Bulk isolation of renal PCT and PST. I. Glucose-dependent metabolic differences.
A new procedure for separately isolating milligram quantities of rabbit renal proximal straight (PST) or convoluted (PCT) tubules is described, and the differential abilities of these segments to utilize glucose as a metabolic substrate are investigated. Separate dissection of the cortical cortices and the outer medullary stripe, followed by collagenase digestion and discontinuous Percoll centrifugation, provide enriched populations (greater than 98% pure) of PCT (37 mg) and PST (14 mg), respectively, per rabbit. The purity of PCT and PST fractions was quantitated morphologically and by comparing the enriched activity of the proximal tubular marker leucine aminopeptidase and deenriched activity of the distal marker hexokinase to previously published values reported from microdissection studies. To investigate glucose-dependent metabolic differences, PCT and PST suspensions (1 mg/ml) were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being incubated for 30 min in buffer with or without glucose as the only available metabolic substrate. In glucose-containing buffer, PST segments maintained their oxygen consumption and ATP contents at levels significantly higher than PCT segments. These differential responses between PST and PCT were glucose-dependent because they were abolished when segments were incubated under glucose-free conditions. Because responses in PCT were glucose-independent, these results suggest that PCT cannot utilize glucose to support oxidative metabolism, whereas PST segments can oxidatively metabolize this substrate. These differences in glucose utilization do not correlate with the distribution of glycolytic enzyme activities, suggesting that differential metabolic regulation of these enzymes may determine the ability of each segment to utilize glucose.
Ruegg
C E
CE
Machu Picchu Research Foundation, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Mandel
L J
LJ
D000227:Adenine Nucleotides / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002469:Cell Separation; D005260:Female; D005947:Glucose / Q000378:metabolism / Q000502:physiology*; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D010101:Oxygen Consumption / Q000502:physiology; D011188:Potassium / Q000378:metabolism; D011817:Rabbits
D000227:Adenine Nucleotides; D000255:Adenosine Triphosphate; D005947:Glucose; D011188:Potassium