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  • 1
    Digitale Medien
    Digitale Medien
    Springer
    Pflügers Archiv 407 (1986), S. 228-234 
    ISSN: 1432-2013
    Schlagwort(e): Adenylate cyclase ; cAMP accumulation ; Oxygen consumption
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract This paper describes a rapid and simple method for isolation of medullary thick ascending limbs (MTAL) from rat kidney. The technique takes advantage of the fact that MTAL represents a high fraction of the inner stripe (IS) tissue in the outer medulla, and that this nephron segment is more resistant than others to mechanical and enzymatic disruption. Special attention was given in the design of each step of the isolation procedure in order to improve purity and yield of the preparation. Major steps are the following: 1) careful dissection of the IS; 2) cutting IS tissue into small pieces of regular size (≈ 1 mm3); 3) mild and brief enzymatic hydrolysis in a 65 U/ml collagenase solution; 4) separation of long MTAL segments from other tubule fragments and cells, and washing of the collagenase solution, on a nylon sieve (100 μm opening). This technique does not require lengthy centrifugations and provides about 6 mg fresh tisue (=1 mg protein) from two rat kidneys in 2 h. Light microscopy and transmission electron microscopy show a good purity (at least 95%) and good preservation of TAL ultrastructural morphology. Adenylate cyclase responsiveness to arginine-vasopressin (AVP), glucagon (GLU) and salmon calcitonin (SCT) of the MTAL suspension is similar to that reported for single microdissected rat MTAL. Viability of the MTALs was demonstrated by the ability to accumulate cyclic AMP in presence of AVP, GLU, SCT and forskolin. Normal oxygen consumption was 45.1±2.4 (SEM) μl·mg protein−1·h−1 (n=8). It was not enhanced with addition of succinate, indicating that integrity of cellular membrane is preserved. It was decreased by half in presence of either furosemide or ouabain. This indicates that electrolyte transport is maintained in the MTAL tubule suspension. This technique represents an easy and quick way in obtaining milligrams of medullary thick ascending limb from small laboratory animals, with a high purity and viability, and maintenance of cell polarity.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    ISSN: 1432-2013
    Schlagwort(e): Antidiuretic hormone ; Thick ascending limb ; Renal medulla ; Morphometry ; Water intake ; Urinary concentrating mechanism ; Kidney adaptation ; Kidney hypertrophy
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Medizin
    Notizen: Abstract Previous experiments in Brattleboro rats with hereditary diabetes insipidus revealed that absence of ADH led to several alterations in kidney anatomy, which could be reversed by chronic ADH treatment. Present experiments were undertaken to determine if similar alterations were observable in normal Wistar rats when endogenous ADH level was varied by manipulating water intake or when exogenous ADH was infused. Water intake was increased by giving food with a high water content ad libitum and offering 5% glucose solution to drink (HWI rats), or decreased by reducing water intake to 1/3 of spontaneous intake (RWI rats). An additional group received chronic ADH infusion with Alzet osmotic minipumps (ADH rats). Results were compared to those obtained in control rats (CON) drinking ad libitum. RWI, CON, and ADH rats ate dry pellets ad libitum. After 6 weeks on these regimens kidneys were perfusion fixed and serial sections were cut for morphometric measurements by light microscopy. Results in the four groups showed that kidney weight relative to body weight was influenced by the operation of urinary concentrating mechanism, with HWI〈CON〈RWI〈ADH. The increase in kidney weight in rats with high urine concentration was not homogeneously distributed throughout the different kidney zones and the different nephron segments. The inner stripe of the outer medulla (IS) increased more in relative height and volume than other kidney zones and, within this zone, the volume of epithelium of thick ascending limb of Henle's loops (TAL) increased more than expected from the whole kidney weight increase. In outer stripe of outer medulla (OS) and in cortex (C), TAL hypertrophy was equal to or lower than expected from whole kidney weight increase. Collecting duct epithelium in C, OS, and IS increased in proportion to whole kidney weight. The MTAL hyperthrophy in IS was due to an increase in size of preexisting cells, except in the ADH group where an increase in cell number was also observed. Internephron heterogeneity with regard to glomerular size was greater in RWI and ADH than in CON and HWI rats. The marked hypertrophy of the deep TAL in the IS of rats in which urine concentration was stimulated could be related to an increase in salt transport in this nephron segment, triggered both by a direct stimulation by ADH, and by an increased salt recycling. The elongation of the inner stripe provides a greater length for the operation of the countercurrent multiplier system responsible for building up of the osmotic pressure gradient in the medulla. These observations show that the mammalian kidney is able to adapt to chronic alterations in water intake and urine concentration status.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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