Isolation of a pure suspension of rat proximal tubules

1981 ◽  
Vol 241 (4) ◽  
pp. F403-F411 ◽  
Author(s):  
P. Vinay ◽  
A. Gougoux ◽  
G. Lemieux
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Periodic Acid ◽  
Vital Staining ◽  
Staining Technique ◽  
Proximal Tubules ◽  
Periodic Acid Schiff ◽  
Homogeneous Population ◽  
Proximal Tubular ◽  
Percoll Density Gradient ◽  
Enzyme Location

A suspension of cortical tissue fragments prepared by collagenase digestion of renal cortex obtained from fed and chronically acidotic (NH4Cl) rats was separated into four bands on a Percoll density gradient. By microscopic examination, vital staining with trypan blue, and histologic staining technique (periodic acid-Schiff) the F4 band was shown to contain only (greater than 98%) proximal tubules, whereas the F1 band was significantly enriched (70%) with distal tubules contaminated by glomeruli and short segments of proximal tubules. Intra/extracellular ratios for PAH of 15 were measured in the F4 band and of 2 in F1 band. ATP was 1.4 and 2.8 mumol/g in the F4 and F1 bands, respectively, and was stable for at least 60 min. The proximal F4 band was shown to be gluconeogenic (L-glutamine or L-lactate 2.5 mM as substrate) and to adapt to metabolic acidosis. The distal F1 band was shown to be glycolytic (glucose 2.5 mM) with no changes with acid-base status. All fractions were shown to metabolize glutamine, but the metabolic fate of this amino acid was different in proximal and distal structures. A F4/F1 activity ratio for the proximal cytoplasmic phosphoenolpyruvate carboxykinase enzyme of 2.6 and 4.3 was observed in normal and acidotic rats, respectively. In contrast, a F4/F1 ratio of 0.13 and 0.22 was observed for the distal cytoplasmic hexokinase enzyme. This preparation, therefore, allows the metabolism of a homogeneous population of proximal tubular fragments to be studied and can be used to obtain information on enzyme location within the nephron.

Carbohydrate metabolism by primary cultures of rabbit proximal tubules

1989 ◽  
Vol 256 (3) ◽  
pp. C532-C539 ◽  
Author(s):  
M. J. Tang ◽  
K. R. Suresh ◽  
R. L. Tannen
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Glycolytic Metabolism ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular

Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.

scholarly journals Epitheliocystis Infection in Cultured White Sturgeon (Acipenser transmontanus): Antigenic and Ultrastructural Similarities of the Causative Agent to the Chlamydiae

1996 ◽  
Vol 8 (2) ◽  
pp. 172-180 ◽  
Author(s):  
Joseph M. Groff ◽  
Scott E. LaPatra ◽  
Robert J. Munn ◽  
Mark L. Anderson ◽  
Bennie I. Osburn
Keyword(s):  
Periodic Acid ◽  
Host Cells ◽  
White Sturgeon ◽  
Acipenser Transmontanus ◽  
Periodic Acid Schiff ◽  
Homogeneous Population ◽  
Cytoplasmic Inclusions ◽  
Individual Host ◽  
Intensive Fish Culture ◽  
Chlamydial Antigen

A mild to moderate branchial epitheliocystis infection was diagnosed in subyearling (11 months old, 250–300 g) white sturgeon ( Acipenser transmontanus) from a private culture facility with a 4–8% mortality in the population. Infected branchial epithelial cells contained the coccoid to coccobacillary epitheliocystis organisms, which appeared as cytoplasmic inclusions composed of a fine, homogeneous, dense, basophilic, granular material. The infected cells were variably enlarged with spherical to oval profiles and were randomly distributed throughout the branchial epithelium. The cytoplasmic inclusions stained positive with Macchiavello stain but negative with Brown and Brenn, periodic acid-Schiff, and Gimenez stains. Expression of chlamydial antigen was demonstrated within the cytoplasmic inclusions using a standard peroxidase-antiperoxidase immunohistochemical technique. Three stages of coordinated intracellular development were recognized by electron microscopy. The reticulate bodies were oval to spherical and 0.4–0.8 × 0.5–1.4 μm but often exhibited a pleomorphic and convoluted appearance because of variable membrane invaginations and evaginations suggestive of uneven fission and budding. Separate host cells contained intermediate bodies that were spherical to oval and 0.2–0.4 × 0.3–0.6 μm although often observed in the process of apparent uneven division. The presence of a cap or plaque composed of hexagonally arrayed fibrillar surface projections was initially recognized in this stage. A homogeneous population of 0.3–0.4 μmoval elementary bodies were observed separately in individual host cells. This developmental stage had a single, dense, compact, eccentrically located cytoplasmic condensation that occurred opposite to the location of the cap of hexagonally arrayed fibrillar surface projections. Morphologic characteristics of the epitheliocystis organism in these white sturgeon were similar to those previously described in other teleosts and expands the species catalogue of epitheliocystis infection. Furthermore, the ultrastructural similarities to the chlamydiae and the immunohistochemical detection of chlamydial antigen provides further evidence that the epitheliocystis agent is related to members of the Chlamydiales. Although the infection was considered mild to moderate and could not be definitively attributed to the mortality in this population, the potential adverse impact of epitheliocystis infection on sturgeon culture should be considered especially in intensive fish culture operations.

Modified technique for periodic acid-Schiff staining of glycoproteins on agarose-film electrophoretograms.

1983 ◽  
Vol 29 (5) ◽  
pp. 836-839 ◽  
Author(s):  
S M Trivedi ◽  
C G Frondoza ◽  
R L Humphrey
Keyword(s):  
Analytical Procedure ◽  
Periodic Acid ◽  
Staining Technique ◽  
Plasma Cell Dyscrasia ◽  
Free Light Chains ◽  
Modified Technique ◽  
Periodic Acid Schiff ◽  
Background Staining ◽  
Ponceau S ◽  
Bence Jones Proteins

Abstract We have modified the periodic acid-Schiff staining technique for glycoproteins for use with thin agarose-film electrophoresis membranes. With this procedure, carbohydrate-rich proteins can be detected with negligible background staining and insignificant staining of nonglycoproteins such as albumin and nonglycosylated Bence Jones proteins (free light chains). On the other hand, carbohydrate-rich M components of immunoglobulins M and A in serum and in cerebrospinal fluid from patients with plasma cell dyscrasia are readily detected. The technique is two- to threefold more sensitive than Ponceau S. Glycoproteins in serum and body fluids can be determined as a routine analytical procedure.

scholarly journals Antioxidant Protection against Pathological Mycotoxins Alterations on Proximal Tubules in Rat Kidney

2011 ◽  
Vol 1 (4) ◽  
pp. 118 ◽  
Author(s):  
Awatef Ali ◽  
Susan Abdu
Keyword(s):  
Serum Creatinine ◽  
Ochratoxin A ◽  
Rat Kidney ◽  
Animal Health ◽  
Periodic Acid ◽  
Relative Weight ◽  
Proximal Tubules ◽  
Protective Effects ◽  
Periodic Acid Schiff ◽  
Ultra Structure

Background: Ochratoxin A (OTA) was one of the mycotoxins and received attention worldwide because of the hazard it posed to human and animal health, where the kidney was the primary target organ for OTA toxicity. In the other hand, dates served as a good source of natural antioxidants and could potentially be considered as a functional food.Methods: The study was performed in the department of biology in King Abdulaziz University. Animals were gavage administrated and divided into four groups: first group received (sodium bicarbonate), second group received (289 µg OTA /kg B.W. /day), third group received (1mg Ajwa/kg B.W. / day) and fourth group received (289 µg OTA /kg B.W./day+ 1mg Ajwa /kg B.W. / day). Serum (creatinine - urea) levels were measured in each group at the time of tissue collection, some biopsies were fixed in 10% buffered formalin solution for light microscopy processing stained with Haematoxylin and Eosin (H& E.), Periodic Acid-Schiff (PAS) and Masson´s Trichrome (M.T.).Other biopsies were immediately collected into electron microscopy processing. Results: After 28 days, a significant decrease in body weight, kidney weight and relative weight was detected in OTA treated group. Also, Serum (creatinine-urea) level were elevated. The normal cyto-architecture of proximal tubules were lost exhibiting damaged bruch border, degenerated, binucleated and karyomegalic cells. The most destructed ultra-structure was the mitochondria which severely swollen with disintegrated membranes. In Ajwa Date extract-group the proximal tubules were normal, whereas in Ajwa date extract + OTA -group the severity of the lesions was significantly reduced. Conclusion: The present results indicated that, Ajwa date have protective effects and ameliorated the lesions of Ochratoxin nepherotoxicity which might lead to kidney failure. Key words: Ochratoxin A., Ajwa date, proximal tubules, light –structure, ultra –structure, biochemical analysis, morphometry.

scholarly journals Cocktail of periodic Acid–Schiff and papanicolaou: Novel staining technique for the identification of leukemic eosinophils – A pilot study

2019 ◽  
Vol 23 (3) ◽  
pp. 476
Author(s):  
Lavanya Mallika ◽  
SV Sowmya ◽  
RoopaS Rao ◽  
Dominic Augustine ◽  
VanishriC Haragannavar ◽  
...  
Keyword(s):  
Pilot Study ◽  
Periodic Acid ◽  
Staining Technique ◽  
Periodic Acid Schiff

Renal functional decline and glomerulotubular injury are arrested but not restored by release of unilateral ureteral obstruction (UUO)

2013 ◽  
Vol 304 (4) ◽  
pp. F432-F439 ◽  
Author(s):  
Wassim Chaabane ◽  
Françoise Praddaude ◽  
Marie Buleon ◽  
Acil Jaafar ◽  
Marion Vallet ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Ureteral Obstruction ◽  
Periodic Acid ◽  
Unilateral Ureteral Obstruction ◽  
Sham Operation ◽  
Glomerular Injury ◽  
Periodic Acid Schiff ◽  
Glomerular Volume ◽  
Atubular Glomeruli ◽  
Proximal Tubular

Murine unilateral ureteral obstruction (UUO), a major model of progressive kidney disease, causes loss of proximal tubular mass and formation of atubular glomeruli. Adult C57BL/6 mice underwent a sham operation or reversible UUO under anesthesia. In group 1, kidneys were harvested after 7 days. In group 2, the obstruction was released after 7 days, and a physiological study of both kidneys was performed 30 days later. Renal blood flow (RBF), glomerular filtration rate (GFR), urine protein, and albumin excretion were measured after ligation of either the left or right ureter. Glomerular volume (periodic acid-Schiff), glomerulotubular integrity and proximal tubular mass ( Lotus tetragonolobus lectin), and interstitial collagen (Sirius red) were measured by histomorphometry. Obstructed kidney weight was reduced by 15% at 7 days but was not different from sham after a 30-day recovery. Glomerular volume and proximal tubular area of the obstructed kidney were reduced by 55% at 7 days, but normalized after 30 days. Interstitial collagen deposition increased 2.4-fold after 7 days of UUO and normalized after release. However, GFR and RBF were reduced by 40% and urine albumin/protein ratio was increased 2.8-fold 30 days after release of UUO. This was associated with a 50% reduction in glomerulotubular integrity despite a 30-day recovery ( P < 0.05 for all data). We conclude that release of 7-day UUO can arrest progression but does not restore normal function of the postobstructed kidney. Although the remaining intact nephrons have hypertrophied, glomerular injury is revealed by albuminuria. These results suggest that glomerulotubular injury should become the primary target of slowing progressive kidney disease.

scholarly journals Postnatal regulation of 15-hydroxyprostaglandin dehydrogenase in the rat kidney

2014 ◽  
Vol 307 (4) ◽  
pp. F388-F395 ◽  
Author(s):  
Ying Liu ◽  
Zhanjun Jia ◽  
Ying Sun ◽  
Li Zhou ◽  
Maicy Downton ◽  
...  
Keyword(s):  
Kidney Development ◽  
Rat Kidney ◽  
Periodic Acid ◽  
Proximal Tubules ◽  
Periodic Acid Schiff ◽  
Qrt Pcr ◽  
Pathological Conditions ◽  
Brush Borders ◽  
Cox 2 ◽  
Established Role

Cyclooxygenase 2 (COX-2) has an established role in postnatal kidney development. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is recently identified as an endogenous inhibitor of COX-2, limiting the production of COX-2-derived prostanoids in several pathological conditions. The present study was undertaken to examine the regulation of renal 15-PGDH expression during postnatal kidney development in rats compared with COX-2. qRT-PCR and immunoblotting demonstrated that 15-PGDH mRNA and protein in the kidney were present in neonates, peaked in the second postnatal week, and then declined sharply to very low level in adulthood. Immunostaining demonstrated that at the second postnatal week, renal 15-PGDH protein was predominantly found in the proximal tubules stained positive for Na/H exchanger 3 and brush borders (periodic acid-Schiff), whereas COX-2 protein was restricted to macular densa and adjacent thick ascending limbs. Interestingly, in the fourth postnatal week, 15-PGDH protein was redistributed to thick ascending limbs stained positive for the Na-K-2Cl cotransporter. After 6 wk of age, 15-PGDH protein was found in the granules in subsets of the proximal tubules. Overall, these results support a possibility that 15-PGDH may regulate postnatal kidney development through interaction with COX-2.

scholarly journals Metabolic substrates alter attachment and differentiated functions of proximal tubule cell culture.

1994 ◽  
Vol 4 (11) ◽  
pp. 1908-1911
Author(s):  
M J Tang ◽  
R L Tannen
Keyword(s):  
Proximal Tubule ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Culture Media ◽  
Proximal Tubules ◽  
Proximal Tubule Cell ◽  
Metabolic Substrates ◽  
Standard Culture ◽  
Leucine Amino Peptidase ◽  
Proximal Tubular

Proximal tubules cultured in vitro gradually lose their differentiated functions. Because standard culture media lacks several substrates important for renal proximal tubule oxidative metabolism, whether a mixture of substrates including butyrate, alanine, and lactate (BAL) would modify growth and/or differentiated function of proximal tubular cells in culture was examined. Tubules cultured in media supplemented with 2 mM butyrate, alanine, and lactate exhibited enhanced attachment but did not exhibit an altered growth rate. Higher levels of phosphoenolpyruvate carboxykinase and leucine-amino peptidase were sustained, although these activities were still diminished in comparison with that in fresh tubules. Sodium-dependent glucose uptake and dome formation--other reflections of epithelial cell differentiated function--also were enhanced. These studies demonstrate that the substrates used to culture proximal tubules can modify both their attachment and their manifestation of differentiated function in culture.

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