Search

Tubular organization and vascular-tubular relations were studied by double injection in canine kidneys. Blood vessels were injected via the artery after perfusion fixation. Tubules were injected by micropipettes inserted into the urinary spaces of selected glomeruli in cleared slices. One hundred proximal convoluted tubules, 16 Henle loops, and 5 distal convoluted tubules were defined. Only subcapsular proximal convolutions were perfused by efferent vessels arising from the same glomerulus (43 of 55). In midcortex, proximal convolutions were generally perfused over less than half their length by the parent efferent (21 of 31). Here tubules entirely perfused by the parent efferent were rare (2 of 31). No inner cortical proximal convolutions were perfused by the efferent from the same glomerulus (0 of 14). Henle's loops were found to be perfused by the efferents of many glomeruli regardless of the cortical position of the parent glomerulus. Distal convolutions shared the perfusion of proximal convolutions of the same glomerulus. Thus, each nephron is apparently functionally dependent on efferent blood from glomeruli of many other nephrons. New synoptic diagrams of canine renal organization are presented.

We modified and improved enzyme digestion and density gradient separation procedures to obtain fractions of proximal and distal renal tubules with high yield and viability. Kidneys from two anesthetized adult Wistar rats were flushed with Krebs-Henseleit buffer (KHB) and then perfused in situ with recirculated KHB containing collagenase and hyaluronidase at 125 mmHg. Cortices were excised, minced, and incubated in KHB containing enzymes for 35 min at 37 degrees C. Dissociated tubules were removed at 10-min intervals, rinsed, and placed in KHB containing 10% calf serum, vitamins, and amino acids at 4 degrees C. Separation was achieved by suspending the tissue in 45% isosmotic Percoll layered over an undiluted Percoll cushion and centrifuging. Proximal tubules sedimented near the cushion. Distal segments were isolated in the uppermost bands of a second 35% Percoll separation. Viability was greater than 95% as measured by lactate dehydrogenase leakage and quantitated by oxygen consumption and ATP content. Basal oxygen consumption was greater than 33 nmol O2 X min-1 X mg protein-1 in all fractions and was stimulated by succinate and inhibited by amiloride and ouabain. Basal ATP content averaged 9.7 nmol/mg ATP. An average 3.3-fold separation for the proximal fraction and 24.5-fold separation for the distal fraction was assessed by the enrichment of six specific enzyme markers, with several of the markers indicating separations up to 32-fold. Isolated tubules also displayed functional responses to parathyroid hormone and vasopressin. Distal, but not proximal, segments demonstrated significantly increased adenosine 3',5'-cyclic monophosphate formation with vasopressin.

Effects of parathyroid hormone (PTH), calcitonin (CT), vasopressin (VP), and glucagon (GL) on adenylate cyclase activity and cyclic AMP (cAMP) levels in isolated cortical thick ascending limbs of Henle's loop (CTAL) of the rat kidney were examined. PTH, CT, and VP each stimulated adenylate cyclase of this nephron segment in a dose-dependent manner. Stimulation of the enzyme activity was greatest with a maximal dose of PTH and least with VP. With maximal doses, the effects of PTH and CT were not additive; whether or not the effects of maximal doses of VP and PTH or CT were additive was not clear. All three hormones increase cAMP in intact CTAL ina dose-dependent manner. Maximal doses of PTH, CT, VP, and GL resulted in comparable rises in cell cAMP, and there was no additive effect. These data suggest that PTH and CT may stimulate the same adenylate cyclase moieties, whereas VP may stimulate distinct enzyme moieties, and that these three peptide hormones as well as GL definitely act on the same cell group in rat CTAL. Thus, it is possible that these hormones may induce qualitatively similar effects on CTAL functions if such effects are mediated by cAMP.

Micropuncture studies were carried out in the rat to evaluate the in situ distensibility characteristics of the proximal and distal tubules under a variety of experimental conditions. In the first phase, we determined the response of tubular diameter (D) to changes in tubular pressure (P) induced by partially obstructing single tubules. The response observed under these conditions (i.e., when interstitial pressure is presumed to be constant) has been defined as the compliance of the tubule. Over the range of tubular pressures studied (10-35 mm Hg for the proximal tubule, 5-25 mm Hg for the distal tubule) the compliance characteristics of the proximal and distal tubule were found to be markedly different; the proximal tubular pressure-diameter relationship was linear, DeltaD/DeltaP = 0.45 mum/mm Hg, whereas the distal pressure-diameter relationship was curvilinear, DeltaD/DeltaP = c(-0.1xP+2.2). In the second phase we used the compliance data to construct a series of theoretical pressure-diameter curves that define the response of the tubule to increments in interstitial as well as intratubular pressure. These curves indicate that changes in distal diameter should provide a sensitive index of a rise in interstitial pressure under conditions in which the transtubular pressure gradient is increased by a small amount, but that proximal diameter should provide a more sensitive index of changes in interstitial pressure when the transtubular pressure gradient is increased by a large amount. In subsequent experiments in which furosemide was administered, we observed that the pressure-diameter relationships for both the proximal and distal tubule were indistinguishable from the compliance curves, a finding consistent with the interpretation that interstitial pressure was not appreciably changed from control. By contrast, when mannitol was administered, both proximal and distal tubular pressure-diameter relationships were significantly altered in a fashion consistent with a large increase in interstitial pressure. Neither with furosemide nor mannitol administration did it appear likely that significant changes in tubular compliance could account for the observed behavior of the tubule.Finally, we propose that a knowledge of tubular compliance will be useful in exploring the interrelationships between tubular and peritubular pressures, tubular anatomy, and transtubular ionic permeability. Recent studies linking changes in the geometry of lateral intercellular spaces of the tubule to changes in passive ion movement suggest that an investigation of such anatomical-functional correlates should be productive.

To determine the sites of tubular phosphate reabsorption in the nephron, microinjection studies were undertaken, utilizing isotonic electrolyte solutions, containing either 1.4 or 8.0 mM phosphate and radioactive PO(4)-(33)P and inulin-(3)H, in rats made mildly diuretic by infusion of mannitol. The injected sites were localized by the technique of latex dissection. The relation between proximal tubular length and per cent (33)P recovery for injections of 1.4 mM phosphate (physiological amounts) suggest that relatively little reabsorption of phosphate occurs in the distal 30% of the proximal tubule compared with the proximal portion of the tubule. The corresponding recoveries for proximal tubular microinjections of 8.0 mM phosphate fall along a smooth curve tending to plateau with essentially complete (33)P recovery (> 95%) beyond 50% of the tubule. Absolute reabsorption of injected phosphate for both concentrations (i.e., absolute efflux per unit tubular length in the proximal tubule) was independent of phosphate delivery, since the relationship between reabsorption and site of injection was no different for the two concentrations. Distal convoluted tubular microinjections for both phosphate concentrations showed complete recovery of (33)P from all injection sites. THE DATA INDICATE THAT: (a) no phosphate reabsorption occurs in the distal convoluted tubule or in the collecting duct, (b) phosphate efflux per unit tubular length is greater in the first one-third of the proximal tubule than in the remaining two-thirds, and (c) in the last two-thirds of the proximal tubule, absolute phosphate reabsorption is relatively small and might be limited by factors other than the amount or concentration of injected phosphate.