Isolation of a pure suspension of rat proximal tubules.
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.
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003102:Colloids; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005972:Glutaminase / Q000378:metabolism; D006593:Hexokinase / Q000378:metabolism; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000201:enzymology; D010732:Phosphofructokinase-1 / Q000378:metabolism; D011205:Povidone; D051381:Rats; D012822:Silicon Dioxide; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D003102:Colloids; C016039:Percoll; D012822:Silicon Dioxide; D005969:Glutamate Dehydrogenase; D005723:gamma-Glutamyltransferase; D006593:Hexokinase; D010732:Phosphofructokinase-1; D000469:Alkaline Phosphatase; D006597:Fructose-Bisphosphatase; D005972:Glutaminase; D011205:Povidone