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Studies were undertaken to examine the mechanism whereby changes in intraluminal flow rates after reabsorption in the isolated perfused proximal tubule of the rabbit. All protocols employed the technique of in vitro perfusion of isolated segments of the proximal convoluted tubule. Stepwise elimination of d-glucose and l-alanine from an artifical perfusate stimulating ultrafiltrate decreased the unidirectional flux of sodium, transtubular potential difference, and net water absorption. Using isosmolal ultrafiltrate as the perfusate, net fluid reabsorption and the unidirectional lumen-to-bath flux of sodium and chloride decreased with a decrease in flow rate below 11 nl/min, but neither net fluid reabsorption nor the unidirectional fluxes of sodium and chloride increased further as the perfusion rate was increased above 11 nl/min. The unidirectional flux of 14C-urea was not affected by changes of perfusion rate from 1.6 to 44 nl/min. The dependence of net fluid reabsorption and unidirectional fluxes of sodium and chloride on flow rate per se, and not on intraluminal hydrostatic pressure or geometry, was established by demonstrating their decrease despite a rise in intraluminal pressure and inside diameter produced by counterpressure at the collecting end of the tubule, while flow was decreased. Ouabain decreased net fluid reabsorption to near zero at all flow rates, but ouabain had no effect on the flow-dependency of unidirectional sodium anf sodium was eliminated with a decrease in bicarbonate concentration and removal of d-glucose and l-alanine from the perfusate. Thus, the present studies demonstrate that net water and unidirectional sodium and chloride fluxes are flow-dependent. At flow rates somewhere below 11 nl/min, unidirectional fluxes decreased with decreasing perfusion rates; however, at perfusion rates greater than 11 nl/min, there was no further effect of perfusion rate on either net water absorption or the unidirectional fluxes of sodium or chloride. These effects may be partly mediated through the flow-dependent changes in the intraluminal concentration of bicarbonate, d-glucose, and 1-alanine.

Our previous in vitro studies have disclosed that the thin ascending limb of Henle (tALH) possesses some unique membrane characteristics. In those studies we failed to demonstrated active transport of sodium chloride by the tALH, although it was shown that the isotopic permeability to sodium and chloride was unusually high. However, we did not examine the mechanisms by which the apparent high permeation of sodium chloride occurs. Thus the purpose of the present studies was to elucidate the mechanism of sodium chloride transport across the isolated tALH of the rabbit by conducting four different types of studies: (1) comparison of the observed chloride and sodium flux ratios to those predicted by Ussing's equation under imposed salt concentration gradients; (2) kinetic evaluation of chloride and sodium fluxes; (3) examination of the effect of bromide on the kinetics of chloride transport; and (4) experiments to test for the existence of exchange diffusion of chloride. In the first set of studies the predicted and the theoretical flux ratios of sodium were identical in those experiments in which sodium chloride was added either to the perfusate or to the bath. However, the observed chloride flux ratio, lumen-to-bath/bath-to-lumen, was significantly lower than that predicted from Ussing's equation when 100 mM sodium chloride was added to the bath. In the second set of experiments the apparent isotopic permeability for sodium and for chloride was measured under varying perfusate and bath NaCl concentrations. There was no statistical change in the apparent sodium permeability coefficient when the NaCl concentration was raised by varying increments from 85.5 to 309.5 mM. However, permeation of 36Cl decrease significantly with an increase in Cl from 73.6 to 598.6 mM. These events could be explained by a two component chloride transport process consisting of simple diffusion and a saturable facilitated diffusion process with a Vmax = 3.71 neq mm-1 min-1. In the third set of studies it was shown that bromide inhibits transport of chloride and that the magnitude of inhibition is dependent on chloride concentrations. The fourth set of studies ruled out the existence of exchange diffusion. In conclusion, these studies indicate that sodium transport across tALH is by simple passive diffusion, while chloride transport across tALH involves at least two mechanisms: (1) simple passive diffusion; and (2) a specific membrane interaction process (carrier-mediated) which is competitively inhibited by bromide.

Rabbit medullary collecting duct (MCD) from inner stripe of outer medulla has been identified as a major distal nephron acidification site. The isolated, perfused tubule technique was used to examine the roles of mineralocorticoid and glucocorticoid in regulation of MCD acidification. Surgical adrenalectomy reduced bicarbonate reabsorptive rate (JHCO3, pmol X mm-1 X min-1) from the normal of 9.79 +/- 1.21 to 0.67 +/- 1.1. Chronic administration of deoxycorticosterone acetate (DOCA) increased JHCO3 of MCD significantly to 18.02 +/- 1.62 whereas chronic dexamethasone administration did not affect JHCO3. The direct effects of aldosterone and dexamethasone upon MCD acidification were examined by perfusing tubules harvested from adrenalectomized rabbits in the presence of aldosterone or dexamethasone. Aldosterone, at 5 X 10(-8) M, increased JHCO3 significantly from 1.27 +/- 0.28 to 3.09 +/- 0.34. At 10(-6) M, aldosterone produced a greater increase in JHCO3 from 0.67 +/- 1.1 to 9.39 +/- 1.59. In vitro dexamethasone treatment had no effect on JHCO3. Studies examining the sodium dependence of aldosterone-stimulated acidification demonstrated that JHCO3 in tubules harvested from normal and deoxycorticosterone acetate-treated animals was unaffected by total replacement of sodium with tetramethylammonium. Likewise, luminal amiloride (5 X 10(-5) M) had no effect on JHCO3 in tubules harvested from adrenalectomized and normal animals. Moreover, the acute, in vitro stimulatory effect of aldosterone was seen to occur in the presence of luminal amiloride. These studies define a mammalian distal nephron segment that possesses major acidifying capacity, which is modulated by mineralocorticoid but independent of luminal sodium.

This study was designed to examine whether prostaglandin E2 can directly affect sodium transport across isolated perfused rabbit renal collecting tubules. Changes in transepithelial potential and isotopic sodium fluxes in response to peritubular prostaglandin E2 were measured. In addition, changes in transepithelial potential of the outer medullary collecting tubule in response to prostaglandin E2 were also measured. With few exceptions, all rabbits received 5 mg/day desoxycorticosterone acetate for 4-11 days before experimentation. The results of the experiments show that: (a) prostaglandin E2 inhibits the negative transepithelial potential in the cortical collecting tubule as well as the outer medullary collecting tubule; (b) prostaglandin E2 inhibits net sodium transport out of the lumen by inhibiting efflux while backflux is unaffected; (c) prostaglandin E2 produces this inhibition within 15 min, and the effects are dose dependent and reversible. These results suggest that prostaglandin E2 may modulate sodium transport in vivo and may contribute to the final regulation of sodium excretion.

Numerous previous studies have proposed a role for angiotensin II (AII) in the renal regulation of salt balance. At least one nephron site, the proximal convoluted segment, has been implicated in this role. We used in vitro microperfusion of rabbit proximal convoluted tubules to further examine this question. To insure use of appropriate in vivo concentrations as well as potency of the hormone in vitro, we measured plasma AII levels by radioimmunoassay in normal, sodium-depleted, and adrenalectomized rabbits, and measured AII activity by bioassay after incubation in various microperfusion baths. Plasma levels ranged from approximately 2 X 10(-11) to 5 X 10(-11) M. AII activity was stable in Ringer's solution plus albumin, but not in rabbit serum or Ringer's solution plus fetal calf serum. In Ringer's solution plus albumin, physiologic concentrations of AII stimulated volume reabsorption (Jv). 10(-11) M AII increased Jv by 16% (P less than 0.01). 10(-10) M AII produced a lesser increase, 7.5% (P less than 0.05). At a frequently studied, but probably pharmacologic dose, 10(-7) M AII inhibited Jv by 24% (P less than 0.001). AII at 10(-11) M did not stimulate Jv in the presence of 10(-7) M saralasin. Though previous studies have suggested agonistic effects of saralasin alone in epithelia, we found no significant effect of 10(-7) M saralasin on Jv. None of the AII doses measurably changed transepithelial voltage. We conclude that AII in physiologic doses directly stimulates Jv in proximal convoluted tubules and this effect is probably receptor mediated and, within the limits of detection, electroneutral.

It has been previously demonstrated that superficial (SF) versus juxtamedullary (JM) proximal convoluted tubules (PCT) of rabbit have different intrinsic transport characteristics: Na is less permeable than Cl in the latter portions of the SFPCT, while Cl is less permeable than Na throughout the JMPCT. These permeability differences have major influences on the mechanism of salt reabsorption across the proximal tubules. However, both populations of PCT have the same embryological origin. Studies therefore were designed to examine whether two distinct populations of PCT exist at birth or whether a second population of tubules develops with delivery as a result of some unidentified acute change in humoral factor affecting epithelial transport properties. Both morphological and electrophysiological studies were conducted on PCTs from rabbits within 36 h of birth. Both transmission and scanning electron microscopy studies clearly disclosed that SFPCT are less mature than the JMPCT. Also the SFPCT had a lower Na permeability than Cl (0.55 +/- 0.06) while the JMPCT had a higher Na permeability than Cl (1.37 +/- 0.11). Thus these studies demonstrate that intrinsic heterogeneity of PCT is present at birth. Since the SF Na to Cl permeability approximates that of free diffusion these studies suggest that epithelial discrimination in PCT is part of a maturation process.

Several hormones induce phosphatidylinositol turnover in cell membranes and thus activate protein kinase C. Activation of protein kinase C can, in turn, have effects on epithelial transport. These experiments were designed to investigate the effects of two activators of protein kinase C, phorbol 12-myristate,13-acetate (PMA) and L-alpha-1,2-dioctanoylglycerol (L-alpha-1,2-DOG), and two inactive analogues, 4 alpha-phorbol and 4-O-methyl phorbol 12-myristate,13-acetate, on sodium, potassium, chloride, and total CO2 transport in the rabbit cortical collecting tubule. Utilizing in vitro microperfusion techniques, we found that activation of protein kinase C with either PMA or L-alpha-1,2-DOG significantly inhibited net sodium absorption, net potassium secretion and transepithelial voltage in a dose-dependent manner. There was no effect on net chloride or total CO2 transport. In contrast, the inactive phorbol analogues did not alter either sodium or potassium transport. These studies demonstrate that in the rabbit cortical collecting tubule sodium and potassium transport can be inhibited by compounds known to activate proteins kinase C. Thus, hormones that induce phosphatidylinositol turnover in the rabbit cortical collecting tubule may lead to inhibition of sodium transport by activation of protein kinase C.

Micropuncture studies have indicated that variation in peritubular oncotic pressure influences net transport of fluid out of the proximal tubule. The present in vitro studies on isolated perfused rabbit proximal convoluted tubules were designed to examine whether protein concentration gradient must act across the peritubular capillary membrane to influence reabsorption, or whether it can exert a direct effect across the tubular basement membrane 71 proximal tubules were perfused with ultrafiltrate made isosmolal to bathing fluids, the latter having identical electrolyte composition as the perfusing ultrafiltrate, but adjusted to three oncotic pressures: hypooncotic, protein 0.0 g/100 ml; control isooncotic serum, protein 6.4 g/100 ml; and hyperoncotic, protein 12.5 g/100 ml. Net volume flux (nl/mm per min), net Na flux (nEq/mm per min), unidirectional Na flux from bath to lumen (nEq/mm per min), and passive permeability coefficient (x 10(-5) cm/sec) for Na (P(Na)), urea (P(urea)), and sucrose (P(sucrose)) were determined using isotopic techniques. When the bath was hypooncotic, there was (as compared with isooncotic serum) a significant decrease in net volume (38%) and net sodium (40%) flux, but no change in P(Na), P(urea), or transtubular potential; however, P(sucrose) increased significantly (78%). In experiments in which hyperoncotic bath was used, there was (compared with isooncotic serum) an increase in net volume (28%) and net sodium (30%) flux, but transtubular potential difference did not change significantly. These data demonstrated that changes in the ambient protein concentration gradient exert direct effects upon proximal tubular reabsorption. Because penetration of sucrose (an index of intercellular movement) but not urea (an index of transcellular movement) varied with changes in tubular reabsorption, it is suggested that oncotic pressure acts by altering the rate of back-leak of reabsorbate through extracellular pathways between tubular cells. It is concluded that an effect of protein concentration on reabsorption can be exerted directly across the basement membrane, without necessary interposition of the capillary bed.