated by collagenase perfusion, followed by percoll isopycnic centrifugation. 14C-alpha-methylglucose uptake and lactate dehydrogenase leakage were used as indices of nephrotoxicity. Surprisingly, CdMT was less toxic than CdCl2 to the cultured rat proximal tubule cells, as well as to cultured LLC-PK1 cells (a pig kidney proximal tubular cell line). Consistent with these observations on nephrotoxicity, 109CdMT uptake into these cultured renal cells was much less than that of 109CdCl2. Transwell cultures of LLC-PK1 cells were also used to examine the toxicity and uptake of CdCl2 and CdMT following basolateral and apical exposure. Uptake of both CdCl2 and CdMT from basolateral exposure was higher than that from apical exposure. Again, more 109CdCl2 was taken up and more cytotoxicity was observed in the CdCl2- than CdMT-exposed cells. In summary, CdCl2 is more toxic than CdMT to cultured rat kidney proximal tubules as well as LLC-PK1 cells. This is in contradiction to the greater in vivo nephrotoxic effects of CdMT than CdCl2. Therefore, cultured renal cells do not appear to be an appropriate model to study the nephrotoxicity of CdMT; transport of CdMT into proximal tubular cells in vivo does not appear to be maintained in vitro.

Analysis of Variance↗

PubMed2001

[Ultrastructural characteristics of reactive changes in the epithelium of the kidney proximal tubules in massive proteinuria in children].

The investigation was designed to analyse the mechanism of the protein transport on the basis of the estimation of ultrastructural organization of the epithelium of proximal convoluted tubules of kidneys of the children, suffering from the nephritic form of glomerulonephritis for a long time. It was established that high tubular proteinuria (from 0.33 to 6.6 g/l) causes many changes in apical, central and basal parts of epithelial cells as well as in intercellular spaces. It was stated that massive proteinuria provoked adaptive processes in epithelial cells of proximal tubules. At the same time signs of exhaustion of adaptive capacities of proximal nephrocytes were demonstrated.

Adolescent↗

PubMed1979

Early cellular effects of circulating cadmium-thionein on kidney proximal tubules.

Circulating cadmium-thionein (Cd-MT) is cleared from the mammalian circulatory system by filtration through the kidney glomerulus with subsequent reabsorption by kidney proximal tubules. Damage to the tubules results following uptake of Cd-MT, which is dependent upon time and the dose level of cadmium administered. Intravenous administration of 109Cd-MT at doses of 0.017 and 0.17 mg Cd/kg body weight with examination of total renal uptake of 109Cd at 0.5, 3, and 24 hr disclosed that the rate of clearance from the blood and uptake by the kidney was significantly more rapid at the 0.017 mg Cd/kg dose. Ultrastructural changes resulting from intravenous injection of either form A or B of Cd-MT were characterized by increased numbers of pinocytotic vesicles and small, dense lysosomal structures. There was no evidence of mitochondrial swelling or cell death at either 3 or 6 hr after injection. The subcellular distribution of cadmium in kidney tissue at various times after administration of Cd-MT was determined by using differential centrifugation techniques with 109Cd and in situ by using x-ray microanalysis. At 30 min after injection of Cd-MT, significant amounts of cadmium were present in lysosomal fractions indicating an interaction between the tubular lysosome system and Cd-MT prior to the onset of overt cellular toxicity. Results suggest that Cd-MT is reabsorbed and broken down by kidney tubule cells in a physiological manner with possible subsequent release of the toxic cadmium ion.

Animals↗

PubMed1996

The basolateral organic cation transport system of rabbit kidney proximal tubules. Influence of anorganic anions.

The influence of different extracellular chloride concentrations of chloride channel blockers and of inorganic anions on renal basolateral organic cation transport was examined on isolated nonperfused S2 segments of superficial proximal tubules of rabbit kidney. Tritium-labeled tetraethylammonium (TEA) with a high specific activity of 13.5 Ci/mmol was used as a model substrate of the organic cation transport system. With the experimental conditions chosen, the TEA accumulation in the cells reflects the TEA transport across the basolateral membrane. A maximum TEA cell to a bath ratio of 711 was observed at a TEA bath concentration of 1.4 x 10(-7) M. Increasing TEA bath concentrations lead to a nonlinear reduction of the TEA cell to the bath ratio. Superficial proximal S2 segments showed a significantly higher accumulation of TEA as compared to the S1 or S3 segments. No significant differences could be found between superficial and juxtamedullary S2 segments. Chloride ions significantly modulated the basolateral cation transport system in the S2 segments. In a chloride-free medium, virtually no TEA transport could be detected. Furthermore, iso-osmotic replacement of chloride in the incubation medium by iodide, sulfate or nitrate significantly reduced tubular TEA accumulation, whereas bromide was without effect. Chloride channel blockers had no effect on TEA transport, and TEA did not significantly influence 36chloride uptake of the S2 segments. Our study demonstrates extensive concentrative TEA accumulation in S2 proximal segments at the low TEA bath concentrations. Extracellular chloride may modulate the tubular TEA uptake, probably indirectly via the basolateral chloride/bicarbonate exchanger. A chloride/TEA cotransport seems to be excluded.

Animals↗