Urinary concentrating mechanism: the role of the inner medulla.
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Animals↗
Biomedical subjects
M A Knepper
Publications and source records attributed to M A Knepper.
Ammonium and bicarbonate transport in rat outer medullary collecting ducts.
Previous in vitro studies have demonstrated spontaneous bicarbonate absorption in the outer stripe portion of the rat outer medullary collecting duct (OMCD) and inner medullary collecting duct, but net acid transport has not been studied in the inner stripe of the rat OMCD (OMCDIS). When we perfused isolated OMCDIS segments with identical bath and perfusate solutions containing HCO-3 and NH4Cl, HCO-3 was spontaneously absorbed, and total ammonia was spontaneously secreted at rapid rates in tubules from both deoxycorticosterone (DOC)-treated and untreated rats. We next measured the NH3 flux due to imposed NH3 concentration gradients. Carbonic anhydrase (CA), when added to the lumen, enhanced the NH3 flux, implying an absence of endogenous CA. The NH3 permeability was 0.0042 +/- 0.0007 cm/s. By measuring the luminal pH in perfused OMCDIS segments with an imposed lumen-to-bath NH3 gradient, we determined the pH at the end of the lumen to be 0.23 units below the equilibrium pH calculated from the simultaneously measured total CO2 concentration in collected fluid, confirming the lack of luminal CA. These results are consistent with the view that ammonium secretion in the OMCDIS occurs predominantly by H+ secretion and parallel NH3 diffusion. A luminal disequilibrium pH due to H+ secretion in the absence of endogenous luminal CA enhances the NH3 entry rate. Spontaneous net acid secretion appears to occur more rapidly in the OMCD than in other parts of the rat collecting duct system.
Ammonia↗
Kinetics of urea and water permeability activation by vasopressin in rat terminal IMCD.
The vasopressin-regulated urea carrier and the vasopressin-regulated water channel are distinct transporters present in the apical membrane of the inner medullary collecting duct (IMCD) cells. To assess whether these transporters may be activated by common mechanisms, we investigated the time course of increase of urea and water permeability in response to vasopressin in isolated perfused terminal IMCD segments. The permeability responses were determined through the use of a specially designed continuous-flow fluorometer for rapid analysis of collected tubule fluid samples. The time courses of activation of the two transporters by vasopressin were virtually identical. Both urea and water permeability displayed a rapid initial increase for the first 10 min followed by a slower secondary response lasting at least 30 additional min. The lag periods between vasopressin addition and the initial rise in permeability were the same for urea (34.2 +/- 8.8 s) and water (34.8 +/- 8.9 s) transport activation. Furthermore, the initial rate of permeability increase (normalized by the total increase) was not significantly different for the two transport processes. The lag periods for the increase in urea permeability in response to 8-bromoadenosine 3',5'-cyclic monophosphate and vasopressin were not significantly different. The results are consistent with the view that the rate-limiting step in vasopressin-induced activation is the same for both the urea carrier and water channel and may lie at a step beyond generation of adenosine 3',5'-cyclic monophosphate.
Animals↗
Time course of renal glutamate dehydrogenase induction during NH4Cl loading in rats.
To study mechanisms involved in renal glutamate dehydrogenase (GDH) regulation in response to systemic acid loading, we have measured blood pH, ammonium excretion, renal GDH mRNA levels, and GDH activity in rats. Acid intake (0.28 M NH4Cl in drinking water for 3 days) increased GDH mRNA levels in the renal cortex, but had no effect in the outer stripe of the outer medulla, inner stripe of the outer medulla, or the inner medulla. Rats were subjected to a step change in acid intake by alkali loading for 3 days (7.2 meq NaHCO3 per day in food slurry) and shifting to acid loading for up to 7 days (7.2 meq NH4Cl in food slurry). Ammonium excretion rose rapidly, increasing by 14-fold in the first 24-h period and 38-fold in the second 24-h period. Cortical GDH mRNA levels were increased relative to alkali-loaded values by 3.7-fold in 24 h, 4.3-fold in 4 days, but only 2.2-fold in 7 days. GDH activity was unchanged after 24 h of acid intake, but was significantly increased after 48 h. We concluded the following: 1) GDH mRNA is present in all regions of the kidney, but levels increase in response to acid loading only in the renal cortex; 2) GDH mRNA levels increase within 1 day after the initiation of acid loading, but the associated increase in functional enzyme activity takes 2 or more days; and 3) the large increases in ammonium excretion that occur in the first day after initiation of acid loading are not dependent on increased GDH activity.
Ammonium Chloride↗
Hormone and autacoid regulation of cAMP production in rat IMCD subsegments.
The inner medullary collecting duct (IMCD) of the rat consists of two structurally and functionally distinct segments, i.e., the initial and the terminal IMCD. To identify factors that may regulate the transport function in the IMCD segments, we assessed whether catecholamines, carbachol, prostaglandin E2 (PGE2), bradykinin, glucagon, calcitonin, parathyroid hormone, or epidermal growth factor affects adenosine 3',5'-cyclic monophosphate (cAMP) production in microdissected tubules in the presence and absence of arginine vasopressin (AVP, 0.1 nM). All experiments were performed in the presence of 3-isobutyl-1-methylxanthine, and cAMP was measured by radioimmunoassay. Epinephrine (greater than or equal to 50 nM) and clonidine (greater than or equal to 1 microM) markedly decreased AVP-induced cAMP levels in both IMCD segments. However, phenylephrine did not show an effect. The inhibitory effect of epinephrine was blocked by yohimbine (50 nM) but not by prazosin (50 nM). In isolated perfused terminal IMCDs, epinephrine inhibited AVP-stimulated urea permeability. Isoproterenol (1 microM), in the absence of AVP, caused a significant increase in cAMP level only in the initial IMCD. Propranolol (1 microM) inhibited this isoproterenol effect, but atenolol did not. Dopamine (less than or equal to 1 microM) had no effect on cAMP levels in either IMCD segment. Carbachol, PGE2, and the various peptide hormones had no effect on cAMP levels (+/- AVP) in either IMCD segment. We conclude that an adrenergic beta 2-receptor is present only in the initial IMCD, where its occupation increases cAMP production. We conclude also that an adrenergic alpha 2-receptor is present in both IMCD segments, where its occupation inhibits AVP-induced cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
In vitro perfusion of chinchilla thin limb segments: segmentation and osmotic water permeability.
The thin limb segments of the long loop of Henle are thought to play important roles in the urinary concentrating mechanism. In this study, we present new approaches to the identification, dissection, and in vitro perfusion of individual thin limb segments from all levels of the chinchilla renal medulla, including the deepest portions of the papilla. We have applied these techniques to the investigation of the osmotic water permeability along the chinchilla long loop of Henle. The results demonstrate that the osmotic water permeability of the thin descending limb is not uniformly high along its length, as previously thought, but that the distal 20% of the long-loop descending limb has a very low water permeability (approximately 50 microns/s). The transition to the low water permeability region of the thin descending limb is accompanied by a relatively abrupt change in morphology (increased cellularity and decreased diameter) that is readily perceptible in the perfused segments and even in the dissection dish. In contrast, the upper part of the chinchilla long-loop thin descending limb had an extremely high osmotic water permeability (greater than 2,000 microns/s) as observed in other species. Thin ascending limbs from deep in the inner medulla had water permeabilities that were indistinguishable from zero, as previously found in thin ascending limbs from near the inner-outer medullary junction. The presence of a low-water-permeability portion of the long-loop thin descending limb in chinchilla may have important implications with regard to the inner medullary concentrating process. A relatively low osmotic water permeability (397 microns/s) was also found in the deep inner medullary portion of the thin descending limb from the rat.
Animals↗
Experimental tests of three-dimensional model of urinary concentrating mechanism.
Recently, a new model of the urinary concentrating process has been proposed that takes into account the three-dimensional architecture of the renal medulla. Under the assumptions of the model, computer simulations predicted significant axial osmolality gradients in the inner medulla without active transport by the inner medullary loop of Henle. Two of the model assumptions (which constitute hypotheses for this study) were: (1) the osmotic water permeability of the initial part of the inner medullary collecting duct (initial IMCD) is very low even in the presence of vasopressin; and (2) there is significant lateral separation of structures such that thin descending limbs are far from the collecting ducts at the same inner medullary level. The first hypothesis was addressed by perfusing rat initial IMCD segments in vitro and measuring osmotic water permeability. With the osmotic gradient oriented as predicted by the model (lumen greater than bath), vasopressin increased the osmotic water permeability from 286 to 852 microns/s. Three additional series of experiments confirmed the high water permeability in the presence of vasopressin. The second hypothesis was addressed by morphometric analysis of histologic cross-sections of the rat renal medulla. Mean distances of descending limbs to the nearest adjacent collecting duct were very small throughout the inner medulla (less than 6 microns) and substantially less than in the outer medulla (28 microns). It was concluded that the data are inconsistent with both hypotheses and therefore do not support the feasibility of the "three-dimensional" model of the renal inner medulla. The axial distributions of loops of Henle and collecting ducts in the rat renal medulla are also reported.
Animals↗
Renal tubular actions of ANF.
Many of the earliest investigations of the renal effects of atrial natriuretic factor (ANF) pointed to the glomerulus as a major site of the peptide's action. More recently, there have been many reports showing various effects of ANF on renal tubular epithelia, including collecting ducts, thick ascending limbs of Henle's loop, thin limbs of Henle's loops, and proximal tubules. The purpose of this review is to summarize the evidence for renal tubular actions of ANF and analyze it from the perspective of the specialized functions of the individual nephron segments, addressing the question: can renal tubule effects of ANF play a significant role in the precise day-to-day regulation of renal NaCl and water excretion? Based on these considerations, we propose that long-term renal tubular action of ANF may be distinct from its short-term natriuretic effect. The short-term action of ANF to accelerate salt and water excretion may play a role in the overall response to acute volume overload. This action of ANF appears to be largely due to an ANF-mediated increase in glomerular filtration rate accompanied by a blunting of the tubuloglomerular feedback mechanism, perhaps with some contribution from ANF-mediated inhibition of fluid absorption in the proximal tubule. In contrast, contributions of ANF to the precise day-to-day regulation of salt and water excretion are likely to be chiefly due to ANF-mediated inhibition of NaCl and water absorption in collecting ducts, but may also involve actions of ANF on the loop of Henle.
Animals↗
Permeabilities of rat collecting duct segments to NH3 and NH4+.
We have measured NH3 and NH4+ permeability coefficients in collecting ducts from the cortex, outer medulla, and inner medulla of the rat kidney. Isolated collecting duct segments of the rat were perfused with bicarbonate-buffered solutions containing carbonic anhydrase to eliminate any pH disequilibrium in the tubule lumen. NH3 or NH4+ concentration gradients were set up between the bath and the lumen. By measuring the total CO2 and total ammonia concentrations in the bath, the perfusate, and collected fluid, the NH3 and NH4+ concentrations were determined. Then, using the flow rate in the tubule and the tubule dimensions, we calculated the apparent permeability in each collecting duct segment for NH3 and NH4+. The NH3 permeabilities were as follows: 0.002 cm/s in the inner medullary collecting duct, 0.012 cm/s in the outer medullary collecting duct, and 0.024 cm/s in the cortical collecting duct. The NH4+ permeabilities for all segments were on the order of 10(-5) cm/s or less. The relative permeability values for the two ammonium species are consistent with the view that the secretion observed in vivo in collecting ducts is due to passive diffusion of NH3 from the interstitium to the lumen of the duct, parallel with H+ secretion.
Ammonia↗
Distribution of luminal carbonic anhydrase activity along rat inner medullary collecting duct.
The isolated perfused tubule technique was utilized to determine whether endogenous luminal carbonic anhydrase is present in the initial or terminal parts of the inner medullary collecting duct (IMCD) of the rat. This was accomplished by measuring the luminal disequilibrium pH in the presence of a large luminal proton source created by perfusing the lumen with a solution containing 10 mM NH4Cl. (NH3 efflux causes H+ to be released from NH+4 in the lumen). The disequilibrium pH was calculated by subtracting the equilibrium pH from the measured pH at the end of the tubule lumen. The end-luminal equilibrium pH was calculated from the total CO2 concentration in the collected fluid, as measured by microcalorimetry. The end-luminal pH was determined by measuring the fluorescent signal from the the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), which was added to the luminal perfusate in its nonesterified form. In the initial IMCD, there was no measurable disequilibrium pH. With the addition of the carbonic anhydrase inhibitor acetazolamide to the luminal fluid, a significant acidic pH disequilibrium was elicited. In the terminal IMCD under control conditions a statistically significant acidic disequilibrium pH was measured. The disequilibrium was obliterated when exogenous carbonic anhydrase was added to the luminal perfusate. These findings were verified by measuring total ammonia flux by ultramicrofluorometry. The results demonstrate endogenous luminal carbonic anhydrase activity in the initial IMCD but a lack of enzyme activity in the terminal IMCD.
Ammonia↗
Regulation of collecting duct water permeability independent of cAMP-mediated AVP response.
We have used the isolated perfused tubule technique, measurements of adenosine 3',5'-cyclic monophosphate (cAMP) content in single tubules, and freeze-fracture electron microscopy to study the basis of high vasopressin-independent (basal) osmotic water permeability (Pf) in the terminal inner medullary collecting duct (IMCD) of the rat. The results confirmed the observation that the basal Pf of the terminal IMCD is considerably higher than that of the initial IMCD. They also showed that the basal Pf of the terminal IMCD is regulated by in vivo factors related to water intake, such that a very high vasopressin-independent Pf can be induced in isolated tubules by prior in vivo thirsting. Tubules from thirsted rats did not display elevated urea permeabilities, nor did they exhibit measurable cAMP levels in the absence of exogenous vasopressin, indicating that the high basal Pf was not due to residual binding of vasopressin to its receptors. Freeze-fracture studies in thirsted rats demonstrated the presence of intramembrane particle (IMP) clusters in both initial and terminal IMCD, with more in the latter. Water loading of the rats suppressed the incidence of clusters almost entirely but did not fully suppress the basal Pf in the terminal IMCD, raising the possibility that a component of transepithelial water transport may occur independently of the vasopressin-regulated IMP clusters. On the basis of these results, we conclude that the vasopressin-independent Pf in the terminal IMCD can be stably elevated to very high levels in response to in vivo thirsting. This elevation appears to be due to a chronic conditioning effect mediated by unknown in vivo factors and is not due to the short-term cAMP-mediated regulatory effect of vasopressin.
Animals↗
RT-PCR microlocalization of mRNA for guanylyl cyclase-coupled ANF receptor in rat kidney.
Microlocalization of mRNA coding for the guanylyl cyclase-coupled atrial natriuretic factor (ANF) receptor was carried out in the rat kidney. We used a combination of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected renal tubule segments, glomeruli, and vasa recta bundles. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P-labeled probe. Among renal tubule segments, the largest signal was found in the terminal inner medullary collecting duct (IMCD). Slightly smaller signals were found in the initial IMCD and in loop of Henle segments from the inner medulla. Readily detectable signals were also seen in the following segments (in descending order): cortical collecting duct, proximal convoluted tubule, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and outer medullary collecting duct. Large signals were also detected in glomeruli and in vasa recta bundles from the inner stripe of the outer medulla. Based on these results, we conclude that 1) renal microlocalization of specific mRNAs coding for hormone receptors is feasible through application of the RT-PCR procedure in microdissected renal tubules and vascular elements, and 2) the gene for the guanylyl cyclase-coupled ANF receptor is broadly expressed along the nephron, raising the possibility that multiple sites of ANF action are present.
Animals↗
Regulation of renal medullary ammonium accumulation.
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Ammonia↗
Mapping ammoniagenic enzyme activities along the rat nephron.
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Ammonia↗
Role of countercurrent multiplication in renal ammonium handling: regulation of medullary ammonium accumulation.
Ammonium (NH3 plus NH4+), produced predominantly in the proximal tubule, is transferred to the final urine by a process involving countercurrent multiplication of ammonium which generates an ammonium concentration gradient in the renal medulla. It was hypothesized that if urinary ammonium excretion rates are controlled in part by the medullary ammonium gradient, changes in hydration and acid-base state should cause changes in the medullary ammonium gradient consistent with expected changes in urinary ammonium concentrations. To test that hypothesis, rats were subjected to water diuresis, water deprivation, water deprivation plus furosemide, and dietary acid and base loads and corticomedullary ammonium gradients in their kidneys were then measured. Sections were cut along the corticomedullary axis to yield slices of cortex, outer stripe of outer medulla, inner stripe of outer medulla, and three levels of the inner medulla. The total ammonia content of homogenized slices was measured by either a membrane ammonia electrode or an enzymatic technique. Kidneys from water-deprived animals showed a distinct ammonium gradient along the corticomedullary axis, with the highest contents found at the tip of the papilla. The gradient was attenuated by water diuresis and abolished by furosemide. Acid loading enhanced the gradient, and base loading abolished it. These results indicate that the corticomedullary ammonium gradient is regulated in response to changes in hydration and acid-base state.
Acid-Base Equilibrium↗
NH4+ transport in the kidney.
We have described the overall process that is responsible for the efficient transfer of ammonium from its production site in the proximal tubule cells to the final urine. The mechanism depends on direct NH4+ transport at a number of sites. There appears to be a predominance of NH3 over NH4+ transport in net total ammonia transport only in the collecting ducts and possibly the descending limbs of Henle's loop. Several examples of physiologically important direct NH4+ transport in the kidney were described. First, coupled Na/NH4/2Cl transport across the apical membrane of the thick ascending limb of Henle's loop mediates secondary active transport of ammonium, which drives countercurrent multiplication of ammonium in the renal medulla. Second, part of the NH4+ uptake across the apical membrane of the thick ascending limb may occur as a result of penetration by NH4+ through apical K+ channels. It is unknown whether NH4+ penetrates K+ channels in other tubule segments. Third, NH4+ can be actively transported into cells by substitution of NH4+ for K+ on the Na-K-ATPase. This NH4+ transport process is likely to be rapid enough to be physiologically significant only in the inner medulla, where NH4+ concentrations are high enough to successfully compete with K+. Fourth, NH4+ penetrates the paracellular pathway in some nephron segments such as the proximal tubule and thick ascending limb. Simple passive diffusion of NH4+ via the paracellular pathway is thought to be physiologically important in the thick ascending limb where it contributes to net NH4+ absorption.
Absorption↗
Microdissection of kidney tubule segments.
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Animals↗
Net acid transport by isolated perfused inner medullary collecting ducts.
The isolated perfused tubule technique was used to study net acid transport in rat terminal inner medullary collecting duct (IMCD) segments. The stop-flow luminal pH [measured fluorometrically with the acidic form of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in the lumen] fell 0.35 units below the bath pH in tubules from control rats and 0.53 units below the bath in tubules from deoxycorticosterone-treated rats. Tubules from control rats absorbed bicarbonate and secreted ammonium against concentration gradients, although at low rates. In control rats, 10(-8) M vasopressin added to the bath increased bicarbonate absorption almost threefold. Treatment of rats in vivo with deoxycorticosterone significantly increased the rate of bicarbonate absorption in vitro. In vivo NH4Cl loading also significantly increased bicarbonate absorption. Staining microdissected tubules with acridine orange confirmed that the perfused segments lacked intercalated cells. We conclude that the terminal IMCD spontaneously acidifies the lumen despite an absence of intercalated cells. Bicarbonate absorption appears to be regulated by the same factors that affect net acidification in other collecting duct segments.
Absorption↗