Characterization and metabolism of canine proximal tubules, thick ascending limbs, and collecting ducts in suspension

Abstract

Preparations of distinct nephron segments were obtained from dog kidneys by collagenase treatment. Four morphologically different tissues were isolated: glomeruli, proximal tubules, thick ascending limbs, and papillary collecting ducts. Each segment possessed a characteristic assay of membrane-bound and cytoplasmic enzymes. Specific metabolic characteristics also were found: gluconeogenesis and ammoniagenesis in proximal tubules, glycolytic aerobic metabolism in thick ascending limbs, and glycolytic anaerobic metabolism in papillary collecting ducts. The assay of Na+–K+ ATPase, H+-ATPase, and Ca2+-ATPase activities in these nephron segments demonstrated a specific enrichment of Na+–K+ ATPase in thick ascending limbs, and of H+-ATPase in proximal tubules and papillary collecting ducts. Tubular respiration in the absence or presence of ouabain, 1,3-dicyclohexylcarbodiimide, or furosemide demonstrated that the respiration of each segment could be correlated to the activity of specific ion motive ATPases. Furthermore, a tight coupling between ion transport, ATP turnover, and substrate oxidation was demonstrated. These isolated tubular structures are thus viable and capable of transepithelial transport. Our preparation provides large amounts of defined population of tubules and are thus useful for the study of biochemical and functional heterogeneity along the nephron.

l-Arginine metabolism in dog kidney and isolated nephron segments

“…Slices were washed 3 times with ice-cold KHS to remove cell debris. Cortical tubules (> 85% PTs), MTALs, and papillary collecting ducts (PCDs) were prepared from relevant tissue by collagenase digestion as previously described [40]. After the digestion, the final suspension adjusted to 60 mg wet weight per milliliter was kept at 4°C in standard KHS fully gassed with 5% CO 2 + 95% O 2 until utilization [40].…”

“…Cortical tubules (> 85% PTs), MTALs, and papillary collecting ducts (PCDs) were prepared from relevant tissue by collagenase digestion as previously described [40]. After the digestion, the final suspension adjusted to 60 mg wet weight per milliliter was kept at 4°C in standard KHS fully gassed with 5% CO 2 + 95% O 2 until utilization [40]. Aliquots of the tubule suspensions were dried to obtain the tissue dry weight after subtraction of the weight due to salts and mannitol contained in KHS.…”

“…The following metabolites were measured enzymatically on the neutralized PCA extract [40,41]: lactate, pyruvate, L-glutamine, L-glutamate, α-ketoglutarate, L-aspartate, L-alanine, NH 4 + , glucose, urea, L-arginine. Therefore, all intracellular and extracellular metabolites were measured together, allowing to calculate net extraction and production of metabolites irrespectively of intracellular concentration or compartmentation.…”

A Brush Border Membrane-Bound H⁺ -ATPase from the Dog Proximal Tubule

“…Suspensions of dog proximal and thick ascending limb tubules were prepared from the cortex and red medulla of the dog kidney, respectively, as already describee [19] and suspended in Krebs-Henseleit sa line modified to contain 0.5 m.V/ calcium and 50 mM mannitol (KHS). Aliquots of both tubule suspensions were preincubated for 5 min at 37 °C in order to achieve thermic equilibrium before a sample (0.5 ml) was introduced in an oxymctric chamber as previously reported [19].…”

“…Aliquots of both tubule suspensions were preincubated for 5 min at 37 °C in order to achieve thermic equilibrium before a sample (0.5 ml) was introduced in an oxymctric chamber as previously reported [19]. The consumption of oxygen by tubules was measured in the presence of 10 mA/ lactate + 1 mM pyruvate + 10 mA/glutamine + 1 mA/glutamate for proximal tubules and in the presence of 10 mA/lac tate + 1 mA/pyruvate for thick ascending limb tubules.…”