Improved renal cortical tubule suspension: spectrophotometric study of O2 delivery

Balaban, Robert S.; Soltoff, Stephen P.; Storey, Janet M.; Mandel, Lazaro J.

doi:10.1152/ajprenal.1980.238.1.f50

Cited by 73 publications

(83 citation statements)

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“…RNA-seq offers important advantages over other approaches to kidney transcriptomics, such as DNA microarrays using biochemically isolated segments 32,33 or SAGE of microdissected renal tubule segments. 2,3 Compared with biochemical isolation techniques, [34][35][36] manual dissection virtually eliminates contamination from neighboring segments. RNA-seq does not depend on hybridization and therefore, eliminates false positivity caused by cross-hybridization in microarray studies.…”

Section: Discussionmentioning

confidence: 99%

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Lee¹,

Chou²,

Knepper³

2015

Journal of the American Society of Nephrology

478

480

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The function of each renal tubule segment depends on the genes expressed therein. High-throughput methods used for global profiling of gene expression in unique cell types have shown low sensitivity and high false positivity, thereby limiting the usefulness of these methods in transcriptomic research. However, deep sequencing of RNA species (RNA-seq) achieves highly sensitive and quantitative transcriptomic profiling by sequencing RNAs in a massive, parallel manner. Here, we used RNA-seq coupled with classic renal tubule microdissection to comprehensively profile gene expression in each of 14 renal tubule segments from the proximal tubule through the inner medullary collecting duct of rat kidneys. Polyadenylated mRNAs were captured by oligo-dT primers and processed into adapter-ligated cDNA libraries that were sequenced using an Illumina platform. Transcriptomes were identified to a median depth of 8261 genes in microdissected renal tubule samples (105 replicates in total) and glomeruli (5 replicates). Manual microdissection allowed a high degree of sample purity, which was evidenced by the observed distributions of well established cell-specific markers. The main product of this work is an extensive database of gene expression along the nephron provided as a publicly accessible webpage (https://helixweb.nih.gov/ESBL/Database/NephronRNAseq/index.html). The data also provide genomewide maps of alternative exon usage and polyadenylation sites in the kidney. We illustrate the use of the data by profiling transcription factor expression along the renal tubule and mapping metabolic pathways.

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Section: Discussionmentioning

confidence: 99%

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Lee¹,

Chou²,

Knepper³

2015

Journal of the American Society of Nephrology

478

480

View full text Add to dashboard Cite

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“…Renal cortical tubules were prepared from female New Zealand White rabbits (Bunny Haven, Durham, NC; 3-4 kg wt) according to previously published methods (20,21). In brief, after the animal was injected with heparin and anesthetized with ether, both kidneys were perfused at 37°C with a medium containing NaCl (I 15 mM), NaHCO3 (25 mM), NaH2PO4 (2 mM), CaCl2 (1 mM), KCI (5 mM), MgSO4 (I mM), glucose (5 mM), lactate (4 mM), alanine (I mM), 0.6% dextran, and mannitol (25 mM) equilibrated with 95% 02/5% CO2 (pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

Mechanisms whereby exogenous adenine nucleotides improve rabbit renal proximal function during and after anoxia.

Mandel

Takano²,

Soltoff³

et al. 1988

J. Clin. Invest.

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When a suspension of rabbit proximal tubules is subjected to anoxia, ATP falls by 80-90% during 40 min of anoxia, and upon reoxygenation (reox) the cells only recover 25-50% of their initial ATP. Addition of Mg-ATP (magnesium chloridetreated ATP), Mg-ADP, or Mg-AMP (five aliquots of 200 nmol/ml added 10 min apart) during anoxia causes complete recovery of ATP levels, and respiratory and transport function after 40 min of reox. Similar additions of adenosine (ADO), or inosine (INO), or Mg-ATP only during reox are less effective. Lactate dehydrogenase (LDH) release after 40 min of anoxia is 30-40% under control conditions, only 10-15% when adenine nucleotides or ADO are added during anoxia, and 20% when INO is added, suggesting that these additions may stabilize the plasma membrane during anoxia and help preserve cellular integrity. During reox, recovery may depend on the entry of ATP precursors and, therefore, we explored the mechanism whereby exogenous ATP increases the intracellular ATP content. Additions of Mg-ATP, Mg-ADP, or Mg-AMP to continuously oxygenated tubules increase cellular ATP content three-to fourfold in 1 h. The added ATP and ADP are rapidly degraded to AMP, and more slowly to ADO, INO, and hypoxanthine. Furthermore, the ATP-induced increase in cellular ATP is abolished by the exogenous addition of adenosine deaminase, which converts extracellular ADO to INO. These results suggest that the increase in cellular ATP requires extracellular ADO. The ADO obtained from the breakdown of AMP may be preferentially transported into the renal cells to be resynthesized into cellular AMP and ATP.

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“…sample) versus a line drawn between two isosbestic reference points (i.e. reference) (26). These wavelength combinations were as follows: cytochrome c (cyto-c) sample:550 nm and cytochrome b (cyto-b) sample:563 nm, both referenced between 535 nm and 575 nm; cytochrome a (cyto-a):sample 605 nm, referenced between 575 nm and 630 nm.…”

Section: Measurements Of Mitochondrial Oxygen Consumption (Mvmentioning

confidence: 99%

Metabolic Network Control of Oxidative Phosphorylation

Bose

French

Evans

et al. 2003

Journal of Biological Chemistry

196

251

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Phosphate (P i ) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that P i controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F 1 F 0 -ATPase in heart and skeletal muscle mitochondria. In these studies the effect of P i was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. P i enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. P i oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of P i was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [P i ]. These results demonstrate that P i signaling results in the balanced modulation of oxidative phosphorylation, by influencing both ⌬G H ؉ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems. Phosphate (P i )1 is the substrate for the phosphorylation of ADP to ATP in oxidative phosphorylation. Because ADP and P i are generated by ATPases in the cytosol, the potential roles of ADP and P i as cytosolic feedback signaling molecules regulating the rate of ATP production was one of the first models of the cytosolic regulation of mitochondrial ATP production (1, 2).However, over the years it has become apparent that the cellular regulation of oxidative phosphorylation is a very complex control network, with numerous potential rate-limiting steps affected by a variety of signaling molecules, including ADP, P i , Ca 2ϩ , creatine, and Mg 2ϩ (3-7). This network results in the ability of tissues to change significantly the rate of ATP generation without significantly modifying the metabolic intermediates coupled to many other processes in the cell. This has been termed an energy metabolism homeostasis (8). Toward a better understanding of this regulatory network, the effects of each putative signaling molecule on oxidative phosphorylation need to be characterized. The purpose of the current work was to further evaluate the effects of P i on different regulatory sites of oxidative phosphorylation in cardiac mitochondria.Phosphate is believed to enter cardiac mitochondria via a neutral phosphate transporter (P t ) in exchange for OH Ϫ or by co-transport with H ϩ (9). Thus, P i transport is linked to the mitochondrial inner membrane pH gradient (⌬pH m ) and the phosphate concentration gradient but not to the membrane potential (⌬⌿). Although P i transport has not been ascribed as a rate-limiting step for phosphate utilization in oxidative phosphorylation, this particular aspect of phosphate metabolism has not been extensively studied, espec...

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Improved renal cortical tubule suspension: spectrophotometric study of O2 delivery

Cited by 73 publications

References 0 publications

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment–Specific Transcriptomes

Mechanisms whereby exogenous adenine nucleotides improve rabbit renal proximal function during and after anoxia.

Metabolic Network Control of Oxidative Phosphorylation

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